Effect of S-adenosyl-L-methionine (SAM), an allosteric activator of cystathionine-β-synthase (CBS) on colorectal cancer cell proliferation and bioenergetics in vitro.

Introduction Several series of in vitro and in vivo data presented at the current Meeting support the conclusion that colon cancer cells selectively overexpress CBS, which produces H 2 S, to maintain cellular bioenergetics, thereby supporting tumor growth and to promote angiogenesis and vasorelaxation. These studies, taken together, identify CBS-derived H 2 S as a tumor promoting factor and a potential future anticancer drug target. The purpose of the current study was to investigate the effect of S-adenosyl- l -methionine (SAM) on the proliferation and bioenergetics of the colon cancer cell line HCT116 in vitro . The non-transformed, the non-tumorigenic colon epithelial cell line NCM356, derived from the normal margin of a rectal cancer specimen, was used as a control. Methods HCT116 cells were cultured in McCoy’s 5A medium, while NCM356 cells were cultured in DMEM supplemented with 10% fetal bovine serum. For assessment of cell proliferation, the xCELLigence system (Roche) was used. For the measurement of bioenergetic function, the XF24 Extracellular Flux Analyzer (Seahorse) was used. Oxygen consumption rate (OCR) after oligomycin (1.5 μg/ml) was used to assess ATP production rate and OCR after FCCP (0.5 μM) to assess maximal mitochondrial respiratory capacity. 2-deoxyglucose (100 mM) was used to estimate cellular glycolytic dependency and antimycin A (2 μg/m) and rotenone (2 μM) were used to inhibit the flux of electrons through complex III and I, to detect residual non-mitochondrial activity. Results Addition of SAM (0.1–1 mM) to HCT116 cells (which show a high expression of CBS) induced a concentration-dependent increase in cell proliferation between 0–50 h, while higher concentrations of SAM (3 mM) inhibited cell proliferation. However, at time periods between 50–100 h, the cells treated with intermediary concentrations of SAM (0.3–1 mM) showed a suppression of cell proliferation compared to the vehicle control; the proliferation-enhancing effect of SAM was only maintained throughout the entire experimental period with the 0.1 mM concentration of SAM. In contrast to HCT116 cells, NCM356 cells (which have only a low-level expression of CBS) were proliferating at a substantially slower rate, and SAM failed to stimulate their proliferation. Extracellular Flux Analysis of the effect of acute exposure of HCT116 cells to SAM (0.1–1 mM) induced a concentration-dependent increase in their oxygen consumption and bioenergetic function, while in NCM356 cells SAM failed to enhance oxygen consumption and cellular bioenergetics. Longer-term exposure of HCT116 cells to both low concentrations of SAM (0.1 mM) and high concentrations of SAM (1 mM) suppressed cellular bioenergetic responses. Conclusions Studies presented at the current meeting demonstrate that the H 2 S-producing enzyme CBS is selectively overexpressed in human colorectal cancer cells, and significantly contributes to their proliferation, migration and invasion in vitro . The present data, demonstrating that short-term exposure of SAM to HCT116 cells exerts proliferative and positive bioenergetic effects that are in line with the effects of exogenously administered H 2 S in the same experimental system further support the view that CBS in colon cancer cells produces H 2 S in a regulated fashion, and that this H 2 S serves as an endogenous cancer cell proliferation and bioenergetic factor. Higher local concentrations of H 2 S are known to be antiproliferative, at least in part due to their inhibitory on mitochondrial function. Therefore, the longer-term inhibition of cell proliferation and bioenergetic function noted with SAM in the present experiments may either be attributed to the adverse autocrine effects of high concentrations of H 2 S, produced when CBS is ‘over-activated’ by SAM, or, alternatively, may be related to additional, CBS-independent pharmacological effects of SAM.