Fig. 1: Simultaneous inhibition of ANT2 and OXPHOS reduces tumorigenesis in PDAC.
A PaTu8902 cells were treated with carboxyatractiloside (CATR; 5μM) for 2 and 4 days. Proliferation was evaluated by Vi-CELL Series Cell Viability Analyzer. PaTu8902 cells were treated with CATR (5μM) for 96 h. B Reactive oxygen species were detected by 2´7´-dichlorofluorescein (DCF; 10 μM, 15 min) and analyzed by FACS. Mitochondrial morphology was documented by (C) confocal microscope following Tomm20 immunofluorescent staining, with DAPI denoting cell nuclei, and (D) by electron microscopy. The scale bar indicates 10 μm (C) and 0.5 μm (D). E Mitochondrial membrane potential (ΔΨm,i) was detected by the fluorescent dye tetramethylrhodamine methyl ester (TMRM; 50 nM, 15 min) and analyzed by FACS. Carbonyl cyanide m-chlorophenyl hydrazine (CCCP; 20 μM) was added 5 min before TMRM to see specific suppression of ΔΨm,i. F PaTu8902 cells (106 in 100 μL of PBS) were grafted subcutaneously into immunodeficient NOD scid gamma (NSG) mice. When tumors reached approximatelly 50 mm3, mice (n = 6) were treated three times per week by intraperitoneal administration of Suramin (10 mg/kg) dissolved in physiological solution or the vehicle. G PaTu8902 cells transfected with shANT2 (clones 4 and 5) were exposed to doxycycline (1 μg/mL) for 72 h, and the levels of ANT1 and ANT2 were analyzed by immunoblot. GAPDH was used as a loading control. H Proliferation was evaluated by hemocytometer at the times indicated (0, 3, 6 and 9 days after the addition of doxycycline - 1 μg/mL). PaTu8902 cells (106 in 100 μL of PBS) transfected with shANT2 cells (mix of clones 4 and 5) pre-treated with doxycycline (1 µg/mL) for 72 h were grafted subcutaneously into NSG mice (n = 6). Mice were subsequently administered doxycycline in water (0.4 mg/mL) throughout the experiment. shANT2 PaTu8902 cells without doxycycline pre-treatment/ administration were used as a control. Six days after engraftment, mice were treated twice per week by intraperitoneal administration of MitoTam (MT; 4 mg/kg) dissolved in 4% EtOH in corn oil or the vehicle (corn oil; CO). I Tumor volume on day 6 after engraftment of tumor cells. J Tumor volume was determined using a caliper at the times indicated. K Tumor weight at the end of the experiment. L Weight of animals during the experiment. Tumor tissue was assessed for expression of M ANT2 and N ANT1 using RT-qPCR. β-Actin was used as a reference gene.
