Differential Selectivity of the Renal Clear Cell Carcinoma Cell Lines to the Antineoplastic Effects of Metformin
Authors: Munusamy Shankar , Pasha Mazhar , Frantz Ronald , Agouni Abdelali 
Published: June 2021

Background

Renal clear cell carcinoma (RCCC) accounts for 3–5% of all cancers among US adults. Mutations in the Von‐Hippel Lindau (VHL) tumor suppresser gene and the resultant overexpression of hypoxia‐inducible factor‐1 alpha (HIF‐1α) protein has been shown to increase cell proliferation and deregulate autophagy in renal cells. Sustained HIF‐1α expression was also shown to promote epithelial‐mesenchymal transition (EMT) and angiogenesis, and ultimately result in RCCC progression and metastasis. Metformin (a widely used antidiabetic drug) and an activator of AMP‐activated kinase (AMPK) was demonstrated to exert anti‐neoplastic effects in various types of cancers. In this study, we investigated the antineoplastic effects and the signaling pathways modulated by metformin in RCCC using two human renal carcinoma cell lines – Caki‐1 and Caki‐2, which express wild‐type and mutated VHL respectively.

Methods

Caki‐1 and Caki‐2 cells treated with metformin (ranging from 1 to 50 mM) for 48 h were assessed for alterations in cell viability (using Alamar blue assay), cell cycle progression (using Tali cell cycle kit), and cell migration (using in vitro scratch migration technique at 6 and 24 h). Protein markers of signaling pathways such as hypoxia (HIF‐1α), autophagy (LC3II), and EMT (alpha‐smooth muscle actin, α‐SMA) were assessed via western blotting followed by densitometric analysis.

Results

Our findings demonstrate a differential response between the two RCC cell lines studied, with Caki‐2 cells being ten‐times more sensitive to metformin's cytotoxic and anti‐proliferative effects compared to Caki‐1 cells, i.e., 2 mM vs. 20 mM metformin to produce 18% cell death, and 5 mM vs. 50 mM metformin to induce a significant cell cycle arrest at G0/G1 phase in Caki‐2 cells as compared to Caki‐1 cells. In contrast, the Caki‐1 cells were more sensitive to metformin‐induced suppression of EMT (evidenced by a reduction in the expression of α‐SMA, an EMT marker) as compared to Caki‐2 cells, i.e., 5 mM vs. 10 mM metformin required to produce 36% reduction in α‐SMA expression in Caki‐1 and Caki‐2 cells respectively. Intriguingly, both cells were equally sensitive to metformin‐induced inhibition of cellular migration and autophagy‐marked by reduced expression of LC3II, a marker of autophagy. In addition, Caki‐2 cells, which constitutively express HIF‐1α, showed a remarkable reduction in HIF‐1α expression following metformin treatment (at 10 mM concentration).

Conclusion

Our study findings not only demonstrate the therapeutic potential of metformin to inhibit the progression of RCCC in vitro but also reveal the differential sensitivities of renal carcinoma cells to the antineoplastic effects of metformin ‐ indicating the complexity of this cancer. Further studies in vivo and humans are required to ascertain the therapeutic efficacy of metformin against RCCC.

Support or Funding Information

This study was supported by the grants to Dr. Shankar Munusamy from the Iowa Space Grant Consortium (2017–2018 Early Career Investigator Research Program ‐ award # 4222051A), Drake University (Kresge endowment and provost funds), and Qatar University (QUUG‐CPH\2017‐2).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.