To confirm this hypothesis, the apoptotic index was compared between TAT-transfected and vector-transfected HCC cells by TUNEL staining. After STS treatment, TUNEL analysis revealed that the apoptotic index in Vec-7703 cells (13.6% ± 0.7%) was significantly lower than that of TAT-c2 (61.7% ± 3.9%, P < 0.05) or TAT-c3 (40% ±
1.4%, P < 0.05), confirming that TAT had a proapoptotic ability (Fig. 5B). Similarly, the apoptotic index in Vec-7402 cells (19.6% ± 1.7%) was also significantly lower Fulvestrant ic50 than that of TAT-7402 (70.1% ± 3.5%, P < 0.05) after STS treatment (Fig. 5C). In addition, immunofluorescence staining showed that TAT was colocalized with mitochondria in TAT-transfected cells (Supporting Fig. 1). To elucidate the molecular basis of apoptosis induced by TAT, we studied its role in the potentially proapoptotic mitochondrial permeability transition (MPT) event, which was measured by loss of mitochondrial ΔΨm using JC-1 dye. Red/orange fluorescence indicates intact mitochondria, selleck chemicals whereas green fluorescence indicates a collapse in mitochondrial ΔΨm. The result showed that the mitochondrial permeability and apoptotic index were similar before STS treatment (Fig. 5D). However, the mitochondrial permeability and apoptotic
index were significantly increased in TAT-7703 cells than that in Vec-7703 cells (67.5% ± 4.5% versus 17.8% ± 4.1%, P < 0.05) after STS treatment (Fig. 5D). Western blot analysis also showed that the release of Cyt-c into cytoplasm and cleavages of caspase-9 and PARP was dramatically increased in TAT-7703 cells after STS treatment compared with Vec-7703 (Fig. 5E). No obvious difference of caspase-8 was detected between the TAT-7703 and Vec-7703 cells. In addition, the mutant TAT could not induce apoptosis after STS treatment (Supporting Fig. 2E,F). To study the importance of TAT in regulating cell apoptosis, RNAi was used to knockdown endogenous TAT expression in PLC8024 cells. The result Prostatic acid phosphatase showed that siRNA against TAT could significantly reduce TAT expression in
PLC8024 cells (Fig. 6A) and MPT assay showed that strong red fluorescence was observed after STS treatment, indicating that these cells obtained an antiapoptotic ability (Fig. 6B). This result was further confirmed by immunoblotting analysis, showing that knockdown of TAT could inhibit the cleavages of caspase-9 and PARP (Fig. 6C). Deletion of 16q is one of the most frequent genetic alterations in HCC, implying the existence of a tumor suppressor gene on 16q.2, 3, 12, 13 Loss of 16q was also frequently detected in other solid tumors including breast,14 lung,15 and gastric cancers,16 suggesting that 16q may harbor one or more TSGs and its inactivation plays a key role in the pathogenesis of many malignancies including HCC. In this study we characterized one candidate TSG, TAT, at 16q22.1. The accurate frequency of loss of TAT allele in 50 HCC cases was characterized by qPCR.