TWI531364B - Use of γ-bisabolene in treating oral cancer - Google Patents

Description

Use of γ-Bisabolene for the treatment of oral cancer

The present invention relates to the use of the compound γ-Bisabolene for the treatment of cancer, in particular, the γ-erythroside is used to inhibit the tissue protein deacetylase 2 (HDAC2) of cancer cells. Phosphorylation, increased acetylation of p53 protein in cancer cells, increased expression of apoptosis-promoting genes in cancer cells, and/or apoptosis-associated proteins that stimulate cancer cells (apoptosis-related) Application of activation of protein). The invention also relates to the use of gamma-erythroside to inhibit the growth of cancer cells.

Tumor (tumor) is a medically abnormal cell lesion. Under the action of various carcinogenic factors, the tumor loses the normal regulation of its growth at the gene level. The cells grow abnormally and accumulate into a mass, which is called a "tumor." Among them, "cancer" is the most common form of tumor. The abnormally proliferating "cancer cells", in addition to being aggregated into a mass, spread and metastasize to other tissues or organs of the body, so it is also called malignant tumor. The proliferation and metastasis of cancer cells can cause serious physiological dysfunction and is difficult to cure. Therefore, cancer has become the leading cause of death in humans in recent years.

Treatments for cancer include surgical treatment, chemotherapy, and Radiation therapy, etc. However, the surgical removal of the mass usually fails to effectively cure the cancer, and the cancer cells that are not completely resected continue to grow, and the patient's condition is worsened. Therefore, it is generally not possible to treat the disease only by surgical resection, but with other treatments such as chemotherapy and/or radiation therapy.

Chemotherapy uses chemical drugs to grow fast-growing cancer cells Toxic effects, but most of the drugs used in chemotherapy also affect normal cells, causing serious side effects to cancer patients, including vomiting, alopecia, fatigue, bleeding and anemia. As for radiotherapy, it uses the principle that rapidly dividing cancer cells are sensitive to radiation than normal cells, causing DNA breaks in cancer cells and killing cancer cells. However, high-energy radiation destroys cancer cells and also illuminates normal cells. Cells, which cause side effects such as reduced white blood cells, fatigue, insomnia, pain, poor appetite. In addition, radiotherapy is not effective for some advanced patients.

For example, head and neck cancer is a Taiwanese male. One of the top ten cancers, including nasopharyngeal carcinoma, oral cancer, oropharyngeal cancer, hypopharyngeal cancer, and laryngeal cancer. More than 90% of head and neck cancers are oral squamous cell carcinoma (OSCC). ), and is highly invasive, metastatic and high mortality. Among them, the incidence of oral cancer in Asia has increased year by year. According to statistics, oral cancer was the fourth highest among women in Taiwan in 2006 (Taiwan, Health Association Cancer Annual Report). The occurrence of oral cancer is related to smoking, drinking, and eating betel nut. These risk factors can cause oral submucosal fibrosis (oral submucous). Fibrosis), leukoplakia, erythema and other precancerous lesions. The treatment of oral cancer includes surgery, chemotherapy and radiation therapy. However, even if treated by the above methods, the survival rate of oral cancer patients within five years after treatment is still less than 50%.

In addition, in childhood cancer, medulloblastoma is the most common childhood malignant brain tumor, mostly located in the cerebellar vermis and protrudes into the fourth ventricle, even filling the cerebellomedullar cisterna. . Under the microscope, the tumor tissue consists of consistent small and undifferentiated tumor cells, which are closely packed into pieces and partially surround the formation of resettes. After surgery, cranial spinal cord radiotherapy, and chemotherapy, patients with medulloblastoma have an overall survival rate of 70 to 80%. However, most surviving patients have long-term complications such as long-term neurocognitive dysfunction and neuroendocrine dysfunction, and recurrent medulloblastoma does not respond well to drug therapy.

In view of the limited effectiveness of clinical medicine for cancer treatment, continuous development of methods or drugs that can effectively treat cancer to reduce morbidity and mortality, improve cure rate, and reduce side effects are quite necessary and urgent. .

The inventors of the present study found that γ-Bisabolene can effectively inhibit the phosphorylation of tissue proteins of cancer cells to acetylase 2, increase the acetylation of p53 protein in cancer cells, and increase the growth of cancer cells. The expression of apoptosis genes and/or the activation of apoptosis-related proteins in cancer cells can induce and/or promote apoptosis of cancer cells, and γ-erythrophylline can also effectively inhibit the growth of cancer cells. Can be used to lift A drug for the treatment of cancer, especially for the treatment of oral cancer, gastric cancer, lung cancer, breast cancer, rhabdomyosarcoma, medulloblastoma, myeloma, lymphoma, bladder cancer, ovarian cancer, prostate cancer, colon cancer, neuroblastoma, And thymic malignancies, especially for oral cancer.

It is an object of the present invention to provide a use of a compound of formula (I) (i.e., gamma-erythrophylline) for the manufacture of a medicament: Wherein, the agent is used for at least one of: inhibiting phosphorylation of tissue protein of the cancer cell to acetylase 2, increasing the acetylation of p53 protein of the cancer cell, and increasing the expression of the apoptosis-promoting gene of the cancer cell. And the activation of apoptosis-related proteins that stimulate cancer cells, especially for diseases related to the above mechanisms, such as oral cancer, myeloma, lymphoma, breast cancer, bladder cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer. , treatment of neuroblastoma, and / or thymic malignancies.

Another object of the present invention is to provide a use of γ-erythrophylline for the manufacture of a medicament, wherein the medicament is for inhibiting the growth of cancer cells, especially Growth of oral cancer cells, gastric cancer cells, lung cancer cells, breast cancer cells, rhabdomyosarcoma cells, and/or medulloblastoma cells.

Another object of the present invention is to provide a method for inhibiting phosphorylation of tissue protein deacetylase 2 of cancer cells, increasing acetylation of p53 protein of cancer cells, increasing expression of proapoptotic genes of cancer cells, and/or A method of stimulating activation of a cell apoptosis-associated protein of a cancer cell comprising administering to a subject in need thereof an effective amount of gamma-erythrophylline and a pharmaceutically acceptable carrier thereof. The method is particularly useful for treating diseases associated with the above mechanisms in the individual, such as oral cancer, myeloma, lymphoma, breast cancer, bladder cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, neuroblastoma, and/or Or treatment of thymic malignancies.

A further object of the present invention is to provide a method for inhibiting the growth of cancer cells in an individual in need thereof, which comprises administering to the individual an effective amount of gamma-erythrophylline and a pharmaceutically acceptable carrier thereof. . Among other things, the method is particularly useful for inhibiting the growth of oral cancer cells, gastric cancer cells, lung cancer cells, breast cancer cells, rhabdomyosarcoma cells, and/or medulloblastoma cells in the individual.

The detailed description of the present invention and the specific embodiments thereof will be described in the following description.

Figure 1 shows the survival of TE671 cells, RD cells, Huh-7 cells, MCF 7 cells, AGS cells, H1299 cells, CA9-22 cells, SAS cells, and OF cells after treatment with γ-erythrophylline. A graph of the rate, wherein the vertical axis represents cell viability, the horizontal axis represents the concentration of γ-erythrophylline; and the 2A and 2B graphs show the treatment of gamma-red myrrhene with 0 and 5 molar concentrations, respectively. After that, the statistical bar graph of the percentage of cells entering the sub-G1 phase of CA9-22 cells, SAS cells, and OF cells, wherein the vertical axis represents the percentage of cells, and the horizontal axis represents the name of each cell line; FIGS. 3A and 3B Shown is a statistical bar graph showing the proportion of CA9-22 cells and OF cells entering early stage of apoptosis (3A) and late stage of apoptosis (3B), where the vertical axis represents the percentage of cells and the horizontal axis represents γ-red myrrh Concentration of alkene; Figure 4 is a statistical bar graph showing the relative expression of apoptosis-related genes in CA9-22 cells by γ-erythroaluminum, wherein the vertical axis represents the relative expression of apoptosis-related genes, The horizontal axis represents the concentration of γ-erythrophylline; the fifth figure shows that γ-erythroarene enhances the activation of CA9-22 cells. Photographs of Western blotting experiments on the relative expression of apoptosis-related proteins; Figures 6A, 6B, and 6C show that γ-erythroside reduces the mitochondrial membrane potential of CA9-22 cells (ie, γ-red Immunofluorescence staining, flow cytometry analysis, and graphs of mycophenolate-induced apoptosis in mitochondria-mediated apoptosis; Figure 7 shows that γ-erythro-alkene inhibits CA9- Phosphorylation of tissue protein of 22 cells to acetylase 2, and photomicrograph of Western blotting test for phosphorylation of p53 protein and extracellular signal-regulated kinase 1/2 (ERK1/2) in CA9-22 cells; Fig. 8 A statistical bar graph showing the relative expression of p53 pathway-associated genes in CA9-22 cells, wherein the vertical axis represents the relative expression of the p53 pathway-associated genes, and the horizontal axis represents the γ-erythrophylline Concentration; Figure 9 is a photograph of a western dot-dot test of gamma-bisabolene increasing the p53 protein acetylation of CA9-22 cells; Figures 10A and 10B show that gamma-erythrophylline increases CA9-22 A statistical bar graph of the relative expression of apoptosis-promoting genes in cells and SAS cells, wherein the vertical axis represents the pro-apoptotic group The amount of relative performance, the horizontal axis represents the concentration of the drug did not γ- alkenyl of red; Fig. 11 lines showed γ- bisabolene CA9-22 cell to induce the apoptosis mechanism of the membrane test photograph of the Western blot (p21 ^WAF1 Represents the p21 ^WAF1 protein; BAD represents the Bcl-2 associated death promoter; Bcl-2 represents the B cell lymphoma protein-2 of cancer cells; Akt represents the protein kinase Akt; mTOR represents the mammalian target of rapamycin; and 12A Figures 12B and 12C show a graph or statistical bar graph of the effect of γ-erythrophylline on tumor volume (12A), tumor weight (12B), and mouse body weight (12C) in mice.

The invention will be described in detail below with reference to the embodiments of the present invention. The present invention may be practiced in various different forms without departing from the spirit and scope of the invention. . In addition, the terms "a", "an" and "the" and "the" "Therapeutically effective amount" means the amount of a compound which, when administered to an individual, is effective to at least partially improve the condition of the suspected individual; the term "individual" means a mammal, which may be a human or a non-human animal.

The present inventors have found that the compound of the following formula (I) (i.e., γ-erythrophylline) inhibits phosphorylation of tissue protein deacetylase 2 of cancer cells, increases acetylation of p53 protein of cancer cells, and increases cancer. The ability of cells to promote the expression of apoptotic genes and to stimulate the activation of apoptosis-related proteins in cancer cells:

Accordingly, the present invention relates to the use of γ-erythrophylline for the manufacture of a medicament, wherein the medicament is for use in at least one of: inhibiting phosphorylation of tissue protein deacetylase 2 of cancer cells, and increasing cancer cells The acetylation of p53 protein, the expression of pro-apoptotic genes in cancer cells, and the ability to stimulate the activation of apoptosis-related proteins in cancer cells. Specific examples of the pro-apoptotic gene include, for example, p53 upregulated modulator of apoptosis (PUMA), NOXA, and Bcl-2 interacting cell death mediator (Bcl-2 interacting) Mediator of cell death, Bim); in addition, specific examples of the apoptosis-related protein include, for example, cysteamine aspartate-specific protease 3 (Caspase 3), cysteamine-aspartate-specific protease 8 (Caspase 8), and cysteine aspartate-specific protease 9 (Caspase 9).

Studies have shown that apoptosis of cancer cells can be induced and/or promoted by increasing the ability of cancer cells to promote the expression of apoptosis genes and/or to stimulate the activation of apoptosis-related proteins in cancer cells. See, for example, The Bad guy cooperates with good cop p53: Bad is transcriptionally up-regulated by p53 and forms a Bad/p53 complex at the mitochondria to suggest apoptosis. Molecular and Cellular Biology .26(23):9071-9082 ( 2006) and p53/p21(WAF1/CIP1)expression and its possible role in G1 arrest and apoptosis in ellagic acid treated cancer cells. Cancer Letters . 136:215-221 (1999), the entire contents of which are hereby incorporated by reference. reference. In addition, it has been confirmed that cancer cells, including oral cancer, myeloma, and lymphoma, can be treated if they inhibit the phosphorylation of the tissue protein of the cancer cells to acetylase 2 and/or increase the acetylation of p53 protein in cancer cells. , breast cancer, bladder cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, glioblastoma, and pleural malignancy, see for example: Histone deacetylases and cancer: causes and therapies. Nature Reviews Cancer .1:194 -202 (2001), Histone deacetylase 2 modulates p53 transcriptional activities through regulation of p53-DNA binding activity. Cancer Res. 67(7): 3145-3152 (2007) and Histone deacetylase (HDAC) inhibitors in recent clinical trials for cancer therapy Clin Epigenet. 1:17-136 (2010), the entire contents of which are hereby incorporated by reference. Therefore, the agent of the present invention can be used for inducing and/or promoting apoptosis of cancer cells, and can be used for treating one or more of the following cancers: oral cancer, myeloma, lymphoma, breast cancer, bladder cancer, ovarian cancer, Prostate cancer, lung cancer, colorectal cancer, glioblastoma, and chest line malignancy. Preferably, the agent is for the treatment of oral cancer.

In addition, the inventor of the present invention found that γ-erythroside can inhibit cancer cells. Growing. Accordingly, the present invention is further directed to the use of gamma-erythrophylline for the manufacture of a medicament for inhibiting the growth of cancer cells. Among them, the agent is particularly suitable for inhibiting the growth of one or more of the following cancer cells: oral cancer cells, gastric cancer cells, lung cancer cells, breast cancer cells, rhabdomyosarcoma cells, and medulloblastoma cells. Preferably, the agent is for inhibiting the growth of oral cancer cells.

The medicament provided by the present invention may be in any form and administered in any convenient manner. For example, but not limited thereto, the drug can be administered to an individual by oral, subcutaneous, nasal or intravenous administration. Depending on the form of use and use, the agent may additionally comprise a pharmaceutically acceptable carrier.

In the case of a dosage form suitable for oral administration, the medicament provided by the present invention may contain any pharmaceutically acceptable carrier which does not adversely affect the desired benefit of gamma-erythrophylline, for example: solvent (water, saline, glucose) (dextrose), glycerin, ethanol or the like, and combinations thereof, oily solvents, diluents, stabilizers, absorption delaying agents, disintegrating agents, emulsifiers, antioxidants, binders, lubricants, moisture absorbents, A solid carrier (such as starch, bentonite) or the like. The agent may be provided in a dosage form suitable for oral administration by any convenient method, for example, a tablet, a capsule, a granule, a powder, a flow extract, a solution, a syrup, a suspension, an emulsion, an expectorant, and the like. Wait.

As for the dosage form suitable for subcutaneous or intravenous injection, one or more e.g. isotonic solutions, such as phosphate buffer or citric acid, may be contained in the medicament produced by the invention using γ-erythrophylline. Intravenous infusion, emulsion intravenous infusion, salt buffer), solubilizer, emulsifier, 5% sugar solution, and other carriers The medicament is provided in the form of a liquid, a dry powder injection, a suspension injection, or a dry powder suspension injection. Alternatively, the agent prepared using γ-erythrophylline is prepared as a pre-injection solid, the pre-injection solid is provided in a dosage form soluble in other solution or suspension, or an emulsifiable dosage form, and is administered to The pre-injection solid is dissolved in the other solution or suspension before the individual in need thereof, or emulsified to provide the desired injection.

If necessary, it may be additionally contained in a medicament manufactured using γ-erythroside There are additives such as flavoring agents, toners, coloring agents, etc., in order to improve the mouthfeel and visual feeling of the medicament when taken; and a suitable amount of preservative, preservative, antibacterial agent, antifungal agent, etc. may be added to improve The storage property of the medicament. In addition, the agent may optionally contain one or more other active ingredients or may be used in combination with the drug containing the one or more other active ingredients to further enhance the efficacy of the agent or increase the flexibility and formulation of the formulation of the formulation, as long as the other The active ingredient does not adversely affect the desired benefit of gamma-red myrrhene. The active ingredient may be an antioxidant (such as vitamin E), an immunomodulator, or the like.

Can be administered differently once a day, multiple times a day, or once a few days. Frequency Administration The agents of the present invention using gamma-erythrophylline vary depending on the needs of the individual to be administered. For example, when used in the human body to treat oral cancer, the amount is from about 0.1 mg/kg body weight to about 50 mg/kg body weight per day, preferably about 1 mg/kg per day. The weight is about 10 mg / kg body weight, wherein the unit "mg / kg body weight" refers to the amount of drug required per kg body weight. However, for acute patients, the amount can be increased according to actual needs, for example, increased to several times or tens of times.

Therefore, the present invention also provides a tissue protein that inhibits cancer cells. Phosphorylation of acetylase 2, increase of acetylation of p53 protein in cancer cells, increase of expression of apoptosis-promoting genes in cancer cells, and/or stimulation of activation of apoptosis-related proteins in cancer cells An effective amount of gamma-erythrophylline and a pharmaceutically acceptable carrier thereof are administered to an individual in need thereof. The method is particularly useful for treating diseases associated with the above mechanisms in the individual, such as oral cancer, myeloma, lymphoma, breast cancer, bladder cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, neuroblastoma, and/or Or treatment of thymic malignancies. Among them, the application form and the applicable dose of γ-erythrophylline are as described above.

The invention further provides a method for inhibiting cancer cells in an individual in need thereof A long method comprising administering to the individual an effective amount of gamma-erythrophylline and a pharmaceutically acceptable carrier thereof. Among other things, the method is particularly useful for inhibiting the growth of oral cancer cells, gastric cancer cells, lung cancer cells, breast cancer cells, rhabdomyosarcoma cells, and/or medulloblastoma cells in the individual. Among them, the application form and the applicable dose of γ-erythrophylline are as described above.

The invention is further illustrated by the following examples. The embodiments are provided by way of illustration only and are not intended to limit the scope of the invention. The scope of the invention is shown in the appended claims.

Example

Example 1: Inhibition of cancer cell growth by γ-erythrophylline

Medulloblastoma cells (TE671 cells), rhabdomyosarcoma cells (RD cells), and liver cancer cells at a concentration of 5×10 ⁴ cells/ml at a concentration of 6×10 ⁴ cells/ml (cell/mL) (Huh-7) cell), breast cancer cells (MCF 7 cells), gastric cancer cells (AGS cells), non-small cell lung carcinoma cells (H 1299 cells), oral cancer cells (CA9-22 cells and SAS cells), and the concentration of ⁴ × 10 4 cells / ML of normal oral fibroblast cells (OF cells) were cultured at 37 ° C under 5% carbon dioxide. Each of the above cell lines was divided into 10 groups, and γ-erythrophylline at different concentrations (0, 0.1, 1, 5, 10, 15, 20, 50, 100, and 200 micromolar concentrations) were separately processed. From Alfa Aesar, model: 495-62-5), for 48 hours, followed by 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is used for staining, and the absorbance of 570/630 nm wavelength is detected by visible light detector (OD _570-630nm ) The absorbance values detected by EXCEL were calculated to analyze the significance of the differences between the groups (i.e., the average and standard deviation of the absorbance values of each group were analyzed), and the results are shown in Fig. 1.

As can be seen from Fig. 1, the CC50 values (i.e., half-toxic concentration) of SAS cells, CA9-22 cells, AGS cells, H1299 cells, MCF 7 cells, RD cells, and TE671 cells after treatment with γ-erythrophylline were added. About 17.92 micromolar concentrations, about 5.6 micromolar concentrations, about 18.67 micromolar concentrations, about 52.08 micromolar concentrations, about 23.6 micromolar concentrations, about 30 micromolar concentrations, and about 9.1 micromoles, respectively. The concentration, as for the OF cells (normal oral fibroblasts) and the CC50 values of Huh-7 cells, could not be calculated. The results showed oral cancer cells, medulloblastoma cells, rhabdomyosarcoma cells, non-small cell lung cancer, breast cancer cells, And gastric cancer cells are highly sensitive to γ-erythrophylline, but γ-erythrophylline does not affect normal oral fibroblasts. By the former As described above, γ-erythroside can effectively inhibit the growth of cancer cells and is not cytotoxic to normal cells, and does not cause side effects due to cytotoxicity, and thus can be used to provide an excellent anticancer drug.

Example 2: Benefits of gamma-erythrophylline promoting cell cycle arrest of cancer cells

Oral cancer cells (CA9-22 cells and SAS cells) and normal oral fibroblasts (OF cells) were treated with γ-erythrophylline (0 or 5 micromolar concentration), and after 48 hours, the cells were collected. The cells were washed twice with phosphate buffered saline (PBS) and fixed in 70% ethanol at -20 ° C for one night, followed by PI (1 μg/ml, Biolegend) and RNase A (10 μg). /ml, Sigma), stained for 30 minutes, and then detected the emission wavelength and excitation wavelength (>575) of the PI by flow cytometry (Becton-Dickinson, BD). Nano and 488 nm), finally using FACSCanto ^TM (Becton-Dickinson, BD) to analyze the sub-G1, G0/G1, S and G2 periods in the cell cycle, the results are shown in 2A, 2B picture.

As can be seen from Figures 2A and 2B, the percentage of cells in the sub-G1 phase of CA9-22 cells and SAS cells increased significantly after treatment with γ-erythrophylline (p<0.05), and the OF cells were not added by γ. - An increase in red myrrhene, which shows that gamma-ubiquinol has the ability to arrest the cell cycle of oral cancer cells and has no effect on normal oral fibroblasts.

Example 3: γ-erythroside induces apoptosis in cancer cells (apoptosis) benefit

CA9-22 cells (oral cancer cells) and OF cells (normal oral fibroblasts) were cultured in 6-well cell culture plates, respectively, at different concentrations of γ-erythrophylline (0.1, 1, and After 5 hours of treatment, the original culture solution was removed and the cells were washed with PBS, and trypsin-EDTA was added to each well to start the cells. After centrifugation, the supernatant was removed, the cells were washed with PBS, and finally cell staining was performed in the dark with Annexin V/PI reagent (BioVision, CA) for 15 minutes, followed by flow cytometry (Becton-Dickinson, USA). Analysis was performed to detect FITC emission wavelength (575 nm), excitation wavelength (488 nm), and detect PI signal (wavelength > 575 nm) and quantify the percentage of apoptosis. The results are shown in Fig. 3A, Fig. 3B, Fig. 3A shows early apoptosis, and Fig. 3B shows late apoptosis.

As can be seen from Figures 3A and 3B, treatment of CA9-22 cells with 5 μmol of γ-erythroside for 48 hours resulted in 47.1% advanced apoptosis and 11.5% early apoptosis, but γ - The treatment of erythroprostol had no effect on the early or late apoptosis of OF cells, indicating that γ-erythrophylline has the ability to induce apoptosis of oral cancer cells and has no effect on normal oral fibroblasts.

Example 4: Benefits of apoptotic pathway of γ-erythroside to activate cancer cells

(A) γ-erythrophylline increases the relative expression of apoptosis-related genes in cancer cells

CA9-22 cells were cultured in 6-well cell culture plates and placed in a 37 °C incubator until the next day, and then cultured at different concentrations of γ-erythrophylline (0.1, 1, and 5 micromolar concentrations). 24 hours. After removing the original culture solution, the cells were washed with PBS, and trypsin-EDTA was added to each well to start the cells. After centrifugation, after removing the supernatant, the cells were washed with PBS, and then RNA RNA (100 ng) was extracted using an RNA reagent (purchased from Invitrogen). Next, the obtained RNA was reverse-transcribed into cDNA using an oligo dT primer and SuperScript III reverse transcriptase (purchased from Invitrogen). Then, the relative expression of cysteamine aspartate-specific protease mRNA in cancer cells was analyzed by an instant reverse transcription polymerase chain reaction, and the following ratio of cDNA mixture was inserted into each tube of 8 serial PCR tubes. : cDNA (2 μL), SYBR Green (200 nm molar concentration, LightCycler TaqMan Master, available from Roche diagnostics), forward primer (1 μL), reverse primer (1 μL), MgCl ₂ (1 Microliters, and ddH ₂ O (12.5 μl), the above cDNA mixture was placed in a real-time reverse transcription polymerase chain reaction machine, and the reaction conditions were set as follows: i) 50 ° C, 2 minutes; ii) 95 ° C, 10 Minutes; iii) A total of 40 cycles were then carried out at 95 ° C, 15 seconds and 60 ° C for 1 minute. Finally, the amount of fluorescence per cycle was measured using a multiple fluorescent gene analysis system (ABI PRISM 7300 sequence detection system, available from PE Appiled Biosystems). It is calculated using the calculated value of _△ Ct Ct and _{△△ Ct (△ Ct = Ct.exp-} Ct.control, △△ Ct = △ Ct.exp- △ Ct.mock), and then calculates 2 ^- _△△ ^Ct, can The fold of each group of genes (i.e., Caspase 3, Caspase 8, and Caspase 9) compared with the control group was calculated, and the results are shown in Fig. 4.

As can be seen from Fig. 4, cysteamine aspartate-specific protease 3 (caspase 3), cysteine aspartate-specific protease 8 (caspase 8), The mRNA relative expression of apoptosis-related genes such as caspase 9 increases with the concentration of γ-erythrophylline. This result shows that γ-erythrophylline can effectively increase the expression of apoptosis-related genes in cancer cells.

(B) Activation of apoptosis-associated proteins by γ-ubiquitin stimulated cancer cells

CA9-22 cells were cultured in 6-well cell culture dishes and placed at 37. The cells were cultured for 24 hours and 48 hours at different concentrations of gamma-red myrrhene (0.1, 1, and 5 micromolar concentrations) from the incubator to the next day. After removing the original culture solution, the cells were washed with PBS, and trypsin-EDTA was added to each well to start the cells. After centrifugation, after removing the supernatant, the cells were washed with PBS, and 100 μl of a phosphatase inhibitor (purchased from Roche diagnostics) was added to each tube of cells (radioimmunoprecipitation buffer, RIPA buffer). ), extracting cellular proteins. Next, 100 μl of sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer solution (2X SDS-PAGE sample loading buffer containing 0.5 mmol) was added to each of the obtained protein supernatants. The ear concentration of Tris-HCl [pH 6.8], 10% sodium lauryl sulfate, 10% glycerol, and 0.5% brilliant blue R) was heated to 100 ° C for 8 minutes and quickly applied to ice. After performing protein electrophoresis analysis on the aforementioned protein sample, the protein was transferred to a nitrocellulose membrane (Nitrocellulose members, purchased from Millipore Corporation, Billerica, Massachusetts, USA), and a western dot ink test was performed to transfer the nitrocellulose after transfer. The membrane was immersed in 5% skim milk (TBS containing 0.1% Tween 20) and reacted at 4 °C. It should be 2 hours. Next, a primary antibody (ie, cysteamine aspartate-specific protease 3 (purchased from Calbiocem), cysteamine aspartate-specific protease 8 (purchased from Upstate), and cysteamine were added. Aspartic acid-specific protease 9 (purchased from Upstate), shaken at 4 ° C until every other day, recover the primary antibody, rinse with 1X TBST, add secondary antibody (IgG antibody), react at 4 ° C for 1 hour Then rinse with TBS (containing 0.1% Tween 20). Then, an ECLTM Western Blotting Detection Reagents (available from GE Healthcare) was added to the nitrocellulose membrane for reaction, and a radioactive development method (X-ray film, available from Kodak, Rochester, NY). The protein was visualized and the results are shown in Figure 5.

As can be seen from Fig. 5, activated caspase 3, activated caspase 8 and activated caspase 8 in cancer cells, and The relative expression of apoptosis-related proteins such as caspase 9 increases with the concentration of γ-erythrophylline. This result shows that γ-erythrophylline can effectively stimulate the activation of apoptosis-related proteins in cancer cells.

Example 5: Benefits of γ-erythroside in inducing cancer cells to mitochondria-mediated apoptosis

CA9-22 cells were cultured in 6-well cell culture plates and placed in a 37 °C incubator until the next day, and then cultured at different concentrations of γ-erythrophylline (0.1, 1, and 5 micromolar concentrations). 48 hours. After removing the original culture solution, the cells were washed with PBS, and trypsin-EDTA was added to each well to start the cells. After centrifugation, the supernatant was removed and the cells were washed twice with PBS. Finally, using JC-1 and DiOC6 dyes on cells The staining was carried out for 1 hour in the dark, and the amount of fluorescence was analyzed by a fluorescence microscope and a flow cytometry, and the data obtained by flow cytometry was quantified into a mitochondria membrane potential (MMP). The percentage is as shown in Figures 6A through 6C.

The JC-1 dye can exist in both monomeric and multimeric states, and can enter the mitochondria of normal cells (granulocyte membrane potential of 530 nm) and aggregate into a red-emitting multimer. Conversely, when the cells go to apoptosis (the mitochondrial membrane potential drops to 488 nm), the inner membrane of the mitochondria will eversion, releasing JC-1 into the mitochondria and becoming a green-emitting monomer. Therefore, when the intracellular mitochondrial potential difference stained by JC-1 is observed by a fluorescence microscope, the red luminescence represents the normal mitochondrial potential difference, and the green fluorescence represents the mitochondrial potential difference of the apoptotic cells. As shown in Figure 6A. On the other hand, DiOC6 dye will be attached to the surface of normal cells (the mitochondrial membrane potential is 530 nm, and the granulocyte inner membrane is filled with H ⁺ ). At this time, the flow cytometer can detect Strong fluorescence; conversely, when the cells go to apoptosis (the mitochondrial membrane potential drops to 488 nm, and there is no H ^{+ in the} mitochondrial inner membrane to attract DiOC6 stain), the fluorescence detected by the flow cytometer Will be reduced, the results are shown in Figures 6B, 6C.

As can be seen from Fig. 6A, the cancer cells cultured with γ-erythrophylline have significantly less red fluorescence and green fluorescence than the cancer cells cultured without γ-erythroside. The green fluorescence system increases as the concentration of gamma-red myrrhene increases. In addition, as can be seen from Figures 6B and 6C, the DiOC6 signal detected on the surface of cancer cells cultured with γ-erythrophylline is significantly lower than that of cancer cells cultured without γ-erythrophylline. (ie, the mitochondrial membrane potential is low), and the signal is associated with the concentration of gamma-red myrrhene The degree is increased and reduced. The above results show that γ-ubiquitin can induce cancer cells to mitochondria-mediated apoptosis.

Example 6: Mechanism of γ-erythroside induced apoptosis in cancer cells

(6-1) Experimental method

(6-1-1) Western blotting (Western blotting)

CA9-22 cells were cultured in 6-well cell culture plates and placed in a 37 °C incubator until the next day, and then cultured at different concentrations of γ-erythrophylline (0.1, 1, and 5 micromolar concentrations). 24 hours and 48 hours. After removing the original culture solution, the cells were washed with PBS, and trypsin-EDTA was added to each well to start the cells. After centrifugation, after removing the supernatant, the cells were washed with PBS, and 100 μl of a phosphatase inhibitor (purchased from Roche diagnostics) was added to each tube of cells (radioimmunoprecipitation buffer, RIPA buffer). ), extracting cellular proteins. Next, 100 μl of sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer solution (2X SDS-PAGE sample loading buffer containing 0.5 mmol) was added to each of the obtained protein supernatants. The ear concentration of Tris-HCl [pH 6.8], 10% sodium lauryl sulfate, 10% glycerol, and 0.5% brilliant blue R) was heated to 100 ° C for 8 minutes and quickly applied to ice. After performing protein electrophoresis analysis on the aforementioned protein sample, the protein was transferred to a nitrocellulose membrane (Nitrocellulose members, purchased from Millipore Corporation, Billerica, Massachusetts, USA), and a western dot ink test was performed to transfer the nitrocellulose after transfer. The membrane was immersed in 5% skim milk (TBS containing 0.1% Tween 20) and reacted at 4 ° C for 2 hours. Next, add primary antibody (ie, phosphate-mTOR (S2448) anti- , phosphate-Akt (S473) antibody, phosphate-ERK1/2 (T202/T204) antibody, p53 antibody, phosphate-p53 (S15) antibody (purchased from Cell signaling), CREB antibody (purchased from SIGMA), Phosphate-CREB (S133) antibody, NF-κB antibody, phosphate-NF-KB (S276) antibody (purchased from Abcam), phosphate-p21 (T145) antibody (purchased from ABGENT), phosphate-BAD (K71) Antibody, phosphate-Bcl-2 (S70) antibody (purchased from SANTA CRUZ), phosphate-HDAC2 (S394) antibody (purchased from Bioss), or β-actin antibody, oscillated at 4 ° C until next day, The primary antibody was recovered, washed with 1X TBST, and then added with secondary antibody (IgG antibody), reacted at 4 ° C for 1 hour, and rinsed with TBS (containing 0.1% Tween 20). Then, an ECLTM Western Blotting Detection Reagents (available from GE Healthcare) was added to the nitrocellulose membrane for reaction, and a radioactive development method (X-ray film, available from Kodak, Rochester, NY). ) Visualize the protein.

(6-1-2) Real-time PCR (RT-PCR)

CA9-22 cells or SAS cells were cultured in 6-well cell culture plates and placed in a 37 °C incubator until the next day, with different concentrations of gamma-red myrrhene (0.1, 1, and 5 micromoles). Concentration) cultured for 24 hours. After removing the original culture solution, the cells were washed with PBS, and trypsin-EDTA was added to each well to start the cells. After centrifugation, after removing the supernatant, the cells were washed with PBS, and then RNA RNA (100 ng) was extracted using an RNA reagent (purchased from Invitrogen). Next, the obtained RNA was reverse-transcribed into cDNA using an oligo dT primer and SuperScript III reverse transcriptase (purchased from Invitrogen). Then, the relative expression of cysteamine aspartate-specific protease mRNA in cancer cells was analyzed by an instant reverse transcription polymerase chain reaction, and the following ratio of cDNA mixture was inserted into each tube of 8 serial PCR tubes. : cDNA (2 μL), SYBR Green (200 nm molar concentration, LightCycler TaqMan Master, available from Roche diagnostics), forward primer (1 μL), reverse primer (1 μL), MgCl ₂ (1 Microliters, and ddH ₂ O (12.5 μl), the above cDNA mixture was placed in a real-time reverse transcription polymerase chain reaction machine, and the reaction conditions were set as follows: i) 50 ° C, 2 minutes; ii) 95 ° C, 10 Minutes; iii) A total of 40 cycles were then carried out at 95 ° C, 15 seconds and 60 ° C for 1 minute. Finally, the amount of fluorescence per cycle was measured using a multiple fluorescent gene analysis system (ABI PRISM 7300 sequence detection system, available from PE Appiled Biosystems). It is calculated using the calculated value of _△ Ct Ct and _{△△ Ct (△ Ct = Ct.exp-} Ct.control, △△ Ct = △ Ct.exp- △ Ct.mock), and then calculates 2 ^- _△△ ^Ct, can The fold of each group of genes (i.e., PKA-R1, PDK1, HDAC2, CK-2α, PUMA, NOXA, and Bim) compared to the control group was calculated.

(6-1-3) Immunoprecipitation (IP)

CA9-22 cells were cultured in 6-well cell culture plates and placed in a 37 °C incubator until the next day, and then cultured at different concentrations of γ-erythrophylline (0.1, 1, and 5 micromolar concentrations). 24 hours and 48 hours. After removing the original culture solution, the cells were washed with PBS, and trypsin-EDTA was added to each well to start the cells. After centrifugation, the supernatant was removed, the cells were washed with PBS, and 100 μl of protein lysate containing phosphatase inhibitor (purchased from Roche diagnostics) was added to each tube. A buffer solution (RIPA buffer) extracts cellular proteins. Then, the protein extract was subjected to immunoprecipitation to analyze the effect of γ-erythroside on the acetylation of p53 protein in cancer cells. Wherein, the steps of the immunoprecipitation reaction are: i) adding 80 μl of protein A beads and 200 μl of p53 antibody, and mixing the reaction at a slow speed of 4 ° C for 24 hours; ii) The p53 antibody was removed and 80 μl of the aforementioned protein extract was added, and the reaction was allowed to mix for 4 hours on a slow-speed day at 4 ° C; iii) centrifugation at 12000 rpm for 2 minutes; iv) removal of the supernatant, And washed twice with PBS buffer; v) adding an appropriate amount of sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer solution, placed at 100 ° C for 5 minutes, quickly on ice, for the West Ink dot analysis (primary antibodies are acetylated antibodies, p53 antibodies, etc.).

(6-2) Results

(6-2-1) γ-erythroside enhances phosphorylation of p53 pathway-associated proteins in cancer cells

The effect of γ-erythrophylline on the p53 pathway-associated protein of cancer cells was analyzed by the (6-1-1) Western blot method, and the results are shown in Fig. 7.

As can be seen from Fig. 7, the expression level of phosphorylated tissue protein deacetylase 2 (HDAC2) in cancer cells is significantly decreased as the concentration of γ-erythrophylline is increased, while in cancer cells. The expression levels of phosphorylated p53 protein and phosphorylated ERK1/2 protein increased significantly as the concentration of γ-erythrophylline increased. This result shows that γ-erythroside can effectively inhibit the phosphorylation of the tissue protein deacetylase 2 (HDAC2) of cancer cells, and can effectively enhance the p53 protein of cancer cells and the extracellular signal-regulated kinase 1/2 (ERK1). Phosphorylation of /2).

(6-2-2) γ-erythroside promotes the expression of p53 pathway-related genes in cancer cells

The effect of γ-erythrophylline on the p53 pathway-associated genes of cancer cells was analyzed by (6-1-2) real-time reverse transcription polymerase chain reaction. The results are shown in Fig. 8.

As can be seen from Fig. 8, the relative expression levels of protein kinase A regulatory subunit 1, PKA-R1 mRNA of cancer cells increased as the concentration of γ-erythrophylline increased. This result shows that γ-erythroside can promote the expression of p53 pathway-related genes in cancer cells.

(6-2-3) γ-erythrophylline increases the p53 protein acetylation in cancer cells

The effect of γ-erythrophylline on the p53 protein acetylation of cancer cells was analyzed by (6-1-3) immunoprecipitation, and the results are shown in Fig. 9.

As can be seen from Fig. 9, when the cancer cells were treated with γ-erythroside, the interaction between the tissue protein (histone) and the p53 protein in the cancer cells increased as the concentration of γ-erythrophylline increased. This result shows that γ-erythrophylline can effectively inhibit the increase of p53 protein acetylation in cancer cells.

(6-2-4) γ-erythrophylline increases the expression of apoptosis-promoting genes in cancer cells

The effect of γ-erythrophylline on the expression level of apoptosis-promoting genes of cancer cells was analyzed by (6-1-2) real-time reverse transcription polymerase chain reaction, and the results are shown in Figures 10A and 10B.

As shown in Figures 10A and 10B, p53 upregulated modulator of apoptosis (PUMA) in CA9-22 cells, The relative expression of pro-apoptotic genes such as NOXA and Bcl-2 mediator of cell death (Bim) increases with the concentration of γ-erythrophylline. . In addition, the relative expression of the p53 positive apoptosis regulatory factor (PUMA) gene in SAS cells also increased as the concentration of γ-erythrophylline increased. These results again show that γ-erythrophylline can effectively increase the expression of pro-apoptotic genes in cancer cells, thereby inducing and/or promoting apoptosis of cancer cells.

(6-2-5) Mechanism of γ-erythroside induced apoptosis in cancer cells

The effect of γ-erythrophylline on the intracellular apoptosis pathway-related genes of cancer cells was analyzed by (6-1-1) Western blotting method, and the results are shown in Fig. 11.

As can be seen from Fig. 11, the expression of phosphorylated p21 ^WAF1 protein and phosphorylated Bcl-2-associated death promoter (Bad) in cancer cells is accompanied by γ-erythrophylline. Increased concentration, phosphorylated B-cell lymphoma 2 (Bcl-2), phosphorylated protein kinase Akt (Akt), and phosphorylated mammalian rapa The amount of expression of the mammalian target of rapamycin (mTOR) decreased as the concentration of γ-erythrophylline increased. This result shows that γ-erythrophylline can induce cancer cells to migrate into the intramembrane apoptosis pathway by activating the expression of the p21 ^WAF1 protein and the Bcl-2 related death promoter of cancer cells.

Example 7: Animal test analysis

Twenty nude mice (purchased from BioLASCO Co., Ltd.) of BALB/c-nu/nu strains aged 4 to 6 weeks were housed under the same conditions for 4 to 6 weeks. The mice were divided into two groups of experimental group and control group, 5 to 7 each. Then, SAS cells (1 × 10 ^{7 /} 0.1 ml DMEM medium) were subcutaneously injected into the back of the experimental group (the control group was not injected with γ-erythrophylline), fed for one week and the tumor volume was measured to reach 100 cubic meters. After MM, 3.065 mg/kg body weight of γ-erythrophylline was injected every two days. Finally, the mice were sacrificed, and the tumors were collected, weighed, and the volume was measured. The results are shown in Figures 12A to 12C.

As can be seen from Figures 12A to 12C, in the case where there was no significant change in the body weight of the mice, the tumor volume and weight of the experimental group were injected with γ- compared with the control group not injected with γ-erythrophylline. After red myrrhene, it decreases with the passage of time. This result shows that γ-erythrophylline is effective in treating cancer.

Claims (3)

Use of a compound of formula (I) for the manufacture of a medicament Wherein the agent is for the treatment of oral cancer. The use of claim 1, wherein the amount of the agent is from about 0.1 mg/kg body weight to about 50 mg/kg body weight per day of the compound of formula (I). The use of claim 1, wherein the amount of the agent is from about 1 mg/kg body weight to about 10 mg/kg body weight per day of the compound of formula (I).