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

Panadda Rojpibulstit; Suthathip Kittisenachai; Songchan Puthong; Sirikul Manochantr; Pornpen Gamnarai; Sarawut Jitrapakdee; Sittiruk Roytrakul

doi:10.1186/s12935-014-0069-9

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

Cancer Cell Int. 2014 Jul 22:14:69. doi: 10.1186/s12935-014-0069-9. eCollection 2014.

Authors

Panadda Rojpibulstit¹, Suthathip Kittisenachai², Songchan Puthong³, Sirikul Manochantr⁴, Pornpen Gamnarai⁴, Sarawut Jitrapakdee⁵, Sittiruk Roytrakul²

Affiliations

¹ Faculty of Medicine, Thammasat University (Rangsit Campus), Khlong Luang 12121, Pathum Thani, Thailand ; Department of Preclinical Science, Faculty of Medicine, Thammasat University, Pathum Thani 12120, Thailand.
² Thailand National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Phahonyothin Road, Khlong Luang 12120, Pathum Thani, Thailand.
³ Antibody Production Research Unit, Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Pathum Wan 10330, Bangkok, Thailand.
⁴ Faculty of Medicine, Thammasat University (Rangsit Campus), Khlong Luang 12121, Pathum Thani, Thailand.
⁵ Molecular Metabolism Research Group, Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.

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.

Keywords: Alpha-enolase; Hepatocellular carcinoma; Monoclonal antibody; Mortalin (HSPA9); Paraptosis-like program cell death; Transmission electron microscopy.