Resveratrol Induces DNA Damage-Mediated Cancer Cellular Senescence Through DLC1-DYRK1A-EGFR Axis

Cellular senescence is an irreversible state of cell cycle arrest,which can be triggered by various stress stimuli such as telomere damage,DNA damage and oxidative stress.Inhibiting proliferation of cancer cells by inducing cellular senescence is considered as a new anti-cancer strategy.The tumor suppressor DLC1 is frequently inactivated or down-regulated in a variety of cancers.Studies have shown that DLC1 plays an anti-tumor role by inhibiting proliferation and migration,inducing apoptosis of cancer cells,but there are few studies on its promotion of cancer cellular senescence.Overexpression of the oncogene EGFR is closely related to the poor prognosis of cancer patients,and DLC1 can prevent cancer progression through targeting suppression of EGFR.In addition,studies have shown that EGFR is positively correlated with the expression of the regulatory kinase DYRK1A,and the inactivation of the DYRK1A-EGFR axis mediates p53-induced cancer cellular senescence.We speculated that there may be some kind of regulatory relationship between DLC1 and DYRK1A-EGFR axis.Resveratrol,a natural polyphenol,plays an important role in preventing and halting cancer progression and induces cancer cellular senescence by up-regulating DLC1 and promoting DNA damage.However,the relationship between DLC1 and DNA damage has not been reported.Therefore,this study aims to investigate whether resveratrol regulates DYRK1A-EGFR axis through DLC1 to mediate DNA damage to induce cancer cellular senescence.First,we examined the effect of resveratrol on the proliferation of cancer cells by MTT and colony formation assay.Human breast cancer cell lines MDA-MB-231 and MCF-7,human liver cancer cell line Hep G2 and human normal liver cell line WRL68 were treated with different concentrations of resveratrol for 24 h and then MTT assay was performed.The results showed that resveratrol inhibited the proliferation of cancer cells in a dose-dependent manner,and reached the half-inhibitory concentration（IC50）at 100μM,while its effect on the proliferation of normal liver cells was small.Colony formation assay was implemented after the three kinds of cancer cells were exposed to 10 or 20μM resveratrol for 10 d.The results showed that resveratrol reduced the number of cancer cell clones in a dose-dependent manner.The enhancement of SA-β-gal activity is a crucial feature of senescence cells.SA-β-gal staining assay results displayed that the proportion of SA-β-gal positive cells aggrandized significantly in the three kinds of cancer cells treated with 20μM resveratrol for 7 d or 100μM resveratrol for24 h.Other senescence markers p53,p21 and lamin B were detected by western blotting assay,and the results showed that the expressions of p53 and p21 increased,and Lamin B expression decreased in cancer cells treated with 100μM resveratrol for 24 h.In order to shorten the experimental period,100μM resveratrol was used to treat cancer cells for 24 h in subsequent experiments to explore the mechanisms of resveratrol induced cancer cellular senescence.We examined the influence of resveratrol on DNA damage in cancer cells by western blotting and immunofluorescence assay.The results showed that resveratrol markedly enhanced the expression and nuclear aggregation of DNA damage proteinγH2AX,and attenuated the expression of DNA repair related proteins p-BRCA1 and RAD51 in cancer cells.Western blotting assay was adopted to explore whether resveratrol promoted DNA damage by regulating DLC1 in cancer cells.The results revealed that resveratrol notably elevated the expression of DLC1 in MDA-MB-231,MCF-7 and Hep G2 cancer cells.In MCF-7 cells,knockdown of DLC1weakened resveratrol induced cancer cellular senescence,and overexpression of DLC1dramatically up-regulatedγH2AX and down-regulated p-BRCA1 and RAD51 protein levels,indicating that resveratrol induced senescence of cancer cells by activating DLC1 to promote DNA damage.Meanwhile,resveratrol significantly suppressed the protein levels of EGFR and DYRK1A in MDA-MB-231,MCF-7 and Hep G2 cancer cells,and depletion of DYRK1A inhibited EGFR expression in MCF-7 cells.When EGFR inhibitor Erlotinib was added to MDA-MB-231 and MCF-7 cells for 24 h,the expression ofγH2AX increased,the expression of p-BRCA1 and RAD51 decreased,and the activity of SA-β-gal improved,suggesting that resveratrol induced senescence of cancer cells by negatively regulating the DYRK1A-EGFR axis to promote DNA damage.We further investigated the regulatory relationship between DLC1 and DYRK1A-EGFR axis.The results showed that silencing of DLC1 reduced the inhibitory effect of resveratrol on DYRK1A-EGFR axis,while overexpression of DLC1 weakened the activation of DYRK1A-EGFR axis in MCF-7 cells,indicating that DLC1 negatively regulated the DYRK1A-EGFR axis.Oxidative stress also can lead to DNA damage to promote senescence of cancer cells.Our results showed that resveratrol prominently elevated the level of ROS in MDA-MB-231,MCF-7 and Hep G2 cancer cells,and DNA damage and cancer cellular senescence promoted by resveratrol were suppressed after ROS scavenging agent NAC was added to MCF-7 and Hep G2 cells.Our previous studies have shown that exogenous supplementation of H₂O₂ aggrandizes the m RNA level of DLC1 in cancer cells.We conjectured that ROS may have an influence on DLC1 and its downstream DYRK1A-EGFR axis.Western blotting results showed that the activation of DLC1 and the inhibition of DYRK1A-EGFR axis induced by resveratrol were alleviated in MCF-7 cells treated with NAC,suggesting that ROS could regulate the DLC1-DYRK1A-EGFR axis.Finally,we evaluated the influence of resveratrol on tumor growth in vivo through a chick embryo allantoic membrane xenograft tumor model.The results showed that the volume of MCF-7 cells xenograft tumors was distinctly reduced after treatment with 0.5 m M or 1 m M resveratrol for 6 d compared with the control group.In order to further research the mechanism of resveratrol inhibiting tumor growth in vivo,we prepared the formed xenografts into frozen sections and then performed SA-β-gal staining and DLC1 immunohistochemical staining.The results showed that 1 m M resveratrol observably enhanced the activity of SA-β-gal and the expression of DLC1 in tumor tissues.We hypothesized that resveratrol suppressed tumor growth via activating DLC1 to induce cancer cellular senescence in vivo.The above results indicated that resveratrol promoted DNA damage to induce cancer cellular senescence through oxidative stress-mediated DLC1-DYRK1A-EGFR axis.In this study,we reported for the first time that DLC1 induced senescence of cancer cells by negatively regulating the DYRK1A-EGFR axis to promote DNA damage,and verified the promoting effect of resveratrol on DLC1 expression and cancer cellular senescence in vivo,which provided a new experimental basis for resveratrol to be used in the treatment of cancer by inducing cellular senescence.