The Effect Of Augmenter Of Liver Regeneration In The Apoptosis Of HepG2Cells Induced By Cisplatin

Augmenter of liver regeneration （ALR） is a newly discoveredregulating factor during the process of liver repair and regeneration. It hasfound that ALR is expressed in many tissues, especially in the tumor tissues.Cell apoptosis is one way that tumor cells loss. Evasion of apoptosis is one ofthe basic features of cancer. So, the induction of apoptosis in tumor cells is amajor goal of cancer chemotherapy. Studies have found that ALR have theability of anti-apoptosis, but it’s still unknown that if ALR which isexpressed highly in Hepatocellular carcinoma has the anti-apoptosis throughinfluencing the efficiency of chemotherapy. In this study,we investigate therole of ALR in the apoptosis of HepG2induced by cisplatin through addingALR exogenously and shut down the expression of ALR.Part1: The effect of exogenous augmenter of liver regeneration in theapoptosis of HepG2cells induced by cisplatinMethod: There are two groups: the control group(DDP-, ALR+), theexperimental group(DDP+, ALR+), the concentration of DDP was10ug/ml, and the concentration of ALR were:0,5,10,20ug/ml, The total time of exposing was24hours.1.Cell proliferation by MTS assay: HepG2cells were incubated in96-well plates, then exposed to different concentration of ALR respectivelyfor24hours, then MTS assay was used to check cell proliferation.2.MTS assay was used to measure the effect of ALR oncisplatin-induced HepG2cell death: HepG2were incubated in96-wellplates, and incubated with DDP alone or with different concentration ofALR for24h. Subsequently, MTS solution was added to each well and theabsorbance was read on a plate reader at490nm.3.The amount of apoptotic cells was measured by flow cytometry:HepG2cells were incubated with ALR alone or with differentconcentration of ALR for24hours; then HepG2cells were incubatedwith DDP alone or with different concentration of ALR for24hours.AnnexinV/FITC double staining was used to detect apoptotic cells.4.The expression of apoptosis-related proteins: HepG2cells wereincubated with DDP alone or with different concentration of ALR for24hours,then Western blot analysis was used to detect the expression ofpro-PARP, cleaved-PARP,pro-caspase8, pro-caspase3,Bax and Bcl-2.Theexpression of apoptosis-related proteins of HepG2without DDP norALR was the control.Results:1.MTS results showed:1)Different concentration of ALR had effect on HepG2cell viability(p<0.05).2) Cell viability of HepG2was inhibitedobviously after treated with DDP only(p<0.05), but this inhibition wasreduced after treated with ALR(p<0.05).2.Data of flow cytometric analysis showed: There were no obviousdifference in apoptotic rates when HepG2cells were with differentconcentration of ALR only (p>0.05), the means of apoptotic rates were（5.20±0.95）%、（5.03±0.37）%、（4.97±0.35）%、（4.85±0.23）%. When HepG2cells were incubated with DDP alone or with differentconcentration of ALR, the mean of apoptotic rates decreased in aconcentration-dependent manner (p<0.05),they were（60.97±6.56）%,（53.03±2.60）%,（40.63±2.02）%,（30.0±3.21）%.3.The results of western blot showed：Compared with the controlone,when incubated with DDP alone or with different concentration ofALR,the expression of pro-PARP, pro-caspase8, pro-caspase3and Bcl-2decreased in a concentration-dependent manner,while cleaved-PARP,Baxincreased in a concentration-dependent manner.Conclusions:1.Exogenous ALR didn’t have obvious effect on apoptotic cells innormal HepG2cell.2. Cisplatin inhibited the proliferation of HepG2significantly, whileexogenous ALR reduced the inhibition of cisplatin on HepG2.3.The apoptosis of HepG2could be induced by cisplatin,while exogenous ALR reduced the rate of apoptotic cells induced by DDP inHepG2cells.4.The mechanism of that exogenous ALR reduced apoptosis inducedby DDP in HepG2might be relevant to the apoptosis proteins regulation. Part2: The effect of decreased expression of the augmenter of liverregeneration in the apoptosis of HepG2cells induced by cisplatinMethod: HepG2, HepG2-NC (lentivirus vector) cells, HepG2-KD(ALR interference lentivirus vector expressing) cells were the experimentalcells.The concentration of DDP was5ug/ml, and the concentration of ALRwas20ug/ml, The total time of exposing are24hours.1.Western blot was used to detect the expression of ALR protein inHepG2, HepG2-NC and HepG2-KD.2.MTS assay was used to measure the rate of cell proliferation:HepG2,HepG2-NC and HepG2-KD were incubated in96-well plates, thenincubated with (1)without DDP nor ALR,(2)with DDP only,(3)with bothDDP and ALR.After24hours, MTS solution was added to each well and theabsorbance was read on a plate reader at490nm.3.The amount of apoptotic cells was measured by flow cytometry:HepG2,HepG2-NC and HepG2-KD were incubated with:(1)without DDP nor ALR,(2)with DDP only,(3)with both DDP and ALR.After24hours,Annexin V-PE/7AAD double staining was used to detect apoptotic cells.4.The expression of apoptosis-related proteins: HepG2-NC andHepG2-KD were incubated with:(1)without DDP nor ALR,(2)with DDPonly,(3)with both DDP and ALR.After24hours, western blot analysis wasused to detect the expression of pro-PARP, cleaved-PARP,pro-caspase8,pro-caspase3,Bax and Bcl-2.Results:1.The expression of ALR protein in HepG2-KD cells wassignificantly lower than that of the HepG2-NC.2.MTS results showed:(1)When incubated without DDP nor ALR,thecell viability of HepG2, HepG2-NC and HepG2-KD were (0.81±0.02),(0.71±0.01),(0.6±0.07) respectively.(2) When incubated with DDPonly,the cell viability of HepG2, HepG2-NC and HepG2-KD were (0.41±0.04),(0.41±0.03),(0.14±0.00)respectively.(3)When incubated with bothDDP and ALR,the cell viability of HepG2, HepG2-NC and HepG2-KDwere (0.69±0.02),(0.69±0.02),(0.47±0.02)respectively. There were noobvious difference in results of each treatment between HepG2andHepG2-NC(p>0.05), when HepG2-NC compared with HepG2-KD,therewere obvious difference (p <0.05).When (DDP-, ALR-) vs (DDP+, ALR-),(DDP+, ALR-) vs (DDP+, ALR+), the difference between them wasobvious (p <0.05). 3.Data of flow cytometric analysis showed: When incubated withoutDDP nor ALR,the means of apoptotic rates of HepG2, HepG2-NC andHepG2-KD were （0.97±0.21）%,（1.08±0.13）%,（5.37±0.7）%respectively.When incubated with DDP only,the means of apoptotic rates ofHepG2, HepG2-NC and HepG2-KD were（20.9±0.62）%,（23.07±2.21）%,（37.53±2.34）%respectively. When incubated with both DDP and ALR,the mean of apoptotic rates of HepG2, HepG2-NC and HepG2-KD were（6.03±0.32）%、（6.57±0.85）%、（25.1±1.93）%respectively.Thestatisticalanalysis of the results was as same as above mentioned.The results ofwestern blot showed：Compared with HepG2-NC，when incubated withoutDDP nor ALR, the expression of pro-PARP, pro-caspase8, pro-caspase3andBcl-2increased, while cleaved-PARP, Bax decreased in HepG2-KD.Afterincubated with DDP only, he expression of pro-PARP, pro-caspase8,pro-caspase3and Bcl-2decreased, while cleaved-PARP, Bax increased inHepG2-KD compared with HepG2-NC.When incubated with both DDP andALR, the expression of pro-PARP, pro-caspase8, pro-caspase3and Bcl-2increased, while cleaved-PARP,Bax decreased in HepG2-KD comparedwith HepG2-NC.Conclusions:1.SiRNA/ALR interference could block the ALR expression efficiently.2.SiRNA/ALR interference could inhibit cell proliferation of HepG2cells,and increased apoptotic rates. 3.HepG2-KD was incubated with DDP, the cell proliferation wasreduced, apoptotic rates was increased significantly, so as to enhance thesensitivity of cisplatin to HepG2.When added ALR exogenously,apoptosis was decreased.4.The mechanism of that siRNA/ALR interference enhanced apoptosisand chemosensitivity might be relevant to the apoptosis relative proteinsregulation.