Analysis IL-24 Induced Apoptosis Of Liver Cancer By The Endoplasmic Reticulum Stress

Background and objectivemda-7 was obtained by Jiang et al during subtraction hybridization(SH) using the cDNA library of actively proliferating and terminally differentiated human HO-1 melanocytes,who preliminarily proved that mda-7 was able to inhibit proliferation of melanoma and promote terminal differentiation.Since then,mda-7 had been studied as a tumor inhibitor.It was not until 2001 when Huang et al found the chromosome location and gene structure of mda-7,and subsequently Caudell et al at defined its cytokine property in a series of experiments that it is named as interleukin-24(IL-24) belonging to the IL-10 family.A series of studies have demonstrated that adenovirus-mediated mda-7/IL-24(Ad.mda-7) selectively induces apoptosis of different kinds of tumor cells without posing significant toxic effects on normal cells.The many kinds of tumor animal model study demonstrated that Ad.mda-7 can suppress in vivo tumor to form and progress.Additionally,Ad.mda-7 has a multiplicity of anti-tumor activities,including antiangiogenesis and radiosensitizing activity.Ad.mda-7 has been currently undergoing phaseⅡclinical trials in American.However,the molecular mechanism of how mda-7/IL-24 selectively induces apoptosis of tumor cells is complex;different types of tumor cells may involve different regulatory pathways.For example,Ad.mda-7 induces apoptosis of ovarian cancer cells through the fas-fasL signal pathway, apoptosis of melanoma cells through the p38MAPK pathway,and apoptosis of lung cancer cells through the PKR pathway.Other studies also showed that intracellular mda-7/IL-24 is located in the endoplasmic reticulum(ER) and Golgi complex,and may also involve the ER stress mediated apoptosis signal transduction pathway.Hepatocellular carcinoma(HCC) is one of the five tumors of the highest mortality and the incidence tends to be rising in recent years.There are few studies reporting mda-7/IL-24 induction of HCC apoptosis.Therefore,our laboratory has been carrying out a series of studies,which are focused on the effect of mda-7/IL-24 on liver cancer cell growth and apoptosis,and its mechanism in recent years.In this study,Ad.mda-7 induced apoptosis of liver cancer was investigated both in vitro and in vivo,and its functional mechanisms of endoplasmic reticulum stress(ER stress) were analyzed.Methods1.Construction,preparation,amplification and purification of Ad.mda-7:The recombinant replication-defective Ad.mda-7 was created in two steps,as described for AdEasy Adenoviral Vector System.Production of infectious virus in 293 cells,analysis of recombinant virus genomes to confirm the recombinant structure,plaque purification,and titration of virus were performed.(All of these were proserved and afforded by our labs)2.Ad.mda-7 induction of liver cancer cell growth suppression and apoptosis:Three human liver cancer cell lines(Hep3B,HepG2 and PLC/PRF/5) and a normal human liver cell line L02 were infected with Ad.mda-7 or Ad.GFP.The cell growth was assayed by MTT,apoptotic cells were examined by DNA fragment analysis and flow cytometry,and the potential mediated mechanisms were examined via western blot.3.Ad.mda-7 gene therapy for liver cancer BALB/c nude mice model: HepG2 cell suspension was injected subcutaneously(s.c.) into the back of each nude mouse.By subcutaneous implantation of HepG2 cells in BALB/c nude mice,Ad.GFP or Ad.mda-7 or ALLN+ Ad.mda-7 was administered by means of intra-tumor single point injection.Body mass and tumor formation of the nude mice were observed daily.And the cleaved caspase-3,apoptosis of cell,cell proliferation-associated antigen ki-67 and microvascular density were detected by immunohistochemistry and TUNEL.Also the potential mediated mechanisms were examined via western blot.Results 1.Ad.mda-7 inhibits growth of liver cancer cells without posing adverse effect on normal liver cellsBefore the experiment,the virus transfection rate of liver cancer cell lines and normal liver cell line was detected as＞70%.Ad.mda-7 or Ad.GFP of 0.01,0.1,1,10 and 100 MOI was used to transfect liver cancer line HepG2, Hep3B and PLC/PRF/5,and normal liver cell line L02.Cell survival was detected by MTT,and untreated cells were used as control.The results showed that Ad.mda-7 or Ad.GFP transfection did not have significant inhibitory effect on normal liver cell line L02(P＞0.05).Compared with Ad.GFP transfection,Ad.mda-7 transfection produced significant inhibitory effects on the growth of liver cancer cell lines(P＜0.05).Of the three liver cancer cell lines,growth inhibition was most significant on HepG2(p53 wild type).At 10 MOI,66%of HepG2 cell growth was inhibited.These results confirmed that Ad.mda-7 had a selective inhibitory effect on the proliferation and growth of liver cancer cells,without posing significant adverse effect on normal liver cells.Later,we explored possible mechanism involving selective inhibition of Ad.mda-7 selectively on the growth of liver cancer cells.We found by flow cytometry that Ad.mda-7 induced apoptosis of liver cancer cells.Compared with Ad.GFP transfection,a significantly high rate of cell apoptosis was found with Ad.mda-7 transfection(P＜0.001),while there was no significant change in L02 normal liver cells either transfected with Ad.mda-7 or with Ad.GFP transfection(15.07%vs 13.65%,P＞0.05).These data suggested that mda-7/IL-24 selectively induced apoptosis of liver cancer cells through some non p53 route.2.Ad.mda-7 induces apoptosis of liver cancer cells by activating the ER stress pathwayHepG2 liver cancer cells that were most sensitive to Ad.mda-7 were used to further study the possible mechanism of inducing cell apoptosis. Previous studies found that intracellular mda-7/IL-24 was located in ER and Golgi complex,and coupled with BiP/GRP78 protein via its C and F ring.In our experiment,it was found by Western blot that HepG2 cells transfected with Ad.mda-7 highly expressed BiP/GRP78.The positive control treated with TG also highly expressed BiP/GRP78,while no BiP/GRP78 expression was detected in PBS and Ad.GFP negative controls.In detecting another ER stress related protein caspase-12,we detected activated degraded caspase-12 fragment(57 kDa) in HepG2 cells transfected with Ad.mda-7 and positive control,while caspase-12 remained to be zymogenic in negative control.In contrast,transfection of normal liver cell L02 with Ad.mda-7did not activate BiP/GRP78 and caspase-12.There was no change in the expression of house-keeping geneβ-actin in all treated cells.Above all,ER stress and activation of BiP/GRP78 degraded caspase-12,finally resulting in cell apoptosis.These results suggested that Ad.mda-7 induced apoptosis of liver cancer cells probably through activating the ER stress pathway of caspase-12.3.Ad.mda-7induces apoptosis of liver cancer cells,upregulates expression of Bax,caspase-3 and p38 MAPKThere were studies demonstrating that activation of caspase through the death receptor pathway(extrinsic pathway) and the mitochondrial pathway (intrinsic pathway) caused cell apoptosis.Bax and other Bcl-2 family members played an important role in the mitochondrial pathway,caspase-3 is the main caspase effector molecule in the mitochondrial apoptosis pathway,while the extrinsic pathway is mediated by the death receptor, where caspase-8 is the main initiator molecule of caspase.In our experiment, Bax was activated 48 h after transfection of HepG2 cells with Ad.mda-7. Likewise,caspase-3 degraded from the zymogenic form(32 kDa) to 17 kDa, while caspase-8 remained zymogenic.Also,we found that p38 MAPK was phosphorylated.No change was found in the proteins in control PBS and Ad.GFP.There was no change in the expression of house-keeping geneβ-actin in the treated cells.These results indicated that Ad.mda-7 induced apoptosis of liver cancer cells through a non-death receptor pathway,and upregulated expression of Bax and caspase-3,and phosphorylation of p38 MAPK. 4.Blocking ER stress inhibits Ad.mda-7 induced apoptosis of liver cancer cellsWe used calpastatin I(ALLN)to block the ER stress pathway and observed whether it affected the growth of Ad.mda-7 transfected HepG2 cells,calpain did not participate in ER stress directly;rather it activated downstream caspase-12 causing cell apoptosis.ALLN may block caspase-12 induced ER stress reaction.ALLN may inhibit Ad.mda-7 induced apoptosis of a variety of tumor cells.Dose dependent experiments showed that 25μM ALLN did not produce significant toxicity to HepG2 cells.The results of MTT showed that the survival rate of HepG2 cells treated with ALLN for 30 min and then transfected with Ad.mda-7(ALLN+Ad.mda-7)was 75%in 3 days,while that of HepG2 cells transfected with Ad.mda-7 (ALLN+Ad.mda-7) without ALLN treatment was 40%(P＜0.05).The survival rate of HepG2 cells transfected with Ad.GFP with ALLN treatment or without ALLN treatment in control groups was 86%and 83%, respectively.There was no significant difference as compared with ALLN+ Ad.mda-7(P＞0.05).A series of experiments showed that ALLN inhibited Ad.mda-7 induced apoptosis of liver cancer cells markedly.No significant DNA gradient was seen in HepG2 cells treated with ALLN+Ad.mda-7,while DNA gradient was obvious in those treated with Ad.mda-7 in 3 days.Likewise,the results of flow cytometry showed that apoptosis of HepG2 cells treated with Ad.mda-7 was 44%,while that of those treated with ALLN+Ad.mda-7 was 28% (P＜0.05).Apoptosis of HepG2 cells treated and Ad.GFP with or without ALLN treatment in the control group was 13%and 11.5%,respective,and apoptosis of HepG2 cell without virus transfection was 9%.There was no statistically significant difference between the three groups(P＞0.05).These results showed that Ad.mda-7 was able to induce apoptosis of liver cancer cells through the ER stress pathway,and this action could be blocked by ALLN.5.Ad.mda-7 downregulates expression of caspase-3,caspase-12 and Bax of liver cancer cells by blocking the ER stress pathwayWestern blot was further performed to see whether caspase-12 played an important role in the Ad.mda-7 mediated ER stress apoptosis pathway. Comparison of Ad.mda-7 transfected HepG2 cells with ALLN treatment and those without ALLN treatment showed that caspase-12 activation was inhibited markedly,and activation of Bax and caspase-3 also decreased markedly,indicating that Ad.mda-7 induced apoptosis of HepG2 cells by activating caspase-12,and then activating Bax and caspase-3.The problem was that expression of BiP/GRP78,either treated with or not with ALLN, did not change.In addition,phosphorylation of p38 MAPK was not affected by ALLN treatment.While in Ad.GFP or ALLN+Ad.GFP control group,no change in expression of the proteins was observed,and expression of house-keeping gene did not change in all treated cells.All these results showed that Ad.mda-7 induced apoptosis of liver cancer cells through the ER stress pathway(including caspase-12),and this effect was closely related to the mitochondrial pathway.6.Ad.mda-7 inhibits growth,proliferation and angiopoiesis of liver cancer in vivoLater,we explored whether Ad.mda-7 had the effect of inhibiting growth of liver cancer cells in vivo.Five days after subcutaneous seeding of HepG2 cells,a subcutaneous nodule about 3 mm was palpated at the site of seeding. When the tumor grew to 4-5 mm in diameter,treatment was started.Seeding was successful in all nude mice and no death occurred during the course of treatment.After intra-tumor injection of the virus,most tumors in Ad.mda-7 treatment group was slower than did the tumors in Ad.GFP control group. The whole tumor was removed 3 weeks later and measured for size and weight.The tumor volume of Ad.mda-7 treatment group and Ad.GFP control group was 312.6±30.24 mm~3 and 20.6±30.00 mm~3 respectively(P=0.001), and the tumor mass was 0.321±0.031 g and 0.534±0.030 g respectively;the difference was statistically significant(P=0.001).Cell proliferation-associated antigen ki-67 is one of the most widely used cell proliferation-associated markers.Most cells in Ad.GFP treated tumor tissue were positively stained,indicating that proliferation of tumor cells was active.While in Ad.mda-7 treated tumor tissue,ki-67 positive cells were decreased markedly,indicating cell proliferation was inhibited. Proliferation index(PI) of tumor cells was 26.67±4.56%for Ad.mda-7 treatment group and 57.14±5.56%for Ad.GFP control group(P=0.002). CD31 factor is one of the markers of neo-vascularization,and its antibody is often used for detection of microvascular density(MVD). Neo-vascularization in Ad.mda-7 treated liver cancer tissue was significantly lower than that in Ad.GFP control group;MVD of the two groups was 19.59±2.50 and 32.27±4.52 respectively(P=0.013).These results showed Ad.mda-7 had the effect of inhibiting growth and proliferation of liver cancer in vivo,and of preventing tumor neo-vascularization.7.Ad.mda-7 induces cell apoptosis of liver cancer tissue in vivoTumor tissue was H-E stained routinely.Large amounts of fibrous septa formation was observed in seeded tumor tissue under the skin of nude mice. The tumor nucleus was large and deeply stained,with irregular mitotic figures.The configuration of tumor cells treated with Ad.mda-7 was different from that treated with Ad.GFP,where nuclear condensation was more evident.In addition,the plasma of most tumor cells in Ad.mda-7 treatment group was deeply stained with brownish yellow color,and highly expressed IL-24 protein,while no IL-24 protein expression was seen in Ad.GFP treatment group.TUNEL is a method of hybridizing bioin or bromine labeled nucleotide onto intracellular split DNA free 3'OH under the action of TdT enzyme and then enzyme or fluorescencin labeled avidin or labeld nucleotide antibody was added for the sake of in situ assay.The number of apoptotic liver cancer cells increased markedly in Ad.mda-7 treatment group,and the apoptotic rate was significantly higher than that in Ad.GFP control group.The difference was statistically significant(P＜0.05). In addition,it was observed that large amounts of caspase-3 was activated in tumor tissue treated with Ad.mda-7,while only a small amounts of caspase-3 was stained in Ad.GFP control group,indicating that Ad.mda-7 was able to induce cell apoptosis of liver cancer tissue in vivo by activating caspase-3.We then injected ALLN into tumor-bearing nude mice intraperitoneally at a dose of 100 mg/kg at a 3-day interval for three times.The animals were sacrificed 24 h after the last injection.Tumor tissue was removed and measured for size and weight.There was no statistically significant difference in size and weight as compared with that of tumor-bearing nude mice without receiving ALLN injection,indicating that ALLN at 100 mg/kg produced no effect on the growth of the tumor-bearing nude mice,and was atoxic.However,the apoptotic rate of tumor-bearing nude mice in ALLN+ Ad.mda-7 treatment group was significantly lower than that in Ad.mda-7control group(P＜0.05),as detected by TUNEL,indicating that ALLN had an effect of preventing Ad.mda-7 from inducing apoptosis of liver cancer cells in vivo.It was further observed by Western blot that in vivo expression of caspase-12,-3 and Bax protein in ALLN+Ad.mda-7 treatment group was lower than that in Ad.mda-7 control group.These results showed that the ER stress pathway that activated caspase-12 played an important role in the process of Ad.mda-7 inducing apoptosis of liver cancer cells in vivo.Conclusions1.Ad.mda-7 could selectively induce growth suppression of human liver cancer cells in vitro.2.Ad.mda-7 could selectively induce apoptosis of human liver cancer cells in vitro,but no toxic to normal cells.3.Ad.mda-7 induces apoptosis of HepG2 cells by activating the ER stress pathway and up-regulating Bax,caspase-3,and p38 MAPK.4.Ad.mda-7 inhibits growth,proliferation,and angiogenesis of HepG2 tumors in vivo. 5.Ad.mda-7 induces HepG2 cell apoptosis in vivo may by the ER stress.