George Joseph, Gondi Christopher S, Dinh Dzung H, Gujrati Meena, Rao Jasti S
Department of Cancer Biology, University of Illinois, College of Medicine at Peoria, Peoria, Illinois, USA.
Clin Cancer Res. 2007 Jun 15;13(12):3507-17. doi: 10.1158/1078-0432.CCR-06-3023.
The induction of apoptotic pathways in cancer cells offers a novel and potentially useful approach to improve patient responses to conventional chemotherapy. Tissue factor pathway inhibitor-2 (TFPI-2) is a protease inhibitor that is abundant in the extracellular matrix and highly expressed in noninvasive cells but absent or undetectable in highly invasive human glioblastoma cells.
Using a recombinant adeno-associated viral vector carrying human TFPI-2 cDNA, we stably expressed TFPI-2 in U-251 cells, a highly invasive human glioblastoma cell line. Our previous studies showed that restoration of TFPI-2 in glioblastomas effectively prevents cell proliferation, angiogenesis, and tumor invasion. In this study, we determined whether TFPI-2 restoration could induce apoptosis through the caspase-mediated signaling pathway.
The results from nuclear chromatin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, and fluorescence-activated cell sorting analysis showed increased apoptosis in U-251 cells after restoration of TFPI-2. Caspase-9 and caspase-3 activity assays showed increased activity, indicating enhanced apoptosis. Immunofluorescence for cleaved caspase-9 and caspase-3 depicted increased expression and colocalization of both molecules. Western blot analysis showed increased transcriptional activities of Fas ligand, tumor necrosis factor-alpha, Bax, Fas-associated death domain, and tumor necrosis factor receptor 1-associated death domain as well as elevated levels of cleaved caspases and poly(ADP-ribose) polymerase. Semiquantitative reverse transcription-PCR depicted increased expression of tumor necrosis factor-alpha and Fas ligand and the related death domains tumor necrosis factor receptor 1-associated death domain and Fas-associated death domain.
Taken together, these results show that restoration of TFPI-2 activates both intrinsic and extrinsic caspase-mediated, proapoptotic signaling pathways and induces apoptosis in U-251 cells. Furthermore, our study suggests that recombinant adeno-associated viral vector-mediated gene expression offers a novel tool for cancer gene therapy.
诱导癌细胞中的凋亡途径为改善患者对传统化疗的反应提供了一种新的且可能有用的方法。组织因子途径抑制剂-2（TFPI-2）是一种蛋白酶抑制剂，在细胞外基质中含量丰富，在非侵袭性细胞中高度表达，但在高侵袭性的人胶质母细胞瘤细胞中不存在或无法检测到。
我们使用携带人TFPI-2 cDNA的重组腺相关病毒载体，在高侵袭性的人胶质母细胞瘤细胞系U-251细胞中稳定表达TFPI-2。我们之前的研究表明，胶质母细胞瘤中TFPI-2的恢复可有效防止细胞增殖、血管生成和肿瘤侵袭。在本研究中，我们确定TFPI-2的恢复是否能通过半胱天冬酶介导的信号通路诱导凋亡。
核染色质染色、末端脱氧核苷酸转移酶介导的dUTP缺口末端标记分析和荧光激活细胞分选分析结果显示，TFPI-2恢复后U-251细胞中的凋亡增加。半胱天冬酶-9和半胱天冬酶-3活性分析显示活性增加，表明凋亡增强。裂解的半胱天冬酶-9和半胱天冬酶-3的免疫荧光显示这两种分子的表达增加且共定位。蛋白质印迹分析显示Fas配体、肿瘤坏死因子-α、Bax、Fas相关死亡结构域和肿瘤坏死因子受体1相关死亡结构域的转录活性增加，以及裂解的半胱天冬酶和聚（ADP-核糖）聚合酶水平升高。半定量逆转录-PCR显示肿瘤坏死因子-α和Fas配体以及相关死亡结构域肿瘤坏死因子受体1相关死亡结构域和Fas相关死亡结构域的表达增加。
综上所述，这些结果表明TFPI-2的恢复激活了内在和外在的半胱天冬酶介导的促凋亡信号通路，并诱导U-251细胞凋亡。此外，我们的研究表明重组腺相关病毒载体介导的基因表达为癌症基因治疗提供了一种新工具。
