Supplementary Figure S1 A synergistic action of the ATO and VC combination on suppressing the growth of human KRAS mutant cancer cells. The combination of VC and ATO synergistically inhibited the growth of HCT116 cancer cells according to the CI plots for the dual-drug combination. HCT116 cells were treated with combinations of VC (0, 0.5, 1, 1.5 or 2 mM) and ATO (0, 2.5, 5, 7.5 or 10 μM) for 48 h. CI values were plotted as a function of fractional inhibition as determined by MTT analysis and computer simulation (CompuSyn) for the Fa (fraction affected) range of 0.10 to 0.95. CIs of <1, 1, and > 1 indicate synergism, additive effect and antagonism, respectively. At least three independent experiments with three replicates were performed. Supplementary Figure S2. Cell line with the deleted KRAS mutant allele is not sensitive to the ATO and VC combination. A, The images of HKH2 human cancer cells after the treatment with PBS (control), 1.5 mM VC, 5 μM ATO or 1.5 mM VC and 5 μM ATO for 48 h. B, The apoptotic assay histograms of the cells described in (A) using Annexin-V and PI staining and flow cytometric analysis. C, Graphical presentation of the apoptotic analysis shown in (B). Supplementary Figure S3. Cell line carrying the KRAS mutant allele is sensitive to the ATO and VC combination. A, The images of HCT116 human cancer cells after the treatment with PBS (control), 1.5 mM VC, 5 μM ATO or 1.5 mM VC and 5 μM ATO for 48 h. B, The apoptotic assay histograms of the cells described in (A) using Annexin-V and PI staining and flow cytometric analysis. C, Graphical presentation of the apoptotic analysis shown in (B). Supplementary Figure S4. Mouse cell line expressing the Kras mutant allele is highly sensitive to the ATO and VC combination. AK192 cells (a mouse PDAC cell line) were incubated in the medium containing doxycycline to maintain expression of the oncogenic KrasG12D transgene. A, Images of AK192 cancer cells after the treatment with PBS (control), 1 mM VC, 5 μM ATO or 1 mM VC and 5 μM ATO for 48 h. B, Apoptotic assay histograms of the cells described in (A) using Annexin-V and PI staining and flow cytometric analysis. C, Graphical presentation of the apoptotic analysis shown in (B). Supplementary Figure S5. Human KRAS mutant cancer cells are sensitive to the ATO and VC combination. A, Images of HPAC human cancer cells after the treatment with PBS (control), 1 mM VC, 5 μM ATO or 1 mM VC and 5 μM ATO for 48 h. B, Apoptotic assay histograms of the cells described in (A) using Annexin-V and PI staining and flow cytometric analysis. C, Graphical presentation of the apoptotic analysis shown in (B). Supplementary Figure S6. HT29 (KRAS wild type) colorectal cancer cells are not sensitive to the ATO and VC combination. A, Images of HT29 human cancer cells after the treatment with PBS (control), 1 mM VC, 5 μM ATO or 1 mM VC and 5 μM ATO for 48 h. B, Apoptotic assay histograms of the cells described in (A) using Annexin-V and PI staining and flow cytometric analysis. C, Graphical presentation of the apoptotic analysis shown in (B). Supplementary Figure S7. RKO (KRAS wild type) colorectal cancer cells are not sensitive to the ATO and VC combination. A, The images of RKO human cancer cells after the treatment with PBS (control), 1 mM VC, 5 μM ATO or 1 mM VC and 5 μM ATO for 48 h. B, Apoptotic assay histograms of the cells described in (A) using Annexin-V and PI staining and flow cytometric analysis. C, Graphical presentation of the apoptotic analysis shown in (B). Supplementary Figure S8. Primary (MRC5) lung fibroblasts are not sensitive to the ATO and VC combination. A, The images of MRC5 fibroblasts after the treatment with PBS (control), 1 mM VC, 5 μM ATO or 1 mM VC and 5 μM ATO for 48 h. B, Apoptotic assay histograms of the cells described in (A) using Annexin-V and PI staining and flow cytometric analysis. C, Graphical presentation of the apoptotic analysis shown in (B). Supplementary Figure S9. NAC blocks the cytotoxic impact of the ATO/VC or ATO/d-VC combination. (A) The images of HCT116 cells after the treatments as in the Fig. 1 or the drug combinations ATO/VC and ATO/d-VC with 5 mM NAC for 48 h. Flow cytometry data for ROS staining by preincubating cells with 5 μM DCFDA for 30 min treated with VC (B) or treated with d-VC (C) shown in A. Supplementary Figure S10. Suppression of the KRAS mutant tumor growth by the ATO and VC combination. Images of xenograft tumors described in Figure 2 after nine drug injections. Supplementary Figure S11. Combinatory treatment of ATO and d-VC induces apoptosis in xenograft tumors. After the drug injections, the HCT116 xenograft tumors were excised and analyzed by immunohistochemical analysis by detecting the Caspase 3 cleavage. Supplementary Figure S12. Graph representing the Caspase 3 cleavage analysis in xenograft tumors after combinatory treatment of ATO and VC. The graph representing the fold change in positivity of Caspase 3 cleavage after the drug treatments compared to the effect of the VC treatment alone in xenograft tumors shown in the Figure S10. Cleaved caspase 3-positive cells were counted at x400 magnification in five or six randomly selected areas in every tumor sample. Supplementary Figure S13. The oxidative drugs and their combinations have no adverse effect on mice. After the drug injections, the mice were sacrificed and their excised livers were analyzed by being stained with hematoxylin and eosin (H&E) for histological analysis. Supplementary Figure S14. The graph data on body weight of mice injected with the oxidative drug combinations. The mice weight has been measured every 3 days after the HCT116 cancer cells implantation into nude mice. The cells were injected on day 0 and weigh was measured after every 3 days. After 10 days the drugs were injected as described in the Fig. 3. with the last injection on the day 27. |
