Effects Of Arginine Specific ADP-ribosyltransferase1on Apoptosis Of Mouse Colon Carcinoma CT26Cells And The Mechanisms

Background and Aim：Colon carcinoma is one of the most common and malignant tumorsin human life throughout the world with prominent morbidity and mortality.Apoptosis is an important biological behavior of tumor cells. There has anintimate relationship between abnormality of apoptosis induced byabnormal gene regulation and the unlimited tumor growth and resistant tovarious anti-tumor treatments. Arginine specific ADP-ribosyltransferase1(ART1), which has a close contact with varied kinds of cell biologybehaviors, is an important and classical mono-ADP-ribosyltransferase. Butresearches about the connection of ART1with diseases are limited.The relations of ART1with colon carcinoma biological behaviorshave been partly invesgated in previous studies of our research team. Wehave demostrated that ART1gene silencing could affect proliferation,invasion, metastasis, cell differentiation and microangiogenesis in mousecolon carcinoma CT26cells. But the influence of ART1on apoptosis incolon carcinoma and its potential mechanisms are not clear yet. The aim of this research is to investigate the influence of ART1onapoptosis induced by CDDP and its mechanisms in mouse colon carcinomaCT26cells via silence ART1or over expression ART1by transfectingCT26cells with lentiviruses carrying ART1-shRNA and ART1-cDNA. Tryto provide experimental basis for ART1as a new treatment target of coloncarcinoma.Methods:This research was conducted through the following two parts.1．Effect of ART1on apoptosis induced by CDDP in mouse coloncarcinoma CT26cells.(1) Obtainment and identification of stable transfected ART1-shRNAand ART1-cDNA CT26cells.Silenced or over expressed ART1via infect CT26cells withART1-shRNA or ART1-cDNA Lentiviruses, respectively. LV-control andUntransfected Control groups were included.1) The transfection efficiency is indentify by observing the greenfluorescence GFP expression of transfected CT26cells throughfluorescence microscope;2) The ART1mRNA expression of each groups was detected withRT-PCR;3) The ART1protein expression of each group was detected with WesternBlot. (2) In vitro study on effect of ART1on apoptosis of mouse coloncarcinoma CT26cells induced with CDDP.ART1-shRNA, ART1-cDNA, LV-control and Untransfected CT26cellswere induced apoptosis with CDDP.1) Apoptosis fragments (Ladder) in each group of CT26cells wereobserved with DNA Laddering detection. LV-control and UntransfectedCT26cells served as control groups, ART1-shRNA and AR1-cDNA CT26cells as experimental groups.2) The Survival rates of each group of CT26cells were detected withCCK8cell counting method in ART1-shRNA and AR1-cDNA CT26cells.LV-control and Untransfected CT26cells treated with CDDP served ascontrol groups.3) Apoptosis rates of each group of CT26cells were detected withAnnexin V-PE/7-AAD flow cytometry and Hoechst33342cell counting.Four CDDP untreated CT26cells served as control groups.(3) In vivo study on effect of ART1on apoptosis of mouse coloncarcinoma CT26cells induced with CDDP.1) ART1-shRNA, ART1-cDNA, LV-control and Untransfected CT26cells transplant tumor at Balb/c mouse right armpit subcutaneous wereestablished, apoptosis of each group of transplant tumor were induced byintraperitoneal injection of CDDP, Balb/c mice intraperitoneal injected with0.9%NaCl served as control groups. The volume and weight of each group of Balb/c mouse CT26cells transplant tumor were measured.2) Cleave level of PARP-1(apoptosis rate) in each group of transplanttumor cells were detected with Western Blot after establishingART1-shRNA, ART1-cDNA, LV-control and Untransfected CT26cellstransplant tumor at Balb/c mouse right armpit subcutaneous and induceingapoptosis by intraperitoneal injection of CDDP. Balb/c mice intraperitonealinjection with0.9%NaCl served as control groups.2. Investigation of mechanisms involved in the effect of ART1onapoptosis of mouse colon carcinoma CT26cells induced with CDDP.(1) Detection of Akt, ERK pathway and NF-κB1) Detection of Akt expression and activityApoptosis of ART1-shRNA, ART1-cDNA, LV-control andUntransfected CT26cells were induced with CDDP. Protein expression ofAkt and phos-AktT308in each group of CT26cells were detected withWestern Blot. CDDP untreated CT26cells served as control groups.2) Influence of PI3K inhibiter LY294002on ART1, Akt expressionand Akt phosphorylation in mouse colon carcinoma CT26cellsProtein expression of ART1, Akt and phos-AktT308in UntransfectedCT26cells were detected with Western Blot after LY294002and CDDPtreatment. LY294002untreated CT26cells served as control group.3) Detection of ERK expression and activityApoptosis of ART1-shRNA, ART1-cDNA, LV-control and Untransfected CT26cells were induced with CDDP. Protein expressions ofERK and phos-ERK in each group of CT26cells were detected withWestern Blot. CDDP untreated CT26groups served as control.4) Influence of MEK inhibiter PD98059on ART1, ERK expression andERK phosphorylation in mouse colon carcinoma CT26cellsProtein expressions of ART1, ERK and phos-ERK in UntransfectedCT26cells were detected with Western Blot after PD98059and CDDPtreatment. PD98059untreated CT26cells as control group.4) Detection of NF-κBp65expression and NF-κBp65nucleartranslocationApoptosis of ART1-shRNA, ART1-cDNA, LV-control andUntransfected CT26cells were induced with CDDP. Total expression ofNF-κB p65and nuclear expression of NF-κB p65in each group of CT26cells were detected with Western Blot. CDDP untreated groups served ascontrol groups.5) Influence of PI3K inhibiter LY294002on NF-κB p65expressionand nuclear translocation in mouse colon carcinoma CT26cells inducedapoptosis with CDDPProtein expressions of total NF-κB p65and nuclear NF-κB p65weredetected in Untransfected CT26cells with Western Blot after LY294002and CDDP treatment. LY294002untreated CT26cells served as controlgroup. 6) Influence of MEK inhibiter PD98059on NF-κB p65expression andnuclear translocation in mouse colon carcinoma CT26cellsProtein expression of total NF-κB p65and nuclear NF-κB p65inUntransfected CT26cells were detected with Western Blot after PD98059and CDDP treatment. PD98059untreated CT26cells served as controlgroup.(2) Detection of Bcl-2family protein Bcl-2, Bcl-xl and Bax1) Detection of Bcl-2, Bcl-xl and Bax expressionApoptosis of ART1-shRNA, ART1-cDNA, LV-control andUntransfected CT26cells were induced with CDDP. Protein expression ofBcl-2, Bcl-xl and Bax in four groups of CT26cells were detected withWestern Blot. CDDP untreated groups served as control groups.2) Influence of PI3K inhibiter LY294002on Bcl-2, Bcl-xl and Baxexpression in mouse colon carcinoma CT26cellsExpression of Bcl-2, Bcl-xl and Bax in Untransfected CT26cells weredetected with Western Blot after LY294002and CDDP treatment.LY294002untreated CT26cells served as control group.3) Influence of MEK inhibiter PD98059on Bcl-2, Bcl-xl and Baxexpression in mouse colon carcinoma CT26cellsExpression of Bcl-2, Bcl-xl and Bax in Untransfected CT26cells weredetected with Western Blot after PD98059and CDDP treatment. PD98059untreated CT26cells served as control group. (3) Influnce of ART1on TUBB3expression in mouse coloncarcinoma CT26cells induced apoptosis by CDDP1) Detection of TUBB3protein expressionApoptosis of ART1-shRNA, ART1-cDNA, LV-control andUntransfected CT26cells were induced with CDDP. Then proteinexpression of TUBB3in four groups of CT26cells were detected withWestern Blot，CDDP untreated groups served as control groups; Meanwhile, right armpit subcutaneous transplant tumor model were establishedwith ART1-shRNA, ART1-cDNA, LV-control and Untransfected CT26cells in Balb/c mouse. Apoptosis of cells were induced by intraperitonealinjection of CDDP. Protein expressions of TUBB3in four groups of CT26cells transplant tumors were also detected with Western Blot. Transplanttumor which intraperitoneal injection with0.9%NaCl served as controlgroups.2) Detection of TUBB3mRNA expressionApoptosis of ART1-shRNA, ART1-cDNA, LV-control andUntransfected CT26cells were induced with CDDP. Then mRNAexpression of TUBB3in four groups of CT26cells were detected withRT-PCR, CDDP untreated groups served as control groups; At the sametime，right armpit subcutaneous transplant tumor model were establishedwith ART1-shRNA, ART1-cDNA, LV-control and Untransfected CT26cells in Balb/c mouse. Apoptosis of transplant tumor cells were induced by intraperitoneal injection of CDDP. Transplant tumor intraperitonealinjection with0.9%NaCl served as control groups. mRNA expression ofTUBB3in four groups of CT26cells transplant tumors were also detectedwith RT-PCR.3) Influence of PI3K inhibiter LY294002on Bcl-2, Bcl-xl and Baxexpression in mouse colon carcinoma CT26cells induced apoptosis byCDDPProtein epression of TUBB3in Untransfected CT26cells was detectedwith Western Blot after LY294002and CDDP treatment. LY294002untreated CT26cells served as control group.4) Influence of MEK inhibiter PD98059on TUBB3expression inmouse colon carcinoma CT26cellsUntransfected CT26cells were treated with PD98059and CDDP. Thenprotein expression of TUBB3was detected with Western Blot. PD98059untreated CT26cells sereved as control group.Results:1. Influence of ART1on apoptosis of mouse colon carcinoma CT26cells induced by CDDP.(1) Stable ART1-shRNA, ART1-cDNA or LV-control transfectedCT26cells with70%-80%fluorescence intensity were successfullyobtained. The expression of ART1mRNA, detected by RT-PCR,significantly reduced in ART1-shRNA CT26cells（P<0.05）and significantly enhanced in ART1-cDNA CT26cells（P<0.05）compared toLV-control or Untransfected CT26cells, respectively. There wasno difference in the expression of ART1mRNA between LV-control andUntransfected CT26cells（P>0.05）. The protein level of ART1, tested byWestern Blot, was significantly reduced in ART1-shRNA CT26cells（P<0.05）and significantly enhanced inART1-cDNACT26cellscompared to LV-control or untransfected groups，respectively（P<0.05）.There was no difference in expression of ART1protein level betweenLV-control and Untransfected CT26cells（P>0.05）. The results suggestedthat ART1silencing and ART1over expression CT26cell lines weresuccessfully obtained.(2) Influence of ART1on CT26cell survival and apoptosis inducedby CDDP in vitro.1) DNA Ladder was detected in ART1-shRNA, ART1-cDNA,LV-control and Untransfected CT26cells treated with CDDP, while noDNA Ladder was observed in four groups of CT26cells without CDDPtreatment.2) By CCK8cell counting method, the survival rate significantlydecreased in ART1-shRNA CT26cells (P<0.05) and significantly increasedin ART1-cDNA CT26cells compared to LV-control CT26cells orUntransfected CT26cells (P<0.05). There was no significant difference insurvival rate between LV-control CT26cells and Untransfected CT26cells (P>0.05).3) The results of Annexin V-PE/7-AAD flow cytometry suggested thatapoptosis rate significantly rised in ART1-shRNA CT26cells (P<0.05)while decreased in ART1-cDNA CT26cells when compared to LV-controlCT26cells or Untransfected CT26cells, respectively (P<0.05). There wasno statistically difference in apoptosis rate between LV-control CT26cellsand Untransfected CT26cells (P>0.05).4) Apoptosis rate, as showed by results of Hoechst33342cellcounting, was significantly elevated in ART1-shRNA CT26cells (P<0.05)while decreased in ART1-cDNA CT26cells compared to LV-control CT26cells or Untransfected CT26cells, respectively (P<0.05). There was nostatistically difference in apoptosis rate between LV-control CT26cells andUntransfected CT26cells (P>0.05).(3) Influence of ART1on the apoptosis of CT26cells induced byCDDP in vivo.1) The transplant tumor volume and weight were significantly reducedin ART1-shRNA group (P<0.05), while statistically enhanced inART1-cDNA group compared to LV-control or Untransfected transplanttumor, respectively (P<0.05), There was no significantly difference involume and weight between LV-control transplant tumor and Untransfectedtransplant tumor (P>0.05).2) Protein expression of PARP-1cleaved peptide, detected by Western Blot, was statistically enhanced in ART1-shRNA transplant tumor whilereduced in ART1-cDNA transplant tumor compared to LV-control orUntransfected transplant tumor, respectively (P<0.05). PARP-1cleavedpeptide did not alter statistically in LV-control transplant tumor comparedto Untransfected group (P>0.05).2. Researches on the mechanisms related to the influence of ART1onCT26apoptosis induced by CDDP.(1) Regulation of ART1on expression and activity of Akt, ERK aswell as expression and nuclear transcription of NF-κBp65.1) The protein expression of phos-AktT308reduced in ART1-shRNACT26cells while statistically enhanced in ART1-cDNA CT26cellscompared to LV-control or Untransfected CT26cells, respectively (P<0.05),both for cells with and without CDDP treatment. There was no statisticallydifference in phos-AktT308expression between LV-control CT26cells andUntransfected CT26cells (P>0.05). The protein expression of Akt did notchange statistically both in ART1-shRNA CT26cells and ART1-cDNACT26cells compared to LV-control or Untransfected CT26cells (P>0.05),There was also no statistically difference in Akt expression betweenLV-control and Untransfected CT26cells (P>0.05).2) The protein expression of ART1and Akt in LY294002treatedCT26cells did not change significantly compared to LY294002untreatedCT26cells (P>0.05), while phos-AktT308significantly reduced compared to LY294002untreated CT26cells (P<0.05), both for cells with and withoutCDDP treatment.3) Protein expression of phos-ERK reduced in ART1-shRNA CT26cells (P<0.05), while enhanced statistically in ART1-cDNA CT26cellscompared to LV-control or Untransfected CT26cells (P<0.05), both forcells with and with out CDDP treatment. Phos-ERK did not changestatistically in LV-control CT26cells compared to Untransfected CT26cells (P>0.05). The protein expression of ERK did not change statisticallyboth in ART1-shRNA CT26cells and ART1-cDNA CT26cells comparedto LV-control or Untransfected CT26cells (P>0.05). ERK expression didnot change statistically in LV-control CT26cells compared toUntransfected CT26cells (P>0.05), both for cells with and with out CDDPtreatment.4) Protein expression of ART1and ERK in PD98059treated CT26cells did not alter significantly compared to PD98059untreated CT26cells(P>0.05), while phos-ERK significantly reduced compared to PD98059untreated CT26cells (P<0.05), both for cells treat or untreat by CDDP.5) Protein expression of NF-κB p65in nuclear significantly reduced inART1-shRNA CT26cells (P<0.05) while statistically enhanced inART1-cDNA CT26cells compared to LV-control or Untransfected CT26cells, respectively (P<0.05), both for cells with or with out CDDP treatment.NF-κB p65in nuclear did not change statistically in LV-control CT26cells compared to Untransfected CT26cells (P>0.05). Total NF-κB p65did notalter statistically both in ART1-shRNA CT26cells and ART1-cDNA CT26cells compared to LV-control or Untransfected CT26cells, respectively(P>0.05). There was no statistically difference in total NF-κB p65expression between LV-control CT26cells and Untransfected CT26cells(P>0.05).6) Protein expression of total NF-κB p65in LY294002treated CT26cells did not change significantly compared to LY294002untreated CT26cells (P>0.05), while NF-κB p65in nuclear significantly reduced comparedto LY294002untreated CT26cells (P<0.05), both for cells treated oruntreated by CDDP.7) Protein expression of both total NF-κB p65and nuclear NF-κB p65in PD98059treated CT26cells did not alter significantly compared toPD98059untreated CT26cells (P>0.05), both for cells treated or untreatedby CDDP.(2) Regulation of ART1on expression of Bcl-2family protein inmouse colon carcinoma CT26cells.1) Protein expression of Bcl-2, Bcl-xl reduced in ART1-shRNA CT26cells and enhanced in ART1-cDNA CT26cells (P<0.05), Bax enhanced inART1-shRNA CT26cells and reduced in ART1-cDNA CT26cells(P<0.05), which compared to LV-control CT26cells or Untransfected CT26cells, respectively (P<0.05). There was no statistically difference in all three proteins between LV-control CT26cells and Untransfected CT26cells (P>0.05), both for cells with or with out CDDP treatment.2) Protein expression of Bcl-2and Bcl-xl both statistically reduced(P<0.05), Bax enhanced significantly (P<0.05) in LY294002treated CT26cells compared to LY294002untreated CT26cells, both for cells treated oruntreated by CDDP.3) There were no statistically difference in Bcl-2, Bcl-xl and Baxprotein expression between PD98059treated CT26cells and PD98059untreated CT26cells (P>0.05), both for cells treated or untreated by CDDP.(3) Regulation of ART1on TUBB3protein and mRNA expression inmouse colon carcinoma CT26cells.1) The protein expression of TUBB3reduced in ART1-shRNA CT26cells while enhanced in ART1-cDNA CT26cells compared to LV-control orUntransfected CT26cells, respectively (P<0.05). There was no statisticallydifference in TUBB3protein expression between LV-control CT26cellsand Untransfected CT26cells (P>0.05) both for cells with or withoutCDDP treatment. Protein expression of TUBB3significantly reduced inART1-shRNA transplant tumor while enhanced in ART1-cDNA transplanttumor compared to two control groups (P<0.05),both for transplant tumorstreated or untreated by CDDP. There was no statistically difference inTUBB3protein expression between LV-control transplant tumor andUntransfected transplant tumor (P>0.05). 2) The mRNA expression of TUBB3, as showed by results of RT-PCR,significantly reduced in ART1-shRNA CT26cells (P<0.05), whilestatistically enhanced in ART1-cDNA CT26cells compared to two controlgroups (P<0.05). There was no statistically difference in TUBB3mRNAbetween LV-control CT26cells and Untransfected CT26cells (P>0.05).TUBB3mRNA expression significantly reduced in ART1-shRNAtransplant tumor (P<0.05), while enhanced in ART1-cDNA transplanttumor compared to LV-control or Untransfected transplant tumor (P<0.05),both for transplant tumors with or without CDDP treatment. There was nostatistically difference in TUBB3mRNA expression between LV-controltransplant tumor and Untransfected transplant tumor (P>0.05).3) The protein expression of TUBB3statistically reduced inLY294002treated CT26cells compared to LY294002untreated CT26cells(P<0.05), both for cells treated or untreated by CDDP.4) The protein expression of TUBB3statistically reduced in PD98059treated CT26cells compared to PD98059untreated CT26cells (P<0.05),both for cells with or without CDDP treatment.Conclusion:1. ART1silencing significantly improves apoptosis level, reduces thesurvival rate of CT26cells, inhibits growth of Balb/c mice subcutaneoustransplant tumor, elevates apoptosis rate of transplant tumor cells, ART1over expression acts oppositely. The results indicate that ART1regulats apoptosis of mouse colon carcinoma CT26cells induced by CDDP both invitro and in vivo.2. ART1silencing inhibits the activity of Akt pathway, reduces theexpression of NF-κB p65in nuclear, Bcl-2and Bcl-xl, meanwhile enhancedexpression of Bax, ART1over expression acts oppositely. In addition, theexpression of NF-κB p65in nuclear, Bcl-2and Bcl-xl are reduced, Bax isenhanced when activity of Akt pathway is inhibited. The results indicatethat ART1might regulate apoptosis of CT26cells induced by CDDP viaAkt/NF-κBp65/Bcl-2, Bcl-xl, Bax approach. Further more, ART1silencinginhibits activity of Akt pathway and expression of TUBB3protein as wellas TUBB3mRNA, while ART1over expression acts oppositely. Theexpression of TUBB3mRNA and TUBB3protein are also reduced byinhibition of Akt pathway activity. The results indicate that ART1couldalso regulate apoptosis of CT26cells induced by CDDP via Akt/TUBB3approach.ART1silencing inhibits ERK pathway activity, NF-κB p65nucleartranscription, reduces Bcl-2and Bcl-xl expression and elevates Baxexpression, while ART1over expression acts oppositely. But inhibition ofERK pathway activity fails to alter NF-κB p65nuclear transcription andexpression of Bcl-2, Bcl-xl and Bax. The results indicat that ERK alsotakes part in the regulation role of ART1on CT26cells apoptosis inducedby CDDP, but NF-κB p65/Bcl-2/Bcl-xl are not involved in this regulation effect. Moreover, ART1silencing inhibits activity of ERK pathway, reducesprotein and mRNA expression of TUBB3, ART1over expression actsoppositely. Inhibition of ERK pathway also leads to reduce of TUBB3protein and TUBB3mRNA expression. The results indicate that ART1might regulate CT26cells apoptosis via ERK/TUBB3approach. All theresults lead us to the conclusion that ART1is a potential synergy anti-tumortherapeutic target and promising therapeutic strategy for colon carcinoma.