TITLE: Regulation of apoptosis inducing factor (AIF) expression in liver tumor cells by hepatic stellate cells present in hepatocellular carcinoma (HCC) microenvironment
Objectives: Tumor cells regulate expression of key cellular survival proteins which help in modulating tumor microenvironment (TME), escaping immune surveillance, and promoting cancer growth. Previously, we demonstrated that hepatic stellate cells (HSCs) present in TME induce expression of heat shock protein 70 (hsp70) in tumor cells. HSP70 upregulation has been associated with poor HCC prognosis and increased protein/enzyme function and stability, which in turn promotes cell survival and drug resistance. Apoptosis inducing factor (AIF) is another important protein initially known as a caspase-independent programmed cell death effector that triggers apoptosis by DNA fragmentation following release from mitochondria and translocation to nucleus; however, recent data shows that it is vital to mitochondrial role in healthy cells. Here, we assessed the effect of HSCs, in addition to hsp70, on AIF expression in liver tumor cells. We also checked the expression of AIF in tumor/non-tumor tissue samples of human and rat livers.
Methods: For cellular analysis, rat tumorigenic cell line N1S1 cultures were exposed to HSC conditioned media (1ml and 3ml concentrates) for 24 hours. AIF expression in tissue and cellular experiments was evaluated by Western blotting and immunocytochemistry. For human tissue analysis, frozen samples of explanted livers from 12 patients with established HCC (who underwent liver transplant without any prior therapy) were used and compared to the non-HCC tissue samples from the same liver. For rat tissue analysis, 16 frozen samples were taken from harvested livers (including 8 healthy livers and 8 hepatoma models).
Results: AIF expression in N1S1 rat hepatoma cells was substantially higher in presence of HSC conditioned media. No meaningful difference was noted in AIF expression between tumor and non-tumor tissue samples in human or rat livers.
Conclusions: HSCs present in HCC microenvironment modulate AIF expression in tumor cells. In human tissue samples, existence of different pathologies such as cirrhosis or pre-cancerous states may explain the lack of any differences in AIF expression between tumor and non-tumor groups. Proper understanding of the mechanisms involved and interactions between AIF and other important proteins including hsp 70 is a crucial step towards development of adjuvants to chemoradiotherapeutic interventions in hepatocellular carcinoma (HCC).