Effect And Possible Mechanism Of Macrophage Colony Stimulating Factor On Proliferation Of Breast Cancer And Cervical Cancer

BackgroundBreast cancer and cervical cancer are common types of gynecologic cancers, and seriously threatening the women's health. Recent research show that the level of macrophage colony stimulating factor(M-CSF) expression is often araised abnormally in many kinds of cancer while it can hardly be detected in physiology tissue or benign tumor, suggesting it may be a kind of biomarker of these cancer, closely linked to the progress of tumorigenesis and development.M-CSF, also named CSF-1, is an important growth factor involved in cellular signalling controlling the survival, proliferation and differentiation of cells belonging to the monocyte-macrophage lineage both in vivo and in vitro. To understand the effect and possible mechanism of M-CSF on the proliferation of breast cancer and cervical cancer so as to provide a new idea of treatment of these disease, we want to study the role of M-CSF in vivo and in vitro and combining with the clinical trial.Partâ… The abnormal expression of M-CSF in the tissue from the breast cancer and cervical cancerAt first, we detect the expression of M-CSF in the tissue from the breast cancer and cervical cancer by immunohistochemistry and enzyme linked immunosorbent assay(ELISA).AIM To detect the expression of M-CSF in the tissue from the breast cancer and cervical cancer. METHODS 10 cases of breast cancer tissues and cervical cancer tissue from the surgical operation and 10 cases of healthy volunteers serum were used to detected the M-CSF expression by the enzyme linked immunosorbent assay(ELISA). Immunohistochemistry was used to detect the M-CSF level in tumor tissues and the nearby physiological tissue by operation.RESULTS M-CSF in the tumor tissues was significantly increased both in breast cancer and in cervical cancer while it could hardly be detected in the nearby physiological tissue. The average M-CSF level in serum of the patients from breast cancer and cervical cancer and healthy volunteers were respectively 620.16, 486.72 and 223.25 pg/ml.CONCLUSION M-CSF expression is araised abnormally both in breast cancer and in cervical cancer.Part II Construction of MCF7 cells and HeLa cells overexpressing cytoplasmic and nuclear M-CSF and the effect of M-CSF on the cell proliferationI Construction and identification of MCF7 cell line and HeLa cell line respectively overexpressing cytoplasmic and nuclear M-CSFAIM To study the role of cytoplasmic and nuclear M-CSF.METHODS pCMV/nuc/M-CSF and pCMV/cyto/myc-M-CSF recombinant vectors were constructed by inserting M-CSF cDNA into pCMV/myc/nuc vector and pCMV/cyto/myc vector, respectively. The resulting vectors were identificated by PCR, Double Digestion and sequencing, and were transfected into HeLa cells and MCF7 cells by liposome. After screening with G418, RT-PCR, western blot and immunofluorescence were used to detect the mRNA and protein expression and localization of M-CSF in MCF7 cells and HeLa cells.RESULTS pCMV/nuc/M-CSF and pCMV/cyto/myc-M-CSF recombinant vectors were constructed successfully. M-CSF in HeLa cells and MCF7 cells were detected at a high level both in the mRNA and protein expressed and localized in cytoplasm or nucleus.CONCLUSION pCMV/nuc/M-CSF and pCMV/cyto/myc-M-CSF recombinant vectors were constructed successfully. A HeLa cell line stably expressed cytoplasmic and nuclear M-CSF was constructed successfully. A MCF7 cell line stably expressed cytoplasmic and nuclear M-CSF was constructed successfully.II The effect of cytoplasmic and nuclear M-CSF on the proliferation of MCF7 cells and HeLa cellsAIM To explore the effect of M-CSF on the proliferationof MCF7 cells and HeLa cells.METHODS The effect of cytoplasmic and nuclear M-CSF on the proliferation was analyzed by counting of cell doubling time, MTT and antisense oligonucleotides.RESULTS The results shew that M-CSF-transfected MCF7 cells in cytoplasm and nucleus had a shorter doubling time and more significantly augmented reproductive activity than either pCMV/cyto/myc or pCMV/nuc/myc transfected MCF7 cells or untransfected MCF7 cells. M-CSF-specific antisense oligonucleotides significantly inhibited the proliferation of the M-CSF-transfected cells but had little effect on the other two groups. So did M-CSF-transfected HeLa cells.CONCLUSION Overexpression of cytoplasmic and nuclear M-CSF could promote the proliferation of MCF7 cells and HeLa cells.Part III The effect of M-CSF expressed at a high level on the growth of human carcinoma implanted tumor in nude miceAIM To observe the effect of M-CSF expressed at a high level on the growth of human carcinoma implanted tumor in nude mice.METHODS The nude mice wer divided into 10 groups. Each group included 5 nude mice. The M-CSF, pCMV/cyto/myc and pCMV/nuc/myc transfected and untransfected Hela cells and MCF7 cells were all subcutaneously inoculated into nude mice respectively. The volume and weight of the implanted tumor were observed.RESULTS The occurrence of tumor in the nude mice implanted M-CSF-transfected HeLa cell or MCF7 cell is significantly earlier than either pCMV/cyto/myc and pCMV/nuc/myc transfected cells or untransfected cells. The tumor volume is larger and tumor weight is heavier in the nude mice implanted M-CSF-transfected HeLa cell or MCF7 cell than the control groups (P<0.05).CONCLUSION Intracellular M-CSF overexpression could promote the growth of human carcinoma implanted tumor in nude mice.Part IV Possible mechanism of intracellular M-CSF on proliferation of MCF7 and HeLa cellsAIM To analyze the possible mechanism of intracellular M-CSF on the proliferation of MCF7 and HeLa cells.METHODS The expression of cyclinD1,p38,p-p38,Akt,p-Akt was detected by western blot. The activity of NADPH oxidase was measured by the reduction of the yellow dye nitroblue tetrazolium(NBT)to insoluble blue-black formazan.RESULTS Overexpression of cytoplasmic and nuclear M-CSF up-regulated the expression of cyclinD1, p-p38 and p-Akt in HeLa and MCF7 cells and increase the activity of NADPH oxidase.CONCLUSION Overexpression of cytoplasmic and nuclear M-CSF could promote the proliferation of MCF7 cells and HeLa cells by up-regulating the protein expression of cyclinD1, p-p38 and p-Akt in HeLa and MCF7 cells and increasing the activity of NADPH oxidase.CONCLUSIONS pCMV/nuc/M-CSF and pCMV/cyto/myc-M-CSF recombinant vectors were constructed successfully. Stably expressed cytoplasmic and nuclear M-CSF in HeLa cells were constructed successfully. Stably expressed cytoplasmic and nuclear M-CSF in MCF7 cells were constructed successfully. Overexpression of cytoplasmic and nuclear M-CSF could promote the proliferation of MCF7 cells and HeLa cells by up-regulating the protein expression of cyclinD1, p-P38 and p-Akt in HeLa and MCF7 cells and increasing the activity of NADPH oxidase.