Studies On Suppressive Effects Of Irradiation And IL-24 On The Growth Of PC-3 And HeLa Cell Lines

Radiotherapy is one of the most important methods for the treatment of tumors, but its application is currently limited by the side-effects such as the radiation-induced damages in the normal tissues nearby and the radiation-resistance of certain tumors. Nowadays, more and more attention has been paid to gene-radiotherapy of malignant tumors. The discovery of radiation-inducible early growth response-1 (Egr-1) promoter represents a novel strategy for effective combination of gene therapy and radiotherapy.Neither human androgen independent prostatic carcinoma cell line (PC-3) and human cervical carcinoma cell line (HeLa) are sensitive to radiotherapy because of their radiation resistance. To solve this problem we need to find an effective way which could decrease the dose of radiation and at the same time increase the suppression of tumors. The IL-24-containing recombinant plasmids, pcDNA-IL-24 and pEgr-IL-24, have been constructed by gene recombination technique. For further enhancing anti-tumor effects, the combined therapy of IL-24 gene and irradiation is particular promising. And the suppression effects of that on PC-3 and HeLa cell lines were observed in this experiment.1. Effects of ionizing radiation on apoptosis in PC-3 and HeLa cell lines The cell line PC-3 was treated with different doses (0,2,4,6,8,12 and 18 Gy) of X-irradiation in vitro. The changes of apoptosis in PC-3 cells were detected with flow cytometry (FCM) 48 hours after X-irradiation using the staining of Annexin-V and PI. The result showed that the increase of apoptosis in PC-3 could not be examined after 2 and 4 Gy X-irradiation because of its probable radiation resistance. The apoptosis percentage was increased significantly after X-irradiation with the doses of 6, 8, 12 and 18 Gy. As compared with sham-irrad,the mean apoptosis percentage was increased by a factor of 1.6 to 3.It has been demonstrated that the apoptosis percentages of HeLa cells were 0.8, 0.8, 1.2, 3.2, 9 and 2 % respectively after high-energy X-irradiation with the doses of 2, 4, 6,8,10 and 20 Gy. It showed that HeLa cell is also resistant to radiation.2. Construction of recombinant plasmids2.1 Construction and identification of recombinant plasmid pcDNA-IL-24Both plasmids pcDNA3.1(+) and pMD18T-IL-24 were treated with restriction enzymes BamH I and Xba I in order to get the fragments of pcDNA3.1(+) carrier and IL-24 gene. Recombinant plasmid pcDNA-IL-24 was constructed successfully with gene recombinant technique after identification with polymerase chain reaction (PCR).2.2 Construction and identification of recombinant plasmid pEgr-IL-24Egr-1 promoter was inserted in plasmid pcDNA3.1(+) instead of CMV promoter. And the fragment of IL-24 gene was connected as the downstream gene. Identifications with restriction enzyme and PCR demonstrated that the recombinant plasmid pEgr-IL-24 was constructed successfully.3. Expression of IL-24 in PC-3 and HeLa cell lines transfected by recombinant plasmidsPlasmid pcDNA3.1(+) and recombinant plasmids pcDNA-IL-24 and pEgr-IL-24 were transferred into PC-3 and HeLa cell lines by polyethyleneimine (PEI) in vitro. The identification of IL-24 gene was detected by RT-PCR 30 hours after transfection. The result showed that the expression of IL-24 gene could be observed in PC-3 and HeLa cell lines transfected by recombinant plasmids pcDNA-IL-24 and pEgr-IL-24 except in the blank control and the group transfected by pcDNA3.1(+).4. Suppressive effects of IL-24 on PC-3 and HeLa cell lines4.1 Effects of IL-24 on apoptosis in PC-3 and HeLa cell linesPlasmid pcDNA3.1(+) and recombinant plasmids pcDNA-IL-24 and pEgr-IL-24 were transferred into PC-3 and HeLa cell lines by PEI in vitro. The changes of apoptosis were detected with FCM 30 hours after transfection using the staining of Annexin-V and PI. As compared with the control, the apoptosis percentages in the groups transfected by recombinant plasmids pcDNA-IL-24 and pEgr-IL-24 (except the group transfected by pcDNA3.1(+)) were increased significantly (P<0.05 or P<0.01). The apoptosis percentages in the groups transfected by recombinant plasmids pcDNA-IL-24 and pEgr-IL-24 were also increased significantly compared with the group transfected by pcDNA3.1(+). It was demonstrated that IL-24 could induce the apoptosis in PC-3 and HeLa cell lines.4.2 Effects of IL-24 on cell proliferation in PC-3 and HeLa cell linesPlasmid pcDNA3.1(+) and recombinant plasmids pcDNA-IL-24 and pEgr-IL-24 were transferred into PC-3 and HeLa cell lines by PEI in vitro. The changes of cell proliferation were detected by MTT assay 30 hours after transfection. The result showed that the cell proliferations in the groups transfected by recombinant plasmids pcDNA-IL-24 and pEgr-IL-24 (except the group transfected by pcDNA3.1(+)) were suppressed significantly compared with the control (P<0.05 or P<0.01). It was demonstrated that IL-24 could suppress the cell proliferation in PC-3 and HeLa cell lines.