ANTI‐NEOPLASTIC EFFECTS OF METFORMIN AGAINST RENAL CLEAR CELL CARCINOMA
Authors: Munusamy Shankar , Pasha Mazhar , Agouni Abdelali 
Published: June 2021

Background and ObjectivesRenal clear cell carcinoma (RCCC) is the most common and lethal form of urological cancer diagnosed globally. Mutation of Von‐Hippel Lindau (VHL) tumor suppresser gene and the resultant overexpression of hypoxia‐inducible factor‐1 alpha (HIF‐1α) protein promotes cell proliferation, angiogenesis and epithelial‐mesenchymal transition (EMT) and leads to RCCC progression and metastasis. Moreover, cancer drug resistance continues to be a major impediment to the treatment of RCCC. Metformin, an activator of AMP‐activated kinase (AMPK), is primarily prescribed for the management of type‐2 diabetes; nonetheless, emerging studies suggest that metformin exerts anti‐neoplastic effects in various types of cancers. Thus, the objectives of this study were to investigate metformin's antineoplastic effects and the signaling pathways modulated by metformin in RCCC using a human renal carcinoma cell line ‐ Caki‐2.MethodsCaki‐2 cells, which lack functional VHL, were treated with metformin (ranging from 1 to 50 mM) for 48 h and assessed for cell viability (using Alamar blue assay), cell cycle progression (using Tali cell cycle kit), cell migration (using in vitro scratch migration technique at 6 and 24 h). Western blotting analysis was performed to assess the effects of metformin on various signaling pathways such as AMPK, mammalian target of rapamycin (mTOR), AKT, HIF‐1α, autophagy (LC3II) pathway and epithelial‐mesenchymal transition or EMT marker (α‐SMA).ResultsFollowing 48 h metformin treatment, a significant and dose‐dependent decrease in cell viability were observed, i.e., 81% in 2 mM, 69% in 5 mM, 60% in 10 mM, 47% in 20 mM and 34% in 50 mM in metformin treated cells compared to control (set as 100%). Similarly, cell cycle analysis revealed a significant and dose‐dependent cell cycle arrest at G0/G1 phase in metformin treated cells compared to control. A prominent decrease in the migration of Caki‐2 cells was noted after 6 and 24 h of metformin treatment (5 mM and 10 mM) compared to control. Western blot analysis revealed a dose‐dependent increase in the levels of phosphorylated (active) AMPK i.e., 115% in 2 mM, 148% in 5 mM and 172% in 10 mM in metformin treated cells in comparison to control (set at 100%). In contrast, a significant decrease in the levels of phosphorylated (active) forms of mTOR (49% in 2 mM, 34% in 5 mM and 20% in 10 mM) and AKT (84% in 5 mM and 66 % in 10 mM) were noted in metformin treated Caki‐2 cells. Moreover, marked downregulation of HIF‐1α expression (62%) and reductions in the levels of EMT marker ‐ α‐SMA (30%) and autophagy marker ‐ LC3II protein (67%) were observed in Caki‐2 cells treated with metformin at 10 mM.ConclusionOur findings indicate that metformin exerts potent anti‐neoplastic and anti‐metastatic effects in RCCC through concerted activation of AMPK pathway and repression of AKT, mTOR, HIF‐1α, LC3II protein and EMT signaling pathways. The modulation of the aforementioned signaling pathways might contribute to the cell cycle arrest at the G0/G1 phase and reductions in cell migration. Together, our study unveils the therapeutic potential of metformin to inhibit the progression of RCCC in vitro.Support or Funding InformationThis study was supported by the intramural research grants from Drake University and Qatar University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.