Nitric oxide-induced cytostasis and cell cycle arrest of a human breast cancer cell line (MDA-MB-231): Potential role of cyclin D1

DETA-NONOate, a nitric oxide (NO) donor, induced cytostasis in the human breast cancer cells MDA-MB-231, and the cells were arrested in the G(1) phase of the cell cycle. This cytostatic effect of the NO donor was associated with the down-regulation of cyclin D1 and hypophosphorylation of the retinoblastoma protein. No changes in the levels of cyclin E or the catalytic partners of these cyclins, CDK2, CDK4, or CDK6, were observed. This NO-induced cytostasis and decrease in cyclin D1 was reversible for up to 48 h of DETA-NONOate (1 mM) treatment. DETA-NONOate (1 mM) produced a steady-state concentration of 0.5 μM of NO over a 24-h period. Synchronized population of the cells exposed to DETA-NONOate remained arrested at the G(1) phase of the cell cycle whereas untreated control cells progressed through the cell cycle after serum stimulation. The cells arrested at the G(1) phase after exposure to the NO donor had low cyclin D1 levels compared with the control cells. The levels of cyclin E and CDK4, however, were similar to the control cells. The decline in cyclin D1 protein preceded the decrease of its mRNA. This decline of cyclin D1 was due to a decrease in its synthesis induced by the NO donor and not due to an increase in its degradation. We conclude that down-regulation of cyclin D1 protein by DETA-NONOate played an important role in the cytostasis and arrest of these tumor cells in the G(1) phase of the cell cycle.

DETA-NONOate作为一氧化氮（NO）供体，可诱导人乳腺癌细胞MDA-MB-231产生细胞生长抑制效应，并使细胞阻滞于细胞周期G₁期。该一氧化氮供体的细胞生长抑制作用，与细胞周期蛋白D1（cyclin D1）的表达下调以及视网膜母细胞瘤蛋白（retinoblastoma protein）的低磷酸化修饰密切相关。未观察到细胞周期蛋白E（cyclin E）或其催化伴侣细胞周期蛋白依赖性激酶2（CDK2）、细胞周期蛋白依赖性激酶4（CDK4）、细胞周期蛋白依赖性激酶6（CDK6）的表达水平发生明显变化。经1 mM DETA-NONOate处理最长达48小时后，这种由一氧化氮诱导的细胞生长抑制及细胞周期蛋白D1水平降低的现象均可逆转。1 mM DETA-NONOate可在24小时内维持稳态浓度为0.5 μM的一氧化氮水平。经DETA-NONOate处理的细胞同步化群体仍阻滞于细胞周期G₁期，而未处理的对照组细胞在血清刺激后可顺利推进细胞周期进程。与对照组细胞相比，经一氧化氮供体处理后阻滞于G₁期的细胞，其细胞周期蛋白D1的表达水平较低；但细胞周期蛋白E与CDK4的表达水平与对照组无显著差异。细胞周期蛋白D1的蛋白水平下降先于其mRNA水平的降低，这种下降是由一氧化氮供体诱导的蛋白合成减少所致，而非其降解速率升高引发。本研究结果显示，DETA-NONOate通过下调细胞周期蛋白D1的蛋白表达，在该肿瘤细胞的细胞生长抑制及G₁期阻滞过程中发挥了重要作用。