Induction Of Apoptosis In Osteogenic Sarcoma Cells By Combination Of TRAIL And Chemotherapeutic Agents

ObjectiveOsteosarcoma is one of the most common primary malignant tumors of bone. It has a bad prognosis. TNF - related apoptosis inducing ligand is a new member of the tumor necrosis factor(TNF) -cytokine family. TRAIL induces cell death in a wide variety of tumor cell lines but doe's not seem to be cytotoxic to many normal cell types in vitro, however, there are some tumor cells such as some of breast cancer, bladder cancer, and colon cancer cell lines are resistant to TRAIL. Some reports show that chemotherapeutic agents can sensitize tumor cells to TRAIL - induced apoptosis. The aim of our study was to investigate the cytotoxic activity of TRAIL and chemotherapeutic agents ( MTX,DOX,CDDP) on established osteogenic sarcoma cell line - OS - 732. Then we combined TRAIL with chemotherapeutic agents. To assess the synergistic effects of combining TRAIL with chemotherapy in osteogenic sarcoma, we examined the cytotoxic effects of TRAIL in combination with the anticancer drugs: MTX , DOX and CDDP. We hope to develop new approaches to the treatment of osteosarcomas which could settle the major problems in using chemotherapy: the frequent acquisition of drug - resistant phenotypes and the occurrence of systemic toxicity side effects.MethodsThe OS - 732 osteogenic sarcoma cells were cultured in the regular condition. The experimental agents were added when the tumor cells were in the ex-ponential growth phase. Chemotherapeutic agents MTX,DOX,CDDP and TRAIL were added to cells at the indicated concentrations either alone or combined and incubated for an additional 24h or 48h. MTT(microculture tetrazolium dye) was used to evaluate the cytotoxic effects, flow Cytometry Assay was also used to test the apoptosis proportion. We observed cellular morphologic changes with phase contrast microscope , scan and transmission electron microscope. Experiments were repeated at 3 times. Results of representative experiments were given as the mean 卤 SD. Statistic analysis were done using SPSS software. Compari-sion between 2 groups were tested by student Newman - Keul's t test. P values less than 0. 05 were considered to be significant.Results1. The result of MTT reductive experiment: Osteosarcoma OS - 732 cells were not responsive to the cytotoxic effects of TRAIL. The rate of killing was 24.44% with TRAIL concentration of 100ng/ml for 24h. The cells were comparatively more responsive to DOX and CDDP with a dose - effect relationship. In OS -732 cells, DOX and CDDP cooperated synergistically with TRAIL, in that the combined treatment resulted in about 58. 36% and 54. 10% respectively. With TRAIL alone or drugs alone, the percent apoptosis was less than 25%. However, the combination of TRAIL and MTX did not present synergistic effects on OS - 732 cells. Compared with TRAIL alone, we did not see significant difference(p>0.05).2. The result of Flow Cytometry Assay(FCA) :By flow cytometry, we obtained apoptosis peaks ahead the diploid peak when the OS - 732 cell line incubating with different agents. With TRAIL alone or drugs alone, the percent apoptosis was less than 25%. DOX and CDDP cooperated synergistically with TRAIL resulted in 60. 88% and 53.29% cell death. The data demonstrated that DOX and CDDP could augment antitumor effects by TRAIL. In contrast, there was no significant augmentation of the apoptosis peaks'value when combining TRAIL with MTX. Perhaps MTX couldn 't augment the antitumor effects of TRAIL. The result of FCA was the same as that of MTT.3. The result of cellular morphologic observation: Under phase contrast microscope, when OS -732 cell line were treated by TRAIL(100ng/ml) or DOX (0.6 Lg/m ) alone for 48h,some cells became small and round with intact cellular membrane. A large number of cells exfoliated and drifted in culture medium when combining TRAIL with DOX. Moreover, margination of condensed chromatin and apoptotic body were observed by electron microscope when the OS -732 ceU line incubating with TRAIL(500ng/ml) for 48h.Conclusion1. Osteosarcoma OS -...