Figure 1.
IL-6/Stat3 signaling in NPC cells.
(A) Whole-cell lysates were prepared from the cells as indicated. -Actin was used as a control for protein loading and integrity. The relative phosphorylated Stat3 (p-Stat3) and total Stat3 (T-Stat3) expression intensity from 5 samples is shown. (B) Treatment with IL-6 (40 ng/mL) activated Stat3, [P-Stat3(Y705)] and such activation was inhibited by the addition of Stattic (20 µM ) in NPC cells. (C). IL-6 (40 ng/mL) promoted CNE1 cell growth, and the growth was inhibited by the addition of Stattic (4 µM ). Data are means ± s.d. for three independent experiments, *P<0.05, **P<0.01. DMSO were used as control in “−” groups.
Figure 2.
Stattic inhibits Stat3 activation in a dose- and time-dependent manner in NPC cells.
NPC cell lines were treated with and without Stattic at the specified concentrations for 2 h (A) or exposed to 20 µM Stattic for different time points (B). Expression of T-Stat3, p-Stat3, cyclin D1, and β-actin were examined by western blot. DMSO were used as control in “0” groups.
Figure 3.
Stattic inhibits cell viability and induces sub-G1 arrest in NPC cells.
(A) Cell viability assay. CNE1, CNE2, HONE1 and C666-1 cells were treated with various doses of Stattic for 48 h. Cell viability was measured by the MTT assay. (B) Stattic inhibits the cell viability of NPC cells in a time-dependent manner. CNE2 cells were treated with 8 µM Stattic for the indicated times, and cell viability was measured by the MTT assay. (C), (Left) Representative results of colony formation assays with NPC cells treated with different doses of Stattic. (Right) Quantification of the relative number of colonies is shown. (D) Measurement of apoptosis by PI staining. (Left) NPC cells were treated with the indicated doses of Stattic for 48 h, followed by PI staining as described in Materials and Methods. (Right) Quantification of PI staining. Data are means ± s.d. for three independent experiments, *P<0.05, **P<0.01. DMSO were used as control in “0” groups.
Figure 4.
Stattic induces apoptosis in NPC cells.
(A) Apoptosis was measured by Hoechst 33342 staining. (Top) NPC cells were treated with 10 µM Stattic for 48 h, nuclei were stained with Hoechst 33342, and imaging analysis was performed as described in the Materials and Methods. The white arrows indicate apoptotic cells. Original magnification, ×200. (Bottom) Quantification of the cell staining. (B) Effect of Stattic on caspase-3 activity. The cells were treated with the indicated concentrations of Stattic for 48 h. The activities were determined as described in Materials and Methods. (C) NPC cells were exposed to the indicated concentrations of Stattic for 48 h; apoptotic cells were measured by western blot analysis of cleaved PARP and cleaved caspase-3. Protein levels were quantified using ImageJ software. Data are means ± s.d. for three independent experiments, *P<0.05, **P<0.01. DMSO were used as control in “0” groups.
Figure 5.
Ectopic Stat3 attenuates Stattic-induced growth inhibition and apoptosis in NPC cells.
(A) NPC cells were transfected with pcDNA, Flag-Stat3 plasmids, control siRNA (Cont-si), or Stat3 siRNA (Stat3-si) and checked by western blot for the flag and Stat3 expression. (B) NPC cells described in (A) were treated with Stattic at 0–16 µM and assayed for cell viability by the MTT assay. Cell viability was calculated by the percentage of surviving cells relative to non-treated controls. (C) CNE2 cells were transfected with pcDNA, Flag-Stat3, control siRNA (Cont-si), or Stat3 siRNA (Stat3-si) and then treated with or without Stattic at 0.1 or 0.3 µM for 48 h, followed by examination for colony formation. (D) CNE1 cells (left) and CNE2 cells (right) were transfected with pcDNA or Stat3 plasmids and then exposed to the indicated doses of Stattic for 48 h, and then apoptotic cells were measured by western blot analysis of cleaved caspase-3. (E) CNE1 cells (left) and CNE2 cells (right) were transfected with control siRNA or Stat3 siRNA and then exposed to the indicated doses of Stattic for 48 h, and then apoptotic cells were measured by western blot analysis of cleaved caspase-3. Protein levels were quantified using ImageJ software. DMSO were used as control in “0” groups.
Figure 6.
Stattic enhances the antitumor effects of cisplatin in NPC.
NPC cells were treated with cisplatin (CP) alone, Stattic alone or both together, 48 h later, cells viability were measured by the MTT assay (A), number of colonies formed were counted in CNE2 cells (B), subG1 cells were analyzed by PI staining (C) and cleaved PARP (C-PARP) and cleaved caspase-3 (C-Caspase 3) was detected by western blot (D). Data are means ± s.d. for three independent experiments. DMSO were used as control in “Cont” groups, PBS were used as control in “0” groups.
Figure 7.
Stattic sensitize NPC to radiotherapy.
NPC cells were treated with 10 Gy of IR alone or together with different doses of Stattic for 48 h, followed by detection of cells viability (A), colony formation (CNE2) (B), PI staining (C) and cleaved caspase-3 (C-Caspase 3) (D). DMSO were used as control in “−” groups.