Orthopedic Center for Musculoskeletal Research, University of Würzburg, Brettreichstrasse 11, 97074 Würzburg, Germany.
Bone. 2012 Mar;50(3):723-32. doi: 10.1016/j.bone.2011.11.025. Epub 2011 Dec 7.
Bisphosphonates (BP) are used for the treatment of osteoporosis and bone metastases due to breast and prostate cancer. Recent clinical studies indicated a benefit in survival and tumor relapse with the supportive treatment of breast cancer using zoledronic acid (ZA), thus stimulating the debate about its putative anti-tumor activity in vivo. MCF-7 breast cancer cells were treated for 3 h (pulse treatment) and 72 h (permanent treatment) with ZA, and apoptosis rates and cell viability, defined as ATP content, were determined after 72 h. Permanent and pulse stimulation with ZA inhibited the viability of MCF-7 cells, which could partly be rescued by atorvastatin (Ator) pre-treatment but not by geranylgeranyl pyrophosphate (GGPP) co-treatment. Microarray analysis of ZA treated MCF-7 cells identified genes of the mevalonate pathway as significantly upregulated, which was verified by qPCR. Additionally the putative tumor suppressors krüppel-like factor 2 and 6 (KLF2 and KLF6) were markedly upregulated, while the classical proliferation marker Ki-67 was clearly downregulated. The expression of all three genes was confirmed by qPCR on mRNA level and by immunocytochemistry or Western blot staining. Expression of target genes were also analyzed in other breast (MDA-MB-231, BT-20, ZR75-1, T47D) and prostate (LNCaP, PC3) cancer cell lines by qPCR. ZA responsiveness of KLF2, KLF6 and Ki-67 could be verified in PC3 and T47D cells, KLF6 responsiveness in LNCaP and KLF2 responsiveness in MDA-MB-231 and BT-20 cells. Here we demonstrate in the apoptosis insensitive MCF-7 cell line a remarkable impact of ZA exposure on cell viability and on the regulation of putative tumor suppressors of the KLF family. The molecular mechanism involved might be the accumulation of isopentenyl pyrophosphate (IPP) and ApppI, since we could partly rescue the ZA effect by Ator pre-treatment and GGPP co-treatment. These data should stimulate further research into both the role of the mevalonate pathway and the accumulation of pyrophosphate compounds like ApppI in tumorigenesis and differentiation and their potential apart from the inhibition of mitochondrial ADP/ATP translocase and apoptosis, since such effects might well be responsible for the adjuvant ZA treatment benefit of patients suffering from breast cancer.
双膦酸盐（BP）用于治疗骨质疏松症和乳腺癌和前列腺癌引起的骨转移。最近的临床研究表明，唑来膦酸（ZA）支持治疗乳腺癌可提高生存率和肿瘤复发率，从而刺激了其在体内潜在抗肿瘤活性的争论。用 ZA 对 MCF-7 乳腺癌细胞进行 3 小时（脉冲处理）和 72 小时（持续处理）处理，并在 72 小时后测定细胞凋亡率和细胞活力（定义为 ATP 含量）。ZA 的持续和脉冲刺激抑制 MCF-7 细胞的活力，阿托伐他汀（Ator）预处理部分可挽救，但 geranylgeranyl pyrophosphate（GGPP）共处理则不能。用 ZA 处理 MCF-7 细胞的微阵列分析鉴定出甲羟戊酸途径的基因显著上调，这通过 qPCR 得到了验证。此外，明显上调了假定的肿瘤抑制因子 kruppel 样因子 2 和 6（KLF2 和 KLF6），而经典增殖标志物 Ki-67 则明显下调。qPCR 证实了所有三个基因在 mRNA 水平上的表达，并通过免疫细胞化学或 Western blot 染色证实。qPCR 还分析了其他乳腺癌（MDA-MB-231、BT-20、ZR75-1、T47D）和前列腺癌（LNCaP、PC3）细胞系中靶基因的表达。在 PC3 和 T47D 细胞中验证了 ZA 对 KLF2、KLF6 和 Ki-67 的反应性，在 LNCaP 和 KLF6 细胞中验证了 KLF6 对 MDA-MB-231 和 BT-20 细胞的反应性。在这里，我们在凋亡不敏感的 MCF-7 细胞系中证明了 ZA 暴露对细胞活力和 KLF 家族潜在肿瘤抑制因子调节的显著影响。涉及的分子机制可能是异戊烯焦磷酸（IPP）和 ApppI 的积累，因为我们可以通过阿托伐他汀预处理和 GGPP 共处理部分挽救 ZA 效应。这些数据应刺激对甲羟戊酸途径的作用以及焦磷酸盐化合物如 ApppI 在肿瘤发生和分化中的积累及其潜力的进一步研究，除了抑制线粒体 ADP/ATP 转运蛋白和凋亡之外，因为这些效应很可能是佐剂 ZA 治疗乳腺癌患者受益的原因。
