Figure 1.
SM-164 induces rapid cIAP-1 degradation and displays modest single-agent effect in HCC cells.
(a–d). BEL-7402, SMMC-7721, HepG2 and Hep3B cell lines were treated with SM-164 at 0.1 µM for 1, 6 and 24 h. Whole cell lysates were examined for the expressions of cIAP-1 and XIAP by western blotting analysis and specific antibodies. β-Actin was used as a loading control. (e-h). BEL-7402, SMMC-7721, HepG2 and Hep3B cell lines were treated with SM-164 for 4 d, cell viability inhibition was determined using an MTT assay.
Figure 2.
SM-164 enhances APO2L/TRAIL-mediated anticancer activity in HCC cells.
(a–d). BEL-7402, SMMC-7721, HepG2 and Hep3B cell lines were treated with APO2L/TRAIL (A/TR) alone, or in combination with SM-164 at 0.1 µM (SM+ A/TR) for 4 d, cell viability inhibition was determined using an MTT assay. (e-f). SMMC-7721 and HepG2 cells were seeded into six-well plates at 600 cells per well in triplicates, and treated with SM-164 alone, A/TR alone or their combination (SM+ A/TR) for 2 weeks, followed by 0.05% methylene blue staining and colony counting. (left panels), representative results show photographs of stained 6-well plates for SMMC-7721 and HepG2, respectively. (right panels), data show means ± S.D. Normal human liver cell line L02 was treated as indicated for 4 d, cell viability inhibition was determined using an MTT assay. *, p<0.05, * *, p<0.01.
Figure 3.
SM-164 promotes APO2L/TRAIL-induced apoptosis and inhibits AKT activation in human HCC cell lines.
(a–b). SMMC-7721 cell line was treated with SM-164 (SM) at 0.1 µM alone, APO2L/TRAIL (A/TR) at 100 ng/ml alone or both for 16 h, cells were stained with Annexin V/PI staining and examined by flow cytometry assay. (a). Representative dot plot of apoptosis assay. (b). data of Annexin V-positive cells show means ± S.D. of from three experiments. **, p<0.01. (c). SMMC-7721 cell line was treated as indicated, whole cell lysates were analyzed with western blotting and specific antibodies as indicated. β-Actin was used as a loading control. (d). SMMC-7721 cell line was pretreated with pan-caspases inhibitor, zVAD (50 µM) for 1 h and followed by treatment with SM at 0.1 µM alone, A/TR at 100 ng/ml alone or both for 24 h, cell death induction was examined with trypan blue exclusion assay. (e). BEL-7402 cell line treated with SM at 0.1 µM alone, A/TR at 10 ng/ml or both for 16 h was examined with Annexin V/PI staining and flow cytometry assay. (f). HepG2 cell line treated with SM at 0.1 µM alone, A/TR at 1000 ng/ml or both for 16 h was examined with Annexin V/PI staining and flow cytometry assay. (g-h). BEL-7402 and HepG2 cell lines were treated as indicated for 12 h. Whole cell lysates were analyzed with western blotting and specific antibodies. (i). Cell lines were treated as indicated for 12 h. Whole cell lysates were analyzed with western blotting and specific antibodies. β-Actin was used as a loading control.
Figure 4.
SM-164 potentiates cell viability inhibition by APO2L/TRAIL in primary HCC cells.
(a). Primary HCC cells freshly isolated from 12 patients’ samples were treated with SM-164 at 0.1 µM alone, APO2L/TRAIL at 100 ng/ml alone or both for 24 h, cell viability was determined with trypan blue exclusion assays. *, p<0.05, * *, p<0.01. (b). Primary HCC cells from Case No.4 and No.12 patients’ samples were treated with SM-164 at 0.1 µM alone, APO2L/TRAIL at 100 ng/ml alone or both for 24 h. Whole cell lysates were probed with anti-cleaved PARP (cl-PARP). β-Actin was used as a loading control.
Table 1.
Clinical data of HCC patients.
Figure 5.
SM-164 potentiates the cytotoxic effect of Doxorubicin in HCC cells.
(a). Hep3B cell line was treated with Doxorubicin (Dox) alone, or in combination with SM-164 at 0.1 µM for 48 h, cell death induction was determined in a trypan blue excluson assay; (b). Hep3B cell line was treated as indicated, whole cell lysates were analyzed with western blotting and specific antibodies. β-Actin was used as a loading control. (c). Hep3B cell line was treated with Dox at 1 µM alone, or SM-164 at 0.1 µM alone for 48 h, representative micrographs were shown. (d-e). SMMC-7721 and BEL-7402 cell lines were treated as indicated, cell death induction was determined in trypan blue exclusion assays;
Figure 6.
SM-164 inhibits AKT activation in HCC cells.
Cells were treated with SM-164 at 0.1 µM alone, Dox alone (1 µM for Hep3B, 10 µM for SMMC-7721 and BEL-7402) for 24 h. Whole cell lysates were probed with anti-phospho-AKT and then re-probed with anti-AKT (unphosphorylated) which served as a loading control.