Table 1.
PI103 sensitizes HT1080 cells to DOX-induced apoptosis.
Figure 1.
PI103 sensitizes RD cells to DOX-induced apoptosis.
A) Levels of phospho-Akt, Akt, phospho-S6 ribosomal protein and S6 ribosomal protein were measured by Western blot analyses in RD, TP5014 and HT1080 cells after treatment with 1 µM or 3 µM PI103 as indicated. B) Upper panel: Proliferation was estimated by BrdU incorporation after treatment with 0.5 µM DOX and/or 3 µM PI103. Middle panel: Apoptosis was analyzed by FACS after treatment with 1 µM DOX and/or 3 µM PI103 or solvent. Data represent mean+SEM of at least three independent experiments performed in triplicates. Comparisons were made with ANOVA/Tukey’s testing. *P<0.05 compared to cells treated with solvent; #P<0.05 compared to cells treated with either drug alone. Lower panel: Protein levels of phospho-Akt, Akt and caspase 3 in cells treated with 1 µM DOX and/or 3 µM PI103 or respective solvents.
Figure 2.
PI103-induced DOX accumulation does not account for cooperative proapoptotic effects in RD cells.
A) Intracellular DOX autofluorescence after treatment with 1 µM DOX (red line), 3 µM PI103 (black line), 2 µM DOX (blue line), 1 µM DOX plus 3 µM PI103 (green line) or solvent (orange line) at 530 nm. The data are expressed as the percentage of the maximum (max) number of positive cells. B) Expression levels of MDR1 (left panel) and MRP1 (middle panel) after treatment with 3 µM PI103 in relation to solvent treated cells (set = 1). Data represent mean+SEM of at least five independent experiments performed in triplicates. *P<0.05 by Students t-test. Protein level of MRP1 after treatment with 3 µM PI103 or the solvent (right panel). C) Annexin V positive RD cells were analysed after treatment with 1 µM DOX, 3 µM PI103, 1 µM DOX plus 3 µM PI103, 2 µM DOX or the solvent. Data represent mean+SEM. Comparisons were made with Students t-test. D) Cells were treated as in C and caspase 3 was assessed by Western blot analyses. E) Annexin V positive RD cells (left panel) and intracellular DOX autofluorescence (right panel) after a 12-hours pretreatment (preinc.) with 3 µM PI103 and subsequent addition of 1 µM DOX for additional 24 h. Annexin V data represent mean+SEM. Comparisons were made with Students t-test.
Figure 3.
PI103 and DOX cooperate in Bax activation and cytochrome c release.
RD cells were treated with 1 µM DOX and/or 3 µM PI103 or the solvent. A) Activation of Bax was analysed by immunoprecipitation. B) Cytochrome c release was assessed by FACS at 530 nm (red line: 1 µM DOX; green line: 3 µM PI103; orange line: 1 µM DOX plus 3 µM PI103; black line: solvent). The data are expressed as the percentage of the maximum (max) number of positive cells and represent one of three independent experiments, measured in duplicates.
Figure 4.
Inhibition of mTOR does not sensitize RD cells to DOX-induced apoptosis.
A) and B) RD cells were treated for 24h with 1 µM DOX and/or 1 µM everolimus (ever; respective left panels) or with 1 µM DOX and/or 20 µM LY294002 (respective right panels) or solvent. A) Annexin V positive RD cells were analysed by FACS. Data represent mean+SEM of two independent experiments performed in duplicates. n.s. not significant by Students t-test. B) Western blot analyses.
Figure 5.
GDC-0941 inhibits tumor growth and increases DOX-mediated effects on caspase 3 activity.
A) Inhibition of RD tumor growth (in %) in nude mice treated with 1.2 mg/kg DOX (i.p. every third day for 22 days; n = 19), 75 mg/kg GDC-0941 (orally every day for 22 days; n = 9), the combination of both drugs (n = 12) at the time points indicated. Vehicle-treated animals served a controls (n = 18). B) Caspase 3 positive cells (in %) in tumors of nude mice isolated after 22 days of treatment with DOX and/or GDC-0941 or solvent. Data represent mean+SEM. *P<0.05 by ANOVA/Tukey’s testing.