Tamoxifen modulates protein kinase C via oxidative stress in estrogen receptor-negative breast cancer cells
Tamoxifen modulates protein kinase C via oxidative stress in estrogen receptor-negative breast cancer cells
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DOI：
10.1074/jbc.271.23.13504
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发表时间：
1996-06-07
影响因子：
4.8
通讯作者：
Gopalakrishna, R
中科院分区：
文献类型：
--
作者：
Gundimeda, U;Chen, ZH;Gopalakrishna, R
非甾体类药物他莫昔芬（Tamoxifen，Tam）是一种乳腺癌的治疗/化学预防剂，可抑制蛋白激酶C（PKC），而PKC被认为是其作用于雌激素受体外的位点之一。（> 100 μ M）浓度以抑制试管中的PKC，而要求较低（1-10 μ M）浓度诱导雌激素受体阴性细胞类型中细胞生长的抑制，为了鉴定Tam对PKC和细胞生长的其他作用机制，已经用MDA-MB-231（一种雌激素受体阴性的乳腺癌细胞类型）进行了研究。在用5-20 μ MTam处理后，在30分钟内发生PKC的胞质溶胶至膜的易位，然后在2小时内下调该酶。在此期间，观察到非钙/脂质依赖性激活形式的PKC的瞬时产生，快速生长的细胞需要近2-3倍的低浓度（2-5 μ M）的Tam比融合细胞诱导PKC的变化。此外，在完整细胞中观察到的佛波酯结合也仅在PKC失活的条件下在Tam处理的细胞中降低。Tam不直接支持PKC的膜结合。花生四烯酸的释放与PKC膜转位有关。用[H-3]Tam进行的研究表明，Tam分配到膜中，并且在此有限的时间段（2 h）内，[3 H]Tam与细胞蛋白没有明显的共价缔合。各种抗氧化剂（维生素E、维生素C、β-胡萝卜素、过氧化氢酶和超氧化物歧化酶）抑制了Tam的所有这些细胞效应。此外，维生素E显著阻断TAM诱导的生长抑制。为了确定Tam的氧化代谢产物是否可以永久影响PKC，用纯化的PKC测试OH-Tam。与Tam相反，它可逆地抑制PKC，OH-Tam通过在较低浓度下修饰催化结构域而使酶永久失活。发现该结构域内存在的邻位硫醇是诱导这种失活所需的。这种作用被各种抗氧化剂部分阻断。这是第一份报告显示氧化应激在介导Tam作用中的作用。综上所述，这些结果表明，谭，通过最初的分配到膜，诱导产生跨膜信号和氧化应激，引发PKC的膜协会，随后由不可逆的激活，和随后的下调，这种酶，这在一定程度上，可能导致细胞生长抑制。
Nonsteroidal agent tamoxifen (Tam), a therapeutic/chemopreventive agent for breast cancer, inhibits protein kinase C (PKC), which is considered tea be one of its extra-estrogen receptor sites of action, This drug is required at higher (> 100 mu M) concentrations to inhibit PKC in the test tube, whereas it is required at lower (1-10 mu M) concentrations to induce inhibition of cell growth in estrogen receptor-negative cell types, To identify additional mechanisms of action of Tam on PKC and cell growth, studies with MDA-MB-231, an estrogen receptor-negative breast carcinoma cell type, have been carried out, Upon treatment with 5-20 mu M Tam, a cytosol to membrane translocation of PKC occurred within 30 min, which was then followed by a down-regulation of the enzyme within 2 h. A transient generation of Ca2+/lipid-independent activated form of PKC was observed during this period, Rapidly growing cells require nearly 2-3-fold lower concentrations (2-5 mu M) of Tam than do confluent cells to induce changes in PKC, Furthermore, phorbol ester binding observed with intact cells also decreased in Tam-treated cells only under the conditions PKC was inactivated, Unlike phorbol esters, Tam did not directly support the membrane association of PKC. The release of arachidonic acid correlated with the PKC membrane translocation. Studies carried out with [H-3]Tam revealed that Tam partitioned into the membrane, and there was no appreciable covalent association of [3H]Tam with cellular proteins within this limited time period (2 h). Various antioxidants (vitamin E, vitamin C, beta-carotene, catalase, and superoxide dismutase) inhibited all these cellular effects of Tam. Moreover, vitamin E strikingly blocked Tam-induced growth inhibition. To determine whether oxymetabolites of Tam can affect PKC permanently, OH-Tam was tested with purified PKC. In contrast to Tam, which reversibly inhibited PKC, OH-Tam permanently inactivated the enzyme by modifying the catalytic domain at lower concentrations. The vicinal thiols present within this domain were found to be required to induce this inactivation. This effect was partially blocked by various antioxidants. This is the first report showing the role of oxidative stress in mediating the actions of Tam. Taken together these results suggest that Tam, by initially partitioning into the membranes, induces a generation of transmembrane signals and an oxidative stress to elicit the membrane association of PKC, followed by an irreversible activation, and subsequent down-regulation of this enzyme, which, in part, may lead to cell growth inhibition.