Synergistic Effect of MC-LR and C-Terminal Truncated HBx on HepG2 Cells and Their Effects on PP2A Mediated Downstream Target of MAPK Signaling Pathway

Abstract

C-terminally truncated hepatitis B virus (HBV) X (ctHBx) infection and exposure to microcystins-LR (MC-LR) can lead to human hepatitis and liver cancer, but the mechanism associated with their synergistically effects not been fully elucidated. The ctHBx (HBx 4 and HBx 32) lentivirus were constructed and transfected into the HepG2 cells. Then we investigated the function of MC-LR and ctHBx using the molecular biology approaches, including enzyme-linked immunosorbent assay, clone formation assay, scratch wound testing, transwell assays, carried out flow cytometry respectively to examine cell cycle and apoptosis in each group, and detected the related proteins of HBx, MEK/ERK/JNK/p38 in mitogen-activated protein kinase (MAPK) pathway and the downstream proteins such as cdc2, cdc25C, and p53 by western blotting. We found that the protein phosphorylase 2A (PP2A) enzyme activity in MC-LR and HBx 32/HBx 4 groups decreased more than in MC-LR and HBx group at the same time point and MC-LR concentration (P < 0.05). Meanwhile the proliferation, migration, invasion and colony formation capability of HepG2 cells were significantly enhanced in MC-LR and ctHBx groups (P < 0.05). In addition the proportion of S stage cells in the MC-LR-treated HBx 32/HBx 4 groups was significantly greater than that in the untreated groups (P < 0.05). Furthermore, the protein expression of MAPK signaling pathway including phospho-MEK1/2, ERKl/2, p38, and JNK were up-regulated by MC-LR and HBx 32, and the expression of cyclin-related proteins, including p53, cdc25C, and cdc2 were also activated (P < 0.05). Taken together, our findings revealed the essential significance of the MC-LR and ctHBx on the PP2A/MAPK/p53, cdc25C and cdc2 axis in the formation and development of HCC and identified MC-LR and ctHBx as potential causal cofactors of hepatocarcinogenesis.

Intracellular antibody targeting HBx suppresses invasion and metastasis in hepatitis B virus‐related hepatocarcinogenesis via protein phosphatase 2A‐B56γ‐mediated dephosphorylation of protein kinase B

“…Consistent with our results, Xi et al suggested that PP2A regulatory subunit B56 could increase HBV core protein C‐terminal domain dephosphorylation and decrease nuclear HBV episomes, thereby inhibiting multiple stages of HBV replication 11 . Xiao et al demonstrated that PP2A protein phosphatase agonist d ‐erythro‐Sphingosine could attenuate microcystins‐LR and C‐terminally truncated HBx‐induced the migration and invasion of HepG2 cells via regulating the dephosphorylation of PP2A/p‐p38 MAPK Thr180/Tyr182 axis 41 . These studies found that although targeted activation of PP2A was found to rescue the malignant phenotype of HBx‐expressing cells, the expression level of PP2A protein itself was not detected, which needs further investigation.…”

“… 11 Xiao et al demonstrated that PP2A protein phosphatase agonist d ‐erythro‐Sphingosine could attenuate microcystins‐LR and C‐terminally truncated HBx‐induced the migration and invasion of HepG2 cells via regulating the dephosphorylation of PP2A/p‐p38 MAPK Thr180/Tyr182 axis. 41 These studies found that although targeted activation of PP2A was found to rescue the malignant phenotype of HBx‐expressing cells, the expression level of PP2A protein itself was not detected, which needs further investigation. Surprisingly, we found that the HBx increased p‐AKT Thr308/Ser473 to drive HCC and then, at the same time, upregulated B56γ in the HBx‐expression cell model.…”

Microcystin‑leucine arginine promotes colorectal cancer cell proliferation by activating the PI3K/Akt/Wnt/β‑catenin pathway

“…However, an increasing number of studies have reported that the concentration of MC-LR in drinking water is much higher than 1 µg/l in certain countries, such as Vietnam and China ( 6 , 7 ). Currently, MC-LR is considered to be a potent carcinogen ( 8 , 9 ). In a previous study, MC-LR activated MMP expression and promoted breast cancer cell migration ( 10 ).…”

“…Old age, dietary patterns and a bad lifestyle are considered to be risk factors for CRC ( 13 , 14 ). Previous studies have demonstrated that MC-LR is a carcinogen and can be transferred to the food chain ( 3 , 8 ). Therefore, MC-LR may be related to the occurrence and development of CRC.…”

Genetic Variant of PP2A Subunit Gene Confers an Increased Risk of Primary Liver Cancer in Chinese

“…It has been reported that the underlying carcinogenic mechanism of MCs was through inhibition of protein phosphatase 2A (PP2A). 9,10 PP2A is a serine/threonine phosphatase that involves the regulation of many cellular processes, including metabolism, cell cycle, DNA replication, growth, and apoptosis. 11,12 The diminished activity of PP2A contributes to the malignant transformation and tumor development.…”