| As the most common subtype of kidney cancer,renal cell carcinoma(RCC)is resistant to radiotherapy and chemotherapy,and surgical resection remains the main therapeutic regimen for early localized RCC.Metastatic RCC has occurred in 30% of patients at the time of RCC diagnosis,and postoperative metastasis still occurs in some patients.Patients with metastatic RCC have a very poor prognosis.Although a great deal of progress has been made in targeted molecular therapy for metastatic RCC,its medicinal performance remains less satisfactory.Thus,to develope new drugs for RCC treatment is of great clinic significance.Natural compounds are valuable to develope new drugs.Resveratrol(Res)is a natural polyphenolic compound,which was widely distributed in a variety of plants.It has been reported that Res could induce cycle arrest,promote apoptosis,and reduce invasion and metastasis of cells,which inhibits multiple types of tumor cells in vitro.Moreover,Res also possesses anti-tumor effect in vivo.At present,studies on Res in RCC are less,and the mechanism of action is not clear because of its complex pharmacological activity.In this study,we observed the effect of Res on renal cell carcinoma,analyzed the related mechanism,and provided a theoretical basis to develope new drugs for RCC treatment.Part Ⅰ Effects of Res on human renal cell human carcinoma 786-O cells Background: It has been established Res possesses anti-tumor activity in multiple tumor cells.However,the effect of Res on renal cell carcinoma is less known,which needs to be further elucidated.Objective: To explore the effect of Res on human renal cell carcinoma 786-O cells.Methods: After indicated treatment of 786-O cells with Res,CCK8 was used to detect cell viability.Flow cytometry was employed to detect cell apoptosis and cell cycle.Ce ll scratch test and transwell test were used to analyze cell migration and invasion,respectively.Western blot was employed to determine the expressions of MMP2(matrix metallopeptidase 2)and MMP9(matrix metallopeptidase 9)proteins.To investigate the effect of Res on the growth of renal cell carcinoma in vivo,transplantation tumor model of 786-O cells was established in nude mice.Results: Res reduced the viability of 786-O cells in a time-and dose-dependent manner.After 48 h of treatment,flow cyto metric analysis showed that Res at 20 μM and 40 μM induced cell apoptosis,while 10 μM Res had no effect on apoptosis,but induced S phase arrest.Cell migration,invasion were decreased and the expressions of MMP2,MMP9 proteins were down-regulated after treatment with Res in 786-O cells.In vivo,the growth of the transplantation tumor of 786-O cells was inhibited,tumor volume and weight were reduced by Res.Conclusion: In vitro and vivo,Res possesses anti-tumor effect on human renal cell carcinoma.Part Ⅱ Resveratrol induces apoptosis of human renal cell carcinoma 786-O cells through ROSBackgroud: Cell poptosis involves the activation,expression and regulation of a series of signal factors.The accumulated evidences have proven that Res could induce apoptosis of tumor cells.Due to its complex pharmacological activity,the molecular mechanism of apoptosis induced by Res still needs to be further elucidated.Objective: To explore relative molecular mechanism of Res-induced apoptosis in 786-O cel s in vitro.Methods: Mitochondrial membrane potential,apoptosis,caspase 3 activity and ROS levels were detected through flow cytometry.Cytochrome C and ROS(reactive oxygen species)were analyzed using immunofluorescence.The expressions of GAPDH,COXⅣ,cytochrome C,PARP,ERK,p-ERK,JNK,p-JNK,p38,p-p38 proteins were analyzed through western blot.Results: Res decreased the mitochondrial membrane potential levels of 786-O cells,promoted cytoplasmic release of cytochrome C,increased caspase3 activity and induced PARP protein cleavage.Z-VAD-FMK,a pan-caspase inhibitor,significantly inhibited Res-induced apoptosis.Further experiments showed that Res promoted ROS production in 786-O cells and NAC(N-acetyl cysteine),an antioxidant,could improve mitochondrial membrane potential,down-regulate caspase 3 activity,reduce PARP cleavage and inhibit Res-induced apoptosis.Res inhibited ERK(extracellular signal-regulated kinase),and activated JNK(c-jun N-terminal kinase),p38(p38 mitogen-activated protein kinase)through ROS.SP600125(a JNK inhibitor)had no significant effect on apoptosis,while SB203580(a p38 inhibitor)attenuated Res-induced apoptosis in 786-O cells.Conclusion: 1.Res can damage mitochondria and induce apoptosis mainly in a caspase-dependent manner in 786-O cells.2.Res up-regulates ROS levels,which is responsible for Res-induced apoptosis in 786-O cells.3.Res activates p38 through ROS,which promotes Res-induced apoptosis in 786-O cells.Part Ⅲ Autophagy protects human renal cell carcinoma 786-O cells from apoptosis induced by resveratrolBackground: Autophagy,as a conservative mechanism,is existed in various cells.Under stress,autophagy can be activated,which maintains cell homeostasis and promotes the survival of cells.However,extreme autophagy can aggravate injury and increase cell death.Studies have shown that Res affects autophagy through many ways,which can promote survival or death.The role of Res-induced autophagy in renal cell carcinoma remains less known.Objective: To investigate the effect of autophagy on Res-induced apoptosis in 786-O cells in vitro.Methods: Flow cytometry and immunofluorescence were employed to detect autophagosomes.The expressions of GAPDH,LC3 BII,P62,S6,p-S6,AMPK,p-AMPK,JNK,p-JNK,BCL2,p-BCL2,Becline1 and PARP proteins were determined through western blot.Apoptosis was detected by flow cytometry.The expression of Beclin1 protein was knocked down using si RNA to analyze the effect of autophagy on Res-induced apoptosis.Results: Res activated autophagy in 786-O cells.ROS was required for Res-induced autophagy,and the antioxidant NAC reduced Res-activated autophagy.JNK inhibitor SP600125 down-regulated autophagy,while p38 inhibitor SB203580 further increased Res-induced autophagy.CQ(chloroquine),an autophagy blocker,aggravated apoptosis,increased PARP cleavage.Moreover,Beclin1 si RNA up-regulated the expression of cleaved PARP protein induced by Res.Conclusion: 1.Res induces autophagy in 786-O cells.2.Res activiates autophagy through ROS-JNK pathway.3.Autophagy protects 786-O cells from apoptosis induced by Res.Part Ⅳ Resveratrol induces senescence of huaman renal cell carcinoma 786-O cells by ROSBackground: Cell senescence refers to the irreversible cell growth arrest.Under certain conditions,chemotherapeutic drugs,ionizing radiation,oxidative damage and other factors can induce premature senescence of tumor cells.Studies have confirmed that Res can induce premature senescence in tumor cells,but whether Res can induce senescence of renal cell carcinoma cel s has not been reported so far.Objective: To investigate the effect of Res on senescence in 786-O cells in vitro.Methods: Cell cycle,apoptosis and ROS levels were analyzed using flow cytometry.Immunofluorescence was employed to detect p-H2 AX.EDU proliferation assay was performed to assay cell proliferation.SA-β-Gal staining was used to assess senescence.Using Western blot analyzed the expressions of GAPDH,p-H2 AX,p-ATM,p-ATR,p-CHK2,p-CHK1,p38,p-p38 and p21 proteins.The expressions of IL-6,IL-8 mRNA were determined using fluorescence quantitative PCR.The secreted IL-6 and IL-8 proteins were detected by ELISA assay.Results: 786-O cells were treated with 10μM Res for 4 d,Res increased the expressions of p-H2 AX,p-ATM,p-ATR,p-CHK2,p-CHK1 and p21 proteins,caused the persistent S-phase arrest,reduced EDU positive cells,up-regulated p-H2 AX and SA-β-Gal staining positive cells,however cell apoptosis was not induced.The increased ROS was required to induce 786-O cells senescence by Res.NAC decreased the expressions of p-H2 AX,p-ATM,p-ATR,p-CHK2,p-CHK1 and p21 proteins up-regulated by Res,rescued cell proliferation and reduced p-H2 AX and SA-β-Gal positive cells.Furthermore,the expressions of IL-6,IL-8 mRNA and secreted proteins were up-regulated in senescent 786-O cells.The up-regulated expressions of IL-6,IL-8 mRNA and secreted proteins required ROS-activated p38,which can be attenuated by p38 inhibitor SB203580.Conclusion: 1.Res can induce senescence in 786-O cells in vitro.2.Increased ROS is required for 786-O cell senescence induced by Res.3.Res promotes SASP(senescence-associated secretory phenotype)through ROS-p38 pathway. |
