Lun Mingyue, Zhang Ping L, Siegelmann-Danieli Nava, Blasick Thomas M, Brown Robert E
Weis Center for Research, Geisinger Medical Center, Danville, Pennsylvania 17822, USA.
Ann Clin Lab Sci. 2005 Winter;35(1):15-24.
Velcade, a proteasome inhibitor, has been shown to inhibit DNA binding activity of nuclear factor-kappaB (NF-kappaB) and to stabilize p53 in vitro. But its impact, in the context of activated (phosphorylated and translocated) NF-kappaB and the expression of p53, has not been studied in breast cancer. It would be desirable to determine whether or not the immunohistochemical (IHC) expressions of activated NF-kappaB and of p53 can predict the effects of Velcade in viable tumor cells. To answer these questions, we selected 3 breast cancer cell lines (SKBR-3, MDA-175, and MDA-231), which are negative for hormonal receptors, but differ in HER-2/neu expression (strong, mild, and minimal, respectively). The 3 cell lines showed different expressions of phosphorylated (p)- NF-kappaB and p53, as evaluated using immunohistochemistry with visual quantification by brightfield microscopy. After being treated with Velcade for 2 days, MDA-231 cells showed markedly reduced proliferation, followed by SKBR-3 cells, and then by MDA-175 cells. There was strong correlation between the nuclear expression of either p-NF-kappaB or p53 and the inhibitory rate of Velcade in the 3 cell lines (r = 0.987 and 0.807, respectively). Western blotting showed an increase in inhibitor-kappaB (I-kappaB) expression in nuclei of MDA-231 and SKBR-3 cells, but not in MDA-175 cells, following exposure to Velcade. Velcade treatment resulted in cleaved caspase-3 expression in MDA-231 cells and in the overexpression of p53 and p21WAF1 in all 3 cell lines, as evaluated using Western blotting. In summary, morphoproteomic analysis of p-NF-kappaB and p53 can be correlated with the inhibitory effect of Velcade in vitro. We propose that this proliferative inhibition is variably associated with blocking p-NF-kappaB function by upregulation of nuclear I-kappaB, stabilization of p53, and induction of p21WAF1.
万珂（硼替佐米）是一种蛋白酶体抑制剂，已被证明在体外可抑制核因子-κB（NF-κB）的DNA结合活性并稳定p53。但在乳腺癌中，其在活化（磷酸化和易位）的NF-κB及p53表达背景下的影响尚未得到研究。确定活化的NF-κB和p53的免疫组化（IHC）表达能否预测万珂对存活肿瘤细胞的作用将是很有必要的。为回答这些问题，我们选择了3种乳腺癌细胞系（SKBR-3、MDA-175和MDA-231），它们激素受体均为阴性，但HER-2/neu表达不同（分别为强、弱和极低）。使用免疫组化结合明场显微镜视觉定量评估，这3种细胞系显示出磷酸化（p）-NF-κB和p53的不同表达。用万珂处理2天后，MDA-231细胞的增殖明显降低，其次是SKBR-3细胞，然后是MDA-175细胞。在这3种细胞系中，p-NF-κB或p53的核表达与万珂的抑制率之间存在很强的相关性（r分别为0.987和0.807）。蛋白质印迹法显示，在接触万珂后，MDA-231和SKBR-3细胞核中抑制剂-κB（I-κB）表达增加，但MDA-175细胞中未增加。使用蛋白质印迹法评估，万珂处理导致MDA-231细胞中裂解的半胱天冬酶-3表达，并在所有3种细胞系中导致p53和p21WAF1过表达。总之，p-NF-κB和p53的形态蛋白质组学分析可与万珂在体外的抑制作用相关联。我们提出，这种增殖抑制与通过上调细胞核I-κB阻断p-NF-κB功能、稳定p53以及诱导p21WAF1存在不同程度的关联。
