WO2011088605A1

WO2011088605A1 - Use of leptin in enhancing therapeutic efficacy of medroxyprogesterone acetate on liver cancer

Info

Publication number: WO2011088605A1
Application number: PCT/CN2010/001662
Authority: WO
Inventors: 王森稔; 叶耀宗; 李金德; 杨翔荏; 杨晓芳
Original assignee: 高雄医学大学
Priority date: 2010-01-19
Filing date: 2010-10-22
Publication date: 2011-07-28

Abstract

Use of leptin in enhancing the effect of medroxyprogesterone acetate on inhibition of hepatocarcinoma cells. When the level of leptin in hepatoma patients is higher, medroxyprogesterone acetate is administrated directly or combined with leptin. When the level of leptin in patients is lower, 5-hydroxy tryptophan is administrated for increasing the level of leptin, and then medroxyprogesterone acetate is administrated.

Description

Application of sputum hormone in strengthening medroxyprogesterone acetate for treatment of liver cancer

Technical field

The invention relates to a method for treating liver cancer by using a thin hormone, in particular to a therapeutic drug, a course of treatment or a screening platform for administering a therapeutically effective amount to a liver cancer patient, in particular to increase or decrease the leptin (Leptin). The physiological concentration of leptin in patients with liver cancer is combined with medroxyprogesterone acetate (MPA) and then contacted with liver cancer cells to kill liver cancer cells.

Background technique

According to US statistics, there has been a significant increase in the incidence of liver cancer in the United States over the past two decades, with three times as many as men and two times as many as blacks. In addition, for the eastern countries such as Taiwan, Japan, South Korea and the mainland, because of the high-risk area of hepatitis B virus and cirrhosis, liver cancer is a common malignant tumor in the eastern countries, and it is found by clinical observation that it contains 5 § ~3⁄4)% of liver cancer is associated with hepatitis and cirrhosis. Among them, the statistics of the top ten causes of death in Taiwan, China, show that since 1982, cancer has leapt to the top, with liver cancer being the first cause of all cancer deaths for men and for women. It is also the second highest, and it kills 4,000 people every year, which is enough to show that it poses a great threat to the health of Chinese people. Therefore, early diagnosis and treatment of liver cancer will be one of the research priorities in the future.

In the current clinical treatment of liver cancer, most of them are treated with surgery to improve the survival rate of patients, but only 10 to 15% of patients with liver cancer can be used for surgery. The rest of patients who cannot be treated with surgery are trying to use radiation therapy or chemotherapy. Or immunotherapy to prolong the survival rate of patients with liver cancer, but the results are not satisfactory, and chemotherapy and radiation therapy will also cause damage to normal human cells, which may lead to side effects of various discomforts.

In view of this, in other related research and reports, it was found that the cytokine secreted by fat cells, leptin, has a close relationship with the occurrence of liver cancer. To date, cancers related to lean hormones have been identified, including endometrial cancer and breast cancer. Studies have shown that by binding to their corresponding membrane surface receptors, estrogen can regulate gene expression through transcriptional signaling of the activator 3 (STAT3) signaling system, many of which are associated with cancer. , such as: Vascular Endothelial Growth Factor (VEGF). However, in recent years, hormone therapy seems to open a new opportunity in the treatment of liver cancer. Therefore, the treatment of liver cancer has gradually tended to treat liver cancer patients with hormone therapy, and clinical and laboratory studies have also found that liver cancer is also found. A sex-dependent hormone type with predominantly androgen, which is significantly associated with changes in steroid hormones and sex receptors. Recently, there is a new treatment for liver cancer patients, Glucocorticoid Antagonists, for example: Progesterone and RU486 can inhibit the expression of a-fetoprotein in human liver cancer. It suggests that progesterone has a potential for hormone therapy in liver cancer. At present, it has been found that progesterone acetate, a progesterone analog, can effectively inhibit the growth of liver cancer cells. The MPA is derived from the luteinizing hormone (17-OH-progesterone) and is administered orally as a synthetic steroid. Its structure is similar to that of the natural luteinizing hormone, except that it has a methyl group at the α - 6 position (Methyl Group). There is an acetate functional group (Acetoxy Group) at position 17. The MPA is a luteinizing hormone with antiestrogenic activity. In postmenopausal breast cancer, it completely inhibits the concentration of plasma estrogen and also inhibits the release of Luteining Hormone (LH). Stimulates the growth of the endometrium and produces typical body hormone changes in breast acinar cells. The MPA action machine is still unclear, but is believed to involve interaction with intracellular hormone receptors. In other studies, it has also been pointed out that MPA binds to receptors of estrogen, progesterone and androgen, and in particular, binding to androgen receptors is particularly important for the cytotoxic effects of this drug. Recently, studies have also indicated that MPA can inhibit the size of liver cancer tumors and prolong the survival rate of liver cancer patients. Related studies have also confirmed that MPA may affect the process of liver cancer evolution and improve the prognosis of patients. Thus, the use of MPA to treat clinical liver cancer patients may be helpful for patient survival, but because MPA has multiple pharmacological biological activities, its mechanism of action needs to be further explored.

In this regard, some clinical trials have shown that MPA has an inhibitory effect on end-stage liver cancer patients, and related studies have also shown that it can inhibit the growth of HepG2 cells. At the same time, in the research results of the applicant's previous study, there was no significant relationship between the expression of leptin in liver cancer tissues and the use of MPA after surgery and the survival time of patients (as shown in Figure 1, P values were respectively 0. 383 and 0. 171). In addition, the microvessel density values in liver cancer tissues and the performance of Ki-67 also showed no significant relationship with the survival time of patients.

In summary, estrogen is a hormone of a single-chain protein component and is generally considered to play an important role in weight maintenance and energy regulation, while MPA is a progesterone-derived synthetic steroid. At present, relevant studies have confirmed that MPA can cause apoptosis of liver cancer cell lines in a system in vivo and in vitro, that is, the MPA system can effectively inhibit the growth of liver cancer cell lines in a cell line system; however, relevant research abroad However, it was found that treatment of liver cancer patients with MPA had no significant effect on prolonging survival. Although MPA is currently on the market for cancer, there is no exhilarating effect on liver cancer patients. Therefore, there is no statistical difference in the survival rate of patients with liver cancer by MPA, and in addition to the lack of clinically valid evidence, there is no consideration of the biomarkers such as estrogen and other biomarkers in liver cancer tissues and liver cancer tissues. To cooperate with the combined administration of MPA. Therefore, the average user has not been able to meet the needs of the user in actual use.

Summary of the invention

The main object of the present invention is to overcome the above problems encountered in the prior art and to provide an application of attenuating MPA with a leptin to inhibit liver cancer cells.

A secondary object of the present invention is to provide a method for directly treating MPC patients with liver cancer patients in the serum of liver cancer patients or in liver cancer tissues, so as to achieve the pharmacological action of enhancing MPA. Another object of the present invention is to provide a method for treating liver cancer patients by combining leptin and MPA into a drug when the expression of lept hormone in the serum of liver cancer patients or liver cancer tissues is high, so as to achieve the pharmacological action of strengthening MPA. .

A further object of the present invention is to provide a method for increasing leptin by 5-hydroxy-trypton (5-HTP) when the expression of leptin in serum or liver cancer tissues of liver cancer patients is low. After the amount, MPA treatment is given to achieve the pharmacological action of strengthening MPA.

In order to achieve the above object, the present invention relates to a method for treating liver cancer by using a lepid hormone, comprising a therapeutic drug, a course of treatment or a screening platform for administering a therapeutically effective amount to a liver cancer patient, characterized in that the hormone is increased or the liver cancer patient is raised. The physiological concentration of lean hormone combined with a MPA is contacted with liver cancer cells, and the interaction of the estrogen and its progesterone receptor through the receptor enhances and accelerates the MPA to kill liver cancer cells, thereby normalizing the liver epithelial cell line and cancer cells. The strain has a specific pharmacological effect, so that it can have no adverse side effects on normal liver cells, and can prolong the overall survival rate of liver cancer patients. Among them, the level of expression of the estrogen can further serve as a predictor of prophylactic factors for the treatment of MPA in patients with liver cancer and a prognostic factor for survival.

DRAWINGS

Figure 1 is a schematic diagram showing the analysis of the prognosis survival time of patients with liver cancer using MPA after surgery.

Figure 2 is a schematic diagram showing the analysis of cell growth by estrogen at different concentrations in the present invention.

Figure 3 is a schematic diagram showing the analysis of cell growth by MPA at various concentrations in the present invention.

Figure 4 is a graphical representation of the analysis of cell growth after treatment with leptin and MPA in the present invention.

Fig. 5 is a schematic view showing the cell type of the present invention after treating cells with leptin and MPA.

Fig. 6 A is a schematic diagram showing the effect of the present invention on cell cycle after treating cells with leptin and MPA.

Fig. 6B is a schematic diagram showing the analysis of cell cycle after treating cells with estrogen and MPA.

Figure 7. Schematic diagram of the effect of the present invention on apoptosis-related proteins after treatment with leptin and MPA.

Fig. 8 A is a schematic diagram showing the interaction of the leptin receptor with the progesterone receptor receptor by immunoprecipitation.

Figure 8B is a schematic representation of the interaction of the leptin receptor with the progesterone receptor receptor by immunofluorescence staining.

Figure 9. Schematic diagram of the analysis of the present invention for inhibiting the reduction of the estrogen receptor and enhancing the action of MPA.

Figure 10 A is a schematic representation of the increase in expression of the long form of the estrogen receptor in the present invention to allow apoptosis of the cells after dosing.

Figure 10B is a schematic diagram showing the analysis of apoptosis of cells after dosing by increasing the amount of progesterone receptor expression. Fig. 1 is a schematic diagram showing changes in the amount of JAK/STAT pathway after drug treatment in the present invention.

Fig. 1 2 is a schematic diagram showing changes in the amount of expression of the MAPK pathway of the present invention after drug treatment.

Fig. 1 3 A is a schematic diagram showing changes in the amount of expression of the JAK/STAT path of the present invention in a short period of time after drug treatment. Fig. 1 3 B is a schematic diagram showing changes in the amount of expression of the MAPK pathway of the present invention in a short time by drug treatment.

Fig. 14 is a schematic diagram showing the degradation of p-STAT3 by inhibiting the activation of ERK1/2 and the expression of PIAS3 in the present invention.

Fig. 15 is a schematic diagram showing the activity analysis of a large amount of leptin in the cells to enhance MPA inhibition of cell growth in the cells.

Fig. 1 6 A is a schematic diagram of the analysis of the STAT3 related message pathway by MPA treatment in the present invention.

Fig. 1 6 B is a schematic diagram of the analysis of the MAPK-related message pathway by MPA treatment in the present invention.

Fig. 17 is a schematic diagram showing the analysis of the growth of normal liver epithelial cell lines by the combination of leptin and MPA in the present invention.

Fig. 1 8 A is a schematic diagram showing the analysis of STAT3 related message pathways after treatment of lean liver cells with MPA by normal liver cells of the present invention.

Fig. 1 8 B is a schematic diagram of the MAPK-related message pathway of normal liver cells of the present invention treated with estrogen and MPA.

detailed description

The invention relates to a method for treating liver cancer by using a lepid hormone, and can be used as statistical data on liver cancer cell lines (HepG2 Cells) and clinical liver cancer patients. The invention comprises a therapeutic drug, a course of treatment or a screening platform for administering a medicinal effective amount to a liver cancer patient, which is characterized by a combination of a leptin (Leptin) or a physiological concentrating agent for increasing the physiological concentration of leptin in a liver cancer patient. After exposure to melanocytogenes (MPA), the interaction of the estrogen and its progesterone receptor through the receptor enhances and accelerates the MPA inhibiting the killing effect of the liver cancer cells, thereby normalizing the liver epithelial cell line. It has a specific pharmacological effect with cancer cell lines. The drug or course of treatment with leptin combined with MPA can cause the cancer cells of liver cancer patients to decrease in size, that is, the drug can respectively reduce the size or quality of liver cancer cells, and is normal. The adverse side effects of non-toxic killing of liver cells can effectively prolong the overall survival rate of liver cancer patients.

The invention has found that the effects of lean hormone and MPA on liver cancer cells and prolonging the survival rate of patients have been found in the study of in vitro and in some clinical patients. Accordingly, the combination of leptin and MAP was designed as a therapeutic drug for the treatment of liver cancer patients, and it was also found that the physiological concentration of lean hormone in normal humans can enhance the efficacy of MPA. Among them, MPA is treated at a dose of 500 mg (mg) per day in a clinical patient in a preferred embodiment of the present invention, and therefore, after the present invention utilizes a combination of MPA to increase the physiological concentration of estrogen or estrogen , can achieve better therapeutic effects. Please refer to FIG. 1 for the analysis of the prognosis survival time of the liver cancer patients after treatment with MPA. As shown in the figure: The present invention compares the results of clinical analysis of clinical liver cancer patients in the above preferred embodiments, wherein the performance of leukemia in liver cancer tissues and the survival time of MPA and patients after surgery can be found. There is no meaningful relationship. The P values shown in the upper left and upper right of Figure 1 are 0. 383 and 0. 171; respectively, there is MPA after surgery. In the case of liver cancer, the analysis showed that the high-performance of the estrogen was longer than the low-performance one, as shown in the lower left of Figure 1, the P value was 0.001.

According to this clinical experiment, the prognosis and survival rate of patients with liver cancer who have high expression of estrogen after surgery are significantly better than those with low expression of estrogen. Therefore, it is sufficient to confirm that the leptin can enhance the effect of MPA on the treatment of liver cancer, and the expression level of the estrogen can further serve as a predictor of the efficacy of the treatment of liver cancer patients with MPA (Predictive Factors) and its survival time. Prognostic Factors, in which the higher the expression of the estrogen, the more obvious the inhibitory effect of the intensive sputum on hepatocellular carcinoma cells, which can be used as a liver cancer patient to receive hormone therapy (ie, sputum) after surgery. Indicators, in turn, can help maintain or prolong the survival of liver cancer patients.

Based on the above results, the prognosis of patients with liver cancer who have higher expression of estrogen is better than that of patients with low expression of estrogen, and the survival rate can be extended for nearly five years. This is among other related technologies. Undiscovered. However, the present invention also demonstrates in an in vitro experiment that leptin enhances the action of sputum to inhibit liver cancer cells through certain mechanisms, and in addition to combining leptin and MAP into a therapeutic drug or treatment, the present invention may also provide A platform for screening for lean hormones to determine whether to perform MPA treatment.

The combination of leptin and MPA is a new treatment that can be used in patients with liver cancer. There are no reports or studies at home and abroad. The difference from other known literatures is that the present invention combines MPA with estrogen as a new treatment method, so that it can effectively improve the shortcomings of the known technique that only MPA is used to treat liver cancer patients without prolonging the survival rate.

The present invention further explores the anti-cancer role of leptin combined with MPA in liver cancer cells and the molecular mechanism of its action. Hepatocellular carcinoma cell lines were treated with leptin and MPA, and their cell viability was observed. The cell cycle, apoptosis and mechanism of action were analyzed. The related message transmission pathways involved in estrogen and MPA were also explored. In addition, the effects of the pharmacological effects of leptin combined with MPA on normal liver epithelial cell lines were examined by observing their drug specificity, in order to further understand the use of hormone therapy in liver cancer.

Example 1: Effect of leptin on the growth of hepatoma cells

Please refer to FIG. 2, which is a schematic diagram of the analysis of cell growth by different hormones in the present invention. As shown in the figure: In order to understand the effect of leptin on liver cancer cells, in this example, cells were treated with two different concentrations of estrogen, wherein the high concentration of lean hormone (LH) was 100 ng (ng), low concentration The lean hormone (LL) is 10 ng. Among them, the above-mentioned determination of the main concentration depends on the range of physiological concentration of 10 ng as a normal person, and the effect of the estrogen on the cells can be observed in the physiological range, and the higher concentration determines whether the higher the concentration of the estrogen is for the cells. The impact will be greater. The experimental results showed that after treatment of cells with high or low concentrations of estrogen for 24 hours, it was found in the XTT assay that leptin had no significant effect on the growth of cancer cells.

Example 2: Effect of MPA on the growth of hepatoma cells Please refer to FIG. 3, which is a schematic diagram of the analysis of cell growth of MPA at different concentrations in the present invention. As shown in the figure: To demonstrate the effect of MPA on the treatment of liver cancer, in this example, two different concentrations of MPA were used to treat the cells, with a high concentration of MPA (MH) of 10 and a low concentration of MPA (ML) of 10 ^→ M. The experimental results showed that after treatment with high concentration of MPA for 24 hours, the cells were significantly inhibited by XTT assay. After 48 hours of treatment, the cell inhibition phenomenon was significantly increased. The low concentration of MPA did not significantly inhibit the effect at 24 or 48 hours. Therefore, it indicates that the inhibitory effect of MPA on cells with increasing concentration and time is better.

Example 3: Effect of leptin on MPA poisoning of liver cancer cells

Please refer to FIG. 4, which is a schematic diagram of the analysis of cell growth after treatment with lean hormone combined with MPA. As shown in the figure: In this example, cells were treated with two different concentrations of estrogen in combination with two different concentrations of MPA. The results of the experiment showed that the high concentration of the estrogen combined with the high concentration of MPA to treat the cells for 24 hours, the XTT assay showed that the inhibition of cell growth was indeed better than the MPA alone, and its P value was 0.001, and The P value of 0.01% is better than that of the MPA alone. However, when high concentration or low concentration of lean hormone combined with low concentration of MPA, there is no obvious inhibitory effect. Therefore, the experimental results show that leptin can indeed enhance the effect of MPA on cell growth, and the higher the concentration of leptin, the more obvious the inhibitory effect.

Example 4: Leptin combined with MPA causes increased apoptosis

Please refer to FIG. 5, which is a schematic diagram of the cell type of the present invention treated with leukemia combined with MPA. As shown in the figure - from the above results, it is known that the estrogen does enhance the cytostatic effect of MPA, and in the present example, the results of the cell type are observed under the microscope, as in the experiments of the above examples, the combination of leptin After MPA treatment, the degree of apoptosis of Apoptosis was significantly greater than that of MPA alone. Furthermore, the present invention further observes the nuclear form by immunofluorescence. After the cells were treated with estrogen and MPA for 24 hours, the nucleus was stained with fluorescent dye (DAPI) and observed under a fluorescence microscope at 40X. The results were also consistent, and the degree of nuclear atrophy was more obvious than that of MPA alone. Therefore, it can be seen from the above results that leptin does enhance and accelerate the apoptosis caused by MPA.

Example 5: Effect of leptin combined with MPA on cell cycle

Please refer to FIG. 6A and FIG. 6B, which are respectively a schematic diagram showing the effect of cell cycle on cell treatment after treatment with leukemia combined with MPA, and the cell cycle analysis of the present invention after treating cells with leptin and MPA. As shown in the figure: To further understand the role of leptin and MPA in liver cancer cells, this example is to collect cells treated with estrogen or MPA and estrogen combined with MPA for 24 hours, by flow cytometry (Flow Cytometry) Analyzes the effect of the drug on the Cell Cycle and counts the portion of Sub-Gl/Apoptosis in the cell cycle as Student's - test. The experimental results showed that the cells treated with MPA had an increasing trend in sub-G1, indicating a marked increase in apoptotic cells, but decreased in the G1 and G2/M phases, but increased in the S phase; After treatment with high concentration of lean hormone and high concentration of MPA for 24 hours, the analysis showed that the increase of sub-G1 was higher than that of MPA alone, and its P value was 0.002; while the low concentration of lean hormone combined with high concentration of MPA Sub- The increase trend of Gl is also higher than that of MPA alone, and its P value is 0.04, which is lower in G1 and G2/M than in MPA alone, and higher in S phase. From the above results, it is known that the ratio of leukemia combined with MPA leads to apoptosis compared with MPA alone. In addition, the effect may be to increase MPA inhibition of G1 and G2/M phases, or to arrest the cell cycle in S phase.

Example 6: Analysis of apoptosis-related proteins

Referring to Fig. 7, the effect of the present invention on apoptosis-related proteins after treatment with leptin and MPA is shown. As shown in the figure: It has been shown that the combination of leptin and MPA increases the apoptosis. The present invention uses Western blotting to analyze whether the apoptosis-related proteins are changed after treatment with estrogen and MPA for 24 hours. As shown in the figure, after treatment with different concentrations of estrogen and different concentrations of MPA for 24 hours, the cell proteins were anti-c leaved caspase 3, anti-cleaved caspase 7 and anti-cleaved PARP (Po 1 y-ADP-r i bose - Polymerase) The antibody was analyzed by Western blotting. It was found that cells treated with MPA alone were significantly activated in cleaved caspase 3/7 and cleaved PARP, and cleaved caspase 3/7 caused by leptin combined with MPA The cleaved PARP activation effect is more pronounced than with MPA alone.

Example 7: The interaction between the leptin receptor (ob-R) and the progesterone receptor (PgR) after dosing is shown in Fig. 8A and Fig. 8B, respectively, which are analyzed by immunoprecipitation according to the present invention. Schematic diagram of the interaction of the leptin receptor with the progesterone receptor receptor, and the present invention by immunofluorescence staining for the interaction of the leptin receptor with the progesterone receptor receptor. As shown in the figure: Further explore whether there is some interaction between the leptin receptor and the progesterone receptor. In this example, the cells are treated with leptin or MPA alone, and lean hormone combined with MPA for 30 minutes. The cell protein was immunoprecipitated with anti-pgR antibody, and the anti-ob-R antibody was used to analyze whether the leptin receptor and the progesterone receptor were combined by the Western blotting method. After 30 minutes of MPA addition, the leptin receptor does bind to the progesterone receptor. After 30 minutes of treatment with estrogen and MPA, the leptin receptor binding to the progesterone receptor was more pronounced, as shown in Figure 8A. The same result was obtained by immunofluorescence staining. After treating the cells with the drug for 10 minutes, FITC stained progesterone receptor, Texas-Red stained leptin receptor, and phalloidin stained cell membrane, observed under fluorescence microscope 40X, when alone When MPA is added, a small amount of the estrogen receptor will bind to the progesterone receptor. After the addition of lean hormone and MPA, the binding is more pronounced at this point, as shown in Figure 8B. Therefore, it has been experimentally found that the leptin receptor and the progesterone receptor do interact to affect cells.

Example 8: Decreasing the expression of the estrogen receptor to reduce the effect of leptin combined with MPA

Please refer to FIG. 9 for a schematic diagram of the analysis of the present invention for inhibiting the reduction of the estrogen receptor to enhance the action of MPA. As shown in the figure - the leptin receptor and progesterone receptors will interact with each other after dosing. In this example, we continue to explore that since leptin increases MPA inhibition of cell effects, and estrogen binding to relative acceptance In order to initiate the action, if the inhibition of the expression of the estrogen receptor is negative, the hormone will lose the effect of strengthening the MPA inhibiting cells, and will not increase the therapeutic effect of MPA. As shown in the figure, the leptin receptor siRNA was transferred to the cell culture day at 20 nM, and the next day was changed. The serum-free (Serum-Free) medium was treated with leptin and MPA for 24 hours, and the effect of cell growth was analyzed by XTT assay. According to the experimental results, when the TNF of the leptin receptor reduces the expression of the estrogen receptor, the leptin can not strengthen the effect of MPA inhibiting cells. It can be seen that the effect of leptin on strengthening MPA is mainly through thin Hormones and their receptors regulate this function.

Example 9: Increasing the expression levels of the leptin receptor and the progesterone receptor, and increasing the apoptosis of the cells after dosing, as shown in Fig. 10A and Fig. 10B, respectively, adding long-type lean hormones to the present invention. The expression of the receptor allows the cells to be analyzed for apoptosis after dosing, and the present invention increases the expression of the progesterone receptor to visualize the apoptosis of the cells after dosing. As shown in the figure: From the above results, it was found that the therapeutic effect of leptin-enhancing MPA did indeed act through the activation of the estrogen receptor. Therefore, in order to reconfirm this study, in this example, ob-Rb plasmid was transferred to cell culture for one day, the next day was replaced with serura-free medium, and then combined with estrogen and MPA for 24 hours, XTT The assay analyzes the effects of cell growth by observing the effects of the cells after dosing by expressing the cells in large amounts of the long-term leptin receptor (ob-Rb). As a result, as shown in Fig. 10A, when the cells express a large amount of long-term leptin receptors, the effect of improving MPA treatment is markedly increased. In addition, long-type leptin receptors appear to increase the performance of progesterone receptors. In view of this, in this example, PgR plasmid was further transferred to cell culture for one day, and the next day was replaced with serum-free medium, and then treated with leptin and MPA for 24 hours, and the effect of cell growth was analyzed by XTT assay. The effect of enhancing the action of MPA is observed by expressing the cells in large amounts of progesterone receptors. The results are shown in Fig. 10B, which is also in line with the expectation of the present invention, and indeed, when the cells express a large amount of progesterone receptors, the therapeutic effect of MPA is also enhanced. In this example, it was found that the action of leptin combined with MPA is due to the indirect increase of progesterone receptor expression by the leptin receptor, thereby improving the therapeutic effect of MPA.

Example 10: Lean Hormone Enhances MPA Inhibition JAK/STAT Message Delivery Path

Please refer to FIG. 1 1 , which is a schematic diagram showing changes in the JAK/STAT path of the present invention after drug treatment. As shown in the figure: In order to find out which related proteins are changed after the combination of leptin and MPA, this example uses western blot method to analyze whether the downstream related proteins are treated by the combination of leptin and MPA for 24 hours. change. According to the experimental results, it was found that p-ob-R, ob-R and PgR were inhibited after 24 hours of treatment with the drug. It was also found that the effect of inhibition by lean hormone combined with MPA was more obvious than that of MPA alone. In addition, in terms of proteins in the JAK2/STAT3 related pathway, the results were also suppressed. Therefore, it was found that MPA inhibits the progesterone receptor and related proteins downstream, so that it loses its original function, and the inhibitory effect of the cells after treatment with leptin and MPA is more obvious.

Example 11: Leptin Enhances MPA Inhibition MAPK Message Delivery Path

Please refer to FIG. 12 for a schematic diagram showing the change of the MAPK pathway of the present invention after the drug treatment. As shown in the figure: After the downstream hormone protein JAK2/STAT3 was inhibited, this example attempts to observe whether the MAPK-related message pathway and the upstream and downstream proteins also change, and the protein expression is analyzed by Western blotting under the same conditions. Real The results showed that the expression levels of the APK-related proteins p-JNK, JNK, p-ERKl/2, ERKl/2, p-p38 and p38 were all inhibited, demonstrating that MPA not only inhibits the downstream proteins associated with the leptin receptor. It also inhibits other message pathways to inhibit the growth of liver cancer cells, and its estrogen also enhances MPA inhibition.

Example 12: Observation of changes in estrogen molecules by MPA on a short time point

Please refer to FIG. 1 3 A and FIG. 1 3 B , which are schematic diagrams showing changes in the JAK/STAT path in the short-term performance of the JAK/STAT path in the present invention, and changes in the performance of the APK path in the short-term period of the present invention. . As shown in the figure - the results of the above experiments showed that the proteins in the JAK2/STAT3 and MAPK-related signal pathways of the drug-treated cells were inhibited in 24 hours, possibly because the cells were in apoptotic state at 24 hours. Related protein degradation does not reveal which of the relevant message pathways changes, so this example uses short time points to observe changes in related proteins. The cells were treated with high concentrations of estrogen and high concentrations of MPA and high concentrations of estrogen combined with high concentrations of MPA for 30 minutes, 3 hours and 6 hours, respectively. Collecting cells according to time points JAK/STAT-related proteins are analyzed by Western blotting methods for protein expression. The results showed that in the 30th minute, the combination of lean hormone and MPA began to degrade p-ob-R, and the degree of degradation became more obvious with time. However, P-STAT3 (Tyr705) was also inhibited at the third hour, and the effect of inhibition was also more obvious than that of leptin combined with MPA, as shown in Fig. 13 A. In addition, under the same conditions, Western blotting method was used to observe the changes in the expression of MAPK and downstream related proteins. p-JN was significantly inhibited at the 3rd hour, while p-ERK1/2 and PIAS3 were at 30th. At the minute and the third hour, MPA and the estrogen combined with MPA showed excessive performance, as shown in Figure 13B.

Example 13: p-ERKl/2 induction PIAS3 activation inhibition P-STAT3

Referring to Figure 14, the present invention is intended to inhibit the activation of ERK1/2 and the expression of PIAS3 to reduce the degradation of p-STAT3. As shown in the figure: The above findings showed that p-ERK1/2 alone was overexpressed with MPA and lean hormone combined with MPA at the 30th minute and the third hour. This example attempts to find a relevant path to clarify p-ERKl/2 activation. Correlation with p-STAT3 (Tyr705) inhibition. As a result, it was found that p-ERKl/2 activation induces the activation of P-STAT3 inhibitor-activated transcriptional activator 3 (PIAS3) and inhibits the expression of P-STAT3 (Tyr705). Apoptosis (as shown in Figure 1 3 B). Therefore, in this example, the cells were added with 10 p-ERKl/2 inhibitor (U0126) 4 hours before the dosing, and then the cells were treated with estrogen and MPA for 3 hours, and the cellular proteins were collected and analyzed by Western blotting. The amount of performance. It was found by experiments that the phosphorylation of p-ERK1/2 was inhibited, and PIAS3 was not induced to activate, and the expression of p-STAT3 (Tyr705) was not inhibited. From the above results, MPA may indirectly activate PIAS3 by activating p-ERK1/2 to inhibit apoptosis of p-STAT3 (Tyr705), which enhances the action of MPA to activate p-ERK1/2. Increased apoptosis.

Example 14: Cell mass expression of leptin enhances MPA activity in inhibiting cell growth Please refer to FIG. 15 to FIG. 1 6B, which are respectively a schematic diagram showing the activity analysis of a large amount of estrogen-enhanced MPA inhibiting cell growth in the cells of the present invention, and the present invention is related to STAT3 by MPA treatment after a large amount of expression of leukemia in the cells. A schematic diagram of the analysis of the message path, and a schematic diagram of the analysis of the MAPK-related message pathway by MPA treatment after the present invention expresses a large amount of lean hormone in the cell. As shown in the figure: In the above examples, the combination of lean hormone and MPA inhibits the hematopoietic cells of the liver cancer cells, and the present invention is in this embodiment. However, in the present embodiment, in order to test whether the liver cancer cells themselves can secrete a large amount of lean hormones, Will enhance the role of MPA in inhibiting liver cancer cells. So after the laser quality thin body (pcLeptin) colonization transferred to the cell culture one day, the next day medium was replaced with serum-free, and then processed to 10- ⁴ M MPA for 24 hours, XTT assay Analysis of the cell growth. As shown in the left half of Figure 15, the cell's transient translocation of the estrogen gene allows the cells to secrete a large amount of estrogen. After adding the MPA for 24 hours, it is found that the secretion of the estrogen by the cells itself also enhances the inhibition of liver cancer cells by MPA. In addition, after a large amount of expression of the leptin gene, the medium was collected and subjected to immunoprecipitation using a leptin antibody, followed by Western blot analysis, as shown in the right half of Fig. 15, when the leptin gene was expressed in a large amount in the cell. , will cause the cells to secrete leptin from the body and can be measured in the medium. Furthermore, in this example, the protein expression of the STAT and MAPK-related message pathways is also analyzed by Western blotting. As shown in FIG. 16A, the pro-liposome is transferred to the cell culture for one day, the next day. After replacing the serum-free medium and treating the cells with Ιθ ΐ MPA for 24 hours, the JAK/STAT-related protein was analyzed by Western blotting. It was found that when the cells expressed a large amount of lean hormone, MPA was added for 24 hours. The degree of STAT3 activation was also more pronounced than that of cells treated with MPA alone, and the cellular MAPK message pathway-related proteins were analyzed by Western blotting under the same conditions, as shown in Figure 16 B, in the MAPK-related protein ERK, JNK. , p38, c-fos and c-jun are also the same result.

Example 15: Effect of leptin combined with MPA on normal liver epithelial cell lines

Please refer to FIG. 1 7 to FIG. 1 8B, which are respectively a schematic diagram of the analysis of the growth of normal liver epithelial cell lines by the combination of lean hormone and MPA, and the STAT3 related message path of the normal liver cells of the present invention treated with lean hormone combined with MPA. The analysis diagram of the analysis, and the analysis of the MAPK-related message pathway after the treatment of the normal liver cells of the present invention with the combination of lean hormone and MPA. As shown in the figure: In this example, after the same conditions were used to treat the normal liver epithelial cell line THLE-3 with MPA for 24 and 48 hours, the cell growth (ie, survival rate) was analyzed by XTT assay after estrogen combined with MPA. ) Impact. As shown in Figure 17, the results of the experiment showed that treatment of leptin and MPA or leptin alone with MPA had no significant effect on the survival rate and toxicity of normal liver epithelial cell lines. Furthermore, as shown in Fig. 18A and Fig. 18B, this example also analyzes the protein expression of STAT and MAPK-related message pathways under the same conditions, and the results show that both leptin and MPA are not significant for related protein. influences.

Therefore, it has been confirmed by the above experiments that the treatment of liver cancer cells with the combination of lean hormone and MPA can indeed enhance the effect of MPA inhibiting liver cancer cells, and is better than using MPA alone; in addition, the present invention displays liver cancer cell lines in large amounts to express hormone proteins. The same effect was also found. Accordingly, it is expected that the present invention will be applied to the treatment of clinical liver cancer patients in the future. On. For example, the present invention can detect the expression amount of leukemia in the serum of liver cancer patients or liver cancer tissues; in a preferred embodiment, for patients with higher performance, direct treatment with MPA can be considered, thereby achieving enhanced MPA inhibition. The effect of liver tumors; in another preferred embodiment, the present invention can also combine leptin and MPA to design a drug for treating liver cancer patients, thereby expanding the scope of use of the drug and enhancing the pharmacological action of MPA; In a preferred embodiment, the present invention may further be administered to MPA after increasing the amount of estrogen in a patient with a low expression of estrogen, such as 5-hydroxy-trypton (5-HTP). Therefore, the present invention can help liver cancer patients maintain a longer survival time. In addition, when the normal liver cell strain is treated under the same conditions in the present invention, it is also found that the estrogen and MPA do not affect the normal liver cell line, indicating that the drug or the course of treatment does not cause adverse side effects to the patient, The known chemotherapeutic and radiotherapy treatments for normal human injuries provide a new opportunity for liver cancer patients.

In summary, the present invention is a method for treating liver cancer by using a thin hormone, which can effectively improve various disadvantages, and includes a therapeutic drug, a course of treatment or a screening platform for administering a therapeutically effective amount to a liver cancer patient. Lean hormone or increase the physiological concentration of leptin in patients with liver cancer combined with medroxyprogesterone acetate and contact with liver cancer cells, and the interaction of the leptin with its progesterone receptor enhances and accelerates the MPA poisoning of liver cancer The cell has a specific pharmacological effect on the normal liver epithelial cell line and the cancer cell line, and the donor can have no adverse side effects on the normal liver cell, and can prolong the overall survival rate of the liver cancer patient, thereby further making the present invention The production can be more progressive, more practical, and more in line with the needs of the user. It has indeed met the requirements for the invention patent application, and loves to file a patent application according to law.

Claims

Claim

1 . A method for treating liver cancer by using a thin hormone, comprising a therapeutic drug, a course of treatment or a screening platform for administering a medicinal effective amount to a liver cancer patient, characterized in that the combination of the lean hormone or the physiological concentration of the lean hormone of the liver cancer patient is combined. After medroxyprogesterone acetate (MPA) is contacted with liver cancer cells, the interaction of the leptin with its progesterone receptor through the receptor enhances and accelerates the MPA to kill liver cancer cells, thereby allowing for normal liver epithelial cell lines. The cancer cell strain has a specific pharmacological effect for the simultaneous side effects of non-toxic killing of normal liver cells, and can prolong the overall survival rate of liver cancer patients;

Among them, the expression level of the estrogen further as a predictor of the efficacy of the treatment of MPA in patients with liver cancer (Predictive Factors) and its survival factor (Prognostic Factors).

The use of leukemia for treating liver cancer according to claim 1, wherein the MPA is a cytotoxic agent.

The use of the leukemia for treating liver cancer according to claim 1, characterized in that the drug causes a reduction in the size of the liver cancer cells or the quality of the liver cancer cells.

The use of leukemia for treating liver cancer according to claim 1, which is useful as statistical data on liver cancer cell lines and clinical liver cancer patients.

The use of the leukemia for treating liver cancer according to claim 1, wherein the liver cancer patient has a higher amount of expression of leukemia in the serum or liver cancer tissue, and the patient is directly treated with MPA to treat the liver cancer patient, To achieve the pharmacological effects of strengthening MPA.

The use of the method for treating hepatocellular carcinoma with a leptin according to claim 1, wherein the liver cancer patient has a higher expression of estrogen in the serum or the liver cancer tissue, and the leukemia and the MPA are combined into one drug treatment. For patients with liver cancer, in order to achieve the pharmacological effects of strengthening MPA.

The use of the method for treating liver cancer with a leptin according to claim 1, wherein the liver cancer patient has a lower expression level of lean hormone in the serum or liver cancer tissue, and is 5-hydroxytryptophan (5). -hydroxy-trypton, 5-HTP) After increasing the amount of estrogen, it is given MPA treatment to enhance the pharmacological effects of MPA.

The use of the leukemia for treating liver cancer according to claim 1, characterized in that the higher the expression of the estrogen, the more effective the effect of enhancing MPA on hepatoma cells is.