The Effets Of NLRP3 Inflammasome On The Proliferation,Migration,Invasion Of A549 Cells And The Underlying Mechanisms

Background and Objective: Primary bronchial lung cancer is one of the most malignant and highly metastatic cancers worldwide;it is the leading cause of cancer death.With the environmental deterioration and air pollution,the morbidity of lung cancer is rising rapidly,and the sufferers trend to be younger.In recent years,although the effects of comprehensive treatments such as surgery,chemotherapy,targeted therapy improve well,the 5-year survival rate of lung cancer is still low.Therefore,in addition to the traditional treatments,immunotherapy aiming at the tumor microenvironment has received great attention.Cancer can be defined by six hallmarks,including limitless replicative potential,sustained ability to develop blood wessels(angiogenesis),evasion of programmed cell death apoptosis,self-sufficiency in growth signals,insensitivity to inhibitors of growth,and tissue invasion and metastasis.The cancer-related inflammation has been recognized as the “seventh hallmark of cancer”.Inflammation is the biological immune response of body to harmful stimuli,such as pathogens,damaged cells,or various chemical and physical damages.Mounting studies emphasize that chronic inflammation is crucial for the development of tumor.Inflammasome is a large cytosolic multi-protein complex that assembles in response to detection of infection-or stress-associated stimuli and results in the activation of inflammatory responses.NLRP3 inflammsome are well characterized and can be activated by many stimuli.It has been demonstrated that NLRP3 inflammsome plays an important role in the development and progression of melanoma,hepatocellular carcinoma,gastric cancer,colon cancer,and prostate cancer.However,the expression and function of inflammasome in cancer development are cell-and tissue-specific.For instance,NLRP3 inflammsome plays beneficial for hepatocellular carcinoma while detrimental for melanoma.The present study aimed to investigate the effets of NLRP3 inflammasome on the proliferation,migration,invasion of A549 cells and the underlying mechanisms.Methods: 1.The association between NLRP3 and ASC was determined by immunofluorescence.The caspase-1 p10 was detested by western blot.The levels of IL-1β and IL-18 in cell culture supernatants were measured by human ELISA kit.2.5-ethynyl-2’-deoxyuridine(Ed U)incorporation proliferation assays were performed to investigate the proliferation of A549 cells using a Cell-Light TM Ed U Imaging Kit.Flow cytometric analysis of annexin V-FITC/PI-staining cells were perfomed to determine the apoptosis of cells.Western blot was performed to detect the signal transduction of p-Akt、p-GSK-3β、p-ERK1/2、p-CREB.The activation of NLRP3 inflammasome was inhibited by: 1)down-regulating NLRP3 expression by sh RNA,2)inhibiting NLRP3 inflammasome activation with caspase-1 inhibitor,or 3)blocking the interleukin-1β(IL-1β)and IL-18 signaling transduction with IL-1 receptor antagonist(IL-1Ra)and IL-18 binding protein(IL-18BP).3.Cell migration was investigated using scratch assays and transwell migration chamber assays.Cell invasion was determined using Matrigel invasion assays.E-cadherin and Snail expressions were determined by western blot.Results: 1.Many ASC and NLRP3 colocalizations were found in punctate spots style after stimulating with LPS+ATP.However,little detectable NLRP3 and ASC colocalization was found in control,LPS,or ATP single group.Consistently,western blot analysis showed that the activation of caspase-1 p10 was significantly increased by LPS+ATP treatment.Moreover,the extracellular levels of IL-18 and IL-1β were significantly increased in the LPS+ATP group rather than other groups.Together,these data demonstrate that LPS+ATP induced the formation and activation of NLRP3 inflammasome in A549 cells.2.Activation of NLRP3 inflammasome enhances the proliferation of A549 cells.Cells treated with LPS+ATP,but not LPS or ATP alone,resulted in a significant increase in Ed U-postive cells.LPS+ATP increased the number of Ed U-positive cells in the sh Ctrl group,but not in the sh NLRP3 group,that is to say sh NLRP3 reversed the LPS+ATP-induced proliferation.The caspase-1 inhibitor z-YVAD-FMK had no effect on the A549 proliferation under baselin conditions,but it inhibited the effects of LPS+ATP on cell proliferation.Moreover,neutralization of IL-18 activity with IL-18 BP or blockage of IL-1β activity with IL-1Ra attenuated the LPS+ATP-induced enhancement of proliferation respectively,and combined use of IL-18 BP and IL-1Ra could abolish pro-proliferative effects of LPS+ATP.The results of western blot showed that activation of NLRP3 inflammasome by LPS+ATP increased the phosphorylation of Akt,GSK-3β,ERK1/2,and CREB.These effects of NLRP3 inflammasome activation were inhibited by the caspase-1 inhibitor z-YVAD-FMK,IL-18 BP or IL-1Ra,and reversed by NLRP3 knockdown,combination of IL-18 BP and IL-1Ra.IL-18 BP played more effect on the Akt/GSK-3β cell signaling pathway,while IL-1Ra played more effect on the ERK1/2 pathway.3.Flow cytometric analysis of annexin V-FITC/PI-staining revealed that LPS,ATP,and LPS+ATP did not alter the apoptosis of A549 cells.4.Activation of NLRP3 inflammasome enhances the migration of A549 cells.Cell migration was investigated using scratch assays and transwell migration chamber assays.Scratch assays demonstrated that LPS+ATP elicited an increase in the migrated distance compared with the control group.The results of transwell migration chamber assay were consistent with the scratch assay.In sh Ctrl A549 cell,LPS+ATP also enhanced cell migration in scratch assays and transwell migration chamber assays.However,this effect was abolished by down-regulating the expression of NLRP3 as in sh NLRP3 cells.In addition,caspase-1 inhibitor z-YVAD-FMK as well as IL-18 BP or IL-1Ra inhibited LPS+ATP-induced migration in both scratch assays and transwell migration chamber assays.Notably,combined treatment of IL-18 BP and IL-1Ra reversed LPS+ATP-induced enhancement of migration.5.Activation of NLRP3 inflammasome enhances the migration of A549 cells.LPS+ATP enhanced invasion of sh Ctrl A549 cells rather than sh NLRP3 A549 cells.The caspase-1 inhibitor decreased LPS+ATP-induced invasion.IL-18 BP or IL-1Ra also inhibited the LPS+ATP-induced invasion and combined use of IL-18 BP and IL-1Ra abolished the effect,maintaining invasion at control levels.6.The cellular adhesion molecule E-cadherin and the transcription factor Snail are two important factors in inflammation-induced cancer cell migration and invasion.Activation of NLRP3 inflammasome by LPS+ATP decreased the expression of E-cadherin and increased the expression of Snail in both wildtype and sh Ctrl A549 cells,but not in sh NLRP3 A549 cells.In addition,these effects were partially attenuated by IL-18 BP and IL-1Ra,and they were reversed by the caspase-1 inhibitor and the combination of IL-18 BP and IL-1Ra.Considering the inconsistent between inhibited effect of scratch assays,transwell migration chamber assays and matrigel invasion assays and the reversed effect of E-cadherin and Snaili by caspase-1 inhibitor z-YVAD-FMK,we hypothsised that there were other signaling pathway involved in migration and invasion by z-YVAD-FMK.It was observed that caspase-1 inhibitor z-YVAD-FMK partically attenuated the p-JNK,p-p38,p-ERK1/2 MAPK pathways.Conclusion: 1.NLRP3 inflammsome activation enhances the proliferation,migration and invasion of A549 cells.The cell signaling pathways of p-Akt、p-GSK-3β、p-ERK1/2、p-CREB、E-cadherin、Snail、MAPK were involved.2.NLRP3 inflammasome plays a vital role in regulating the features of A549 cells and it could be a novel target for the treatment of lung cancer.