| Osteosarcoma is one of the most common primary malignant tumors of bone with the high malignant degree, the tendency to early pulmonary metastasis and a bad prognosis. Osteosarcoma is not sensitive to chemotherapy and drugs alone may be difficult to produce desired effects. Although recently various antitumor drugs have been applied in combination, the prognosis is still poor. Furthermore , the side effects and resistance of chemotherapeutic agents remain to be a major obstacle. Nowadays by studying the transformation of genes and abnormality of apoptotic signal transduction in tumor pathogenesis, it has been discovered that the promotion of apoptosis induced by PTEN, Fas and TRAIL may kill various tumor cells and the application has acquired exciting results in chemotherapy of some tumors. The research in the field of osteosarcoma will provide new theoretical evidence for the choice of osteosarcoma chemotherapeutic methods and show important value in clinical application.PTEN gene is one of the tumor suppressor genes that is discovered after P53 gene and related to the abnormal apoptosis of various tumors. The low expression of PTEN in tumor tissues often weakens the apoptosis induced by PTEN, which results in the infinite growth of tumor. Some study indicated that the expression of PTEN in various malignant tissues reduced, but it has not been reported whether the expression of ITEN in human osteosarcoma tissues would reduce or not. Fas is a death receptor expressed on cellular surface. The expression of Fas in malignant tumor reduces, which inhibits the apoptosis induced by Fas. Therefore , how to enhance the expression of Fas on tumor cells and promote the apoptosis induced by Fas has become of major concern in the research of tumorchemotherapy. TRAIL(TNF -related apoptosis inducing ligand) is the same as Fas system, belonging to the TNF - cytokine family. Compared with Fas system, the advantage is that TRAIL only induces apoptosis of tumor cells and has no obvious toxicity to normal cells, while the disadvantage is that some tumor cells are not sensitive or completely resistant to the apoptosis - induced effect of TRAIL. Therefore, how to enhance the sensitivity of these tumor cells to TRAIL has drawn extensive attention of scholars. Some study revealed that the combination of TRAIL and chemotherapeutic agents would enhance the sensitivity of these tumor cells to TRAIL and reduce the dose and toxicity of chemotherapeutic a-gents. At present, no such reports about the combination therapy of osteosarco-ma with TRAIL and chemotherapeutic agents have been seen, and the research in this field may result in thorough changes in the chemotherapeutic methods of osteosarcoma as Evdokiou said.It shows important significance that the apoptosis induced by PTEN, Fas and TRAIL is studied together. On one hand, besides Fas and TRAIL, TNF that has been extensively used in the treatment of tumor also induces apoptosis of tumor cells via the pathway of death receptors. But for the strong side effects of TNF, how to replace TNF with Fas and TRAIL of fewer side effects has become the consensus of scholars. From the combined study of Fas and TRAIL, we may further understand the mechanism of apoptosis induced via the pathway of death receptors. On the other hand, the relationship between PTEN and death receptors has just drawn attention in recent years. In 1999 SCIENCE magazine first reported that in PTEN - hybridzed mice, the apoptosis induced by T lymphocyte weakened, the proliferation enhanced, and the blocker of PI3K could recover Fas reaction, which indicated that PTEN was the necessary mediator in Fas reaction. Therefore, the importance of multiple pathway research has been advanced for the nature of multiple pathways blocked in tumor cellular apoptosis. On the basis of above views, our research first examined the expression of PTEN and Fas whether reduced or not in osteosarcoma tissues, investigated their relationship with osteosarcoma and the possible relationship between them in the apoptosis - regulating net. Then, INF - y was applied on the osteosarcoma OS -732 cells cultured in vitro to observe whether it could induce the apoptosis of os-teosarcoma cells and prove whether the apoptosis was related with the upregulat-ing expression of Fas on osteosarcoma cells. Finally, TRAIL and chemothera-peutic agents such as DOX, CDDP and MTX et al alone or in combination were used on osteosarcoma cell lines and antitumor effects were compared between different medication methods to provide new evidence for clinical chemothera-peutic method of osteosarcoma.Materials and methodsThe postoperative paraffined tissues of osteosarcoma from thirty - eight cases in First Affiliated Hospital of China Medical University were selected. The SP immunohistochemical method was used to examine the expression of PTEN and Fas in osteosarcoma tissues, and twenty cases of osteochondroma were selected as the control group.The OU/ml, 10U/ml, 100U/ml, 1000U/ml of INF - 7 was applied on the osteosarcoma OS -732 cells cultured in vitro respectively. After 24 hours SP immunohistochemical method and RT - PCR technique were used to examine the expression of Fas on osteosarcoma cells respectively. The morphologic changes of osteosarcoma cells were observed with inverted phase contrast microscope and fluorescence microscope after Hoechst33258 staining. At the same time, the inhibition rates and apoptotic rates of osteosarcoma cells were measured respectively with MTT reductive experiment and flow cytometry assay.Different concentrations of MTX, DOX, CDDP and TRAIL were applied a-lone or jointly on the osteosarcoma OS -732 cells cultured in vitro. The inhibition rates of osteosarcoma cells were measured with MTT method, simultaneously the cellular apoptotic proportion was measured with flow cytometry assay and the apoptotic cellular morphology was respectively observed with inverted phase contrast microscope, fluorescence microscope, scanning and transmitted electron microscope.Results1. The positive expression rate of PTEN in 38 cases of osteosarcoma was 55. 3% , while that in the control group of 20 cases of chrodroma was 90.0% indicating significant difference ( p <0.01). The positive expression rate of Fas in 38 cases of osteosarcoma was 73.7% , while that in the control group of 20 cases of chrodroma was 95. 0% indicating significant difference ( p < 0. 05 ). The expression of PTEN and Fas was positively correlated( r =0.424,p < 0.01).2. After the application of the 10U/ml, 100U/ml, 1000U/ml of INF - 7 on osteosarcoma OS - 732 cells cultured in vitro for 24 hours, the inhibition rates were 13.45 ±3.58, 35. 83 ±5.09 and 64.37 ±9.46 respectively and the apop-totic rates were 7.56 ± 1.09, 25. 64 ± 5. 53 and 45. 24 ± 6. 73 respectively. It showed significant concentration - dependence, indicating the obvious inhibition and apoptosis - inducing effects of INF - 7. Under inverted phase contrast microscope, when OS -732 cell lines were treated with lOU/ml of INF - 7 only some cells became small and round; while with 1000U/ml of INF - 7 a large number of cells exfoliated and drifted in culture fluid. Under fluorescence microscope , when OS - 732 cell lines were treated without INF - 7, fluorescence was evenly distributed with light stained normal cells mostly; while without INF - 7 the intracellular condensed and bright fluorescence staining may be seen indicating the existence of a large number of apoptotic cells. The measurement of im-munocytochemical method and RT - PCR technique both showed that the expression of Fas on OS - 732 cells was significantly enhanced with the increase of INF- 7 concentration ( P < 0.01).3. MTT reductive experiment showed the osteosarcoma OS - 732 cells were not very sensitive to TRAIL, and relatively sensitive to DOX and CDDP in a dose - dependent cytotoxic effect, but relatively insensitive to MTX. When TRAIL (100ng/ml) and DOX or CDDP (1PPC) were used in combination for 24 hours, the inhibition rates of tumor were obviously increased, to 69. 38% and 61.46% respectively. While the agent or TRAIL was used alone, the apoptotic cells were not more than 25%. But when TRAIL and MTX were used in... |
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