Gypenoside L and Gypenoside LI Inhibit Proliferation in Renal Cell Carcinoma via Regulation of the MAPK and Arachidonic Acid Metabolism Pathways

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  FIGURE 1. Gypenoside L (Gyp L) and gypenoside LI (Gyp LI) inhibit proliferation and induce apoptosis of clear cell renal cell carcinoma (ccRCC) cells. (A) CCK8 assays were performed to detect the cell viability of 769-P and ACHN cells after different doses of Gyp L and Gyp LI treatment for 48 h. (B) The Colony formation assays were used to detect clonogenicity of 769-P and ACHN cells after Gyp L and Gyp LI treatment. (C) The apoptosis of 769-P and ACHN cells treated with Gyp L and Gyp LI was analyzed via flow cytometry. (D) Hoechst 33258 was used for ACHN and 769-P cell apoptosis detection, stained, and observed with a fluorescence microscope. (E) The protein levels of Bax, Bcl2, and cytochrome C were detected by Western blotting experiments. Data are presented as the mean ± SD of expression levels from three independent experiments (*p < 0.05, vs. control group).

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  FIGURE 2. Gyp L and Gyp LI induced cell cycle arrest in 769-P and ACHN cells. (A) Gyp L and Gyp LI arrest cell cycle at G2/M phase in 769-P, while G1/S phase arrest after Gyp L and Gyp LI treatment in ACHN, were determined by flow cytometry. (B) Statistical graph of three independent repeated experiments. (C) The protein levels of cyclin A and B1, CDK1, and CDK2 were detected by Western blotting. Data are expressed as the mean± SD of expression levels from three independent experiments (*p < 0.05, vs. control group).

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  FIGURE 3. Analyses of the key targets and pathways of Gyp L and Gyp LI inhibiting ccRCC cells through network pharmacology and RNA-seq. (A) Venn diagram summarizing the intersection targets between Gyp L, Gyp LI, and renal cell carcinoma (RCC). (B) Protein interaction network diagram of 49 intersection targets was constructed through the cytoscape platform. (C) Gene ontology (GO) analysis of 49 targets in terms of biological processes, cell components, and molecular functions. (D) The KEGG pathway that Gyp L and Gyp LI affect the RCC process analyzed by R language. (E, F) Unsupervised hierarchical clustering of mitogen-activated protein kinase (MAPK) (E,F) arachidonic acid.

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  FIGURE 4. Expression of MAPK and arachidonic acid metabolism signaling-related genes and the reduced content of AA after Gyp L and Gyp LI treatments. (A, B) The expressions of arachidonic acid metabolism signaling-related genes COX7A, PLA2G4, ALOX5, CYP2E1 and MAPK pathway-related genes DUSP1, FOS, JUN in 769-P (A) and ACHN (B) cells were detected by RT-qPCR. (C) The expression of MAPK-related proteins after Gyp L and Gyp LI treatments were detected via Western blotting. (D) The expression of arachidonic acid metabolism signaling-related proteins after Gyp L and Gyp LI treatments were detected via Western blotting. (E) The expression of cPLA2 after Gyp L and Gyp LI treatment was detected by immunofluorescence assays. (F) The levels of arachidonic acid in 769-P and ACHN treated with Gyp L and Gyp LI and in tumor tissues after gypenoside treatment were measured by liquid chromatography-mass spectrometry (LC/MS). Data are presented as the mean ± SD of expression levels from three independent experiments (*p < 0.05, vs. control group).

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  FIGURE 5. The supplement of AA rescued the killing effect of Gyp L and LI. (A) Cell viability of 769-P and ACHN cells following treatment with different doses of Gyp L and Gyp LI for 48 h, supplemented with or without AA. (B) Clonogenic assays of 769-P and ACHN cells treated with different doses of Gyp L and Gyp LI under conditions, supplemented with or without AA. (C) Cell apoptosis of 769-P and ACHN after treatment with Gyp L and LI, with or without AA detected by flow cytometry. Data are presented as the mean ± SD of expression levels from three independent experiments (n.s., not significant; *p < 0.05, vs. control group).

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  FIGURE 6. Gypenosides suppressed the growth of RCC cell xenograft tumors in vivo. (A) Nude mice were treated with saline or gypenosides for 28 days, and pictures of nude mice and tumors were obtained. (B) Changes in tumor weight after gypenoside administration, compared with the control group. (C) Changes in tumor volume after administration of gypenosides, compared with the control group. (D) The body weight of the two groups of mice during the entire experiment. (E) HE staining of mice liver and tumors. (F) The differences in the protein expression levels of Ki67, cPLA2, CYP1A1, and COX2 between the two tumor groups were detected by immunohistochemistry.

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  FIGURE 7. The molecular mechanism of gypenoside L and gypenoside LI inducing apoptosis in renal cell carcinoma.