WO2006128378A1

WO2006128378A1 - Use of ganoderic acid in treating tumour

Info

Publication number: WO2006128378A1
Application number: PCT/CN2006/001171
Authority: WO
Inventors: Jianjiang Zhong; Jianwen Liu; Wen Tang; Yingbo Hong
Original assignee: East China University Of Science And Technology; Shanghai Ying-Xing Biotechnology Co., Ltd.
Priority date: 2005-06-01
Filing date: 2006-05-31
Publication date: 2006-12-07

Abstract

A pharmaceutical composition comprising ganoderic acid T and/or ganoderic acid Me, as well as its application for inhibiting tumor growth or proliferation.Ganoderic acid T and/or ganoderic acid Me have obvious action for inhibiting human tumor cell growth or proliferation, at the same time its toxicity for normal cells is low.

Description

Application of Ganoderma Acid in Cancer Therapy

The present invention relates to new uses of ganoderic acid, in particular to the new use of Me (Ganoderic acid Me) and Ganoic acid T (Ganoderic acid T). Background technique

Ganoderma lucidum is a precious medicinal fungus that has been used for thousands of years. Ancient medical scientists have fully recognized the medicinal value of Ganoderma lucidum. They believe that Ganoderma lucidum has the function of "supporting the body and strengthening the body" and can be used to treat a variety of diseases. _D pharmacological research proves that Ganoderma lucidum has a wide range of pharmacological effects, such as anti-tumor, anti-HIV. Virus, immune regulation, anti-myocardial ischemia, regulation of blood lipids, blood sugar lowering, sedative effect, liver protection, anti-radiation and anti-chemotherapy, anti-hypoxia and anti-aging effects.

Triterpenoids are one of the main chemical constituents of Ganoderma lucidum, and many triterpenoids have important physiological activities. Since the first separation of such compounds by T Kubota (Helv. Chim. Acta, 65 (2), 611-619, 1982) in 1982, more than 100 similar compounds have been isolated to date.

Ganoderma lucidum Me (GA-Me) or Ganoderma lucidum T (GA- Τ) are extracted from the fermentation of Ganoderma lucidum mycelium, respectively (Chem. Pharm. Bull., 34 (5), 2282-2285, 1986; Agric Biol. Chem., 51 (2), 619-622, 1987) The technique of the cockroach is prepared for extraction with a purity greater than 99%, which was first isolated and identified in 1983 (Tetrahedron Lett., 24 (10), 1081 -1084, 1983) and 1987 (Agric. Biol. Chem., 51 (2), 619-622, 1987). There are already patents and literatures on the production of ganoderic acid, ganoderma lucidum anti-tumor and ganoderic acid in the prior art (see, for example, Japanese Patent Laid-Open Publication No. Hei-4-304890 s Chinese Patent Publication No. CN1264743A, CN1483423, CN 1480208, CN 1286119 and CN 1181271, etc.). However, there is no report on the application of ganoderic acid monomer and its application in the prior art. Tumor chemotherapy is an important means for treating tumors at present, and the main problem affecting the therapeutic effect is that tumors can form multidrug resistance. Existing sensitizing and potentiating drugs have drawbacks in use, and finding new drug resources capable of simultaneously anti-tumor and sensitizing and increasing efficiency has become an urgent problem to be solved. The ideal MDR reversal agent should have the following conditions: 1 safe, less toxic to normal tissues; 2 can achieve effective concentration in vitro and in tumor cells; 3 itself has certain anti-tumor activity; 4 stable, long half-life in vivo; Metabolites are also effective.

Therefore, the urgent problem to be solved in the field of tumor chemotherapy is to find new anticancer drugs that are low in toxicity, high in efficiency, and difficult to produce multidrug resistance.

1

Confirmation Summary of the invention

The inventors have found that Ganoderma lucidum Me or Ganoderma lucidum T can inhibit tumor proliferation and growth, and a small dose produces an effect of inhibiting the growth of various tumor cells in a dose-dependent manner, which is different for different human cancer cells and human normal cells. Cytotoxicity, especially IC _5D for cancer cells is much lower than normal cells 1 (: _{5 ()} , and the difference between the two is significant (p < 0.05). Moreover, low doses of Ganoderma lucidum Me or Ganoderma lucidum T It can increase the sensitivity of multidrug-resistant tumors to chemotherapeutic drugs that have developed resistance. High-dose use has the effect of inhibiting the growth of multiple multidrug-resistant tumor cells and is dose-dependent, so they can also be used as Drugs and sensitizing synergists for the treatment of multidrug resistant tumors are used in the chemotherapy of tumors.

In particular, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of Ganoderma lucidum Me or Ganoderma lucidum T and a pharmaceutically acceptable carrier. The carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, such as a diluent, an excipient such as water, a filler such as starch, sucrose, etc., a binder such as a cellulose derivative gelatin, polyvinylpyrrolidone, etc., a lubricant , such as talcum powder. 9%。 The 9%, the content of the ginseng acid is 0. 5-99. 9%. In one embodiment, the pharmaceutical composition consists essentially of Ganoderma lucidum Me or Ganoderma lucidum T and a pharmaceutically acceptable carrier. The pharmaceutical composition can be used not only to inhibit tumor proliferation and growth (especially proliferation and growth of multidrug resistant tumor cells), but also to increase multidrug resistant tumor-resistant chemotherapy drugs. Sensitivity. Accordingly, in a preferred embodiment, the composition further comprises the chemotherapeutic agent, such as doxorubicin, vinblastine, vincristine, verapamil, and the like.

The invention further relates to a method of treating a tumor comprising administering a therapeutically effective amount of the above composition of the invention to a subject in need of such treatment. The present invention also provides the use of Ganoderma lucidum Me and/or Ganoderma lucidum T in the preparation of a medicament for the treatment of tumors.

Ganoderma lucidum Me or Ganoderma lucidum T and a pharmaceutically acceptable carrier may be administered to a patient in need of treatment in the form of a composition, generally at a dose of 1-100 mg / (kg body weight per day), depending on the patient Age, condition, etc. change.

The composition of the ganoderic acid Me or ganoderic acid T of the present invention and the above carrier can be applied to a patient in need of such treatment by oral, nasal, intravenous or subcutaneous injection. When used orally, it can be prepared into a conventional tablet, powder or oral solution; when it is used for injection, it can be prepared into an injection solution by a method conventional in the art; the meic acid Me or the ganoderic acid T of the present invention Various preparations, including powders, pills, tablets, capsules and the like, solid preparations, ointments, patches, injections and the like can be prepared by a conventional method in the pharmaceutical field.

The composition or preparation provided by the present invention has the effect of enhancing the sensitivity of multidrug resistant tumor cells to chemotherapeutic drugs at a low concentration (the low concentration means less than l-10 mg / (kg body weight per day) Dose), at At high concentrations, it has the effect of inhibiting the proliferation of multidrug resistant tumor cells (the high concentration refers to a dose higher than 10-100 mg / (kg body weight · day)), indicating that it not only has tumor cell killing The role of the tumor cells can also inhibit the infinite proliferation of tumor cells, and thus can play a greater and greater degree of treatment of tumors.

The anticancer agent and the sensitizing synergist in the multidrug resistant tumor chemotherapy provided by the invention have the same cytotoxicity to the multidrug resistant cancer cells and the non-multidrug resistant cancer cells, and the difference therebetween Not significant (p<0.05).

The Ganoderma lucidum Me (GA-Me) or Ganoderma lucidum T (GA- Τ) mentioned herein is inhibited in the prior art and extracted from the fermentation of Ganoderma lucidum mycelium, and can be used in the literature (Chem. Pharm. Bull., 34 (5), 2282-2285, 1986 ; Agric. Biol. Chem., 51 (2), 619-622, 1987) The disclosed technique for preparative extraction with a purity greater than 99%, which was first isolated and identified in 1983 (Tetrahedron Lett., 24 (10), 1081-1084, 1983) and 1987 (Agric. Biol. Chem., 51 (2), 619-622, 1987).

The tumor of the present invention includes various types of tumors medically belonging, including benign tumors and malignant tumors. The malignant tumors include: malignant melanoma, malignant lymphoma, tumors of the digestive organs (gastric cancer, colon cancer, liver cancer, gallbladder cancer, biliary cancer, pancreatic cancer), lung cancer, breast cancer, testicular cancer, ovarian cancer, uterine cancer , prostate cancer, upper jaw cancer, tongue cancer, oral cancer, throat cancer, thyroid cancer, brain tumor, various sarcoma, osteosarcoma, leukemia, nervous system tumor, bladder tumor, skin cancer, skin accessory organ cancer and skin metastases . In a preferred embodiment, the tumor is a multi-drug resistant tumor.

The animal test proves that the anticancer agent and the sensitizing synergist of the present invention are low in toxicity and are suitable for oral or parenteral administration of human and mammalian animals. The anticancer agent and the sensitizing synergist in the multidrug resistant tumor chemotherapy provided by the invention can usually be combined with the auxiliary material which does not affect the pharmacological action to prepare a preparation for oral or parenteral administration.

Further studies have shown that ganoderic acid inhibits the growth of cancer cells by inducing apoptosis. The present invention also investigates the related apoptotic pathways in which cancer cells are induced after GAs treatment. In the experiment, DNA degradation was observed, the membrane potential of mitochondria decreased, and cytochrome C was released from mitochondria. In addition, there was no significant change in the activity of caspase-8, and the activity of caspase-3 was rapidly increased. An increase in the expression of p53 and an increase in the expression of Bax were observed, while the expression of Bel-2 did not change. Taken together, the above results indicate that GAs inhibit high-metastasis lung growth mainly through intracellular P53-associated mitochondrial pathway.

Drug resistance of chemotherapy drugs is a common cause of ineffective cancer treatment. Finding new drug leaders that can simultaneously anti-tumor and enhance sensitization has become an urgent problem in tumor chemotherapy. The present invention also investigates the possibility that ganoderic acid increases the sensitivity of multidrug resistance KB-A-1 cells to doxorubicin and its possible mechanism of action, and the results indicate that ganoderic acid is relatively high in concentration. Doxorubicin-sensitive and insensitive cancer cell lines are highly cytotoxic, and the multidrug resistance induced by doxorubicin does not affect the inhibitory effect of ganoderic acid on cancer cells. At lower concentrations, the multidrug resistance induced by doxorubicin can be reversed The sensitivity of the hormone increases. Further studies have shown that cells treated with ganoderic acid can increase the absorption of doxorubicin by multidrug resistance. The absorption test of Rhodanminl23 showed that ganoderic acid treatment may affect the activity of Pgp protein. Animal experiments showed that: Ganoderma lucidum inhibited the proliferation of multidrug-resistant solid tumors in nude mice and increased the sensitivity to doxorubicin. The combination of doxorubicin and ganoderic acid did not cause animal death, indicating that ganoderic acid did not cause Increased toxic side effects.

Based on the above results, it can be found that ganoderic acid acts as a triterpene molecule to inhibit tumor proliferation by inducing apoptosis, and apoptosis of cancer cells is induced by the mitochondrial pathway. Further studies have found that inhibiting the activity of Topoisomerase l is the cause of apoptosis. At the same time, ganoderic acid has certain sensitization and synergistic effects on multidrug resistant cells. Combined with the application history of Ganoderma lucidum for thousands of years, it can be speculated that they may be an effective tumor suppressor or lead compound. In summary, the present invention provides two natural compounds having the functions of simultaneous anti-tumor and sensitization and synergy, overcoming the drawback of traditionally using Ganoderma lucidum mixed extract as a medicine, and using modern biotechnology as a means of invention, clearly The active ingredient, using pure compounds as anti-tumor drugs and sensitizing synergists, reduces the possibility of negative effects caused by unknown components. These two compounds have significant simultaneous anti-tumor and sensitizing effects (in vitro and in vivo experiments), and have less cytotoxicity to human normal cells, and have good application prospects. DRAWINGS

Figure 1 shows the inhibitory effects of different concentrations of GA-Me and GA-T on multidrug resistance KB-A-1 cells. Figure 2 shows that different concentrations of GA-Me and GA-T increase the sensitivity of KB-A-1 cells to doxorubicin.

Figure 3 shows that different concentrations of GA-Me and GA-T increase the content of doxorubicin in KB-A-1 cells.

Figure 4 shows that different concentrations of GA-Me and GA-T increase the content of Rhodanminl23 in KB-A-1 cells. Figure 5 shows the sensitivity of GA-Me and GA-T to the anticancer drug doxorubicin in nude mice.

Figure 6 shows the inhibition of proliferation of multidrug resistant solid tumors in nude mice by GA-Me and GA-T.

Figure 7 shows the inhibitory effects of different concentrations of GA-Me and GA-T on HeLa cells.

Figure 8 shows the inhibitory effects of different concentrations of GA-Me and GA-T on HeLa cells.

Figure 9 shows the inhibitory effects of different concentrations of GA-Me and GA-T on 95-D cells.

Figure 10 is a comparison of IC _5D of several tumor cells and normal cells L02 by GA-Me and GA-T.

Figure 11 shows the comparison of different drugs and concentrations to inhibit tumor growth in nude mice. detailed description

The contents of the present invention will be further described below by way of examples. The examples given do not limit the protection of the present invention. Range of protection.

The supplier of 95-D, Hela, SMMC-7721, L02 and KB-A-1 cell lines in the examples is the Shanghai Cell Bank of the Chinese Academy of Sciences. Among them, 95-D is a human high metastatic lung cancer cell; Hela is a human cervical cancer cell; SMMC-7721 is a human liver cancer cell; L02 is a human liver cell; KB-A-1 cell Chinese medical name is an oral epidermoid carcinoma cell line, English medical name for cell line of epidermal carcinoma of the mouth 0 which is a derived from a human oral epithelial cancer cell lines, the cell lines showed inducible multidrug resistance properties.

Experimental BALB/c nude mice were provided by the Experimental Animal Center of the Chinese Academy of Sciences. Rating: SPF. Quality certificate number: SCXK (Shanghai) 2003-0003. The animal experiment process follows the relevant animal experiments and ethics regulations of the People's Republic of China. Example 1 Toxicity of GA-Me and GA-T to KB-A-1 cells

Both GA-Me and GA-T are pure natural products isolated from the fermented mycelium of Ganoderma lucidum, with a purity greater than 99%, and diluted to the desired concentration with DMEM medium.

The toxic effects of GA-Me and GA-T on HeLa cells were determined by MTT rapid colorimetry. Logarithmic growth phase cells (

Inoculate in a 96-well culture plate, 0.2 ml per well, and add a certain concentration of GA-Me and GA-T respectively. Each concentration is paralleled with 4 wells, and the control group is added with an equal volume of the culture solution at 37 ° C, 5%. Incubate C0 ₂ and saturated humidity incubator for 1-4 days, add Smg'mr ¹ MTT ΙΟμΙ to each well 4 hours before the end of the experiment, and add 0.04 Ν dimethyl sulfoxide (DMS0) per well after the end of the culture. After 150 μ 1 , shaking for 10 min, the MTT reduction product was completely dissolved. The absorbance was measured with a BioRad 550 microplate reader at 550 nm and the reference wavelength of 655 nra, and the inhibition rate of the drug was calculated. The concentration of the drug and the inhibition rate of the cells were plotted to determine the half-inhibitory concentration of the cells (IC _5Q ). The experimental results are shown in Figure 1 below.

The above examples demonstrate that both GA-Me and GA-T have a good effect on killing KB-A-1 tumor cells, and this inhibitory effect is also dose-dependent. Example 2 GA-Me and GA-T increase the sensitivity of KB-A-1 cells to the anticancer drug doxorubicin

Both GA-Me and GA-T are pure natural products isolated from the fermented mycelium of Ganoderma lucidum, with a purity greater than 99%, and diluted to the desired concentration with RPMI-1640 medium. The toxic effects of GA-Me and GA-T on 95-D cells were determined by thiazole blue (MTT) rapid colorimetry. Logarithmic growth phase cells (

Inoculated in a 96-well culture plate, 0.2 ml per well, in the presence of 5 _g / ml of ganoderic acid, a certain concentration of doxorubicin was added, each concentration was parallel 4 wells, and the control group was added with an equal volume of the culture solution. , set 37 ° C, 5% C0 ₂ N2006/001171 and the incubator of saturated humidity were cultured for 3 days, and δι^'ηύ^ΜΤΤ ΙΟμΙ was added to each well 4 hours before the end of the experiment. After the completion of the culture, 0. 04 N DMS0 was added to each well, and 150 μl per well was shaken for 10 min. The MTT reduction product was completely dissolved. The absorbance was measured with a BioRad 550 microplate reader at 550 rnn as the experimental wavelength and 655 nm as the reference wavelength. The inhibition rate of doxorubicin on cancer cells was calculated, and the drug concentration and cells were determined. The inhibition rate is plotted to determine IC ₅ . . The experimental results are shown in Figure 2.

The above examples demonstrate that both GA-Me and GA-T have a good increase in the sensitivity of KB-A-1 cells to doxorubicin. Example 3 GA-Me and GA-T increase the content of doxorubicin in KB-A-1 cells

The intracellular doxorubicin content was measured by high performance liquid chromatography (HPLC). Sensitive logarithmic growth phase cells were seeded in 24-well plates at a concentration of 10 ⁵ cells/ml. Cells were treated with a combination of doxorubicin and ganoderic acid for 4 hours. The control was treated with doxorubicin alone. After the treated cells were washed three times with cold PBS, the cells were suspended, the concentration of each group was adjusted, and 0.5 ml of water was added thereto, and the cells were repeatedly thawed and centrifuged for 30 minutes at 12,000 rpm, and the supernatant was used for doxorubicin detection. The analytical conditions are: The detector is a fluorescence monitor with excitation and emission wavelengths of 495 and 560 dishes. The flow rate is 0. 8 ml/min. The flow rate is 0. 8 ml/min. The flow rate is 0. 8 ml/min. The results are shown in Figure 3.

The above examples demonstrate that GA-Me and GA-T increase the concentration of anticancer drugs in cancer cells and increase the sensitivity of multidrug resistant cancer cells to doxorubicin. Example 4 GA-Me and GA-T increase the content of Rhodanminl23 in KB-A-1 cells

The intracellular accumulation of Rhodamine 123 was detected by a fluorescence spectrophotometer. Sensitive logarithmic growth phase cells were seeded in 96-well plates at a concentration of 10 ⁵ cells/well. The cells were first adherently cultured for 4 hours, and then the cells were treated with ganoderic acid for 4 hours. The control was doxorubicin alone (0. 75 μΜ) Treated and treated with ganoderic acid alone. Then add Rodin name 123, the final concentration is 20 μΜ, and continue to culture for 60 minutes. After the treated cells were washed three times with cold PBS, the cells were suspended, the concentration of each group was adjusted, and 0.5 ml of water was added thereto, and the cells were repeatedly thawed and centrifuged for 30 minutes at 12,000 rpm, and the supernatant was used for Rhodamine 123 detection. The fluorescence spectrophotometer has excitation and emission wavelengths of 485 nm and 530 nm, respectively. The result is shown in Figure 4.

The above examples demonstrate that GA-Me and GA-T increase the concentration of anticancer drugs in cancer cells and increase the sensitivity of multidrug resistant cancer cells to anticancer drugs. Example 5 GA-Me and GA-T increase the sensitivity of tumor-bearing nude mice to doxorubicin

Experiments were performed using nude mice. Sensitive logarithmic growth phase KB-A-1 cells were inoculated into 6 nude mice. The cell concentration was 10 ⁵ / 0.2 ml, and growth was carried out for 15 days under SPF conditions. The nude mice were sacrificed, and the solid tumors were removed. The cells were cut to a size of 0.5 mm under aseptic conditions, and the nude mice were used to enter the experimental nude mice, one on each side. Five rats in each group were administered with a control drug and a positive drug three days later. Both the control drug and the positive drug were administered by intraperitoneal injection. Five hours after the administration of the control drug, the positive drug was administered. After four days, the positive drug was administered again, and the drug was administered twice. The control drug was administered continuously for 12 days. After stopping the drug for 6 days, the nude mice were sacrificed and the solid tumor was removed. Weigh and analyze the inhibition rate of solid tumors.

Tumor inhibition rate (%) = [ (the average weight of the solid tumor in the control group - the average weight of the solid tumor in the administration group)

I control group of solid tumors average weight] X 100

The results are shown in Figure 5 of the accompanying drawings, in Figure 5:

Positive drug doxorubicin (1mg/kg) group;

The positive drug doxorubicin (lrag/kg) + ganoderic acid T (25 mg/kg) group;

The positive drug doxorubicin (1 mg/kg) + ganoderic acid Me (25 rag/kg) group.

The above examples demonstrate that GA-Me and GA-T can increase the sensitivity of tumor-bearing nude mice to doxorubicin, and have a sensitizing and synergistic effect. Example 6 Inhibition of multidrug resistance solid tumors in nude mice by GA-Me and GA-T

Experiments were performed using nude mice. KB-A-1 cells in logarithmic growth phase were prepared under aseptic conditions, and about 2.5 X lOVmL cell suspension was prepared. 0.2 mL/mouse was inoculated subcutaneously in the lower limbs of two nude mice. After 15 days of growth, the animals were sacrificed. The solid tumor of the mouse was dissected under the conditions of the bacteria, cut open, the dead cells at the center of the tumor were removed, and the living tissue was cut to a size of 0.5 mm, and washed with PBS. The nude mice were inoculated subcutaneously in the lower limbs of the experimental nude mice. After 3 days of growth, the size of the rice grains was allowed to grow, and the hard solid tumors that could be touched were administered according to the experimental design. After 5 weeks, the animals in each group were sacrificed, and the mice in each group were dissected. For solid tumors, the weight of each solid tumor was measured. The tumor inhibition rate was calculated according to the following formula: Tumor inhibition rate (%) = [(Average weight of control solid tumors - Average weight of solid tumors in the administration group) I Control entity Average tumor weight] X 100

The results are shown in Figure 6, in Figure 6:

Ganoderma acid T (25mg/kg) group;

Ganoderma lucidum Me (25 mg/kg) group.

The above examples demonstrate that GA-Me and GA-T inhibit the growth of multidrug resistant solid tumors in nude mice and have the activity of inhibiting the proliferation of multidrug resistant tumors. Example 7

1 part by weight of GA-Me or GA-T was uniformly mixed with 199 parts of distilled water, and the mixture was dispensed to obtain an oral solution. PT/CN2006/001171 Example 8

One part by weight of GA-Me or GA-T was mixed with 199 parts by weight of physiological saline for injection to obtain an injection. Example 9

The toxic effects of GA-Me and GA-T on KB, HeLa and 95-D tumor cells and HLF human normal cells were determined by MTT rapid colorimetry. Will be a variety of cells in the logarithmic growth phase (

Inoculate in a 96-well culture plate, 0.2 ml per well, and add 50, 100, 150, and 200 μ _§ ml- ¹ concentrations of GA-Me and GA-T, respectively. Each concentration is parallel to 4 wells, and the control group is added with an equal volume. The culture medium was placed in an incubator at 37 ° C, 5% ∞ ₂ and saturated humidity for 1-4 days, and δπ^πιΓ^ΜΤΤ ΙΟμΙ was added to each well 4 hours before the end of the experiment, and 0. 04 每 was added to each well after the end of the culture. Dimethyl sulfoxide (DMS0), 150 μl per well, shaking for 10 min, until the MTT reduction product was completely dissolved, and the absorbance was measured using a BioRad 550 microplate reader with 550 nm as the experimental wavelength and 655 nm as the reference wavelength. Drug inhibition rate of the cell, and a cell half maximal inhibitory concentration (IC ₅₀₎ plotted. The results of statistical experimental data are shown in Figure 。.

The above examples demonstrate that both GA-Me and GA-T have a good killing effect on KB-A-1, HeLa and 95-D tumor cell lines, but less on normal human cell lines. Example 10 Toxicity of GA-Me and GA-T to HeLa cells

Both GA-Me and GA-T are pure natural products isolated from the fermented mycelium of Ganoderma lucidum, with a purity greater than 99%, and diluted to the desired concentration with DMEM medium. The toxic effects of GA - Me and GA-T on HeLa cells were determined by MTT rapid colorimetry. The logarithmic growth phase cells (106 cells. ml-1) were inoculated into 96-well culture plates at 0.2 ml per well, and treated with a certain concentration of GA-Me and GA-T, respectively, each concentration was parallel 4 wells, the control group was added, etc. The volume of the culture solution was incubated at 37 ° C, 5% C02 and saturated humidity in an incubator for 1-4 days. 5 mg. ml-1 MTT ΙΟμΙ was added to each well 4 hours before the end of the experiment, and 0 was added to each well after the completion of the culture. 04 Ν Dimethyl sulfoxide (DMS0), 150 μl per well, shaking for 10 min, until the MTT reduction product is completely dissolved, using BioRad 550 microplate reader, 550 nm as the experimental wavelength, 655 nm as the reference wavelength The absorbance, the inhibition rate of the tumor cells and the inhibition rate of the drug to the cells were calculated, and the half-inhibitory concentration (IC50) of the cells was determined by plotting the different concentrations of the drug and the inhibition rate of the cells. The experimental results are shown in Figure 8 below.

The above examples show that both GA-Me and GA-T have a good effect of killing HeLa tumor cells, and this inhibitory effect is also dose-dependent. Example 11 Toxicity of GA-Me and GA-T to 95-D cells

Both GA-Me and GA-T are pure natural products isolated from the fermented mycelium of Ganoderma lucidum, with a purity greater than 99%, and diluted to the desired concentration with RPMI-1640 medium. The toxic effects of GA-Me and GA-T on 95-D cells were determined by MTT rapid colorimetry. The logarithmic growth phase cells (106 cells. ml-1) were inoculated into a 96-well culture plate at 0. 2 ml per well, and a certain concentration of GA-Me and GA-T were added respectively. Each concentration was parallelized with 4 wells, and the control group was added. The volume of the culture medium was cultured in an incubator at 37 ° C, 5% CO 2 and saturated humidity for 1-4 days. 5 mg. ml-1 MTT ΙΟμΙ was added to each well 4 hours before the end of the experiment, and 0 was added to each well after the completion of the culture. 04 N DMS0, 150μ1 per well, shaking for 10 min, until the MTT reduction product is completely dissolved, the absorbance is determined by the BioRad 550 microplate reader with 550 nm as the experimental wavelength and 655 nm as the reference wavelength, and the inhibition rate of the tumor cells is calculated. And the inhibition rate of the drug to the cells, and the IC50 is determined by plotting the different concentrations of the drug and the inhibition rate of the cells. The experimental results are shown in Figure 9.

The above examples show that both GA-Me and GA-T have a good effect of killing 95-D tumor cells, and this inhibitory effect is dose-dependent. Example 12 Comparison of cytotoxicity of GA-Me and GA-T against several tumor cells and normal cells L02

Both GA-Me and GA-T are pure natural products isolated from the fermented mycelium of Ganoderma lucidum, with a purity greater than 99%, and diluted to the desired concentration with DMEM or RPMI-1640 medium. The toxic effects of GA-Me and GA-T on several cells were determined by MTT rapid colorimetry. The logarithmic growth phase cells (106cell.ml-l) were inoculated into 96-well culture plates at 0.2 ml per well, and treated with a certain concentration of GA-Me and GA-T, each concentration was parallel 4 wells, the control group was added, etc. The volume of the culture medium was cultured in an incubator at 37 ° C, 5% CO 2 and saturated humidity for 1-4 days. 5 mg. ral-1 MTT ΙΟμΙ was added to each well 4 hours before the end of the experiment, and 0 was added to each well after the completion of the culture. 04 DMSO DMSO (dimethyl sulfoxide), 150 yl per well, shaking for 10 min, until the MTT reduction product is completely dissolved, using BioRad 550 microplate reader, 550 nm as the experimental wavelength, 655 nm as the reference wavelength to determine its absorption Degree, calculate the inhibition rate of tumor cells and the inhibition rate of the drug on the cells, determine the IC50 and map, the results are shown in Figure 10.

The above examples demonstrate that GA-Me and GA-T are more cytotoxic to human tumor cells and less cytotoxic to normal cells, ie, GA-Me and GA-T have certain targeting to certain human tumor cells. Inhibition. Example 13 Comparison of GA-Me and GA-T inhibition of tumor growth activity in nude mice

Both GA-Me and GA-T are pure natural products isolated from the fermented mycelium of Ganoderma lucidum, with a purity greater than 99%, formulated in PBS and diluted to the desired concentration. Two male nude mice, forefoot subcutaneous injection 95-D fine Cells (1 x 106 cells/ml) were sacrificed after four weeks of feeding. Solid tumors of nude mice were stripped, cut and incubated with PBS (7.4), and filtered through an 80 mesh screen. Formulated as a cell suspension for use. Male nude mice were randomly divided into groups according to their body weight. The 95-D cell suspension (1x106 cells/ml) was injected into the forelimbs for one week. After the solid tumors were grown, different drugs were injected intraperitoneally every other day. The negative control was normal saline (PBS). . The anticancer drug was used as a positive control. After 2 weeks of injection, the drug was stopped, and then it was sacrificed for 10 days. The patient was sacrificed and the solid tumor of the nude mice was weighed. The inhibition rate was calculated in comparison with the control. The experimental results of the effects of different concentrations of the drug on the inhibition rate of the tumor are shown in FIG.

The above examples demonstrate that GA-Me and GA-T also inhibit tumor growth in vivo and are also dose dependent. Example 14 Molecular Pathway of Ganoderma Acid-induced Apoptosis of 95-D Tumor Cells

Materials and Method

Cell cycle detection

After the logarithmic growth phase cells were treated with 25, 5 (^g/mL two concentrations of the drug for a specific period of time, the various treated cells were trypsinized, centrifuged to collect the cells (1500 rpm x 10 min), and washed twice with PBS. The cell concentration was adjusted to 10 ⁶ /mL with PBS, RNASE was added, the final concentration was 100 U/mL, 37 ^G c was kept for 30 min, PI was added, the final concentration was 50 g/mL, and the reaction was carried out in the dark at room temperature for 30 min, then 300 mesh filtration. Membrane filtration, flow cytometry analysis of apoptosis and cell cycle, recording 10,000 cells each time.

Detection of apoptosis

The cells were stimulated with Ganoderma lucidum T at 25, 50 g/mL, washed twice with PBS, trypsinized, serum stopped, and centrifuged to collect cells (1500 rpm, 10 min) with Binding solution (see 3.3). Into a single cell suspension, the cell concentration was 2x10 ⁵ / mL. Aiuiexin V-FITC solution was added to a final concentration of 10 ng/mL, incubated for 15 minutes at room temperature in the dark, and then stained with PI to a final concentration of 5 g/mL. Flow cytometry was used for analysis.

Results and analysis

1) Ganoderma acid inhibits the proliferation of highly metastatic lung cancer cells by inducing apoptosis.

MTT assay and colony formation experiments. It was found that Ganoderma lucidum T has strong cytotoxicity against high metastatic lung cancer cells. Although the MTT assay can show the effect of drugs on cell proliferation, it does not provide further explanation. That is, it is impossible to determine whether affecting cell proliferation is achieved by inhibiting an increase in the number of cells or promoting cell death. To explain the reason for inhibiting proliferation, apoptosis detection was performed. It was found that the cancer cells treated with Ganoderma lucidum T showed apoptosis, and the degree of apoptosis increased with the increase of the concentration of ganoderic acid. Acting for 8 hours at a concentration of 50 μ _§ , resulted in more than 40% apoptosis. The most important feature of apoptosis is the fragmentation of DNA. As the apoptosis increases, the DNA fragment in increments of 180 bp gradually increases. The lipogel gel appears as a ladder-like electrophoresis band. Cancer cells treated with ganoderic acid also showed significant DNA ladders.

Table 1 Ganoderma acid limit cell cycle of 95-D cancer cells

G ₂ -M

Cell line concentration ^g/mL) GQ-GJ (%) S (%)

(%)

0 26. 59 25. 53 47. 88

95-D 25 45 21. 51 33. 48

50 60. 67 16. 99 22. 35 In vitro cancer cells treated with ganoderic acid T cause apoptosis and also limit the cell growth cycle in G1 phase. At the same time, the higher the concentration of ganoderic acid, the cumulative G1 The more cells there are.

These results indicate that ganoderic acid can induce apoptosis and limit cell cycle in cancer cells, and is concentration dependent. It can be speculated that ganoderic acid mainly expresses its cytotoxicity by inducing apoptosis of cancer cells and limiting cell cycle.

2) Ganoderma lucidum Τ affects the function of cancer cell mitochondria

There are two main pathways for apoptosis, and the mitochondrial pathway is generally accompanied by the leakage of cytochrome c. Normally, it exists in the lumen between the mitochondrial inner membrane and the outer membrane. The apoptotic signal stimulates it to release from the mitochondria to the cytosol, and initiates the caspase cascade after binding with apaf-1 (apoptotic protease activating factor-1). : The cytochrome C/Apaf-1 complex activates caspase_9, which in turn activates caspase-3 and other downstream caspase. The increase in cytochrome C content was observed in the cytoplasm of cancer cells treated with ganoderic acid, as shown in Figure 4.2. After 4 hours of treatment, the cytochrome C content was increased by a factor of 2 compared to the control, and after 8 hours of treatment, the content was increased by a factor of 3. As a signal substance, cytochrome C plays an important role in apoptosis. Ganoderma acid treatment significantly stimulated the leakage of cytochrome c. Therefore, it can be speculated that ganoderic acid has a direct or indirect effect on mitochondria.

Induction of tumor cell apoptosis is associated with functional status of mitochondria. Such as changing the integrity of mitochondrial membranes. Flow cytometry was used to detect the effect of ganoderic acid on the mitochondrial inner membrane transmembrane potential of cancer cells. The results are shown in Figure 4.3. The decrease in fluorescence intensity in the mitochondria decreased with increasing processing time, and after 8 hours of treatment, the fluorescence intensity decreased by approximately 20%. The results suggest that ganoderic acid affects the transmembrane potential of cancer cells, causing the membrane potential to decrease, leading to leakage of cytochrome c.

3) Ganoderma acid T stimulates the production of reactive oxygen species

The amount of intracellular reactive oxygen species (R0S) in cancer cells treated with ganoderic acid varies with treatment time The extension has increased. After 8 hours of treatment, it increased by 40% compared to the control. There are many possible ways to increase free radicals. First, because ganoderic acid affects the membrane potential of mitochondria, free radicals inside the mitochondria leak into the cytoplasm, causing an increase in intracellular free radicals. It is the result of apoptosis. Secondly, ganoderic acid may interfere with the redox balance in the cell, leading to the dysregulation of the free radical scavenging mechanism, thereby accumulating free radicals and further causing apoptosis. The increase in free radicals in this case is the cause of apoptosis.

4) Ganoderma acid affects the activity of Caspase-3, but has no effect on the activity of Caspase-8

Ganoderma acid treatment of cancer cells causes leakage of cytochrome C, which in turn activates caspase-3 and other downstream caspa _Se . The process of apoptosis is allowed to proceed. By detecting the Caspase-3 of the treated cells, caspase-3 activity was also significantly increased as the treatment time increased. After 8 hours of treatment, the activity increased nearly threefold. Activation of Caspase-3 occurs in both pathways of apoptosis. That is, the caspase-8 of the FAS pathway can be linked to the mitochondrial pathway via the BID protein. Therefore, we tested the activity of caspase-8 to determine if the addition of the FAS pathway resulted in the activation of caspase-3. The results are shown in the figure. After treatment with ganoderic acid, the activity of caspase-8 did not change within 8 hours. Thus, the influence of the FAS pathway can be ruled out, and it can be inferred that the activation of caspase-3 is caused by the complex of cytochrome C.

In order to further verify the effect of ganoderic acid on the activity of caspase in the induction of apoptosis in cancer cells, a specific caspase inhibitor treatment was used. The results showed that Caspase 3 inhibitors or pan- caspase inhibitors significantly reduced apoptosis induced by ganoderic acid, whereas caspase 8 inhibitors did not inhibit drug-induced apoptosis. These data further demonstrate that ganoderic acid induces apoptosis in cancer cells by activating caspase 3 activity rather than activating caspase 8 activity. Because caspase8 is a key enzyme in the external pathway, this suggests that the external pathway is not involved in the ganic acid-induced apoptosis pathway. Therefore, ganoderic acid may activate downstream mitochondrial-related apoptotic events through cells, including activation of casp _aS e3, and induction of apoptosis in cancer cells by activating the mitochondrial pathway of _CaS pa _S e3.

5) Ganoderma acid T affects the expression of apoptosis-related proteins

The occurrence of intracellular apoptosis is accompanied by the expression of apoptosis-related proteins. The key factor regulating apoptosis is

The Bel-2 family, which includes the apoptosis-promoting factors Bax, Bak, Bid, and the apoptosis inhibitors Bel-2 and Bel-x. These promoting factors and inhibitors maintain an equilibrium state, and once the balance is broken, it will cause the occurrence and suppression of apoptosis. Another important protein that affects apoptosis is P53. When DNA damage occurs, more P53 is released for maintenance of genomic DNA stability. It can also guide P53 extranuclear output by mediating P53 monoubiquitination. Mitochondria, which directly activate Bax, initiate mitochondrial non-transcription-dependent apoptosis. When DNA damage has been repaired, active P53 is rapidly downregulated to restore normal cell homeostasis.

The results of the effect of ganoderic acid on the expression of related proteins are shown in Figure 4. 8. First, after treatment with ganoderic acid, intracellular The expression level of P53 is increased. In our experiments, it has been found that ganoderic acid can induce DNA fragmentation, and the increased expression of P53 may be mainly caused by DNA damage. In the Bel-2 family of proteins, there was no significant change in the expression of BCL-2, but the expression of BAX increased. Thus the ratio of BCL-2/BAX decreases. Consistent with the results of most anti-cancer studies, but the effect of ganoderic acid monomer on this ratio was first discovered. While the invention has been described with respect to the specific embodiments, it will be apparent to those skilled in the art Therefore, the appended claims are intended to cover all such modifications within the scope of the invention. All publications, patents and patent applications cited herein are hereby incorporated by reference.

Claims

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A pharmaceutical composition comprising a therapeutically effective amount of Ganoderma lucidum Me and/or Ganoderma lucidum T and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition according to claim 1, wherein it further comprises a chemotherapeutic agent.

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4. Ganoderma lucidum Me is used in the preparation of inhibitors of tumor growth and proliferation, multidrug resistant cancer chemotherapy sensitizers and inhibitors.

5. The application of ganoderic acid T in the preparation of inhibitors of tumor growth and proliferation, multidrug resistant cancer chemotherapy sensitizers and inhibitors.

6. The use according to claim 4 or 5, wherein the tumor is selected from the group consisting of malignant melanoma, malignant lymphoma, tumor of the digestive organ (gastric cancer, intestinal cancer, liver cancer, gallbladder cancer, biliary cancer, pancreatic cancer), lung cancer , breast cancer, testicular cancer, ovarian cancer, uterine cancer, prostate cancer, upper jaw cancer, tongue cancer, oral cancer, throat cancer, thyroid cancer, brain tumor, various sarcoma, osteosarcoma, leukemia, nervous system tumor, bladder tumor, Skin cancer, skin accessory organ cancer and skin metastases.

A method of inhibiting tumor proliferation or growth in a subject, the method comprising administering a therapeutically effective amount of the composition of claim 1 to a subject in need of such treatment.

8. The method according to claim 7, wherein the tumor is selected from the group consisting of malignant melanoma, malignant lymphoma, tumor of the digestive organ (gastric cancer, intestinal cancer, liver cancer, gallbladder cancer, biliary cancer, pancreatic cancer), lung cancer, breast cancer , testicular cancer, ovarian cancer, uterine cancer, prostate cancer, upper jaw cancer, tongue cancer, oral cancer, throat cancer, thyroid cancer, brain tumor, various sarcoma, osteosarcoma, leukemia, nervous system tumor, bladder tumor, skin cancer , skin accessory organ cancer and skin metastases.

9. The method of claim 7, wherein a therapeutically effective amount of the composition of claim 1 in combination with a chemotherapeutic agent is administered to a subject in need of such treatment.

10. A method of enhancing the sensitivity of a tumor in a subject to a chemotherapeutic drug that has developed resistance, the method comprising administering an effective amount of the composition of claim 1 to the subject in need thereof.