Blocking Autophagy Enhances Meloxicam Lethality For Hepatocellular Carcinoma Through Promotion Of ER Stress-related Apoptosis

Background:Hepatocellular carcinoma(HCC)is the sixth most prevalent cancer in the world and the second leading cause of cancer-related death.In present,HCC patients who are amenable to curative therapy only account for 20%of the patients.Unfortunately,the development of effective therapy for the advanced-stage of HCC has been rather slow.Sorafenib,a tyrosine kinase inhibitor with abroad inhibitory profile,is the most effective drug currently approved to treat advanced-stage disease.However,it has not been widely accepted as it only prolongs 2-3 month of survival of advanced HCC patients compared to placebo.Therefore novel targeted molecular therapeutic strategies are required and may greatly contribute to the treatment of advanced-stage HCC.Accumulated evidence has demonstrated that COX-2 expression influences proliferation,invasion and apoptosis resistance in many malignant diseases.Meloxicam,a selective COX-2 inhibitor and non-steroidal anti-inflammatory drug(NSAID),is widely used for inflammation and can induce growth inhibition and apoptosis through suppressing COX-2 expression.However,the mechanisms involved in meloxicam anti-hepatocellular carcinoma effects remain unclear.Evidences suggested that the high rate proliferation of HCC cells usually result in overload of endoplasmic reticulum,leading to accumulation of misfolded and/or unfolded proteins in endoplasmic reticulum,a condition referred to"endoplasmic reticulum stress(ER stress)".Therefore,it is very important to reveal mechanism of HCC proliferation and apoptosis under ER stress.Autophagy is a highly regulated process that allows cells to sequester cytoplasmic material by forming double-membrane vesicles(autophagosomes)and to deliver material to lysosomes for degradation.Previous studies have reported that some anti-neoplastic therapies could lead to autophagy and apoptosis in cancer cells.Targeting autophagy could enhance chemotherapy sensitivity for many cancers.Therefore,we designed the present study to investigate whether targeting autophagy could enhance meloxicam lethality for hepatocellular carcinoma through promotion of ER stress-related apoptosis.Methods:Five common human hepatocellular carcinoma cells HepG2?Bel-7402?Huh-7?SMMC-7721 and SMMC-7402 were selected.HCC cells were exposed to meloxicam for 24 h and cell viability was determined using the CCK-8 assay.Because HepG2 and Bel-7402 cells were more sensitivity to meloxicam than other HCC cells,we chose these two cells for the following experiments.We used flow cytometry(FACS)with propidium iodide(PI)and annexin V-FITC staining to analyze apoptotic cells.We determined levels of the ER stress markers of IRE1,GRP78/Bip and p-e1F2? by western blot and immunofluorescence staining.Western blot was used to detect the effects of meloxicam on the level of caspase-12,caspase-3 and PARP.The effects of meloxicam on caspase-12 mRNA was also detected by real-time RT-PCR.In addition,an apoptosis inhibitor(Z-VAD-FMK,Z-VAD)was used to assess the cell death by meloxicam.A specific siRNA to down-regulate GRP78 was introduced in both HCC cell lines.Cell viability was determined using the CCK-8 assay.The extent of GRP78 was determined using the western blot assay.Apoptosis was used flow cytometry(FACS)with propidium iodide(PI)and annexin V-FITC staining.(-)-Epigallocatechin-3-gallate(EGCG),a flavonoid component of Green Tea,has been shown to block unfolded conformations of the GRP78 ATPase domain and then inhibit its functions.In the current work,we used flow cytometry and western blot to explore the properties of EGCG in regard to the induction of cell death in combination with meloxicam in HepG2 and Bel-7402 cells.To further explore the antitumor mechanism of meloxicam treatment,in this current study,we used western blot to detect the effect of meloxicam on the autophagy target genes of Beclin-1,Atg5,Atg7,LC3 as well as p62.To further confirm the observations that LC3 was increased in HCC cells after treatment with meloxicam,LC3 was visualized by immunofluorescence staining.To further investigate whether the autophagy exerts a crucial role in cell death,HepG2 and Bel-7402 cells in which autophagy were inhibited by 3-MA or Atg5 siRNA were treated with meloxicam.Annexin V-FITC/PI double staining was employed to quantify the apoptosis of HCC cells.Western blot was used to detect the expression of LC3,Atg5 and caspase-12.Several studies have revealed that the GRP78 signaling pathway is required for stress-induced autophagy.In the present study,we investigated whether GRP78 is required for activation of meloxicam-induced autophagy in HCC.Inhibition of GRP78 by siRNA or EGCG was used to detect expression levels of GRP78 and LC3 in HepG2 and Bel-7402 cell lines after meloxicam treatment.Previous studies have revealed that the AMPK-mTOR signaling pathway plays a crucial role in regulating autophagy.Therefore,we assumed whether phosphorylation of AMPK and mTOR was involved in GRP78 mediated autophagy during meloxicam treatment.Inhibition of GRP78 by siRNA or EGCG was used to detect expression levels of AMPK and mTOR.To further investigate the mechanism of the AMPK-mTOR signaling pathway in meloxicam induced autophagy,compound C(a chemical inhibitor of AMPK)and rapamycin(an inhibitor of m-TOR)was employed before treating with meloxicam.Western blot was used to detect the expression of LC3.Results:CCK-8 cell proliferation assay showed that the viability of the HCC cells exposed to meloxicam was significantly lower compared with the control cells.A sharp decrease in cell viability was present at 80?M which led to the highest inhibitory rates.These results showed that meloxicam cytotoxicity increased in a concentration-dependent manner.Annexin V-FITC/PI double staining showed that meloxicam markedly increased cellular apoptosis in a concentration-dependent manner.Western blot analysis showed that meloxicam increased protein expression levels of IRE1 and GRP78/Bip as well as eIF2a phosphorylation in a concentration-dependent manner.To further confirm the observations that GRP78 was increased in HepG2 and Bel-7402 cells after treatment with meloxicam,GRP78 was visualized by immunofluorescence staining.We observed that immunofluorescence staining of GRP78 was notably increased after meloxicam treatment.Previous studies demonstrated that caspase-12 induced apoptosis was associated with ER-stress.After treatment with meloxicam for 24h,we observed that the activation of caspase-12 was significantly increased in HepG2 and Bel-7402 cell lines which were consistent with cell death assays.We also observed that expression of cleaved PARP and cleaved caspase-3 significantly increased.Furthermore,as measured by RT-PCR,meloxicam treatment induced increased expression of caspase-12 mRNA.In addition,an apoptosis inhibitor(Z-VAD-FMK,Z-VAD)was used to assess the cell death by meloxicam.Annexin V-FITC/P1 double staining showed that Z-VAD notably down-regulated meloxicam induced apoptosis in HCC cell lines.These results revealed that ER impairment by meloxicam can trigger the process of apoptosis and that activations of caspases are involved in meloxicam-induced apoptosis.A specific siRNA to down-regulate GRP78 was introduced in both HCC cell lines.CCK-8 cell proliferation assay showed that knockdown of GRP78 by siRNA significantly reduced cell viability.Furthermore,Western blot analysis and Annexin V-FITC/PI double staining showed that down-regulation of GRP78 by siRNA notably decreased protein expression of GRP78 and significantly enhanced the increase of cell apoptosis and the cleavage of caspase-3 and PARP in meloxicam treated HepG2 and Bel-7402 cell lines.Next,we explored the properties of EGCG in regard to the induction of cell death in combination with meloxicam in HepG2 and Bel-7402 cells.Annexin V-FITC/PI double staining showed that EGCG promoted meloxicam-induced cell death in HepG2 and Bel-7402 cells to varying degrees.Western blot analysis showed that treatment with EGCG down-regulated the level of GRP78 which suggested that EGCG enhances meloxicam lethality in both HCC cells.Western blot analysis showed that meloxicam notably increased the levels of Beclin-1,Atg5,Atg7 and activated LC3 whereas p62 was significantly decreased after treatment with meloxicam.To further confirm the observations that LC3 was increased in HCC cells after treatment with meloxicam,LC3 was visualized by immunofluorescence staining.We found that immunofluorescence staining of LC3 was notably increased after meloxicam treatment.Moreover,we investigated whether the autophagy exerts a crucial role in cell death,HepG2 and Bel-7402 cells in which autophagy were inhibited by 3-MA were treated with meloxicam.Annexin V-FITC/PI double staining and Western blot analysis showed that the 3-MA-treated cells had significantly inhibited autophagy whereas increased levels of cell death and expression of caspase-12 cleavage after meloxicam treatment was enhanced.Similarly,Annexin V-FITC/PI double staining and Western blot analysis showed that knockdown of Atg5 by siRNA notably increased ER stress-mediated apoptotic cell death.In the present study,Western blot analysis showed that blocking of GRP78 by siRNA led to down-regulation of LC3-? in HCC cells treated with meloxicam.These data revealed that inhibition of GRP78 could suppress autophagy activation induced by meloxicam in HCC cells.Previous studies have revealed that the AMPK-mTOR signaling pathway plays a crucial role in regulating autophagy.Therefore,we assumed whether phosphorylation of AMPK and mTOR was involved in GRP78 mediated autophagy during meloxicam treatment.Western blot analysis showed that meloxicam significantly activated AMPK but inhibited the phosphorylation of mTOR in HepG2 and Bel-7402 cells.However,GRP78 siRNA or EGCG suppressed the changes of p-AMPK and p-mTOR induced by meloxicam.To further investigate the mechanism of the AMPK-mTOR signaling pathway in meloxicam induced autophagy,compound C,a chemical inhibitor of AMPK,was employed before treating with meloxicam.Our results revealed that treating HepG2 and Bel-7402 cells to compound C reduced meloxicam-induced activation of AMPK.In addition,we also found that inhibition of the activation of AMPK notably reduced the expression of LC3.However,rapamycin,an inhibitor of m-TOR,attenuated the effect of compound C on the expression of LC3 during meloxicam treatment.To further support the importance of the AMPK-mTOR pathway in HCC cells treatment with meloxicam,we evaluated for the effect of compound C on meloxicam-induced apoptosis.Annexin V-FITC/PI double staining showed that compound C significantly enhanced meloxicam lethality in HepG2 and Bel-7402 cells.These data suggested that AMPK-mTOR is involved in autophagy activation during meloxicam treatment.GRP78 may induce autophagy activation via AMPK-mTOR signaling pathway.Conclusions:1.ER impairment by meloxicam can trigger the process of apoptosis and that activations of caspases are involved in meloxicam-induced apoptosis.2.Autophagy induced by meloxicam conferred protection to HCC cells against apoptosis.Meloxicam-induced autophagy alleviates ER stress and inhibition of autophagy could enhance ER stress-related apoptosis.3.The GRP78 is involved in autophagy activation induced by meloxicam may through AMPK-mTOR signaling pathway in HCC cell lines.GRP78 knockdown increased meloxicam-induced apoptosis in HCC cell lines.