Survivin Antisense Oligonucleotide Transfection By Liposome Sensitizes Cervical Cancer Cells To Chemotherapy And Radiotherapy

The gene encoding the IAP survivin was cloned recently,and the protein was characterized.Unlike other IAP family members,survivin expression is found during embryonic and fetal development,but is undetectable in terminally differentiated adult tissues.It subsequently becomes re-expressed in most common human tumors and avariety of human cancer cell lines.Previous studies demonstrated that survivin expression in cancer was strongly correlated with a poor prognosis in gastric,colorectal and breast cancer.Because survivin inhibits processing of downstream effector caspase-3 and –7 in cells receiving an apoptotic stimulus,its overexpression in gastric and pancreatic cancer cell lines are implicated in the resistance to a variety of apoptotic stimuli,such as chemotherapy and radiation.But in lung cancer cell line A549,after exposing to cisplatin(DDP) and VP16,survivin is downregulated and may play a important role in the apoptosis.Then,does it increase in cervical cancercell induced by cisplatin,taxol or X ray?Does antisense oligonucleotide targing survin result in an increased sensitivity to chemotherapy and radiation in cervical cancer?In present study,we investigate the possible mechanisms and effects of antisense oligonucleotide targing survin on increasing sensitivity to chemotherapy and radiation in cervical cancer cell line,and provide new ideas for treating cervical cancer. Methods: 1. The cervical cancer cell line Hela was chosed in this experiment. The inhibitory effects of different dose of DDP or Taxol on Hela cell line were assayed with MTT test. The expression of survivin was determined by RT-PCR and Western-blot on 24h,48h and 72h after exposing to DDP. Apoptosis and cell cycle changes were determined by flow cytometry. 2. MTT assay was used to analyze inhibition of cell growth after 2 Gy X ray. Flow cytometry was used to analyzed the changes of apoptosis index (AI) of Hela cell after having been treated with 2Gy X ray after 24h,48h and 72h.RT-PCR and Western blot were used to analyze the changes in expression levels of survivin mRNA and survivin protein. 3. Immunohistochemical S-P staining method were used to detect the expression of survivin in 54 cervical cancer specimens,to study the relationship between the expression of apoptotic inhubitor gene survivin protein and clinic categorization and prognosis in cervical cancer. 4. Anti-survivin phosphorothioate ASODN was transfected into Hela cells by lipofectin.MTT assay was used to detect cytotoxicity.Apoptosis was observed by flow cytometry.survivin expression was determined by RT-PCR and Western-blotting. 5. A survivin antisense oligonucleotide was transfected into Hela cells by lipofectin.MTT assay were performed to evaluate the sensitivity of the transfected cells to DDP.Apoptosis was detected by Flow cytometry. 6. A survivin antisense oligonucleotide was transfected into Hela cells by lipofectin. MTT assay were performed to evaluate the sensitivity of the transfected cells to X ray.Apoptosis was detected by Flow cytometry. 7. Hela cell line was implanted subcutaneously into nude mice.3 weeks after implantation,the mice were divided into 3 groups:control group,DDP group,survivin ASODN and DDP combination treatment group.After 15 days,the tumor volume and weight were measured,expression changes of survivin mRNA and proteion of transplanted tumor were deteced by RT-PCR and Western-blotting. 8. Hela cell line was implanted subcutaneously into nude mice.2 weeks after implantation,the mice were divided into 3 groups:control group,X ray group,survivin ASODN and X ray combination treatment group.After 15 days,the tumor volume and weight were measured,expression changes of survivin mRNA and proteion of transplanted tumor were deteced by RT-PCR and Western-blotting Results: 1. DDP and Taxol obviously inhibited the cervical cancer cell growth in a manner of dose and time dependent.Apoptosis induced by chemotherapeutical drug was also changed with time rising and dose increasing. As time gone on,survivin mRNA and protein level were increased after exposing to 0.01,0.1,1,2 mg·L-1 DDP and 0.01,0.1,0.5 mg·L-1Taxol. As time gone on,survivin mRNA and protein level were decreased after exposing to 4,10mg·L-1 DDP and 4,10,40 mg·L-1Taxol.when exposing to 0.1 mg·L-1 DDP after 24,48,72h, survivin mRNA and protein level were respectively increased as 2.5,3.6,5.9 times and 1.6,2.7,3.9 times as control. when exposing to 0.1 mg·L-1 Taxol after 24,48,72h, survivin mRNA and protein level were respectively increased as 1.5,3.7,3.9 times and 1.4,2.6,3.5 times as control. 2. After exposing to 2 Gy X ray for 24,48,72h,the cell apoptosis ratioes were 1.85%,2.16% and 2.19%; After exposing to 0.67 Gy 252Cf neutron ray for 24,48,72h,the cell apoptosis ratioes were 5.36%,8.12% and 14.21%.survivin mRNA and protein level were increased after exposing to 2 Gy X ray.At 24h, survivin mRNA and protein level were viability increased as 3.2 and 2.3 times as control;at 48h,3.4 and 2.2 times as control;at 72h,2.2 and 2.0 times as control.There were no significantly different among the expression of survivin mRNA and protein level after exposing to 0.67 Gy 252Cf as control. 3. The positive rates of expression of survivin protein were 41.7%(5/12) in Grade Ⅰ,66.7%(12/18) in Grade Ⅱ,87.5% (21/24) in Grade Ⅲ,respectively and there was significant differences among them(P<0.05).The positive rates of survivin protein were 53.3%(16/30) and 91.7%(22/24) in stage Ⅱand Ⅲ,respectively with a significant difference between them(P<0.01).The 3 year survival rat was recurrence rate was 81.5%(stage Ⅱ86.7%, stageⅢ75%),the local control rate of 3 year was 92.6%.The 3 year survival rat was significantly lower in patients expressing survivin(73.7%) as compared with the survivin negative group(100%).The positive expression rate of survivin protein in cervical cancer patiens was significantly lower in the survival period > 3 years group(63.6%) than in < 3year group(100%). 4. Survivin ASODN inhibited the cells proliferation in a dose and time dependent manner. Survivin ASODN can induce apoptosis in Hela cell.The expression of survivin mRNA and protein significantly decreased after treatment with survivin ASODN. 5. There were significant increase of cell inhibition rate and apoptosis rate after exposure to the combination of survivin ASODN and DDP as compared to DDP alone. 6. There were significant increase of cell inhibition rate and apoptosis rate after exposure to the combination of survivin ASODN and X ray as compared to X ray alone. 7.Growth of cervical cancer in nude mice was significantly inhibited in the survivin ASODN and DDP combination treatment group as compared with those in control groupand DDP group.The growth inhibitory rate was higher in the combination treatment group than in the DDP group.The expression of survivin mRNA and protein significantly decreased in the combination treatment group,wherwas in DDP group there was a significant increase in contrast with control group. 8. Growth of cervical cancer in nude mice was significantly inhibited in the survivin ASODN and X ray combination treatment group as compared with those in control group and X ray group.The growth inhibitory rate was higher in the combination treatment group than in the X ray group.The expression of survivin mRNA and protein significantly decreased in the combination treatment group,whereas in X ray group there was a significant increase in contrast with control group. Conclusions: 1. A high expression of survivin was induced in Hela cells by low concentration of DDP and Taxol,and it may play a critical role on chemoresistence of cervical carcinoma cells induced by chemotherapy agent. 2. 252Cf neutron radiation can induce apoptosis in Hela cell.Compared with X-ray, apoptosis rate induced by 252Cf neutron is larger than that by X-ray . There were no significantly different among the expression of survivin mRNA and protein level after exposing to 0.67 Gy 252Cf as control.Expression of survivin mRNA and protein increases in Hela cells after 2 Gy X ray treatment. Survivin may enhance the radioresistence of cervical cancer cells after 2 Gy X ray treatmen. 3. Expression rate of survivin protein is related to the tumor grade and clinical stage. Survivin expression in cervical carcinoma is a useful prognostic marker. 4. Survivin ASODN is able to inhibit the proliferation of Hela cells via specific down-regulation of survivin expression. Survivin ASODN can induce apoptosis in Hela cell. 5. Survivin ASODN is able to enhance the sensitivity of Hela cells to DDP in vitro and in vivo. 6. Survivin ASODN is able to enhance the sensitivity of Hela cells to X ray in vitro and in vivo. These results indicate the expression rules of survivin mRNA and protein in Hela cells after DDP,Taxol, X ray or 252Cf neutron treatment,and find that survivin ASODN is able to enhance the sensitivity of Hela cells to DDP and X ray in vitro and in vivo.Thesestudies provide scientific evidence for the clinical treatment of survivin ASODN in cervical cancer.252Cf brachytherapy has advantages of high 3 year local control rate(92.6%) and survival rate(81.5%),it is worth spreading and studing more.