Effects of 17β-estradiol and 1,25-dihydroxyvitamin D3 on the proliferation and differentiation of osteoblastic MC3T3-E1 cells / 中华内分泌代谢杂志

Objective To assess the coordinated regulation and the molecular mechanisms of 17β-estradiol and 1,25-dihydroxyvitamin D3 [ 1,25-( OH ) 2 D3 ] on the proliferation and differentiation of osteoblastic MC3T3-E1 cells.Methods MC3T3-E1 cells were cultured in phenol-red free α-MEM medium supplemented with 10％ FBS,MTT assay was performed to determine the effects of 17β-estradiol and 1,25-( OH )2 D3 on MC3T3-E1 cells proliferation.After cells were treated with different agents,cell cycle related genes [ cyclin E,proliferation cell nuclear antigen ( PCNA ),and cyclin-dependent kinase inhibitor 2b ( Cdkn2b ) ] and markers of osteoblastic differentiation [ type Ⅰ collagen ( COL Ⅰ ),alkaline phosphatase ( ALP),osteopontin ( OPN ) ] were detected with SYBR green-based quantitative PCR.ALP activity was detected with BCIP/NBT method.Results 17β-estradiol could promote proliferation of MC3T3-E1 cells,which was accordant to its ability to increase cyclin E and PCNA and to inhibit Cdkn2b mRNA expression in MC3T3-E1 cells.However,1,25-( OH)2D3 had no effect on the proliferation of MC3T3-E1 cells and also did not enhance the proliferation of MC3T3-E1 cells stimulated by 17β-estradiol.On the other hand,17β-estradiol promoted the gene expression of differentiation markers Col Ⅰ,ALP,and OPN,and 1,25-(OH) 2 D3 synergistically increased the expression of these genes with 17 β-estradiol.Conclusion As two of the most important hormones which regulate bone metabolism,estrogen and vitamin D may coordinately promote osteoblast differentiation,but may not regulate osteoblasts proliferation synergistically.

Mechanism Underlying NaF-Induced Apoptosis in Human Oral Squamous Cell Carcinoma

Few studies have evaluated the apoptosis-inducing efficacy of NaF on cancer cells in vitro but there has been no previous investigation of the apoptotic effects of NaF on human oral squamous cell carcinoma cells. In this study, we have investigated the mechanisms underlying the apoptotic response to NaF treatment in the YD9 human squamous cell carcinoma cell line. The viability of YD9 cells and their growth inhibition were assessed by MTT and clonogenic assays, respectively. Hoechst staining, DNA electrophoresis and TUNEL staining were conducted to detect apoptosis. YD9 cells were treated with NaF, and western blotting, immunocytochemistry, confocal microscopy, FACScan flow cytometry, and MMP and proteasome activity assays were performed sequentially. The NaF treatment resulted in a time- and dose-dependent decrease in YD9 cell viability, a dose-dependent inhibition of cell growth, and the induction of apoptotic cell death. The apoptotic response of these cells was manifested by nuclear condensation, DNA fragmentation, the reduction of MMP and proteasome activity, a decreased DNA content, the release of cytochrome c into the cytosol, the translocation of AIF and DFF40 (CAD) into the nucleus, a significant shift of the Bax/Bcl-2 ratio, and the activation of caspase-9, caspase-3, PARP, Lamin A/C and DFF45 (ICAD). Furthermore, NaF treatment resulted in the downregulation of G1 cell cyclerelated proteins, and upregulation of p53 and the Cdk inhibitor p27KIP1. Taken collectively, our present findings demonstrate that NaF strongly inhibits YD9 cell proliferation by modulating the expression of G1 cell cycle-related proteins and inducing apoptosis via mitochondrial and caspase pathways.

Mechanism Underlying NaF-Induced Apoptosis in Human Oral Squamous Cell Carcinoma

Few studies have evaluated the apoptosis-inducing efficacy of NaF on cancer cells in vitro but there has been no previous investigation of the apoptotic effects of NaF on human oral squamous cell carcinoma cells. In this study, we have investigated the mechanisms underlying the apoptotic response to NaF treatment in the YD9 human squamous cell carcinoma cell line. The viability of YD9 cells and their growth inhibition were assessed by MTT and clonogenic assays, respectively. Hoechst staining, DNA electrophoresis and TUNEL staining were conducted to detect apoptosis. YD9 cells were treated with NaF, and western blotting, immunocytochemistry, confocal microscopy, FACScan flow cytometry, and MMP and proteasome activity assays were performed sequentially. The NaF treatment resulted in a time- and dose-dependent decrease in YD9 cell viability, a dose-dependent inhibition of cell growth, and the induction of apoptotic cell death. The apoptotic response of these cells was manifested by nuclear condensation, DNA fragmentation, the reduction of MMP and proteasome activity, a decreased DNA content, the release of cytochrome c into the cytosol, the translocation of AIF and DFF40 (CAD) into the nucleus, a significant shift of the Bax/Bcl-2 ratio, and the activation of caspase-9, caspase-3, PARP, Lamin A/C and DFF45 (ICAD). Furthermore, NaF treatment resulted in the downregulation of G1 cell cyclerelated proteins, and upregulation of p53 and the Cdk inhibitor p27KIP1. Taken collectively, our present findings demonstrate that NaF strongly inhibits YD9 cell proliferation by modulating the expression of G1 cell cycle-related proteins and inducing apoptosis via mitochondrial and caspase pathways.

Effects of Extremely Low Frequency Elecromagnetic Fields on Thyroid Carcinogenesis Induced by N-bis(2-hydroxypropyl)nitrosamine and Sulfadimethoxine

PURPOSE: Long-term exposure to extremely low-frequency (60 Hz) electromagnetic fields (ELF-EMF) raises the questions of the induction of biological effects including tumorigenesis. One mechanism through which ELF-MFS could influence neoplastic development is the imbalance of cellular proliferation and cell apoptosis. The present study investigated the effect of ELF-EMF on chemically-induced thyroid carcinogenesis in a rat. METHODS: We examined cellular proliferation index measured by anti-Ki-67 antigen, apoptosis, apoptosis related proteins such as caspase 3 and p53, and cell cycle-related proteins (cyclin D1 and p21(WAF1/Cip1)). Forty Male F344 rats received a subcutaneous N-bis(2-hydroxypropyl)nitrosamine (DHPN, 2,800 mg/kg) injection, and 1 week later were allowed free access to drinking water containing sulfadimethoxine (0.1%) for 12 weeks. Twenty rats were exposed by ELF-EMF. During the carcinogenesis, sequential histological changes from hyperplasia, adenoma, and ultimately to overt carcinomas were noted. RESULTS: The exposure group of ELF-EMF, significantly increases the number size of carcinomas. Also, the proliferative and apoptotic indices were significantly increased in the ELF-EMF exposure group than in the control group. The caspase 3 protein expression did not show any significant changes between ELF-EMF group and control group. The p53 protein was not detected in both ELF-EMF exposure and control group. Among the cell cycle related proteins, cyclin D1, not p21(WAF1/Cip1), was significantly increased in adenomas and carcinomas in ELF-EMF exposure group compared with the control group. CONCLUSION: Exposure of ELF-EMF effects on chemically-induced rat thyroid carcinogenesis as results of altered increase of cellular proliferation, apoptosis, and cyclin D1 expression.

Significance of Expression of Cell Cycle Related Proteins and Apoptosis Related Proteins in Follicular Adenoma and Follicular Carcinoma of Thyroid

PURPOSE: To explore the role of cell cycle regulators and apoptosis regulators in carcinogenesis of thyroid, the expression of cell cycle related proteins (cyclin D1, Ki-67) and apoptosis related proteins (survivin, caspase 3, bcl-2, p53) were investigated in follicular adenoma and follicular carcinoma of thyroid. METHODS: The following formalin-fixed paraffin embedded surgical specimens were immunohistochemically stained by avidin-biotin complex method for cyclin D1, Ki-67, survivin, caspase 3, bcl-2, p53; 15 cases of follicular adenoma (FA), 31 cases of minimally invasive follicular carcinoma (MIFC) and 12 cases of widely invasive follicular carcinoma (WIFC). RESULTS: The overexpression of six gene products in follicular neoplasms of thyroid was noted in varying frequency. Among them, increased Ki-67, caspase 3 index and overexpression of bcl-2 were noted in statistically significant, widely invasive follicular carcinoma than that of follicular adenoma and minimally invasive follicular carcinoma. CONCLUSION: These results suggest that the overexpression of Ki-67, caspase 3, bcl-2 appear to play an important role during follicular carcinogenesis of thyroid. In addition, the overexpression of these proteins is related to the differentiation of MIFC and WIFC. However, further molecular genetic studies are required to determine the interrelationships between the expression of cell cycle related proteins and apoptosis related proteins.