Kang Y, Cortina R, Perry R R
Division of Surgical Oncology, Eastern Virginia Medical School, Norfolk, USA.
J Natl Cancer Inst. 1996 Mar 6;88(5):279-84. doi: 10.1093/jnci/88.5.279.
The antiestrogen tamoxifen (TAM) is effective in the treatment of estrogen receptor (ER)-positive as well as some ER-negative breast cancers. However, the precise mechanism of action of TAM, especially in estrogen-independent cells, remains unclear. Previous work by our laboratory has demonstrated that TAM induces the morphologic and biochemical changes that are characteristic of apoptosis in both ER-positive and ER-negative cells.
We compared the effect of TAM at a clinically achievable concentration on cell growth and apoptosis with the effect of TAM on c-myc (also known as C-MYC) messenger RNA (mRNA) and protein expression in ER-negative MDA-231 cells.
MDA-231 cells were treated for up to 72 hours with 1.0 microM TAM alone or in the presence of 50 microM c-myc antisense or nonsense oligonucleotides. c-myc mRNA expression was determined by northern blot analysis, protein expression by western blot analysis, cell growth inhibition counts, and DNA cleavage by agarose gel electrophoretic analysis. Differences between the mean values from different treatment groups were compared with the use of the two-sided Wilcoxon Ranksum test.
TAM treatment for 72 hours increased c-myc mRNA five-fold (from a relative radiolabeled hybridization signal intensity of 17 +/- 4 up to 93 +/- 10; P<.05) and c-MYC protein threefold (from a relative immunofluorescence signal intensity of 28 +/- 7 up to 83+/-21; P< .05). The induction of c-myc by TAM was accompanied by internucleosomal DNA cleavage characteristic of apoptotic cell death. Addition of c-myc antisense oligonucleotide (5'CACGTTGAGGGGCAT-3') to MDA-231 cells resulted in a nearly twofold decrease of basal c-myc mRNA (P< .05) and a sevenfold decrease of basal c-Myc protein (P< .05) expression. Addition of c-myc antisense oligomer also antagonized the TAM-induced increase in c-myc mRNA (P< .05) and protein expression (P< .05) and inhibited TAM-induced cytostasis (P< .01) and apoptosis. In parallel experiments, addition of the nonsense oligomer had no effect on any of the measured parameters.
These results indicate that the effects of TAM on ER-negative MDA-231 cells may be mediated through c-myc overexpression. c-myc may play a critical role in the growth and progression of MDA-231 breast cancer cells.
抗雌激素药物他莫昔芬（TAM）对雌激素受体（ER）阳性以及某些ER阴性乳腺癌的治疗有效。然而，TAM的确切作用机制，尤其是在雌激素非依赖性细胞中的作用机制仍不清楚。我们实验室之前的研究表明，TAM可诱导ER阳性和ER阴性细胞发生凋亡特有的形态学和生化变化。
我们比较了临床可达到浓度的TAM对细胞生长和凋亡的影响，以及TAM对ER阴性的MDA-231细胞中c-myc（也称为C-MYC）信使核糖核酸（mRNA）和蛋白质表达的影响。
MDA-231细胞单独用1.0微摩尔/升TAM处理长达72小时，或在存在50微摩尔/升c-myc反义或无义寡核苷酸的情况下处理。通过Northern印迹分析测定c-myc mRNA表达，通过Western印迹分析、细胞生长抑制计数和琼脂糖凝胶电泳分析DNA裂解来测定蛋白质表达。使用双侧Wilcoxon秩和检验比较不同处理组平均值之间的差异。
TAM处理72小时使c-myc mRNA增加了五倍（相对放射性标记杂交信号强度从17±4增加到93±10；P<0.05），使c-MYC蛋白增加了三倍（相对免疫荧光信号强度从28±7增加到83±21；P<0.05）。TAM诱导c-myc增加的同时伴有凋亡细胞死亡特有的核小体间DNA裂解。向MDA-231细胞中加入c-myc反义寡核苷酸（5'CACGTTGAGGGGCAT-3'）导致基础c-myc mRNA下降近两倍（P<0.05），基础c-Myc蛋白表达下降七倍（P<0.05）。加入c-myc反义寡聚物还拮抗了TAM诱导的c-myc mRNA增加（P<0.05）和蛋白质表达增加（P<0.05），并抑制了TAM诱导的细胞生长停滞（P<0.01）和凋亡。在平行实验中，加入无义寡聚物对任何测量参数均无影响。
这些结果表明，TAM对ER阴性的MDA-231细胞的作用可能是通过c-myc过表达介导的。c-myc可能在MDA-231乳腺癌细胞的生长和进展中起关键作用。
