Use of p2ry2 receptor inhibitors for the preparation of a medicament for the treatment of colorectal cancer and for the sensitization of chemotherapy

Abstract

The application discloses application of a P2RY2 receptor inhibitor in preparation of a drug for treating colorectal cancer and for chemotherapy sensitization, and belongs to the field of biological medicines. The application first discloses that the P2RY2 receptor is specifically highly expressed in colorectal cancer tissues, and drives tumor lipid metabolism remodeling through activation of a SRC-mTOR-STAT3-SREBP1 signal axis, thereby promoting malignant progression of the colorectal cancer and FOLFIRI chemotherapy resistance. Based on this, the application provides a new medical use of the P2RY2 receptor inhibitor alone, in combination with an mTOR inhibitor, in combination with a FOLFIRI chemotherapy scheme, or in combination of the three. Experiments show that the combined medication can significantly synergistically inhibit proliferation of colorectal cancer cells, reverse abnormal accumulation of lipid droplets, effectively reverse FOLFIRI resistance mediated by high expression of P2RY2 in a transplanted tumor model in vivo, and does not increase systemic toxicity of chemotherapy. The application has great significance in the field of colorectal cancer treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of P2RY2 receptor inhibitors in the preparation of drugs for treating colorectal cancer and for enhancing chemotherapy sensitivity. Background Technology

[0002] Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide, with extremely poor clinical prognosis for patients with advanced or metastatic disease. Current core treatments for CRC include platinum-based chemotherapy regimens (such as FOLFOX) and non-platinum-based chemotherapy regimens (such as FOLFIRI). Although these regimens are widely used, primary and acquired chemotherapy resistance, and distant metastasis (especially liver metastasis) remain the core reasons for treatment failure.

[0003] Recent studies have confirmed that lipid metabolism reprogramming is a key metabolic feature of malignant progression in colorectal cancer, which can drive tumor cell proliferation, invasion and metastasis, and chemotherapy resistance. However, there is still a lack of intervention targets in clinical practice that can precisely target lipid metabolism in colorectal cancer and have both high specificity and low toxicity, and an effective combined treatment strategy of "targeting lipid metabolism + chemotherapy sensitization" has not been established.

[0004] P2RY2 receptor is a classic G protein-coupled receptor. Current research has only confirmed its involvement in the regulation of inflammatory responses, and there is no application of it in the treatment of colorectal cancer in clinical practice or scientific research. There are mature small molecule inhibitors of P2RY2 (such as AR-C118925XX), but they are only used as laboratory research tools. Their anti-tumor and chemosensitizing value in colorectal cancer has not been fully explored.

[0005] In summary, there are three major technological gaps in the current clinical treatment of colorectal cancer that urgently need to be addressed: First, the high incidence of resistance to traditional chemotherapy limits the efficacy of first-line chemotherapy regimens, making it impossible to effectively curb the progression and metastasis of advanced tumors; second, there is a lack of specific lipid metabolism targets and drugs suitable for colorectal cancer, making it impossible to precisely intervene in the malignant metabolic characteristics of tumors and achieve chemotherapy sensitization; third, there is a complete lack of new applications for the treatment of colorectal cancer using P2RY2 receptors and their specific inhibitors, and there is currently no technology to utilize P2RY2 receptor inhibitors to target and regulate lipid metabolism in colorectal cancer, reverse chemotherapy resistance, and use them for the treatment of colorectal cancer and to enhance the efficacy of chemotherapy.

[0006] Therefore, there is an urgent need in this field to develop a novel drug that can target and regulate lipid metabolism in colorectal cancer, effectively inhibit the malignant progression of colorectal cancer, and reverse chemotherapy resistance, in order to solve the technical problems of limited efficacy of existing chemotherapy for colorectal cancer, high incidence of drug resistance, and lack of targeted intervention methods. Summary of the Invention

[0007] To address the aforementioned problems in existing technologies, this invention reveals for the first time the specific high expression of the P2RY2 receptor in colorectal cancer tissues. This receptor drives tumor lipid metabolism remodeling by activating the SRC-mTOR-STAT3-SREBP1 signaling axis, thereby promoting malignant progression of colorectal cancer and resistance to FOLFIRI chemotherapy. Based on this, this invention provides novel pharmaceutical uses for P2RY2 receptor inhibitors (preferably AR-C118925XX) alone, in combination with mTOR inhibitors (preferably rapamycin), in combination with FOLFIRI chemotherapy regimens, or in combination of all three. Experimental evidence shows that the combined treatment can significantly and synergistically inhibit colorectal cancer cell proliferation, reverse abnormal lipid droplet accumulation, and effectively reverse P2RY2-mediated FOLFIRI resistance in in vivo xenograft models without increasing systemic toxicity of chemotherapy. This invention provides a precise, highly effective, and low-toxicity combined treatment strategy for colorectal cancer, especially FOLFIRI-resistant colorectal cancer.

[0008] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One of the objectives of this invention is to provide the application of P2RY2 receptor inhibitors in the preparation of drugs for treating colorectal cancer.

[0009] Furthermore, the P2RY2 receptor inhibitor includes AR-C118925XX.

[0010] Furthermore, the drug also contains an mTOR inhibitor.

[0011] Furthermore, the mTOR inhibitors include rapamycin.

[0012] Furthermore, the drug also includes chemotherapy drugs.

[0013] Furthermore, the chemotherapy drug is selected from irinotecan, 5-fluorouracil, or combinations thereof.

[0014] Furthermore, the drug also comprises an mTOR inhibitor and a chemotherapy drug, wherein the mTOR inhibitor includes rapamycin and the chemotherapy drug is selected from irinotecan, 5-fluorouracil, or a combination thereof.

[0015] Furthermore, the drug is an oral or injectable formulation.

[0016] Furthermore, the colorectal cancer includes colorectal cancer that is resistant to or at risk of developing resistance to FOLFIRI chemotherapy regimens.

[0017] A second objective of this invention is to provide the application of reagents for detecting P2RY2 expression levels in the preparation of products for assessing the prognosis of colorectal cancer patients or their responsiveness to FOLFIRI chemotherapy regimens.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) A novel mechanism by which P2RY2 serves as a key target for lipid metabolism in colorectal cancer is revealed for the first time. This invention is the first to discover that the P2RY2 receptor is specifically highly expressed in colorectal cancer tissues, and its expression level is significantly negatively correlated with overall survival (OS), recurrence-free survival (RFS), and progression-free survival (PFS), with the expression level further increasing in metastatic lesions. Mechanistic studies show that P2RY2 activates the SRC-mTOR-STAT3-SREBP1 signaling axis, upregulates SCD1 expression, and induces abnormal lipid droplet accumulation, thereby driving malignant progression of colorectal cancer and resistance to FOLFIRI chemotherapy, providing a novel intervention target for precision treatment of colorectal cancer.

[0019] (2) P2RY2 inhibitors, used alone or in combination, have significant anti-tumor effects. In vivo animal experiments showed that targeted knockdown of P2RY2 significantly inhibited the growth of subcutaneous xenografts in nude mice, resulting in a significant decrease in tumor volume and weight, and a marked reduction in the positive rate of the proliferation marker Ki-67; while P2RY2 overexpression significantly accelerated tumor growth and liver metastasis. The P2RY2-specific inhibitor AR-C118925XX showed moderate inhibitory activity against tumor growth when used alone, and produced a significant synergistic effect when combined with the mTOR inhibitor rapamycin, with a synergistic index CI < 0.7, and the tumor-suppressing effect was significantly better than that of the single-drug group.

[0020] (3) Effective reversal of FOLFIRI chemotherapy resistance in colorectal cancer. Clinical transcriptome data analysis confirmed that high P2RY2 expression was significantly associated with FOLFIRI resistance, but not with FOLFOX resistance. In a P2RY2-overexpressing resistant xenograft model, the antitumor efficacy of FOLFIRI monotherapy was significantly weakened; however, the addition of AR-C118925XX and / or rapamycin significantly restored and enhanced the in vivo antitumor efficacy of FOLFIRI. Among them, the tumor volume reduction rate of the FOLFIRI + AR-C118925XX + rapamycin combination therapy group was more than 70% higher than that of the FOLFIRI monotherapy group, and the proportion of Ki-67 positive cells was significantly reduced, completely reversing the chemotherapy resistance phenotype mediated by high P2RY2 expression.

[0021] (4) The combination therapy regimen has good safety and clinical translation potential. In vivo systemic safety assessment showed that after introducing AR-C118925XX and / or rapamycin into the FOLFIRI chemotherapy regimen, there were no statistically significant differences in body weight changes, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine (Cr) levels in nude mice compared with the FOLFIRI monotherapy group. This confirms that the combination therapy regimen of the present invention does not aggravate the hepatotoxicity and nephrotoxicity of chemotherapy drugs and has good in vivo tolerability and biosafety.

[0022] (5) It expands the new pharmaceutical applications of known small molecule compounds. AR-C118925XX is a reported P2RY2-specific small molecule inhibitor with relatively well-defined pharmacokinetic properties. This invention is the first to apply it to the treatment of colorectal cancer and chemotherapy sensitization. Combined with rapamycin (a marketed mTOR inhibitor) and the FOLFIRI clinical standard chemotherapy regimen, it can significantly reduce development risks and costs, and has high clinical translational value and application prospects. Attached Figure Description

[0023] Figure 1 This study demonstrates that upregulation of P2RY2 expression promotes the progression of colorectal cancer, including: (AB) GO-MF (gene ontology-molecular function) analysis of normal and tumor tissues in the UNION cohort (A) and TCGA and GTEx databases (B). (C) Extracellular ATP levels in tissues. (D) Venn diagram of the intersection of differentially expressed genes (DEGs) associated with P2 purinergic receptors with differentially expressed genes identified by RNA-seq in the UNION cohort (cancer tissue vs. normal tissue). (E) RNA sequencing results of five P2 purinergic receptor subtypes. (F) P2RY2 mRNA expression levels in the UNION cohort and GEO dataset. (G) Representative H&E and IHC (immunohistochemical) staining images of P2RY2 in the UNION cohort, and corresponding IHC scores. (H) Kaplan-Meier curves of OS (overall survival, left panel) and RFS (recurrence-free survival, middle panel) based on P2RY2 expression levels in the GEO database, and PFS (progression-free survival, right panel) in the UNION cohort. (I) Representative IHC staining and corresponding quantitative IHC scores of P2RY2 in different tissues in the UNION cohort. (JK) Images of xenografts, tumor volume, tumor weight, and Ki-67 IHC analysis. (L) Schematic diagram of the spleen injection metastasis model (top). Representative liver images (left), small animal in vivo imaging images (middle), and liver H&E staining (right) at 6 weeks post-injection, as well as quantitative analysis of relative fluorescence levels and metastatic nodules.

[0024] Figure 2This study demonstrates the beneficial effects of P2RY2 inhibitors combined with mTOR inhibitors in improving the chemosensitivity of colorectal cancer, including: (A) the effect of specific P2RY2 inhibitor AR-C, mTOR inhibitor RAPA (rapamycin), or their combination on the viability of a group of human colorectal cancer (CRC) cell lines (SW480, HT-29, HCT116, SW620) using the CCK-8 assay. (B) representative images and quantitative analysis of SW480 colorectal cancer cell colony formation assays after treatment, as shown in Figure A. (C) a heatmap of the inhibition rate of AR-C and RAPA in SW480 colorectal cancer cells, and synergistic effect analysis based on the Bliss independent model and the Loewe additivity model. (D) the accumulation of lipid droplets in SW480 colorectal cancer cells after treatment with AR-C, RAPA, or their combination, detected by BODIPY 493 / 503 staining. (E) Using the public GEO (Gene Expression Comprehensive Database) dataset, the correlation between P2RY2 expression levels and clinical response to FOLFOX and FOLFIRI chemotherapy regimens was analyzed. (F) Response rates of patients to FOLFOX and FOLFIRI chemotherapy regimens were analyzed after stratification based on high and low P2RY2 expression levels.

[0025] Figure 3 This study demonstrates that combined inhibition of P2RY2 and blockade of mTOR can enhance the antitumor efficacy in colorectal cancer, including: (A) Cell viability of SW480 colorectal cancer cells after treatment with SN-38 (the active cytotoxic metabolite of irinotecan IRI) alone, or in combination with AR-C and RAPA alone and in combination, as detected by CCK-8 assay. (B) Representative images and quantitative analysis of SW480 colorectal cancer cell colony formation assay after treatment with the SN-38-based combination therapy, as shown in Figure A. (C) Heatmap of the inhibition rate of the three drugs SN-38, AR-C, and RAPA in SW480 colorectal cancer cells, and synergistic effect analysis based on the Bliss independent model and the Loewe additive model. (D) Nude mouse subcutaneous xenograft models were constructed using SW480 cells overexpressing P2Y2. Representative tumor images, quantitative analysis of tumor weight, and tumor growth curves (tumor volume) were obtained after treatment with solvent (control group), AR-C, RAPA, AR-C+RAPA, FOLFIRI, FOLFIRI+AR-C, FOLFIRI+RAPA, or FOLFIRI+AR-C+RAPA (n = 6 per group). (E) Representative H&E (hematoxylin-eosin) staining and Ki-67 IHC (immunohistochemistry) staining images of SW480 xenograft tissues overexpressing P2Y2, and quantitative analysis of the proportion of Ki-67 positive proliferating cells in each treatment group (n = 9 per group).

[0026] Figure 4This study presents the in vivo antitumor efficacy and systemic safety assessment results of the P2RY2 / mTOR dual inhibition combined with the FOLFIRI regimen in a colorectal cancer xenograft model, including: (A) Representative tumor images, quantitative analysis of tumor weight, and tumor growth curves (n = 6 per group) of SW480 cells constructed using an empty vector control. (B) Representative H&E (hematoxylin and eosin) staining and Ki-67 IHC (immunohistochemistry) staining images of SW480 xenograft tissues, and quantitative analysis of the proportion of Ki-67 positive proliferating cells in each treatment group (n = 9 per group). (C) Weight change curves of nude mice bearing SW480 xenografts after receiving the above-mentioned treatments throughout the in vivo experiment (n = 6 per group). (DF) At the end of the experiment, the levels of aspartate aminotransferase (AST) (D), alanine aminotransferase (ALT) (E) and creatinine (Cr) (F) in the serum of nude mice bearing SW480 xenografts in each treatment group (n = 6 per group). Detailed Implementation

[0027] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0028] I. Core Content of the Invention This invention is the first to demonstrate, through multi-dimensional experiments involving clinical samples, cells, and animals, that the P2RY2 receptor is specifically and highly expressed in colorectal cancer tissues, and that the expression level is significantly positively correlated with poor patient prognosis, tumor metastasis, and chemotherapy resistance. It also elucidates the molecular mechanism for the first time: P2RY2 induces lipid metabolism remodeling in colorectal cancer cells (upregulation of SCD1 expression and abnormal accumulation of lipid droplets) by activating the SRC-mTOR-STAT3-SREBP1 signaling axis, thereby driving tumor proliferation, liver metastasis, and FOLFIRI chemotherapy resistance.

[0029] Based on the above novel findings, this invention proposes novel pharmaceutical applications for P2RY2 receptor inhibitors, specifically including: 1. P2RY2 receptor inhibitors can be prepared alone as drugs for treating colorectal cancer, used to inhibit the growth of colorectal cancer tumors; 2. P2RY2 receptor inhibitors can be used in combination with mTOR inhibitors (such as rapamycin) to prepare synergistic antitumor drugs; 3. P2RY2 receptor inhibitors can be used in combination with chemotherapeutic drugs (such as irinotecan and 5-fluorouracil) to prepare chemotherapy sensitizers for colorectal cancer and reverse chemotherapy resistance; 4. P2RY2 receptor inhibitors can be combined with mTOR inhibitors and chemotherapy drugs to prepare highly effective and low-toxicity combination therapy drugs for colorectal cancer.

[0030] The preferred P2RY2 receptor inhibitor in this invention is AR-C118925XX.

[0031] II. Technical Details and Experimental Support of the Invention This invention provides comprehensive experimental support for its technical solutions through a complete system of clinical sample validation, molecular mechanism analysis, in vitro cell experiments, in vivo animal experiments, and safety evaluation. Specific technical details are as follows: 1. Expression characteristics and prognostic value of P2RY2 in colorectal cancer Clinical samples: Cancer tissue, paired adjacent normal tissue, and metastatic lesions from 47 colorectal cancer patients (UNION cohort) were included and validated using the TCGA, GTEx, and GEO public databases; Detection methods: RT-qPCR, immunohistochemistry (IHC), and bioinformatics analysis; Experimental results: ① The mRNA and protein levels of P2RY2 in colorectal cancer tissues were significantly higher than those in adjacent normal tissues, and the expression level was further increased in metastatic lesions; ② Patients with high P2RY2 expression had significantly shorter overall survival (OS), recurrence-free survival (RFS), and progression-free survival (PFS), making it an independent biomarker for poor prognosis and metastasis risk in colorectal cancer.

[0032] 2. Synergistic effect of combining P2RY2 inhibitors with mTOR inhibitors Experimental models: colorectal cancer cell lines and nude mouse xenograft models; Combination therapy: AR-C118925XX + rapamycin; Verification method: IC 50 Detection, Bliss independent model, Loewe adduct model, lipid droplet staining; Experimental results: The combination of the two drugs exhibited a strong synergistic antitumor effect, with a synergistic index (CI) < 0.7, and significantly reduced the cellular IC50. 50 It blocks lipid droplet accumulation and has a significantly better tumor-suppressing effect than single drugs.

[0033] 3. P2RY2 inhibitors reverse chemosensitizing effects in patients with resistance to FOLFIRI chemotherapy. Clinical relevance: The GEO database confirmed that high expression of P2RY2 was significantly associated with resistance to FOLFIRI chemotherapy, but not with the FOLFOX regimen; In vitro experiments: The combination of AR-C118925XX, rapamycin, and FOLFIRI (irinotecan / 5-FU / leucovorin) significantly reduced the SN-38 (irinotecan active metabolite) IC50. 50 Completely reverses chemotherapy resistance mediated by P2RY2 overexpression; In vivo experiments: In a drug-resistant xenograft model with P2RY2 overexpression, the tumor volume reduction rate of the three-drug combination group was more than 70% higher than that of the FOLFIRI monotherapy group, and the positive rate of the proliferation marker Ki-67 was significantly reduced.

[0034] 4. In vivo safety of the combination therapy regimen Detection indicators: nude mouse body weight, serum liver function (AST / ALT), kidney function (Cr), and histopathology; Experimental results: The combination of AR-C118925XX with rapamycin and FOLFIRI regimen did not increase the liver and kidney toxicity of chemotherapy, and there was no significant weight loss. It has good safety and has clinical translation potential.

[0035] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0036] Example 1: Upregulation of P2RY2 expression promotes the progression of colorectal cancer. I. Methods 1. Experimental Materials 1.1 Clinical Samples A total of 47 patients (UNION cohort) diagnosed with primary colorectal cancer at Union Hospital affiliated with Tongji Medical College of Huazhong University of Science and Technology and the First Affiliated Hospital of Shihezi University between August 2019 and July 2025 were included in this study. Of these, 36 were used for immunohistochemical (IHC) staining, and 6 were used for RNA sequencing. 26 patients provided paired cancerous and adjacent normal tissues. Inclusion criteria: histopathological diagnosis; complete clinicopathological and follow-up data; no prior neoadjuvant therapy (RNA-seq). Exclusion criteria: coexisting primary malignancies; history of inflammatory bowel disease or familial adenomatous polyposis; insufficient tissue sample size.

[0037] 1.2 Public Database TCGA-COAD colorectal cancer dataset, GTEx normal colon tissue dataset, and GEO dataset (GSE6988 and GSE12032 were used for expression validation; GSE28702 and GSE72970 were used for chemotherapy response analysis).

[0038] 1.3 Main Reagents TRIzol reagent (TAKARA, 9108), reverse transcription kit (Vazyme, Q225), SYBR Green qPCR kit (Vazyme, Q311), P2RY2 antibody (Abcam, ab272891), Ki-67 antibody (Cell Signaling, 9449), HRP-labeled secondary antibody (Cell Signaling, 7074).

[0039] 2. Experimental Apparatus

[0040] 3 Experimental Methods 3.1 RNA extraction and real-time quantitative PCR (RT-qPCR) Take 50 mg of frozen tissue, add 1 mL of TRIzol reagent homogenate, and extract total RNA according to the instructions; take 1 μg of total RNA and reverse transcribe it into cDNA; perform qPCR in a 20 μL reaction system, with the following reaction program: 95℃ pre-denaturation for 15 s, then 95℃ for 15 s, 58℃ for 15 s, and 72℃ for 15 s, for a total of 45 cycles. Using ACTB as an internal control, the relative expression level of P2RY2 mRNA was calculated using the 2^-ΔΔCt method. The primer sequences are as follows: P2RY2-F (SEQ ID NO.1): 5'-CGAGGACTTCAAGTACGTGCTG-3' P2RY2-R (SEQ ID NO.2): 5'-GTGACGCATTCCAGGTCTTGA-3' ACTB-F (SEQ ID NO.3): 5'-CACCATTGGCAATGAGCGGTTC-3' ACTB-R (SEQ ID NO.4): 5'-AGGTCTTTGCGGATGTCCACGT-3' 3.2 Immunohistochemical staining and scoring (1) After the tissue was fixed with 4% paraformaldehyde and embedded in paraffin, it was cut into 5μm sections; dewaxed to water, antigen retrieval was performed at high temperature with citrate buffer, endogenous peroxidase was blocked with 3% H2O2, and 5% BSA was blocked at room temperature for 1h; P2RY2 primary antibody (1:200 dilution) was added and incubated at 4℃ overnight; the next day, HRP-labeled secondary antibody (1:1000 dilution) was added and incubated at room temperature for 1h; DAB staining was performed, hematoxylin counterstaining was performed, dehydrated and cleared, and neutral resin was mounted.

[0041] (2) Semi-quantitative analysis was performed by two independent pathologists using the Immunological Response Score (IRS): Positive cell percentage score (0: no positive cells; 1: <10%; 2: 10%-50%; 3: 51%-80%; 4: >80%) × staining intensity score (0: no staining; 1: pale yellow; 2: brownish yellow; 3: brownish brown).

[0042] 3.3 Construction of a subcutaneous xenograft model in nude mice (1) Construction of recombinant vector: pLenti-CMV-Puro was selected as the backbone plasmid and the full-length coding sequence of human P2RY2 (Accession: NM_176072.3) was directionally inserted into the multiple cloning site to construct the overexpression vector (named P2RY2-OE), with the original empty vector plasmid as the control. The recombinant clones were bidirectionally sequenced using the Sanger method to confirm that the inserted sequence was highly consistent with the target template.

[0043] (2) Lentiviral packaging and titer enrichment: When HEK-293T cells are in the logarithmic growth phase (confluence approximately 70%~80%), PEI-mediated three-plasmid co-transfection technology is used to package HCFC1. OE Alternatively, control plasmids and helper packaging elements (pMD2.G, psPAX2) were introduced into cells. The culture medium was replaced 6 hours after transfection to reduce cytotoxicity, and the culture was maintained for 72 hours. Subsequently, the virus-rich culture medium was collected, centrifuged at 3000 rpm, clarified using a 0.45 μm pore size filter, and the virus was enriched by ultracentrifugation sedimentation. Construction of the P2RY2 knockdown lentiviral vector: The shRNA sequence targeting the human BCLAF1 gene was designed and synthesized by Shanghai Jikai Biotechnology Co., Ltd., and inserted into the GV112 lentiviral vector. The target sequence is shown in the table below. Simultaneously, a nonsense sequence negative control lentivirus (scramble shRNA) was constructed.

[0044] (3) Screening and enrichment of stable cell lines: HT-29 and SW480 colon cancer cells were selected as target cells, and lentiviruses were introduced at a confluence of 40%–50%. To enhance infection efficiency, 10 μg / mL polybrene was added to the transfection system. 72 hours after transfection, long-term resistance screening was performed using 4 μg / mL puromycin. After approximately 7 days of continuous pressure culture and medium replacement, a cell population with stable expression characteristics was finally obtained.

[0045] (4) SPF-grade male BALB / c-nu / nu nude mice (4-6 weeks old, weighing 18-22g) were selected and randomly divided into experimental and control groups after 5-7 days of acclimatization. After successful screening of stable transgenic strains, cells were collected by digestion with 0.25% trypsin-EDTA, washed 2-3 times with PBS, and resuspended in sterile PBS to adjust the cell density to 2×10⁻⁶. 7 Cells / mL. Each nude mouse was subcutaneously inoculated with 100 μL of cell suspension (2 × 10⁶ cells / mL) in the right or left axilla. 6 (1 cell), insert the needle at a 30° angle to ensure subcutaneous penetration, and slowly inject to prevent extravasation. After needle removal, gently press the puncture site with a sterile dry cotton swab for 1 minute. Observe the nude mice's mental state, weight, and injection site daily after inoculation. Starting from the 7th day after inoculation, measure the tumor's long diameter (a) and short diameter (b) every 2-3 days using electronic calipers, and calculate using the formula V = 0.5 × a × b. 2 Tumor volume was calculated, and the experiment continued until the tumor volume reached 800-1500 mmHg. 3 The experiment ended when the nude mice showed obvious signs of weight loss.

[0046] 3.4 Survival Analysis Patients were divided into high-expression and low-expression groups based on the median P2RY2 expression. The Kaplan-Meier method was used to plot the curves for overall survival (OS), recurrence-free survival (RFS), and progression-free survival (PFS), and the log-rank test was used to compare the differences between the groups.

[0047] II. Results To investigate the molecular alterations in colorectal cancer (CRC), this invention utilizes a clinical cohort (UNION cohort) and public databases (TCGA, GTEx) to conduct comparative transcriptomic analyses of normal colon tissue and colorectal cancer tissue. Gene ontology-molecular function (GO-MF) enrichment analysis revealed significant dysregulation of molecular functions related to adenosine triphosphate (ATP) binding in colorectal cancer tissue. Figure 1 AB). Extracellular ATP (eATP) level detection showed that the eATP concentration in the extracellular matrix of colorectal cancer cells was significantly higher than that in normal colon tissue. Figure 1 C). Given that P2 purinergic receptors mediate exogenous ATP binding, we systematically analyzed their subtype expression patterns and screened for significantly differentially expressed subtypes: P2RX2, P2RX5, P2RY2, P2RY10, and P2RY13. Among them, only P2RY2 was significantly upregulated in colorectal cancer tissues, while P2RX2, P2RX5, P2RY10, and P2RY13 were all significantly downregulated. Figure 1 DE).

[0048] In the UNION cohort, the P2RY2 mRNA level in colorectal cancer tissues was significantly higher than that in paired adjacent normal tissues; this multicenter expression feature was validated in an independent dataset (GEO database GSE6988). Figure 1 F). Clinical phenotype association analysis showed that patients with colorectal cancer who had distant metastases or postoperative recurrence had significantly higher levels of P2RY2 expression in their tumor tissues than patients without metastases or recurrence. Figure 1 F). Immunohistochemical (IHC) staining showed that P2RY2 was strongly positive in colorectal cancer tissue, and its semi-quantitative histological score was significantly higher than that of adjacent normal tissue. Figure 1 G). Survival analysis showed that patients with high P2RY2 expression had significantly shorter overall survival (OS), relapse-free survival (RFS), and progression-free survival (PFS). Figure 1 H). Furthermore, high levels of P2RY2 protein expression are also maintained in metastatic colorectal cancer tissues (H). Figure 1 I). The above results confirm the stability and specificity of P2RY2 as a biomarker for poor prognosis and malignant metastasis.

[0049] To elucidate the direct regulatory effect of P2RY2 on tumor growth in vivo, this invention used previously constructed P2RY2-knockdown HT29 cells and P2RY2-overexpressing SW480 stable transgenic cells, along with their control cells, to subcutaneously in nude mice to establish a xenograft model. In vivo experimental results showed that, compared to the control group, targeted knockdown of P2RY2 in human colorectal cancer HT-29 cells (a P2RY2-overexpressing cell line) significantly inhibited the growth of xenografts in vivo, resulting in a substantial decrease in tumor volume and weight, and a significant reduction in the positive rate of the proliferation marker Ki-67. Figure 1 J). Conversely, overexpression of P2RY2 in human colorectal cancer SW480 cells (a P2RY2-low expression strain) significantly accelerated tumor growth, dramatically increased tumor volume and weight, and enhanced cell proliferation activity. Figure 1 K), and liver metastases (K), and liver metastases ( Figure 1 These results confirm, at the in vivo level, the technical effect of P2RY2 in promoting the malignant progression and cell proliferation of colorectal cancer.

[0050] Example 2: Beneficial effects of P2RY2 inhibitors combined with mTOR blockers in improving chemosensitivity in colorectal cancer. I. Methods 1. Experimental Materials 1.1 Cell lines Normal human colonic epithelial cells NCM460, human colorectal cancer cell lines HT-29 (high expression of P2RY2), SW480 (low expression of P2RY2), HCT116, SW620, and human embryonic kidney cells HEK-293T were all purchased from ATCC in the United States.

[0051] 1.2 Main Reagents DMEM medium (Gibco), fetal bovine serum (Gibco), puromycin (MCE, HY-B1743), CCK-8 kit (Dojindo, CK04), Transwell chamber (Corning, 8μm), BODIPY 493 / 503 (MCE, HY-W090090), triglyceride assay kit (Solarbio, BC0625), AR-C118925XX (P2RY2 inhibitor, MCE, HY-110126), rapamycin (RAPA, mTOR inhibitor, MCE, HY-10219), irinotecan (IRI, MCE, HY-16562), SN-38 (irinotecan active metabolite, MCE, HY-13704), 5-fluorouracil (5-FU, MCE, HY-90006), leucovorin calcium (MCE, HY-13664).

[0052] 1.3 Lentiviral vector pLKO.1-TRC knockdown vector (Addgene #10878), plenti-CMV-Puro overexpression vector (Addgene #17448), packaging plasmid pMD2.G (Addgene #12259), and psPAX2 (Addgene #12260).

[0053] 2 Experimental Methods 2.1 Cell Culture HEK-293T, SW480, HCT116, HT-29, and SW620 cells were cultured in DMEM complete medium under the following conditions: 37°C, 5% CO2, and saturated humidity.

[0054] 2.2 Construction of stable cell lines (1) Knockdown cell line: The shRNA sequence targeting P2RY2 was cloned into the pLKO.1-TRC vector to construct LV-shP2RY2; the non-targeting shRNA (Sigma, SHC016) was used as a negative control (LV-shNC).

[0055] (2) Overexpression cell line: The full-length CDS sequence of human P2RY2 was cloned into the plenti-CMV-Puro vector to construct LV-OE-P2RY2; the empty vector was used as a negative control (LV-Vector).

[0056] 2.3 Lentiviral Packaging and Infection HEK-293T cells were co-transfected with the target plasmid and the packaging plasmid at a ratio of 4:3:1. After 72 hours, the viral supernatant was collected and filtered through a 0.45 μm filter membrane. 10 μg / mL Polybrene was added when infecting target cells. After 48 hours, stable cell lines were obtained by screening with 4 μg / mL puromycin for one week.

[0057] 2.4 Cell viability assay (CCK-8) Logarithmic growth phase cells were fed at 3 × 10 3 Cells were seeded per well in 96-well plates and cultured for 24 h. Then, different concentrations (AR-C and RAPA intervention concentration gradients: 0.0064, 0.032, 0.16, 0.8, 4, 20, 100 μmol / L) of drugs were added for 72 h. 10 μL of CCK-8 reagent was added to each well and incubated at 37 °C for 1 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0058] 2.5 Cloning Experiment Cells were loaded at a rate of 1×10 3 Clones were inoculated per well in a 24-well plate. After 24 hours of drug treatment, the medium was replaced with fresh medium, and the medium was changed every 3 days. After 14 days, the cells were fixed with 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet for 20 minutes. The number of clones was counted using ImageJ software.

[0059] 2.6 Lipid metabolism detection (1) Lipid droplet staining: Cells were seeded on coverslips and cultured to 70% confluence; fixed with 4% paraformaldehyde for 15 min and washed 3 times with PBS; 5 μmol / L BODIPY 493 / 503 was added and incubated at 37℃ in the dark for 30 min; nuclei were stained with Hoechst 33342 for 10 min; lipid droplet morphology and number were observed under a confocal microscope.

[0060] (2) Triglyceride quantification: Collect 1×10 6 For each cell, add 1 mL of chloroform-methanol mixture (2:1) to homogenize and extract lipids; follow the instructions of the triglyceride assay kit to detect the absorbance value at 540 nm and calculate the triglyceride concentration.

[0061] II. Results To confirm the effect of targeted inhibition of P2RY2 on the chemosensitivity of colorectal cancer (CRC), this invention combined the P2RY2-specific inhibitor AR-C118925XX (AR-C) with the mTOR signaling pathway inhibitor rapamycin (RAPA) in a group of human colorectal cancer cell lines. In vitro experimental results showed that, compared with AR-C or RAPA monotherapy, the combined inhibition of P2RY2 and mTOR exhibited a significant synergistic antitumor effect: the half-maximal effector concentration (IC50) of the tested cells was significantly reduced, and long-term clonogenic ability was severely inhibited. Figure 2 AB). Mathematical evaluation using Bliss independent models and Loewe additive models further confirmed a strong synergistic biological effect between AR-C and RAPA. Figure 2 C). Furthermore, cellular biochemical assays confirmed that dual inhibition of P2RY2 / mTOR was significantly more effective than either drug alone in blocking lipid droplet accumulation in colorectal cancer cells. Figure 2 D).

[0062] To elucidate the correlation between P2RY2 expression levels and response to first-line chemotherapy regimens in colorectal cancer, this invention employed a systematic analysis of clinical transcriptomic data from the public GEO database. The results showed no statistically significant difference in P2RY2 transcription levels between patients treated with the FOLFOX regimen in the sensitive and resistant groups; however, in colorectal cancer patients insensitive to the FOLFIRI regimen (resistant), P2RY2 expression was significantly pathologically upregulated. Figure 2 E). Stratified analysis of clinical response rates consistently showed that patients in the high P2RY2 expression group had a significantly lower response rate to FOLFIRI chemotherapy than those in the low expression group ( Figure 2 F). The above clinical facts confirm that high expression of P2RY2 is a key molecular indicator and potential intervention target that mediates clinical resistance to the FOLFIRI regimen in colorectal cancer.

[0063] Example 3: Combined inhibition of P2RY2 and blocking of mTOR can enhance the anti-tumor efficacy of colorectal cancer. I. Methods 1. Main Reagents AR-C118925XX (P2RY2 inhibitor, MCE, HY-110126), Rapamycin (RAPA, mTOR inhibitor, MCE, HY-10219), Irinotecan (IRI, MCE, HY-16562), SN-38 (irinotecan active metabolite, MCE, HY-13704), 5-fluorouracil (5-FU, MCE, HY-90006), leucovorin calcium (MCE, HY-13664).

[0064] 2 Experimental Methods 2.1 Cell viability assay (CCK-8) Logarithmic growth phase cells were fed at 3 × 10 3 Cells were seeded per well in 96-well plates and cultured for 24 h. Different concentrations of the drug were then added for 72 h of treatment. The SN-38 intervention concentration gradient was: 0.5, 1, 2, 5, 10, 50, 100 nmol / L. 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37℃ for 1 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0065] 2.2 Cloning Experiment Cells were loaded at a rate of 1×10 3 Clones were inoculated per well in a 24-well plate. After 24 hours of drug treatment, the medium was replaced with fresh medium, and the medium was changed every 3 days. After 14 days, the cells were fixed with 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet for 20 minutes. The number of clones was counted using ImageJ software.

[0066] 2.3 In vitro drug synergistic experiment: (1) SW480, HT-29, HCT116, and SW620 cells were seeded in 96-well plates and treated with different concentrations of AR-C, RAPA, and SN-38, either alone or in combination, for 72 h. The concentration gradient of SN-38 was 0.5, 1, 2, 5, 10, 50, and 100 nmol / L. Cell viability was detected by the CCK-8 assay, and IC50 was calculated. 50 value.

[0067] (2) The synergistic effect of drugs was evaluated by using the Bliss independent model and the Loewe additive model. The synergistic index CI<1 indicates the existence of a synergistic effect.

[0068] 2.4 In vivo pharmacodynamic experiments 2×10 6 One OE-P2RY2 SW480 cell was injected into the right back of a nude mouse; when the tumor volume reached 200 mm², the tumor was induced to rupture. 3 At that time, stratified randomization was used to divide the data into 8 groups (n=6): Control group: Intraperitoneal injection of 0.9% saline, once every 3 days; AR-C group: 10 mg / kg, intraperitoneal injection, once every 3 days; RAPA group: 2 mg / kg, intraperitoneal injection, once daily; AR-C+RAPA group: AR-C 10mg / kg once every 3 days + RAPA 2mg / kg once daily; FOLFIRI group: 5-FU 50mg / kg + IRI 25mg / kg + leucovorin calcium 90mg / kg, intraperitoneal injection, once every 3 days; FOLFIRI+AR-C group: FOLFIRI regimen + AR-C 10mg / kg once every 3 days; FOLFIRI+RAPA group: FOLFIRI regimen + RAPA 2mg / kg once daily; FOLFIRI+AR-C+RAPA group: FOLFIRI regimen + AR-C 10mg / kg once every 3 days + RAPA 2mg / kg once daily.

[0069] The intervention period was 2 weeks, with tumor volume and mouse weight measured every 2 days. At the end of the experiment, the mice were sacrificed, the tumors were removed and weighed, and the tumor tissues were stained with H&E and Ki-67 IHC.

[0070] II. Results To confirm whether targeted inhibition of P2RY2 can reverse resistance to the FOLFIRI regimen in colorectal cancer (CRC), this invention conducted combination tests in human colorectal cancer cell lines. Cell viability assays showed that, compared to the single use of irinotecan hydrochloride (IRI)'s active cytotoxic metabolite SN-38, the combination of SN-38 with the P2RY2-specific inhibitor AR-C118925XX (AR-C) or the mTOR inhibitor rapamycin (RAPA) significantly reduced the half-maximal effective concentration (IC50) of SN-38; and the three-drug combination regimen consisting of SN-38, AR-C, and RAPA achieved the most potent growth-inhibiting activity in colorectal cancer cells. Figure 3 A). Clonogenic assays further confirmed that the long-term clonogenic capacity of the test cells in the three-drug combination group was reduced to the lowest level. Figure 3 B); Mathematical pharmacodynamic synergistic model analysis also confirmed that the three-drug combination regimen exhibits strong synergistic sensitization activity within the system. Figure 3 C).

[0071] To verify in vivo whether P2RY2 overexpression directly mediates clinical resistance to FOLFIRI chemotherapy in colorectal cancer, this invention constructed a subcutaneous xenograft model of SW480 cells overexpressing P2RY2 in nude mice. In vivo pharmacodynamic experiments showed that in xenografts with high P2RY2 expression, the antitumor efficacy of conventional FOLFIRI monotherapy was significantly reduced. Figure 3 (D) confirmed the causal relationship between upregulated P2RY2 expression and FOLFIRI resistance. However, the introduction of AR-C and / or RAPA into the drug delivery system significantly restored and enhanced the in vivo antitumor efficacy of the FOLFIRI regimen. Among them, the three-drug combination therapy group consisting of FOLFIRI + AR-C + RAPA exhibited the strongest tumor growth inhibition effect, effectively reversing the chemotherapy resistance phenotype mediated by P2RY2 overexpression (see appendix). Figure 3 D). Immunohistochemical (IHC) staining of tumor tissue sections further showed that the proportion of Ki-67-positive cells in the triple-drug combination therapy group was dramatically downregulated. Figure 3 (E) In vivo pathological studies have confirmed that this combination therapy regimen can effectively block the proliferation and evolution of drug-resistant tumor cells. These results confirm that the combination therapy strategy described in this invention has significant clinical efficacy in enhancing chemosensitivity and reversing drug resistance.

[0072] Example 4: Evaluation of in vivo antitumor efficacy and systemic safety of P2RY2 / mTOR dual inhibition combined with FOLFIRI regimen in a colorectal cancer xenograft model. I. Methods 1. Experimental Materials 1.1 Laboratory Animals Specific pathogen-free (SPF) grade BALB / c male thymus-deficient nude mice, aged 4-6 weeks and weighing 18-22g, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were housed in an SPF-grade animal facility at an ambient temperature of 22±1℃ and a relative humidity of 55%±5%, with a 12h light / 12h dark cycle, and were allowed free access to sterilized feed and sterile water.

[0073] 1.2 Cell line construction and culture (1) Construction of recombinant vector: pLenti-CMV-Puro was selected as the backbone plasmid and the full-length coding sequence of human P2RY2 (Accession: NM_176072.3) was directionally inserted into the multiple cloning site to construct the overexpression vector (named P2RY2-OE), with the original empty vector plasmid as the control. The recombinant clones were bidirectionally sequenced using the Sanger method to confirm that the inserted sequence was highly consistent with the target template.

[0074] (2) Lentiviral packaging and titer enrichment: When HEK-293T cells are in the logarithmic growth phase (confluence approximately 70%~80%), PEI-mediated three-plasmid co-transfection technology is used to package HCFC1. OE Alternatively, control plasmids and helper packaging elements (pMD2.G, psPAX2) could be introduced into cells. The culture medium was refreshed 6 hours after transfection to reduce cytotoxicity, and the cells were maintained for 72 hours. Subsequently, the virus-rich culture medium was collected, centrifuged at 3000 rpm, clarified by filtering through a 0.45 μm pore membrane, and the virus was enriched by ultracentrifugation sedimentation.

[0075] (3) Screening and enrichment of stable cell lines: SW480 colon cancer cells were selected as target cells, and lentivirus was introduced at a confluence of 40%–50%. To enhance infection efficiency, 10 μg / mL polybrene was added to the transfection system. 72 hours after transfection, long-term resistance screening was performed using 4 μg / mL puromycin. After approximately 7 days of continuous pressure culture and medium replacement, a cell population with stable expression characteristics was finally obtained.

[0076] Cells were cultured in DMEM medium containing 10% fetal bovine serum and placed in a 37°C, 5% CO2 saturated humidity incubator. Cells in the logarithmic growth phase were used for animal inoculation.

[0077] 1.3 Main Reagents AR-C118925XX (P2RY2 inhibitor, MCE, HY-110126), Rapamycin (RAPA, mTOR inhibitor, MCE, HY-10219), Irinotecan (IRI, MCE, HY-16562), SN-38 (irinotecan active metabolite, MCE, HY-13704), 5-Fluorouracil (5-FU, MCE, HY-90006), Leucovorin (MCE, HY-13664) Tissue fixation and staining reagents: 4% paraformaldehyde fixative, hematoxylin staining solution, eosin staining solution, DAB staining kit; Antibodies: Rabbit anti-human Ki-67 monoclonal antibody and horseradish peroxidase (HRP)-labeled goat anti-rabbit universal secondary antibody.

[0078] 1.4 Biochemical Detection Kit Serum aspartate aminotransferase (AST) assay kit, serum alanine aminotransferase (ALT) assay kit, serum creatinine (Cr) assay kit.

[0079] 1.5 Main Instruments Electronic vernier calipers, 1 / 100,000 electronic analytical balance, high-speed refrigerated centrifuge, fully automated biochemical analyzer, optical microscope, paraffin sectioner, tissue dehydrator, embedding machine, and constant temperature incubator.

[0080] 2 Experimental Methods 2.1 In vitro drug synergistic experiment (1) SW480, HT-29, HCT116 and SW620 cells were seeded in 96-well plates and treated with different concentrations of AR-C, RAPA and SN-38 alone or in combination for 72 h; cell viability was detected by CCK-8 assay and IC50 was calculated. 50 value.

[0081] (2) The synergistic effect of drugs was evaluated by using the Bliss independent model and the Loewe additive model. The synergistic index CI<1 indicates the existence of a synergistic effect.

[0082] 2.2 Construction of a subcutaneous xenograft model in nude mice (1) Take SW480-Vector cells in the logarithmic growth phase, digest them with 0.25% trypsin-EDTA, wash them twice with sterile PBS, centrifuge and resuspend them, and count them; adjust the cell concentration to 2×10⁶ cells. 6 One hundred μL of DMEM cell suspension was injected subcutaneously into the right lateral back of each nude mouse. The first seven days after cell injection were the tumor incubation period, during which no drug intervention was applied, and the condition of the nude mice and the formation of subcutaneous nodules were routinely observed.

[0083] (2) Tumor volume measurement and stratified randomization: Observe tumor growth daily, measure the long diameter (L) and short diameter (W) of the tumor using electronic calipers, and calculate the tumor volume (V) using the following formula: When the average volume of subcutaneous xenografts reaches 200 mm 3 (Recorded as day 0 after drug administration and day 7 after cell injection), stratified randomization was performed using a computer-generated random number table to divide tumor-bearing nude mice into 8 groups of 6 mice each, ensuring that there was no statistically significant difference in initial tumor volume among the groups (P>0.05).

[0084] 2.3 Dosing regimen All groups received intraperitoneal injection (ip) for 14 consecutive days. The specific dosage and frequency of administration are as follows: Control group (Vehicle): 0.9% sterile saline solution, once every 3 days; AR-C monotherapy group: AR-C118925XX (10 mg / kg), once every 3 days; RAPA monotherapy group: rapamycin (2 mg / kg), once daily; AR-C+RAPA combination group: AR-C (10 mg / kg, once every 3 days) + RAPA (2 mg / kg, once daily). FOLFIRI chemotherapy group: 5-FU (50mg / kg) + irinotecan hydrochloride (25mg / kg) + leucovorin calcium (90mg / kg), once every 3 days; FOLFIRI+AR-C combination group: standard FOLFIRI regimen + AR-C (10 mg / kg, once every 3 days); FOLFIRI+RAPA combination group: standard FOLFIRI regimen + RAPA (2 mg / kg, once daily). The three-drug combination group consisted of the standard FOLFIRI regimen plus AR-C (10 mg / kg every 3 days) plus RAPA (2 mg / kg once daily).

[0085] 2.4 Monitoring of tumor growth kinetics and systemic tolerance During the drug administration intervention, the weight of nude mice was measured every 2 days to plot the dynamic change curve of weight and assess the systemic tolerance of the drug; at the same time, the long and short diameters of the tumor were measured every 2 days to calculate the volume and plot the tumor growth curve.

[0086] 2.5 Specimen Collection at Experimental Endpoint On day 14 of drug administration (day 22 after cell injection), nude mice were deeply anesthetized, and whole blood was collected using either the retro-orbital venous plexus method or the cardiac blood collection method. After the whole blood was allowed to coagulate naturally at room temperature, it was centrifuged at 3000 rpm for 15 minutes at 4°C, and the supernatant serum was separated and stored at -80°C for later use. After blood collection, the nude mice were euthanized humanely using cervical dislocation, and the subcutaneous transplanted tumor tissue was completely dissected. After gross photography, the net weight of the tumor was measured using a 1 / 100,000 electronic analytical balance.

[0087] 2.6 Serum biochemical toxicology tests Frozen serum from each group was collected, and the biochemical test kit instructions were strictly followed. The levels of AST, ALT, and Cr in the serum were detected using a fully automated biochemical analyzer to quantitatively assess the systemic effects of the drug on the liver and kidney function of nude mice.

[0088] 2.7 Tumor histopathological examination (1) Tissue processing and section preparation Tumor tissue was cut into small pieces, fixed in 4% paraformaldehyde solution for 48 hours, dehydrated by gradient ethanol, cleared with xylene, and embedded in paraffin to prepare continuous paraffin sections with a thickness of 5 μm.

[0089] (2) Hematoxylin-eosin (H&E) staining Paraffin sections were dewaxed with xylene, hydrated with graded ethanol, stained with hematoxylin and eosin, dehydrated, cleared, and mounted with neutral resin; the pathological morphology, cell necrosis and tissue structure integrity of the tumor tissue were observed under an optical microscope.

[0090] (3) Ki-67 immunohistochemical (IHC) staining Paraffin sections were dewaxed to water and subjected to high-temperature, high-pressure antigen retrieval using citrate buffer. Incubation with 3% H2O2 at room temperature for 10 min blocked endogenous peroxidase activity; Blocking with 5% bovine serum albumin (BSA) at room temperature for 30 minutes reduces non-specific binding; Add rabbit anti-human Ki-67 monoclonal primary antibody (1:200 dilution) and incubate overnight at 4°C; After rewarming, wash with PBS, add HRP-labeled universal secondary antibody, and incubate at room temperature for 1 h; DAB developing solution for color development in the dark, hematoxylin for light counterstaining, and then dehydrated, clear, and mounted with neutral resin.

[0091] (4) Quantitative analysis and statistical processing of pathological images The results were interpreted by two independent pathologists using a double-blind method; nine 400x high-power fields were randomly selected from each slide, and the number of Ki-67 positive cells and the total number of cells were counted using professional image analysis software, and the percentage of Ki-67 positive cells was calculated.

[0092] Quantitative data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, and Tukey's multiple comparison test was used for multiple comparisons among groups. P < 0.05 was considered statistically significant.

[0093] II. Results To confirm in vivo the antitumor efficacy of the P2RY2 inhibitor AR-C118925XX (AR-C), the mTOR inhibitor rapamycin (RAPA), and their respective combinations with the FOLFIRI chemotherapy regimen, this invention constructed a nude mouse subcutaneous xenograft model using empty vector control SW480 cells. In vivo pharmacodynamic results showed that AR-C or RAPA monotherapy had moderate inhibitory activity against tumor growth, while the combination of AR-C and RAPA exhibited a significantly superior in vivo antitumor effect compared to the monotherapy group. Compared to FOLFIRI monotherapy, the introduction of AR-C or RAPA into the FOLFIRI regimen further enhanced its antitumor therapeutic effect; among them, the three-drug combination therapy group consisting of FOLFIRI + AR-C + RAPA achieved the most potent in vivo tumor growth inhibitory activity. Figure 4 AB).

[0094] In vivo systemic safety assessments showed that, compared with the solvent control group and the AR-C or RAPA monotherapy groups, all FOLFIRI-containing groups of nude mice exhibited mild but statistically significant weight loss, and moderately elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. However, no statistically significant differences were observed in weight change, serum AST, and ALT levels between the FOLFIRI monotherapy group and the various FOLFIRI-based combination therapy groups. Figure 4 CE). Meanwhile, biochemical tests showed that serum creatinine levels in all treatment groups remained within the normal physiological range, and no signs of nephrotoxicity were detected. Figure 4F). The above facts confirm that the introduction of AR-C and / or RAPA into the FOLFIRI chemotherapy regimen does not exacerbate systemic toxicity caused by chemotherapy drugs in vivo, and demonstrate that the combination therapy regimen described in this invention has excellent tolerability and biosafety in vivo.

[0095] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of P2RY2 receptor inhibitors in the preparation of drugs for treating colorectal cancer.

2. The application according to claim 1, characterized in that, The P2RY2 receptor inhibitors include AR-C118925XX.

3. The application according to claim 1, characterized in that, The drug also contains an mTOR inhibitor.

4. The application according to claim 3, characterized in that, The mTOR inhibitors include rapamycin.

5. The application according to claim 1, characterized in that, The drug also includes chemotherapy drugs.

6. The application according to claim 5, characterized in that, The chemotherapy drug is selected from irinotecan, 5-fluorouracil, or a combination thereof.

7. The application according to claim 1, characterized in that, The drug also contains an mTOR inhibitor and a chemotherapy drug, wherein the mTOR inhibitor includes rapamycin and the chemotherapy drug is selected from irinotecan, 5-fluorouracil, or a combination thereof.

8. The application according to claim 1, characterized in that, The drug is an oral or injectable formulation.

9. The application according to claim 1, characterized in that, The colorectal cancers mentioned include those resistant to or at risk of developing resistance to FOLFIRI chemotherapy regimens.

10. Application of reagents for detecting P2RY2 expression levels in the preparation of products for assessing the prognosis of colorectal cancer patients or their response to FOLFIRI chemotherapy regimens.

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