Application of MK2206 in preparation of medicine for treating colorectal cancer

Abstract

The invention relates to the field of tumor treatment, in particular to application of a small molecule compound in preparation of a medicine for treating colorectal cancer. The AKT pathway specific inhibitor MK2206 disclosed by the invention can inhibit the proliferation of colorectal cancer, induce cell cycle arrest, induce apoptosis and enhance the sensitivity of colorectal cancer cells to oxaliplatin and 5-fluorouracil. The MK2206 is used for inhibiting the activity of an AKT pathway, so that the effects of inhibiting proliferation of colorectal cancer cells, inducing apoptosis of the colorectal cancer cells and improving the sensitivity of the colorectal cancer cells to chemotherapeutic drugs oxaliplatin and 5-fluorouracil are achieved, and the MK2206 can be used for preparing drugs for treating the colorectal cancer.

Description

Technical Field

[0001] The present invention relates to the field of tumor treatment, and specifically refers to the application of the small molecule compound MK2206 in the preparation of drugs for treating colorectal cancer. Background Art

[0002] Colon cancer is the third most common digestive tract malignancy in the world, and ranks third and second in the incidence of colon cancer in male and female cancer patients worldwide, respectively. 20% of patients have already developed metastasis and lost the opportunity for surgery when they are first diagnosed with colon cancer, while other patients often have local or distant metastasis of the tumor even after surgical treatment. In addition, colon cancer is highly malignant and the tumor progresses rapidly, so its 5-year survival rate is only 64-67%. So far, there is a strong demand for new targeted drugs that can solve the problem of colorectal cancer in clinical practice, and traditional chemotherapy drugs oxaliplatin and 5-fluorouracil often encounter the problem of insufficient response in clinical treatment. Therefore, finding potential drugs for the treatment of colorectal cancer is of great clinical significance. Similarly, finding drugs that can enhance the sensitivity of colorectal cancer to chemotherapy drugs oxaliplatin and 5-fluorouracil is also of great clinical value.

[0003] At present, studies have confirmed that the AKT / GSK3β / β-catenin pathway can regulate the occurrence and development of tumors. Tang et al.'s study confirmed that regulating the AKT / GSK3β / β-catenin signaling pathway in glioma cells U87 and U251 can accelerate the cell cycle progression and cell proliferation of glioma cells in vitro and in vivo. In colon cancer, the activation of the AKT / GSK3β / β-catenin pathway causes β-catenin to translocate to the nucleus, directly bind to DNA and activate CCND1 expression. CCND1 is an important transcription factor that regulates cell proliferation and can inhibit DNA replication and synthesis. This may be a factor in the regulation of colon cancer progression by the AKT / GSK3β / β-catenin pathway, but there are currently few studies on the AKT / GSK3β / β-catenin pathway, and there are few studies on upstream genes that regulate this pathway. So far, it is still unclear whether the AKT pathway-specific inhibitor MK2206 can regulate the proliferation of colorectal cancer cells, affect the cell cycle of colorectal cancer cells, induce apoptosis of colorectal cancer cells, and affect the sensitivity of colorectal cancer cells to chemotherapeutic drugs. The present invention will evaluate the effects of AKT pathway specific inhibitor MK2206 on the proliferation of colorectal cancer cells, the cell cycle of colorectal cancer cells, the apoptosis of colorectal cancer cells, and the sensitivity of colorectal cancer cells to chemotherapeutic drugs through in vivo and in vitro models, confirm its effects, and propose technical solutions related to the invention. Summary of the invention

[0004] The purpose of the present invention is to reveal the effects of MK2206, a specific inhibitor of the AKT pathway, on the proliferation of colorectal cancer cells, the cell cycle of colorectal cancer cells, the apoptosis of colorectal cancer cells, and the sensitivity of colorectal cancer cells to chemotherapeutic drugs through in vitro studies. Therefore, the main contents of the present invention are summarized as follows: the method of inhibiting the AKT pathway using the small molecule compound MK2206 can inhibit the proliferation of colorectal cancer cells, induce cell cycle arrest of colorectal cancer cells, induce apoptosis of colorectal cancer cells, and increase the sensitivity of colorectal cancer cells to chemotherapeutic drugs, and the small molecule compound MK2206 can be used to prepare drugs for treating colorectal cancer.

[0005] The details of the present invention are as follows:

[0006] 1. We first revealed that MK2206 inhibits AKT pathway activation and counteracts MDFI-induced colon cancer cell proliferation;

[0007] 2. We first revealed that the number of colony cells in the MDFI overexpression group after MK2206 treatment was significantly reduced compared with the untreated group

[0008] 3. We revealed for the first time that after MK2206 treatment, the protein expression levels of p-AKT473, p-GSK3β, and β-catenin in colon cancer cells in the MDFI overexpression group were significantly decreased compared with those in the control group, while there was no significant difference in the total AKT expression level.

[0009] Based on the above results, we propose the following solution:

[0010] 1. Application of MK2206 in the preparation of drugs for the treatment of colorectal cancer.

[0011] 2. Application of MK2206 in the preparation of drugs for enhancing the sensitivity of colorectal cancer to chemotherapeutic drugs.

[0012] 3. The chemotherapy drugs are oxaliplatin and 5-fluorouracil.

[0013] 4. Use of a combination of MK2206 and oxaliplatin in the preparation of drugs for treating colorectal cancer.

[0014] 5. Use of a combination of MK2206 and 5-fluorouracil in the preparation of drugs for treating colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 KEGG gene enrichment analysis KEGG gene enrichment analysis. generatio: the total number of genes enriched in a certain pathway after analysis / the total number of genes known to exist that are closely related to the pathway. Count: the total number of genes enriched in a certain pathway after analysis. p.adjust: the significance of enrichment in a certain pathway.

[0016] Figure 2 Effect of knockdown of MDFI on AKT / GSK3β / β-catenin pathway in colon cancer cells (A) After knockdown of MDFI gene in HCT116 and SW620 for 48h, total cell protein was extracted, and Western blot was used to detect the expression levels of MDFI, ITGB4, LAMB3, total AKT (t-AKT), phosphorylated AKT (p-AKT473), phosphorylated GSK3β (p-GSK3β) and β-catenin. (B) Quantitative bar graph of relative expression levels of the tested proteins, with the relative expression levels of the tested proteins in shMDFI#1 and shMDFI#2 as reference. *P<0.05; **P<0.01; ***P<0.001.

[0017] Figure 3 Effect of overexpression of MDFI gene on AKT / GSK3β / β-catenin pathway in colon cancer cells (A) Total protein of HCT116 and SW620 cells was extracted after 48h of MDFI overexpression, and the expression levels of MDFI, ITGB4, LAMB3, total AKT (t-AKT), phosphorylated AKT (p-AKT473), phosphorylated GSK3β (p-GSK3β), and β-catenin were detected by Western blot. (B) Quantitative bar graph of relative expression levels of the tested proteins, with the relative expression levels of the tested proteins in lvMDFI as reference. *P<0.05; **P<0.01; ***P<0.001.

[0018] Figure 4 HURI verifies protein interactions between MDFI and ITGB4 and LAMB3. The HuRI database searches for MDFI and ITGB4 and LAMB3 gene information, and directly displays the interaction relationship diagram between the three. The endogenous protein-protein interactions between MDFI and ITGB4 and LAMB3 were determined by yeast two-hybrid assay (HuRI).

[0019] Figure 5 The CCLE database verifies the correlation between MDFI and ITGB4 and LAMB3 expression in colon cancer. In colon cancer cell lines, the Pearson correlation coefficient was calculated between MDFI and the expression of ITGB4 and LAMB3 in different colon cancer cells to obtain the correlation of gene expression. Each circle represents a colon cancer cell, ***P < 0.001.

[0020] Figure 6Interaction between MDFI, ITGB4 and LAMB3 in colon cancer Immunoprecipitation experiments verified the protein binding levels of MDFI and ITGB4, and MDFI and LAMB3 in HCT116 cells.

[0021] Figure 7 Phosphorylation level of AKT in colon cancer tumor tissues overexpressing MDFI

[0022] Figure 8 Effect of MK2206 on the viability of colon cancer cells HCT116 (A) and SW620 (B) cells stably overexpressing MDFI were treated with different concentrations of MK2206 for 24 hours and then CCK8 was detected. The relative cell survival level at each concentration was obtained by taking the OD value of the control group under 0 μM treatment as a reference. *P<0.05; **P<0.01; ***P<0.001.

[0023] Fig. 9 Effect of MK2206 on AKT / GSK3β / β-catenin pathway in colon cancer cells (A) HCT116 and SW620 cells stably overexpressing the MDFI gene were exposed to MK2206 (1μM) for 24h, proteins were extracted and Western blot was used to detect the expression levels of total AKT (t-AKT), phosphorylated AKT (p-AKT473), phosphorylated GSK3β (p-GSK3β), and β-catenin proteins. (B) Quantitative bar graph of the relative expression levels of the tested proteins, with the relative expression levels of the tested proteins in lvMDFI as the reference. **P<0.01; ***P<0.001.

[0024] Fig.10 Effect of MK2206 on colony formation ability of colon cancer cells HCT116 and SW620 cells stably overexpressing MDFI were obtained by lentiviral transfection for 72 hours, and clone formation experiments were performed. HCT116 (A) and SW620 (B) were uniformly plated at a density of 600 cells / well and 1000 cells / well, respectively, and treated with 1μM MK2206 for 48 hours before switching to ordinary culture medium. After the experiment was completed, the colonies were fixed, stained, photographed and counted. Each group of cell samples was inoculated into 3 replicate wells, and the experiment was repeated. *P<0.05; **P<0.01; ***P<0.001.

[0025] Fig.11Effect of MK2206 on colony formation ability of colon cancer cells after oxaliplatin treatment HCT116 and SW620 cells stably overexpressing MDFI were obtained by lentiviral transfection for 72 hours, and clone formation experiments were performed. HCT116 and SW620 were uniformly plated at a density of 600 cells / well and 1000 cells / well, respectively. The control virus cells were divided into blank treatment and oxaliplatin treatment. HCT116 overexpressing MDFI was divided into blank treatment, oxaliplatin treatment and MK2206 and oxaliplatin treatment. After 48 hours, ordinary culture medium was used. After the experiment, the colonies were fixed, stained, photographed and counted. Each group of cell samples was inoculated into 3 replicates, and the experiment was repeated. *P<0.05; **P<0.01; ***P<0.001.

[0026] Fig.12 Effect of MK2206 on colony formation ability of colon cancer cells after oxaliplatin treatment HCT116 and SW620 cells stably overexpressing MDFI were obtained by lentiviral transfection for 72 hours, and clone formation experiments were performed. HCT116 and SW620 were uniformly plated at a density of 600 cells / well and 1000 cells / well, respectively. The control virus cells were divided into blank treatment and treatment with pentafluorouracil. HCT116 overexpressing MDFI was divided into blank treatment, treatment with pentafluorouracil, and treatment with MK2206 and pentafluorouracil. After 48 hours, ordinary culture medium was used. After the experiment, the colonies were fixed, stained, photographed and counted. Each group of cell samples was inoculated into 3 replicate wells, and the experiment was repeated. *P<0.05; **P<0.01; ***P<0.001.

[0027] Fig.13 Effect of MK2206 on the growth of subcutaneous tumors of colon cancer HCT116 cells stably overexpressing MDFI and HCT116 cells transfected with control virus were obtained by lentiviral transfection for 72 hours, and subcutaneous tumor drug administration experiments were performed. HCT116 cells were 1×10 7 The cells were injected subcutaneously into nude mice. The control virus mice were divided into blank treatment, oxaliplatin treatment, and HCT116 overexpressing MDFI were divided into blank treatment, oxaliplatin treatment, and MK2206 and oxaliplatin treatment. The tumor size was calculated every 3 days until the subcutaneous tumor volume of mice was greater than 1000mm 3 The mice were euthanized, and the tumors were removed and photographed. *P<0.05; **P<0.01; ***P<0.001. DETAILED DESCRIPTION

[0028] Experimental materials and methods involved in the examples

[0029] 2.1. Experimental Materials and Reagents

[0030] 2.1.1. Cell source:

[0031] Human colon cancer cell lines HCT116 and SW620 were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0032] 2.1.2. Experimental animals:

[0033] The experimental mice were 5-6 weeks old male Balb / c-nu nude mice purchased from the Model Animal Institute of Nanjing University. The experimental mice were fed with experimental mouse maintenance feed and raised in the Animal Experiment Center of Nanjing University Medical School.

[0034] 2.1.3. Main reagents:

[0035]

[0036]

[0037]

[0038] 2.1.4. Main instruments and facilities:

[0039]

[0040]

[0041] 2.1.5. Experimental consumables

[0042] Cell culture dishes, cell cryopreservation tubes, pipettes, centrifuge tubes, multi-well cell culture plates and other consumables were purchased from CORNING, USA; micropipette tips were purchased from Axygen, USA.

[0043] 2.1.6. Main reagent formula:

[0044] 1) MK2206 solution:

[0045] Weigh 0.5 mg of MK2206 powder solution and fully dissolve it in 1.04 ml of DMSO to obtain 1 mM MK 2206 mother solution, and store it in a freezer at -20°C. In the experiment, use the gradient dilution method from high to low concentration to obtain 40 μM, 20 μM, 10 μM, 5 μM, and 1 μM MK2206 solutions in sequence, which are prepared and used immediately.

[0046] 2.2 Experimental methods:

[0047] For detailed experimental methods such as cell recovery, culture, passaging, freezing, cell counting, shRNA construction and transfection, lentivirus construction and transfection, CCK8 assay, clone formation assay, flow cytometry, qPCR, immunoblotting and tumor xenotransplantation, please refer to 2.2 in Chapter 2.

[0048] 2.2.1. Bioinformatics analysis:

[0049] With the help of gseKEGG function (cluster Profiler package), we performed KEGG pathway analysis on the differentially expressed biomarker genes annotated in Chapter 1

[16] The HuRI and CCLE sources are annotated in the results section, and the gene expression correlation analysis was performed using Pearson correlation coefficient analysis.

[0050] 2.2.2. Co-immunoprecipitation:

[0051] 2.2.2.1. Preparation of protein samples

[0052] See Chapter 2 Experimental Methods for details.

[0053] 2.2.2.2. Protein quantification by BCA method

[0054] See Chapter 2 Experimental Methods for details.

[0055] Co-immunoprecipitation

[0056] 1) Take about 1 mg of protein sample, add appropriate amount of antibody to the protein sample according to the instructions in the antibody manual, and set up input control group and IgG control group. Then place the protein in a shaker at 4°C overnight.

[0057] 2) Shake Protein A+G Agarose evenly, take 20 μL and add it to the protein sample, and shake the protein on a shaker in a 4°C refrigerator for 2 hours.

[0058] 3) Pre-cool the centrifuge and centrifuge the protein at 12000 rpm for 1 minute. Carefully remove the supernatant and do not aspirate the lower layer of Agarose.

[0059] 4) Wash the Agarose-containing precipitate with 1 mL of NP40 solution and repeat 3 times.

[0060] 5) Add 30 μL 2×SDS-PAGE protein loading buffer, shake evenly, and centrifuge at 12,000 rpm.

[0061] 6) Heat the protein sample in a metal bath heater at 95°C for 5 minutes. The sample can then be used for SDS-PAGE gel electrophoresis. For details, refer to the experimental methods in Chapter 2.

[0062] Example 1 AKT pathway levels in colon cancer cells

[0063] In order to explore the potential reasons why MDFI regulates the malignant phenotype of colon cancer cells and the effect of chemotherapy, this study performed differential gene analysis on colon cancer tumor samples and non-tumor samples of GSE144735, and performed pathway enrichment analysis (Kyoto Encyclopedia of Genes and Genomes, KEGG) on the obtained differential genes. The analysis results showed that the PI3K / AKT pathway in colon cancer tumor samples was significantly activated compared with non-tumor samples, verifying that the PI3K / AKT pathway is a key pathway that promotes the progression of colon cancer ( Figure 1 ).

[0064] Example 2 Relationship between MDFI and AKT / GSK3β / β-catenin pathway in colon cancer cells

[0065] According to the results of the second chapter, the expression of MDFI in colon cancer promotes the progression of malignant phenotypes of colon cancer. Studies have shown that the AKT / GSK3β / β-catenin pathway can promote the progression of colon cancer. [6] In addition, MDFI has a regulatory effect on β-catenin

[11] We can't help but speculate that MDFI may promote colon cancer proliferation and progression by regulating the AKT / GSK3β / β-catenin pathway. In addition, literature shows that ITGB4 and LAMB3 can promote colon cancer progression by regulating AKT phosphorylation. [14,15] This chapter knocked down the MDFI gene in HCT116 and SW620 cells and used Western blot experiments to detect changes in related proteins to explore the specific mechanism by which MDFI regulates the AKT / GSK3β / β-catenin pathway in colon cancer cells.

[0066] Western blot results showed that the expression level of t-AKT in the MDFI knockdown group of HCT116 and SW620 cells was not significantly different from that in the control group; the expression levels of ITGB4, LAMB3, p-AKT473 (ser473), p-GSK3β, and β-catenin proteins in the MDFI knockdown group were significantly downregulated compared with those in the control cells ( Figure 2 ). The results showed that knocking down the MDFI gene could inhibit the expression levels of ITGB4 and LAMB3 and the activation of the AKT / GSK3β / β-catenin pathway in colon cancer cells.

[0067] In order to further confirm the regulatory effect of MDFI on the AKT / GSK3β / β-catenin pathway, this study overexpressed MDFI in HCT116 and SW620 cells, and then used Western blot experiments to detect the expression of AKT / GSK3β / β-catenin pathway-related proteins and upstream proteins ITGB4 and LAMB3. Western blot results showed that the t-AKT expression level in the MDFI overexpression group of HCT116 and SW620 cells was not significantly different from that in the control cells; the expression levels of ITGB4, LAMB3, p-AKT473, p-GSK3β, and β-catenin proteins in the MDFI overexpression group were significantly increased compared with the control group ( Figure 3 ). The results showed that MDFI gene overexpression could promote the expression of ITGB4 and LAMB3 proteins and the activation of AKT / GSK3β / β-catenin pathway in colon cancer cells.

[0068] Example 3 Interaction between MDFI and ITGB4 and LAMB3 in colon cancer

[0069] The Human Reference Interactome (HuRI) contains about 53,000 different protein interactions involving more than 8,000 proteins. http: / / interactome-atlas.org ) provided data to obtain the protein interaction relationship between MDFI and ITGB4 and LAMB3 genes. HuRI showed that there was endogenous protein-protein interaction between MDFI and ITGB4 and LAMB3 as identified by yeast two-hybrid assay ( Figure 4 ).

[0070] To further verify the interaction between MDFI and ITGB4 and LAMB3, this experiment was based on the CCLE database ( https: / / sites.broadinstitute.org / ccle ) provided information to analyze the correlation between MDFI expression and ITGB4 and LAMB3 expression in colon cancer cell lines. The experimental results showed that in colon cancer cell lines, the gene expression of MDFI was highly significantly correlated with the gene expression of ITGB4 and LAMB3 (p<0.001, Figure 5 ). The co-expression relationship between MDFI and ITGB4 and LAMB3 was preliminarily verified.

[0071] The interaction between MDFI, ITGB4 and LAMB3 proteins shown by HuRI and the expression correlation between MDFI, ITGB4 and LAMB3 shown in CCLE suggest that there is a direct interaction between MDFI, ITGB4 and LAMB3. Therefore, this experiment verified the co-expression relationship between MDFI and ITGB4 and LAMB3 in colon cancer cells by immunoprecipitation experiment. The experimental results showed that in HCT116 cells, MDFI can bind to both ITGB4 and LAMB3 proteins, verifying their direct interaction ( Figure 6 ). These results comprehensively verified that MDFI and ITGB4, and MDFI and LAMB3 proteins have direct interactions in colon cancer, indicating that the three jointly regulate the downstream AKT / GSK3β / β-catenin pathway.

[0072] Example 4 Activation Levels of AKT in Colon Cancer Xenografts Overexpressing MDFI

[0073] Previous studies have shown that AKT can induce GSK3 kinase activity and thus regulate the intracellular expression level of β-catenin. Based on the literature and existing results, it is speculated that MDFI may regulate the AKT / GSK3β / β-catenin pathway by regulating AKT. In this part of the study, an HCT116 xenograft model with stable overexpression of the MDFI gene was established, and immunohistochemistry was used to detect changes in the expression level of phosphorylated AKT in tumor tissues. The results of immunohistochemistry showed that the expression level of phosphorylated p-AKT473 in nude mouse tumors in the MDFI overexpression group was significantly increased compared with that in the control group, and there was no significant difference in the expression level of t-AKT between the two groups. The experimental results showed that overexpression of MDFI can promote the activation of AKT in colon cancer tumors in nude mice ( Figure 7 ).

[0074] Example 5 Relationship between AKT phosphorylation in colon cancer cells and AKT / GSK3β / β-catenin pathway regulated by MDFI

[0075] In order to further verify the role of AKT activation in the process of MDFI regulating the AKT / GSK3β / β-catenin pathway, this study selected the AKT-specific inhibitor MK2206 for further exploration.

[0076] By treating colon cancer cells stably overexpressing MDFI with different concentrations of MK2206, the relative survival levels of HCT116 and SW620 cells under the action of 0, 1, 5, 10, 20, and 40 μM MK2206 were obtained. The results showed that when the concentration of MK2206 was 1 μM, its drug toxicity was weak, and HCT116 and SW620 cells could survive more stably ( Figure 8).

[0077] Subsequently, the experiment used MK2206 (1 μM) to treat HCT116 and SW620 cells for 24 hours and extract proteins, and the expression levels of AKT / GSK3β / β-catenin signaling pathway proteins were detected by Western blot. Under the action of MK2206, the protein expression levels of p-AKT473, p-GSK3β and β-catenin in the lvMDFI group and lvcontrol group of HCT116 cells were significantly decreased compared with the corresponding groups without MK2206, and there was no significant difference in the expression level of t-AKT between the groups with and without MK2206; consistent results were also obtained in SW620 cells ( Fig. 9 ). The experimental results showed that MDFI could induce the activation of AKT / GSK3β / β-catenin signaling pathway in colon cancer cells, while AKT specific inhibitor MK2206 could significantly inhibit this effect, indicating that MDFI regulates AKT / GSK3β / β-catenin signaling pathway by affecting AKT phosphorylation in colon cancer cells.

[0078] Example 6 MK2206 inhibits the proliferation of colon cancer cells induced by MDFI gene

[0079] Taking the cell survival rate of the HCT116 control group without MK2206 treatment as a reference, the cell survival rate of the MDFI overexpression group of HCT116 without MK2206 treatment was 114.8±1.6%, and the cell survival rate decreased significantly after treatment with 20μM and 40μM MK2206 for 24 hours, falling to 69.8±9.6% and 4.9±0.6%, respectively; similarly, taking the cell survival rate of the SW620 control group without MK2206 treatment as a reference, the cell survival rate of the MDFI overexpression group of SW620 without MK2206 treatment was as high as 136.3±1.3%, and the cell survival rate decreased significantly after treatment with 5, 10, 20 and 40μM MK2206 for 24 hours, falling to 119.8±4.2%, 81.4±4.9%, 14.3±0.3%, and 3.1±0.1%, respectively. The results showed that a certain concentration of MK2206 treatment could significantly inhibit the proliferation effect of MDFI on colon cancer cells ( Fig.10 ).

[0080] To further verify the inhibitory effect of MK2206 on the proliferation of MDFI-overexpressing colon cancer cells, this study exposed HCT116 and SW620 cells with stable MDFI overexpression to 1 μM MK2206 for 48 hours and performed a clone formation experiment. The results showed that the survival rate of the MDFI-overexpressing group of HCT116 was significantly higher than that of the control group cells, and the cell proliferation ability of the HCT116 control group and the MDFI-overexpressing group after MK2206 treatment was significantly decreased compared with the corresponding groups not treated with MK2206 ( Fig.10 ). The experimental results showed that MDFI can promote the proliferation of colon cancer cells, while MK2206 significantly inhibited the excessive proliferation of colon cancer cells induced by MDFI, indicating that MK2206 reversed the carcinogenic effect of MDFI on colon cancer.

[0081] Example 7 MK2206 inhibits MDFI gene-induced resistance of colon cancer cells to oxaliplatin

[0082] In this study, HCT116 and SW620 cells with stable MDFI overexpression were exposed to oxaliplatin treatment, and 1 μM MK2206 was added to treat with oxaliplatin at the same time. The cells were cultured for 48 hours and clone formation experiments were performed. The results showed that the survival rate of the MDFI overexpression group of HCT116 with MK2206 under oxaliplatin treatment was significantly lower than that of the MDFI overexpression group of HCT116 without MK2206, and the survival rate of the MDFI overexpression group of SW620 with MK2206 under oxaliplatin treatment was significantly lower than that of the MDFI overexpression group of SW620 without MK2206 ( Fig.11 ). The experimental results showed that MK2206 significantly inhibited the resistance of colon cancer cells to oxaliplatin induced by MDFI, indicating that MK2206 reversed the oxaliplatin resistance of colon cancer cells enhanced by MDFI.

[0083] Example 8 MK2206 inhibits the resistance of colon cancer cells induced by MDFI gene to 5-fluorouracil

[0084] In this study, HCT116 and SW620 cells with stable MDFI overexpression were exposed to pentafluorouracil treatment, and 1 μM MK2206 was added to treat pentafluorouracil at the same time. The cells were cultured for 48 hours and clone formation experiments were performed. The results showed that the survival rate of the MDFI overexpression group of HCT116 with MK2206 was significantly lower than that of the MDFI overexpression group of HCT116 without MK2206 under pentafluorouracil treatment, and the survival rate of the MDFI overexpression group of SW620 with MK2206 under pentafluorouracil treatment was significantly lower than that of the MDFI overexpression group of SW620 without MK2206 ( Fig.12). The experimental results showed that MK2206 significantly inhibited the resistance of colon cancer cells to 5-fluorouracil induced by MDFI, indicating that MK2206 reversed the 5-fluorouracil resistance of colon cancer cells enhanced by MDFI.

[0085] Example 9 MK2206 inhibits the resistance of MDFI gene-induced subcutaneous colon cancer tumors to chemotherapeutic drugs

[0086] Taking the proliferation curve of HCT116 subcutaneous tumors overexpressing MDFI treated with the chemotherapy drug oxaliplatin without adding MK2206 as a reference, the growth rate of HCT116 subcutaneous tumors overexpressing MDFI after adding MK2206 was significantly slower than that of HCT116 subcutaneous tumors overexpressing MDFI without adding MK2206. The tumor volume of HCT116 subcutaneous tumors overexpressing MDFI treated with the chemotherapy drug oxaliplatin with MK2206 on day 24 was 123±11.7, which was significantly lower than the volume of HCT116 subcutaneous tumors overexpressing MDFI treated with the chemotherapy drug oxaliplatin without adding MK2206 (875±44.3mm). 3 ( Fig.13 ), the results showed that a certain concentration of MK2206 treatment can significantly inhibit the resistance of MDFI gene-induced colon cancer cells to chemotherapy drugs ( Fig.13 ).

Claims

1. Application of MK2206 in the preparation of drugs for the treatment of colorectal cancer.

2. Application of MK2206 in the preparation of drugs for enhancing the sensitivity of colorectal cancer to chemotherapeutic drugs.

3. Use of MK2206 as claimed in claim 2 in the preparation of a drug for enhancing the sensitivity of colorectal cancer to chemotherapeutic drugs, characterized in that the chemotherapeutic drugs are oxaliplatin and 5-fluorouracil.

4. Application of the combination of MK2206 and oxaliplatin in the preparation of drugs for treating colorectal cancer.

5. Application of the combination of MK2206 and 5-fluorouracil in the preparation of drugs for the treatment of colorectal cancer.

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