Twaddle G M, Turbov J, Liu N, Murthy S
Department of Surgery, Evanston Hospital, Illinois 60201, USA.
J Surg Oncol. 1999 Feb;70(2):83-90. doi: 10.1002/(sici)1096-9098(199902)70:2<83::aid-jso4>3.0.co;2-l.
Receptor tyrosine kinase (RTK) activation is critical for growth factor-mediated cell proliferation. Blockade of RTK activation inhibits growth factor-induced cell proliferation. A panel of RTK inhibitors (tyrphostins) have been tested and compared for their antiproliferative effects on the hormone-dependent human breast cancer cell line, MCF-7, in vitro.
MCF-7 cells (10(4)/well) were seeded into 96 well plates and maintained in DMEM with 1% bovine serum albumin (BSA), 200-pg/mL estrogen, or 10% fetal bovine serum. After a defined time interval, the cells were exposed to RTK inhibitors and a non-RTK-inhibitory analog of tyrphostins (0 to 400 microM). After 3 days, the number of viable cells in each well was estimated by an MTT assay and the results expressed as percent of controls. Using a representative tyrphostin, A47, the validity of MTT assay as a measure of cell proliferation was tested by a colony formation assay and by immunostaining with Ki-67 antibodies.
MCF-7 cells maintained in DMEM containing 1% BSA without E2 or serum showed a minimal increase in cell number. Supplementation with E2 stimulated cell proliferation in a dose-dependent manner. This E2-mediated growth stimulation was completely inhibited (cytostatic effects) by the epidermal growth factor receptor (EGFR)-selective tyrphostins A47, B48, RG13022, and B50. These same tyrphostins also decreased the cell numbers to below control numbers in cultures maintained in 1% BSA or in serum containing medium (cytostatic/cytotoxic effects). B44 (EGFR-selective tyrphostin), AG1295 (platelet-derived growth factor receptor [PDGFR]-selective tyrphostin), and A1 had no inhibitory effects on cells with or without E2 treatments. However, A1 inhibited cell growth under serum supplementation. Genistein, a phytoestrogen, stimulated the autonomous, E2-induced as well as serum-induced growth of MCF-7 cells. Cell proliferation results derived from the MTT assay were corroborated by both the colony formation assay as well as the Ki-67 assay.
Of the agents tested, only EGFR-selective tyrphostins blocked E2-stimulated tumor cell proliferation, as opposed to the PDGFR-selective tyrphostin, RTK noninhibitory agent, or the phytoestrogen, genistein, which did not exert such an effect. These findings suggest that epidermal growth factor (EGF) is an important mediator of E2-induced proliferation of MCF-7 cells. Thus, tyrphostins may be selectively used to prevent the growth of hormone-dependent breast cancers, particularly regrowth of residual tumor in postmenopausal breast cancer survivors receiving estrogen replacement therapy.
受体酪氨酸激酶（RTK）激活对于生长因子介导的细胞增殖至关重要。阻断RTK激活可抑制生长因子诱导的细胞增殖。已对一组RTK抑制剂（曲磷胺）进行了测试，并比较了它们在体外对激素依赖性人乳腺癌细胞系MCF-7的抗增殖作用。
将MCF-7细胞（10⁴/孔）接种到96孔板中，在含有1%牛血清白蛋白（BSA）、200 pg/mL雌激素或10%胎牛血清的DMEM中培养。在规定的时间间隔后，将细胞暴露于RTK抑制剂和曲磷胺的非RTK抑制类似物（0至400 μM）。3天后，通过MTT法估计每孔中活细胞的数量，并将结果表示为对照的百分比。使用代表性的曲磷胺A47，通过集落形成试验和用Ki-67抗体进行免疫染色来测试MTT法作为细胞增殖测量方法的有效性。
在不含E2或血清的含有1% BSA的DMEM中培养的MCF-7细胞显示细胞数量仅有微小增加。补充E2以剂量依赖性方式刺激细胞增殖。这种E2介导的生长刺激被表皮生长因子受体（EGFR）选择性曲磷胺A47、B48、RG13022和B50完全抑制（细胞生长抑制作用）。在含有1% BSA或含血清培养基中培养的细胞中，这些相同的曲磷胺也将细胞数量降低至对照数量以下（细胞生长抑制/细胞毒性作用）。B44（EGFR选择性曲磷胺）、AG1295（血小板衍生生长因子受体[PDGFR]选择性曲磷胺）和A1对有无E2处理的细胞均无抑制作用。然而，A1在补充血清的情况下抑制细胞生长。染料木黄酮，一种植物雌激素，刺激MCF-7细胞的自主生长、E2诱导的生长以及血清诱导的生长。集落形成试验和Ki-67试验均证实了MTT法得出的细胞增殖结果。
在所测试的药物中，只有EGFR选择性曲磷胺阻断E2刺激的肿瘤细胞增殖，而PDGFR选择性曲磷胺、RTK非抑制性药物或植物雌激素染料木黄酮则没有这种作用。这些发现表明表皮生长因子（EGF）是E2诱导MCF-7细胞增殖的重要介质。因此，曲磷胺可选择性用于预防激素依赖性乳腺癌的生长，特别是接受雌激素替代疗法的绝经后乳腺癌幸存者中残留肿瘤的再生长。
