The Roles Of Endoplasmic Reticulum Stress In PA-mediated Hepatocellular Carcinoma Cells Apoptosis

Objective: To investigate the effects and mechanisms of endoplasmic reticulum stress on palmitic acid(PA)induced apoptosis in human hepatocellular carcinoma cells.Methods: Human hepatocelluar carcinoma SMMC-7721 cells were treated with PA to induce hepatocyte lipoapoptosis.4-phenyl butyric acid(PBA)was used to inhibit endoplasmic reticulum stress response.Glucose-regulated protein 78(GRP78)vectors transfection was used to mimic the over-expression of GRP78 in SMMC-7721 cells upon PA treatment.Reverse transcription polymerase chain reaction(RT-PCR)and real-time polymerase chain reaction were used to detect the molecule markers of ER stress activation,and Tun-treated SMMC-7721 cells were used as positive control.Western blot and Annexin V-FITC flow cytometry were performed to analyze PA induced apoptosis in SMMC-7721 cells.The mRNA levels of XBP1,CHOP and GRP78 were detected to investigate the difference between PA-and Tun-induced endoplasmic reticulum stress in SMMC-7721 cells.Furthermore,GRP78 over-express was used to analyze the effects of GRP78 on PA-induced apoptosis in human hepatocellular carcinoma SMMC-7721 cells.Results:(1)PA treatment resulted in the activation of endoplasmic reticulum stress in human hepatocellular carcinoma SMMC-7721 cells.RT-PCR results showed that PA not only induced the splice of XBP1,but also increased the expression of GRP78 and CHOP at transcription level.The data of real-time PCR also confirmed the effect of PA in inducting endoplasmic reticulum stress molecule markers XBP1,CHOP and GRP78.(2)PA induces human hepatocellular carcinoma SMMC-7721 cells death.Light microscope data showed that PA treatment exerts obviously cytotoxicity in SMMC-7721 cells in a time-dependent manner.(3)PA initiates SMMC-7721 cells apoptosis.Western blot analysis revealed that PA treatment induced the cleavage of PARP in SMMC-7721 cells in a time-dependent manner.Furthermore,Annexin V-FITC staining confirmed that PA treatment can induce SMMC-7721 cells apoptosis in a time-dependent manner(P < 0.001).(4)Endoplasmic reticulum stress promotes PA-induced SMMC-7721 cells apoptosis.Western blot and annexin V-FITC flow cytometry data showed that PBA pre-incubation obviously decreased the apoptosis of SMMC-7721 cells upon PA treatment(P < 0.001).(5)SMMC-7721 cells resistant to Tun-induced apoptosis.Western blot and annexin V-FITC flow cytometry data showed that SMMC-7721 cells were relatively resistant to endoplasmic reticulum stress-induced apoptosis triggered by Tun treatment for a relatively long time.(6)PA differently activates endoplasmic reticulum stress signal pathways.The results showed that the mRNA levels of spliced XBP1 began accumulating within 3 h and decreased after 12 h in SMMC-7721 cells,when exposed to Tun.In contrast,the up-regulating of spliced XBP1 mRNA levels can’t be observed within 6 h in SMMC-7721 cells upon PA treatment.However,PA-induced up-regulating of spliced XBP1 mRNA levels sustained more than 24 h.Upon PA and Tun treatment,no obvious difference of CHOP mRNA levels was observed in SMMC-7721 cells.Moreover,Tun-induced up-regulating of GRP78 m RNA levels are not only higher but also earlier than that of PA in SMMC-7721 cells(P< 0.05)(7)GRP78 over-expression inhibits PA-induced hepatocellular carcinoma SMMC-7721 cells apoptosis.The Western blot data showed that GRP78 over-expression obviously inhibited PA-mediated SMMC-7721 cells apoptosis.The effects of GRP78 on apoptosis inhibition in PA-treated SMMC-7721 cells were reconfirmed by annexin V-FITC apoptosis analysis(P < 0.001).Conclusion: This study suggested that endoplasmic reticulum stress plays an important role in PA-induced hepatocellular carcinoma cell apoptosis.The insufficient induction of GRP78 exerts the pro-apoptotic effects of PA-induced endoplasmic reticulum stress in hepatocellular carcinoma cells.