J Cancer 2020; 11(13):3701-3712. doi:10.7150/jca.36372

Research Paper

Metformin in combination with JS-K inhibits growth of renal cell carcinoma cells via reactive oxygen species activation and inducing DNA breaks

Yuwan Zhao*, Qiuming Luo*, Jierong Mo*, Jianwei Li, Dongcai Ye, Zhixian Ao, Lixin Chen, Jianjun Liu

Laboratory of Urology, Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong 524001, China
*Contributed equally

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Citation:
Zhao Y, Luo Q, Mo J, Li J, Ye D, Ao Z, Chen L, Liu J. Metformin in combination with JS-K inhibits growth of renal cell carcinoma cells via reactive oxygen species activation and inducing DNA breaks. J Cancer 2020; 11(13):3701-3712. doi:10.7150/jca.36372. Available from https://www.jcancer.org/v11p3701.htm

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Abstract

Metformin (MET) is taken as a principal medication for remedying Type 2 diabetes mellitus. Its anti-tumor effect has been reported increasingly, but the precise mechanism of it remains unclear. This study aims to explore the efficacy of MET and MET combined with nitric oxide donor prodrug JS-K on the proliferation, apoptosis, and DNA damage in human renal cell carcinoma (RCC) cells, and investigate the possible molecular mechanism involved. The cell proliferation was tested through methyl-tetrazolium assay and cell apoptosis was ascertained by flow cytometry. The dihydroethidium and JC-1 fluorescent methods were used to detect Reactive oxygen species (ROS) and mitochondrial transmembrane potential (Δψm), respectively. Proteins associated with apoptosis and DNA damage were evaluated by Western blotting. Results showed that MET and JS-K could suppress cell growth, and the inhibition concentration 50 of treatment with MET combined with JS-K (MET + JS-K) showed more toxicity than individual agents on RCC cells. This augmented toxicity was associated with intracellular reactive oxygen species (ROS) level, mitochondrial membrane potential alteration, and induced DNA breaks. The results of Western blotting showed that the expression level of pro-apoptotic proteins, such as Bax, Bak, caspase-3, and caspase-9, was up-regulated, and the anti-apoptotic protein Bcl-2 was down-regulated after treatment using MET alone and MET + JS-K, correspondingly. Moreover, MET + JS-K inhibited the expression of cellular PCNA and Rad51, and immunofluorescence analysis of γH2AX proved that MET + JS-K enhanced DNA damage. In summary, the results of this research indicated that MET and JS-K inhibited RCC cell growth by activating ROS, targeting mitochondria-dependent apoptotic pathways, and inducing DNA breaks.

Keywords: metformin, JS-K, renal cell carcinoma, ROS, DNA breaks