Figure 1.
Cell proliferation of A2780 (A–B) and A2780/CP70 (C–D) cells on treatment with Dox and WFA, both alone or combination of WFA/DOX using MTT assays.
A2780 and A2780/CP cells were plated into 96 well plates. After 24 h of plating, cells were treated with various concentrations of Dox and WFA, both alone or in combination. After 48 h of treatment, cell viability was assayed using MTT assays. Values shown are mean ±SD of four independent experiments. *P<0.05 compared to control, #p<0.05 compared to Dox or WFA alone. (E) Isobologram analysis of A2780 cells (n = 4) and (F) A2780/CP70 (n = 4) using 7 doses of Dox and WFA maintained at a constant ratio and cell death was assayed by MTT assays. Results were analyzed with CalcuSyn software.
Table 1.
IC50 values of doxorubicin, Withaferin A, on treatment of A2780, A2780/CP70 and CAOV3 cells for 48 h with DOX and WFA, both alone or combination of DOX and WFA.
Figure 2.
ROS generation in A2780 cells.
A2780 cells were plated into glass bottom dishes. After 24 h of plating, cells were treated with Dox and WFA, both alone or combination of WFA/DOX as described in Figure 1. After 24 h of treatment, medium was replaced with fresh medium containing H2DCFDA and incubated for 30 min. Cells were rinsed with PBS and examined under confocal microscope. (A) ROS positive cells (green color) were counted based on 3 low power fields. Mean ±SD. P values were determined by ANOVA analysis followed by Student-Newman-Keuls test for multiple comparisons. *P<0.05 from control, $P<0.05 compared to Dox, &P<0.05 compared to WFA. (B) Confocal microscopy analysis of cells indicating generation of ROS (green color).
Figure 3.
Effect of non-enzymatic ROS antioxidant NAC on A2780 cell proliferation after 48 h of treatment.
A2780 cells were co-treated with NAC and Dox, WFA or combination of WFA/WFA for 48 h. Cell proliferation was determined using MTT assays. Values shown are mean ±SD of three independent experiments. P<0.05 compared to control, #P<0.05 compared to no NAC and NAC.
Figure 4.
Effect of enzymatic antioxidant SOD (100 units/ml) on A2780 cell proliferation after 48 h of treatment. A2780 cells were co-treated with SOD and Dox, WFA or combination of WFA/Dox for 48 h.
Cell proliferation was determined using MTT assays. Values shown are mean ±SD of three independent experiments. *P<0.05 compared to control, #P<0.05 compared to no SOD and SOD.
Figure 5.
DNA damage (TUNEL) assay of A2780 cells after 24 h of treatment.
A2780 cells were treated with Dox, WFA both alone or combination of WFA/Dox. After 24 h of treatment, DNA damage was analyzed using TUNNEL assays. Images were obtained using confocal microscopy at 20X magnification.
Figure 6.
Analysis of autophagy using transmission electron microscope (TEM).
A2780 cells were treated with Dox and WFA, both alone or combination of WFA/Dox. After 24 h of treatment, cells were rinsed with PBS, fixed and processed for TEM analysis. Electron microscopic images at 5,600X magnification are shown.
Figure 7.
Western blot analysis of autophagy pathway of A2780 cells treated for 24 hr. A2780 cells treated with Dox and WFA, both alone or combination of WFA/Dox.
After 24 h of treatment, cells were washed with PBS and lysed. Western blot analysis was performed for LC3B, Caspase 3 and GAPDH proteins.
Figure 8.
In vitro analysis of tumor growth using 3D tumor model.
(A) A2780 Cells were combined with Hubiogel® in a 1∶4 ratio and grown from 10 µL beads. Tumors were treated with Dox and WFA, both alone or combination of WFA/Dox. Tumors were treated twice/week by replacing the medium with medium containing fresh agent. Tumor growth was measured using MTT assays after day 3 or 7 of treatment. (B–C) Tumors after treatment were incubated with calcein AM for 30 min, and images were taken using fluorescence microscope after 3 days of treatment (B) or 7 days of treatment (C).
Figure 9.
S.C. tumors were generated in nude mice and treated with PBS, Vehicle, Dox 1 mg/kg, Dox 9 mg/kg, WFA 2 mg/kg or Dox 1 mg/kg plus WFA 2 mg/kg.
(A) Tumors growth was measured from day 20–32 (post-cell injection) and treated every other day *P<0.05. (B) Tumor weight at day 32 collected immediately after sacrificing the animals.
Figure 10.
Immunohistochemistry compilation of tumor tissues developed with DAB (brown) and counterstained with hematoxylin to stain nuclei (blue).
Negative control samples were tissues without primary antibody. TUNEL assay was performed using ApopTag Plus Peroxidase Apoptosis Detection Kit.
Figure 11.
Schematic summary of mechanisms of cell death induced by Dox/WFA combination treatment.