The study of Hep88 mab mediated program cell death through paraptosis-like and/or apoptosis in Hepg2 call line

Name: Thantip Mitupatum

keyword: Hepatocellular carcinoma

Othors: Carcinoma, Hepatocellular

; Monoclonal antibody

Othors: Antibodies, Monoclonal

; Caspase

Othors: Apoptosis

; Apoptosis

Othors: Liver neoplasms

; Paraptosis

; Bax

; Bcl-2

; Cathepsin B

; p53

Abstract: The hepatocellular carcinoma (HCC) is the most common cancer worldwide, which is high incidence and high mortality rate. HCC becomes a crucial cancer because it is low detected at the early diagnosis and the remedies are still not specific to late stage of HCC. Interestingly, the targeted therapy via monoclonal antibodies (mAbs) specific to tumor-associated antigen is continuously developed in treatment and diagnosis of the disease. After establishing and consequently explored, it was found that Hep88 mAb has been shown anticancer effects against a HepG2 cell line. In this study, the gene expression at mRNA level of caspase-3, caspase-8 and caspase-9, the caspases enzyme activities in IC50 dose of Hep88 mAb-treated HepG2 cells and IC50 dose of doxorubicin-treated HepG2 cells were investigated to compare ability of them. After that, the concentration of Hep88 mAb treatment was increased to cytotoxic dose to detect the hallmarks of apoptosis by flow cytometry. Hep88 mAb-treated HepG2 cells were stained with different methods to examine the apoptotic cell population, the cell cycle progression and active caspase-3 determination. Afterwards, the mechanisms of program cell death (PCD) of IC50 and cytotoxic dose of Hep88 mAb were determined through the gene expression at mRNA level of Bax, Bcl-2, p53, cathepsin B, caspase-3 and caspase-9. Finally, the proteomic analysis of proteins involved in the anticancer activities of Hep88 mAb in HepG2 cells were explored by GeLC-MS/MS. The results were demonstrated that Hep88 mAb induced HepG2 cell line by the activation and expression of caspase-3, caspase-8 and caspase-9 in 24 hours of treatment. When compare with doxorubicin, Hep88 mAb treated cells were revealed higher enzyme activation and higher gene expression. Moreover, the increasing of G2/M phase cell cycle arrest, subsequent increasing of apoptotic cells population and more active caspase-3 detection were found in cytotoxic dose of Hep88 mAb-treated HepG2 cells in time- and dose-dependent manner. The expression of Bax, Bcl-2, p53, cathepsin B, caspase-3 and caspase-9 were used to evaluate the paraptosis and apoptosis PCD. Up-regulation of Bax, p53, caspase-3 and caspase-9 as well as the down-regulation of Bcl-2 could be responsible for the apoptotic effects of Hep88 mAb observed in this study. While, up-regulation of cathepsin B (CTSB) might contribute to paraptosis effects. Both paraptosis and apoptosis were occurred after treated with IC50 and cytotoxic dose of Hep88 mAb, which were depended on the strongest expression. Furthermore, the protein profile of Hep88 mAb-treated HepG2 cells had shown 323 differentially expressed proteins. From all of differentially expressed proteins, 29 proteins were found after Hep88 mAb. Two of these proteins were only found in IC50 dose of treatment, they were spindlin-1 (SPIN1) and ankyrin repeat domain (CLIP4). Spindlin-1 might contribute to paraptosis effects and ankyrin repeat domain might relate to the inhibitory effects on apoptosis cell death of Hep88 mAb. While, the apoptosis induction in this study might associated with 29 proteins that involved apoptotic process and apoptotic signaling pathway. These findings represent new insights into the molecular mechanisms underlying the anticancer properties of Hep88 mAb in liver cancer cells

Thammasat University. Thammasat University Library

Address: BANGKOK

Email: preserv@tu.ac.th

Name: Panadda Rojpibulstit

Role: advisor

Name: Kalaya Aree

Role: co-advisor

Name: Sasichai Kangsadalampai

Role: co-advisor

Created: 2014

Modified: 2022-01-31

Issued: 2022-01-31

วิทยานิพนธ์/Thesis

application/pdf

DOI: 10.14457/TU.the.2014.13

eng

DegreeName: Doctor of Philosophy

Level: Doctoral Degree

Descipline: Biochemistry and Molecular Biology

Grantor: Thammasat University

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