Cytochrome c oxidase is activated by the oncoprotein Ras and is required for A549 lung adenocarcinoma growth - PubMed (original) (raw)
Cytochrome c oxidase is activated by the oncoprotein Ras and is required for A549 lung adenocarcinoma growth
Sucheta Telang et al. Mol Cancer. 2012.
Abstract
Background: Constitutive activation of Ras in immortalized bronchial epithelial cells increases electron transport chain activity, oxygen consumption and tricarboxylic acid cycling through unknown mechanisms. We hypothesized that members of the Ras family may stimulate respiration by enhancing the expression of the Vb regulatory subunit of cytochrome c oxidase (COX).
Results: We found that the introduction of activated H-Ras(V12) into immortalized human bronchial epithelial cells increased eIF4E-dependent COX Vb protein expression simultaneously with an increase in COX activity and oxygen consumption. In support of the regulation of COX Vb expression by the Ras family, we also found that selective siRNA-mediated inhibition of K-Ras expression in A549 lung adenocarcinoma cells reduced COX Vb protein expression, COX activity, oxygen consumption and the steady-state concentration of ATP. We postulated that COX Vb-mediated activation of COX activity may be required for the anchorage-independent growth of A549 cells as soft agar colonies or as lung xenografts. We transfected the A549 cells with COX Vb small interfering or shRNA and observed a significant reduction of their COX activity, oxygen consumption, ATP and ability to grow in soft agar and as poorly differentiated tumors in athymic mice.
Conclusion: Taken together, our findings indicate that the activation of Ras increases COX activity and mitochondrial respiration in part via up-regulation of COX Vb and that this regulatory subunit of COX may have utility as a Ras effector target for the development of anti-neoplastic agents.
Figures
Figure 1
Introduction of oncogenic H-Ras V12 into immortalized human bronchial epithelial cells increases COX Vb protein expression, COX activity and oxygen consumption. hT/LT and H-RasV12 cells were examined for H-Ras and COX Vb protein expression by Western blot analysis (A) and COX Vb mRNA expression by real-time RT-PCR (D). Densitometry of H-Ras (B) and COX Vb (C) protein expression relative to β-Actin expression was determined. Enzyme activity assays of COX (E) and oxygen consumption (F) were performed as described. Data are expressed as the mean ± SD of three experiments. *p value < 0.05 compared to hT/LT.
Figure 2
Selective inhibition of eIF4E reduces COX Vb protein expression but has no effect on β-Actin protein expression. hT/LT, H-RasV12 and A549 cells were transfected with transfection reagent alone, control siRNA or siRNA specific for eIF4E for 48 hours and then examined for eIF4E and COX Vb mRNA expression by real-time RT-PCR (A) and COX Vb and β-actin protein expression using Western blot analysis (B). Protein expression was quantified by densitometric analyses (C) and expressed as percentage of transfection reagent. Data are expressed as the mean ± SD of three experiments. * p value <0.05.
Figure 3
Selective inhibition of K-Ras expression by A549 lung adenocarcinoma cells reduces COX activity, oxygen consumption and ATP. A549 cells were transfected with transfection reagent (TR) alone, control siRNA or siRNA specific for K-Ras for 72 hours and then examined for COX Vb protein expression (A and B), COX Vb mRNA expression (C), COX activity (D), oxygen consumption (E) and ATP (F). Data are expressed as the mean ± SD of three experiments. * p value <0.05.
Figure 4
Selective inhibition of COX Vb by A549 cells reduces COX activity, oxygen consumption and ATP. A549 cells were transfected with transfection reagent alone, control siRNA or siRNA specific for the ORF (Vb1) or 3’-UTR (Vb2) of COX Vb for 48 hours and then examined for COX Vb protein expression (A and B), COX activity (C), oxygen consumption (D), ATP (E) and NAD+/NADH ratios (F). Data are expressed as the mean ± SD of three experiments. * p value <0.05.
Figure 5
Selective inhibition of COX Vb expression by A549 cells suppresses anchorage-independent growth in soft agar. A. 105 A549 cells transfected with COX Vb or control siRNA were cultured for 48 hours and viable cells were enumerated using light microscopy. B. After the 48 hour transfection, A549 cells were plated on 6 cm dishes containing DMEM with 0.6% agar. Cells were fed with 0.2% agar in media every three days. After 21 days, soft agar colonies were enumerated. C. Representative photomicrographs of the soft agar colonies. Data are expressed as the mean ± SD of five (A) and three (B) experiments. * p value <0.05.
Figure 6
Stable COX Vb shRNA expression in A549 cells reduces the outgrowth of A549 xenografts and inhibits multiple histopathological indicators of poor differentiation in athymic mice. 5x106 A549 cells stably transfected with control or COX Vb-specific shRNA were subjected to Western blot analysis for COX Vb or β-actin expression (A) and then injected subcutaneously into athymic mice. After 4 weeks, tumors were measured weekly for a total of 3 additional weeks (timepoints 0, 7, 14 and 21) using microcalipers. Tumor mass was calculated based on bidimensional measurements and data are expressed as the mean ± SEM of two experiments (B). After 7 weeks of growth, mice were euthanized and tumors were excised, fixed in formalin, paraffin-embedded, sectioned and stained with hematoxylin/eosin. Light micrographs demonstrate that, relative to the COX Vb1 shRNA-transfected A549 tumors (F-H), the control A549 tumors were higher grade, poorly differentiated and invasive (C-E), contain large areas of tumor necrosis (D; white arrows), pleomorphic nuclei (E, black arrows), and numerous mitotic figures (E, white arrows).
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