Application of epidermal growth factor receptor inhibitor combined with mevalonic acid pathway inhibitor in preparation of medicine for treating KRAS mutation colorectal cancer

Abstract

The invention discloses application of an epidermal growth factor receptor inhibitor combined with a mevalonic acid pathway inhibitor in preparation of a medicine for treating KRAS mutation colorectal cancer, and belongs to the technical field of biological medicine. A lipid metabolism pathway-mevalonic acid pathway participates in a drug resistance process of KRAS mutation colorectal cancer to an EGFR (epidermal growth factor receptor) inhibitor, and a plurality of KRAS mutation colorectal cancer cells and a mouse in-vivo model prove that the drug resistance of the KRAS mutation colorectal cancer to the EGFR inhibitor can be effectively overcome by inhibiting the mevalonic acid pathway. Therefore, by combining the EGFR inhibitor and the mevalonic acid pathway inhibitor, synergistic interaction can be achieved, the treatment effect of EGFR is improved, and precise treatment of KRAS mutation colorectal cancer is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of an epidermal growth factor receptor inhibitor combined with a mevalonate pathway inhibitor in the preparation of a drug for treating KRAS mutant colorectal cancer. Background Art

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors worldwide. KRAS mutation is one of the most common oncogenic mutations in CRC. KRAS mutations are observed in approximately 40% of CRC and 60% of metastatic colorectal cancer (mCRC) patients, leading to tumor cell resistance, easy metastasis and poor prognosis. Patients with KRAS-mutated CRC have poor progression-free survival and overall survival, and have a higher rate of liver and lung metastasis. Existing targeted treatment options for KRAS-mutated CRC are limited. For example, small molecule inhibitors that directly target KRAS have poor clinical effects and are prone to induce drug resistance. Therefore, the development of new treatment strategies to overcome the drug resistance problem of KRAS-mutated CRC is a clinical challenge that needs to be urgently addressed.

[0003] Epidermal growth factor receptor (EGFR) inhibitors bind to epidermal growth factor (EGF) and block its signal transduction pathway, thereby inhibiting the proliferation, invasion and metastasis of tumor cells and promoting tumor cell apoptosis. They are currently one of the important treatment strategies for patients with metastatic CRC. Gefitinib is a commonly used EGFR inhibitor. However, patients with KRAS-mutated colorectal cancer have resistance to gefitinib, resulting in poor efficacy of existing treatments for KRAS-mutated mCRC.

[0004] The mevalonate (MVA) pathway is a core pathway of cellular metabolism, mainly responsible for the synthesis of cholesterol and isoprenoid compounds (such as farnesyl pyrophosphate and geranyl pyrophosphate). These products are essential for cell membrane structure, protein modification (isoprenylation), signal transduction and energy metabolism. Mevalonate pathway inhibitors are key enzymes that inhibit the MVA pathway and can regulate the pathological processes of various diseases. Common mevalonate pathway inhibitors include atorvastatin, simvastatin and zoledronic acid, which are mainly used clinically to lower cholesterol and prevent cardiovascular diseases. At present, there are no reports that mevalonate pathway inhibitors can be used to treat colorectal cancer, and there are no reports that mevalonate pathway inhibitors are combined with EGFR inhibitors to treat KRAS mutant colorectal cancer. Summary of the invention

[0005] In order to overcome the disadvantages of the above-mentioned prior art, the object of the present invention is to provide the application of an epidermal growth factor receptor inhibitor combined with a mevalonate pathway inhibitor in the preparation of a drug for treating KRAS-mutated colorectal cancer, so as to solve the technical problem that existing KRAS-mutated CRC patients are resistant to epidermal growth factor receptor inhibitors.

[0006] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0007] In the first aspect of the present invention, the application of an epidermal growth factor receptor inhibitor combined with a mevalonate pathway inhibitor in the preparation of a drug for treating KRAS-mutated colorectal cancer is disclosed.

[0008] Preferably, the mass ratio of the epidermal growth factor receptor inhibitor to the mevalonate pathway inhibitor is 10:1.

[0009] Preferably, the in-vivo administration dose of the epidermal growth factor receptor inhibitor is 100 mg / kg, and the in-vivo administration dose of the mevalonate pathway inhibitor is 10 mg / kg.

[0010] Preferably, the epidermal growth factor receptor inhibitor and the mevalonate pathway inhibitor synergistically inhibit the growth of KRAS-mutated colorectal cancer tumors.

[0011] Preferably, the drug is a drug that overcomes the resistance of KRAS-mutated colorectal cancer to EGFR inhibitors by inhibiting the mevalonate pathway.

[0012] Preferably, the drug is a drug that inhibits the proliferation of KRAS-mutated colorectal cancer cells, induces the apoptosis of KRAS-mutated colorectal cancer cells, and inhibits the growth of KRAS-mutated colorectal cancer tumors.

[0013] Preferably, the epidermal growth factor receptor inhibitor is gefitinib, and the mevalonate pathway inhibitor is atorvastatin.

[0014] In the second aspect of the present invention, a composition for treating KRAS-mutated colorectal cancer is disclosed, which comprises an epidermal growth factor receptor inhibitor and a mevalonate pathway inhibitor with a mass ratio of 10:1.

[0015] Preferably, the epidermal growth factor receptor inhibitor is gefitinib, and the mevalonate pathway inhibitor is atorvastatin.

[0016] Preferably, it further comprises a pharmaceutically acceptable excipient and / or carrier.

[0017] Preferably, the dosage form of the composition is tablets, capsules, granules, oral solutions or injections.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The application of the epidermal growth factor receptor inhibitor combined with the mevalonate pathway inhibitor in the preparation of a drug for treating KRAS mutant colorectal cancer. Based on the fact that the lipid metabolism pathway - the mevalonate pathway is involved in the resistance process of KRAS mutant colorectal cancer to EGFR inhibitors, the EGFR inhibitor is combined with the mevalonate pathway inhibitor. Experiments have shown that the combination of the EGFR inhibitor and the mevalonate pathway inhibitor exhibits significant tumor suppression effects in in vitro or in vivo KRAS mutant colorectal cancer models, including: inhibiting the proliferation of KRAS mutant colorectal cancer cells, inducing apoptosis of KRAS mutant colorectal cancer cells, and inhibiting tumor growth in a KRAS mutant colorectal cancer mouse model. By inhibiting the mevalonate pathway, the resistance of KRAS mutant colorectal cancer to EGFR inhibitors is reversed. This method has the following advantages when applied: reducing the systemic toxicity of the mevalonate pathway inhibitor, improving the therapeutic effect of the EGFR inhibitor, and achieving precise treatment of KRAS mutant colorectal cancer. Therefore, on the one hand, the combination of the EGFR inhibitor and the mevalonate pathway inhibitor can target and inhibit the mevalonate pathway of tumors to overcome the resistance of KRAS mutant CRC to EGFR inhibitors. On the other hand, it can effectively increase the drug effect of the EGFR inhibitor, having both high targeting and better tumor killing effects, reducing the side effects brought by the combination of the two drugs, lowering the health economic cost, and being able to be used to treat KRAS mutant colorectal cancer, especially for overcoming the resistance of KRAS mutant colorectal cancer to EGFR inhibitors, and having potential clinical transformation value. Description of the Drawings

[0020] Figure 1 It is the experimental result diagram of gefitinib activating the mevalonate pathway in SW837; among them, (a) principal component analysis of lipid metabolites in the control group and SW837 treated with gefitinib; (b) KEGG enrichment analysis of differential metabolites; (c) gene expression heat map related to the cholesterol metabolism pathway; (d) qPCR detection of transcriptional changes of genes related to the mevalonate pathway in SW837 treated with gradient concentrations of gefitinib, GE - 0.1 is 0.1 μM gefitinib, GE - 1 is 1 μM gefitinib, GE - 10 is 10 μM gefitinib; (e) Western blotting detection of the expression changes of GGPS1 and FDPS in SW837 treated with gradient concentrations of gefitinib; *P < 0.05, **P < 0.01, ***P < 0.001;

[0021] Figure 2Experimental result graphs of gefitinib activating the mevalonate pathway in SW480 and HCT116; among them, (a) gefitinib-treated SW480, (b) gefitinib-treated HCT116, and in (a) and (b), from top to bottom are the changes in lipid metabolites detected by targeted lipidomics, and the transcriptional and expression changes of genes related to the mevalonate pathway detected by qPCR and Western blotting, *P<0.05, **P<0.01, ***P<0.001, GE vs DMSO;

[0022] Figure 3 Experimental result graphs of gefitinib combined with atorvastatin inhibiting cell viability; among them, (a) CCK8 cell viability experiment comparing cell viability after different drug treatments, and from top to bottom are HCT116, DLD1, and SW480 cells; (b) representative pictures of colony formation of HCT116 cells; (c) quantitative results of three independent replicates of colony formation. *P<0.05, **P<0.01, ***P<0.001; ns: P>0.05;

[0023] Figure 4 Experimental result graphs of gefitinib combined with atorvastatin inducing apoptosis sensitivity in HCT116; among them, (a) flow cytometry detecting the apoptosis ratio of HCT116 after different drug treatments; (b) quantitative results of three independent replicates of apoptosis detection; (c) WB detecting the expression of apoptosis proteins such as PARP1 and CASPASE3 in HCT116 cells after different drug treatments; (d) WB detecting the expression of EGFR, P-EGFR, ERK, and P-ERK in HCT116 cells after different drug treatments, ***P<0.001;

[0024] Figure 5 Experimental result graphs of gefitinib combined with atorvastatin inducing apoptosis sensitivity in DLD1; among them, (a) flow cytometry detecting the apoptosis ratio of DLD1 cells after different drug treatments; (b) quantitative results of three independent replicates of apoptosis detection; (c) WB detecting the expression of PARP1, BCL-XL, BAX, and CASPASE3 in DLD1 cells after different drug treatments; (d) WB detecting the expression of EGFR, P-EGFR, ERK, and P-ERK in DLD1 cells after different drug treatments, **P<0.01;

[0025] Figure 6In vivo verification experimental result graph for targeting the mevalonate pathway to overcome the resistance of KRAS - mutated colorectal cancer to EGFR inhibitors; among them, (a) photographs of tumors of subcutaneous tumor model mice in each group at the treatment endpoint when an anti - EGFR drug is combined with a mevalonate pathway inhibitor, significantly inhibiting the growth of subcutaneous tumors in mice with colon cancer; (b) tumor weights of subcutaneous tumor model mice; (c) tumor growth curves of subcutaneous tumor model mice in different treatment groups; (d) pictures of Tunel immunofluorescence staining of tumor tissues of subcutaneous tumor model mice in each group at the treatment endpoint; (e) survival curve analysis of subcutaneous tumor model mice in different treatment groups; (f) quantitative analysis of Tunel immunofluorescence staining of tumor tissues of subcutaneous tumor model mice in each group at the treatment endpoint, Scalebar = 20μm; *P < 0.05, **P < 0.01, ***P < 0.001; ns: P > 0.05. Detailed implementation manners

[0026] To enable those skilled in the art to understand the features and effects of the present invention, the following provides only a general description and definition of the terms and phrases mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein shall have the ordinary meaning understood by those skilled in the art with respect to the present invention. In case of conflict, the definition in this specification shall prevail.

[0027] The theories or mechanisms described and disclosed herein, whether right or wrong, shall not in any way limit the scope of the present invention, that is, the content of the present invention can be implemented without being limited by any specific theory or mechanism.

[0028] In this article, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are only for the sake of brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be regarded as having covered and specifically disclosed all possible sub - ranges and individual numerical values (including integers and fractions) within the range.

[0029] In this article, unless otherwise specified, terms such as "comprising", "including", "containing", "having", or similar terms cover the meanings of "consisting of" and "consisting essentially of". For example, "A comprises a" covers the meanings of "A comprises a and others" and "A consists only of a".

[0030] In this article, for the sake of concise description, all possible combinations of all technical features in each embodiment or example are not described. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each embodiment or example can be combined arbitrarily, and all possible combinations should be considered as the scope described in this specification.

[0031] This experiment found that KRAS mutant CRC resists epidermal growth factor receptor (EGFR) inhibitor therapy by abnormally activating the mevalonate (MVA) metabolic pathway. Given that the abnormal activation of the mevalonate pathway is a key factor in the drug resistance of KRAS mutant tumors, using statins to inhibit this pathway to reverse the drug resistance of KRAS mutant CRC to EGFR drugs is an important strategic idea for conquering KRAS mutant tumors. Therefore, the application of epidermal growth factor receptor inhibitors combined with mevalonate pathway inhibitors in the preparation of drugs for treating KRAS mutant colorectal cancer is provided.

[0032] The following, in combination with the following examples, uses Gefitinib as an EGFR inhibitor and Atorvastatin as a mevalonate pathway inhibitor to further elaborate the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0033] In the following examples, conventional instrument equipment in the art is used. For the experimental methods without specific conditions noted in the following examples, they are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Various raw materials are used in the following examples, and unless otherwise stated, commercially available products with conventional specifications in the art are used.

[0034] Example 1 EGFR Inhibitor Upregulates the Mevalonate Pathway in KRAS Mutant Colorectal Cancer

[0035] 1. Cell Culture and Treatment

[0036] The KRAS mutant colorectal cancer cell lines used are HCT116, SW480, and SW837. HCT116 cells are cultured in DMEM medium containing 10% FBS, and SW480 and SW837 cells are cultured in L-15 medium containing 10% FBS. Cells are recovered from liquid nitrogen, centrifuged and resuspended after thawing, and inoculated into 6-cm culture dishes. After the cells adhere, the fresh medium is replaced. When the cell confluence reaches 80%, the cells are digested and passaged with 0.25% trypsin-EDTA. HCT116 cells are seeded at a density of 2.5×10 5 cells / 4 mL or 1×10 5 cells / 2 mL in 6-cm culture dishes or 6-well plates, and drug treatment is carried out 24 hours later.

[0037] 2. Drug Treatment and Sample Collection

[0038] The cells in each group were divided into a control group and an experimental group (gefitinib, 10 μM). The control group was treated with 10 μM DMSO for 48 hours. In the experimental group, cells were treated with gradient concentrations of gefitinib, namely 0.1 μM, 1 μM, 5 μM, and 10 μM gefitinib for 48 hours. Then, the SW837 cell pellet was collected using a cell scraper, and 1 mL of TRIzol was added to lyse the SW837 cells. After thorough mixing, the lysate was divided into two portions, one for transcriptome sequencing and the other for metabolomics analysis, and stored at -80 °C.

[0039] 3. Transcriptome and metabolomics analysis

[0040] Total RNA was extracted using TRIzol and sent for sequencing after reverse transcription. Differentially expressed metabolites were screened based on the OPLS-DA model with the screening criteria of VIP ≥ 1 and fold change ≥ 2 or ≤ 0.5.

[0041] 4. qPCR verification

[0042] SRE BF1, HMGCR, MVK, MVD, DHCR, LSS, and SQLE were used as target genes, and qPCR primers were designed according to these target genes. The reaction system was prepared using the 2×RealStarFastSYBR qPCR Mix kit. The qPCR instrument was set for pre-denaturation at 95 °C for 2 minutes, followed by 40 cycles of cycling reactions at 95 °C for 15 seconds and 60 °C for 30 seconds. The CT values were recorded and the gene expression changes were calculated.

[0043] 5. Statistical analysis

[0044] VIP ≥ 1 and fold change ≥ 2 or ≤ 0.5 were used as the criteria for significant differences. The results were expressed as mean ± SEM, and a two-tailed t-test was used. A P value < 0.05 was considered statistically significant.

[0045] The experimental results are as Figure 1 and Figure 2 shown. Lipid metabolites in cells treated with gefitinib changed significantly compared to those in the control group cells ( Figure 1 (a) in Figure 1 ). Further KEGG pathway analysis of the differentially expressed metabolites revealed that the metabolites with significantly changed abundances were concentrated in pathways such as vitamin digestion and absorption, cholesterol metabolism, and fat digestion and absorption ( Figure 1 (b) in Figure 1(d) below. The WB results further showed that gefitinib significantly increased the expression of mevalonate pathway-related genes GGPS1 and FDPS in SW837 cells ( Figure 1 (e) below.

[0046] Two other KRAS mutant colorectal cancer cell lines (SW480 and HCT116) were further treated with gefitinib, and the treated cells were subjected to targeted lipid metabolism sequencing. Analysis of the sequencing results showed that sterol lipids and prenol lipids metabolites were significantly consumed in SW480 and HCT116 after gefitinib treatment, and the level of cholesterol also changed significantly in SW480 ( Figure 2 (a) and the first line of (b) below.

[0047] Sterol lipids and prenol lipids metabolites are mainly produced through the mevalonate pathway. Further, qPCR was used to detect the gene changes related to the mevalonate pathway in SW480 and HCT116 after gefitinib treatment. The results were consistent with those of SW837 and the metabolomics results. The mevalonate pathway-related genes were significantly upregulated in HCT116 and SW480 after gefitinib treatment, and increased with the increase in gefitinib concentration ( Figure 2 (a) and the second line of (b) below.

[0048] Further Western blotting results also showed that the protein expression of mevalonate pathway-related genes: SREBF1, HMGCS1, FDFT, GGPS1, FDPS was significantly increased in SW480 and HCT116 after gefitinib treatment ( Figure 2 (a) and the third line of (b) below.

[0049] In summary, the abundance of metabolites produced by the mevalonate pathway in KRAS mutant colorectal cancer after anti-EGFR drug treatment changed significantly, and the mevalonate pathway was abnormally activated.

[0050] Example 2 Overcoming Resistance of KRAS Mutant Colorectal Cancer Cells to EGFR Inhibitors by Targeting the Mevalonate Pathway

[0051] 1. Cell culture

[0052] The KRAS mutant colorectal cancer cell lines HCT116, DLD1, and SW480 were used. HCT116 cells were cultured in DMEM medium containing 10% FBS, and DLD1 and SW480 cells were cultured in L-15 medium containing 10% FBS.

[0053] 2. Drug group setting

[0054] The drug groups were set as follows: gefitinib treatment group (GE; 0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 5 μM, 8 μM, 10 μM, 16 μM gefitinib); atorvastatin treatment group (S; at a concentration of 1 / 5 of atorvastatin IC 50 ); gefitinib + atorvastatin treatment group (GE + S; 0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 5 μM, 8 μM, 10 μM, 16 μM gefitinib combined with 1 / 5 IC 50 of atorvastatin respectively); blank control group (DMSO).

[0055] Among them, HCT116 cells were treated with 5 μM of the drug, SW480 with 10 μM of the drug, and DLD1 with 5 μM of the drug.

[0056] 3. Cell viability and colony formation assay

[0057] Tumor cell suspensions (HCT116, DLD1, and SW480) were respectively inoculated into 96-well plates. After attachment, different drugs were added for treatment. Each treatment was set with 3 - 5 replicates. After 48 h, CCK-8 solution was added, and incubation continued for 1 - 4 h. The OD value at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader for cell viability determination.

[0058] Cells in the logarithmic growth phase were digested with trypsin, resuspended and counted, and then inoculated into 6-well plates. After attachment, different drugs were added for treatment for 48 - 72 h. The culture medium was changed regularly for observation. After colony formation, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. After washing and air drying, photographs were taken for analysis.

[0059] 4. Flow cytometry for detecting cell apoptosis

[0060] Equal amounts of cells (HCT116, DLD1, and SW480) were respectively plated in 6-cm culture dishes. After overnight attachment, different drugs were added for treatment for 48 h. The cell culture supernatant and adherent cells were collected, and cell pellets were obtained by centrifugation. The cells were washed twice with PBS and resuspended with binding buffer. Single-label tubes and negative tubes were set up. AnnexinV-EGFP and Propidium Iodide were added to the remaining cells, and the reaction was carried out at room temperature in the dark for 10 - 20 min, followed by flow cytometry analysis.

[0061] 5. Western blotting for detecting apoptosis-related proteins

[0062] Remove the cell culture medium in a 6-cm petri dish, wash it twice with pre-cooled PBS, add RIPA lysis buffer containing PMSF (add phosphatase inhibitors as needed), lyse on ice, sonicate, centrifuge at 13,000 rpm for 15 min at 4°C, and take the supernatant. Quantify with a BCA kit, add 5× loading buffer to the remaining supernatant, denature it in a metal bath at 100°C, and store it at -20°C. Clean and dry the glass plates and install them on the gel-making rack. Prepare the separating gel and stacking gel with the kit. During electrophoresis, fix the gel plate in the electrophoresis tank. After loading the samples, first keep the voltage constant at 80 V, and adjust it to 120 V constant voltage after the protein enters the separating gel. For wet transfer, assemble the "sandwich" in sequence and transfer the membrane at a constant current of 300 mA for 90 min. After transfer, wash the PVDF membrane with TBST, block it with 5% skim milk, incubate with the primary antibody, wash the membrane, incubate with the secondary antibody, wash the membrane, and finally expose it with HRP-ECL luminescent solution, and analyze the results with ImageJ software.

[0063] 6. Statistical analysis

[0064] Statistical processing and analysis were performed using GraphPad Prism 9. The t-test or Mann-Whitney test was used for inter-group difference analysis. The experimental results were expressed as mean ± SEM, and P < 0.05 represented statistically significant differences.

[0065] The experimental results are as Figures 3 to 5 shown. The results showed that compared with gefitinib monotherapy, the combination treatment group of gefitinib + atorvastatin significantly inhibited the growth of KRAS mutant colorectal cancer cells HCT116, SW480, and DLD1, and the inhibitory effect increased with the increase in gefitinib concentration ( Figure 3 (a) in the figure). The colony formation assay based on HCT116 cells further demonstrated that after inhibiting the mevalonate pathway with atorvastatin, the inhibitory effect of gefitinib on cell growth was significantly enhanced ( Figure 3 (b) and (c) in the figure).

[0066] Furthermore, the effects of the combination of the two drugs on inducing apoptosis in KRAS mutant colorectal cancer cells were detected by flow cytometry and WB. The results showed that when the mevalonate pathway was inhibited with atorvastatin, the ratio of HCT116 cells induced to apoptosis by gefitinib increased significantly ( Figure 4 (a) and (b) in the figure; the WB results showed that in the case of the combination of gefitinib + atorvastatin, the expression of pro-apoptotic proteins such as PARP1 and Cleaved Caspase 3 increased significantly and increased with the increase in gefitinib concentration ( Figure 4 (c) in the figure).

[0067] Similarly, compared with the control group and the monotherapy group, the apoptosis rate of DLD1 cells induced by the combination treatment of gefitinib + atorvastatin also increased significantly ( Figure 5in (a) and (b)), and gefitinib + atorvastatin increased the expression of pro-apoptotic proteins such as active PARP1, BAX, and Cleaved Caspase3 in DLD1 cells, and decreased the expression of anti-apoptotic BCL-XL protein ( Figure 5 in (c)).

[0068] Subsequently, the activity (phosphorylation) of the EGFR-KRAS-ERK pathway was detected by WB. Although gefitinib alone could inhibit the expression of P-EGFR, it could not inhibit the downstream P-ERK. However, when atorvastatin and gefitinib were used to treat cells in combination, the expression of P-ERK in cells could be significantly inhibited ( Figure 4 in (d), Figure 5 in (d))

[0069] In summary, after inhibiting the mevalonate pathway in KRAS-mutant colorectal cancer cells, the EGFR inhibitor can exert its classical anti-tumor drug activity, that is, induce apoptosis of cells by inhibiting the EGFR-KRAS-ERK signaling pathway, that is, targeting the mevalonate pathway to overcome the resistance of KRAS-mutant colorectal cancer cells to EGFR inhibitors.

[0070] Example 3 In vivo verification of targeting the mevalonate pathway to overcome the resistance of KRAS-mutant colorectal cancer to EGFR inhibitors

[0071] 1. Construction of mouse subcutaneous tumor model

[0072] Male BALB / c mice aged 4-5 weeks and weighing 18-20 g were used and raised under SPF conditions. KRAS-mutant colorectal cancer cells CT26 were cultured in RPMI1640 medium containing 10% FBS. CT26 cells in the logarithmic growth phase were collected and resuspended in PBS to 5×10 7 cells / mL. 100 μL of cell suspension (5×10 6 cells) was subcutaneously inoculated into the right iliac fossa of each mouse. 1-2 weeks after inoculation, tumor growth was visible to the naked eye. The longest axis (a) and the shortest axis (b) of the tumor were measured with a vernier caliper, and the tumor volume was calculated (V = 1 / 2 × a × b 2 ). When the tumor volume reached 100 mm 3 , drug treatment was started.

[0073] 2. Drug treatment

[0074] The tumor-bearing mice were randomly divided into four groups: control group (CON; 100 mg / kg DMSO), gefitinib group (Gefitinib; the dosage of gefitinib was 100 mg / kg), atorvastatin group (Atorvastatin; the dosage of atorvastatin was 10 mg / kg), and combination drug group (GE+S; the dosage of atorvastatin was 10 mg / kg and the dosage of gefitinib was 100 mg / kg). They were given drugs by gavage once a day. The tumor volume of the mice was measured every 3 days after the start of drug administration. After continuous drug administration for 10 days, the tumors of the mice were harvested, and the gross pictures, volume, and weight of the tumors were photographed and recorded, and the tumor growth curve was recorded.

[0075] 3. Analysis of treatment endpoint

[0076] After the treatment was completed, the mice were sacrificed, and the tumors were dissected and weighed. Part of the tumor tissue was fixed with 4% paraformaldehyde for HE staining and Tunel immunofluorescence staining.

[0077] 4. Tunel immunofluorescence staining

[0078] The fixed tumor tissue was paraffin-embedded, and the section thickness was 4 μm. After dewaxing and hydration, the sections were treated with proteinase K for 20 minutes. The Tunel reaction mixture was added and incubated at 37°C for 1 hour. After washing with PBS, the nuclei were stained with DAPI, and the slides were sealed. Observation and photography were performed under a fluorescence microscope, and the proportion of apoptotic cells was quantitatively analyzed.

[0079] 5. Survival analysis

[0080] After the treatment was completed, the survival of the mice was continuously observed, and the survival time was recorded. The Kaplan-Meier method was used to plot the survival curve, and the Log-rank test was used to compare the differences between groups.

[0081] 6. Statistical analysis

[0082] Statistical analysis was performed using GraphPad Prism 9.0. The tumor volume, weight, and proportion of apoptotic cells were expressed as mean ± SEM, and the two-tailed t-test or one-way ANOVA was used. The Log-rank test was used for survival analysis, and P < 0.05 was considered statistically significant.

[0083] The experimental results are as Figure 6 shown. Compared with the control group, there was no significant change in the tumor volume in the atorvastatin and gefitinib single-drug treatment groups, while the tumor volume of the mice treated with the combination drug was significantly reduced ( Figure 6 in (a) and (b)). The recorded results of the tumor volume during the experiment showed that compared with the control group, gefitinib group, and atorvastatin group, the combination drug significantly inhibited the tumor growth rate ( Figure 6In (c)). After the administration was completed, the harvested tumor tissues were fixed with formalin, embedded in paraffin and sectioned, and subjected to Tunel immunofluorescence staining. The results showed that in the tumors derived from the mice in the combination treatment group, the ratio of Tunel-positive cells increased significantly, indicating an increase in the number of apoptotic tumor cells ( Figure 6 In (d) and (f)). The survival curves showed that the average survival time of the control group mice was 27.3 days, that of the mice treated with atorvastatin alone was 25.5 days, that of the mice treated with gefitinib alone was 26.5 days, while the average survival time of the mice treated with the combination of the two drugs was 35.6 days, and the therapeutic effect was significantly better than that of the other three groups of mice ( Figure 6 In (e)).

[0084] In summary, inhibiting the mevalonate pathway can effectively overcome the resistance of KRAS mutant colorectal cancer to EGFR inhibitors, and the combination treatment strategy shows excellent in vivo therapeutic effects.

[0085] The above content is only to illustrate the technical idea of the present invention, and the protection scope of the present invention cannot be limited thereby. Any modification made on the basis of the technical solution according to the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims

1. Application of epidermal growth factor receptor inhibitors combined with mevalonate pathway inhibitors in the preparation of drugs for the treatment of KRAS mutant colorectal cancer.

2. The use according to claim 1, characterized in that: The mass ratio of epidermal growth factor receptor inhibitor to mevalonate pathway inhibitor is 10:

1.

3. The use according to claim 1 or 2, characterized in that: Epidermal growth factor receptor inhibitors synergistically inhibit KRAS-mutant colorectal cancer tumor growth with mevalonate pathway inhibitors.

4. The use according to claim 1 or 2, characterized in that: The drug overcomes the resistance of KRAS mutant colorectal cancer to EGFR inhibitors by inhibiting the mevalonate pathway.

5. The use according to claim 1 or 2, characterized in that: The drug is a drug that inhibits the proliferation of KRAS mutant colorectal cancer cells, induces apoptosis of KRAS mutant colorectal cancer cells, and inhibits the growth of KRAS mutant colorectal cancer tumors.

6. The use according to claim 1 or 2, characterized in that: The epidermal growth factor receptor inhibitor is gefitinib, and the mevalonate pathway inhibitor is atorvastatin.

7. A composition for treating KRAS mutant colorectal cancer, characterized in that: It includes an epidermal growth factor receptor inhibitor and a mevalonate pathway inhibitor in a mass ratio of 10:

1.

8. The composition for treating KRAS mutation colorectal cancer according to claim 7, characterized in that: The epidermal growth factor receptor inhibitor is gefitinib, and the mevalonate pathway inhibitor is atorvastatin.

9. The composition for treating KRAS mutation colorectal cancer according to claim 7 or 8, characterized in that: Pharmaceutically acceptable excipients and / or carriers are also included.

10. The composition for treating KRAS mutation colorectal cancer according to claim 7 or 8, characterized in that: The dosage form of the composition is tablet, capsule, granule, oral solution or injection.

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