Roles Of EP₁-Gαq Protein-Ca²⁺ Signal Transduction Pathway In Hepatocellular Carcinoma Invasion And The Effect Of (-)-epigallocatechin-3-gallate

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third most common cause of death from cancer worldwide. In China HCC accounts for 55% of all cases worldwide, and is the second leading cause of cancer death. So far the tumorigenic mechanism of HCC remains undefined. And the antineoplastic agents often achieve low antineoplastic activity at the expense of close to unacceptable toxicity. Therefore,there is a tremendous interest and urgency to search for recurrence/metastasis related biomarkers and new agents with high effectiveness and low toxicity for inhibiting HCC invasion and metastasis , which would better provide novel measures for intervention. (-)-epigallocatechin-3-gallate (EGCG) is a polyphenolic compound from tea and natural plants that has been shown to have anti-tumor activities. However, the delicate mechanisms and signaling pathways underlying the potential anticancer effects of EGCG in HCC cells remain unclear. HCC often arises from a background of long-standing chronic inflammatory liver diseases. Recent studies reavealed that intrahepatic recurrence and metastasis were associated with a unique inflammation response signature in the peritumoral liver tissue. In hepatitis C virus (HCV)-related HCC cyclooxygenase-2 (COX-2) expression was highly correlated with the presence of active inflammation, and was also closely related to the postoperative relapse of HCC. More and more evidences revealed that COX-2-derived prostaglandin E2 (PGE2) plays pivotal roles in tumor invasion. PGE2 exerts its biological actions primarily via their respective G protein-coupled receptors (GPCR) superfamily of prostaglandin E receptor (EP) on the cell surface membrane vascular endothelial growth factor(VEGF), one of the most potent angiogenic factors, has been shown to play an important role in tumor growth, proliferation, angiogenesis, and invasion. VEGF secretion in cancer cells might be mediated through PGE2-EP2-cyclic adenosine monophosphate (cAMP) signaling pathway.Therefore, the first part of this study aimed to investigate the significance of the expressed COX-2 level in the noncancerous liver as a prognostic indicator for hepatitis B virus (HBV)-related HCC patients. The second part of this study is designed to investigate the possible mechanism of PGE2-EP1-G protein-Ca2+ signal transduction in HCC and the effect of EGCG on this pathway. Thus, these findings might provide the novel target for HCC treatment and the rationale for EGCG served as a novel therapeutic agent to inhibit HCC invasion for HCC patients, and future study on its clinical application might be worthwhile.AIMS1. To investigate the significance of the expressed COX-2 level in the noncancerous liver as a prognostic indicator for HBV-related HCC patients.2. To investigate the effect of EGCG on PGE2/ONO-DI-004-induced proliferation, adhesion, migration and invasion abilities of MHCC97L cell in vitro. 3. To investigate the effect of EGCG on PGE2/ONO-DI-004-induced VEGF production levels in MHCC97L cell in vitro.4. The MHCC97L cell was adopted for the study of PGE2-EP1-Gαq-Ca2+ signal transduction pathway. We further explored the effects of EGCG on the PGE2-EP1-Gαq-Ca2+ signal transduction pathway.METHODS1. Immunohistochemistry was used to evaluate the expression of COX-2 in the noncancerous tissue.2. Any significant differences in the recurrence-free survival rates were determined with the use of log-rank test. All variables were put into a Cox proportional hazard model to identify any independent variables that were related closely to the recurrence-free survival rates3. MTT is performed to determine the proliferation and adhesion of MHCC97L cell.4. Migration and invasion were evaluated with transwell chambers.5. ELISA was used to measure the level of PGE2 and VEGF in MHCC97L cell.6. The expression of EP1 and Gαq was detected by Western blot analysis in MHCC97L cell.7. Laser confocal scanning microscope was used to measure the intracellular concentration of Ca2+.RESULTS1. The 5-year recurrence rate in the HCC patients with COX-2 overexpression in the background cirrhotic liver was significantly higher than that in the patients with COX-2 low expression.In a univariate analysis of recurrence-free survival, significant differences were observed in the following 11 variables: COX-2 express level, intrahepatic metastasis, ALT level, total bilirubin level, albumin level, capsular formation, vascular invasion, AFP level, number of nodules, maximal tumor dimension, blood infusion. The results of multivariate analysis showed COX-2 expression level, vascular invasion were selected as independent and significant indicators for recurrence-free survival.2. Treatment of PGE2/ONO-DI-004-induced MHCC97L cell with EGCG inhibited the proliferation, adhesion, migration and invasion abilities.Treatment of MHCC97L cell with EGCG concentration- dependently (12.5, 25, 50 and 100 mg·L-1) and time-dependently (24, 48 and 72 h) inhibited the proliferation. Treatment of PGE2 (10μM)/ONO-DI-004 (1μM)-induced MHCC97L cell with EGCG concentration-dependently (12.5, 25, 50 and 100 mg·L-1) inhibited the adhesion, migration and invasion abilities compared with no EGCG treatment.3. Treatment of PGE2/ONO-DI-004-induced MHCC97L cell with EGCG inhibited the production of VEGF.Treatment of MHCC97L cell with EGCG concentration-dependently (12.5, 25, 50 and 100 mg·L-1) inhibited the secretion of VEGF compared with no EGCG treatment. Treatment of PGE2 (10μM)/ONO-DI-004 (1μM)-induced MHCC97L cell with EGCG concentration-dependently (12.5, 25, 50 and 100 mg·L-1) suppressed the secretion of VEGF compared with no EGCG.4. Attenuation of the disorders of the PGE2-EP1-Gαq-Ca2+ signal transduction is probablely one of the most important mechanisms of EGCG for the inhibition of HCC invasion.Previous studies revealed that EP1 is involved in the invasion of HCC. In the present study, EGCG caused a decline in the secretion of PGE2 and VEGF, a decrease in the expressions of EP1 and Gαq proteins, and intracellular Ca2+ concentration. These findings provide the evidence that EGCG exerted its functions by modulating PGE2-EP1-Gαq-Ca2+ signal transduction pathway, which might be one of the most important mechanisms of EGCG for the inhibition of HCC invasion. CONCLUSIONS1. The 5-year recurrence rate in the HCC patients with COX-2 overexpression in the background cirrhotic liver was significantly higher than that in the patients with COX-2 low expression2. EGCG inhibited PGE2/ONO-DI-004-induced proliferation, adhesion, migration and invasion abilities of MHCC97L cell in vitro3. EGCG decreased PGE2/ONO-DI-004-induced VEGF production, which is an important characteristic of EGCG suppressing invasion for MHCC97L cell.4. EGCG obviously inhibited the expression of EP1 and Gαq, decreased the intracellular concentration of Ca2+. These results suggested that regulating the PGE2-EP1-Gαq-Ca2+ signal transduction pathway might be one of the most important mechanisms of EGCG for the inhibition of HCC invasion.