Gypenoside LI modulates autophagy-associated apoptosis in clear cell renal cell carcinoma by PPARG/PTEN/PI3K/AKT/mTOR/ULK1 signaling network

Objective This study was conducted to comprehensively evaluate the anti-clear cell renal cell carcinoma (ccRCC) activity of gypenoside LI (Gyp LI) through in vitro and in vivo models; elucidate the specific molecular pathways mediating the induction of apoptosis and autophagy in Gyp LI-treated ccRCC cells, focusing on the regulatory role of Gypenoside LI in upregulating PPARG and coordinately modulating the PTEN/PI3K/AKT/mTOR-ULK1 signaling network; and render experimental evidence and theoretical support to develop Gyp LI as a novel anti-ccRCC agent. Materials and Methods Human ccRCC cell lines (769-P and ACHN) and a BALB/c nude mouse xenograft model were used for this study. The suppressive effect of Gyp LI on ccRCC cell proliferation was assessed by colony formation assay, Cell Counting Kit-8 (CCK-8) assay, wound healing assay, and Hoechst staining. Autophagy-related ultrastructural changes in treated cells were observed by transmission electron microscopy. Western blot and quantitative real-time PCR (RT-qPCR) were conducted to evaluate fluctuations in the expression levels of autophagic and apoptotic process markers. Putative therapeutic targets associated with Gyp LI were predicted utilizing a network pharmacology framework. Concurrently, the transcriptional control exerted by PPARG over the PTEN gene was validated by dual-luciferase reporter systems and chromatin immunoprecipitation combined with quantitative PCR. To clarify the regulatory relationships within the signaling pathway, we performed in vitro rescue experiments using target gene knockdown. Additionally, a subcutaneous xenograft model in nude mice was established for assessing the in vivo antitumor activity and biosafety of Gyp LI. Apoptosis-, autophagy-, and pathway-related protein expression in tumor tissues was assessed by Western blotting (WB) assay. Results CCK-8 reagent-based cell viability assay revealed that Gyp LI dose-dependently inhibited 769-P and ACHN cell growth, with 24-h IC 50 values of 62.57 and 79.30 μM, respectively. Colony formation, wound healing, and Hoechst staining assay results collectively showed that Gyp LI suppressed cell proliferation and migration and exerted apoptotic effects. Morphological and molecular assays showed that Gyp LI-treated ccRCC cells exhibited prominent autophagosomes and autolysosomes, with upregulation of the autophagy-related proteins Beclin1, ATG5, and LC3 I/II and downregulation of P62. The antiapoptotic factor Bcl-2 exhibited substantially reduced expression, while the proapoptotic mediators caspase-3 and Bax were upregulated, these findings indicated concurrent autophagy-related alterations and apoptosis in Gyp LI-treated ccRCC cells. WB and RT-qPCR assays revealed that Gyp LI upregulated the mRNA and protein expression of the transcription factor PPARG. PTEN transcription was stimulated by the specific binding of PPARG to its promoter region. The resulting intracellular accumulation of PTEN effectively blocked abnormal activation associated with the PI3K/AKT/mTOR signaling axis and ultimately modulated autophagy-associated apoptosis through the phosphorylation-mediated activation of ULK1. ULK1 knockdown reversed the autophagic phenotype modulated by Gyp LI. Moreover, PPARG knockdown reversed Gyp LI-mediated PTEN upregulation, PI3K/AKT/mTOR pathway inhibition, and ULK1 phosphorylation, while attenuating Gyp LI-modulated autophagy and apoptosis. In vivo experiments showed that oral administration of Gyp LI significantly inhibited xenograft tumor growth but did not affect body weight in nude mice. WB assay further confirmed increased expression of proapoptotic and autophagy-related proteins and decreased expression of phosphorylated AKT/mTOR pathway proteins in Gyp LI-treated tumor tissues. However, further studies are required to fully elucidate the autophagic flux and the direct binding targets. Conclusion This study revealed that Gyp LI promotes cell death associated with autophagic alterations in ccRCC cell lines. Mechanistically, this phenomenon is driven by upregulating PPARG and coordinately modulating the PTEN/PI3K/AKT/mTOR-ULK1 signaling network, but the exact causal interdependencies and comprehensive biosafety profile warrant further investigation before any clinical translation. The in vivo efficacy suggests that Gyp LI could serve as promising candidates for plant-derived therapeutic interventions for treating RCC.