Application of AKT inhibitor or STAT3 inhibitor in overcoming drug resistance of bladder cancer to MEK inhibitor

Abstract

The invention discloses application of an AKT inhibitor or an STAT3 inhibitor in preparation of a medicine for overcoming drug resistance of bladder cancer to an MEK inhibitor, and belongs to the field of biological medicine. Detecting the cell activity through an MTT method; a Western blot experiment is adopted to detect a key target protein or gene expression level, a bladder cancer drug-resistant cell ERK abnormal activation + STAT3 / Akt compensation activation mechanism is defined, p-ERK, p-STAT3 and p-Akt are locked as targets, the drug resistance of bladder cancer to an MEK inhibitor is overcome through an AKT inhibitor or an STAT3 inhibitor, the scheme can accurately inhibit abnormal pathways, reverse drug resistance and improve treatment sensitivity, and the method is safe and suitable for clinical application. A new direction is provided for patients with drug-resistant bladder cancer, and the clinical bottleneck of the MEK inhibitor is helped to be broken through.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the use of AKT inhibitors or STAT3 inhibitors to overcome the drug resistance of bladder cancer to MEK inhibitors. BACKGROUND

[0002] Bladder cancer is a common malignant tumor of the urinary system worldwide. The treatment options for patients with metastatic or advanced bladder cancer are limited, and the prognosis is poor. MEK inhibitors, as important drugs targeting the MAPK signaling pathway, have shown certain efficacy in the treatment of bladder cancer. By inhibiting MEK kinase activity, it blocks the activation of the ERK signaling pathway, thereby inhibiting tumor cell proliferation.

[0003] However, it is found in clinical application that some bladder cancer patients will gradually develop drug resistance after receiving MEK inhibitor treatment for a period of time, resulting in decreased or even ineffective drug efficacy, which becomes a key bottleneck restricting the treatment of bladder cancer with MEK inhibitors. Existing studies have shown that after MEK inhibitor treatment, the ERK signaling pathway in bladder cancer tumor cells is inhibited, while the STAT3 and Akt signaling pathways are activated compensatorily, which together maintain the survival and proliferation of tumor cells and weaken the therapeutic effect of MEK inhibitors. At present, there are few solutions to the resistance of MEK inhibitors to bladder cancer, and the effect is limited. Therefore, it is urgent to develop a method that can effectively overcome the drug resistance to improve the treatment and prognosis of bladder cancer patients. SUMMARY

[0004] The present application provides the use of AKT inhibitors or STAT3 inhibitors in combination with MEK inhibitors in the preparation of a drug for treating bladder cancer. The present application first determines that the STAT3 and Akt signaling pathways in bladder cancer drug-resistant cells will be activated compensatorily under the action of MEK inhibitors. These abnormally activated signaling pathways together cause drug resistance. Therefore, the related signaling pathway proteins are used as regulatory targets. Then, based on the characteristics of the targets, the abnormal activation of the signaling pathways is inhibited to weaken their maintenance effect on tumor cell survival and proliferation, thereby reversing the drug resistance of bladder cancer drug-resistant cells to MEK inhibitors, significantly improving the treatment sensitivity, and improving the treatment effect.

[0005] The components (or effective components) of the drug of the present application are AKT inhibitors and MEK inhibitors, or STAT3 inhibitors and MEK inhibitors. One or more pharmaceutically acceptable adjuvants can also be added, or other active ingredients can be compounded to play an inhibitory effect. The preparation can be prepared into tablets, pills, capsules, granules, oral liquids, and injection solutions, etc. in addition to tablets, and other pharmaceutically acceptable forms.

[0006] Compared with the prior art, the present application has the following advantages: The application detects cell activity by MTT method; adopts Western blot experiment to detect key target protein or gene expression level; the application locks the key molecular mechanism of "ERK abnormal activation + STAT3 / Akt compensatory activation" in bladder cancer cells, takes p-ERK, p-STAT3 and p-Akt as core regulation targets, directly intervenes in the drug resistance source, and avoids the low efficiency problem caused by the "general pathway inhibition" of the prior art.

[0007] The application provides a new direction for bladder cancer patients (especially metastatic or advanced patients) with MEK inhibitor resistance to improve the treatment sensitivity of drug-resistant cells by regulating the expression of key signal pathway proteins, which is expected to improve the poor prognosis of patients under limited treatment options; from the perspective of drug application, it helps to break through the clinical application bottleneck of MEK inhibitors in bladder cancer treatment due to drug resistance, and improves its treatment effectiveness; from the mechanism research, further research is carried out around the abnormal activation of ERK, STAT3, Akt and other signal pathways in drug-resistant cells, which provides support for deepening the understanding of bladder cancer drug resistance mechanism, solving the problem of few and limited effect of current related solutions. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 is the influence result of NB on the protein expression of MAPK pathway in T24 cells, the upper graph is a Western blot graph, and the lower graph is an expression amount statistical graph; Figure 2 is the influence result of NB on the protein expression of MAPK pathway in T24CDDP cells, the upper graph is a Western blot graph, and the lower graph is an expression amount statistical graph; Figure 3 is the influence result of NB on the protein expression of MAPK pathway in UM-UC-3 cells, the upper graph is a Western blot graph, and the lower graph is an expression amount statistical graph; Figure 4 is the influence result of NB on the protein expression of MAPK pathway in TCCSUP cells, the upper graph is a Western blot graph, and the lower graph is an expression amount statistical graph; Figure 5 is the influence result of CB on the protein expression of MAPK pathway in T24CDDP cells, the upper graph is a Western blot graph, and the lower graph is an expression amount statistical graph; Figure 6 is the influence result of CB on the protein expression of MAPK pathway in UM-UC-3 cells, the upper graph is a Western blot graph, and the lower graph is an expression amount statistical graph; Figure 7 is the influence result of TB on the protein expression of MAPK pathway in T24CDDP cells, the upper graph is a Western blot graph, and the lower graph is an expression amount statistical graph; Figure 8Figure 6 is the result of the effect of TB on the protein expression of the MAPK pathway in UM-UC-3 cells, the upper panel is a Western blotting diagram, and the lower panel is a statistical diagram of the expression amount; Figure 9 Figure 7 is the result of the effect of NB and Stattic on the protein expression of the STST3 pathway in T24CDDP cells, the upper panel is a Western blotting diagram, and the lower panel is a statistical diagram of the expression amount; Figure 10 Figure 8 is the result of the effect of NB and Stattic on the protein expression of the STST3 pathway in UM-UC-3 cells, the upper panel is a Western blotting diagram, and the lower panel is a statistical diagram of the expression amount; Figure 11 Figure 9 is the result of the effect of CB and Stattic on the protein expression of the STST3 pathway in T24CDDP cells, the upper panel is a Western blotting diagram, and the lower panel is a statistical diagram of the expression amount; Figure 12 Figure 10 is the result of the effect of CB and Stattic on the protein expression of the STST3 pathway in UM-UC-3 cells, the upper panel is a Western blotting diagram, and the lower panel is a statistical diagram of the expression amount; Figure 13 Figure 11 is the result of the effect of TB and Stattic on the protein expression of the STST3 pathway in T24CDDP cells, the upper panel is a Western blotting diagram, and the lower panel is a statistical diagram of the expression amount; Figure 14 Figure 12 is the result of the effect of TB and Stattic on the protein expression of the STST3 pathway in UM-UC-3 cells, the upper panel is a Western blotting diagram, and the lower panel is a statistical diagram of the expression amount; Figure 15 Figure 13 is the result of the effect of NB and MK2206 on the protein expression of the AKT pathway in T24CDDP cells, the upper panel is a Western blotting diagram, and the lower panel is a statistical diagram of the expression amount; Figure 16 Figure 14 is the result of the effect of NB and MK2206 on the protein expression of the AKT pathway in UM-UC-3 cells, the upper panel is a Western blotting diagram, and the lower panel is a statistical diagram of the expression amount; Figure 17 Figure 15 is the result of the effect of CB and MK2206 on the protein expression of the AKT pathway in T24CDDP cells, the upper panel is a Western blotting diagram, and the lower panel is a statistical diagram of the expression amount; Figure 18 Figure 16 is the result of the effect of CB and MK2206 on the protein expression of the AKT pathway in UM-UC-3 cells, the upper panel is a Western blotting diagram, and the lower panel is a statistical diagram of the expression amount; Figure 19 Figure 17 is the result of the effect of TB and MK2206 on the protein expression of the AKT pathway in T24CDDP cells, the upper panel is a Western blotting diagram, and the lower panel is a statistical diagram of the expression amount; Figure 20Figure 6 is the result of the effect of TB, MK2206 on the protein expression of AKT pathway in UM-UC-3 cells, the upper panel is a Western blotting diagram, and the lower panel is a statistical diagram of the expression amount; Figure 21 Figure 7 is the result of the effect of NB, Stattic or MK2206 on the cell death of T24CDDP, UM-UC-3, the upper panel is for T24CDDP cells, and the lower panel is for UM-UC-3 cells; Figure 22 Figure 8 is the result of the effect of CB, Stattic or MK2206 on the cell death of T24CDDP, UM-UC-3, the left panel is for T24CDDP cells, and the right panel is for UM-UC-3 cells; Figure 23 Figure 9 is the result of the effect of TB, Stattic or MK2206 on the cell death of T24CDDP, UM-UC-3, the left panel is for T24CDDP cells, and the right panel is for UM-UC-3 cells. DETAILED DESCRIPTION

[0009] 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 conventional methods unless otherwise specified. In the following examples, the test materials are commercially available unless otherwise specified; after reading the description of the present application, various modifications, modifications and modifications of the equivalents thereof by the skilled person in the art are within the scope defined by the claims of the present application.

[0010] In the examples, Nilotinib (NB), Cobimetinib (CB), Trametinib (TB), Cisplatin (CDDP) MK2206, Stattic are purchased from MedChemExpress (MCE) Biotech Co., Ltd. in the United States; MTT reagent (Solarbio, Beijing, China); RPMI 1640 medium (Gibco, USA), fetal bovine serum (Guangzhou Sike Biotechnology Co., Ltd.), trypsin (Gibco, USA), Opti-MEM medium (Gibco, USA).

[0011] Cell lines: human bladder cancer cell T24, UM-UC-3, TCCSUP, purchased from Guangzhou Sike Biotechnology Co., Ltd.

[0012] Example 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 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 culturing until the cells could stably grow and continuing to culture and passage for 5 times, the CDDP concentration was gradually increased until the cells could stably grow and smoothly passaged for 20 times in the culture medium with a CDDP concentration of 10 μmol / L. Thus, a cisplatin-resistant human bladder cancer cell model was obtained, which was named cisplatin-resistant human bladder cancer cells (T24CDDP cells).

[0013] Example 2: Detection of the regulation of key signaling proteins in T24, T24CDDP, UM-UC3, and TCCSUP cells by the MEK inhibitor Nintedanib (NB) 1. Logarithmic growth phase T24, T24CDDP, UM-UC3, and TCCSUP cells were inoculated in 6-well plates, and placed in a cell incubator for culture at 37°C and 5% CO2 for 24 hours. After the cells were completely adherent, the cells were treated with 1.25 μmol / L, 2.5 μmol / L, 5 μmol / L, 10 μmol / L, and 20 μmol / L of NB for 24 hours, respectively. Then, the total protein of the cells was extracted, and Western blotting was performed to detect the expression levels of p-ERK, p-STAT3, and p-Akt proteins. A control group without the addition of NB was also set. The results are shown in Figures 1-4. As can be seen from the figures, compared with the control group, NB down-regulated the expression of p-ERK in T24 cells, T24CDDP cells, UM-UC3 cells, and TCCSUP cells, while up-regulated the levels of p-STAT3 and p-Akt.

[0014] The results showed that NB inhibited the activity of the Akt pathway (manifested as a decrease in the phosphorylation level of p-ERK, accompanied by an increase in the expression of p-STAT3 and p-Akt).

[0015] 2. Detection of the regulation of key signaling proteins in T24CDDP and UM-UC3 cells by the MEK inhibitor Cobimetinib (CB) Logarithmic growth phase T24CDDP and UM-UC3 cells were inoculated in 6-well plates, and placed in a cell incubator for culture at 37°C and 5% CO2 for 24 hours. After the cells were completely adherent, the cells were treated with 1.25 μmol / L, 2.5 μmol / L, 5 μmol / L, 10 μmol / L, and 20 μmol / L of CB for 24 hours, respectively. Then, the total protein of the cells was extracted, and Western blotting was performed to detect the expression levels of p-ERK, p-STAT3, and p-Akt proteins. A control group without the addition of CB was also set. The results are shown in Figures 5-6. As can be seen from the figures, compared with the control group, CB down-regulates the expression of p-ERK in T24CDDP cells and UM-UC3 cells, while feedback up-regulating the levels of p-STAT3 and p-Akt; The results show that CB inhibits the activity of the Akt pathway (manifested as a decrease in the phosphorylation level of p-ERK, accompanied by an increase in the expression of p-STAT3 and p-Akt), 3. Detection of the regulation of key signaling proteins in T24CDDP and UM-UC3 cells by the MEK inhibitor trametinib (TB) The method is the same as step 2, except that the cells are treated with TB; the results are shown in Figures 7-8 As can be seen from the figures, compared with the control group, TB down-regulates the expression of p-ERK in T24CDDP cells and UM-UC3 cells, while feedback up-regulating the levels of p-STAT3 and p-Akt.

[0016] The results show that TB inhibits the activity of the Akt pathway (manifested as a decrease in the phosphorylation level of p-ERK, accompanied by an increase in the expression of p-STAT3 and p-Akt).

[0017] Example 3: STAT3 inhibitor Stattic attenuates the feedback activation of the STAT3 pathway in T24CDDP and UM-UC3 cells by the MEK inhibitor 1. Stattic attenuates the feedback activation of the STAT3 pathway in T24CDDP and UM-UC3 cells by the MEK inhibitor Logarithmic growth period bladder cancer cells T24CDDP and UM-UC-3 were evenly inoculated in 6-well plates and placed in a cell culture incubator at 37°C, 5% CO2 for 24 hours. After the cells were completely adherent, total protein was extracted from the cells treated with 20 μmol / L NB, 20 μmol / L Stattic, and NB+Stattic for 24 hours, and Western blotting was performed to detect the expression level of p-STAT3 protein, with a blank (no addition of NB or Stattic) set as a control. The results are shown in Figures 9-10. As can be seen from the figures, compared with the control group, Stattic down-regulates the increase in the expression of p-STAT3 in T24CDDP cells and UM-UC-3 cells caused by NB.

[0018] The results show that the STAT3 inhibitor Stattic can effectively overcome the drug resistance of bladder cancer cells to NB (the MEK inhibitor) (manifested as a decrease in p-STAT3).

[0019] 2. Stattic attenuates the feedback activation of STAT3 pathway by cobimetinib (CB) in T24CDDP, UM-UC3 cells Method: Step 1, except for adding 20 μmol / L CB and / or 20 μmol / L Stattic; Results: See Figs. 11-12, which show that, compared with the control group, Stattic down-regulates the expression of p-STAT3 in T24CDDP cells and UM-UC-3 cells caused by CB.

[0020] The results show that the STAT3 inhibitor Stattic can effectively overcome the drug resistance of bladder cancer cells to CB (MEK inhibitor) (manifested as a decrease in p-STAT3).

[0021] 3. Stattic attenuates the feedback activation of STAT3 pathway by cobimetinib (CB) in T24CDDP, UM-UC3 cells Method: Step 1, except for adding 20 μmol / L CB and / or 20 μmol / L Stattic; Results: See Figs. 11-12, which show that, compared with the control group, Stattic down-regulates the expression of p-STAT3 in T24CDDP cells and UM-UC-3 cells caused by CB. Figure 1

[0022] The results show that the STAT3 inhibitor Stattic can effectively overcome the drug resistance of bladder cancer cells to CB (MEK inhibitor) (manifested as a decrease in p-STAT3).

[0023] Example 4: AKT inhibitor MK2206 attenuates the feedback activation of AKT pathway by MEK inhibitors in T24CDDP, UM-UC3 cells 1. MK2206 attenuates the feedback activation of AKT pathway by cobimetinib (CB) in T24CDDP, UM-UC-3 cells Logarithmic growth period bladder cancer cells T24CDDP, UM-UC-3 were inoculated in 6-well plates and placed in a cell culture incubator at 37°C, 5% CO2 for 24 hours. After the cells were completely attached, total cell protein was extracted after treatment with 20 μmol / L NB and / or 20 μmol / L MK2206 for 24 hours, and Western blotting was performed to detect the expression level of p-STAT3 protein. A blank (without adding NB, MK2206) was also set up. Results: See Figs. 11-12, which show that, compared with the control group, Stattic down-regulates the expression of p-STAT3 in T24CDDP cells and UM-UC-3 cells caused by CB. Figures 15-16 The results show that the STAT3 inhibitor Stattic can effectively overcome the drug resistance of bladder cancer cells to CB (MEK inhibitor) (manifested as a decrease in p-STAT3).​

[0024] Results show that AKT inhibitor - MK2206 can effectively overcome the drug resistance of bladder cancer cells to CB (MEK inhibitor) (manifested as the decrease of p-AKT).

[0025] 2. MK2206 attenuates the feedback activation of AKT pathway in T24CDDP, UM-UC3 cells by cobimetinib (CB) Method: same as step 1, except for adding 20 μmol / L CB and / or 20 μmol / L MK2206; results are shown in Figures 17-18. As can be seen from the figures, compared with the control group, MK2206 down-regulates the increase of p-AKT expression in T24CDDP cells, UM-UC-3 cells caused by CB.

[0026] Results show that AKT inhibitor - MK2206 can effectively overcome the drug resistance of bladder cancer cells to CB (MEK inhibitor) (manifested as the decrease of p-AKT).

[0027] 3. MK2206 attenuates the feedback activation of AKT pathway in T24CDDP, UM-UC3 cells by cobimetinib (CB) Method: same as step 1, except for adding 20 μmol / L CB and / or 20 μmol / L MK2206; results are shown in Figures 17-18. As can be seen from the figures, compared with the control group, MK2206 down-regulates the increase of p-AKT expression in T24CDDP cells, UM-UC-3 cells caused by CB.

[0028] Results show that AKT inhibitor - MK2206 can effectively overcome the drug resistance of bladder cancer cells to CB (MEK inhibitor) (manifested as the decrease of p-AKT).

[0029] Example 11: MK2206 or Stattic overcomes the drug resistance of MEK inhibitors against bladder cancer 1. MK2206, Stattic overcomes the drug resistance of nintedanib (NB) against bladder cancer The bladder cancer cell lines T24CDDP and UM-UC-3 cells were uniformly spread in 96-well plates (4000 cells / well, 100 μL per well), and were placed in a cell culture box and cultured at 37°C, 5% CO2 for 24 hours. After the cells were completely adhered, 10 μmol / L of NB, MK2206 (2, 4 μmol / L), Stattic (2, 4 μmol / L), NB+MK2206, and NB+Stattic were used for treatment for 48 hours. Then, 30 μL of MTT solution (5 mg / mL) was added to the 96-well plates, and was placed in a culture box and reacted 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 was shaken on a shaker for 10 minutes (20 revolutions per minute). The absorbance was detected at 570 nm, and the cell survival rate was calculated. The results are shown in Figure 21 As can be seen from the figure, MK2206 and Stattic can significantly inhibit the proliferation of bladder cancer cells when used in combination with NB, that is, MK2206 and Stattic can effectively overcome the drug resistance of nintedanib (NB) against bladder cancer.

[0030] 2. MK2206 or Stattic overcomes the drug resistance of cabozantinib (CB) against bladder cancer The method is the same as step 1, except that CB is studied. The results are shown in Figure 22 As can be seen from the figure, MK2206 and Stattic can significantly inhibit the proliferation of bladder cancer cells when used in combination with CB, that is, MK2206 and Stattic can effectively overcome the drug resistance of cabozantinib (CB) against bladder cancer.

[0031] 3. MK2206 and Stattic overcome the drug resistance of trametinib (TB) against bladder cancer The method is the same as step 1, except that TB is studied. The results are shown in Figure 23 As can be seen from the figure, MK2206 and Stattic can significantly inhibit the proliferation of bladder cancer cells when used in combination with TB, that is, MK2206 and Stattic can effectively overcome the drug resistance of trametinib (TB) against bladder cancer.

[0032] 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 substitutions within the technical scope 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. Use of an AKT inhibitor or a STAT3 inhibitor in the manufacture of a medicament for overcoming resistance of bladder cancer to a MEK inhibitor, characterized in that: AKT inhibitors or STAT3 inhibitors, MEK inhibitors.

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