ATRA Inhibits The Malignant Behaviors Of Hepa1-6 Hepatocarcinoma Cells By Regulating Autophagy

Objective: To investigate the effects of all-trans-retinoic acid(ATRA)on the differentiation and autophagy level of Hepa1-6 cells,and to explore the role and possible mechanism of autophagy in the regulation of proliferation,apoptosis,migration,invasion and differentiation of Hepa1-6 cells during ATRA induction,providing an important experimental basis for the clinical application of ATRA on the treatment of hepatocellular carcinoma.Methods: 1 Hepa1-6 cells were treated with ATRA at concentrations of 0.1,1,10 ?mol/L,respectively.Real-time PCR was used to detect the mRNA level of hepatocyte-related markers including albumin(ALB),cytokeratin 18(CK18),tyrosine aminotransferase(TAT),apolipoprotein B(ApoB)and alpha fetoprotein(AFP).Western blot analysis was performed to detect the protein level of ALB,CK18 and AFP.Indocyanine green(ICG)uptake and periodic acid-schiff(PAS)staining were used to measure the mature hepatic function of Hepa1-6 cells.Cell-cell junctions and autophagosomes were observed under transmission electron microscopy(TEM).According to the results above,10 ?mol/L of ATRA was selected as the most suitable concentration.Then Western blot was used to detect the expressions of autophagy related marker proteins including LC3,Beclin1,RAB7 and P62.Dual fluorescene mRFP-GFP-LC3 plasmid(ptfLC3)was transfected into Hepa1-6 cells to observe the autophagic flux.2 1)3-Methyladenine(3-MA)and Bafilomycin(Baf)were used to inhibit the autophagy,and PAS staining was performed to screening out the most effective concentration of 3-MA and Baf.Then the autophagosomes were observed by TEM,the autophagic flux was measured by confocal microscope,the mRNA level of LC3 and BECN1 were detected by real-time PCR,the protein level of LC3,Beclin1 and P62 were detected by Western blot.2)Typan blue staining and colony formation were performed to measure the proliferation of Hepa1-6 cells,flow cytometry and Hoechst staining were used to evaluate cell apoptosis,wound healing assay and Transwell assay were used to detect cell migration and invasion.Immunofluorescence staining and real-time PCR were carried out to detect the expression of hepatocyte-related markers.ICG uptake and PAS staining and were performed to measure metablism and storage function of Hepa1-6 cells.3)To further explore the potential effect of autophagy on ATRA's regulated the malignant behaviors of Hepa1-6 cells,we evaluated the relationship between autophagy and epithelial mesenchymal transformation(EMT)of Hepa1-6 cells.After autophagy was inhibited,real-time PCR and Western blot were performed to detect the mRNA level and protein level of the mesenchymal markers including Snail,Vimentin,Twist,and N-cadherin and the expression of epithelial marker such as E-cadherin.At the same time,Western blot was used to detect the expression of p-PI3K(including p85 and p110),p-Akt,mTOR,p-mTOR,Bcl-2,p-Bcl-2,JNK and p-JNK,and immunofluorescence was further performed to detect the expressions of Bcl-2 and p-Bcl-2,so as to explore the possible molecular mechanism of ATRA in autophagy regulation in Hepa1-6 cells.Results: 1 Compared with Control group,ATRA treated Hepa1-6 cells showed a decrease in the expression of AFP and an increase in the expressions of ALB,CK18,TAT and ApoB in a concentration-dependent manner.ICG uptake and PAS staining revealed significantly increased number of positive stained cells after ATRA treatment.After ATRA treatment,the cells exhibited obviously increased tight junctions,cytoskeleton and more autophagosomes under transmission electron microscopy,golgi bodies were found in the 10 ?mol/L group.In conclusion,10 ?mol/L of ATRA was selected as the optimal concentration.In addition,Western blot also showed that the expression of autophagy-related markers such as LC3,Beclin1 and RAB7 were significantly increased after ATRA treatment,the expression of P62 were slightly increased as well.Confocal microscopy revealed obviously increased green and red spots in the cells after ATRA treatment.2 1)PAS staining showed that the number of positive cells of 3-MA and Baf treated groups significantly reduced compared with ATRA group.Considering the effects on cell function and cell condition,4 mmol/L of 3-MA and 50 nmol/L of Baf were selected as the optimal concentrations.After pretreated with 3-MA and Baf,less autophagosomes were found in the 3-MA+ATRA group compared with ATRA group under TEM,while more autophagosomes were found in the Baf+ATRA group.Similarly,confocal microscopy revealed that the 3-MA+ATRA group exhibited a reduction of green spots,yet Baf+ATRA group exhibited more green spots and less red spots compared with ATRA group.In addition,the protein level of LC3,Beclin1 and P62 decreased in the 3-MA+ATRA group,and autophagy-related genes BECN1 was down-regulated.On the other hand,protein level of LC3 and P62 were significantly increased,and the expression of Beclin1 was decreased in Baf+ATRA group compared with ATRA group,and the mRNA level of BECN1 was decreased as well.However,there is no significant change in the mRNA level of LC3 between 3-MA+ATRA or Baf+ATRA group and ATRA group.These results suggested that 3-MA and Baf could effectively inhibit ATRA induced autophagy in Hepa1-6 cells.2)Typan blue staining showed that ATRA could obviously inhibit the proliferation of Hepa1-6 cells,and the inhibition was attenuated when cells were exposed to autophagy inhibitors 3-MA and Baf,but there is no significant defferences in colony formation among different ATRA treated groups.Flow cytometry and Hoechst results showed that the apoptosis rate was increased after ATRA treatment,while the apoptosis rate of 3-MA+ATRA and Baf+ATRA groups were statistically lower than that of ATRA group.The results of wound healing assay and Transwell assay revealed that ATRA could significantly inhibit the migration and invasion of Hepa1-6 cells,however,after the autophagy was inhibited by 3-MA and Baf,the Hepa1-6 cells healed faster and the number of migrating and invasive cells in transwell were markedly increased.ICG uptake and PAS staining showed that the number of positive cells significantly reduced with the inhibition of autophagy compared with ATRA group,and both the mRNA level and protein level of ALB and CK18 in 3-MA+ATRA and Baf+ATRA group were decreased compared with ATRA group,while the expression of AFP was increased.3)Real-time PCR and Western blot indicated that the mesenchymal markers including Snail,Vimentin,Twist,and N-cadherin were all expressed at lower levels in ATRA group compared with Control group,nevertheless,the ATRA increased expression levels of above genes were inhibited in 3-MA+ATRA and Baf+ATRA groups.In the contrast,the expression of epithelial markers such as CK18 was upregulated after ATRA treatment and downregulated after cotreated with autophagy inhibitors.The expression of E-cadherin was increased after ATRA treatment but there is no statistic difference among ATRA,3-MA+ATRA and Baf+ATRA groups.In addition,Western blot also revealed that the expression of p-PI3 K,p-Akt,p-mTOR had no obvious change after ATRA treatment,but the expression of Bcl-2 was downregulated after ATRA treatment,while the expression of p-Bcl-2,JNK and p-JNK were increased after ATRA treatment.Immunofluorescence results showed that Bcl-2 was localized in the cytoplasm and its active form p-Bcl-2 was localized in the nucleus.After ATRA treatment,the expression of Bcl-2 decreased,while the expression of p-Bcl-2 increased,and translocated into the nucleus.Conclusion: 1 ATRA induced the differentiation and autophagy of Hepa1-6 cells in a concentration-dependent manner.2 ATRA-induced autophagy was involved in the inhibition functions of ATRA on the malignant behaviors of Hepa1-6 cells by reversing EMT process.ATRA may induce autophagy by activating the expression of Beclin1 through JNK/Bcl-2 pathway rather than PI3K/Akt/mTOR signal.