The Role And Mechanism Of Alpinetin In Regulating The Proliferation And Migration Of Renal Clear Cell Carcinoma

Objective:This study aims to analyze the network pharmacology of alpinetin(ALP)in treating renal clear cell carcinoma(ccRCC),focusing on the target and molecular mechanism.Method:The Swiss Target Prediction database and Pharmapper database were employed to investigate the relationship between ALP and ccRCC,and to identify the core target of ALP intervention in ccRCC.Based on R software,GO and KEGG enrichment analysis of key target genes was carried out using Bioconductor bioinformatics package.Results:Database analysis yielded 987 ccRCC disease targets,341 alpinetin drug targets,and 82 drug-disease common targets.Additionally,we generated a "disease-target-component" interaction network diagram and a drug-disease common target protein interaction network.).PPI topology analysis revealed GAPDH,HRAS,SRC,EGFR,and AKT1 as the primary alpinetin targets.GO analysis of the 82 common targets demonstrated an intersection gene set enrichment in 1721 biological processes,52 cellular components,and 111 molecular functions.KEGG signaling pathway enrichment analysis was performed on the 82 common targets,resulting in 145 signaling pathways.KEGG enrichment analysis indicated that the PI3K/AKT signaling pathway is the core signaling pathway for alpinetin treatment of ccRCC.Consequently,the PI3K/AKT signaling pathway was selected as the core pathway for cellular and animal experimental studies.Conclusion:Network pharmacology analysis identified HRAS,SRC,EGFR,and AKT1 as the primary ALP targets in ccRCC treatment,with ALP exhibiting an antitumor role through the PI3K/AKT signaling pathway.Objective:This study investigates the signaling pathway through which alpinetin(ALP)modulates the proliferation and migration of renal clear cell carcinoma(ccRCC)cells in vitro,and uncovers the specific regulatory mechanism.Method:Flow cytometry and Cell Counting Kit-8(CCK-8)were utilized to assess cell proliferation and the cell cycle.Cell migration analysis was performed using a 24-well transwell chamber and ibidi scratch insertion.Western blotting was employed to detect the protein expression of signaling pathways and related target molecules.Results:Alpinetin(Oμm,50μm,100μm,and 200μm)was found to inhibit ccRCC cell proliferation in a dose-dependent manner,with higher concentrations exhibiting stronger inhibitory effects.The CCK-8 assay results also demonstrated a similar time-and dose-dependent inhibition effect.Compared to the Oμm group,alpinetin at a concentration of 100μm reduced the proliferation of 786-O and OS-RC-2 cells to approximately 20%on day 4,significantly inhibiting ccRCC cell proliferative capacity.The transwell experiment showed a significant reduction in the number of cells penetrating the transwell membrane after alpinetin treatment in 786-O and OS-RC-2 cells.Likewise,the wound-healing(scratch)assay results indicated a significant decrease in the migration abilities of 786-O and OS-RC-2 cells after alpinetin treatment.Alpinetin treatment was found to substantially promote apoptosis,with more pronounced effects at higher concentrations.Concurrently,cell cycle results demonstrated that alpinetin treatment could block ccRCC cell division in the G1 phase,thus inhibiting cell cycle progression and cell proliferation.To determine whether alpinetin’s effect on ccRCC is associated with the PI3K/AKT/mTOR pathway,we employed western blotting to detect changes in the protein expression of the PI3K/AKT/mTOR pathway in ccRCC cells after alpinetin treatment.The results revealed that the expressions of p-PI3K,p-AKT,and p-mTOR were significantly decreased in 786-O and OS-RC-2 cells following alpinetin treatment,with more pronounced decreases at higher concentrations.Conclusion:In vitro cell experiments demonstrated that alpinetin induces tumor cell apoptosis,negatively regulates the proliferation and migration of tumor cells,and operates through the PI3K/AKT/mTOR signaling pathway.Objective:To investigate the antitumor effect and mechanism of alpinetin in nude mice.Method:Six-week-old mice were utilized in the tumor xenotransplantation experiment(six per group)and a control group and an experimental group.In the experimental group,106 OSRC-2 cells in 200μL PBS were subcutaneously injected into the mice,while the control group received a 200 μL PBS injection.Mice in the experimental group were intraperitoneally injected with a dose of 100 mg/kg alpinetin every three days,and tumor volume was measured with a caliper.Four weeks after OSRC-2 cell injection,the mice were euthanized.The tumor was weighed,and immunohistochemical staining was performed with paraformaldehyde.Western blotting was employed to detect the expression of signaling pathway proteins in extracted tissue proteins.Results:Following the successful establishment of the in vivo tumorigenic model,gross specimen analysis and tumor growth curve comparisons revealed a 66.67%reduction in tumor volume and a 50%decrease in tumor weight after alpinetin injection compared to the NC group.Immunohistochemical experiments were conducted on the samples,showing a significant decrease in the expression of tumor proliferation marker Ki-67 and a significant increase in the expression of tumor suppressor marker p27 after alpinetin treatment.Finally,tissue proteins were analyzed using western blotting,which demonstrated that the expression of phosphorylated PI3K,phosphorylated AKT,and phosphorylated mTOR in OS-RC-2 cell tissue was significantly reduced in the experimental group after alpinetin injection,consistent with the in vitro results from earlier experiments.In vivo experiments indicated that alpinetin can inhibit tumor growth through the PI3K/AKT/mTOR signaling pathway.Conclusion:Animal experiments confirmed that alpinetin negatively regulates the proliferation and migration of tumor cells through the PI3K/AKT/mTOR signaling axis.