PPARγ Mediates The Effects Of WIN On The Proliferation And Apoptosis Of The BEL-7402Hepatocarcinoma Cells

In recent years, researches showed that cannabinoids had anti-tumor effects.Therewas study showing that WIN55,212-2(WIN), an synthetic cannabinoid,could reduce cellviability and induce apoptosis of HepG2hepatocarcinoma cells,and that peroxisomeproliferator activated receptor γ(PPARγ) played an important role in these effects.Atpresent,however, there is no study showing if WIN will exhibit similar effects in otherhepatocarcinoma cells such as BEL-7402cells, and the mechanism of WIN’s anti-tumoreffect has not been fully elucidated.In addition, so far,the role of PPARγ in cancersremains controversial.ObjectiveTo study the effects and the preliminary mechanism of WIN on the proliferation andapoptosis of BEL-7402hepatocarcinoma cells.MethodsCell counting kit-8and trypan blue staining were used to detect the cell viability ofBEL-7402cells.Propidium iodide (PI) single staining was used to analyze the cellcycle.DAPI staining and Hoechst33258staining were used to observe the changes ofnuclear morphology of BEL-7402cells.Mitochondrial membrane potential assay kit withJC-1and cell apoptosis rhodamine123detection kit were used to detect the mitochondrialmembrane potential. Annexin V-FITC and PI were used to detect the apoptotic rate ofBEL-7402cells.Spectrophotometry was used to detect the activity of caspase-3, caspase-8 and caspase-9.Real-time quantitative PCR was used to detect the mRNA expression levelof PPARγ,Bax and Bcl-2.Western blot assay was used to detect the expression level ofsome proteins.ResultsWIN treatment resulted in cell viability decrease, proliferation inhibition andapoptosis of BEL-7402cells. After the cells were treated by WIN,the apoptotic rate wasincreased.Western blot assay showed that after WIN treatment,the cleavage of polyADP-ribose polymerase(PARP) and caspase-3appeared, the expression level of PPARγand Bax were up-regulated and the expression level of Bcl-2was down-regulated.WINtreatment resulted in increase of caspase-3, caspase-8and caspase-9activity,upregulationof PPARγ and Bax mRNA level and downregulation of Bcl-2mRNA level.PPARγantagonist GW9662weakened the effect of proliferation inhibition and apoptosis causedby WIN in BEL-7402cells.PPARγ agonist15-deoxy-Δ12,14-prostaglandin J2(15d-PGJ2)induced apoptosis of BEL-7402cells.15d-PGJ2could reduce cell viability of BEL-7402cells. The proliferation inhibition effect was more obvious when BEL-7402cells weretreated by WIN and15d-PGJ2simultaneously.PPARγ antagonist GW9662attenuated WINinduced inhibition of c-myc expression. AM630,the selective antagonist of cannabinoidreceptor2(CB2), attenuated WIN induced proliferation inhibition and apoptosis.Inaddition,AM630reduced WIN induced PPARγ expression.ConclusionWIN can inhibit the proliferation and induce apoptosis of BEL-7402cells, andPPARγ plays an important role in these effects. CB2plays an important role in WINinduced upregulation of PPARγ expression,proliferation inhibition and apoptosis ofBEL-7402cells.PPARγ plays a role in WIN induced inhibition of c-myc expression.