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. 2022 Jan 12;11(1):2. doi: 10.1038/s41389-022-00378-7

Fig. 5. A combination of αCT1 and TGX-221 overcomes TMZ resistance in vitro and in vivo.

Fig. 5

A The effect of the αCT1/TGX/TMZ combo in Cx43/p110β-high/MGMT–/TMZ-resistant SF295 and VTC-103 cells. Cells were treated with 50 μM TMZ, 20 μM TGX-221, and/or 30 μM αCT1 including single agents, double combinations, and the αCT1/TGX/TMZ combo. This scheme has been repeated in experiments presented hereafter. Cell viability was determined using the MTS viability assay. Percentage of cell viability was obtained by normalizing the MTS reading of treatment groups to that of the DMSO group. B EOB scores were calculated using the Bliss Independence model. The drug combination is synergistic if EOB is more than 0%, additive if EOB equals to 0%, or antagonistic if EOB is less than 0%. C The effect of the αCT1/TGX/TMZ in Cx43/p110β-low/MGMT–/TMZ-sensitive LN229 and TMZ-resistant VTC-001 cells. D EOB scores of drug combinations in LN229 and VTC-001 cells. E Caspase 3/7 activity in VTC-103 and VTC-001 cells. The activity of cleaved caspase 3/7 (active) was determined using a luminescence assay as described in Methods. Shown are luminescence readings. F EOB scores of drug combinations in VTC-103 and VTC-001 cells. G The effect of αCT1/TGX/TMZ combo on SF295 xenograft tumors. SF295 cells were subcutaneously injected into immuno-deficient mice. 8 days later, mice were treated with TMZ, TGX-221, or αCT1 through intraperitoneal or intratumoral injection every other day until day 18. Tumor volumes are shown. H EOB scores of drug combinations in SF295 tumors at different days. I Immunoblotting of PI3K signaling in SF-295 tumors. Proteins were directly extracted from homogenized tumor tissues and analyzed using western-blotting as described in Methods. ACTB is the loading control. Two repeated experiments are shown. J The effect of shRNA of Cx43 or PI3K catalytic subunits on the TMZ sensitivity of SF295 cells. Cells were transfected with NS shRNA or shRNA of Cx43, PIK3CA, PIK3CB, or PIK3CD followed by the treatment of 50 μM TMZ. Cell viability was determined using the MTS viability assay. Percentages of cell viability were obtained by normalizing the MTS readings of treatment groups to that of shNS group. K EOB scores of drug combinations in SF295 cells. One-way ANOVA with Dunnett test for correcting multiple comparisons or Student’s t test was used to determine statistical significance. *P < 0.05; ns not significant. Drug combinations with strong synergistic effect were marked in red. Three independent experiments were performed to yield standard deviations (error bars).