Resveratrol Promotes P38MAPK And DLC1 Expression To Drive ER Stress Mediated Cancer Cell Senescence

Resveratrol is a kind of plant polyphenol,which has attracted more and more attention due to its outstanding qualities such as easy acquirement,low price,brilliant anti-tumor potential and lower side effects.Pro-senescence strategy is a newly discovered mechanismfor anti-tumor therapy.It has been recently suggested that resveratrol can induce cancer cell senescence to execute a suppression effect on tumor development.However,the specific mechanisms are still obscure.Endoplasmic reticulumstress(ER stress)is involved not only in the progress of tumor,but also in the regulation of cell senescence.So,we propose that ER stress mediates resveratrol-induced cancer cell senescence.In this study,MCF-7 and NCI-H1299 cancer cell lines were used to explore the tumor suppressive mechanisms of resveratrol.First,we employed SA-β-Gal staining analysis,a widely used technology for senescent cell detection,to reflect the senescence-inducing effects of resveratrol on cancer cells.Treatment with resveratrol at the concentration of 20 μM for 7 days or100 μM for 1 day could both effectively induce cancer cell senescence,which is in accordance with our previous work.Then colony forming cell assay was performed to assess the effect of resveratrol on cancer cell clone formation ability and result showed that resveratrol reduced number of clones in cancer cells in a dose-dependent manner.To further investigate the specific molecular mechanisms involved,we detected the expression levels of ER stress related genes,Perk,IRE-1α,ATF-6α and GRP78,by quantitative PCR and western blot.The results showed that all of the ER stress related genes were upregulated under 100 μM resveratrol treatment.Besides,increased calciumrelease by ER was found by using the specific calciumprobe.Correspondingly,inhibition of ER stress by 4-PBA could partially rescue cell senescence induced by resveratrol.Resveratrol is an agonist of SIRT1 and it has suggested that depletion of SIRT1 can reduce the expression of p-p38 MAPK,the active formof p38 MAPK.p38MAPK was demonstrated to involve in regulation of ER stress in cancer cells.To investigate the possible role of SIRT1/p38 MAPK signaling pathway in ER stress regulation,EX527 and SB203580,which can exclusively inhibit SIRT1 and p38 MAPK expression respectively,were introduced.Compared with control group,100 μM resveratrol promoted the protein expression of SIRT1,p-p38 MAPK and p38 MAPK,and the up-regulation of p-p38 MAPK and GRP78 induced by resveratrol was partially restored after EX527 was added to the culture media.Moreover,GRP78 expression was impeded by additional treatment with SB203580.Delete in Liver Cancer 1(DLC1),a tumor suppressor gene,is usually found downregulated or depleted in cancer cells.Our previous studies have shown that resveratrol promotes ROS generation to drive DLC1 mediated cancer cell senescence.Another active molecule nitrogen oxide(NO)which also involves in oxidative stress has been shown to negatively regulate Rho A,a downstreammolecule of DLC1.Researches have shown that resveratrol can inhibit NO production in cells.In addition,it’s been demonstrated that inhibition of Rho A/C signaling can promote unfolded protein response.Thus,we supposed that resveratrol induces cancer cell senescence by activating ER stress via NO/DLC1 signaling pathway.By NO probe detection and western blot,we demonstrated that treatment with 100 μM resveratrol for 24 h significantly suppressed the expression level of inducible NO synthase(i NOS)to impede the production of NO.Replenishing NO donor,sodiumnitroprusside(SNP),reversed DLC1 expression promoted by resveratrol.Knockdown and overexpression of DLC1 gene were executed and the expression level of GRP78 was respectively downregulated and upregulated as the level of DLC1 changed.DNA damage is a classical marker of cell senescence.To investigate whether ER-stress promotes cancer cell senescence via inducing DNA damage,we detected the expression level of DNA damage-associated protein p-CHK1 and found that the protein was upregulated by 100 μM resveratrol in cancer cells,while application of 4-PBA impeded the resveratrol-induced p-CHK1 upregulation.Mitochondrial dysfunction is another factor triggering cell senescence,we wondered whether mitochondrial dysfunction also involves in the ER stress-induced senescent phenotype.We determined mitochondrial membrane potential using specific detection kit,and found that there was significant reduction in mitochondrial membrane potential with the treatment of resveratrol.Western blot result showed that expression level of peroxisome proliferator-activated receptorgamma coactivator 1α(PGC-1α),a mitochondrial biogenesis related protein,was downregulated by resveratrol treatment in cancer cells.Correspondingly,the specific ER stress inhibitor,4-PBA,rescued the drop of mitochondrial membrane potential and the downregulation of PGC-1αexpression.All the above results proved the essential role of DNA damage and mitochondrial dysfunction in resveratrol-induced cancer cell senescence.In this study,we demonstrated that SIRT1 was upregulated under the treatment of resveratrol and promoted p38 MAPK expression.Resveratrol could also impede the NO synthesis through inhibiting i NOS expression,resulting in increased DLC1 protein level in cancer cells.Both p38 MAPK and DLC1 were able to promote ER stress-induced DNA damage and mitochondrial dysfunction,finally contributing to cancer cell senescence.It’s the first time to confirma regulatory role of SIRT1 on the expression of p38 MAPK in cancer cells and find the tight relationship between resveratrol and NO/DLC1 signaling,which is a significant factor to regulate ER stress.This study provides a new signaling pathway and experimental basis for the antitumor effects of resveratrol.