| Objective: As two important members of the inhibitor of apoptosis protein(IAP)family,survivin and XIAP are significantly overexpressed in many tumor tissues,including colorectal cancer.They participate in many biological processes of tumorigenesis and development and have been considered as potential targets for cancer treatment.Previous study reported that survivin can form an IAP-IAP complex with XIAP to stabilize XIAP and then synergistically inhibit Caspase 9 activity and play anti-apoptotic effects.Therefore,this study aims to develop a novel anticancer peptide targeting survivin-XIAP complex and explore its antitumor mechanisms in colorectal cancer cells.Methods: 1.Online databases Oncomine and GEPIA were used to analyze the expression of IAPs in various cancer tissues,including colorectal cancer,and normal tissues.2.The anticancer peptide Sur-X,targeting survivin-XIAP complex,was designed and synthesized,with Con as a negative control.3.Co-immunoprecipitation was used to detect the formation of survivin-XIAP complex in colorectal cancer cells and the effect of Sur-X on the binding of survivin and XIAP.4.The effect of Sur-X on the growth of colorectal cancer cells were determined by MTT assay and colony formation assay.5.Subcutaneous xenograft tumor models were established in nude mice to detect the anticancer activity and safety of Sur-X in vivo.6.Real-time apoptosis assay and Caspase 3 activity assay were performed to detect apoptosis in colorectal cancer cells with the treatment of Sur-X.7.The effect of Sur-X on apoptosis-related proteins expression levels was detected by Western blot.8.The mRNA expression levels of survivin and XIAP were detected by qRT-PCR.9.Cycloheximide(CHX)was used to determine the half-life of protein,and the effect of SurX on the degradation rate of survivin and XIAP was examined.10.Coimmunoprecipitation was used to detect the effect of Sur-X on the binding of XIAP to Caspase 9.11.Giemsa-staining,real-time necrosis assay and Annexin V/7-AAD double staining were used to detect the effect of Sur-X on cell membrane integrity.12.Western blot was used to detect the effect of Sur-X on the expression levels of necroptosis-related proteins.13.Colorectal cancer cells were pretreated by z-VAD and Nec-1s,then Annexin V/7-AAD double staining and flow cytometry was used to confirm Sur-X-induced apoptosis and necroptosis in colorectal cancer cells.14.Molecular dynamics simulation was used to predict the binding of Sur-X to XIAP.15.PDB database was used to find proteins that bind to XIAP.16.The formation of XIAP-TAB1-TAK1 complex in colorectal cancer cells and Sur-X's effect on the binding of XIAP to TAB1 were determined by Coimmunoprecipitation.17.Western blot was used to detect the effect of Sur-X on the expression of TAB1,TAK1,and p-TAK1.18.CHX was used to determine the half-life of the protein,and the effect of Sur-X on the degradation rate of TAK1 was examined.19.Jet-mediated transfection of siRNAs and Plasmids was used to knock down TAK1 or overexpress TAB1.20.Western blot was used to detect the effect of Sur-X on NF-?B pathway.21.ELISA was used to detect the effect of Sur-X on secretion of TNF-? by colorectal cancer cells.22.Protein expression levels of survivin,XIAP,and MLKL in tumor tissues from nude mice was detected by immunohistochemical staining.23.TUNEL staining was used to detect apoptosis in tumor tissues of nude mice.Results: 1.Survivin and XIAP were overexpressed in colorectal cancer.Oncome analysis showed that survivin was the most widely studied member of IAP family.GEPIA analysis showed that among all IAPs,only survivin and XIAP were highly expressed in colorectal cancer compared with normal tissues.2.The anti-cancer peptide Sur-X,targeting survivin XIAP complex in colorectal cancer cells,was designed.The results of coimmunoprecipitation showed that there was survivin-XIAP complex in HCT116 and RKO cells.Based on the findings of previous study that the smallest segment of survivin that can bind with XIAP was K15-M38,we designed and synthesized the peptide Sur-X.After treated with Sur-X for 1 hour,the interaction between survivin and XIAP was significantly reduced in HCT116 cells.3.Sur-X can specifically and effectively inhibit the growth of colorectal cancer cells.Four kinds of human colorectal cancer cells(HCT116,RKO,HCT15 and HT29)and one human normal peritoneal mesothelial cell HMrSV5(SV5)were treated by a series of concentrations of Sur-X and Con,the cell viability was detected by MTT assay.The results showed that Sur-X could significantly inhibit the viability of colorectal cancer cells,but had no effect on the normal cell SV5.The colony formation assay also confirmed the significant inhibition of Sur-X on colorectal cancer cells.Western blot analysis showed that survivin and XIAP were highly expressed in four colorectal cancer cell lines,but not in normal SV5 cells,suggesting that the specific anticancer effect of Sur-X may depend on the expression of survivn and XIAP.4.Sur-X can significantly inhibit the growth of colorectal cancer cells in nude mice without significant toxicity.In order to study the antitumor activity of peptide Sur-X in vivo,we first established subcutaneous tumor model by HCT116 cells in nude mice,which then received intratumoral injection of peptides.The results showed that Sur-X could significantly inhibit the growth of tumor and had no significant effect on the weight of nude mice.In order to further confirm the efficacy and safety of Sur-X in vivo,we next established the subcutaneous tumor model of nude mice which then received intravenous injection of peptides.The results showed that Sur-X could significantly inhibit the growth of tumor in nude mice,and had no significant toxicity to vital organs.5.Sur-X induced apoptosis of colorectal cancer cells by targeting Survivin-XIAP complex and promoting the degradation of survivin and XIAP.The results of real-time apoptosis detection and Caspase-3 activity detection showed that Sur-X rapidly induced apoptosis and increased Caspase 3 activity in colorectal cancer cells.Western blot analysis showed that Sur-X could reduce the expression of survivin and XIAP in HCT116 and RKO,and increase cleavages of Caspase 9,Caspase 3,Caspase 7 and PARP.Sur-X had no significant inhibition on the mRNA expression of survivin and XIAP,but could significantly accelerate the protein degradation of survivin and XIAP.No effect of Sur-X on the interaction between XIAP and Caspase 9 was detected by co-immunoprecipitation.ZVAD could only partially rescue Sur-X-induced cell death in colorectal cancer cells.6.Sur-X promoted necroptosis in colorectal cancer cells by inhibiting the binding of XIAP and TAB1 and promoting the degradation of TAK1.The results of Giemsa staining,realtime necrosis detection and Annexin V/7-AAD flow cytometry showed that colorectal cancer cells treated by Sur-X rapidly lost cell membrane integrity.Western blot analysis showed that Sur-X could significantly increase the expression of p-RIP1,p-RIP3 and pMLKL in HCT116 and RKO.Nec-1s,an inhibitor of necroptosis,could partially rescue Sur-X-induced colorectal cancer cell death,especially decrease the percentage of 7-AADpositive cells.Molecular dynamics simulation showed that Sur-X mainly binds to the BIR1 domain of XIAP.The results of searching “XIAP BIR1” in PDB database showed that TAB1 could bind to the BIR1 domain of XIAP.The XIAP-TAB1-TAK1 complex was detected in colorectal cancer cells,and Sur-X was found to be able to interfere with the binding of XIAP and TAB1.The results of Western blot analysis showed that Sur-X could significantly reduce the expression of TAK1 and p-TAK1 in colorectal cancer cells,but had no significant effect on the expression of TAB1.CHX was used to determine the halflife of TAB1 and it was found that Sur-X could accelerate the degradation of TAK1.In order to verify how the expression of TAB1 and TAK1 could affect the sensitivity of colorectal cancer cells to Sur-X-induced necroptosis,plasmids and siRNA were used to overexpress TAB1 and knock down TAK1 respectively,and the influence of Sur-X on the viability of transfected cells was detected by MTT assay,and Western blot analysis was used to evaluate the expression of necroptosis-related proteins.The results showed that the knockdown of TAK1 enhanced the inhibition of Sur-X on colorectal cancer cells and promoted the expression of p-RIP1 and p-MLKL,the overexpression of TAB1 weakened the inhibition of Sur-X on colorectal cancer cells and decreased the expression of p-MLKL,however,the knockdown of TAK1 restored the inhibition of Sur-X on colorectal cancer cells overexpressed by TAB1,as well as the expression of p-MLKL.Western blot analysis showed that Sur-X only transiently activated NF-?B pathway,accordingly,it was found that TNF-? content in supernatant was the highest when HCT116 cells were treated by Sur-X for 30 minutes.7.Sur-X induced both apoptosis and necroptosis in vivo.HE,immunohistochemistry and TUNEL staining were used to analyze the indexes related to apoptosis and necroptosis in the tumor tissue of the nude mice receiving intravenous administration.The results showed that the expression of survivin and XIAP in the tumor tissue of the Sur-X group was lower than that of the Con group,and the TUNEL staining was significantly stronger in Sur-X group.The tumor tissue of the Sur-X group showed extensive necrosis area,and the expression of MLKL was significantly lower than that of the Con group.Conclusion: 1.The peptide Sur-X can inhibit the formation of survivin XIAP complex in colorectal cancer cells.2.Sur-X can specifically and effectively inhibit the growth of colorectal cancer cells both in vitro and in vivo.3.Sur-X can promote the apoptosis of colorectal cancer cells by interfering with the formation of survivin-XIAP complex and accelerating the degradation of Survivin and XIAP.4.Sur-X can induce necroptosis by inhibiting the binding of XIAP and TAB1 and promoting the degradation of TAK1.5.SurX plays anticancer effects by inducing both apoptosis and necroptosis in colorectal cancer cells. |
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