Studies On The Synergistic Effects Of TRAIL With A Derivative From Heat Modified Pectin And Ginsenoside CK On Human Colon Cancer Cells

Colorectal cancer(CRC)is the third most common malignancy globally,and it is a major cause of cancer related death.It is a heterogemeous disease.CRCs differ in molecular characteristics,response to radiotherapy,chemotherapy,clinical presentation,and disease prognosis.The incidence of CRC trends to raise by reason of the changing of people’s lifestyles and diets.Tumor necrosis factor-(TNF)-related apoptosis-inducing ligand(TRAIL)is a member of the TNF superfamily.TRAIL is considered to be an attractive agent against cancer.One of the most important problems using TRAIL in CRC treatment is resistant to TRAIL-mediated cell death.Fortunately,discovering drugs or natural products which can increase the susceptibility of CRC cells to TRAIL might be an effective method to solve this problem.DHCP((Trans)4,5-dihydroxy-2-cyclopentene-l-one)is a small molecule isolated from heat-modified citrus pectin and displayed cytotoxic effects in cancer cells.Here,we tested the synergistic effects of DHCP combine with TRAIL on CRC suppression.The results of cell viability assay,flow cytometry assay and western-blot assay indicated that the combination treatment with DHCP and TRAIL could synergistically decrease cell viability and induce apoptosis though caspase-dependent pathway.We further investigated the mechanisms by which DHCP enhanced TRAIL-induced apoptosis.The results showed that DHCP could increase the expression of proapoptotic proteins Bax,t Bid and cytochrome C,and decrease the expression of anti-apoptotic proteins Mcl-1,Bcl-2,XIAP,survivn,livin and c FLIP.To our excitment,we found that DHCP can significantly induce the up-regulation of DR5.We transfected HCT116 cells with DR5 si RNAs,and then the synergistic effect of DHCP and TRAIL was tested.The results showed that the synergistic effect of DHCP and TRAIL was inhibited significantly in DR5 si RNAs transfected cells.These data indicated that DR5 played an important role in the synergy in combination treatment of TRAIL with DHCP.We further investigated the mechanisms by which DHCP increase DR5 expression.We found that DHCP could upregulate DR5 expression through p53-CHOP,ERK,and JNK pathway by detecting related signaling molecules that regulate DR5 expression.The ROS generatio induced by DHCP is the upstream mechanism of the three pathways.Then we studied the mechanism of DHCP-induced generation of ROS in cells and the results indicated that mitochondria are the main source of ROS.The data of seahorse cell energy metabolism analyz and mitochondrial complex activity assay indicated that DHCP have an effect of inhibiting mitochondrial complex II function significantly.It is known that mitochondrial complex II(SDH)contains four subunits: SDHa,SDHb,SDHc,and SDHd.To determine the specificity of complex II inhibition by DHCP,the SQR and SDH activities of complex II were tested.Our results indicated that DHCP inhibits complex II activity by primarily binding to the ubiquinonebinding site in SDHc and SDHd.We next tested combination treatment effect of DHCP and TRAIL in vivo.Mice bearing xenografted HCT116 and HT-29 colon tumors were divided into four groups and treated with saline,intraperitoneal dose of DHCP(4.5 μmol/kg),intravenous dose of TRAIL(100 μg),and therapeutic combination of TRAIL and DHCP(100 μg/TRAIL + 4.5 μmol/kg DHCP)ever other day.The body weight and tumor size of each group were measured followed by ten consecutive cycles of combination therapy regimen.The expression of Ki67 and cleaved-caspase-3 in tumor tissues was detected by immunohistochemistry.The results showed that DHCP and TRAIL inhibited the growth of xenografts and the expression of Ki67 synergistically,and greatly promoted the expression of cleaved-caspase-3.CK,absent naturally,could be generated from other protopanaxadiol-type ginsenosides via various methods.It was reported that ginsenoside Rg3 could sensitize cancer cells to TRAILinduced cell death.In this paper,we investigated wheather CK could enhance TRAIL-induced apoptosis.The cell viability assay,flow cytometry assay and western-blot assay were used to detect the synergistic effect of CK and TRAIL on colon cancer.The results indicate that the combination treatment with CK and TRAIL could synergistically decrease cell viability and induce apoptosis though caspase-dependent pathway.We further investigated the mechanisms by which CK enhanced TRAIL-induced apoptosis.The results showed that CK could significantly induce up-regulation of DR5,and the synergistic effect of CK and TRAIL was inhibited significantly in DR5 si RNAs transfected cells.The results indicated that DR5 plays an important role in the synergy in combination treatment of CK and TRAIL.In addition,CK increased the expression of proapoptotic proteins Bax,t Bid and cytochrome C,and decreased the expression of anti-apoptotic proteins Mcl-1,Bcl-2,XIAP,survivn,livin and c FLIP.The mechanisms by which CK induce DR5 expression was investigated.we first found that CK upregulates DR5 expression through autophagy pathway.When we pretreated cells with autophagy inhibitors 3-MA,LY294002 or tranfected with Atg7 si RNAs can significantly inhibit CK induced upregulation of DR5 expression,and the synergistic effect of CK and TRAIL is also significantly decreased.We also tested ROS,p53,CHOP,JNK,ERK,and p38 in CK treated cells.The results indicated that CK induced DR5 upragulation through ROSJNK-autophagy and p53-CHOP two independent signaling pathways,respectively.In conclusion,we found that both DHCP and CK exhibited synergistic effects with TRAIL on inhibiting colon cancer.Our reseach data will provide a theoretical basis for the clinical application of TRAIL in colon cancer therapy.