Cerbera odollam fruit extracts enhance anti-cancer activity of sorafenib in HCT116 and HepG2 cells

Figures

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  Fig. 1. Appearance of C. odollam. (A) Tree, (B) flower, (C) leaves, (D) green whole fruit, (E) cross section of fruit and (F) chopped fruit. (G) Phytochemical content of extracts from fruits of C. odollam: total cardiac glycoside (mg DXE/g extract), total triterpenoid content (mg UAE/g extract), total saponin content (mg OLE/g extract), total phenolic content (mg GAE/g extract), and total flavonoid content (mg RTE/g extract). COEtOH, CODCM, COEtOAc and COW were ethanolic crude extract, dichloromethane fraction, ethyl acetate fraction, and water fraction of fruits of C. odollam, respectively. The data, presented as mean ± SD from three independent experiments, were analyzed to investigate significant differences using One-way ANOVA and t-test, unequal variance with Bonferroni correction. aP < 0.05 vs COEtOH group, bP < 0.05 vs CODCM group, cP < 0.05 vs COEtOAc group, and dP < 0.05 vs COW group.

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  Fig. 2. Cytotoxicity of extracts from C. odollam fruits against HCT116 and HepG2 cells was assessed after 24 h of incubation using MTT technique to determine cell viability. (A–D) Demonstrating percentage of cell viability for HCT116 cells treated with COEtOH, CODCM, COEtOAc, and COW, respectively. (E–H) Showing percentage of cell viability for HepG2 cells treated with COEtOH, CODCM, COEtOAc, and COW, respectively. The vehicle control was represented by treatment with 0.8% DMSO. The data, presented as mean ± SD from at least three different experiments, were analyzed to investigate significant differences using One-way ANOVA with Tukey’s HSD test. *P < 0.05 vs vehicle control.

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  Fig. 3. Cytotoxicity of combination of COEtOAc or CODCM with sorafenib against HCT116 and HepG2 cells was assessed using MTT technique to measure cell viability. (A–D) Demonstrating percentage of cell viability in HCT116 cells treated with COEtOAc combined with sorafenib after 24 and 48 h of incubation, respectively. (E–H) Showing percentage of cell viability in HepG2 cells treated with CODCM combined with sorafenib after 24 and 48 h of incubation, respectively. The vehicle control was represented by treatment with 0.8% DMSO. The data, presented as mean ± SD from at least three different experiments, were analyzed to investigate significant differences using One-way ANOVA with Tukey’s HSD test. *P < 0.05 vs vehicle control, aP < 0.05 vs a single COEtOAc or CODCM treatment at their respective doses, bP < 0.05 vs a single sorafenib treatment.

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  Fig. 4. Flow cytometric analysis was conducted to assess apoptotic effect, MMP, cell cycle distribution, and ROS levels following treatment with COEtOAc at 20 μg/mL combined with sorafenib at 6 μmol/L for 24 h in HCT116 cells. The data demonstrate: (A–B) apoptotic cells after double labeling with annexin V and PI, (C–D) MMP levels labeled with JC-1, (E–F) cell cycle distribution labeled with PI, and (G–H) ROS levels labeled with H2DCFDA. The vehicle control was represented by treatment with 0.8% DMSO. The data, presented as mean ± SD from at least three different experiments, were analyzed to investigate significant differences using One-way ANOVA with Tukey’s HSD test. ΔP < 0.05 vs vehicle control, *P < 0.05 vs vehicle control (red), #P < 0.05 vs vehicle control (green), aP < 0.05 vs a single COEtOAc treatment, bP < 0.05 vs a single sorafenib treatment.

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  Fig. 5. Flow cytometric analysis was conducted to assess apoptotic effect, MMP, cell cycle distribution, and ROS levels following treatment with CODCM at 30 μg/mL combined with sorafenib at 2 μmol/L for 24 h in HepG2 cells. The data demonstrate: (A–B) apoptotic cells after double labeling with annexin V and PI, (C–D) MMP levels labeled with JC-1, (E–F) cell cycle distribution labeled with PI, and (G–H) ROS levels labeled with H2DCFDA. The vehicle control was represented by treatment with 0.8% DMSO. The data, presented as mean ± SD from at least three different experiments, were analyzed to investigate significant differences using One-way ANOVA with Tukey’s HSD test. ΔP < 0.05 vs vehicle control, *P < 0.05 vs vehicle control (red), #P < 0.05 vs vehicle control (green), aP < 0.05 vs a single CODCM treatment, bP < 0.05 vs a single sorafenib treatment.

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  Fig. 6. Fluorescence images of MMP and ROS formation following treatment with COEtOAc at 20 μg/mL combined with sorafenib at 6 μmol/L for 24 h in HCT116 cells. The data demonstrates: (A) MMP, labeled with JC-1, and (B) ROS, labeled with H2DCFDA. Nuclear DNA was labeled with Hoechst 33342. The treatment with 0.8% DMSO served as vehicle control. Scale bar = 20 μm under × 20 magnification, visualized by fluorescence microscopy.

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  Fig. 7. Fluorescence images of MMP and ROS formation following treatment with CODCM at 30 μg/mL combined with sorafenib at 2 μmol/L for 24 h in HepG2 cells. The data demonstrates: (A) MMP, labeled with JC-1, and (B) ROS, labeled with H2DCFDA. Nuclear DNA was labelled Hoechst 33342. The treatment with 0.8% DMSO served as vehicle control. Scale bar = 20 μm under × 20 magnification, visualized by fluorescence microscopy.

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  Fig. 8. Levels of protein SOD2 and catalase were assessed following treatment with COEtOAc or CODCM combined with sorafenib in HCT116 and HepG2 cells for 24 h. For HCT116 cells: (A–C) show representative western blot images and histograms depicting SOD2 and catalase levels following treatment with COEtOAc at 20 μg/mL combined with sorafenib at 6 μmol/L. For HepG2 cells: (D–F) show representative western blot images and histograms depicting SOD2 and catalase levels following treatment with CODCM at 30 μg/mL combined with sorafenib at 2 μmol/L. (G–H) Cell viability of HFF-1 cells following COEtOAc or CODCM treatment in 24 h. The vehicle control was represented by treatment with 0.8% DMSO. The data, presented as mean ± SD from at least three different experiments, were analyzed to investigate significant differences using One-way ANOVA with Tukey’s HSD test. *P < 0.05 vs vehicle control, aP < 0.05 vs a single COEtOAc or CODCM treatment at their respective doses, bP < 0.05 vs a single sorafenib treatment.