Application of tripterine in preparation of medicine for enhancing anti-bladder cancer activity of MEK inhibitor

Abstract

The invention discloses an application of tripterine in preparation of a medicine for enhancing bladder cancer resisting activity of an MEK inhibitor, the Western blot experiment proves that the tripterine can effectively reduce p-STAT3 and p-Akt compensatory expression increase caused by the MEK inhibitor for the first time, so that STAT3 and Akt signal channel compensatory activation caused by the MEK inhibitor is reversed; in-vivo animal experiments show that the combination of the tripterine and the MEK inhibitor has a remarkable synergistic anti-tumor effect, and the anti-tumor effect of the tripterine is superior to that of any single drug; the pharmaceutical composition provided by the invention can effectively block the escape path of tumor cells by simultaneously inhibiting the three key signal paths of MEK / ERK, STAT3 and PI3K / Akt, overcomes or delays the drug resistance of the MEK inhibitor mediated by compensatory pathway activation, and provides a new drug combination strategy for the treatment of malignant tumors.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to application of celastin in preparation of a medicine for enhancing the activity of a MEK inhibitor against bladder cancer. BACKGROUND

[0002] Cancer, also known as malignant tumor, is caused by cell malignant proliferation, has invasiveness and metastasis, and has become one of important diseases threatening human life and health. Bladder cancer is listed as one of the top ten common cancers in the world, and its high recurrence rate and poor prognosis are the causes of difficulties in clinical treatment. Clinically, the combination of surgery, radiotherapy and chemotherapy is mainly used, which is expensive and has great side effects on the human body. In the occurrence and development of tumors, a variety of signal pathways play a key role. Among them, STAT3 signal pathway and PI3K / Akt signal pathway are two key oncogenic pathways, which drive tumor progression by promoting cell proliferation, inhibiting apoptosis, inducing angiogenesis and mediating immune escape. Therefore, simultaneous inhibition of the activation of STAT3 and Akt phosphorylation has become a promising anticancer strategy.

[0003] The mitogen-activated protein kinase (MAPK) signal pathway, i.e. Ras / Raf / MEK / ERK signal pathway, is one of the most characteristic kinase cascades in cancer cell biology. MEK inhibitors are a class of targeted therapy drugs for MAPK signal pathway, which have been approved for the treatment of malignant tumors such as BRAF mutant melanoma. They mainly inhibit the activity of MEK1 / 2, block the phosphorylation of downstream ERK1 / 2, and interrupt signal transduction to exert antitumor activity. However, MEK inhibitors have problems such as poor effect, easy drug resistance and large side effects in clinical monotherapy. Among them, the resistance mechanism usually involves feedback activation of compensatory signal pathways, i.e. when the MEK / ERK pathway is inhibited, tumor cells will activate STAT3 or PI3K / Akt pathway to maintain their survival and proliferation, which greatly limits the clinical application of MEK inhibitors.

[0004] Celastin is extracted from the plant Tripterygium wilfordii Hook.f. of Celastraceae Tripterygium wilfordiiA monomer component extracted from the dried root bark of Hook. f. Modern medical research shows that it has biological activities in anti-inflammatory, immunosuppression and anti-tumor, etc. Celastro red can inhibit tumor through inducing apoptosis, autophagy, ferroptosis, regulating cell cycle and inhibiting angiogenesis, etc. One of its mechanisms has been confirmed, that is, inducing tumor cell apoptosis by inhibiting the phosphorylation of STAT3 and Akt (p-STAT3, p-Akt). However, there are certain limitations in the application of celastro red alone, such as poor water solubility, low bioavailability, etc. These factors limit its wide application in clinical. At present, there is no related report on the combination of celastro red and MEK inhibitor for treating bladder cancer. SUMMARY

[0005] The present application provides a new use of celastro red (CST), that is, the application of CST in the preparation of a drug for enhancing the activity of MEK inhibitor against bladder cancer, wherein CST reduces the feedback activation of MEK inhibitor on STAT3 pathway and PI3K / Akt pathway by inhibiting STAT3 pathway and PI3K / Akt pathway, so as to enhance the activity of MEK inhibitor against bladder cancer.

[0006] The chemical structural formula of the celastro red (CST) is as follows:

[0007] The MEK inhibitor in the present application is Nedometinib (NB, Nilotinib), Trametinib (TB, Trametinib) and Cobimetinib (CB, Cobimetinib).

[0008] The present application proves by Western blot detection that CST can dose-dependently inhibit the expression of key proteins p-STAT3 and p-Akt in STAT3 pathway and PI3K / Akt pathway, and then evaluates the synergistic effect of the two drugs through in vitro and in vivo experiments. The in vitro experiment proves that CST can reduce the increase of the expression level of p-STAT3 and p-Akt proteins caused by MEK inhibitor, and CST can overcome the compensatory activation of STAT3 and Akt signaling pathway caused by MEK inhibitor. The in vivo experiment proves that CST and MEK inhibitor produce strong synergistic effect.

[0009] The components (or effective components) of the drug of the present application are celastro red and MEK inhibitor, and one or more pharmaceutically acceptable adjuvants can be added, or the drug can be compounded with other active ingredients to play an inhibitory effect. The preparation can be prepared into tablets, pills, capsules, granules, oral liquids and injection solutions and other forms acceptable in pharmacy.

[0010] Compared with the prior art, the present application has the following advantages: The present application first discovers that CST can effectively reverse the compensatory activation of STAT3 and Akt signaling pathways caused by the use of MEK inhibitors alone. The synergistic effect is significant: the pharmaceutical composition of the present application shows stronger ability to inhibit tumor cell proliferation and induce tumor cell apoptosis than either single drug in in vitro cell experiments and in vivo animal models. The present application overcomes or delays the development of drug resistance: by simultaneously inhibiting the MEK / ERK, STAT3 and PI3K / Akt three key signaling pathways, the composition of the present application can maximally block the escape path of tumor cells, thereby effectively overcoming or delaying the drug resistance mediated by compensatory pathway activation, providing a new solution to overcome the clinical resistance of MEK inhibitors. The present application provides a new strategy for clinical treatment: the present application combines traditional Chinese medicine active ingredients with modern targeted drugs, fully utilizes the advantages of multi-target therapy, and provides a new combination drug regimen and drug selection for the treatment of malignant tumors that are not sensitive or have developed resistance to MEK inhibitors. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 Figure 1 is the experimental results of CST inhibiting STAT3 pathway and PI3K / Akt pathway in T24CDDP cells in a dose-dependent manner, the upper graph is a protein electrophoresis graph, and the lower graph is a statistical graph of expression amount; Figure 2 Figure 2 is the experimental results of CST inhibiting STAT3 pathway and PI3K / Akt pathway in UM-UC-3 cells in a dose-dependent manner, the upper graph is a protein electrophoresis graph, and the lower graph is a statistical graph of expression amount; Figure 3 Figure 3 is the effect of CST and NB on the protein expression of STAT3 pathway and PI3K / Akt pathway in T24CDDP cells, the upper graph is a protein electrophoresis graph, and the lower graph is a statistical graph of expression amount; Figure 4 Figure 4 is the effect of CST and NB on the protein expression of STAT3 pathway and PI3K / Akt pathway in UM-UC-3 cells, the upper graph is a protein electrophoresis graph, and the lower graph is a statistical graph of expression amount; Figure 5 Figure 5 is the effect of CST and NB on the death of T24CDDP cells; Figure 6 Figure 6 is the effect of CST and NB on the death of UM-UC-3 cells; Figure 7 Figure 7 is the inhibitory effect of CST, NB single drug and the combination of the two drugs on the volume and weight of UM-UC-3 transplanted tumors, the left graph is a schematic diagram of tumor size, the middle graph is a statistical graph of tumor weight, and the right graph is a statistical graph of tumor volume; Figure 8Figure 6 is the result of CST and CB affecting the protein expression of STAT3 pathway and PI3K / Akt pathway in T24CDDP cells, the upper graph is a protein electrophoresis graph, and the lower graph is an expression amount statistical graph; Figure 9 Figure 7 is the result of CST and CB affecting the protein expression of STAT3 pathway and PI3K / Akt pathway in UM-UC-3 cells, the upper graph is a protein electrophoresis graph, and the lower graph is an expression amount statistical graph; Figure 10 Figure 8 is the result of CST and TB affecting the protein expression of STAT3 pathway and PI3K / Akt pathway in T24CDDP cells, the upper graph is a protein electrophoresis graph, and the lower graph is an expression amount statistical graph; Figure 11 Figure 9 is the result of CST and TB affecting the protein expression of STAT3 pathway and PI3K / Akt pathway in UM-UC-3 cells, the upper graph is a protein electrophoresis graph, and the lower graph is an expression amount statistical graph; Figure 12 Figure 10 is the result of CST and CB affecting the cell death of T24CDDP cells; Figure 13 Figure 11 is the result of CST and CB affecting the cell death of UM-UC-3 cells; Figure 14 Figure 12 is the result of CST and TB affecting the cell death of T24CDDP cells; Figure 15 Figure 13 is the result of CST and TB affecting the cell death of UM-UC-3 cells. DETAILED DESCRIPTION

[0012] The technical solutions of the present application will be further described below in combination with the drawings and specific embodiments, but these embodiments shall not be used to explain the limitation of the present application. In the following examples, the experimental methods are all conventional methods unless otherwise specified. In the following examples, the test materials are all commercially available unless otherwise specified; after reading the description of the present application, various modifications, changes and modifications of the equivalents thereof by the skilled person in the art all belong to the range defined by the claims of the present application.

[0013] Reagents: MTT reagent (Solarbio, Beijing, China), RPMI 1640 medium (Guangzhou Sike Biotechnology Co., Ltd.), MEM medium (Guangzhou Sike Biotechnology Co., Ltd.), fetal bovine serum (Guangzhou Sike Biotechnology Co., Ltd.), trypsin (Gibco, USA).

[0014] Cell lines: bladder cancer cells T24 and UM-UC-3, purchased from Guangzhou Sike Biotechnology Co., Ltd.

[0015] Example 1: Detection of the regulation of key signal proteins in T24CDDP, UM-UC-3 cells by celastrol (CST) 1. Preparation of cisplatin (CDDP) -resistant strain of human bladder cancer cell T24 Human bladder cancer T24 cells were inoculated in T25 culture bottles containing 10% fetal bovine serum in high-sugar DMEM medium and placed in a cell culture incubator for culture at 37°C and 5% CO2. Cisplatin-resistant cells were induced by gradually increasing the drug dose, using 0.5 μmol / L of CDDP as the initial concentration. After the cells stably grew and were continuously cultured and passaged for 5 times, the CDDP concentration was gradually increased until the cells stably grew and were smoothly passaged in the culture medium containing 10 μmol / L of CDDP for 20 times, obtaining a cisplatin-resistant human bladder cancer cell model, named cisplatin-resistant human bladder cancer cell (T24CDDP cell).

[0016] 2. Logarithmic growth phase T24CDDP and UM-UC-3 cells were inoculated in 6-well plates and placed in a cell culture incubator for culture at 37°C and 5% CO2 for 24 hours. After the cells were completely adherent, the cells were treated with 1 μmol / L, 2 μmol / L and 4 μmol / L of CST for 12 hours, respectively. Then, the total protein of the cells was extracted for Western blotting to detect the expression levels of p-STAT3 and p-Akt proteins. A control group without CST was also set. The results are shown in Figure 1-2 From Figure 1 it can be seen that, compared with the control group, CST down-regulated the expression levels of p-STAT3 and p-Akt proteins in T24CDDP cells.

[0017] From Figure 2 it can be seen that, compared with the control group, CST down-regulated the expression levels of p-STAT3 and p-Akt proteins in UM-UC-3 cells.

[0018] The above results show that CST inhibits the activities of STAT3 and PI3K / Akt pathways in a dose-dependent manner (manifested as the decreased phosphorylation levels of p-STAT3 and p-Akt).

[0019] Example 2: Detection of the regulation of key signal proteins in T24CDDP, UM-UC-3 cells by CST and MEK inhibitor NB in combination T24CDDP, UM-UC-3 cells in logarithmic growth phase were inoculated in 6-well plates, and were placed in a cell incubator and cultured at 37°C, 5% CO2 for 24 hours. After the cells were completely adherent, they were treated with single drug NB (20 μmol / L), CST (4 μmol / L), NB (20 μmol / L) + CST (4 μmol / L) for 12 hours, and then total cell protein was extracted for Western blotting to detect the expression levels of p-STAT3 and p-Akt proteins. A blank control group without drug addition was also set up; Results are shown in Figure 3-4 As can be seen from the figure, NB (20 μmol / L) alone can promote the increase of p-STAT3 and p-Akt protein levels, and CST (4 μmol / L) alone can inhibit the increase of p-STAT3 and p-Akt protein levels. Compared with NB alone, the use of NB (20 μmol / L) + CST (4 μmol / L) can significantly reduce the levels of p-STAT3 and p-Akt proteins, indicating that the combined use of NB (20 μmol / L) + CST (4 μmol / L) can effectively reverse the compensatory activation of STAT3 and Akt signaling pathways caused by the use of NB alone, and further promote the apoptosis of human bladder cancer cells.

[0020] Example 3: Inhibition experiment of CST and NB combination on T24CDDP, UM-UC-3 cells T24CDDP, UM-UC-3 cells were evenly plated in 96-well plates (4000 cells / well, 100 μL / well), and were placed in a cell incubator and cultured at 37°C, 5% CO2 for 24 hours. After the cells were completely adherent, they were treated with different concentrations (0.1 μmol / L, 0.2 μmol / L, 0.4 μmol / L) of CST and different concentrations (3.125 μmol / L, 6.25 μmol / L, 12.5 μmol / L, 25 μmol / L, 50 μmol / L, 100 μmol / L) of MEK inhibitor (NB) for 48 hours. Then 30 μL of MTT solution (5 mg / mL) was added to the 96-well plate, which was placed in a cell incubator and cultured at 37°C, 5% CO2 for 5 hours. Then the liquid in the well plate was discarded, 100 μL of DMSO solution (purity 99.99%) was added, and the shaking bed was shaken for 10 minutes (180 rpm). The absorbance was detected at 570 nm, the cell survival rate was calculated, and the dose-effect curve was drawn; Results are shown in Figure 5-6As can be seen from the figure, under the action of NB combined with CST, the proliferation of T24CDDP and UM-UC-3 cells is inhibited, and CST inhibits the proliferation of cancer cells in a concentration-dependent manner. Compared with NB alone, CST enhances the cytotoxicity of NB on T24CDDP and UM-UC-3 cells.

[0021] Example 4: Evaluation of the in vivo anti-tumor effect of CST single-agent and combination with NB on UM-UC-3 xenograft BALB / c nu / nu male mice (13-17 g; 4-5 weeks) were purchased from the Animal Research and Resource Center of Yunnan University (Kunming, Yunnan).

[0022] Mice were raised at 25°C under a 12 / 12 hour light-dark cycle. After one week of adaptation to standard rodent chow, they were randomly divided into four groups: blank control, CST, NB, and CST+NB combination. UM-UC-3 cells were resuspended in PBS containing 50% Corning Matrigel (Corning, New York, USA) and then injected subcutaneously into the right inguinal region of each mouse (3x10 6 cells per mouse). The long diameter (L; mm) and short diameter (W; mm) of the xenograft were measured regularly, and the tumor volume (V; mm 3 ) was calculated according to the formula (V=0.5xLxW 2 ). When the volume of the xenograft reached 50-100mm 3 , the mice were injected intraperitoneally with drug-loaded (PBS containing 50% Corning Matrigel, blank control, once every 2 days), CST (1 mg / kg, once every 2 days), NB (1 mg / kg, once every 2 days), and CST+NB treatment. On day 14, the body weight of the mice was measured. The mice were sacrificed (carbon dioxide anesthesia), dissected, and the tumors were removed, measured for volume and weighed.

[0023] The results are shown in Figure 7 . Animal experiments showed that in the UM-UC-3 xenograft model, CST single-agent treatment and NB single-agent treatment showed significant tumor growth inhibition, and when combined with NB, a significant synergistic anti-tumor effect was produced, with significant reductions in tumor volume and weight.

[0024] Example 5: Detection of the regulation of key signaling proteins by CST and CB in combination with T24CDDP and UM-UC-3 cells The T24CDDP and UM-UC-3 cells in the logarithmic growth phase were uniformly inoculated in a 6-well plate and placed in a cell incubator for culture at 37°C and 5% CO2 for 24 hours. After the cells were completely adhered, they were treated with single drug CB (20 μmol / L), CST (4 μmol / L), CB (20 μmol / L) + CST (4 μmol / L) for 12 hours, and then total cell protein was extracted for Western blotting to detect the expression levels of p-STAT3 and p-Akt proteins. A blank control group without drug addition was also set up. The results are shown in Figure 8-9 As can be seen from the figure, CB (20 μmol / L) alone can promote the increase of p-STAT3 and p-Akt protein levels, and CST (4 μmol / L) alone can inhibit the increase of p-STAT3 and p-Akt protein levels. Compared with single drug administration, the combination of CB (20 μmol / L) + CST (4 μmol / L) can significantly reduce the levels of p-STAT3 and p-Akt proteins, indicating that the combination of CB (20 μmol / L) + CST (4 μmol / L) can effectively reverse the compensatory activation of STAT3 and Akt signaling pathways caused by CB alone, and further promote the apoptosis of human bladder cancer cells.

[0025] Example 6: Regulation of key signaling proteins by the combination of CST and TB in T24CDDP and UM-UC-3 cells The T24CDDP and UM-UC-3 cells in the logarithmic growth phase were uniformly inoculated in a 6-well plate and placed in a cell incubator for culture at 37°C and 5% CO2 for 24 hours. After the cells were completely adhered, they were treated with single drug CB (20 μmol / L), CST (4 μmol / L), CB (20 μmol / L) + CST (4 μmol / L) for 12 hours, and then total cell protein was extracted for Western blotting to detect the expression levels of p-STAT3 and p-Akt proteins. A blank control group without drug addition was also set up. The results are shown in Figure 10-11 As can be seen from the figure, TB (20 μmol / L) alone can promote the increase of p-STAT3 and p-Akt protein levels, and CST (4 μmol / L) alone can inhibit the increase of p-STAT3 and p-Akt protein levels. Compared with single drug administration, the combination of TB (20 μmol / L) + CST (4 μmol / L) can significantly reduce the levels of p-STAT3 and p-Akt proteins, indicating that the combination of TB (20 μmol / L) + CST (4 μmol / L) can effectively reverse the compensatory activation of STAT3 and Akt signaling pathways caused by TB alone, and further promote the apoptosis of human bladder cancer cells.

[0026] Example 7: Inhibition experiment of T24CDDP, UM-UC-3 cells by combination of CST and CB T24CDDP, UM-UC-3 cells were evenly spread in 96-well plates (4000 cells / well, 100 μL per well), and placed in a cell incubator for 24 hours at 37°C, 5% CO2. After the cells were completely adherent, different concentrations (0.1 μmol / L, 0.2 μmol / L, 0.4 μmol / L) of CST and different concentrations (3.125 μmol / L, 6.25 μmol / L, 12.5 μmol / L, 25 μmol / L, 50 μmol / L, 100 μmol / L) of MEK inhibitor (CB) were combined for 48 hours. Then 30 μL of MTT solution (5 mg / mL) was added to the 96-well plates, which were placed in the incubator at 37°C, 5% CO2 for 5 hours. Then the liquid in the well plates was discarded, 100 μL of DMSO solution (purity 99.99%) was added, and the shaking bed was shaken for 10 minutes (180 rpm). The absorbance was detected at 570 nm, the cell survival rate was calculated, and the dose-effect curve was drawn. The results are shown in Figure 12-13 As can be seen from the figure, under the action of CB combined with CST, the proliferation of T24CDDP, UM-UC-3 cells was inhibited, and CST inhibited the proliferation of cancer cells in a concentration-dependent manner. Compared with CB alone, CST enhanced the cytotoxicity of CB on T24CDDP, UM-UC-3 cells.

[0027] Example 8: Inhibition experiment of T24CDDP, UM-UC-3 cells by combination of CST and TB T24CDDP, UM-UC-3 cells were evenly spread in 96-well plates (4000 cells / well, 100 μL per well), and placed in a cell incubator for 24 hours at 37°C, 5% CO2. After the cells were completely adherent, different concentrations (0.1 μmol / L, 0.2 μmol / L, 0.4 μmol / L) of CST and different concentrations (3.125 μmol / L, 6.25 μmol / L, 12.5 μmol / L, 25 μmol / L, 50 μmol / L, 100 μmol / L) of MEK inhibitor (TB) were combined for 48 hours. Then 30 μL of MTT solution (5 mg / mL) was added to the 96-well plates, which were placed in the incubator at 37°C, 5% CO2 for 5 hours. Then the liquid in the well plates was discarded, 100 μL of DMSO solution (purity 99.99%) was added, and the shaking bed was shaken for 10 minutes (180 rpm). The absorbance was detected at 570 nm, the cell survival rate was calculated, and the dose-effect curve was drawn. The results are shown in Figure 14-15As can be seen from the figure, under the action of TB combined with CST, the proliferation of T24CDDP and UM-UC-3 cells is inhibited, and CST inhibits the proliferation of cancer cells in a concentration-dependent manner. Compared with TB alone, CST enhances the cytotoxicity of TB on T24CDDP and UM-UC-3 cells.

[0028] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. The use of celadixine in the preparation of a drug for enhancing the activity of a MEK inhibitor against bladder cancer.

2. Use according to claim 1, characterized in that: Celadixine is used in combination with a MEK inhibitor, and celadixine enhances the effect of the MEK inhibitor against bladder cancer by inhibiting the STAT3 and PI3K / Akt signaling pathways.

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