Pyroptosis Is Involved In The Antitumor Effect Of FL118 On Colorectal Cancer

Objective Pyroptosis is a newly discovered inflammatory programmed cell death,but the effect of it in colorectal cancer requires in depth studies.Furthermore,relevant findings highlighted the critical role of FL118,which is a novel agent that governs the inhibitory effect on the growth and metastasis of colorectal cancer.However,the molecular mechanism on the correlation of FL118 and pyroptosis requires further exploration.This study mainly evaluated whether FL118 could affect the anti-neoplastic effect through pyroptosis.Methods First,we detected the expression of caspase-1 in colorectal cancer.The expression of Caspase-1 in colorectal cancer was analyzed by bioinformatics,and the clinical samples of colorectal cancer patients were estimated by Immunohistochemistry,so as to understand the correlation with the expression of caspase-1 protein and colorectal cancer.Secondly,the antitumor effects of FL118 on human colorectal cancer cells(HT29 and SW480)were detected.MTT test was used to examine the effect of FL118 on cell viability;Wound healing and Transwell assay was used to detect the effect of FL118 on cell migration and invasion.In order to explore the role of pyroptosis in FL118's antitumor effect,the morphological changes of cells were observed by light microscope,and the phenomenon of cell death was further proved by TUNEL,and the occurrence of pyroptosis was verified by LDH release experiment.To further verify the role of caspase-1-mediated pyroptosis in antitumor process of FL118,the expression of caspase-1 and IL-18 and IL-1? genes were tested by qRT-PCR and the expression of caspase-1 and its downstream proteins were detected by Western Blot and Immunofluorescence.Complementally,the expression of IL-18 and IL-1? in the supernatant of FL118 treated cells was confirmed by Elisa.Finally,VX-765,the specific inhibitor of Caspase-1,was screened by MTT,and the appropriate concentration and action time were selected.On this basis,LDH release assay was used to detect the effect of VX-765 on FL118's ability to perforate colorectal cancer;qRT-PCR assay was used to detect the effect of VX-765 on caspase-1,IL-18 and IL-1 ? expression at mRNA level;Western blot was used the effects of VX-765 on Caspase-1 and its downstream protein expression were analyzed by Immunofluorescence assay;the effects of VX-765 on the expression of IL-18 and IL-1 ? protein were verified by Elisa assay;Lastly,the effects of VX-765 on the migration and invasion of FL118 were verified by scratch and Transwell assay.It is suggested that pyroptosis may be involved in the inhibition of FL118 on the growth and metastasis of colorectal cancer cells in vitro.Complementally,mice were subcutaneously injected and caudal vein with colorectal cancer cell HCT116/HT29 to construct tumor xenograft model and lung metastasis model.The nude mice were randomly divided into three groups: the vehicle group,the low dose group(4 mg / kg / week)and the high dose group(8 mg / kg / week).Different doses of FL118 solution were given by gavage,and the body weight and tumor weight of allogeneic mice were measured regularly.At the end of the experiment,the tumor tissues and various organs(heart,liver,spleen,lung,kidney,brain)of allogeneic transplantation model mice were frozen,and the lung tissues of lung metastasis model mice were embedded and preserved.HE staining experiment was used to examine the effect of FL118 on the morphology of mouse heart,liver,spleen,lung,kidney,brain and pulmonary surface nodules.TUNEL staining experiment was used to detect the effect of FL118 on animals.The effect of FL118 on the pyroptosis related proteins of colorectal cancer cells was detected by Immunohistochemistry.It can be proved that FL118 can inhibit the growth and metastasis of colorectal cancer in vivo.Results Bioinformatics statistical analysis showed that the expression of NLRP3 and caspase-1 was negatively correlated with the occurrence of colon cancer,and the expression of caspase-1 in normal samples was more than that in colorectal cancer samples;Immunohistochemistry analyzing suggested that the expression of caspase-1 in adjacent tissues was higher than that in colorectal cancer tissues.In vivo experimental results are as follows.MTT test displayd that FL118 significantly reduced the activity of colorectal cancer cells in a dose-dependent manner,and had no toxic effect on normal colon epithelial cells;Wound healing assay and Transwell showed that FL118 decreased the migration and invasion ability of colorectal cancer cells in a dose-dependent manner.The typical morphological characteristics of the colorectal cancer cells treated with FL118 were observed under the microscope.The results of TUNEL staining and LDH release exhibited that the number of cell death and the rate of LDH release in FL118 group were significantly increased compared with those in the control group,which further proved the occurrence of pyroptosis.The results of qRT-PCR indicated that with the increase of drug concentration,the expression of pyroptosis related genes increased significantly;Western Blot test and Immunofluorescence test also showed that the expression of protein level increased,and then Elisa analyzing suggested that the expression of secretory protein IL-18 and IL-1? increased with the augment of FL118 concentration.In MTT experiment,when FL118 and VX-765 was co-cultured,the cell viability was significantly higher than that of FL118 alone;LDH experiment showed that VX-765 weakened the ability of FL118 to perforate colorectal cancer cells;in mRNA and protein level,the expression of Caspase-1,IL-18 and IL-1? in FL118 + VX-765 group was significantly lower than that of FL118 alone;Wound healing assay and Transwell experiment declared that VX-765 reversed the ability of FL118 to inhibit cell migration and invasion.In vivo experimental results are as follows.The weight of subcutaneous tumor in mice decreased with the increase of FL118 concentration,but there was no significant difference among different groups.The results of staining suggested that there was no significant change in the morphology of each organ,and the number of pulmonary surface nodules in the lung metastasis model decreased with the increase of FL118 concentration;TUNEL analysis showed that the number of cell death increased in a dose-dependent manner;Immunohistochemistry indicated that the expression of NLRP3,Caspase-1 and their downstream proteins IL-18,IL-1? raised with the increase of FL118 concentration.Conclusion FL118 exhibits the growth and metastasis of colorectal cancer by inducing caspase-1-mediated pyroptosis,which provides important evidence in the relationship of pyroptosis and different tumors.