Article Figures & Data
Figures
- Figure 1.
Immunohistochemical analysis of Smac in ESCC and normal epithelial tissues on TMAs. A and B, downregulation of Smac in ESCC. Expression of Smac was analyzed using TMAs containing 68 cases of ESCC and adjacent normal epithelial tissues, with duplicate cores for each case. The TMAs were stained with Smac polyclonal antibodies. The majority of normal esophageal epithelia showed positive staining in the cytoplasm. Lower cytoplasmic Smac expression was detected in the corresponding esophageal cancer tissues. C–F, Smac expression in ESCC with differential chemotherapeutic response. The esophageal cancers with CR (C) and PR (D) responses to cisplatin chemotherapy expressed higher levels, whereas those with SD (E) and PD (F) responses expressed lower levels of Smac. Magnification, 200×; inserts, 400×.
- Figure 2.
Apoptosis induced by cisplatin in esophageal cancer cells. A, apoptosis induced by cisplatin was quantified following nuclear staining. EC0156 cells were treated with 10 μmol/L cisplatin for 24 hours. Nuclei were stained with DAPI. The appearance of apoptotic cells was significant 24 hours after exposure to cisplatin. B, dose- and time-dependent apoptosis induced by cisplatin. EC0156 cells were treated with the indicated concentration of cisplatin for 24 or 48 hours. C, apoptosis was quantified following annexin V–PI staining. EC0156 cells were treated with cisplatin (10 and 20 μmol/L) for 24 hours, stained with annexin V–PI, and analyzed with flow cytometry. Four subpopulations are indicated, including necrotic cells (R1), late apoptotic cells (R2), viable cells (R3), and early apoptotic cells (R4). D, EC0156 cells were treated with 10 μmol/L cisplatin for 24 hours, stained with Mito Tracker Red CMXRos, and analyzed by flow cytometry. E, EC0156 cells were treated with 10 μmol/L cisplatin for 6 hours. Long-term cell viability was assessed with the colony formation assay. F, cisplatin-induced caspase activation and Smac and cytochrome c release in ESCC cells. EC0156 and KYSE510 cells were treated with 10 μmol/L cisplatin for 12 or 24 hours. Top, mitochondrial and cytosolic fractions were isolated from the treated cells and analyzed for the indicated proteins with Western blotting. α-tublulin was used as a control for fraction and loading. Bottom, total cell lysates were probed for the cleavage of caspase-3 with Western blotting. Arrow indicates the 17 kDa caspase-3 cleavage fragment.
- Figure 3.
Inhibition of cisplatin-induced apoptosis in Smac-KD EC0156 cells. A, RNAi knockdown of Smac in EC0156 cells. EC0156 cells were transfected with a Smac shRNA construct. Smac knocked down puromycin-resistant clones were identified with Western blotting, β-actin was the loading control. B, quantification of Smac expression in (A) using the Quantity One program. C, inhibition of apoptosis in Smac-KD cells. Parental cells, Smac-KD cells (G10 and E1 clones), and a control puromycin-resistant EC0156 clone with Smac expression (D7) were treated with 10 μmol/L cisplatin for 24 hours. Apoptosis was quantified by fluorescence microscopy after nuclear staining with DAPI. D, parental and Smac-DK-G10 EC0156 cells were treated with the indicated concentrations of cisplatin for 24 hours to induce apoptosis. Apoptosis was analyzed by nuclear staining as in (C). Results are the averages of 3 independent measurements, with a minimum of 200 cells counted for each measurement. E, parental and Smac-DK-G10 EC0156 cells were treated with 10 μmol/L cisplatin for 24 hours stained with annexin V–PI and analyzed by flow cytometry. Four subpopulations are indicated, including necrotic cells (R1), late apoptotic cells (R2), viable cells (R3), and early apoptotic cells (R4). F, long-term cell viability in parental and Smac-KD cells. After parental and Smac-KD-G10 EC0156 cells were treated with 10 μmol/L cisplatin for 6 hours, long-term cell viability was assessed by the colony formation assay. Representative pictures are shown.
- Figure 4.
Smac mediates cisplatin-induced apoptosis through the mitochondrial pathway; cisplatin sensitivity of ESCC tumors in vivo. A, cisplatin-induced caspase activation and cytochrome c release in Smac-KD cells. Parental and Smac-KD-G10 EC0156 cells were treated with 10 μmol/L cisplatin for 12 or 24 hours. Smac and cytochrome c release were analyzed by Western blotting of lysates of the treated cells. α-tubulin was used as a loading and fractionation control. Mitochondrial fractions were isolated from the treated cells and analyzed for cytochrome c and COX IV with Western blotting. Total cell lysates were assayed for the cleavage of caspase-9 and caspase-3 with Western blotting. The cleavage fragments were indicated by arrows. β-actin was the loading control. B, mitochondrial membrane potential in Smac-KD cells. Parental and Smac-KD-G10 cells were treated with 10 μmol/L cisplatin for 24 hours, harvested, and stained with Mito Tracker Red CMXRos, and analyzed by flow cytometry. C, suppression of the antitumor effects of cisplatin in Smac knockdown in mice. Parental and Smac-KD-G10 EC0156 cells suspended in 0.2 mL PBS were injected into nude mice (female, 6 -weeks old) to establish xenograft tumors. After the tumor volume reached 100 to 200 mm3, mice were treated with 3 mg/kg cisplatin in 0.1 mL PBS, or with vehicle alone, by i.p. injection once every other day for a week. Tumor volume was measured every 2 to 3 days after treatment. Cisplatin treatment significantly decreased the growth of the parental tumors (P < 0.001), but had no significant effects (P > 0.05) on Smac-KD tumors. Values are means ± SD (n = 5 in each group). **, P < 0.001; #, P > 0.05.
- Figure 5.
The Smac mimetic LBW242 restores cisplatin-induced apoptosis in Smac-KD cells. A and B, apoptosis induced by cisplatin combined with LBW242. Parental EC0156 and Smac-KD-G10 cells were treated with 10 μmol/L cisplatin with or without 10 μmol/L LBW242 for 24 hours. Apoptosis was quantified by fluorescence microscopy after nuclear staining with DAPI. C, following treatment as in (B), cells were stained with annexin V–PI, and apoptosis was quantified by flow cytometry. Four subpopulations are indicated, including necrotic cells (R1), late apoptotic cells (R2), viable cells (R3), and early apoptotic cells (R4). D, caspase-9 activation in Smac-KD cells treated with cisplatin and LBW242. After treatment as in (B), cells were lysed and assayed for caspase-9 with Western blotting. β-actin was the loading control. E, cytochrome c release in Smac-KD cells treated with cisplatin and LBW242. Following treatment as in (B), cytosolic fractions were isolated from the treated cells and analyzed for cytochrome c with Western blotting. α-tubulin was the loading and fractionation control.
Tables
- Table 1.
Correlation of chemotherapeutic response and clinicopathologic features with Smac expression
Variable Total N = 31 Expression of Smac P OR (95% CI) Positive (%) Negative (%) Gender Male 26 11 (42.3) 15 (57.7) Female 5 1 (20) 4 (80) 0.364 2.9 (0.28–30.0) Age ≤60 18 9 (50) 9 (50) 60 13 3 (23.1) 10 (76.9) 0.137 3.3 (0.68–16.3) Histologic grade Well+Mod 18 5 (27.8) 13 (72.2) Poor 13 7 (53.8) 6 (46.2) 0.062 0.23 (0.049–1.078) Chemosensitivity CR + PR 10 7 (70) 3 (30) SD + PD 21 5 (23.8) 16 (76.2) 0.019 7.47 (1.385–40.245) NOTE: Values are expressed as n (%). Statistically significant at P < 0.05.
Supplementary Data
Files in this Data Supplement:
- Supplementary Tables 1-3 - PDF file - 155K
Volume 17, Issue 16
