A novel role of tissue factor pathway inhibitor-2 in apoptosis of malignant human gliomas

Tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa serine protease inhi bitor found at high levels in extracellular matrix. Recombinant human TFPI- 2 has recently been shown to be a strong inhibitor of trypsin, plasmin, pla sma kallikrein, and factor XIa amidolytic activity. Earlier studies in our laboratory showed that the expression of TFPI-2 is lost during tumor progre ssion in human gliomas. We stably transfected this protease inhibitor in mu ltiform glioblastoma cell line (SN13-19) and in low-grade glioma cell line (Hs683) in sense and antisense orientation respectively. This confirmed tha t the upregulation/down-regulation of TFPI-2 plays a significant role in th e invasive behavior of human gliomas both in vitro and in vivo models. Coll ectively, these results suggested an idea to determine whether TFPI-2 is ne cessary for cell survival and inhibition of tumor formation in nude mice, d ue to apoptosis of intracerebrally injected SNB-19 cells. In the present st udy we determined p-ERK levels and found that they are decreased in TFPI-2 overexpressed clones (SNB-19) and increased in TFPI-2 downregulated clones (Hs683). We also checked the levels of BAX/BCl-2, caspases (for e.g., 9, 7, 3, 8), PARP, cytochrome-c and Apaf-1. Moreover, the increase of apoptosis in vitro is associated with increased and decreased expression of apoptotic protein BAX in sense clones (SNB-19) and antisense clones (Hs683) respecti vely, when compared to controls and vice versa with Bcl-2 the anti-apoptoti c protein. Caspases (9, 7 and 3), cytochrome-c, Apaf-1 and PARP levels are increased in SNB-19 and decreased in Hs683. Caspase 8 was not expressed in either cell line. Caspases 9 and 3 activity assay revealed higher activity in sense clones (SNB-19) but lesser in antisense clones (Hs683) compared to controls. This is the first report of TFPI-2 playing a novel role in cell survival in human gliomas.