CN109316483B - Medical application of isotoosendanin - Google Patents
Medical application of isotoosendanin Download PDFInfo
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- CN109316483B CN109316483B CN201811280279.8A CN201811280279A CN109316483B CN 109316483 B CN109316483 B CN 109316483B CN 201811280279 A CN201811280279 A CN 201811280279A CN 109316483 B CN109316483 B CN 109316483B
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Abstract
The invention relates to the field of medicine, in particular to medical application of isotoosendanin. The toosendanin of the invention can be used for preventing and treating breast cancer.
Description
Technical Field
The invention relates to the field of medicines, in particular to a medical application of isotoosendanin.
Background
Malignant tumor is a worldwide public problem, has great influence on national economy, social development and health of people, and tumor prevention and control have become important points of global health strategy. An important feature of tumor cells is the inability to differentiate normally, initiating normal apoptotic processes, and therefore, the development of drugs for preventing and treating tumors would benefit humans.
Breast cancer is the most frequent malignancy in women. World Health Organization (WHO) international cancer research institute (International Agency for Research on Cancer, IARC) documents and publishes by constantly collecting, storing, collating, analyzing and evaluating tumor registration data worldwide: in 2012, 1409 ten thousand new malignant tumor cases are worldwide, 820 ten thousand deaths are caused, more than 167 ten thousand new cases of breast cancer are caused in worldwide women, the new cases account for the first place of all malignant tumor diseases of women, and 1 case is breast cancer case in almost every 4 female malignant tumor cases; about 52 ten thousand female patients who die from breast cancer at the same time are also located at the first place of death of female malignant tumors. In recent years, the global female breast cancer incidence and mortality level have been increasing year by year, and the global female malignant tumor incidence and mortality composition has been increasing.
Triple negative breast cancer is one of the more serious types of breast cancer. Triple negative breast cancer refers to breast cancer that is negative for both Estrogen Receptor (ER), progestogen Receptor (PR) and protooncogene Her-2 as a result of cancer tissue immunohistochemical examination. The breast cancer accounts for 10.0% -20.8% of all pathological types of breast cancer, has special biological behaviors and clinical pathological characteristics, has poorer prognosis and higher death risk than other types. The clinical manifestation of triple negative breast cancer is an invasive disease course, the risk of distant metastasis is higher, the chance of visceral metastasis is higher than that of bone metastasis, and the probability of brain metastasis is also higher. The risk of distant metastasis of triple negative breast cancer peaks at 3 years, after which it may decline. Prognosis of triple negative breast cancer is not greatly related to tumor size and lymph node condition, and recurrence is rapid. Because of lack of endocrine and anti-HER 2 therapeutic targets, no specific therapeutic guidelines for triple negative breast cancer exist at present, and thus the treatment is generally carried out according to the conventional standard of breast cancer treatment.
In the treatment of cancer, although the traditional radiotherapy and chemotherapy have been greatly progressed, the traditional radiotherapy and chemotherapy also produce different degrees of toxic and side effects on human bodies while killing cancer cells, wherein mainly myelosuppression and low immune function can cause leucopenia and the like, and sometimes even the treatment has to be interrupted, thereby limiting the curative effect of the medicament. In order to overcome a series of disadvantages caused by traditional radiotherapy and chemotherapy, in recent years, the continuous exploration of pharmaceutical workers and the development of safer and more effective herbal medicines for preventing and treating cancers become the hot spot field of current researches.
Fructus Toosendan is a dry mature fruit of Melia toosendan Sieb. Et Zucc. Of Meliaceae, and is mainly distributed in Henan, gansu, hunan, guangxi, sichuan, guizhou, yunnan, etc. in Shennong BenCao Jing (Shennong's herbal medicine). Bitter taste and cold nature, small toxicity, fire purging, pain relieving, and insect killing effects, and can be used for treating stomach ache, abdominal pain due to insect accumulation, hernia pain, dysmenorrhea, etc. Liver soothing, heat expelling, qi moving, pain relieving, and insect killing effects. Is used for treating liver depression transforming into fire, chest and hypochondrium, abdominal distention and pain, hernia pain, and abdominal pain due to accumulation of parasites. Isoosendanin (iso-chuanliansu) is an isolated and purified chinaberry alkane type triterpene compound from fructus Toosendan, and the pharmacological activity of Isoosendanin is not clearly studied at present.
Disclosure of Invention
The invention aims to provide a novel medical application of isotoosendanin.
In particular, the first aspect of the invention provides an application of isotoosendanin in preparing medicines for preventing and treating breast cancer.
In a preferred embodiment, the isotoosendanin is used as the only active ingredient for preparing the medicine for preventing and treating breast cancer.
In another preferred embodiment, the breast cancer is triple negative breast cancer.
The second aspect of the invention provides an application of the isotoosendanin in preparing medicines for preventing and treating breast cancer metastasis.
In a preferred embodiment, the isotoosendanin is used as the only active ingredient for preparing the medicine for preventing and treating breast cancer metastasis.
In another preferred embodiment, the breast cancer is triple negative breast cancer.
Details of the various aspects of the invention will be described in detail in subsequent sections. The features, objects, and advantages of the invention will be apparent from the description, and from the claims.
Drawings
FIG. 1 killing effect of Isocyanochinaberry element on various breast cancer cells
(a)MDA-MB-231 (b)MCF-7 (c)4T1
FIG. 2 Effect of Isocyanochinin administration on 4T1 solid tumors in female Balb/c mice
FIG. 3 Effect of Isocyanochinin administration on serum Biochemical index of female Balb/c 4T1 tumor-bearing mice
FIG. 4 scratch test result showing the inhibition effect of the azadirachtin administration on breast cancer cell migration
(a)MDA-MB-231 (b)4T1
The model group, the low dose group of the isotoosendanin, the medium dose group of the isotoosendanin and the high dose group of the isotoosendanin are respectively arranged from left to right
FIG. 5 results of a cell migration experiment, which shows the inhibition of breast cancer cell migration by azadirachtin
(a)MDA-MB-231 (b)4T1
The model group, the low dose group of the isotoosendanin, the medium dose group of the isotoosendanin and the high dose group of the isotoosendanin are respectively arranged from top to bottom
FIG. 6 Effect of Isocyanochinin administration on 4T1 solid tumors in female Balb/c mice
FIG. 7 Effect of Isocyanochinin administration on mortality of female Balb/c tumor-bearing mice 4T1
FIG. 8 effect of Isocyanochinin administration on metastasis of 4T1 solid tumor in female Balb/c mice-in vivo imaging results
FIG. 9 Effect of Isocyanochinin administration on metastasis Rate of 4T1 solid tumor in female Balb/c mice
FIG. 10 effect of azadirachtin administration on metastasis of 4T1 solid tumors in female Balb/c mice-pulmonary HE staining results A: blank B: model set C: isocyanochinin 0.1mg/kg D: 1mg/kg of azadirachtin E: 5-fluorouracil
Detailed Description
The present invention is based in part on the unexpected discovery that: the toosendanin has a certain inhibiting effect on most tumors, especially triple negative breast cancer. Therefore, the compound can be hopefully developed into a medicine for preventing or treating triple negative breast cancer.
Furthermore, the invention provides application of the toosendanin in preparing medicines for preventing and treating breast cancer and breast cancer metastasis. The Isotoosendanin (iso-chuanlisu) is colorless fine needle crystal, and has a melting point of 270-273 ℃, is easy to dissolve in pyridine, slightly dissolve in methanol, acetone and dioxane, and is difficult to dissolve in chloroform, benzene and water. The molecular formula of the isotoosendanin is C 30 H 38 O 11 The structural formula is as follows:
the azadirachtin of the present invention may be obtained commercially from Shanghai source leaf biotechnology Co., ltd, or may be obtained by extraction from Toosendan by a method conventional in the art, for example: pulverizing Toosendan, and reflux extracting with water, 20-95% ethanol, acetone, ethyl acetate, gasoline or petroleum ether.
The azadirachtin of the present invention may be used alone or in the form of a pharmaceutical composition. The pharmaceutical composition comprises the isotoosendanin of the present invention as an active ingredient and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition of the present invention contains 0.1 to 99.9% by weight of the isotoosendanin of the present invention as an active ingredient. The pharmaceutically acceptable carrier does not destroy the pharmaceutical activity of the isotoosendanin, and the effective dosage of the isotoosendanin can play the role of the pharmaceutical carrier, so that the dosage is nontoxic to human bodies.
The pharmaceutically acceptable carrier includes, but is not limited to: soft phospholipids, aluminum stearate, aluminum oxide, ion exchange materials, self emulsifying drug delivery systems, tween or other surfactants, serum proteins, buffer substances such as phosphates, glycine, sorbic acid, water, salts, electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, magnesium silicate, saturated fatty acid partial glyceride mixtures and the like.
Other commonly used pharmaceutical excipients such as binders (e.g. microcrystalline cellulose), fillers (e.g. starch, glucose, lactose anhydrous and lactose beads), disintegrants (e.g. crospvp, croscarmellose sodium, low substituted hydroxypropylcellulose), lubricants (e.g. magnesium stearate) and absorption promoters, adsorption carriers, fragrances, sweeteners, excipients, diluents, wetting agents, etc.
The azadirachtin and pharmaceutical compositions thereof of the present invention may be prepared according to conventional methods in the art and may be administered by enteral or parenteral or topical routes. The oral preparation comprises capsule, tablet, oral liquid, granule, pill, powder, pellet, paste, etc.; parenteral formulations include injection solutions and the like; topical formulations include creams, patches, ointments, sprays and the like. Preferably an oral formulation.
The administration route of the azadirachtin and the pharmaceutical composition of the present invention may be oral, sublingual, transdermal, intramuscular or subcutaneous, skin mucosa, vein, urethra, vagina, etc.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. All percentages, ratios, proportions, or parts are by weight unless otherwise indicated.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.
The above-mentioned features of the invention, or of the embodiments, may be combined in any desired manner. All of the features disclosed in this patent specification may be combined with any combination of the features disclosed in this specification, and the various features disclosed in this specification may be substituted for any alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are merely general examples of equivalent or similar features.
Example 1 killing of Isocyanochinaberry to multiple breast cancer cells
The invention firstly examines the relation between the survival rate of the azadirachtin on various breast cancer cells of different species and the concentration of the azadirachtin by using an MTT method.
The MTT method is also called MTT colorimetric method, and is a method for detecting the survival and growth of cells. The detection principle is to detect the number of living cells by utilizing the principle that mitochondrial dehydrogenase in living cells can convert tetramethyl azoazole blue into blue-violet derivative formazan. After the formazan crystals were dissolved with a triple reagent (10% SDS-5% isobutanol-0.01M HCl), absorbance was measured at 570nm and 630nm using an enzyme-labeled instrument. Can indirectly reflect the number of living cells. The amount of MTT crystals formed is proportional to the number of cells over a range of cell numbers.
1.1 Experimental materials
1.1.1 cell lines
MDA-MB-231 cell line; MCF-7 cell line (human breast cancer cell line); 4T1 cell strain (mouse breast cancer cell strain)
1.1.2 test Agents and preparation thereof
Isocyanochinin (available from Shanghai Secoides BioCo., ltd.) was dissolved in DMSO (dimethyl sulfoxide) to prepare a mother solution of 0.1M.
1.1.3 major reagents
DMEM, RPMI1640, fetal bovine serum (GIBCO), dimethyl sulfoxide (DMSO) (GIBCO), MTT (Sigma), other reagents are homemade analytically pure, etc.
1.1.4 major instrumentation
Laminar flow super clean bench; a carbon dioxide incubator available from Thermo company; inverted phase contrast microscope, available from Olympus corporation; microplate reader, available from Biotek company.
1.2 Experimental methods
MCF-7, MDA-MB-231, 4T1 and other cells are inoculated into a 96-well plate at a cell density of 6000 cells/well, 100 mu L of cell suspension is added into each well, and ITSN (0,0.1,0.3,1,3,10 mu M) with different concentrations is added after about 12 hours until the cell adhesion growth state is good. After 48h, 10. Mu.L MTT (final concentration 0.5 mg/ml) was added to each well and incubated at 37℃in a 5% CO2 incubator for 4h, and then 50. Mu.L of triple agent was added to solubilize formazan crystals. After 12h of reaction, the reaction mixture was placed on an ELISA reader, and absorbance (OD) values at 570nm and 630nm were read. The value of the administration group divided by the control group was defined as the cell viability, calculated according to the following formula:
cell viability (%) = dosing OD (570 nm-630 nm)/control OD (570 nm-630 nm) ×100%
1.3 statistical treatment
The experimental data are all expressed by mean value + -standard error, analyzed by SPSS17.0 statistical software, and analyzed by One-Way ANOVA, and P <0.05 is the standard with statistically significant difference.
1.4 experimental results
As shown in figure 1, the azadirachtin can obviously inhibit the growth of human breast cancer cells MCF-7, MDA-MB-231 and mouse breast cancer cells 4T1, and suggests that the azadirachtin can be used for treating various breast cancers. In addition, the human breast cancer cells MDA-MB-231 and the mouse breast cancer cells 4T1 are triple-negative breast cancer cell lines, the MCF-7 is an ER-positive breast cancer cell line, and from the MTT experimental result, the killing effect of the isocapraxin on the triple-negative breast cancer cells is obviously larger than that of the non-triple-negative breast cancer cell line under the same dose, so that the isocapraxin can be used as a potential treatment drug for the triple-negative breast cancer.
TABLE 1 Effect of Isocyanochinaberry on growth (%) of various breast cancer cells
P <0.01, P <0.001 compared to the blank group
Example 2 in vivo anti-triple negative breast cancer solid tumor action of Isocyanochinaberry
After determining that the isotoosendanin has stronger killing effect on the breast cancer cells in vitro, especially determining that the isotoosendanin has stronger killing effect on triple negative breast cancer cell lines in vitro, the invention firstly further examines the in vivo triple negative breast cancer solid tumor resisting effect of the isotoosendanin.
2.1 Experimental materials
2.1.1 test animals
Female Balb/c mice
2.1.2 test Agents and preparation thereof
Isocyanochinaberry extract was dissolved in 0.5% CMC-Na (sodium carboxymethylcellulose) solution. 5-Fu (Shanghai bang) was dissolved in physiological saline.
2.2 Experimental methods
2.2.1 in vivo tumor-bearing mice anti-tumor experiments
Culturing a certain number of 4T1 cells by in vivo experiment of tumor-bearing mice, centrifuging after digestion to remove pancreatin, and diluting to 1.0X10 by serum-free culture medium 7 cells/mL were placed in centrifuge tubes, ice-on, and the concentration of tumor cells was inoculated by subcutaneous injection (sc) into the right underarm of Balb/c mice at a dose of 0.1 mL/mouse. The animals were randomly grouped according to body weight, and the animals were model group (0.5% CMC-Na), positive drug 5-fluorouracil group, low dose of isotoosendanin group (1 mg/kg), and high dose of isotoosendanin group (3 mg/kg). The drug was dissolved to the desired concentration with 0.5% CMC-Na, and daily gavage (ig) dosing was started at 3 weeks post-inoculation with body weight, model group ig was given an equivalent volume of 0.5% CMC-Na, 5-fluorouracil (dissolved to 2.5mg/mL with dd water) group i.p. (i)p) injecting once every other day; the administration was continued for 14d. Tumor diameter was measured with vernier calipers every other week, and length and width (mm) of the tumor outside the body were recorded. 24 hours after the last administration, taking the eyeball to collect blood for measuring the activity of serum ALT and AST after centrifugally separating the serum; tumor tissue is taken, weighed and photographed. Tumor volume (Tumor volume) and Tumor inhibition (Tumor inhibition ratio) were calculated as follows.
Tumor volumn(mm 3 ) =a×b×b/2, where a denotes the length of the tumor and b denotes the width of the tumor. With significant differences between tumor volumes and model groups, P<0.05 is the standard of the antitumor activity of the medicine.
Tumor inhibition ratio (%) = [ (C-T)/C ] ×100, where C refers to CMC-Na control group mean tumor volume and T refers to administration group mean volume. The tumor volume is obviously different, and the tumor inhibition rate is more than 30 percent, which is the standard of the medicine with anti-tumor activity.
2.3 statistical treatment
The experimental data are all expressed by mean value + -standard error, analyzed by SPSS17.0 statistical software, and analyzed by One-Way ANOVA, and P <0.05 is the standard with statistically significant difference.
2.4 experimental results
2.4.1 Effect of Isocyanochinin administration on 4T1 solid tumors in female Balb/c mice
Compared with the model group, as shown in table 2 and fig. 2, the tumor volume of the 4T 1-inoculated mice is significantly reduced (P < 0.05) after 14d of continuous administration of ITSN (1 mg/kg), which indicates that ITSN (1 mg/kg) is effective against 4T1 solid tumors, and the tumor inhibition rate exceeds 30%, which indicates that isotoosendanin has significant in vivo anti-triple negative breast cancer activity. Compared with 5-Fu group toosendanin, the dosage is lower, and the anti-tumor effect is similar, which shows that the toosendanin has certain advantage in dosage for resisting triple negative breast cancer.
TABLE 2 Effect of Isocyanochinin administration on 4T1 solid tumors in female Balb/c mice
Compared with model group, P <0.05
2.4.2 Effect on serum Biochemical index
From table 3 and fig. 3, the pharmaceutical composition of the invention has no significant hepatotoxicity to tumor-bearing mice, which suggests that the invention has no obvious influence on liver injury index while maintaining curative effect, thus indicating that the invention does not induce obvious liver injury on anti-tumor dose.
TABLE 3 Effect of Isocyanochinin administration on serum Biochemical index of female Balb/c 4T1 tumor-bearing mice
EXAMPLE 3 migration inhibition of Isocyanochinaberry element on various breast cancer cells
After determining that the isotoosendanin has stronger killing effect on the breast cancer cells in vitro, the invention firstly further examines the effect of the low dose of the isotoosendanin in vitro on inhibiting the migration of the triple negative breast cancer cell line.
3.1 Experimental materials
3.1.1 cell lines
MDA-MB-231 cell line (human breast cancer cell line)
4T1 cell strain (mouse breast cancer cell strain)
3.1.2 test Agents and preparation thereof
Isocyanochinin (available from Shanghai Secoides BioCo., ltd.) was dissolved in DMSO (dimethyl sulfoxide) to prepare a mother solution of 0.1M.
3.1.3 major reagents
RPMI1640, fetal bovine serum (GIBCO Co.), dimethyl sulfoxide (DMSO) (GIBCO Co.), crystal violet solution (Life Co.), and other reagents are homemade analytically pure.
3.1.4 major instrumentation
Laminar flow super clean bench; a carbon dioxide incubator available from Thermo company; inverted phase contrast microscope, olympus IX81 fluorescence microscope, available from Olympus corporation; the microplate reader, available from Biotek company; camera, available from Nikon corporation.
3.2 Experimental methods
3.2.1MDA-MB-231, 4T1 cell scratch assay
Cells such as MDA-MB-231 and 4T1 were digested with pancreatin, and resuspended to a density of 3X 10 5 Each/mL of the cell suspension was seeded into a 24-well plate with 1mL of the cell suspension per well. When the cell fusion rate is over 90%, the original culture medium is sucked, 200 mu L of DPBS is added to clean the residual culture medium, and 200 mu L of DPBS is added. After washing the detached cells with 200. Mu.L of sterilized gun head and center of plate bottom, washing with DPBS was repeated twice, 1mL of medium without FBS was added to each well, and different doses of azadirachtin (MDA-MB-231: 100nM, 300nM, 1000nM;4T1:10nM, 30nM, 100 nM) were added to each of the drug administration. Two fields of view are taken per hole, the width of the scratch is recorded by using a cellSens Dimension image acquisition workstation, and the position of the shooting part is marked by using a marker. The width changes of scratches at 0h, 24h and 48h after the labeling positions were photographed, the data was recorded, and the relative cell mobility was calculated (Migration percentage). Relative cell mobility was defined as the ratio of the difference in scratch width between the control and dosing groups at each time point to the scratch width of the control, as follows:
migration percentage (%) = (administration group scratch width-control group scratch width)/control group scratch width×100%
3.2.2MDA-MB-231, 4T1 cell migration experiments
Standing cells were placed in 24-well plates, the cells were coated with 0.5% gelatin at 37deg.C for 30min, and cells starved for one hour in advance were digested and resuspended to a density of 1.5X10 with serum-free RPMI1640 medium 5 Individual/ml cell suspension. MDA-MB-231, 4T1 and other cells were pre-incubated with different concentrations of isotoosendanin (MDA-MB-231: 100nM, 300nM, 1000nM;4T1:10nM, 30nM, 100 nM) for 15min, and inoculated into cells, each cell suspension of 100. Mu.L, and 600. Mu.L of RPMI1640 medium containing 10% fetal bovine serum was added to the lower chamber. After 24h, the culture medium in the small chamber and the lower chamber is sucked, 1mL of pre-cooled methanol at-20 ℃ is added into each hole, the methanol is sucked after being fixed in a refrigerator at-20 ℃ for 30min, the small chamber is taken out, inverted and dried, and the small chamber is dyed by 0.1 percent crystal violet solution15min. And washing off crystal violet staining solution in PBS solution, wiping off cells in the cells by using a wet cotton swab, airing, photographing the cells stained with purple at the bottom of the cells under an inverted phase contrast microscope, taking 5 views from each cell, and photographing. After photographing, the cells were placed in 33% glacial acetic acid solution, the eluate was transferred to a 96-well plate, and the absorbance value (OD) at 570nm was read. The difference between the control group minus the dosing group divided by the value of the control group was defined as the cell migration inhibition rate, and was calculated as follows:
cell migration inhibition ratio (%) = (administration group OD value-control group OD value)/control group OD value×100%
3.3 statistical treatment
The experimental data are all expressed by mean value + -standard error, analyzed by SPSS17.0 statistical software, and analyzed by One-Way ANOVA, and P <0.05 is the standard with statistically significant difference.
3.4 experimental results
3.4.1 Isocyanochinaberry to inhibit MDA-MB-231, 4T1 cell migration-scratch test results
The scratch results are shown in Table 4 and FIG. 4, and after the administration of the composition in a dose without obvious toxicity, the crawling distance of cells in a medium-high dose group is obviously shortened, which indicates that the toosendanin obviously inhibits the cell migration, and the toosendanin has a certain effect of inhibiting the cell migration of triple negative breast cancer.
TABLE 4 Effect of Isocyanochinin administration for 48h on migration of triple negative breast cancer cells MDA-MB-231 and 4T 1-scratch test results
3.4.2 Isocyanochinaberry extract inhibits MDA-MB-231 and 4T1 cell migration-laboratory migration experiment result
The cell migration results are shown in Table 5 and FIG. 5, and the results show that the migration cells are obviously reduced in the visual field of the middling and high-dose azadirachtin after the azadirachtin is administered at a dose without obvious toxicity, so that the azadirachtin has a certain effect of inhibiting the migration of triple negative breast cancer cells.
TABLE 5 Effect of Isocyanochinin administration for 48h on migration of triple negative breast cancer cells MDA-MB-231 and 4T 1-results of laboratory migration experiments
Example 4 in vivo anti-breast cancer solid tumor metastasis with Isocyanochinaberry
After determining that the isotoosendanin has stronger killing effect on the tumor cells in vitro, the invention firstly further examines the in vivo effect of inhibiting breast cancer cell metastasis of the isotoosendanin.
4.1 Experimental materials
4.1.1 test animals
Female Balb/c mice
4.1.2 test Agents and preparation thereof
Isocyanochinaberry extract was dissolved in 0.5% CMC-Na (sodium carboxymethylcellulose) solution. 5-Fu (Shanghai bang) was dissolved in physiological saline.
4.2 Experimental methods
4.2.1 in vivo tumor-bearing mice anti-tumor experiments
Culturing a certain amount of inoculated 4T1-Luc-GFP cells by in vivo experiment of tumor-bearing mice, centrifuging after digestion to remove pancreatin, and diluting to 1.0X10 by weight of serum-free culture medium 7 cells/mL were placed in centrifuge tubes, ice inserted, and the tumor cells were inoculated at this concentration by subcutaneous injection (sc) at 2mm under the #4 pad on the left side of Balb/c mice, at an injection rate of 0.1 mL/mouse. The animals were randomly grouped according to body weight, namely Model (0.5% CMC-Na), 5-Fu, low dose of isotoosendanin (0.1 mg/kg) and high dose of isotoosendanin (1 mg/kg). The drug was dissolved to the required concentration with 0.5% cmc-Na, and the drug was administered by daily gavage (ig) on body weight beginning 1 week after inoculation, and the Model group ig was given the same volume of 0.5% cmc-Na,5-Fu (dissolved to 2.5mg/mL with dd water) group i.p. (ip) once every other day; the administration was continued for 5 weeks. In vivo imaging experiments were performed weekly 2 weeks after inoculation to see if metastasis occurred. Tumor diameter was measured every 3 days with vernier calipers and length and width (mm) of the tumor in vitro was recorded. 24 hours after the last administration, the eyeballs are picked up for blood sampling for centrifugal separationMeasuring ALT and AST activity of serum after separating serum; tumor tissue is taken, lung is taken, liver is taken, tissue beside cancer is taken, weighing is carried out, photographing is carried out, and whether the lung and the liver are effective in tumor nodule. Tumor volume (Tumor volume) and Tumor inhibition (Tumor inhibition ratio) were calculated as follows.
Tumor volumn(mm 3 ) =a×b×b/2, where a denotes the length of the tumor and b denotes the width of the tumor. With significant differences between tumor volumes and model groups, P<0.05 is the standard of the antitumor activity of the medicine.
Tumor inhibition ratio (%) = [ (C-T)/C ] ×100, where C refers to CMC-Na control group mean tumor volume and T refers to administration group mean volume. The tumor volume is obviously different, and the tumor inhibition rate is more than 30 percent, which is the standard of the medicine with anti-tumor activity.
4.3 statistical treatment
The experimental data are all expressed by mean value + -standard error, analyzed by SPSS17.0 statistical software, and analyzed by One-Way ANOVA, and P <0.05 is the standard with statistically significant difference.
4.4 experimental results
4.4.1 Effect of Isocyanochinin administration on 4T1 solid tumors in female Balb/c mice
Compared with the model group, as shown in table 6, fig. 6 and fig. 7, the mice inoculated with 4T1-luc-GFP cells had significantly reduced tumor volume (P < 0.01) in the high dose group after 5 weeks of continuous administration of ITSN (0.1 mg/kg and 1 mg/kg), indicating that the ITSN high dose was effective against 4T1 solid tumors and the high dose tumor suppression rate exceeded 30%; as shown in fig. 7, after administration, the mortality of tumor-bearing mice was significantly reduced, indicating that isotoosendanin had significant in vivo anti-triple negative breast cancer activity. Compared with 5-Fu group toosendanin, the dosage of the toosendanin is lower, and the anti-tumor effect is similar, which shows that the toosendanin has certain advantage in dosage.
TABLE 6 Effect of Isocyanochinin administration on 4T1 solid tumors in female Balb/c mice
P <0.001 compared to model group
4.4.2 Effect of Isocyanochinin administration on metastasis of 4T1 solid tumors in female Balb/c mice
Compared with the model group, as shown in FIGS. 8, 9 and 10, the transfer rate of the high dose group was significantly reduced after continuous administration of ITSN (0.1 mg/kg and 1 mg/kg) for 5 weeks in the mice vaccinated with 4T1-luc-GFP cells. The results of the pulmonary HE staining in fig. 8 show a significant reduction in the number of pulmonary nodules in the high dose group relative to the model group. Compared with 5-Fu group isotoosendanin, the dosage is lower, and the anti-tumor metastasis effect is similar, which shows that the isotoosendanin has certain advantage in dosage.
The various aspects of the invention have been described above. It will be understood, however, that equivalent changes and modifications may be made thereto by those skilled in the art without departing from the spirit of the invention, which changes and modifications likewise fall within the scope of the appended claims.
Claims (3)
1. Application of isotoosendanin in preparing medicine for preventing and treating breast cancer metastasis is provided.
2. The use according to claim 1, wherein the isotoosendanin is used as the sole active ingredient for the preparation of a medicament for the prevention and treatment of breast cancer metastasis.
3. The use of claim 1, wherein the breast cancer is a triple negative breast cancer.
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