The increased TRAIL by one clone can be secreted into the local microenvironment, thus giving the soluble TRAIL an opportunity to exhibit its bystander effect to trigger apoptosis in the otherwise metformin-insensitive clones. expression significantly attenuated metformin-induced apoptosis. Studies with a tumor xenograft model revealed that metformin not only significantly inhibited tumor growth but also elicited apoptosis and enhanced TRAIL expression and models of solid tumors, including breast cancer and NSCLC.21 Despite its encouraging preclinical results, dulanermin has failed to demonstrate significant efficacy in clinical trials.22,23 This failure is in part due to dulanermins short half-life and weak activity to induce clustering of TRAIL-Rs.24,25 In addition, recombinant TRAIL, including dulanermin, has the potential to develop anti-drug antibody (ADA) responses, which may be responsible for liver toxicity.26,27 Thus, induction of endogenous TRAIL is believed to be able to overcome the limitations, and it has become a new strategy to harness the TRAIL-TRAIL-R system for identifying more effective treatments for human cancers.26 Previous studies have reported that induction of endogenous TRAIL triggers tumor-selective death and sensitizes cancer cells to chemotherapy in various human cancers.28, 29, 30 Metformin, a safe and Bz-Lys-OMe commonly prescribed drug for type II diabetes, possesses promising therapeutic activity Bz-Lys-OMe in various Bz-Lys-OMe human cancers, including TNBC and NSCLC.31,32 Nonetheless, the mechanism of action of metformin in suppressing tumor growth remains elusive.33,34 We have reported that metformin selectively induces apoptosis in TNBC cells, likely through a caspase-8-initiated caspase cascade, 35 suggesting that metformin might trigger extrinsic apoptosis signaling in TNBC cells. In the current study, we have explored the capability of metformin to enhance endogenous TRAIL expression in TNBC and NSCLC cells and investigated whether TRAIL-induced apoptosis plays a critical role in metformin-mediated antitumor activity. Results Metformin inhibited viability of TNBC and NSCLC cells via induction of apoptosis. To investigate whether metformin would exhibit a similar anti-proliferative/anti-survival effect on NSCLC cells, as we observed in TNBC cells,35 both short-term cell proliferation (MTS) and long-term clonogenic assays were performed in three NSCLC cell lines (H460, H1650, and A549) treated with different concentrations of metformin. First, we confirmed our previous results showing that metformin potently inhibited cell proliferation and colony formation in TNBC cells (Figure?S1). Then, we found that metformin significantly inhibited proliferation of NSCLC cells in a dose-dependent manner (Figure?1A). In clonogenic assays, metformin dramatically suppressed NSCLC colony formation at concentrations as low as 0.5?mM. Colony numbers decreased upon metformin treatment in a dose-dependent manner (Figure?1B). Therefore, TNBC and NSCLC cells seemed to show a similar sensitivity to metformin-mediated inhibition regarding cell viability. Open in a separate window Figure?1 Metformin inhibited proliferation and survival of NSCLC cells (A) NSCLC cells (H460, H1650, and A549) were plated onto 96-well plates with 0.1?mL RPMI 1640 medium containing 10% FBS. After 24 h, the culture medium was replaced with fresh medium with 5% FBS as control, or the same medium with 5% FBS containing indicated concentrations of metformin, and incubated for an additional 48 h. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by MTS assays. Data show a representative of three independent experiments. Bars, SD. ?p? 0.05, ??p? 0.01, ???p? 0.001. (B) NSCLC cells (H460, H1650, and A549) were plated onto 6-well plates in triplicate in 2?mL p54bSAPK of medium containing 10% FBS. After 24 h, the culture medium was replaced with fresh medium with 5% FBS as control or the same medium with 5% FBS containing indicated concentrations of metformin. The culture medium was changed every 3?days for 2?weeks. Representative images of the clonogenic assay for each cell line were taken by a digital Bz-Lys-OMe camera on day 14 (top panel) and its relevant quantification of the number of colonies was performed using the ImageJ Software (bottom panel). Bars, SD. ?p? 0.05, ??p? 0.01,.
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