Shalli Kawan, Brown Iain, Heys Steven D, Schofield Andrew C
School of Medicine, College of Life Sciences and Medicine, University of Aberdeen, Medical School, Foresterhill, Aberdeen, UK.
FASEB J. 2005 Aug;19(10):1299-301. doi: 10.1096/fj.04-3178fje. Epub 2005 Jun 9.
Docetaxel is one of the most active drugs used to treat breast cancer. The cellular target of docetaxel is the microtubule, specifically the beta-tubulin subunit, that comprises a series of isotypes and that can modulate function. This study has examined the role of alteration in beta-tubulin isotypes in vitro and has sequenced the beta-tubulin gene to determine if there were mutations, both of which may represent important mechanisms of acquired resistance to docetaxel. Breast cancer cells, MCF-7 (oestrogen-receptor positive) and MDA-MB-231, (oestrogen-receptor negative) were made resistant to docetaxel in vitro. Expression of beta-tubulin isotypes (class I, II, III, IVa, IVb, and VI) was determined at the RNA and protein level using RT-PCR and western analysis, respectively. DNA sequencing evaluated the beta-tubulin gene. At the mRNA level, class I, II, III, and IVa beta-tubulin mRNA isotypes were over-expressed in docetaxel-resistant MCF-7 cells when compared with the docetaxel-sensitive parental cells. However, class VI beta-tubulin mRNA isotype expression was decreased in resistant cells. In MDA-MB-231 cells, there was a decrease in expression of the class I and class IVa beta-tubulin mRNA. However, there were increased expressions in class II, IVb, and VI beta-tubulin mRNA isotypes in resistant cells. Western analysis has confirmed corresponding increases in beta-tubulin protein levels in MCF-7 cells. However, in MDA-MB-231 cells, there were decreased protein levels for class II and class III beta-tubulin. This study demonstrates that altered expression of mRNA beta-tubulin isotypes and modulation of beta-tubulin protein levels are associated with acquired docetaxel resistance in breast cancer cells. This allows further understanding and elucidation of mechanisms involved in resistance to docetaxel.
多西他赛是用于治疗乳腺癌的最有效的药物之一。多西他赛的细胞靶点是微管，具体是β-微管蛋白亚基，它包含一系列同种型并且能够调节功能。本研究检测了体外β-微管蛋白同种型改变的作用，并对β-微管蛋白基因进行了测序以确定是否存在突变，这两者都可能是获得性多西他赛耐药的重要机制。体外使乳腺癌细胞MCF-7（雌激素受体阳性）和MDA-MB-231（雌激素受体阴性）对多西他赛产生耐药。分别使用逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析在RNA和蛋白质水平测定β-微管蛋白同种型（I类、II类、III类、IVa类、IVb类和VI类）的表达。DNA测序评估β-微管蛋白基因情况。在mRNA水平，与多西他赛敏感的亲本细胞相比，I类、II类、III类和IVa类β-微管蛋白mRNA同种型在多西他赛耐药的MCF-7细胞中过表达。然而，VI类β-微管蛋白mRNA同种型在耐药细胞中的表达降低。在MDA-MB-231细胞中，I类和IVa类β-微管蛋白mRNA的表达降低。然而，耐药细胞中II类、IVb类和VI类β-微管蛋白mRNA同种型的表达增加。蛋白质免疫印迹分析证实了MCF-7细胞中β-微管蛋白水平相应升高。然而，在MDA-MB-231细胞中，II类和III类β-微管蛋白的蛋白质水平降低。本研究表明，mRNAβ-微管蛋白同种型表达改变和β-微管蛋白水平调节与乳腺癌细胞获得性多西他赛耐药有关。这有助于进一步理解和阐明多西他赛耐药的相关机制。
