目的:研究1α,25-二羟基维生素D3(1,25VD3)通过对表皮生长因子受体和细胞周期的调控调节人卵巢癌细胞的生长。方法:用1,25VD3处理人卵巢癌细胞株OVCAR3后,MTT法测定细胞生长情况,流式细胞仪分析细胞周期,Western blot检测EGFR表达。用pcDNA3-EGFRvIII质粒转染OVCAR3细胞,筛选出稳定过表达EGFR的克隆株EGFR—OVCAR3。用1,25VD3及EGFR抑制剂分别处理OVCAR3和EGFR—OVCAR3细胞,Westernblot检测p27、CyclinE和GADD45蛋白表达。结果:1,25VD3下调OVCAR3细胞EG—FR蛋白表达,对细胞生长有明显抑制作用,使其G0/G1和G2/M期增多,S期明显减少,而对EGFR—OVCAR3细胞无明显作用。Western blot显示,1,25VD3上调OVCAR3细胞p27和GADD45蛋白表达,下调CyclinE蛋白表达,而对EGFR—OVCAR3细胞三种蛋白表达无调节作用,用EGFR抑制剂处理EGFR—OVCAR3细胞后,又能调节p27和cyclinE蛋白的表达。结论:1,25VD3可以调节EGFR蛋白表达,并进而调节下游分子,包括p27和GADD45,最后调控细胞周期,抑制细胞G1/S期转换和G2/M期转换,从而抑制卵巢癌细胞的生长。
Objective: To investigate the regulation of 1α,25 - dihydroxyvitamin D3 on the expression of epidermal growth factor receptor(EGFR)and cell cycle in human ovarian cancer cells( OVCAR3 ). Methods: Western blot was used to determine the expression of EGFR, p27, Cyclin E and GADD45 protein. Cell growth assay was used to measure cell growth, and flow cytometry was used to determine the cell cycle distribution. OVCAR3 cells were transfected with pcDNA3- EGFRvIII plasmids ,for the establishment of stable clones( EGFR- OVCAR3 ) that highly express EGFR constitutively. Results: 1,25 (OH)2D3 decreased EGFR protein expression in OVCAR3 cells. EGFR- OVCAR3 ceils resisted to 1,25 ( OH)2D3 - induced growth suppression of OVCAR3 cells. 1,25 ( OH)2D3 increased I)27 and GADD45 protein expression and decreased Cychn E protein expression in OVCAR3. EGFR - OVCAR3 cells resisted to 1,25 (OH) 2 D3 - induced regulation of p27, Cyclin E and GADD45. Inhibitor of EGFR regulated p27 and Cyclin E protein expression in EGFR - OVCAR3 cells. Conclusion: 1,25 (OH) 2 D3 down regulates the expression of EGFR and then induces cell cycle arrest in growth suppression of ovarian cancer cells.