Hayakawa J, Ohmichi M, Kurachi H, Kanda Y, Hisamoto K, Nishio Y, Adachi K, Tasaka K, Kanzaki T, Murata Y
Department of Obstetrics and Gynecology, Osaka University Medical School, Suita, Japan.
Cancer Res. 2000 Nov 1;60(21):5988-94.
We studied the roles of the phosphatidylinositol 3-kinase (PI-3K)-protein kinase B/Akt-BAD cascade in both cisplatin-resistant Caov-3 and -sensitive A2780 human ovarian cancer cell lines. Treatment of both Caov-3 and A2780 cells with cisplatin but not with the trans-diaminodichloroplatinum (transplatin) isomer stimulated the activation of Akt, and the PI-3K inhibitor wortmannin blocked the cisplatin-induced activation of Akt. Treatment of both Caov-3 and A2780 cells with cisplatin but not with the trans-diaminodichloroplatinum isomer also stimulated the phosphorylation of BAD at both the Ser-112 and Ser-136 sites. Whereas the phosphorylation of BAD at Ser-136 was blocked by treatment with wortmannin, its phosphorylation at Ser-112 was blocked by a MAP/ERK kinase inhibitor, PD98059. Exogenous expression of a dominant-negative Akt in both Caov-3 and A2780 cells decreased the cell viability after treatment with cisplatin. In contrast, no sensitization to cisplatin was observed in cells expressing wild-type Akt. We further examined the role of BAD in the viability after cisplatin treatment using BAD mutants. Exogenous expression of each of the singly substituted BADS112A or BADS136A in both Caov-3 and A2780 cells decreased the viability after treatment with cisplatin to a degree intermediate between that caused by exogenous expression of wild-type BAD and doubly substituted BAD2SA. Cisplatin did not stimulate the phosphorylation of BAD Ser-136, but did stimulate the phosphorylation of BAD Ser-112 in cells expressing a dominant-negative Akt, suggesting that BAD Ser-136 but not Ser-112 was phosphorylated by Akt. Our findings suggest that cisplatin-induced DNA damage causes the phosphorylation of both BAD Ser-112 via an extracellular signal-regulated protein kinase (ERK) cascade and BAD Ser-136 via a PI-3K-protein kinase B/Akt cascade and that inhibition of either of these cascades sensitizes ovarian cancer cells to cisplatin.
我们研究了磷脂酰肌醇3激酶（PI-3K）-蛋白激酶B/Akt-BAD级联反应在顺铂耐药的Caov-3和敏感的A2780人卵巢癌细胞系中的作用。用顺铂而非反式二氨基二氯铂（反铂）异构体处理Caov-3和A2780细胞，可刺激Akt的激活，而PI-3K抑制剂渥曼青霉素可阻断顺铂诱导的Akt激活。用顺铂而非反式二氨基二氯铂异构体处理Caov-3和A2780细胞，也可刺激BAD在丝氨酸112和丝氨酸136位点的磷酸化。虽然渥曼青霉素处理可阻断BAD在丝氨酸136位点的磷酸化，但其在丝氨酸112位点的磷酸化可被丝裂原活化蛋白激酶/细胞外信号调节激酶（MAP/ERK）抑制剂PD98059阻断。在Caov-3和A2780细胞中外源表达显性负性Akt可降低顺铂处理后的细胞活力。相反，在表达野生型Akt的细胞中未观察到对顺铂的敏感性增加。我们进一步使用BAD突变体研究了BAD在顺铂处理后细胞活力中的作用。在Caov-3和A2780细胞中外源表达单取代的BADS112A或BADS136A中的每一种，均可使顺铂处理后的细胞活力降低至介于野生型BAD和双取代的BAD2SA外源表达所导致的程度之间。顺铂未刺激表达显性负性Akt的细胞中BAD丝氨酸136的磷酸化，但刺激了BAD丝氨酸-112的磷酸化，这表明BAD丝氨酸136而非丝氨酸112是由Akt磷酸化的。我们的研究结果表明，顺铂诱导的DNA损伤通过细胞外信号调节蛋白激酶（ERK）级联反应导致BAD丝氨酸112磷酸化，并通过PI-3K-蛋白激酶B/Akt级联反应导致BAD丝氨酸136磷酸化，并且抑制这些级联反应中的任何一个均可使卵巢癌细胞对顺铂敏感。
