Application of combination of lovastatin and DHCR7 inhibitor AY9944 in preparation of medicine for treating colorectal cancer

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of lovastatin combined with DHCR7 inhibitor AY9944 in preparation of medicine for treating colorectal cancer. The DHCR7 protein expression in the drug-resistant tumor tissue is obviously improved compared with that of normal tumor cells, and verification finds that the DHCR7 inhibitor AY9944 can specifically inhibit the drug resistance of colorectal cancer cells generating drug resistance, and further finds that when AY9944 and lovastatin are combined for use at a specific concentration ratio, the drug resistance of the colorectal cancer cells generating drug resistance can be inhibited, and the drug resistance of the colorectal cancer cells generating drug resistance can be inhibited. The two can play an obvious synergistic effect, so that the sensitivity of intestinal cancer cells to oxaliplatin is more effectively improved. Therefore, the combined medication mode provided by the invention can provide a new strategy reference for clinically overcoming the chemotherapy resistance of colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of lovastatin in combination with the DHCR7 inhibitor AY9944 in the preparation of a drug for treating colorectal cancer. Background Technology

[0002] Currently, treatment strategies for colorectal cancer mainly include surgical resection, chemotherapy, radiotherapy, and targeted therapy. For early-stage patients, surgical resection is the preferred treatment; while for advanced-stage patients, a combination of chemotherapy, radiotherapy, and targeted therapy is necessary. However, despite continuous advancements in treatment methods, tumor drug resistance and recurrence remain major factors contributing to poor prognosis in colorectal cancer patients. Therefore, exploring novel treatment strategies to overcome drug resistance and improve efficacy has become a key focus of current colorectal cancer research.

[0003] Studies have shown that cholesterol and its metabolites play a crucial role in the pathogenesis of colorectal cancer (CRC). A high-cholesterol diet can increase the proliferation of cancer cells and promote the development of colorectal cancer. Recent research has discovered that 7-dehydrocholesterol reductase (DHCR7), a key enzyme catalyzing cholesterol metabolism, can regulate the growth and apoptosis of colorectal cancer cells. DHCR7 can serve as a potential therapeutic target for colorectal cancer. As a therapeutic target, the primary consideration for DHCR7 is to develop an economical, effective, and safe DHCR7 inhibitor to truly realize its value in clinical practice.

[0004] AY9944 (trans-1,4-bis(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride) is a widely used, highly selective inhibitor of DHCR7. This inhibitor inhibits the conversion of 7-dehydrocholesterol into cholesterol, thereby lowering cholesterol levels. In the 1990s, AY9944 was first discovered to restore normal mitotic responses and cytokine profiles in peripheral blood mononuclear cells (PBMCs) from HIV patients in vitro. Subsequently, it has been widely used in research on antiviral, immune, and metabolic diseases. Interestingly, research on AY9944 in malignant tumors, especially its inhibitory effect on tumor stemness in colorectal cancer, is rarely reported.

[0005] Lovastatin is a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, widely used clinically to lower cholesterol levels and prevent cardiovascular disease. Recent studies have shown that lovastatin has potential applications in cancer treatment, with its anti-tumor effects primarily achieved through the following mechanisms: (1) inhibiting the mevalonate pathway, reducing the production of isoprene metabolites, thereby inhibiting tumor cell proliferation; (2) inducing tumor cell apoptosis; and (3) inhibiting tumor angiogenesis and metastasis. However, current research on lovastatin in colorectal cancer is mainly focused on the preclinical stage, with clinical research still in its initial exploratory phase. Furthermore, the anti-cancer dose of lovastatin may be higher than its cholesterol-lowering dose, therefore further research is needed on its safety and tolerability. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide the application of lovastatin combined with the DHCR7 inhibitor AY9944 in the preparation of drugs for treating colorectal cancer, so as to effectively inhibit the tumor drug resistance phenotype of colorectal malignant tumor cells with high DHCR7 expression, and to effectively improve the therapeutic effect of first-line chemotherapy drugs when used in combination with them.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0008] In a first aspect, the present invention provides the use of lovastatin in combination with a DHCR7 inhibitor in the preparation of a medicament for treating colorectal cancer.

[0009] Preferably, the DHCR7 inhibitor includes AY9944.

[0010] Preferably, the colorectal cancer includes oxaliplatin-resistant colorectal cancer.

[0011] In previous studies, the inventors used Western blotting to analyze the expression level of DHCR7 in drug-resistant and drug-resistant colorectal cancer cells. The results showed that DHCR7 protein expression was significantly higher in drug-resistant tumor tissues than in normal tumor cells. Based on this finding, the inventors further treated human colorectal cancer cell lines with different concentrations of AY9944 in vitro and analyzed its effects on cell viability and related protein expression using MTS assays and Western blotting. The results showed that with increasing AY9944 concentration, the activity of colorectal cancer cells was gradually inhibited, and the expression levels of markers related to tumor drug resistance, such as SOX2 and CD133, also decreased. These results reveal that abnormally elevated DHCR7 expression levels are significantly correlated with the activity and drug-resistant phenotype of colorectal cancer cells.

[0012] Based on this, the inventors combined AY9944 with lovastatin to try to treat colorectal cancer cells. The effect was tested again by MTS test and Western Blot. The results showed that the combined use of AY9944 and lovastatin could more effectively reduce the resistance of colorectal cancer cells to oxaliplatin.

[0013] Therefore, this invention is the first to demonstrate that the combination of DHCR7 inhibitor AY9944 and lovastatin can significantly inhibit the drug resistance phenotype of colorectal cancer cells, providing a combination therapy with clinical translational potential for targeted therapy of colorectal cancer. This discovery not only expands the research perspective on tumor drug resistance mechanisms, but also provides a new strategy reference for overcoming chemotherapy resistance in colorectal cancer in clinical practice.

[0014] Preferably, the ratio of the effective concentration of AY9944 to that of lovastatin is (1-10):(40-100).

[0015] Preferably, the ratio of the effective concentration of AY9944 to that of lovastatin is 8:80.

[0016] The inventors designed experiments with different concentration ratios of AY9944 and lovastatin to explore the effect of combined drug therapy on reversing tumor drug resistance. The experiments revealed that when the effective concentrations of AY9944 and lovastatin were 8 μM and 80 μM, respectively, i.e., the ratio of their effective concentrations was 8:80, the combination of the two drugs could maximally inhibit the activity of colorectal cancer cells. Furthermore, through synergistic effect index analysis, it was found that the combination of lovastatin and AY9944 could exert a synergistic effect, thereby effectively increasing the drug sensitivity of colorectal cancer cells to oxaliplatin.

[0017] In a second aspect, the present invention provides a medicament for treating colorectal cancer, the medicament comprising lovastatin and / or a DHCR7 inhibitor.

[0018] Preferably, the DHCR7 inhibitor includes AY9944.

[0019] Preferably, the drug comprises lovastatin and AY9944, wherein the effective concentration ratio of AY9944 to lovastatin is (1-10):(40-100).

[0020] More preferably, the ratio of the effective concentration of AY9944 to that of lovastatin is 8:80.

[0021] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention, through cellular-level experiments, clarifies that AY9944 can specifically inhibit the drug resistance of drug-resistant colorectal cancer cells, filling a gap in the field. Furthermore, it was discovered that when AY9944 and lovastatin are used in combination at specific concentrations, they exhibit a significant synergistic effect, thereby more effectively reducing the resistance of colorectal cancer cells to oxaliplatin. This combination therapy not only significantly enhances the drug sensitivity of colorectal cancer cells to oxaliplatin, breaking down the drug resistance barrier of tumor cells, but also provides a novel and highly effective strategy for the clinical treatment of drug-resistant colorectal cancer. Compared to traditional single-treatment methods, the combination therapy of this invention can enhance the killing effect of chemotherapy drugs on drug-resistant colorectal cancer cells, potentially further improving the efficacy of chemotherapy for patients. Attached Figure Description

[0024] Figure 1 This image shows the expression of DHCR7 protein in oxaliplatin-resistant colorectal cancer cells.

[0025] Figure 2 The graph shows the results of different concentrations of AY9944 inhibiting drug resistance-related proteins in colorectal cancer cells;

[0026] Figure 3 The effect of AY9944 alone on the sensitivity of colorectal cancer cells to oxaliplatin is shown in the figure.

[0027] Figure 4 Figure showing the effect of AY9944 combined with lovastatin on the activity of colorectal cancer cells;

[0028] Figure 5 The figure shows the effect of AY9944 combined with lovastatin on the oxaliplatin sensitivity of colorectal cancer cells;

[0029] Figure 6 The figure shows the effect of the combination of AY9944 and lovastatin on the sensitivity of colorectal cancer cell lines to oxaliplatin. Detailed Implementation

[0030] The following detailed embodiments illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments.

[0031] Unless otherwise specified, all reagents used in the examples are conventional reagents available in the art and can be purchased commercially. Experimental procedures not specifically described in the examples are conventional procedures in the art or can be understood or known by those skilled in the art based on their prior knowledge or common general knowledge.

[0032] Example 1. Detection of DHCR7 protein expression in oxaliplatin-resistant colorectal cancer cells.

[0033] In this embodiment, Western blotting was used to analyze the expression level of DHCR7 in drug-resistant and drug-resistant colorectal cancer cells. The specific method is as follows:

[0034] Approximately 20,000 cells were seeded per well in a 6-well plate. These cells included oxaliplatin-resistant DLD1 and wild-type DLD1 cells, as well as oxaliplatin-resistant HCT116 and wild-type HCT116 cells. After incubating the different cell types overnight at 37°C and 5% CO2, the expression level of DHCR7 in each group of colorectal cancer cells was detected using standard Western blotting methods.

[0035] Figure 1 The results showed that DHCR7 expression was significantly upregulated in cells resistant to colorectal cancer compared to normal tumor cells.

[0036] Example 2. Investigating the effect of the DHCR7 selective inhibitor AY9944 on inhibiting the expression of drug resistance-related proteins in colorectal cancer cells.

[0037] In this embodiment, different concentrations of AY9944 were used to treat human colorectal cancer cell lines in vitro to analyze its effects on cell viability and the expression of drug resistance-related proteins. The specific methods are as follows:

[0038] Approximately 200,000 DLD1 wild-type cells were seeded in each well of a 6-well plate. The cells were treated with AY9944 at concentrations of 0, 10 nM, 100 nm, 1 μM, and 10 μM for 48 hours. The expression of drug resistance-related proteins SOX2 and CD133 in the cells was then analyzed using standard Western blotting methods.

[0039] Figure 2 The results showed that as the concentration of the selective DHCR7 inhibitor AY9944 increased, the expression of drugs resistance-related proteins such as CD133 and SOX2 decreased, indicating that high DHCR7 expression is associated with the tumor drug resistance phenotype of colorectal malignant tumor cells. Inhibiting DHCR7 expression with AY9944 can effectively reverse the drug resistance of colorectal cancer cells.

[0040] Example 3. Investigating the effect of the DHCR7 selective inhibitor AY9944 on oxaliplatin resistance in colorectal cancer cells.

[0041] This embodiment uses the MTS assay to detect the effect of using the DHCR7 selective inhibitor AY9944 alone on the sensitivity of colorectal cancer cells to oxaliplatin. The specific method is as follows:

[0042] First, 500,000 cells were seeded per well in a 6-well plate. These cells were DLD1 and HCT116 cells, resistant to oxaliplatin. Control and experimental groups were set up for DLD1 and HCT116 cells, respectively. The control group received no treatment, while the experimental group was treated with 10 μM AY9944 for 48 hours. Subsequently, approximately 2000 cells from each of the control and experimental groups were seeded per well in a 96-well plate. All cells were treated with oxaliplatin and incubated overnight at 37°C and 5% CO2. Cell viability was then assessed using the standard MTS assay to determine the IC50.

[0043] Figure 3 The results showed that, when IC50 was measured, the colorectal cancer cells in the AY9944-treated group showed a significant decrease in resistance to oxaliplatin compared to normal colorectal cancer cells. This further indicates that AY9944 can effectively reverse the drug resistance of colorectal cancer cells, thereby enhancing the sensitivity of cells to oxaliplatin chemotherapy and improving the efficacy of chemotherapy.

[0044] Example 4. Investigating the effect of AY9944 combined with lovastatin on the drug-resistant phenotype of colorectal cancer cells.

[0045] This embodiment uses the MTS assay to detect the effect of AY9944 combined with lovastatin on the drug resistance phenotype of colorectal cancer cells. The specific method is as follows:

[0046] First, 500,000 cells were seeded per well in a 6-well plate, including DLD1 and HCT116 cells. Cells were treated for 48 hours with different concentrations (0, 8 μM) of AY9944 and different concentrations (0, 60 μM, 100 μM for HCT116; 0, 40 μM, 80 μM for DLD1). Then, approximately 2000 cells from each of the control and experimental groups were seeded per well in a 96-well plate. All cells were treated with oxaliplatin and incubated overnight at 37°C and 5% CO2. Cell viability and IC50 were then measured using standard MTS assays.

[0047] Figure 4 The results showed that, as indicated by cell viability measurements, the combination of AY9944 and lovastatin effectively inhibited the cell viability of wild-type colorectal cancer cells. Furthermore, measuring the IC50 value provided a more direct indication of this effect. Figure 5Compared to experimental groups treated with AY9944 or lovastatin alone, or those not treated with a combination of AY9944 and lovastatin, the IC50 of oxaliplatin in wild-type colorectal cancer cell lines treated with the combination of AY9944 and lovastatin was significantly reduced. This indicates that the combination of AY9944 and lovastatin can more effectively reverse the resistance of wild-type colorectal cancer cells to oxaliplatin, thereby further enhancing the therapeutic effect of oxaliplatin chemotherapy against drug-resistant colorectal cancer. Furthermore, the IC50 value was lowest when the concentration of AY9944 was 8 μM and the concentration of lovastatin was 80 μM, meaning that at this specific concentration ratio, colorectal cancer cells showed the best drug sensitivity to oxaliplatin, thus achieving better chemotherapy efficacy.

[0048] Furthermore, in this embodiment, cell viability data was standardized and uploaded to the SynergyFinder online platform, and the combination index was calculated using the Bliss independence model. Synergistic effect index analysis was performed on the combined use of lovastatin and AY9944, revealing that the combination of lovastatin and AY9944 exerts a synergistic effect on colorectal cancer cells, thereby increasing the drug sensitivity to oxaliplatin. Figure 6 This is consistent with the above conclusion.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of lovastatin combined with DHCR7 inhibitors in the preparation of drugs for the treatment of colorectal cancer.

2. The application as described in claim 1, characterized in that, The DHCR7 inhibitor includes AY9944.

3. The application as described in claim 1, characterized in that, The colorectal cancers mentioned include oxaliplatin-resistant colorectal cancers.

4. The application as described in claim 2, characterized in that, The ratio of the effective concentration of AY9944 to that of lovastatin is (1-10):(40-100).

5. The application as described in claim 4, characterized in that, The effective concentration ratio of AY9944 to lovastatin is 8:

80.

6. A drug for treating colorectal cancer, characterized in that, The drugs include lovastatin and / or DHCR7 inhibitors.

7. The drug as described in claim 6, characterized in that, The DHCR7 inhibitor includes AY9944.

8. The drug as described in claim 7, characterized in that, The drug includes lovastatin and AY9944, wherein the effective concentration ratio of AY9944 to lovastatin is (1-10):(40-100).

9. The drug as described in claim 6, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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