Effect of membrane cholesterol on oxaliplatin-induced cell death patterns in HepG2 cells

Abstract

Hepatocellular carcinoma (HCC) is known to be resistant to chemotherapy. However, recently several oxaliplatin-based combinatorial treatments have shown a promising anti-tumor activity in patients with HCC. Previously, our group demonstrated oxaliplatin triggered massive necrosis with some apoptosis in HepG2 HCC cells, while it induced mostly apoptosis in HCT116 and HT29 colon cancer cells. As necrosis is closely associated with inflammation, causing tumor progression and malignancy, the regulatory mechanisms underlying death mode determination were investigated. In the present study, we observed that the mild and excessive ROS production due to oxaliplatin caused apoptotic and necrotic death and N-acetyl-L-cysteine (NAC) and superoxide scavengers, BHA and Tiron attenuated necrosis, but the simple alleviation of ROS production did not switch necrosis to apoptosis. On the other hand, addition of cholesterol switched oxaliplatin-induced necrosis to apoptosis, which was reverted by methyl beta cyclodextrin (MBCD), a membrane cholesterol depleting agent, suggesting membrane cholesterol level is a critical factor of cell death mode determination. Apoptosis induced by the combined treatment was evident by HO/PI double staining, semi-thin epon sections, and caspase 8, caspase 3 and parp cleavages. Combined treatment-induced apoptosis was prevented by p53 inhibitor. In addition, cholesterol/oxaliplatin-induced caspase 8 activation, and caspase 3 and parp cleavages were suppressed by CD95/Fas interference. Moreover cholesterol/oxaliplatin-induced CD95/Fas colocalization with lipid rafts was detected by immunofluorescence technique and lipid rafts isolation. In conclusion, ROS plays critical roles in oxaliplatin-induced cell death mode and increased membrane cholesterol level switches oxaliplatin-induced necrosis to apoptosis by caspase 8 activation via p53 pathway and triggering CD95/Fas relocalization and death inducing signaling complex (DISC) formation in HepG2 cells. Since necrosis and subsequent inflammation are implicated in tumor progression and malignancy, our results suggest a potential improved efficacy of oxaliplatin-based chemotherapy by shifting cell death mode from necrosis to apoptosis through enhancing membrane rafts formation.