Melatonin suppresses Akt/mTOR/S6K activity, induces cell apoptosis, and synergistically inhibits cell growth with sunitinib in renal carcinoma cells via reversing Warburg effect

Metabolic alteration drives renal cell carcinoma (RCC) development, while the impact of melatonin (MLT), a neurohormone secreted during darkness, on RCC cell growth and underlying mechanisms remains unclear. We detected concentration of metabolites through metabolomic analyses using UPLC-MS/MS, and the oxygen consumption rate was determined using the Seahorse Extracellular Flux analyzer. We observed that MLT effectively inhibited RCC cell growth both in vitro and in vivo. Additionally, MLT increased ROS levels, suppressed antioxidant enzyme activity, and induced apoptosis. Furthermore, MLT treatment upregulated key TCA cycle metabolites while reducing aerobic glycolysis products, leading to higher oxygen consumption rate, ATP production, and membrane potential. Moreover, MLT treatment suppressed phosphorylation of Akt, mTOR, and p70 S6 Kinase as well as the expression of HIF-1α/VEGFA in RCC cells; these effects were reversed by NAC (ROS inhibitors). Conversely, MLT synergistically inhibited cell growth with sunitinib and counteracted the Warburg effect induced by sunitinib in RCC cells. In conclusion, our results indicate that MLT treatment reverses the Warburg effect and promotes intracellular ROS production, which leads to the suppression of Akt/mTOR/S6K signaling pathway, induction of cell apoptosis, and synergistically inhibition of cell growth with sunitinib in RCC cells. Overall, this study provides new insights into the mechanisms underlying anti-tumor effect of MLT in RCC cells, and suggests that MLT might be a promising therapeutic for RCC.

代谢重编程驱动肾细胞癌（renal cell carcinoma, RCC）的发生发展，而黑暗时段分泌的神经激素褪黑素（melatonin, MLT）对肾癌细胞生长的影响及其潜在机制仍不明确。本研究采用超高效液相色谱-串联质谱（UPLC-MS/MS）进行代谢组学分析以检测代谢物浓度，并使用海马细胞外通量分析仪（Seahorse Extracellular Flux analyzer）测定细胞耗氧率。研究结果显示，褪黑素可在体外及体内有效抑制肾癌细胞的增殖。此外，褪黑素可升高活性氧（ROS）水平、抑制抗氧化酶活性并诱导细胞凋亡。进一步研究发现，褪黑素处理可上调三羧酸（TCA）循环关键代谢物的水平，同时降低有氧糖酵解产物的含量，进而提升细胞耗氧率、ATP生成量与膜电位。此外，褪黑素处理可抑制肾癌细胞中Akt、mTOR及p70 S6激酶的磷酸化水平，同时下调HIF-1α/VEGFA的表达；上述效应可被NAC（活性氧抑制剂）逆转。与之相反，褪黑素与舒尼替尼（sunitinib）协同抑制肾癌细胞增殖，并抵消舒尼替尼诱导的肾癌细胞沃伯格效应（Warburg effect）。综上，本研究结果表明，褪黑素处理可逆转沃伯格效应、促进细胞内活性氧生成，进而抑制Akt/mTOR/S6K信号通路、诱导细胞凋亡，并与舒尼替尼协同抑制肾癌细胞增殖。总而言之，本研究为褪黑素在肾癌细胞中的抗肿瘤效应机制提供了新的见解，并提示褪黑素有望成为肾细胞癌的潜在治疗手段。