Abstract
Metformin (Met) exhibits anticancer ability in various cancer cell lines. This report aims to explore the exact molecular mechanism of Met-induced apoptosis in HCT116 cells, a human colorectal cancer cell line. Met-induced reactive oxygen species (ROS) increase and ROS-dependent cell death accompanied by plasma membrane blistering, mitochondrial swelling, loss of mitochondrial membrane potential, and release of cytochrome c. Western blotting analysis showed that Met upregulated Bak expression but downregulated Bax expression. Most importantly, silencing Bak instead of Bax inhibited Met-induced loss of mitochondrial membrane potential, indicating the key role of Bak in Met-induced apoptosis. Live-cell fluorescence resonance energy transfer (FRET) analysis showed that Met unlocked the binding of Mcl-1 to Bak, and enhanced the binding of Bim to Bak and subsequent Bak homo-oligomerization. Western blotting analysis showed that Met enhanced AMPK phosphorylation and Bim expression, and compound C, an inhibitor of AMPK, inhibited Met-induced Bim upregulation. Although Met increased the expression of Bcl-xL, overexpression of Bcl-xL did not prevent Met-induced apoptosis. In summary, our data demonstrate for the first time that Met promotes ROS-dependent apoptosis by regulating the Mcl-1-Bim-Bak axis.
Highlights
Metformin (Met), a biguanide derivative derived from herbal medicine and being used as the first-line oral drug for the treatment of type 2 diabetes, has been demonstrated to prevent a variety of tumors [1]
Our observations that silencing Bak but not Bax by shRNA significantly prevented Met-induced loss of mitochondrial membrane potential (Fig. 3D) demonstrate that Bak, but not Bax, plays a key role in Met-induced apoptosis, which is further verified by Met-increased Bak homo-oligomerization (Fig. 3F)
Microscopic imaging of cells expressing CFP-Bax showed that Met did not induce Bax translocation from cytosol to mitochondrion, which suggests that Bax is not involved in Met-induced apoptosis
Summary
Metformin (Met), a biguanide derivative derived from herbal medicine and being used as the first-line oral drug for the treatment of type 2 diabetes, has been demonstrated to prevent a variety of tumors [1]. Epidemiological and clinical studies have shown that Met is associated with the reduction of cancer incidence and cancer-related death [1, 2]. Met is thought to have two potential anti-tumor pathways: AMPKdependent and AMPK-independent pathways. In the AMPKdependent pathway, Met directly inhibits mitochondrial respiratory chain complex I to limit oxidative phosphorylation, thereby reducing the amount of ATP production, and increasing AMP/ATP ratio and AMPK phosphorylation and activation. In the AMPKindependent pathway, Met reduces insulin and insulin-like growth factor-1 [5, 6]. The exact mechanism by which Met reduces tumor growth is still unclear
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