Study On The Changes Of Nuclear Matrix Proteins During The Differention Of The Osteosarcoma MG-63 Cells Induced By Effective Ingredients From Chinese Materia Medica

In this study, the combination of effective ingredients from Chinese materia medica (33μg/mL ginsenoside Rg1, 2mmol/L cinnamic acid and 0.3μg/mL tanshinone II A, shortened form RCT in this paper) was used to induce the osteosarcoma MG-63 cells into terminal differentiation, and its effects were investigated by cellular biology and immunocytochemistry. The differentially expressed nuclear matrix proteins were analyzed by subcellular proteomics methods and laser Confocal scanning microscope in order to find out a new way to explore the molecular mechanisms of carcinogenesis and malignant phenotypic reversion in a systematic level.The results revealed that the proliferation of MG-63 cells were inhibited, as the percentage of cells increased from 47.5% to 72.5% in G0/G1 phase while decreased from 20.0% to 7.3% in S phase. The malignant morphological and ultrastructural characteristics were reversed towards the normal related cells, e.g., the morphology of the cells were regular and inclined to the same volume, the nucleo-cytoplasm ratio lessened, the heterochromatin decreased while euchromatin increased, the organelle increased and their structure appeared well-developed ,typical and polarized, microvilli were rare on the cellular surface. The expression level of the phenotype markers of osteoblast such as collagen, osteocalcin and osteonectin, was upregulated and the typical mineralized bone nodules could be observed under light microscopy in the treated cells. Immunocytochemistry assay revealed that the expression level of oncogene including c-myc, c-fos was downregulated, while the expression level of tumor suppressor genes including p27, Rb was upregulated. The configuration of nuclear matrix-intermediate filament was altered towards the normal related cells as the filament was more abundant, the distribution was regular and the filaments of nuclear matrix, nuclear lamina and intermediatefilment connected tightly into a compact meshwork. Nuclear matrix proteins, selectively extracted from MG-63 cells treated with or without RCT, were subjected to Proteomic analysis. Seventeen differentially expressed proteins were identified, including up-regulated proteins Msh3, Annexin A1, Vimentin, mago nashi homolog, Cep290 and Pseudouridylate synthase 7 homolog ; down-regulated proteins Prohibitin, Nucleophosmin, hnRNP A2/B1, Actin, TRIP-1, Lamin-B1, Lamin-A/C and Mitosin. Most of them are related to DNA replication and repair, cell cycle regulation, gene expression and regulation. Some proteins were confirmed by western blot and immunofluorescence analysis. Prohibitin and nucleophosmin, two of the specific nuclear matrix proteins, were colocalized with oncogene c-fos and c-myc products, as well as tumor suppressor gene p53 and Rb products. The expression level and location of them were changed following the proliferation and differentiation of MG-63 cells.Our study showed that the osteosarcoma MG-63 cells were induced into terminal differentiation after treatment with RCT, as the proliferation of MG-63 cells were inhibited, the cell cycle were arrested in G0/G1, the malignant morphological and ultrastructural characteristics were reversed, the configuration of nuclear matrix-intermediate filament was altered, the expression of the osteoblat- like phenotype markers were highly increased. Some nuclear matrix proteins which play an important role in DNA replication and repair, cell cycle regulation, gene expression and regulation, have changed in expression level and location during the differentiation. The colocalization of prohibitin and nucleophosmin with products of osteosarcoma associated oncogenes c-myc, c-fos and tumor suppressor genes Rb, p53, suggested the pathway and mechanisms in which specific nuclear matrix proteins regulated the proliferation and differentiation of the human osteosarcoma MG-63 cells. These results indicated that RCT can induce MG-63 cells into terminal differentiation by regulating the activities of some oncogenes and antioncongenes, altering the expression of some key nuclear matrix proteins, arresting cell cycle, and facilitating the expression of osteoblat-like phenotype markers. Our study provides the proofs and a new way to explore the mechanisms and the relationships of a series of life activities during the differentiation in tumor cells induced by effective ingredients from Chinese materia medica, e.g., the expression of oncogenes and tumor suppressor genes, signal transduction, and cell cycle regulation. Besides, this study provides several potential target proteins for cancer diagnosis and cancer therapy.