Hep88 mAb-initiated paraptosis-like PCD pathway in hepatocellular carcinoma cell line through the binding of mortalin (HSPA9) and alpha-enolase

Abstract

Background: Hepatocellular carcinoma (HCC) is the most prevalent hepatic cancer worldwide. Currently, a targeted therapy via monoclonal antibodies (mAbs) specific to tumor-associated antigen is undergoing continual development in HCC treatment. Methods: In this regard, after establishing and consequently exploring Hep88 mAb's tumoricidal effect on hepatocellular carcinoma cell line (HepG2 cell line), the Hep88 mAb's specific antigens from both membrane and cytoplasmic fractions of HepG2 cell line were identified by 2-D gel electrophoresis and western blot analysis. After in-gel digestion and subsequent analysis by liquid chromatography-mass spectrometry (LC-MS), mortalin (HSPA9) and alpha-enolase were identified. The recombinant proteins specific to Hep88 mAb were cloned and expressed in E. coli BL21(DE3). Moreover, alteration of HepG2 and Chang liver cell line after being induced by Hep88 mAb for 1-3 days was investigated using a transmission electron microscope. Results: The result demonstrated that Hep88 mAb can bind to the recombinant mortalin (HSPA9) and alpha-enolase. In addition, the gradual appearing of mitochondria vacuolization and endoplasmic reticulum dilatation were observed. Those characteristics might be explained by the paraptosis-like program cell death (PCD), which is induced by the binding of Hep88 mAb to mortalin (HSPA9). Mortalin depletion resulting from the formation of Hep88 mAb-mortalin (HSPA9) complex might initiate transcription-independence of p53-mediated apoptosis. Additionally, Hep88mAb-alpha- enolase complex might initiate HepG2 cells energy exhaustion by glycolysis pathway obstruction. Conclusion: These fascinating results imply that Hep88 mAb might be a promising tool for the development of an effective treatment of HCC in the next decade.