Meloxicam Suppresses The Migration Of Hepatocellular Carcinoma Cells Via Inhibition Of COX-2/PGE2/?-catenin Signaling Pathway

ObjectivesThe incidence and mortality of hepatocellular carcinoma(HCC)rank the top of malignant solid tumors,which characterizes the rapid progression and hidden onset.Surgical resection is the first choice for HCC,and combination with trans arterial chemoembolization(TACE),radiofrequency ablation,and target molecular drugs are also applied in subsequent treatment.However,HCC is prone to invasion and migrate,which is one of the most important factors affecting the clinical benefit and prognosis of HCC patients.So,to further explore the biological characteristics and potential mechanisms of HCC is vital for recognizing completely the clinical procedure and establishing the related treatment schemes.Cyclooxygenase-2 is not expressed in most normal cells or in low expression,while cyclooxygenase-1 is in high expression in kidney and stomach.However,some inflammatory factors and cancer inducers,such as IL-1,TNF-a and ultraviolet,could improve the COX-2 expression,which involved in the inflammatory response and tumor progression.A mass of studies have demonstrated the high expression of COX-2 in most malignant solid tumors,such as HCC,colon cancer,and breast cancer.COX-2 modulates the tumor progression via angiogenesis,tumor apoptosis,and ability of invasion and migration mediated by related signaling pathway such as Akt.Based on previous studies,COX-2 is a vital no negligible target in tumor treatment and the modulation mechanisms of COX-2 owe important studies value in tumor cells.Prostaglandin E2(PGE2)is the main metabolite of COX-2,and PGE2 is involved in the pathological procedure mediated by COX-2.Abundant previous studies have demonstrated that PGE2 is positively related to the tumor volume and pathological grade.Meanwhile,some researches indicated that the serum level of PGE2 in colon cancer patients with liver metastasis is higher than that with no metastasis.The effects of PGE2 in tumor progression,invasion,and migration have been recognized followed with related deep researches.However,more and more molecules and related mechanisms involved in tumor migration have been found accompanied with the improvement of oncology.So,to further study the molecular mechanisms involved in tumor migration mediated by PGE2 is vital for the recognition of PGE2 biological functions and the choice of molecular target in tumor treatment.Meloxicam(Mel)is a nonsteroidal anti-inflammatory drugs used for treatment of rheumatoid arthritis,however lots of studies have demonstrated the important role of Mel in tumor treatment.Our previous studies also indicated that Mel executes its anti-tumor effect via COX-2 dependent/independent pathway and autophagy in HCC cells.In lung cancer and gastrointestinal malignant tumors,Mel executes its anti-tumor activity via modulation of tumor cells apoptosis,viability,and migration ability.COX-2/PGE2 is one of the most important pathways mediating the activity of Mel.Based on previous studies,to further explore the mechanisms of Mel modulating tumor migration and the effect of COX-2/PGE2 in anti-tumor activity of Mel could support the experimental evidences for the research and development of Mel similar drugs and the study in clinical target.Tumor internal environment is complex,and the various cells and factors in microenvironment interact mutually.To explore the related mechanisms and factors involved in tumor development is in great significance.Meloxicam,the COX-2 specific inhibitor,was our study object.We discussed the effect of COX-2/PGE2 pathway in HCC cells migration and mediating the anti-HCC activity of Mel.Based on above studies,to further explore the down-stream signaling pathway and molecular proteins involved in COX-2/PGE2 pathway.We hope to support some experimental and theoretical evidences for the recognition of HCC biological activity,the choice of novel treatment target,the clinical experimental study of Mel or similar COX-2 inhibitors.Methods(1)Five human HCC cell lines,SMMC-7402,Bel-7402,Huh-7,SMMC-7721,and HepG2 were incubated for 24 h,and western blot was used to detect the COX-2 expression,and the HCC cell lines with high COX-2 expression were the study objects.(2)Based on above experiment,HCC cell lines SMMC-7721 and HepG2 with high expression of COX-2 were used for subsequent experiment.We incubated the cells with various concentrations of Mel(20?M,40?M.and 80?M)for 24 h,and then obtained the culture supernatants.ELISA was used to detect the level of PGE2 in supernatants.(3)To determine the effect of Mel on cell viability,we incubated SMMC-7721 and HepG2 with various concentrations of Mel(20?M.40?M,80?M).Cell Counting Kit-8 was applied to observe the cell viability at different time check point(24 h,48 h,and 72 h).(4)We continually incubated SMMC-7721 and HepG2 with Mel(80?M)for 24 h.Then we used the cell cycle analysis kit to detect the effect of Mel on cell cycle.To determine the mechanisms of Mel affecting cell cycle,we cultured the cells without change,then we extracted the cell protein,and western blot was applied to detect the expression levels of cyclin-dependent kinases inhibitor p21 and p27.(5)To determine the role of Mel and PGE2 in migration of HCC cells,SMMC-7721 and HepG2 cells were incubated in upper chamber of a transwell plate.HCC cells were dealt as follow:control group,PGE2 group(10?M),Mel group(80?M),PGE2+Mel group,and the lower chamber were the basic medium with 10%FBS.After 48 h,we fixed cells with 95%ethanol and then stained with crystal violet,and the number of migrated cells was observed under microscope.(6)To explore the role of COX-2 in mediating the modulation of Mel in HCC cells migration,we incubated the cells with various concentrations of Mel(40?M,80?M),and the expression of COX-2 was detected by western blot assay.Then we used the COX-2 siRNA to transfect the HCC cells,and trans well assay was to observe the effect of COX-2 siRNA on cell migration ability.(7)To determine the mechanisms of PGE2 in migration of HCC mediating by Mel,we grouped SMMC-7721 and HepG2 cells into control group,PGE2 group(10?M),Mel group(80?M),PGE2+Mel group,and incubated for 24 h.Western blot detected the expression of MMP2,MMP9,and E-cadherin in HCC cells.In the subsequent experiment,we extract the total RNA,and RT-PCR was applied to detect the mRNA expression of MMP2,MMP9,and E-cadherin.(8)For exploring whether some signaling pathway or proteins mediate the expression of MMPs and E-cadherin modulated by PGE2 and Mel,we grouped as same as(7),and western blot detected the nuclear ?-catenin expression,an key protein of Wnt signaling pathway,meanwhile the expression of p-GSK-3? was also detected.In the subsequent experiments,we used ?-catenin siRNA and scramble siRNA to transfect the HepG2 cells respectively,and the two kind of HepG2 cells transfected with siRNA were grouped into control group,PGE2 group(10?M),Mel group(80?M),PGE2+Mel group for 24 h culture,then we extracted the total protein to detect the expression of MMP2,MMP9,and E-cadherin by western blot assay.(9)To determine the role of ?-catenin in HCC migration mediating by PGE2,we incubated SMMC-7721 and HepG2 cells in upper chamber of a trans well plate,and grouped into control group,PGE2 group(10?M),FH535 group(5?M),PGE2+FH535 group,and the lower chamber was the basic medium with 10%FBS.After 48 h,we fixed cells with 95%ethanol and then stained with 1%crystal violet,and the number of migrated cells was observed under microscope.(10)We incubated the cells as same as(9)for 24 h,and extracted total protein to observe the expression of MMP2,MMP9,and E-cadherin in SMMC-7721 and HepG2 cells.And then RT-PCR was applied to detected the mRNA expression of MMP2,MMP9,and E-cadherin.Results(1)COX-2 expressed in the five HCC cell lines,while SMMC-7721 and HepG2 in high expression level.ELISA indicated that Meloxicam,a specific inhibitor of COX-2,could decrease the PGE2 level in the cell supernatants in concentration dependent manner(P<0.05).(2)CCK-8 indicated that Mel could significantly decrease the viability of HCC cells.At the same time point,the viability of HCC cells decreased in concentration dependent manner.And at the same concentration of Mel(20?M,40?M,or 80?M),we found that Mel could inhibit the viability in time dependent manner.(3)Mel was proved to own the cell cycle arrest effect,and the HCC cells were suppressed in the G1 period(P<0.01),and the cells in S and M period were decreased.Western blot assay demonstrated that cyclin-dependent kinase inhibitors p21 and p27 were downregulated significantly by Mel(P<0.01).(4)Transwell assay indicated that PGE2 could increase the migration ability of SMMC-7721 and HepG2(P<0.01),while Mel decreased significantly the migration ability(P<0.01).The data demonstrated that PGE2 mediated the HCC cells migration modulated by Mel.Western blot indicated that Mel downregulated the COX-2 expression in concentration dependent manner(P<0.01).And further transwell assay indicated that COX-2 siRNA could decrease the migration ability of HCC cells(P<0.01).(5)Western blot observed that ectogenic PGE2 could upregulate the MMP2 and MMP9 expression in SMMC-7721 and HepG2,while the key protein E-cadherin of EMT was downregulated significantly(P<0.01).Mel decreased the expression levels of MMP2 and MMP9 accompanied with the upregulation of E-cadherin(P<0.01).RT-PCR indicated that PGE2 and Mel could regulate the mRNA expression as same as proteins(P<0.01).However,we found that combination PGE2 with Mel could partly reverse the modulation effect of PGE2 or Mel alone.(6)Western blot indicated that PGE2 could increase the expression of nuclear P-catenin and p-GSK-3?,while Mel could reverse the process.Meanwhile,we found that ectogenic PGE2 could partly reverse the downregulation of nuclear ?-catenin and p-GSK-3? mediated by Mel.Blocking the expression of ?-catenin could inhibit the modulation effect of PGE2 and Mel on MMP2,MMP9,and E-cadherin.(7)In SMMC-7721 and HepG2,transwell found that FH535,a specific inhibitor of P-catenin,could significantly decrease the cell migration ability(P<0.05).Combination PGE2 with FH535 could reverse the enhanced migration induced by PGE2.Western blot indicated that FH535 could downregulate the expression of MMP2 and MMP9,while upregulate the E-cadherin expression(P<0.01).And FH535 could partly reverse the effect of PGE2 on MMP2,MMP9,and E-cadherin expression.RT-PCR indicated that PGE2 and FH535 could regulate the mRNA expression of MMP2,MMP9,and E-cadherin as same as proteins(P<0.01).Conclusions(1)COX-2 is highly expressed in HCC cell lines,and Meloxicam could inhibit the protein levels of COX-2 and PGE2,a main metabolite of COX-2.(2)Meloxicam suppresses the proliferation and migration of HCC cells via inhibition of cell viability and cell cycle.(3)COX-2/PGE2 pathway mediates the HCC cells migration inhibited by Meloxicam.(4)Meloxicam suppresses HCC cells migration via modulating the expression MMPs and E-cadherin mediating by PGE2.(5)?-catenin pathway is involved the intracellular signal transduction and modulation of downstream proteins of COX-2/PGE2,and further mediates the inhibition of HCC cells migration modulated by Meloxicam.Significances(1)We demonstrated the vital role of COX-2/PGE2 in invasion and migration of HCC.We supply more abundant experimental and theoretical evidences for researches based on the target.(2)We demonstrated that EMT is involved in the migration of HCC cells modulated by Meloxicam.(3)?-catenin signaling pathway mediates signal transduction of COX-2/PGE2,which could support research basis for choice of novel target in the future tumor treatment.(4)We support experimental and theoretical evidences for the clinical application of Meloxicam in tumor treatment and the study of similar drugs.