CN110801449A - Application of benzisoselenazole derivative in preparation of tumor treatment drug - Google Patents

Abstract

The invention belongs to the technical field of tumor treatment, and discloses an application of benzisoselenazole derivatives in preparing a tumor treatment drug. The benzisoselenazole derivative has a structure of a compound shown as a formula A, can inhibit TrxR activity in mouse serum and tumor tissues, has the tumor inhibition rate on postoperative liver cancer, lung cancer and breast cancer of over 60 percent, has the tumor growth rate of less than 40 percent, and shows the effect of effectively inhibiting postoperative recurrence of tumors. Furthermore, the benzisoselenazole derivative also has a broad-spectrum tumor inhibition effect, shows excellent inhibition effect on various tumors (such as liver cancer, lung cancer, colon cancer, esophageal cancer and the like), is suitable for being used as a postoperative tumor control drug, and has popularization and application values;

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Description

Application of benzisoselenazole derivative in preparation of tumor treatment drug

Technical Field

The invention belongs to the technical field of tumor treatment, and particularly relates to an application of a benzisoselenazole derivative in preparation of a tumor treatment drug.

Background

The incomplete treatment of cancer causes the survival of cancer cells in the body, which is a major cause of cancer cell recurrence. The main treatment means of cancer is surgery, radiotherapy and chemotherapy, the surgery is a mechanical means, the local treatment is thorough, but the cancer cell is dropped after the cancer disease is caused by the metastasis of the cancer cell, and the cancer cell is proliferated or metastasized to the periphery and far away through the ways of local diffusion, blood vessels, lymphatic vessels and the like. According to the monitoring data of the cancer condition of the disease pre-control center in China, the recurrence and metastasis rate of cancer patients in China after operation for 3 months is 50%, the recurrence and metastasis rate of cancer patients in China after operation for 6 months is as high as 69%, and the recurrence or metastasis rate of cancer patients after operation for five years is as high as more than 90%.

Overall, recurrent cancer is more difficult than initial cancer treatment, mainly because: 1. the initial local treatment, such as surgery or radiotherapy, has been performed to remove the cancer cells as much as possible, but some very small cancer cell population may remain, which has long migrated out of the scope of surgery and radiotherapy. These cancer cell populations tend to be very aggressive (rapid growth and rapid spread), i.e., recurring tumors are a very high malignancy subpopulation, and treatment difficulties are greatly increased for tumors of further increased malignancy. 2. Cancer is resistant to the initial chemoradiotherapy treatment and the side effects of the initial treatment can also lead to difficulties in the re-treatment of the recurrent tumor.

Therefore, the technical problem to be solved is to find a drug which can inhibit or eliminate the growth of cancer cells from the root, so as to overcome the defects of the operation and effectively inhibit the postoperative tumor recurrence.

Disclosure of Invention

The invention provides an application of benzisoselenazole derivatives in preparing medicaments for treating tumors.

According to the invention, the benzisoselenazole derivative has a structure of a compound shown as a formula A, and is selected from at least one of the compound shown as the formula A, a precursor, an active metabolite, a stereoisomer, a pharmaceutically acceptable salt, a prodrug and a solvate thereof,

wherein R is₁、R₂Identical or different, independently of one another, from H or the following radicals: c_1-12Alkyl radical, C_3-20A cycloalkyl group; preferably, selected from H, orThe following groups: c_1-6Alkyl radical, C_3-10A cycloalkyl group; illustratively, R₁、R₂Is selected from H.

Wherein R is selected from C_1-12Alkylene, phenylene, biphenylene, triphenylene, or

Wherein M represents Pt, Pd or Rh; preferably, R represents C_1-6Alkylene radicals, e.g. C_1-4Alkylene, for example R is butylene.

Preferably, in the compound shown in the formula A, R₁、R₂Identical or different, independently of one another, from H, -CH₃、-CH₂CH₃、 -CH(CH₃)₂、-C(CH₃)₃、-CH(CH₂)₄or-CH (CH)₂)₅(ii) a R is selected from-CH₂-、-C₂H₄-、-C₄H₈-, phenylene-C₆H₄-。

According to an exemplary embodiment of the present invention, the benzisoselenazole derivative is selected from 1, 2-bis [2- (1, 2-benzisoselenazol-3 (2H) -one) ] -butane, the structure of which is shown below:

according to the invention, the tumors include solid tumors and non-solid tumors, and may be benign and malignant. The tumor includes an initial tumor and a postoperative recurrent tumor. For example, the tumors include, but are not limited to: liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal carcinoma, gastric cancer, skin cancer, bladder cancer, ovarian cancer, prostate cancer, bone cancer, brain cancer, rectal cancer, esophageal cancer, tongue cancer, lymph cancer, oral epithelial cancer, epithelial cervical cancer or chronic myelogenous leukemia. Preferably, the tumor is selected from liver cancer, lung cancer, breast cancer.

Furthermore, the invention also provides application of the benzisoselenazole derivative in inhibiting tumor cell proliferation. For example, the use of benzisoselenazole derivatives for inhibiting the proliferation of tumor cells in vitro or in vivo; preferably, for use in inhibiting the in vitro proliferation of tumor cells; for example, TrxR (thioredoxin oxidoreductase) activity can be inhibited.

For example, the tumor cells include human cancer cells and mouse cancer cells. Such human cancer cells include, but are not limited to: human liver cancer cell HepG2, human liver cancer cell Bel-7402, human liver cancer cell Huh7721, human colorectal cancer cell LoVo, human colorectal cancer cell RKO, human colorectal cancer cell SW480, human lung cancer cell A549, human lung cancer cell H1299, human lung cancer cell SPCA-1, human epithelial cervical cancer cell HeLa, human breast cancer cell MCF-7, human chronic myelogenous leukemia cell k562, human esophageal cancer cell KYSE150, human esophageal cancer cell KYSE450 or human esophageal cancer cell KYSE 510. According to the technical scheme, the tumor cell is selected from human liver cancer cell Bel-7402, human colorectal cancer cell LoVo, human epithelial cervical cancer cell HeLa and human lung cancer cell A549.

The mouse cancer cells include, but are not limited to: mouse liver cancer cell H22, mouse liver cancer cell Hepa 1-6, mouse erythroleukemia cell MEL, mouse kidney cancer cell Renca, mouse lymphoma cell EL-4, mouse lung cancer cell Lewis, and mouse breast cancer cell 4T 1. Illustratively, mouse liver cancer cells H22, mouse lung cancer cells Lewis and mouse breast cancer cells 4T1 are selected.

According to the technical scheme of the invention, the benzisoselenazole derivative is applied to preparing a medicament for treating liver cancer. Preferably, the benzisoselenazole derivative can inhibit the occurrence and the formation of liver cancer; for example, the occurrence and formation of liver cell damage, liver cirrhosis, liver cancer are inhibited. Further, the benzisoselenazole derivative can inhibit abnormal proliferation of liver cells; for example, the expression of TR/Trx can be inhibited.

Preferably, the benzisoselenazole derivative is used in an amount of 1-500 mg/kg based on the weight of a subject (such as a mouse); for example, 1 to 360mg/kg, 50 to 180mg/kg, 50 to 100 mg/kg; illustratively, the amounts are 9, 18, 36, 72, 144 mg/kg.

Preferably, the concentration of the benzisoselenazole derivative is 1-100 mu M; such as 5 to 60 μ M, 10 to 50 μ M; illustratively, the concentration is 10, 20, 30, 40 μ M.

According to the technical scheme of the invention, the benzisoselenazole derivative is applied to preparing a medicine for treating postoperative recurrence of tumor.

Preferably, the tumor is a postoperative recurrent tumor, which has the meaning as described above.

Preferably, the benzisoselenazole derivative can inhibit postoperative recurrence of a tumor by inhibiting the activity of TrxR (thioredoxin oxidoreductase) of the tumor cell.

Preferably, the tumor cell is at least one selected from human liver cancer cell Bel-7402, human colorectal cancer cell LoVo, human epithelial cervical cancer cell HeLa, human lung cancer cell A549, mouse liver cancer cell H22, mouse lung cancer cell Lewis and mouse breast cancer cell 4T 1.

Preferably, the benzisoselenazole derivative is used in an amount of 1-500 mg/kg based on the weight of a subject (such as human tumor cells); for example, 50 to 400 mg/kg; illustratively, the amount is 90, 180, 360 mg/kg.

Preferably, the concentration of the benzisoselenazole derivative is 1-100 mu M; such as 5 to 60 μ M, 10 to 50 μ M; illustratively, the concentration is 10, 20, 30, 40 μ M.

Further, the invention also provides a medicament for preventing and/or treating tumors, which at least comprises the benzisoselenazole derivative. Preferably, the benzisoselenazole derivative has the meaning as described above.

Preferably, the prevention and/or treatment of a tumor comprises prevention and/or treatment in postoperative recurrence of a tumor.

Preferably, the medicament may also optionally comprise at least one pharmaceutically acceptable excipient.

Preferably, the pharmaceutically acceptable excipients are various excipients commonly used or known in the pharmaceutical field, including but not limited to: diluents, binders, antioxidants, pH adjusters, preservatives, lubricants, disintegrants, and the like.

For example, the diluent is selected from lactose, starch, cellulose derivatives, inorganic calcium salts, sorbitol and the like. The binder is, for example: starch, gelatin, sodium carboxymethylcellulose, polyvinylpyrrolidone, and the like. For example, the antioxidant is selected from vitamin E, sodium bisulfite, sodium sulfite, butylated hydroxyanisole, and the like. For example, the pH adjusting agent is selected from hydrochloric acid, sodium hydroxide, citric acid, tartaric acid, Tris, acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, and the like. For example, the preservative is selected from methyl paraben, ethyl paraben, m-cresol, benzalkonium chloride, and the like. For example, the lubricant is selected from magnesium stearate, aerosil, talc, and the like. The disintegrant is, for example: starch, methyl cellulose, xanthan gum, croscarmellose sodium, and the like.

The dosage form of the medicament can be in the form of oral preparations, such as tablets, capsules, pills, powders, granules, suspensions, syrups and the like; it can also be administered by injection, such as injection solution, powder for injection, etc., by intravenous, intraperitoneal, subcutaneous or intramuscular route. All dosage forms used are well known to those of ordinary skill in the pharmaceutical arts.

Further, the present invention also provides a method for treating tumor with the above-mentioned medicament, and a therapeutically effective amount of the medicament is administered to an individual in need thereof.

Preferably, the subject may be a mammal, such as a human, a mouse.

Preferably, the tumor has the meaning as described above.

The invention has the beneficial effects that:

the invention unexpectedly discovers that the BS can inhibit the activity of TrxR in the serum and the tumor tissue of the mouse, the tumor inhibition rate of the BS on the liver cancer, the lung cancer and the breast cancer of the mouse after operation reaches more than 60 percent, the tumor growth rate is lower than 40 percent, and the BS shows the effect of effectively inhibiting the relapse of the tumor after operation.

Furthermore, the invention also unexpectedly discovers that the BS has a broad-spectrum tumor inhibition effect, shows excellent inhibition effect on various tumors (such as liver cancer, lung cancer, colon cancer, esophageal cancer and the like), is suitable for being used as a postoperative tumor control medicament, and has popularization and application values.

The mechanism of the BS as the postoperative control drug for the tumor:

BS can exhibit an excellent inhibitory action on tumor proliferation as a thioredoxin reductase (TrxR) inhibitory drug. Since post-operative tumor growth plays an important role in tumor recurrence, direct inhibition of the regulatory enzyme (TrxR) associated with tumor growth is a substantial and persistent need for tumor growth. In order to meet the requirement, other common chemotherapeutic drugs are adopted, so that the toxicity of the drugs is high, the cost of a patient is high, and the growth initiation of tumorigenesis is not controlled, so that the continuous tumorigenesis growth initiation still can cause new tumor promotion to the patient, and the patient can be trapped in a new pair of already-formed tumor killing treatment. While the BS shows a good mechanism for preventing the formation of key links of liver tumor occurrence, the invention unexpectedly discovers that the BS also has the control effect of the key links of tumor recurrence on the postoperative recurrence of the liver cancer. It is known that recurrence of liver cancer and basic diseases such as liver cirrhosis and hepatitis are closely related, and therefore, a control effect on the progress of cirrhosis formation and hepatitis is also a key factor for postoperative recurrence of liver cancer. In general, the BS has a variety of functions, including: the medicine has the protection effect on the development of cirrhosis and the promotion of hepatitis, has the multiple functions of inhibiting the abnormal proliferation of tumor cells (TrxR inhibition), promoting the apoptosis of the tumor cells and the like, and realizes the effective medicine effect on the control of the postoperative recurrence of the tumor.

Definition and description of terms

Unless otherwise indicated, the definitions of groups and terms described in the specification and claims of the present application, including definitions thereof as examples, exemplary definitions, preferred definitions, definitions described in tables, definitions of specific compounds in the examples, and the like, may be arbitrarily combined and coupled with each other. The definitions of the groups and the structures of the compounds in such combinations and after the combination are within the scope of the present specification.

The term "C_1-12Alkyl is understood to preferably mean a straight-chain or branched, saturated monovalent hydrocarbon radical having from 1 to 12 carbon atoms, preferably C_1-6An alkyl group. "C_1-6Alkyl "is understood to preferably mean a straight-chain or branched, saturated monovalent hydrocarbon radical having 1,2, 3, 4, 5, 6 carbon atoms. The alkyl group is, for example, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, an isopropyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an isopentyl group, a 2-methylbutyl group, a 1-ethylpropyl group, a1, 2-dimethylpropyl group, a neopentyl group, a1, 1-dimethylpropyl group, a 4-methylpentyl group, a 3-methylpentyl group, a 2-methylpentyl group, a 1-methylpentyl group, a 2-ethylbutyl group, a 1-ethylbutyl group, a3, 3-dimethylbutyl group, a2, 2-dimethylbutyl group, a1, 1-dimethylbutyl group, a2, 3-dimethylbutyl group, a1, 3-dimethylbutyl group or a 1. In particular, the radicals have 1,2, 3, 4, 5, 6 carbon atoms ("C)_1-6Alkyl groups) such as methyl, ethyl, propyl, butyl, isopropyl, isobutyl, sec-butyl, tert-butyl, more particularly groups having 1,2, 3 or 4 carbon atoms ("C)_1-4Alkyl groups) such as methyl, ethyl, n-propyl, isopropyl or butyl.

The term "C_3-20Cycloalkyl is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 20 carbon atoms, preferably "C_3-10Cycloalkyl groups ". The term "C_3-10Cycloalkyl "is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. Said C is_3-10Cycloalkyl groups may be monocyclic hydrocarbon groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl, or bicyclic hydrocarbon groups such as decalin rings.

The term "C_1-12Alkylene is understood to mean preferably a straight-chain or branched saturated monovalent hydrocarbon radical having from 1 to 12 carbon atoms, preferably C, with the loss of two hydrogen atoms_1-4An alkylene group. "C_1-4Alkylene "is understood to preferably mean a straight-chain or branched saturated monovalent hydrocarbon radical having 1,2, 3, 4 carbon atoms which has lost two hydrogen atoms. The alkylene group is, for example, methylene, ethylene, propylene, butylene.

The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound of the present invention sufficient to effect the intended use, including but not limited to the treatment of a disease as defined below. The therapeutically effective amount may vary depending on the following factors: the intended application (in vitro or in vivo), or the subject and disease condition being treated, such as the weight and age of the subject, the severity of the disease condition and the mode of administration, etc., can be readily determined by one of ordinary skill in the art. The specific dosage will vary depending on the following factors: the particular compound selected, the dosing regimen to be followed, whether to administer in combination with other compounds, the timing of administration, the tissue to be administered and the physical delivery system carried.

Drawings

FIG. 1 is a comparison of the body weight change and liver coefficient of mice in the cancer induction process of example 1. A: weight change of mice in the cancer inducing process; b: and comparing the liver coefficients of the mice in the cancer induction process, wherein the liver injury stage is at week 6, the liver cirrhosis stage is at week 16, and the liver cancer stage is at week 24. Data values are mean ± sd, n is 6. P <0.05, indicating a statistical difference compared to the control group.

FIG. 2 is the results of observation of liver tissues of control and model mice in example 2: (1) observing with naked eyes; (2) and (5) observing pathology under a mirror. A: a control group; b: cancer-inducing week 6 model group; c: cancer-inducing model group at week 16; d: cancer induction 24 week model group. HE × 200.

FIG. 3 shows the body weight changes of the mice in each group during cancer induction in example 2. A: a change in body weight; b: relative body weight change. Data are presented as mean ± standard deviation, n ═ 6.^*P<0.05, which indicates a statistical difference from the control group;^#P<0.05, representing a statistical difference compared to the model group.

FIG. 4 is the histopathological structure of the liver (HE staining) of each group of mice at the stage of liver injury (cancer-inducing week 6) in example 2. A: a control group; b: a model group; c: BSL group (BS 9 mg/kg); d: BSM group (BS 18 mg/kg); e: BSH group (BS36 mg/kg). Magnification: (1) x 100; (2) x 200. Black arrows: a focally necrotic area.

FIG. 5 is a graph showing the results of the liver injury stage (cancer-inducing week 6) of example 2And (5) detecting the liver function of mouse serum.^*P<0.05, which indicates a statistical difference compared to the control group;^#P<0.05, representing a statistical difference compared to the model group.

FIG. 6 shows the appearance of liver, HE staining and Masson staining of each group of mice in the stage of cirrhosis (cancer-inducing week 16) in example 2. A: a normal control group; b: a model group; c: BSL group (BS 9 mg/kg); d: BSM group (BS 18 mg/kg); e: BSH group (BS36 mg/kg). HE staining magnification: x 200, Masson staining magnification: x 100.

FIG. 7 shows the appearance of liver and HE staining of mice in the liver cancer stage (cancer-inducing week 24) in example 2. A: a normal control group; b: a model group; c: BSL group (BS 9 mg/kg); d: BSM group (BS 18 mg/kg); e: BSH group (BS36 mg/kg). HE staining magnification: x 100 and x 200. Black arrows: b: hepatocellular carcinoma; c: hepatocellular adenoma; d: a hepatocyte dysplasia lesion AHF; e: liver fibrosis.

FIG. 8 is serum AFP levels in mice from each group at the hepatocarcinoma stage (cancer-inducing week 24) of example 2. Data are presented as mean ± standard deviation, n ═ 10.^*P<0.05, which indicates a statistical difference from the control group;^#P<0.05, representing a statistical difference compared to the model group.

FIG. 9 is the inhibitory effect of different concentrations of BS on TrxR and Trx in liver tissues of mice in example 2. A: (ii) a TrxR activity; b: protein expression levels of TrxR and Trx; b: relative protein expression of TrxR; c: relative protein expression of Trx. Data are presented as mean ± standard deviation, n ═ 3.^*P<0.05, which indicates a statistical difference from the control group;^#P<0.05, representing a statistical difference from the model group.

FIG. 10 is a graph of the inhibition of tumor growth in H22-loaded mice by the BS-lead intervention at different doses and at different dosing intervals of example 3.

FIG. 11 is the mouse peripheral blood TR activity at the end of example 3 dosing.

FIG. 12 shows tumor growth in the groups of mice of example 4. Mouse tumor size was measured every two days using a vernier caliper according to the formula: tumor length x tumor width²×0.5236Tumor volume was calculated. (a) The method comprises the following steps Photographs of each group of tumors at the end of the experiment; (b) the method comprises the following steps Tumor volume changes in mice of each group. Data are presented as mean ± standard deviation (n ═ 6). P<0.05, which indicates a statistical difference from the control group.

FIG. 13 shows tumor growth in the groups of mice of example 5. Mouse tumor size was measured every two days using a vernier caliper according to the formula: tumor length x tumor width²X 0.5236 calculate tumor volume. (a) The method comprises the following steps Photographs of each group of tumors at the end of the experiment; (b) the method comprises the following steps Tumor volume changes in mice of each group. Data are presented as mean ± standard deviation (n ═ 6). P<0.05, which indicates a statistical difference from the control group.

FIG. 14 shows tumor growth in the groups of mice of example 6. Mouse tumor size was measured every two days using a vernier caliper according to the formula: tumor length x tumor width²X 0.5236 calculate tumor volume. a: photographs of each group of tumors at the end of the experiment; b: tumor volume changes in mice of each group. Data are presented as mean ± standard deviation (n ═ 6). P<0.05, which indicates a statistical difference from the control group.

FIG. 15 shows the growth inhibition rates of BS on LoVo, RKO, SW480 cells 24, 48, 72h in example 7. (a) The method comprises the following steps BS acts on the growth inhibition rate of LoVo cells for 24h, 48 h and 72 h; (b) the method comprises the following steps BS acts on growth inhibition rate of RKO cells for 24, 48, 72 h; (c) the method comprises the following steps The growth inhibition rate of BS on SW480 cells for 24, 48 and 72 h. Data are presented as mean ± standard deviation (n ═ 3).

FIG. 16 shows the growth inhibition rates of BS in example 7 on A549, H1299, SPCA-1 cells 24, 48, 72H. (a) The method comprises the following steps The growth inhibition rate of the BS acting on the A549 cells for 24, 48 and 72 hours; (b) the method comprises the following steps The growth inhibition rate of BS acting on H1299 cells for 24, 48 and 72 hours; (c) the method comprises the following steps BS acts on the growth inhibition rate of SPCA-1 cells for 24, 48 and 72 h. Data are presented as mean ± standard deviation (n ═ 3).

FIG. 17 is the growth inhibition rate of example 7BS on KYSE150, KYSE450, KYSE510 cells 24, 48, 72 h. (a): the growth inhibition rate of BS on KYSE150 cells for 24, 48 and 72 h; (b) the method comprises the following steps The growth inhibition rate of BS on KYSE450 cells for 24, 48 and 72 h; (c) the method comprises the following steps BS acts on the growth inhibition rate of KYSE510 cells 24, 48 and 72 h. Data are presented as mean ± standard deviation (n ═ 3).

FIG. 18 shows the growth inhibition rate of example 7BS on MCF-7, HeLa, k562 cells 24, 48, 72 h. (a) The method comprises the following steps The growth inhibition rate of the BS acting on the MCF-7 cells for 24, 48 and 72 hours; (b) the method comprises the following steps The growth inhibition rate of BS acting on HeLa cells for 24, 48 and 72 hours; (c) the method comprises the following steps BS acts on the growth inhibition rate of k562 cells for 24, 48 and 72 h. Data are presented as mean ± standard deviation (n ═ 3).

Detailed Description

The technical solution of the present invention will be further described in detail with reference to specific embodiments. The following examples are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of the invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

The experimental method comprises the following steps:

1. test drugs, cells and animals:

BS: the chemical name of the product is 1, 2-bis [2- (1, 2-benzisoselenazol-3 (2H) -ketone) ] -butane, which is referred to Chinese patent No. 02158917.8.

Mouse hepatoma cell line H22: presented by a Chinese toon researcher in the institute of tumor prevention and treatment of Beijing university.

Mouse lung cancer cell line Lewis: purchased from basic medical college of cooperative medical university.

Mouse breast cancer cell line 4T 1: purchased from basic medical college of cooperative medical university.

Human hepatoma cell line HepG 2: human hepatoma cell line Bel 7402: human hepatoma cell line Huh 7721: human colorectal cancer cell line LoVo: human colorectal cancer cell line RKO: human colorectal cancer cell line SW 480: human lung cancer cell line a 549: human lung cancer cell line H1299: human lung cancer cell line SPCA-1: all purchased from basic medical colleges of the university of self-supporting and medicine; human epithelial cervical cancer cell line HeLa: human breast cancer cell line MCF-7: human chronic myelogenous leukemia cell line k 562: purchased from cell center of basic medical college of the university of medical science; human esophageal cancer cell line KYSE150, human esophageal cancer cell line KYSE450 and human esophageal cancer cell line KYSE510, which are given by Beijing tumor hospitals.

Kunming inbred mice (KM mice): male, 4 weeks, with a weight of 18-22 g, purchased from the experimental animals center of the department of medicine of Beijing university, the license number of experimental animals: SYXK (Kd) 2012 and 0036. The breeding environment is clean, the breeding temperature is 25 +/-2 ℃, the illumination is 12 hours and the darkness is 12 hours after 12 hours, and the food and water supply is sufficient.

Balb/c mice: balb/c mice: the experimental animal is purchased from the center of experimental animals of department of medicine of Beijing university, and the production license number SYXK (Jing) 2012-0036 of the experimental animals, wherein the experimental animal is 4 weeks old, the weight of the experimental animal is 16-18 g, and male (lung cancer and postoperative recurrence of liver cancer) and female (postoperative recurrence of breast cancer). The feeding environment is clean, the feeding temperature is 25 +/-2 ℃, the illumination is 12h and the darkness is 12h, and the food and water supply is sufficient.

2. The test method comprises the following steps:

2.1 mouse H22 liver cancer/Lewis lung cancer/4T 1 breast cancer transplantable tumor excision model

Respectively taking out H22 (liver cancer) cryopreserved cells/Lewis (lung cancer) cryopreserved cells/4T 1 (breast cancer) cryopreserved cells, taking out after the cells become liquid at 37 ℃ for 1-2 min; and (4) centrifuging after resuspension, discarding the supernatant after centrifuging at 1200rpm/min for 2min, resuspending in 1mL serum-free medium, and injecting cells into the abdominal cavity.

Approximately one week later, when the abdomen of the mouse was touched to have a swelling sensation, ascites was extracted. The abdominal cavity outer skin of the mouse is slightly reduced with a small opening by scissors and tweezers, then the two sides of the hemostatic forceps are slightly pulled open to expose the abdomen, and the ascites is extracted by a syringe.

Preparing ascites of Balb/c mice into single cell suspension, and counting the number of living cells by trypan blue dye exclusion, wherein the cell survival rate is more than 95 percent; injecting the tumor cell suspension into the right axilla of male Balb/c mice with the weight of 16g-18g, 10g each⁶And (4) cells.

2.1.1 mouse H22 model for excision of liver cancer transplantable tumor

Balb/c mice subcutaneously transplanted with mouse hepatoma cells H22 were subjected to local tumor resection as follows: when the right axillary tumor of the mouse reaches about 500mm³When necessary, 5% chloral hydrate solution is injected into the abdominal cavity(0.08mL/10g) were anesthetized and prepared for hair. After the mice enter the general anesthesia state, a 1-2cm incision is cut near the tumor, the tumor tissue is blunt stripped and cut off 90%, and the residual is about 50mm³Finally, the tumor tissue is sutured by using sterile medical silk threads, and the wound is coated with iodophor for disinfection. The above operations are all completed in a super clean bench. Mice were placed in a clean, warm place and given sterile aqueous glucose for their consumption after awakening. The day of surgery is considered to be 0 days post-surgery. After the mice were fully awakened, the mice were randomly divided into 4 groups of 6 mice each, and the drug treatment protocol was as follows:

a blank control group (5% CMC-Na, i.g., q.d.);

BS Low dose group (90 mg. kg)^-1,i.g.,q.d.)；

BS Medium dose group (180 mg. kg)^-1,i.g.,q.d.)；

BS high dose group (360 mg. kg)^-1,i.g.,q.d.)。

Mice body weight, tumor volume and activity were recorded at intervals during the experiment. Eight days after administration, mice were sacrificed, and the eyeballs were removed to obtain blood for the detection of TrxR activity in serum. Tumors, liver, kidney, thymus and spleen were rapidly isolated, observed and weighed. Tumor tissues were kept in liquid nitrogen for subsequent experiments.

2.1.2 mouse Lewis Lung cancer transplantation tumor excision model

As above, Balb/c mice subcutaneously transplanted with murine lung carcinoma cells Lewis were subjected to local tumor resection as follows: when the right axillary tumor of the mouse reaches about 500mm³In this case, 5% chloral hydrate solution (0.08mL/10g) was intraperitoneally injected for anesthesia and hair preparation. After the mice enter the general anesthesia state, a 1-2cm incision is cut near the tumor, the tumor tissue is blunt stripped and cut off 90%, and the residual is about 50mm³Finally, the tumor tissue is sutured by using sterile medical silk threads, and the wound is coated with iodophor for disinfection. The above operations are all completed in a super clean bench. Mice were placed in a clean, warm place and given sterile aqueous glucose for their consumption after awakening. The day of surgery is considered to be 0 days after surgery. The mice are randomly divided into 4 groups of 6 mice after being fully recovered, and the drug treatment formula isThe scheme is as follows:

a blank control group (5% CMC-Na, i.g., q.d.);

BS Low dose group (90 mg. kg)^-1,i.g.,q.d.)；

BS Medium dose group (180 mg. kg)^-1,i.g.,q.d.)；

BS high dose group (360 mg. kg)^-1,i.g.,q.d.)。

Mice body weight, tumor volume and activity were recorded at intervals during the experiment. Eight days after administration, mice were sacrificed, and the eyeballs were removed to obtain blood for the detection of TrxR activity in serum. Tumors, liver, kidney, thymus and spleen were rapidly isolated, observed and weighed. Tumor tissues were kept in liquid nitrogen for subsequent experiments.

2.1.3 mouse 4T1 Breast cancer graft tumor excision model

Meanwhile, Balb/c mice subcutaneously transplanted with mouse mammary cancer cells 4T1 were subjected to local tumor resection as follows: when the right axillary tumor of the mouse reaches about 500mm³In this case, 5% chloral hydrate solution (0.08mL/10g) was intraperitoneally injected for anesthesia and hair preparation. After the mice enter the general anesthesia state, a 1-2cm incision is cut near the tumor, the tumor tissue is blunt stripped and cut off 90%, and the residual is about 50mm³Finally, the tumor tissue is sutured by using sterile medical silk threads, and the wound is coated with iodophor for disinfection. The above operations are all completed in a super clean bench. Mice were placed in a clean, warm place and given sterile aqueous glucose for their consumption after awakening. The day of surgery is considered to be 0 days after surgery. After the mice were fully awakened, the mice were randomly divided into 4 groups of 6 mice each, and the drug treatment protocol was as follows:

a blank control group (5% CMC-Na, i.g., q.d.);

BS Low dose group (90 mg. kg)^-1,i.g.,q.d.)；

BS Medium dose group (180 mg. kg)^-1,i.g.,q.d.)；

BS high dose group (360 mg. kg)^-1,i.g.,q.d.)。

Mice body weight, tumor volume and activity were recorded at intervals during the experiment. Eight days after administration, mice were sacrificed, and the eyeballs were removed to obtain blood for the detection of TrxR activity in serum. Tumors, liver, kidney, thymus and spleen were rapidly isolated, observed and weighed. Tumor tissues were kept in liquid nitrogen for subsequent experiments.

2.2 establishment of mouse liver cancer model

Reference is made to Butaselen precursors hepatocarcinogenesis and regression thereof through thiohinhibitting

thioredoxin reductase activity，《Redox Biology》,2017,14:237-249。

2.3 establishment of animal model of mouse subcutaneous transplantation tumor

Feeding Kunming mice at 25 + -2 deg.C under 12 hr illumination for 12 hr in dark condition, and supplying sufficient diet. H22 hepatoma tumor cells were inoculated by conventional methods: the recovered H22 cells were injected into the abdominal cavity of mice for subculture growth. After 3 generations of ascites are fully transmitted, ascites H22 cells which grow well for 6-8 days are taken, diluted to corresponding concentration by serum-free culture medium, and inoculated to the subcutaneous tissue of the right axilla of the mouse at a rate of 0.1 mL/mouse, and an evaluation model is established.

After 3 days of animal adaptive feeding, the qualified animals were randomly divided into 4 groups of 5 animals each, and the drug treatment regimen was as follows: the administration is carried out 3 days in advance: a blank control group (5 per mill of CMC-Na, intragastric, once a day); BS 72mg/kg qd group (36mg/kg, gavage, once daily); BS 72mg/kg bid group (72mg/kg, gavage, twice daily); BS 144mg/Kg group (144mg/Kg, gavage, once daily); 3 days later, the right axilla of the mice were inoculated subcutaneously with H22 cells, 1X 10⁶This was the day 0 of treatment, and mice were treated as before. During the treatment period, the animal body weight was measured daily, the general condition and tumorigenesis of the animals were recorded, and the time of tumorigenesis was recorded. The average tumor volume of the tumor of the control group reaches 2000mm³The experiment was ended: mouse eyeball is removed and blood is taken, cervical vertebra is removed to kill the animal, tumor, liver, spleen and kidney are quickly separated, whether the internal organs of the animal have pathological changes or not is observed, the internal organs and tumor tissues are weighed, the length and the short diameter of the tumor are measured, and according to the formula: calculating the tumor volume by the long diameter multiplied by the short diameter multiplied by 2 multiplied by 0.5236; the whole mouse blood is centrifuged at 6000r/min for 10min, and the plasma is taken for TR activity detection.

All experiments were in compliance with animal ethics requirements set by the ethics committee of the department of medicine of Beijing university.

Statistical treatment:

statistical analysis was performed using the SPSS 17.0 statistical software package and data are presented as mean. + -. standard deviation (X (). + -. SD). Student's t-test was used for comparison between two groups of values, analysis of variance (ANOVA) was used for pairwise comparison between three or more groups of values, and P <0.05 was statistically significant for differences.

2.4 assay of inhibition of cell proliferation (SRB method):

(1) digesting the wall-attached cultured cells into a single-cell suspension, and adjusting the cell concentration to be 2 multiplied by 10⁴one/mL, and was inoculated uniformly in a 96-well plate (Corning), 180. mu.L per well;

(2) placing the 96-well plate in an incubator for 24h, and adding 20 mu L of compound per well according to the specified concentration after the cells adhere to the wall;

(3) at the end of the drug action, 100 μ L of 10% (v/v) trichloroacetic acid precooled at 4 ℃ is added into each well, and a 96-well plate is fixed for 1h at 4 ℃;

(4) carefully discarding the fixative, slowly washing with deionized water from one side of the 96-well plate for 4 times, blow-drying with electric heating blower or naturally drying at room temperature (the dried 96-well plate can be stored at room temperature for a long time);

(5) adding 100 μ L of 0.057% (w/v) SRB staining solution into each well, and staining for 30min at room temperature;

(6) carefully discarding the dye solution, washing each well with 150 μ L of 1% (v/v) acetic acid for 4 times, drying by blowing with electric heat or naturally drying at room temperature (the dried 96-well plate can be stored at room temperature for a long time);

(7) accurately adding 200 mu L of 10mM Tris destaining solution into each hole, standing at room temperature to dissolve the Tris destaining solution naturally or putting the Tris destaining solution on a shaking table to dissolve the Tris destaining solution;

(8) the absorbance of the sample at 492nm was measured using a microplate reader (Thermo Fisher Scientific). The cell proliferation inhibition rate was calculated according to the following formula:

wherein, the control well is inoculated with 180 μ L of cell suspension, and the compound is replaced by medium (20 μ L) of the same volume; for the inoculation of the blank wells, 180. mu.L of medium was added, and for the administration, 20. mu.L of medium was added. Three duplicate wells were set for each drug concentration.

2.5Western Blot:

extracting total tissue protein, determining protein concentration (30-50 μ g), adding 5 × loading buffer, adding RIPA lysate to make up volume, mixing, and denaturing at 95 deg.C for 10 min. After fully and uniformly mixing, adding a clean 1.5mm rubber plate to a proper position, and sealing with n-butanol. Adding 1mL of 1 XSEPARATION GEL buffer solution, and standing at room temperature overnight; inserting a 1.5mm sample adding comb, and standing at room temperature for 2h to wait for gelation.

Pulling out the sample adding comb, and loading the sample by a conventional method; and (4) performing constant voltage electrophoresis at 80V until the front edge of the bromophenol blue enters the separation gel, increasing the voltage to 160V, continuing the constant voltage electrophoresis, and stopping the electrophoresis until the bromophenol blue migrates to the end of the separation gel.

Accurately shearing 6 pieces of filter paper with the same size as the separation gel and a 0.2 mu m PVDF membrane, soaking the PVDF membrane in methanol for 10s, soaking in deionized water for 3min, and soaking in an electrotransformation buffer solution for 3 min;

the electricity changes sandwich and lays by the order of positive pole to negative pole in proper order: one sponge, three pieces of filter paper, a PVDF membrane, separation glue, three pieces of filter paper and one sponge (all soaked in an electric rotating buffer solution in advance), wherein air bubbles are expelled from the layers by a glass rod, and the electric rotating clamp and the ice box are clamped and put into an electric rotating groove;

and (5) placing the electric rotating tank in an ice-water bath, and rotating for 2 hours at a constant current of 250 mA.

After the electrotransfer is finished, taking out the PVDF membrane, and placing the PVDF membrane in a 5% closed state; washing the membrane with TBST for 3 times, sealing the PVDF membrane and a diluted secondary antibody solution with a proper proportion in a hybridization bag, incubating for 1h at room temperature, washing the membrane with TBST for 3 times, and developing;

and opening a gel imaging system, uniformly mixing the solution A and the solution B of the ECL luminescent solution in equal volume, uniformly distributing the mixture on the PVDF membrane, and performing exposure analysis.

2.6 determination of thioredoxin reductase Activity by DTNB reduction

1. Adding 50 μ g protein sample into 96-well plate, adding 0.1M sodium phosphate buffer to make up volume to 80 μ L, and incubating at 37 deg.C for 30 min;

2. add 20. mu.L of 5mM NADPH solution to each sample well, add an equal volume of 0.1M sodium phosphate buffer to the control well;

3. after 100. mu.L of 10mM DTNB solution was added to each well by a line gun, the absorbance at 405nm was measured immediately with a microplate reader, and the wells were shaken for 10 seconds before the first reading, and measured every 15 seconds for 30 times. The maximum reaction rate was taken as an indicator of enzyme activity. The experiment was independently repeated three times.

2.6 flow cytometry assay:

1) cells from logarithmic growth phase were seeded in six-well plates (Corning), 5X 10⁴Each well contains 2mL of cell suspension, and the culture dish is placed in an incubator for culture. After 24 hours of cell adherence, removing the culture medium, adding 2mL of liquid medicine with corresponding concentration, and adding the culture medium with the same volume to the control group;

2) after 24h of drug action, the recovery medium is centrifuged at 1200rpm for 2min in a 5mL centrifuge tube, and the supernatant is discarded. Rinsing adherent cells in the culture dish once by using PBS precooled at 4 ℃, adding 300 mu L of pancreatin, and placing in an incubator for digestion for 3 min;

3) and (3) blowing the cells into single cells by using 1mL of culture medium, re-suspending the precipitated cells in a centrifuge tube by using 1mL of PBS, combining the precipitated cells with the adherent cells in the culture dish of the corresponding group, centrifuging at 400rpm for 2min, and discarding the supernatant. Resuspend the cells with 1mL PBS, screen through 300 mesh cell screen;

4) resuspending the precipitated cells with 190. mu.L of binding solution, adding 10. mu.l of Annexin V-FITC, incubating at room temperature in the dark for 10min, centrifuging at 4000rpm for 2min, and discarding the supernatant;

5) the cells were resuspended in 195. mu.L of binding solution, 5. mu.L of PI solution was added and immediately detected on a flow cytometer (BDFACSCalibur).

Example 1 successful establishment of mouse liver cancer model

1.1 general growth and liver coefficient changes in mice

The 25 mice in the control group had good mental status, smooth body hair, normal activity, normal diet and water, and sustained weight gain over 24 weeks, with the average weight gradually increasing from about 19g to about 35g without natural death, as shown in fig. 1A.

1-2 weeks in cancer induction, due to DEN and CCl₄Acute toxicity ofIn effect, most mice in the model group showed signs of hair erection, lassitude, decreased activity, decreased appetite, etc., to varying degrees, and began to die in large numbers (6 mice). This was most severe with re-administration of DEN at weeks 3-4, with some mice dying (3). After week 4, the model mice began to improve, the spirit improved, the activity increased and the weight increased. Most mice in the model group at weeks 4-8 were well-maintained, had a normal diet, vigorous energy and a significant weight gain, but 1 mouse still died at day 30 of the experiment. After week 8, due to CCl₄The dosage is increased, the mice in the model group have the symptoms of hair uprightness, lassitude, reduced activity, reduced appetite and the like in different degrees, but the degree is obviously weaker than that in the previous 4 weeks, and 2 mice die. The mice then developed a slow increase in body weight until CCl at week 12₄The frequency of administration is increased, the mice in the model group have the symptoms of fur erection, lassitude, activity reduction, appetite reduction and the like in different degrees, at the moment, the weight is obviously reduced, and 1 mouse in total dies. After 14 weeks, although the body weight of the model group mice slowly increased, the activity of the mice was obviously inferior to that of the control group, the mice had hair uprighted, the spirit was poor, the physique was poor, the appetite was poor, and the body weight of the mice continuously decreased at 18-24 weeks. The body weight of mice in the model group from 2 weeks of cancer induction was significantly lower than that in the control group (P)<0.05), the total mortality was 21.7%.

We compared the liver coefficients of model and control mice at week 0, week 6, week 16 and week 24 of cancer induction, and the results are shown in fig. 1B. The liver coefficients of the two groups were significantly different at week 6 and week 24 (P <0.05), which were 1.37 and 1.58 times of those of the control group, respectively, and the liver coefficients of the mice in the initial group (week 0) and week 16 model groups were not significantly different from those of the control group (P > 0.05).

1.2 pathological changes in the liver in mice

After cancer induction, the liver of the mice was picked, and the control group mice had no special change in liver visual observation, smooth envelope, dark red color, soft texture and normal liver performance (fig. 2. A1). Under the observation of the microscope, the mouse liver cells are normal in structure, regular in shape, eosinophilic in cytoplasm, basophilic in nucleus, dense and uniform in arrangement, neat in liver cell cable, radial around central vein, complete and clear in liver lobule structure, and free of other pathological changes except slight edema of the liver cells (fig. 2. A2).

The liver of the model group mice was harvested at 6 weeks, and the liver was observed with naked eyes to be obviously swollen and rough, fine particles were observed on the surface, and the envelope was tense (fig. 2. B1). The liver tissues of the mice were observed under a microscope to have different degrees of drug-induced liver injury, liver cell edema and vesicular steatosis (10%), liver lobules are intact, but liver cells around lobules and central veins have hemorrhage and multifocal necrosis with a small amount of scattered neutrophils in infiltration (fig. 2. B2).

At 16 weeks of cancer induction, the liver of the model mice became lighter in color, hard in texture, rough in surface, and began to appear as diffuse nodular changes of varying sizes and numbers (fig. 2. C1). Mouse liver cells were observed under a microscope for severe edema, mixed steatosis of large and small vacuoles (10%), multifocal hepatocyte necrosis and multiple mononuclear lymphocytic multifocal infiltrates. Severe inflammation in the zone of the tract, infiltration of multiple mononuclear lymphocytes, destruction of the majority (> 50%) of the hepatocyte boundary plate; normal structure of the liver lobules disappears, fibrosis is bridged between the areas of the collector and the central vein, and the liver lobules are divided by extensively proliferating connective tissue and wrapped into masses of round or quasi-round liver cells of unequal size, i.e. typical pseudolobules are formed (fig. 2. C2).

At 24 weeks of cancer induction, the liver of the model mice is obviously hardened and presents with multifocal lesions, and a plurality of single or multiple grey white nodules with different sizes are visible on the surface of the liver, and the diameter of the single large nodule can reach 0.8cm (fig. 2. D1). Observing mouse liver cell edema, fatty degeneration and inflammatory cell infiltration under a mirror; most of the liver structure is destroyed, and false lobules are formed; typical pathological changes of liver cancer can be seen, the differentiation degree of cancer cells is low, the arrangement of the structure of the liver cells is disordered, the cellular heterogeneity is obvious, such as multinucleate, vacuole and the like, the nucleus-to-cytoplasmic ratio is increased, the nucleolus is increased, pathological nuclear division can be seen, the cancer nest is often pushed or infiltrated into surrounding tissues, the cancer diameter is different from 0.3 cm to 0.8cm, and the cancer rate is 80 percent (8/10) (figure 2. D2).

In conclusion, it can be seen that,the research successfully establishes DEN/CCl₄Ethanol induced mouse liver cancer model. In the cancer induction process, pathological changes of different stages of the mouse are very similar to the generation process of human liver cancer, and the pathological processes from hepatocyte injury (6 weeks), hepatic fibrosis and liver cirrhosis (16 weeks) to liver cancer (24 weeks) are successively carried out, so that conditions are provided for the protection effect of the follow-up research medicament in the mouse canceration process, and a model basis is provided.

Example 2 protective role of BS in the development of liver cancer in mice

The dosage of BS in the low, medium and high dosage groups is respectively 9mg/kg, 18mg/kg and 36 mg/kg.

2.1 general growth of mice

In the cancer inducing process, the control group mice have good mental state, smooth body hair, normal activity and normal diet and drinking water; the mice in the model group and the drug administration group have the manifestations of hair erection, lassitude, activity reduction, appetite reduction and the like in different degrees, wherein the conditions of the mice in the model group are the most serious, the conditions of the mice in the groups with low, medium and high doses of BS are all relieved, and the adverse symptoms of the mice are relieved more and more obviously along with the increase of the dosage of the BS.

2.2 weight Change in mice

TABLE 1 cancer inducing conditions of body weight change in mice at different stages

Data are presented as mean ± standard deviation, n ═ 6. P <0.05, representing a statistical difference compared to the control group; # P <0.05, which indicates a statistical difference compared to the model group.

FIG. 3 summarizes the body weight changes of the mice in each group during cancer induction, and the body weight changes of the mice in each group are shown in Table 1. The body weights of the mice in each group at the beginning of cancer induction (day 0) are similar, and no significant difference exists (P)>0.05). The weight of the control mice increased continuously over 25 weeks, and the average body weight increased from about 19g initially to about 35 g. At week 1 of cancer induction, due to DEN and CCl, compared to control group₄Toxic effects of (1), model group and administration groupThe mice have weight loss expressions of different degrees, wherein the weight loss of the mice in the model group is the most obvious and is negative growth; the BSH group decreased the least, the body weight was still slowly increasing, and the two had significant difference (P)<0.05). Over time, the mean body weight of mice in each cancer-inducing group gradually increased until CCl at week 8₄Increase in dosing concentration and week 12 CCl₄The frequency of administration is increased, so that the weight of the mice of each cancer inducing group is reduced again, and then the weight of the mice of each BS administration group is continuously increased, which is similar to the growth speed of the control group, while the weight of the mice of the model group shows negative growth. At the end of the 24-week experiment, the normal control mice gained 1.85 times the initial body weight, while the model mice had a final body weight that was only 1.09 times the initial body weight (P)<0.05). Although the weight of mice in each treatment group of BS was reduced in comparison with the normal control group (P)<0.05), but the weight gain of the mice in the BS-treated group was significant (P) compared with the model group<0.05). At the end of the experiment, the body weight of the mice in the low, medium and high dose treatment groups of BS increased to 1.25, 1.34 and 1.50 times the initial body weight, respectively.

2.3 mouse mortality

The death of mice in each group during cancer induction is shown in FIG. 4 and Table 2, and the control group of mice has no natural death. Due to large dose of DEN and CCl₄The 1 st to 2 nd week of cancer induction is the peak period of death of mice in each experimental group, 6 mice in the model group die, and the number of deaths of the mice in the BS administration group is reduced along with the increase of concentration, namely 3, 2 and 1 mice respectively. Mice died significantly at weeks 3-4 due to re-administration of DEN. After week 8, due to CCl₄With the increased dose, mice died again in the model group and the BSL group. After week 12 although CCl₄The administration frequency is increased, but no mouse death occurs in each group of BS administration, only 1 mouse death occurs in the model group, mainly due to the reduction of physique and cachexia, the liver surface is scattered on nodules and necrotic lesions occur in the lung when the model group is dissected. At the end of the experiment, the total mortality rates of the mice in the normal control group, model group, BSL, BSM, BSH group were 0% (0/25), 21.7% (13/60), 17.5% (7/40), 10.0% (4/40), 7.5% (3/40), respectively. The mortality rate of the BSM and BSH groups was significantly lower than that of the model group (P)<0.05), no difference from the control groupDisplay (P)>0.05)。

TABLE 2 cancer-inducing death in mice at different stages

P <0.05, representing a statistical difference compared to the control group; # P <0.05, which indicates a statistical difference compared to the model group.

2.4 Effect of BS on the mouse immune System

At the end of the experiment, the thymus and spleen of the immune organs of the mice are picked up for observation and weighing, and the number of white blood cells, red blood cells and platelets in peripheral blood is detected, and the results are shown in table 3.

TABLE 3 mouse organ coefficient and blood routine test

Data are presented as mean ± standard deviation, n ═ 10. P <0.05, representing a statistical difference compared to the control group; # P <0.05, which indicates a statistical difference compared to the model group.

In the process of dissection, the thymus of the model group mice is obviously atrophied, the spleen is enlarged, the result of organ coefficient is consistent with the observed phenomenon, compared with the control group mice, the thymus coefficient of the model group mice is obviously reduced, the spleen coefficient is obviously increased, the corresponding peripheral blood leukocyte is increased, the number of red blood cells and blood platelets is reduced, and the number of red blood cells and blood platelets is respectively 0.5, 2.0, 2.1, 0.9 and 0.7 times of that of the control group, and the differences are significant (P is less than 0.05). The low, medium and high dosage BS treatment groups can obviously increase the thymus coefficient, the number of red blood cells and blood platelets of mice and reduce the spleen coefficient and the number of white blood cells, and each index of the high dosage BS treatment group has no statistical difference (P is more than 0.05) compared with the normal control group.

2.5 protective Effect of BS in different stages of the development of liver cancer in mice

(1) Protection of BS in liver injury stage in mice-first stage protection

● Effect of BS on mouse liver

In the liver cell damage-repair period of 6 weeks of cancer induction, 6 mice were randomly selected from each group, livers were extracted, and changes in livers were visually observed. The liver of the control mouse has no special change, the coating is smooth, the color is dark red, the texture is soft, and the normal liver performance is shown. The liver of the model group mouse is obviously swollen and rough, fine particles can be seen on the surface, and the envelope is tense. The livers of mice treated by low, medium and high doses of BS are obviously reduced, and the surface graininess is reduced.

HE staining of paraffin sections of liver was used to assess the effect of drugs on liver histopathology. As shown in FIG. 4, the normal control group mice had normal hepatocyte structure, regular shape, dense and uniform arrangement, and intact and clear lobular structure (FIG. 4. A). Compared with the normal control group, the model group had liver cell edema, vesicular steatosis, multifocal necrosis of 60% liver cells in liver lobules with a small amount of neutrophils scattered in the infiltration (fig. 4. B). Compared with the model group, the necrotic areas of the liver of the mice treated by the low, medium and high BS doses are obviously reduced, the degree of drug-induced liver injury is reduced, and particularly the group treated by the high BS dose is most obvious (fig. 4. C-E).

TABLE 4 hepatic necrosis score in groups of mice at the stage of hepatic injury (cancer-inducing week 6)

P <0.05, representing a statistical difference compared to the control group; # P <0.05, which indicates a statistical difference compared to the model group.

The hepatocyte necrosis score of each group of mice is shown in table 4. The hepatic cell necrosis score of the model group is significantly increased compared with that of the normal control group (P)<0.05), suggesting DEN/CCl due to₄The toxic effect of ethanol causes drug-induced liver injury, and the mouse liver cells have serious necrosis; the low, medium and high dose groups of BS all reduced the hepatic cell necrosis score, and the medium and high dose groups of BS had significant differences (P) compared with the model group<0.05), prompt BS to DEN/CCl₄The liver injury of mice caused by ethanol has certain protection effect.

● BS Effect on mouse liver coefficients

TABLE 5 liver coefficient changes in groups of mice at the stage of liver injury (cancer-inducing week 6)

Data are presented as mean ± standard deviation, n ═ 6. P <0.05, representing a statistical difference compared to the control group; # P <0.05, which indicates a statistical difference compared to the model group.

The body weight, liver weight and liver coefficient of each group of mice at 6 weeks of cancer induction are shown in table 5. Compared with the normal control group, the model group mice have reduced weight, increased liver weight and obviously increased liver coefficient which is about 1.4 times of the liver coefficient of the normal control group mice (P < 0.05). Compared with the model group, the weight of the mice in the BS treatment group is increased, the weight of the liver is reduced, and the liver coefficient is obviously reduced. Liver coefficients of the low, medium and high-dose groups of BS are respectively reduced by 4.9%, 11.5% (P <0.05) and 17.6% (P <0.05), and the liver coefficients of the high-dose group of BS have no statistical difference (P >0.05) compared with the liver coefficients of the normal control group.

● Effect of BS on Biochemical indicators of mouse serum

The liver function test result is shown in fig. 6, compared with the normal control group, the ALT and AST activities in the serum of the model group mouse are obviously improved (P is less than 0.05), and are respectively improved to 9.9 times and 5.3 times, which shows that the DEN/CCl 4/ethanol treatment causes the liver of the mouse to generate obvious liver injury; compared with the model group, the indexes of the BS treatment group are obviously reduced (P is less than 0.05), ALT activities of the low, medium and high dosage groups of the BS are respectively reduced by 24.2%, 33.9% and 55.6%, and AST activities are respectively reduced by 18.6%, 45.4% and 55.9%.

(2) Protective action of BS in liver cirrhosis stage-second stage action in mice

● Effect of BS on mouse liver

In the 16 th week liver cirrhosis stage of cancer induction, 6 mice were randomly selected from each group, livers were extracted, and changes in livers were visually observed. The control mice had no specific change in liver, smooth coating, dark red color, soft texture, and normal liver performance (fig. 7.a 1). The liver of the model group mice became light in color, hard in texture, rough in surface, and began to change in size and number of diffuse nodular changes (fig. 7. B1). The symptoms of the liver of the mice treated by the low, medium and high BS dose are relieved, particularly the group treated by the high BS dose is most obvious (FIG. 7. C1-E1).

The result of liver HE staining is shown in FIG. 7, and the normal control mice had normal hepatocyte structure, dense and uniform arrangement, and intact and clear lobular structure (FIG. 7. A2). Compared with a control group, the liver cells of the model group mice are severely edematous, large and small vacuole mixed fatty degeneration, multifocal hepatocyte necrosis and multiple mononuclear lymphocyte multifocal infiltration; severe inflammation in the area of the manifold, destruction of the majority (> 50%) of the hepatocyte boundary plate, disappearance of normal hepatic lobule structure, extensive formation of pseudolobules in the liver, and fibrous repartition phenomenon visible in the pseudolobules (fig. 7. B2). Compared with the model group, the mice treated by the low, medium and high BS dose have reduced hepatocyte necrosis and inflammation, reduced fibrosis degree of the junction area, the central vein and the periphery thereof, especially the group treated by the high BS dose has the most obvious dose-effect relationship (FIG. 7. C2-E2).

Liver Masson staining was used to evaluate the effect of the drug on liver collagen deposition, the results of which are shown in fig. 7 (collagen fibers stained in blue-green). The normal control group had only a very small amount of collagen fibrils present around the zone of the sink (fig. 7.a 3). Model groups bridging fibrosis between the zone of the manifold and central vein, fibroseptal formation, suggested that the mice reached the stage of cirrhosis (fig. 7. B3). Compared with the model group, the mice treated by the low, medium and high BS dose have obviously reduced collagen fibers in the liver manifold area, the central vein and the periphery thereof, particularly the group treated by the high BS dose has the most obvious dose-effect relationship (FIG. 7. C3-E3).

TABLE 6 hepatic fibrosis scoring in groups of mice at the stage of cirrhosis (cancer-inducing week 16)

P <0.05, representing a statistical difference compared to the control group; # P <0.05, which indicates a statistical difference compared to the model group.

The hepatic fibrosis scores of the mice in each group are shown in Table 6. The hepatic cell necrosis score of the model group is significantly increased compared with that of the normal control group (P)<0.05), suggesting DEN/CCl due to₄The chronic toxic effect of ethanol causes severe hepatic fibrosis of mice, and reaches the liver cirrhosis stage; the hepatic fibrosis scores of the low, medium and high dosage groups of BS are all reduced, and have significant difference (P) compared with the model group<0.05), prompt BS to DEN/CCl₄The liver cirrhosis of mice caused by ethanol has a certain protective effect.

(3) Protective action of BS in liver cancer stage of mouse-third stage action

● Effect of BS on mouse liver

In the 24 th week liver cancer stage, 10 mice were randomly selected from each group, livers were extracted, and changes in livers were visually observed. The control mice had no specific change in liver, smooth envelope, dark red color, soft texture, and normal liver performance (fig. 7.a 1). The liver of the mouse in the model group is obviously hardened compared with the prior liver, the mouse is in multifocal lesion, a plurality of single or multiple gray nodules with different sizes can be seen on the surface of the liver, and the diameter of a single large nodule can reach 0.8 cm. (FIG. 7. B1). The symptoms of the liver of the mice treated by the low, medium and high BS dose are relieved, the appearance is more regular, and the gray-white nodules become smaller and less, particularly the group treated by the high BS dose is most obvious (figure 7. C1-E1).

The result of liver HE staining is shown in FIG. 7, and the normal control mice had normal hepatocyte structure, dense and uniform arrangement, and intact and clear lobular structure (FIG. 7. A2/3). Compared with a normal control group, the liver cell edema, the steatosis and the inflammatory cell infiltration of the model group mouse; most of the liver structure is destroyed, and false lobules are formed; typical pathological changes of liver cancer can be seen, differentiation degree of cancer cells is low, arrangement of liver cell structures is disordered, cellular heterogeneity is obvious, such as multinucleate, vacuole and the like, nuclear-to-cytoplasmic ratio is increased, nucleolus is increased, pathological nuclear division can be seen, cancer nests are often pushed or infiltrated into surrounding tissues, and the diameter of cancer is different from 0.3 cm to 0.8cm (fig. 7. B2/3). Compared with the model group, the liver lobule damage degree and the cellular heterotypia of the mice of the low, medium and high-dose treatment group of the BS are reduced, particularly the group of the high-dose BS is most obvious and has a dose-effect relationship (figure 7. C2/3-E2/3).

TABLE 7 evaluation of the degree of abnormal liver cell type in the liver cancer stage (24 th week of cancer induction) in each group of mice

^*P<0.05, which indicates a statistical difference from the control group;^#P<0.05, representing a statistical difference compared to the model group.

The degree of hepatocyte allotype scores for each group of mice are shown in table 7. Compared with a normal control group, the abnormal degree score of the liver cells in the model group is obviously increased (P <0.05), the canceration rate is 80 percent (8/10), and the rest 2 cases are liver cell adenomas. The liver cell allotype degree scores of the low, medium and high dose treatment groups of the BS are obviously reduced, wherein the medium and high dose groups of the BS have significant difference compared with the model group (P < 0.05). The rate of malignancy in the BSL group was 40% (4/10), in addition 4 cases of hepatocellular adenomas, 2 cases of common hepatocellular dysplastic foci (AHF); the rate of carcinoma in the BSM group was 10% (1/10), in addition 3 hepatocellular adenomas, 4 common AHFs, 1 sporadic AHFs; the canceration rate in the BSH group was 0(0/10), in addition to 2 hepatocellular adenomas, 2 common AHFs, 3 occasional AHFs, and 3 no hepatocellular dysplasia. The BS is suggested to reduce the DEN/CCl 4/ethanol induced liver cancer incidence rate of mice.

● BS Effect on mouse liver coefficients

TABLE 8 liver coefficient changes in groups of mice at the liver cancer stage (cancer induction week 24).

Data are presented as mean ± standard deviation, n ═ 10. P <0.05, representing a statistical difference compared to the control group; # P <0.05, which indicates a statistical difference compared to the model group.

The body weight, liver weight and liver coefficient of each group of mice at 24 weeks of cancer induction are shown in table 8. Compared with the normal control group, the body weight of the mice in the model group is obviously reduced, and the liver coefficient is obviously increased and is about 1.6 times of that of the mice in the control group (P < 0.05). Compared with the model group, the weight of the mice in the BS treatment group is obviously increased (P is less than 0.05), the weight of the liver is reduced, and the liver coefficient is obviously reduced. Liver coefficients of the low, medium and high-dose groups of BS are respectively reduced by 14.1%, 24.4% (P <0.05) and 30.9% (P <0.05), and the liver coefficients of the high-dose group of BS are not statistically different from those of the normal control group (P > 0.05).

● Effect of BS on mouse serum AFP levels

Serum AFP levels are the most common serological biomarker for detection of liver cancer (Yim, Chung, 2010). At the end of the experiment, the serum AFP levels of each group of mice were measured by ELISA, as shown in figure 9. The results show that the AFP level in the serum of the model group mice is obviously increased compared with the normal control group, and is about 4.1 times of that of the control group (P < 0.05). Compared with the model group, the serum AFP level of the mice in the BS treatment group is obviously reduced, the serum AFP level of the mice in the BS low, medium and high dose groups is respectively reduced by 15.0 percent, 37.8 percent (P <0.05) and 59.8 percent (P <0.05), and the serum AFP level of the mice in the BS high dose group is not statistically different (P >0.05) compared with the normal control group.

● influence of BS on TrxR of liver cancer model mouse

Finally, the activity of the liver TrxR of each group of mice is detected by a DTNB reduction method, and the result is shown in figure 9. Compared with a normal control group, the activity of the liver TrxR of the model group mouse is obviously improved (P <0.05), which is 5.4 times of that of the control group, and is consistent with the conclusion that the activity of the liver cancer tissue TrxR is obviously improved in literature report [ BBA ]. The activity of TrxR in the BS treatment group is obviously reduced compared with that in the model group (P is less than 0.05), and the activity of TrxR in the BS low, medium and high dose groups is respectively reduced by 19.3%, 48.1% and 67.9%, which indicates that the BS can inhibit the abnormal proliferation of mouse liver cells, inhibit the progress of liver cancer and reduce the cancer rate by reducing the activity of TrxR.

The BS can respectively and remarkably inhibit the first stage, the second stage and the third stage of the model, and totally inhibit the occurrence and the formation of the liver cancer; has no influence on animal body weight, immune system, biochemical index, organ coefficient, etc. (no significant difference)

The BS medicine realizes the mechanism of inhibiting the liver cancer generation process, including the process of inhibiting the expression mechanism of TR/Trx.

EXAMPLE 3 preventive Effect of BS on tumor formation in H22-bearing mice at different doses and at different administration times

3.1 tumor volume and tumor inhibition Rate

The experiment is administrated by adopting different dosages and different administration intervals of BS for 3 days of early intervention, and as shown in Table 9, the average tumor volume of a blank tumor-bearing group is 2317.87 +/-232.52 mm at the 14 th day (17 days in total) of the end of administration³The mean tumor volumes of the 1-day 1-dose and 1-day 2-dose groups of the BS-administered dose of 72mg/kg were 1504.19. + -. 427.06mm, respectively³、1188.36±140.04mm³The average tumor volume of the administration group is 710.47 +/-33.82 mm when the administration dose is 144mg/kg for 1 day and 1 time³. The tumor photograph of FIG. 11 is a very intuitive observation that the tumor volume decreased after the early dry-dose treatment, and the effect of inhibiting tumor cell proliferation increased with the increase of the dose concentration, and the dose was positively correlated, especially in the high dose group. As shown in fig. 10 and 11, tumor growth was inhibited by 35.10% (P ═ 0.044), 48.73% (P ═ 0.010) and 69.35% (P ═ 0.001), respectively, compared to the tumor-bearing blank group, which were significantly different from the tumor-bearing blank group. When the administration dose is fixed to 72mg/kg, the administration times are increased, namely the tumor inhibition rate of the 1-day 2-time administration group is higher than that of the 1-day 1-time administration group, and the tumor inhibition rate is far lower than that of the one-time administration 144mg/kg group, so that the relation between the effect of inhibiting the tumor cell growth by performing BS administration intervention on the mice in advance and the single administration dose is closer.

TABLE 9 analysis of the inhibition of tumor growth in H22-bearing mice by the early intervention of BS at different doses and different administration times

Note: d0: cage dividing time; dn: the optimal time of the actual treatment effect; TV: tumor volume.

3.2 Effect of BS on peripheral blood TR Activity in mice

As shown in Table 10, the peripheral blood TR activity of the mice at the end of the administration was examined, and the mean TR activity of the peripheral blood of the blank tumor-bearing group was 16.58. + -. 0.40U/mL, the mean TR activity of the 1-day 1-dose group and the mean TR activity of the 1-day 2-dose group of the BS-dose 72mg/kg group were 14.75. + -. 0.90 and 13.95. + -. 0.68U/mL, respectively, and the mean TR activity of the 1-day 1-dose group of the BS-dose 144mg/kg group was 13.15. + -. 0.34U/mL. Compared with the control group, the intervention of the BS administration in advance at different doses and different administration times for inhibiting the peripheral blood TR activity is respectively 11.02% (P ═ 0.045), 12.73% (P ═ 0.035) and 20.67% (P ═ 0.001), and the intervention is significantly different from that of the blank tumor-bearing group. When the dosage of the BS is 72mg/kg, the inhibition effect on the peripheral blood TR activity of the mice cannot be obviously improved by increasing the administration frequency, and the relationship between the TR inhibition degree and the single dosage of the BS is more compact.

TABLE 10 Effect of different doses, different dosing times of BS-mediated prior intervention on peripheral blood TR activity in H22-bearing mice

Note: d0: cage dividing time; dn: the optimal time of the actual treatment effect; U/mL: the amount of reduced DTNB per unit time.

Example 4 inhibition of BS on postoperative recurrence of H22 hepatoma mice and preliminary mechanism study thereof

4.1BS inhibition of postoperative tumor recurrence in H22 mice

Balb/c mice were selected to establish a mouse H22 liver cancer graft tumor resection model and sacrificed eight consecutive days after administration on the second day of surgery. The results are shown in FIG. 12. BS all showed potent ability to suppress postoperative tumor recurrence after administration and exhibited time and dose dependence. After the experiment is finished, the average tumor volume of the tumor-bearing mice of the control group is 856.1 +/-282.6 mm, and the average tumor volume of the tumor-bearing mice of the BS low, medium and high dose administration groups is 468.9 +/-123.3 mm³，385.6±189.9mm³，274.4±100.3mm³The tumor growth inhibition rates are 45.2%, 55.0% and 67.9%, respectively, with significant differences (P)<0.05). By the end of the treatment, the relative tumor volume of the control group is 17.12 +/-5.65, and the relative tumor volumes of the BS low, medium and high dose administration groups are 9.38 +/-2.47, 7.71 +/-3.80 and 5.49 +/-2.01 respectively; compared with the control group, the relative tumor proliferation rate of each dose administration group is 54.8 percent, 45.0 percent and 32.1 percent respectively, and the significant difference (P) is shown<0.05) (see table 11).

After the experiment was completed, the mice were dissected, tumors were completely detached, and weighed. The average tumor weight of the tumor-bearing mice of the control group is 766.6 +/-152.1 mg, the average tumor weight of the tumor-bearing mice of the BS low, medium and high dose administration groups is 534.9 +/-183.8 mg, 411.3 +/-116.4 mg and 254.8 +/-29.4 mg, the tumor growth inhibition rates are respectively 30.2%, 46.4% and 66.8%, and significant differences (P <0.05) exist (see Table 12).

TABLE 11 tumor volumes in groups of mice at the end of the experiment

Data are presented as mean ± standard deviation (n ═ 6). P <0.05, indicating a statistical difference compared to the control group.

TABLE 12 tumor weights of groups of mice at the end of the experiment

Data are presented as mean ± standard deviation (n ═ 6). P <0.05, indicating a statistical difference compared to the control group.

4.2 preliminary mechanism study of H22 hepatocarcinoma-inhibiting mice postoperative tumor recurrence by BS

The TrxR activity was measured in mouse serum and tumor tissue, respectively, and the results are shown in tables 13 and 14. In mouse serum, compared with a control group, the TrxR activities of the low, medium and high dose groups of the BS are 45.56 +/-5.58U/mg, 33.18 +/-6.92U/mg, 26.83 +/-5.71U/mg and 12.70 +/-3.73U/mg respectively; the activity of TrxR is reduced by 27.18 percent, 41.11 percent and 72.13 percent respectively, the obvious difference is realized (P is less than 0.05), and the inhibition effect of BS on TrxR is dose-dependent. In tumor tissues, the TrxR activity of the control group was considered as 100%, and the TrxR activity of the low, medium and high dose groups of BS was reduced by 12.74%, 46.78% and 53.03%, respectively, with significant differences (P <0.05) compared to the control group. The results indicate that BS can inhibit postoperative recurrence of tumors by inhibiting TrxR activity.

TABLE 13 Effect of different doses of BS on TrxR Activity in mouse serum

Data are presented as mean ± standard deviation (n ═ 6). P <0.05, representing a statistical difference compared to the control group;^#p <0.05, which indicates a statistical difference from the BSL group.

TABLE 14 Effect of different doses of BS on TrxR Activity in mouse tumor tissues

Data are presented as mean ± standard deviation (n ═ 3). P <0.05, representing a statistical difference compared to the control group;^#p <0.05, which indicates a statistical difference from the BSL group.

Example 5 inhibition of BS on postoperative recurrence of Lewis Lung cancer mice and preliminary mechanism study thereof

5.1BS inhibition of Lewis Lung cancer mice postoperative tumor recurrence

To investigate whether porselenoline has the effect of inhibiting tumor recurrence after mouse lung cancer surgery, we selected Balb/c mice to establish a mouse Lewis lung cancer transplantation tumor resection model and killed eight days after continuous administration on the next day of surgery.

The results are shown in fig. 13, where BS all showed an effective ability to suppress postoperative tumor recurrence and exhibited time and dose dependence. After the experiment is finished, the average tumor volume of the tumor-bearing mice of the control group is 568.2 +/-121.1 mm, and the average tumor volume of the tumor-bearing mice of the BS low, medium and high dose administration groups is 391.1 +/-59.5 mm³，283.3±45.7mm³，225.3±30.1mm³The tumor growth inhibition rates are 31.2%, 50.1% and 60.4%, respectively, and have significant differences (P)<0.05). By the end of the treatment, the relative tumor volume of the control group is 11.36 +/-2.42, and the relative tumor volumes of the BS low, medium and high dose administration groups are 7.82 +/-1.19, 5.67 +/-0.91 and 4.51 +/-0.60 respectively; compared with the control group, the relative tumor increment rates of the dose groups are respectively 68.8%, 49.9% and 39.6%, and the dose groups have dose correlation.

After the experiment was completed, the mice were dissected, tumors were completely detached, and weighed. The average tumor weight of tumor-bearing mice of a control group is 476.5 +/-169.6 mg, the average tumor weight of tumor-bearing mice of a BS low, medium and high dose administration group is 339.0 +/-97.0 mg, 275.0 +/-73.8 mg and 177.4 +/-53.1 mg, the tumor growth inhibition rates are 28.9%, 42.3% and 62.8% respectively, and the significant difference is realized (P < 0.05).

TABLE 15 tumor volume at the end of the experiment in each group of mice

Data are presented as mean ± standard deviation (n ═ 6). P <0.05, indicating a statistical difference compared to the control group.

TABLE 16 tumor weights of groups of mice at the end of the experiment

Data are presented as mean ± standard deviation (n ═ 6). P <0.05, indicating a statistical difference compared to the control group.

5.2 preliminary mechanism study of inhibition of Lewis Lung cancer mice postoperative tumor recurrence by BS

The TrxR activity was measured in serum and tumor tissues of mice given different doses of BS, respectively, and the results are shown in tables 17 and 18. In mouse serum, the TrxR activities of the low, medium and high dose groups of BS are respectively 29.55 +/-6.05U/mg, 25.76 +/-5.71U/mg, 17.63 +/-6.59U/mg and 13.19 +/-1.04U/mg compared with the control group; the activity of TrxR is respectively reduced by 12.85%, 40.34% and 55.37%, the significant difference is that P is less than 0.05, and the inhibition effect of BS on TrxR is dose-dependent. In tumor tissues, the TrxR activity of the control group is set as 100%, and compared with the control group, the TrxR activity of the low, medium and high dose groups of the BS is respectively reduced by 12.74%, 46.78% and 53.03%, and has significant difference (P < 0.05). The results indicate that BS can inhibit postoperative recurrence of tumors by inhibiting TrxR activity.

TABLE 17 Effect of different doses of BS on TrxR Activity in mouse serum

Data are presented as mean ± standard deviation (n ═ 6). P <0.05, representing a statistical difference compared to the control group; # P <0.05, which indicates a statistical difference compared to the BSL group.

TABLE 18 Effect of different doses of BS on TrxR Activity in mouse tumor tissues

Data are presented as mean ± standard deviation (n ═ 3). P <0.05, representing a statistical difference compared to the control group;^#p <0.05, which indicates a statistical difference from the BSL group.

Example 6 inhibition of BS on postoperative recurrence of 4T1 Breast cancer mice and preliminary mechanistic studies thereof

6.1BS inhibition of postoperative tumor recurrence in 4T1 Breast cancer mice

Balb/c mice were selected to establish a mouse 4T1 breast cancer graft tumor resection model and sacrificed eight consecutive days after the administration on the second day of surgery.

The results are shown in fig. 14, where BS all showed an effective ability to suppress postoperative tumor recurrence and exhibited time and dose dependence. After the experiment is finished, the average tumor volume of the tumor-bearing mice in the control group is 808.9 +/-84.6 mm, and the average tumor volume of the low, medium and high-dose BS tumor-bearing mice is 808.9 +/-84.6 mm³,575.3±54.5mm³,462.5±78.6mm³，291.6±37.5mm³The tumor growth inhibition rates are respectively 28.9%, 42.8% and 64.0%, and significant differences (P) are observed<0.05). By the end of the treatment, the relative tumor volume of the control group is 16.18 +/-1.69, and the relative tumor volumes of the BS low, medium and high dose administration groups are 11.51 +/-1.09, 9.25 +/-1.57 and 5.83 +/-0.75 respectively; compared with the control group, the relative tumor increment rates of the dosing groups are 71.1%, 57.2% and 36.0%, respectively, and have dose correlation.

After the experiment was completed, the mice were dissected, tumors were completely detached, and weighed. The average tumor weight of the tumor-bearing mice of the control group is 603.1 +/-118.9 mg, the average tumor weight of the tumor-bearing mice of the BS low, medium and high dose administration groups is 457.8 +/-115.0 mg, 388.8 +/-69.8 mg and 215.1 +/-42.9 mg, the tumor growth inhibition rates are 24.1%, 35.5% and 64.3% respectively, and the significant difference is realized (P < 0.05).

TABLE 19 tumor volume at the end of the experiment in each group of mice

Data are presented as mean ± standard deviation (n ═ 6). P <0.05, indicating a statistical difference compared to the control group.

TABLE 20 tumor weights of groups of mice at the end of the experiment

Data are presented as mean ± standard deviation (n ═ 6). P <0.05, indicating a statistical difference compared to the control group.

6.2 preliminary mechanistic study of the inhibition of postoperative tumor recurrence in 4T1 Breast cancer mice by BS

TrxR activity was measured in mouse serum and tumor tissues, respectively, and the results are shown in tables 21 and 22. In mouse serum, the TrxR activities of the low, medium and high dose groups of BS are respectively 25.74 +/-3.21U/mg, 20.71 +/-3.46U/mg, 16.87 +/-2.81U/mg and 13.26 +/-4.80U/mg compared with the control group; the activity of TrxR is respectively reduced by 19.54 percent, 34.46 percent and 48.51 percent, the significant difference is realized (P is less than 0.05), and the inhibition effect of BS on TrxR is dose-dependent. In tumor tissues, the TrxR activity of the control group is set as 100%, and compared with the control group, the TrxR activity of the low, medium and high dose groups of the BS is respectively reduced by 12.74%, 46.78% and 53.03%, and has significant difference (P < 0.05). The results indicate that BS can inhibit postoperative recurrence of tumors by inhibiting TrxR activity.

TABLE 21 Effect of different doses of BS on TrxR Activity in mouse serum

Data are presented as mean ± standard deviation (n ═ 6). P <0.05, representing a statistical difference compared to the control group;^#p <0.05, which indicates a statistical difference from the BSL group.

TABLE 22 Effect of different doses of BS on TrxR Activity in mouse tumor tissues

Data are presented as mean ± standard deviation (n ═ 6). P <0.05, representing a statistical difference compared to the control group; # P <0.05, which indicates a statistical difference compared to the BSL group.

Examples 4-6 establish mouse liver cancer, lung cancer and breast cancer postoperative tumor recurrence models, and evaluate the inhibition ability of low, medium and high dose of BS on mouse tumor recurrence, the highest dose of BS on liver cancer, lung cancer and breast cancer inhibition rates are 67.9%, 60.4% and 64%, respectively, and the tumor growth rate is lower than 40%, showing effective inhibition of tumor postoperative recurrence effect. The BS is a targeted TrxR inhibitor, and after the detection of the TrxR activity in the serum and the tumor tissue of the mouse, the BS has the capability of inhibiting the TrxR activity in the serum and the tumor tissue of the mouse in a dose-dependent manner, which indicates that the BS can inhibit the postoperative recurrence of the tumor by inhibiting the Trx/TrxR system.

Example 7 the inhibitory effect of BS drugs on the growth of various tumor cells can be widely used for the postoperative recurrence control of various tumors

7.1 study of inhibition of BS on proliferation of human colorectal cancer cell lines

Firstly, researching the inhibition effect of BS on the proliferation of a human colorectal cancer cell line, inoculating LoVo, RKO and SW480 cells in a logarithmic growth phase into a 96-well plate, using BS with different concentrations to act for 24 hours, 48 hours and 72 hours respectively after the cells are attached to the wall, and detecting the inhibition rate of the cell proliferation by an SRB method. Data were processed using graphpad6.0 software and the results are shown in fig. 15, with increasing dosing and duration of drug action, BS showed inhibitory effect on all human colorectal cancer cell lines LoVo, RKO and SW480 cells.

In LoVo cell line, BS acts on half inhibitory concentrations IC of 24, 48, 72h₅₀Respectively 10.28 +/-1.27, 8.28 +/-0.95 and 6.79 +/-0.34 mu M; the maximum inhibition rates of the BS on 24h, 48 h and 72h are 73.21 +/-3.67 h, 84.33 +/-5.80 h and 100% respectively.

Half maximal inhibitory concentration IC of BS on 24, 48, 72h in RKO cell lines₅₀11.76 +/-0.37, 7.20 +/-0.58 and 6.06 +/-0.31 mu M respectively; the maximum inhibition rates of the BS for 24h, 48 h and 72h are respectively 62.46 +/-0.78, 78.72 +/-2.71 and 87.68 +/-1.26 percent.

Half maximal inhibitory concentration IC of BS on 24, 48, 72h in SW480 cell line₅₀Respectively is 18.51 +/-1.78, 9.79 +/-1.25 and 9.51 +/-0.64 mu M; the maximum inhibition rates of BS for 24h, 48 h and 72h are 61.48 +/-8.34, 90.79 +/-5.50 and 84.40 +/-7.59 respectively.

7.2 study of the inhibitory Effect of BS on the proliferation of human Lung cancer cell lines

The human lung cancer cell lines A549, H1299 and SPCA-1 cells in logarithmic growth phase are inoculated into a 96-well plate, after adherence, BS with different concentrations is used for respectively acting for 24H, 48H and 72H, and then an SRB method is adopted to detect the cell proliferation inhibition rate. The data were processed using graphpad6.0 software and the results are shown in figure 16, with increasing dose and duration of drug action, BS showed inhibitory effects on all human lung cancer cell lines a549, H1299 and SPCA-1 cells.

Half maximal inhibitory concentration IC of BS on 24, 48, 72h in A549 cell line₅₀9.40 +/-0.97, 5.68 +/-1.49 and 5.47 +/-0.96 mu M respectively; the maximum inhibition rates of 24, 48 and 72 hours of BS action are 73.73 +/-7.22, 94.35 +/-4.89 and 87.70 +/-2.67 percent respectively.

Half maximal inhibitory concentration IC of BS on 24, 48, 72H in H1299 cell line₅₀11.17 +/-0.71, 8.14 +/-0.23 and 8.07 +/-1.40 mu M respectively; the maximum inhibition rates of BS for 24h, 48 h and 72h are respectively 76.44 +/-1.79, 90.12 +/-2.30 and 92.86 +/-0.95 percent.

Half maximal inhibitory concentration IC of BS on 24, 48, 72h in SFCA-1 cell line₅₀20.61 +/-5.03, 19.01 +/-1.11 and 9.12 +/-1.67 mu M respectively; the maximum inhibition rates of the BS for 24h, 48 h and 72h are 60.67 +/-9.64, 77.98 +/-0.69 and 92.27 +/-6.46 percent respectively.

7.3 study of the inhibitory Effect of BS on the proliferation of human esophageal carcinoma cell lines

Inoculating human esophageal cancer cell lines KYSE150, KYSE450 and KYSE510 in logarithmic growth phase into a 96-well plate, respectively acting for 24h, 48 h and 72h by using BS with different concentrations after adherence, and detecting the cell proliferation inhibition rate by adopting an SRB method. Data were processed using graphpad6.0 software and as a result, BS showed inhibitory effect on human esophageal cancer cell lines KYSE150, KYSE450 and KYSE510 cells as dose and duration of drug action increased as shown in fig. 17.

Half inhibitory concentration IC of BS on 24, 48, 72h in KYSE150 cell line₅₀23.22 +/-2.74, 9.74 +/-0.23 and 8.23 +/-0.31 mu M respectively; the maximum inhibition rates of the BS for 24h, 48 h and 72h are 51.87 +/-4.72, 96.20 +/-0.39 and 88.67 +/-4.88 percent respectively.

Half inhibitory concentration IC of BS on 24, 48, 72h in KYSE450 cell line₅₀Are respectively10.10 +/-1.05, 8.78 +/-0.64 and 8.42 +/-0.34 mu M; the maximum inhibition rates of 24, 48 and 72 hours of BS action are 88.82 +/-2.18, 89.61 +/-1.45 and 93.34 +/-1.77 respectively.

Half inhibitory concentration IC of BS on 24, 48, 72h in KYSE510 cell line₅₀11.63 +/-0.64, 8.75 +/-0.07 and 6.61 +/-0.19 mu M respectively; the maximum inhibition rates of BS for 24h, 48 h and 72h are respectively 76.64 +/-2.05, 94.54 +/-1.58 and 100 percent.

7.4 investigation of the inhibitory Effect of BS on the proliferation of other cancer cell lines in humans

Inoculating a human breast cancer cell line MCF-7, a human cervical cancer cell line HeLa and a human chronic myelogenous leukemia cell k562 which are in a logarithmic growth phase into a 96-well plate, respectively acting for 24 hours, 48 hours and 72 hours by using BS with different concentrations after attaching to the wall, and detecting the proliferation inhibition rate of the cells by adopting an SRB method. Data were processed using graphpad6.0 software, and as a result, as shown in fig. 18, BS showed inhibitory effects on all of the human breast cancer cell line MCF-7, the human cervical cancer cell line HeLa, and the human chronic myelogenous leukemia cell k562 cells as the administration dose and the drug action time were increased.

Half maximal inhibitory concentration IC of BS on 24, 48, 72h in MCF-7 cell line₅₀Respectively 17.38 +/-2.38, 11.53 +/-0.71 and 8.18 +/-0.15 mu M; the maximum inhibition rates of BS for 24h, 48 h and 72h are respectively 80.65 +/-4.29, 92.85 +/-0.95 and 95.71 +/-0.73 percent.

Half maximal inhibitory concentration IC of BS on 24, 48, 72h in HeLa cell line₅₀20.19 +/-5.12, 15.58 +/-2.31 and 12.49 +/-1.35 mu M respectively; the maximum inhibition rates of BS for 24h, 48 h and 72h are respectively 56.20 +/-5.58%, 62.76 +/-3.04% and 80.79 +/-7.49%.

Half maximal inhibitory concentration IC of BS on 24, 48, 72h in K562 cell line₅₀Respectively 10.56 +/-0.94, 5.97 +/-0.45 and 6.20 +/-0.33 mu M; the maximum inhibition rates of BS in 24h, 48 h and 72h are respectively 83.08 +/-1.07, 94.87 +/-3.20 and 97.31 +/-1.08%.

BS exhibits proliferation inhibitory effects on cells of various human cell lines. The research shows that the BS has broad-spectrum and high-efficiency tumor cell proliferation inhibition effect and time and dose dependence, and has stronger inhibition effect on lung cancer, colorectal cancer and esophageal cancer. As a post-operative control drug for tumors, various tumors may have an effective effect.

TABLE 23 growth inhibition of BS on various tumor cells

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. Application of benzisoselenazole derivatives in preparing medicaments for treating tumors.

2. The use according to claim 1, wherein the benzisoselenazole derivative has the structure of a compound represented by formula A, which is selected from at least one of a compound represented by formula A, a precursor thereof, an active metabolite, a stereoisomer, a pharmaceutically acceptable salt, a prodrug, and a solvate thereof,

wherein R is₁、R₂Identical or different, independently of one another, from H or the following radicals: c_1-12Alkyl radical, C_3-20A cycloalkyl group;

wherein R is selected from C_1-12Alkylene, phenylene, biphenylene, triphenylene, orWherein M represents Pt, Pd or Rh.

3. The use according to claim 2, wherein in the compound of formula a, R is₁、R₂The same or different, and the same or different,independently of each other, selected from H, -CH₃、-CH₂CH₃、-CH(CH₃)₂、-C(CH₃)₃、-CH(CH₂)₄or-CH (CH)₂)₅(ii) a R is selected from-CH₂-、-C₂H₄-、-C₄H₈-, phenylene-C₆H₄-。

4. Use according to any one of claims 1 to 3, wherein the benzisoselenazole derivative is selected from 1, 2-bis [2- (1, 2-benzisoselenazol-3 (2H) -one) ] -butane, the structure of which is shown below:

5. the use according to any one of claims 1 to 4, wherein the tumour comprises solid tumours and non-solid tumours, both benign and malignant;

preferably, the tumor comprises an initial tumor and a postoperative recurrent tumor;

preferably, the tumor includes, but is not limited to: liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, gastric cancer, skin cancer, bladder cancer, ovarian cancer, prostate cancer, bone cancer, brain cancer, rectal cancer, esophageal cancer, tongue cancer, lymph cancer, oral epithelial cancer, epithelial cervical cancer or chronic myelogenous leukemia.

6. The use according to any one of claims 1 to 5, wherein the benzisoselenazole derivative is used for inhibiting tumor cell proliferation; preferably, the tumor cells include human cancer cells and mouse cancer cells.

7. The use according to any one of claims 1 to 5, wherein the benzisoselenazole derivative is used for preparing a medicament for treating liver cancer;

preferably, the dosage of the benzisoselenazole derivative is 1-500 mg/kg based on the weight of an administration object;

preferably, the concentration of the benzisoselenazole derivative is 1-100 mu M.

8. Use of the benzisoselenazole derivative according to any one of claims 1 to 4 for the preparation of a medicament for the treatment of postoperative recurrence of a neoplasm;

preferably, the tumor cell is at least one selected from human liver cancer cell Bel-7402, human colorectal cancer cell LoVo, human epithelial cervical cancer cell HeLa, human lung cancer cell A549, mouse liver cancer cell H22, mouse lung cancer cell Lewis and mouse breast cancer cell 4T 1.

9. The use according to claim 8, wherein the benzisoselenazole derivative is administered in an amount of 1-500 mg/kg, based on the weight of the subject;

preferably, the concentration of the benzisoselenazole derivative is 1-100 mu M.

10. A drug for preventing and/or treating tumors, which comprises at least the benzisoselenazole derivative according to any one of claims 1 to 4;

preferably, the prevention and/or treatment of a tumor comprises prevention and/or treatment in post-operative recurrence of a tumor according to claim 8;

preferably, the medicament may also optionally comprise at least one pharmaceutically acceptable excipient.

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