Figure 1.
NSC745885 inhibits SAS cell growth in vitro.
(A) Morphological changes in SAS cells. Cells were treated with indicated dosages of NSC745885 for 24 hours. Dosages higher than 3 µΜ caused cell death in SAS cells. The survival of NSC745885 treated SAS cells was assessed by MTT assay at various times and dosages. (B) At 24 hours post treatment, the survival of SAS cells showed a significant difference at the dosages higher than 1 µM. (C) At 48 hours, treatment at 1 µM or higher than that dosages of NSC745885 exhibited a significant anti-cancer effect. (D) After 72 hours of treatment, the IC50 of NSC745885 was 0.85 µM in SAS cells. Survival proportion (%) indicated the relative value compared with vehicle control groups in SAS (n = 3; *, P<0.05; **, P<0.01; ***, P<0.001; ns., non-significant).
Figure 2.
NSC745885 induces early apoptosis in SAS cells.
SAS cells were treated with (A to E) indicated doses of NSC745885 for 24 hours. Harvested cells were stained with FITC-conjugated annexin V and analyzed by flow cytometry. (F) The percentages of annexin V positive cells under NSC745885 treatment were compared with vehicle controls (0 µM) (n = 3; **, P<0.01; ***, P<0.001; ns., non-significant).
Figure 3.
NSC745885 treatment exhibits a cancer cell specific cytotoxicity.
Oral cancer cells (OECM-1, SCC4, and SCC25) and normal lung fibroblast MRC-5 cell were treated with NSC745885 for 24 or 48 hours with indicated concentrations. The viability of these cell lines were assessed by MTT assay.The results indicated that low micromolar concentrations of NSC745885 induced (A) SAS cell death but did not affect the growth of (G and H) MRC-5 cells (n = 3; *, P<0.05; **, P<0.01; ***, P<0.001; ns., non-significant).
Figure 4.
NSC745885 treatment enhanced the expression and activation of caspase-3 in SAS cells.
(A) The relative RNA expression level of caspase-3 in SAS cell treated with indicated dosages of NSC745885 for 24 hours was evaluated using real-time RT-PCR. The expression of caspase-3 significantly increased with NSC745885 treatment in a dosage-dependent manner. (B) The caspase-3 and cleaved caspase-3 protein levels in the SAS cell treated with NSC745885 for 48 hours was quantified using western blot. (C) The results of the western blot in caspase-3 were evaluated in 3 independent experiments and normalized with β-actin (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns., non-significant). The protein level of caspase-3 was highest in 1.5 µΜ of NSC745885 treatment. (D) The results of the western blot in cleaved caspase-3 were evaluated in 3 independent experiments (*, P < 0.05; ns., non-significant).
Figure 5.
NSC745885 treatment decreased the RNA and protein levels of XIAP in SAS cells.
The relative RNA expression level of XIAP in SAS cells treated with indicated dosages of NSC745885 was evaluated at 24 hours using real-time RT-PCR. The expression of XIAP significantly decreased with NSC745885 treatment in a dose-dependent manner. (B) The expression of XIAP in SAS cells treated with NSC745885 was quantified using western blot at 48 hours of treatment. (C) The western blot results were evaluated in 3 independent experiments and normalized with β-actin (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The protein level of XIAP decreased in a dose-dependent manner.
Figure 6.
NSC745885 treatment inhibited SAS cell growth in vivo.
(A) The cancer cell implanted NOD/SCID mice were administered with NSC745885 (2 mg/kg/d). Engrafted tumors were isolated on Day 10 after drug administration. NSC745885 treatment significantly reduced tumor size when compared with the vehicle control. (B) The average tumor weight was compared between the NSC745885-treated and PBS-treated tumor-bearing mice on Day 10 (n = 9; *, P < 0.01). (C) The body weight of the experimental mice did not significantly decrease with NSC745885 treatment compared with the PBS treated controls.
Figure 7.
NSC745885 treatment induced apoptosis in xenografted tumors.
(A) The histological changes in tumor grafts were assessed using HE staining. Comparing the PBS control with the NSC745885-treated groups revealed a marked tumor necrosis in the NSC745885-treated group. (B) The expression level of caspase-3 in the isolated tumor grafts were evaluated using immunohistochemical staining. (C) The expression level of XIAP in isolated tumor grafts were also evaluated using immuno-histochemical staining. The expression scores for caspase-3 (D) and XIAP (E) were quantified, revealing an increase in the expression of caspase-3 with NSC745885 treatment (P < 0.05). By contrast, the expression of XIAP in tumor grafts decreased with this treatment (P < 0.05).
Figure 8.
The effects of NSC745885 treatment in the experimental mice.
(A) NOD/SCID mice were treated with PBS or indicated daily dosages of NSC745885, and the body weights of these mice were measured. The influence of NSC745885 treatment on the organs of the experimental mice was assessed using a histological assay and HE staining on Day 14. No obvious differences existed in the (B) spleen, (C) lung, (D) liver, or (E) heart of PBS-treated and NSC745885-treated mice.
Figure 9.
Comparisons of the safety and the anti-tumor efficiency between NSC745885 and doxorubicin.
The cancer cell implanted NOD/SCID mice were administered with NSC745885 (NSC, 2 mg/kg/d) or doxorubicin (Dox, 2 mg/kg/d) on day 3. (A) The survival rates between NSC745885 and doxorubicin treated mice were assessed. There are no experimental mice were dead under NSC745885 treatment by day 11. By contrast, 50% of doxorubicin treated mice were dead (n = 10). (B) The body weights of doxorubicin treated mice were significant decreased when compared to NSC745885-treated group from day 3 to day 11 after the beginning of drug treatment. (C) SAS cells were inoculated into NOD/SCID mcie, and the treatments of doxorubicin or NSC745885 were started at day 3 after cell inoculation. Engrafted tumors were isolated on day 11 after SAS cell inoculation (n = 9). (D) There are no significant differences between NSC745885-treated and doxorubicin-treated groups (n = 9).
Figure 10.
Pathohistological analysis in doxorubicin-treated and NSC745885-treated mouse.
The histological changes in (A) tumors, (B) hearts, (C) spleens, (D) liver and (E) lungs which isolated from doxorubicin-treated and NSC745885-treated mouse were assessed by using HE staining.