Prostanoid EP₁Receptor As A Novel Target Of(-)-Epigallocatechin-3-galIate Against Hepatocellular Carcinoma

(Institute of Clinical Pharmacology, Anhui Medical University, Key Laboratory ofAnti-inflammatory and Immunopharmacology, Anhui Medical University, Ministryof Education, Anhui Key Laboratory of Research and Development of ChineseMedicine, Engineering Technology Research Center of Anti-inflammatory andImmunodrugs in Anhui Province, Scientific and Technological Team ofAnti-inflammatory and Immunopharmacology of Chinese Medicine in AnhuiProvince, the115industial innovation team of basic and applied research intoanti-inflammation and immunodrugs in anhui province, Hefei,230032)Hepatocellular carcinoma (HCC) is the fifth most common cancer and the thirdmost common cause of death from cancer worldwide. In China HCC accounts for55%of all cases worldwide, and is the second leading cause of cancer death. So farthe tumorigenic mechanism of HCC remains undefined. And the antineoplastic agentsoften achieve low antineoplastic activity at the expense of close to unacceptabletoxicity. Therefore, there is a tremendous interest and urgency to search forrecurrence/metastasis related biomarkers and new agents with high effectiveness andlow toxicity for inhibiting HCC invasion and metastasis, which would better providenovel measures for intervention. The study found that the well-differentiated HCCexpressed COX-2more frequently and strongly than less-differentiated HCC,indicating that COX-2may participate in the early onset of HCC process. The level ofCOX-2expression and Akt phosphorylation was correlated positively in culturedHCC cells and human liver cancer tissues. In vitro and in vivo experiments showedthat COX-2inhibited the activity of caspases3, and caspases9, reduced theexpression of p53and Bax, enhanced Akt activity. COX-2expression was closely related to HCC occurrence and development. More and more evidences revealed thatCOX-2-derived prostaglandin E2(PGE₂) plays pivotal roles in tumor invasion. InHCC the level of PGE₂was highest compared with other PG productions. PGE₂exertsits biological actions primarily via their respective G protein-coupled receptors(GPCR) superfamily of prostaglandin E receptor (EP). Reports showed that EP₁expression was most abundant in HCC.(-)-epigallocatechin-3-gallate (EGCG) is apolyphenolic compound from tea and natural plants that has been shown to haveanti-tumor activities. However, the delicate mechanisms and signaling pathwaysunderlying the potential anticancer effects of EGCG in HCC cells remain unclear.This study is designed to investigate the possible mechanism of PGE₂/EP₁signaling pathway in HCC and the effects of EGCG on this signaling. Thus, thesefindings might provide the novel target for HCC treatment and the rationale forEGCG served as a novel therapeutic agent to treat HCC patients, and future study onits clinical application might be worthwhile.AIMS:To observe EP1expression in HCC cells and human hepatocyte L02and theserum PGE₂levels of patients with HCC, to analysis of the effects of PGE₂and theEP1agonist ONO-DI-004on proliferation and migration of HCC, to investigate theeffects of EGCG on cell cycle, apoptosis, PGE₂, EP1and Bcl-2expression of HCC, todiscuss the anti-HCC mechanism of EGCG.METHODS:HepG2cells were designed into several groups: control group, PGE₂(0,4,40,400,4000,40000nM) group, EGCG (12.5,25,50,100μg/mL) group, PGE₂(4,40,400,4000)+EGCG(100μg/mL) group, ONO-DI-004(4,40,400,4000nM) group,ONO-DI-004+EGCG (100μg/mL) group, ONO-8711(210nM,1,5,10μM) group.MTT assay was performed to determine the proliferation of HepG2cells; Would healing assay and Transwell filter cell migration assay were used to determine themigration of HepG2cells; Western blot and Immunofluorescence microscopy wereused to determine the expression of EP1, Gq and Bcl-2protein in HepG2cells; Elisaassay was performed to determine the PGE₂production in serum from patients withHCC or in HepG2cells; Flow cytomertry was performed to determine the cell cycleand apoptosis of HepG2cells.Results1. Serum PGE₂level was higher in HCC patientsCompared with the serum PGE₂level from normal people, serum PGE₂level washigher in HCC patients.2. That EGCG reduced PGE₂and VEGF levels, and lowered the expression ofEP1and Bcl-2is the important role of EGCG on HCC inhibitionCompared with control group, EGCG (12.5,25,50,100μg/mL) reduced PGE₂and VEGF levels in HepG2in dose-dependent way (r=0.758, r=0.958). EP₁expression was higher in the MHCC-97L and HepG2cells compared with humanhepatocytes L02cells. EGCG significantly inhibited EP1expression andONO-DI-004-induced Bcl-2expression in HCC cells.3. EGCG significantly inhibited PGE₂and ONO-DI-004-induced proliferationand migration of HepG2cells.Elisa assay was used to detected the effects of EGCG (12.5,25,50,100μg/mL)on proliferation of HepG2.100μg/mL EGCG significantly inhibited HepG2growthafter24h incubation, the mean inhibition ratio was41%(r=0.8). EGCG significantlyinhibited HepG2growth. Wound healing assay and Transwell assay were used to testEGCG and ONO-8711suppressed migration of HepG2and EGCG inhibited PGE₂orONO-DI-004-induced migration of HepG2(**P<0.01). 3.3EGCG induced the apoptosis of HepG2cells.HepG2cells were incubated with ONO-DI-004(400nM),PGE₂(4μM),EGCG(100μg/mL), EGCG (100μg/mL)+ONO-DI-004(400nM) or EGCG (100μg/mL)+PGE₂(4μM) after24h. The apoptosis ratio in control group was(2.36±2.51)%, that was (16.8±1.73)%in EGCG (100μg/mL) group. EGCGsignificantly induced HepG2cell apoptosis (**P<0.01). The apoptosis ratio inONO-DI-004(400nM) was (0.6±1.86)%, that was (12.25±2.64)%in EGCG (100μg/mL)+ONO-DI-004(400nM) group.Conclusion1. High serum PGE₂levels in HCC patients indicated the important role of PGE₂inHCC patients;2. EP1expression in HCC cell lines compared with normal liver cells was abnormalelevation of PGE₂, which indicated that PGE₂might promote HCC by binding to EP₁receptors.3. EGCG inhibited PGE₂or ONO-DI-004-induced proliferation and migration ofHepG2, and induced cell cycle arrest and apoptosis. These finding suggested thatinducing cell apoptosis and inhibiting proliferation, migration and cell cycle were theimportant role of EGCG on anti-HCC.4. EGCG reduced VEGF and PGE₂levels in HepG2cells, and inhibited EP₁expression and ONO-DI-004-induced Bcl-2expression but almost no effects on Gqprotein. That reducing PGE₂, VEGF and EP1and Bcl-2expression was one ofmechanisms of EGCG on proliferation and migration of HepG2cells.