Effects Of Blocking Ubiquitin-proteasome Pathway On Osteosarcoma Cell Line MG-63

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. It is a highly aggressive mesenchimal neoplasm typically composed of spindle cells producing osteoid. Before the advent of adjuvant multidrug chemotherapy, the treatment of osteosarcoma was high amputation or local wide excision and radiotherapy. Eighty percent of these patients went on to die from disseminated pulmonary disease, with high local recurrences and 2-year overall survival rates of only 15%-20% . The prognosis of patients with osteosarcoma has greatly improved, with 5-year overall survival rates increasing from 15% to 70%, and limb-salvage has become the standard of care. This improvement is attributed to the introduction of preoperative chemotherapy or neoadjuvant chemotherapy. However, many studies have showed that resistance to chemotherapy and metastatic spread are the most important mechanisms responsible for the failure of current multimodal therapeutic programs. Higher dose of conventional regimens is often associated with significant toxicity affecting the kidney, heart damage, and cochlea, which may affect quality of life and can be life threatening, in addition to toxicity a major problem associated with medicine resistance. Thus, research for new alternatives for the therapy of osteosarcoma is greatly required.Apoptosis, or programmed cell death, is a highly regulated and conserved form of cellsuicide that plays a critical role in embiyogenesis, tissue homeostasis and in the development of various disorders such as autoimmunity, cancer and neurodegenerative disease. Induction of apoptosis has been recognized as the major cytotoxic mechanism of anticancer therapies, including chemotherapy and radiotherapy. Apoptosis-based drug screening has been an important method to detect compounds selectively toxic to tumor cells and evaluate the efficiency of chemotherapy. How to raise the apoptotic rates of tumor cells without marked toxicity on normal cells and avoid resistance to chemotherapy have stimulated great interest.The ubiquitin-proteasome pathway is a non-lysosome proteolytic mechanism by which intracellular proteins are rapidly hydrolyzed, and this pathway plays an important role in the ordered elimination of these proteins. This pathway is mainly responsible for the degradation of a variety of short-lived functional regulators, including various cell cycle regulators, tumor suppressors and transcription factors such as p53,cyclins, nuclear factor-κB/IκB, c-Myc and c-Jun. Blockage of the ubiquitin-proteasome pathway can result in the disruption of normal proteolytic destruction, and disturbing normal cellular processes, and leads to cell apoptosis. The ability of blocking the ubiquitin-proteasome pathway to counteract cell proliferation and to selectively induce apoptosis in cancer cells but not in normal cells makes it an attractive new pathway in cancer therapy.In the presented study, we selected proteasome inhibitor, Z-LLL-CHO, which could efficiently blocked ubiquitin-proteasome pathway in eucaryotic cells. MTT assay, fluorescence microscopy, DNA fragmentation, and flow cytometry and Western blot were performed to analyze the effects of blocking ubiquitin-proteasome pathway on human osteosarcoma MG-63 cells. Our findings demonstrate that proteasome inhibitors possess anti-tumorigenic and induction of apoptosis effects on osteosarcoma cells. Blockage of the ubiquitin-proteasome pathway may be used as a possible target of novel therapy for patients with osteosarcoma.Part one:The growth inhibitory effect of blocking ubiquitin-proteasome pathway on human osteosarcoma cell line MG-63Objective: To observe the anticancer effect of blocking ubiquitin-proteasome pathway on human osteosarcoma cell line MG-63, and to study the relationship of anticancer effect and cell apoptosis.Methods: The human osteosarcoma cells MG-63 and human diploid fibroblastic cells WI-38 were cultured in vitro and exposed to varying concentrations of Z-LLL-CHO for the indicated times, and then the cytotoxicity was determined by a MTT...