Establishment And Proteomics Study Of Human Multidrug Resistant Osteosarcoma Cell Line

Part I. Establishment and Characters of Human Multidrug Resistant Osteosarcoma Cell Line.Background: Osteosarcoma is the most common malignant bone tumor with poor prognosis in children and teenagers. At present, the acceptable treatment of osteosarcoma is the combined therapy with surgery and chemotherapy. However, the resistance to chemotherapeutics is the most important reason for the failure of chemotherapy. Therefore, the deep research is needed to explore the mechanism of multidrug resistance(MDR) of osteosarcoma. Only in this way, the phenomenon of MDR could be reversed and osteosarcoma be treated reasonably.Objective: To establish and analyze the characters of the human MDR osteosarcoma cell line.Methods: Human osteosarcoma cell line MG-63 was induced by increasing dose of Doxorubicin for six months and the MDR model was established successfully. It was named MG-63/DXR100 because it could be cultured stablely in the high glucose DMEM solution with 100ng/ml Doxorubicin. The characters of MDR MG-63/DXR100 were analyzed as follows: the change of morphology was observed by light microscope and transmission electron microscope; the proliferation ability was measured by growth curve; the cell cycle was analyzed by flow cytometry; the sensibility to Doxorubicin, Methotrexate, Cyclophosphamide and Vincristine were measured by MTT assay; the MDR related protein P-gp and MRP3 were detected in mRNA and protein level by RT-PCR and immunocytochemistry respectively.Results: 1. MG-63/DXR100 appears ununiformity with larger volume and poor clarity in light microscope. More granula and vacuolus could also be found in cytoplasm. Further more, Lots of glucogen vacuolus, lipid droplet and rough endoplasmic reticulum in cytoplasm could be found in transmission electron microscope. The protrusion on cell surface was also more than its parental cell MG-63. 2. Growth curves of two cell lines were all typical S shape. The logarithmic growth phase of MG-63 started on the second day and the platform phase on the fifth day. While the logarithmic growth phase and the platform phase of MG-63/DXR100 started on the third and sixth day respectively.MG-63/DXR100 grew more slowly than its parental cells. 3. The MG-63/DXR100 cells of S phase were more than its parental cells significantly while the changes of G2 and G1 phases were not obvious. 4. MG-63/DXR100 had strong resistance not only to Doxorubicin but also to Methotrexate, Cyclophosphamide and Vincristine. The resistance indexes were 45.26, 3.14, 3.23 and 1.88 fold respectively. 5. P-gp and MRP3 were all expressed positively in MG-63/DXR100 while negtively in MG-63.Conclusion: The multidrug resistant osteosarcoma cell line was established successfully by stepwise selection on exposure to increasing doses of Doxorubicin for six months. Part II. Proteomics Study on Human Multidrug Resistant Osteosarcoma Cell Line.Background: At present, the mechanism of MDR in osteosarcoma are: 1. The proteins with function of drug-pump such as permeability glycoprotein(P-gp), multidrug resistance associated protein(MRP), breast cancer resistance protein (BCRP) and lung resistance protein(LRP). 2. The detoxication system and DNA restoration system, such as glutathione S-transferase(GST). 3. Target enzyme of anticancer drugs such as DNA topoisomeraseâ…¡(Topoâ…¡). 4. Protein kinase C(PKC) accelerating phosphorylation of P-gp. 5. Disorder of tumor cells apoptosis. The mechanism of multidrug resistance of osteosarcoma has been investigated for a long time, but the phenomenon of MDR still can not be explained thoroughly and be reversed completely. Proteomics is a powerful screening method detecting agnoprotein which may be missed by conventional biomedical techniques. Comparative proteomic analysis of human MDR osteosarcoma cell line could identify new proteins involved in MDR.Objective: To detect the differential proteins in MG-63/DXR100 and MG-63 with comparative proteomics and to explore the mechanism of MDR.Methods: The total protein of MG-63/DXR100 and MG-63 were separated and purified with two-dimensional electrophoresis(2-DE). Then the proteins of differential expression chosen by image scanning and DeCyder software were identified in MALDI-TOF Pro and Mascot database.Results: It showed that thirty kinds of proteins expressed differently in proteomic analysis. Eighteen of them that matched well were chosen to be identified in MALDI-TOF Pro. Five of them were identified successfully including alcohol dehydrogenase(ADH6) with function of detoxication, matrix metalloproteinases 1(MMP1) related with tumor cell metastasis, FERM domain containing protein 3(FRMD3) which belongs to anti-oncogenes, and two agnoproteins composed of 128 and 300 amino acid residues. ADH6, MMP1 and the two agnoproteins expressed higher in MG-63/DXR100 while FRMD3 lower in the MDR cell line.Conclusion: Five proteins including ADH6, MMP1, FRMD3 and two agnoproteins can be identified in MDR osteosarcoma cell with comparative proteomics. It is possible that they played important roles in MDR.