Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Feb;22(2):399-412.
doi: 10.1038/cr.2011.145. Epub 2011 Aug 30.

K-ras(G12V) transformation leads to mitochondrial dysfunction and a metabolic switch from oxidative phosphorylation to glycolysis

Yumin Hu  1 , Weiqin Lu, Gang Chen, Peng Wang, Zhao Chen, Yan Zhou, Marcia Ogasawara, Dunyaporn Trachootham, Li Feng, Helene Pelicano, Paul J Chiao, Michael J Keating, Guillermo Garcia-Manero, Peng Huang
Affiliations

K-ras(G12V) transformation leads to mitochondrial dysfunction and a metabolic switch from oxidative phosphorylation to glycolysis

Yumin Hu et al. Cell Res. 2012 Feb.

Abstract

Increased aerobic glycolysis and oxidative stress are important features of cancer cell metabolism, but the underlying biochemical and molecular mechanisms remain elusive. Using a tetracycline inducible model, we show that activation of K-ras(G12V) causes mitochondrial dysfunction, leading to decreased respiration, elevated glycolysis, and increased generation of reactive oxygen species. The K-RAS protein is associated with mitochondria, and induces a rapid suppression of respiratory chain complex-I and a decrease in mitochondrial transmembrane potential by affecting the cyclosporin-sensitive permeability transition pore. Furthermore, pre-induction of K-ras(G12V) expression in vitro to allow metabolic adaptation to high glycolytic metabolism enhances the ability of the transformed cells to form tumor in vivo. Our study suggests that induction of mitochondrial dysfunction is an important mechanism by which K-ras(G12V) causes metabolic changes and ROS stress in cancer cells, and promotes tumor development.

PubMed Disclaimer

Figures

Figure 1
Figure 1
K-rasG12V activation caused mitochondrial dysfunction. (A) 20 ng/ml doxycycline induced ectopic K-ras expression in T-Rex/K-ras cells in a time-dependent manner. The same doxycycline treatment in T-Rex/Vector control cells caused no significant changes. Protein expression of K-rasG12V was detected by immunoblotting with specific antibody for K-ras. β-Actin was used as a loading control. (B) Loss of mitochondrial transmembrane potential in T-Rex/K-ras after K-ras induction by doxycycline for 24 h, measured by fluorescent probe Rho-123. The same concentration of doxycycline caused no effect on transmembrane potential in vector control cells. (C) K-ras activation caused a decrease of mitochondrial transmembrane potential in a time-dependent manner. Transmembrane potential levels of induced cells were normalized to the level of cells without induction. (D) Oxygen consumption rate of T-Rex-293 cells with (Tet/on) and without (Tet/off) K-ras activation for 24 h. (E) K-ras activation inhibited oxygen consumption in a time-dependent manner. (F) K-ras activation did not affect mitochondrial DNA contents. (G) Effect of K-ras activation on cell viability measured by annexin V-PI assay. Data in C and E are shown as mean ± SD. n = 3, *P < 0.05, **P < 0.01.
Figure 2
Figure 2
Mitochondrial dysfunction induced by K-rasG12V activation led to metabolic alterations. (A) Experimental rationale of analyzing mitochondrial complex I and II activities and representative oxygen consumption curve of T-Rex-293 cells before and after K-ras induction for 48 h. The numbers indicate oxygen consumption rate (nmol/ml/min) of mitochondrial complex I to IV and II to IV. Arrows indicate the time points when reagents were added. Rotenone: 100 nM; digitonin: 30 μg/ml; succinate: 5 μM. (B) Quantitative analysis of oxygen consumption rates of complex I-IV and II-IV after K-ras induction. Data are mean ± SD. n = 3, *P < 0.05, **P < 0.01. (C) K-rasG12V induction caused a decrease of complex I 20 kD (Com I 20 kD) subunit and increase of complex II 30 kD (Com II 30 kD) subunit of mitochondrial respiratory chain and increase of phospho-Akt (S473). SOD2 and catalase were also inhibited 24 h after induction. (D) K-ras activation for 48 h caused a significant increase of ROS production measured by fluorescent probe DCF-DA. (E) K-ras activation caused an increase of ROS generation in a time-dependent manner. (F) Effect of K-ras activation on oxygen consumption, glucose uptake and lactate production. The metabolic parameters were measured 24 h and 72 h after K-ras induction.
Figure 3
Figure 3
Translocation of K-RAS protein to the mitochondria and its role in causing mitochondrial dysfunction. (A) Mitochondrial fraction was isolated from T-Rex cells before and after doxycycline induction for 12 h. Protein lysates of cytosol and mitochondria (Mito) were analyzed for the presence of K-ras. HSP60 and tubulin were used as mitochondrial and cytosolic markers, respectively. (B) Isolated mitochondria from T-Rex cells with K-rasG12V induction (Control) were treated with 100 μg/ml trypsin for 30 min at 4 °C, 24 °C and 37 °C. Western blotting analysis was used to reveal K-ras, hexokinase II (HKII) and cytochrome c. (C) Confocal microscopic analysis of the localization of K-ras. HEK293 cells before and after K-ras induction for 12 h were labeled with MitoTracker Red, K-ras-FITC (green), and DAPI for nuclei (blue) as described in Materials and Methods. (D) Immunoblotting of mitochondrial lysates from T-Rex cells without doxycycline induction (lane 1), with doxycycline induction for 12 h (lane 2), and with doxycycline induction plus 25 μM H-7 (lane 3). Translocation of K-ras to the mitochondria was partially prevented by PKC inhibitor H-7. (E) K-ras activation (Tet/on) caused a decrease of mitochondrial transmembrane potential by 50% compared to control (off). Decrease of transmembrane potential was rescued by simultaneous treatment of H-7 (on + H-7). (F) K-ras-induced decrease of transmembrane potential was rescued by treatment with 5 μM cyclosporin A (on + CysA). (G) Quantitative analysis of transmembrane potential in K-ras-expressing cells in the presence or absence of cyclosporin A. Data are shown as mean ± SD. n = 3, *P < 0.05, **P < 0.01.
Figure 4
Figure 4
Reversibility of K-rasG12V-induced ROS generation and mitochondrial dysfunction. (A) T-Rex cells were treated with 20 ng/ml doxycycline for 24 h (Tet/on), followed by withdrawal of doxycycline from cell culture for 1, 2 and 3 days. Expression of K-ras, SOD2, catalase and β-actin was measured by western blot analysis. (B) K-ras induction (Tet/on) caused an increase of ROS. Removal of doxycycline for 2 days reversed ROS increase. (C) K-ras induction caused a decrease of mitochondrial transmembrane potential. Removal of doxycycline for 2 days reversed such decrease to baseline comparable to that of the control (Tet/off). (D) Morphology of HEK293 cells without K-rasG12V expression (Tet/off), with K-rasG12V expression for 1 day (Tet/on), and after withdrawal of doxycycline for 2 days.
Figure 5
Figure 5
Effect of long-term expression of K-rasG12V on cellular metabolism and tumor formation capacity. (A) T-Rex/K-ras cells were induced to express K-RAS by continuous culture with 20 ng/ml doxycycline for over 1 month. Oxygen consumption, glucose uptake and lactate production were measured in comparison with the Tet/off control cells. (B) Western blot analysis of Akt and hexokinase II (HKII) in T-Rex/K-ras cells with or without long-term expression of K-ras. (C) Comparison of mitochondrial mass in Tet/off and long-term Tet/on cells, measured by flow cytometry analysis after staining with MitoTracker Green. (D) ROS production remained elevated after induction of K-rasG12V for over a month (long-term Tet/on). (E) Analysis of glutathione (GSH) levels in T-Rex/K-ras cells without or with K-rasG12V induction for short term (48 h) and long term (> 1 month). (F) Comparison of T-Rex/K-ras cells with or without long-term pre-induction of K-rasG12V for colony formation in soft agar. The same number of cells were seeded in soft agar suspension in a six-well plate described in Materials and Methods. After incubation for 15 days, colonies were stained with iodonitroterazolium violet and counted. Data are shown as mean ± SD from triplicate experiments. (G) Representative mice showing tumors grown from inoculated T-Rex/K-ras cells. Cells with and without long-term pre-induction of K-rasG12V expression were inoculated into the left flank and right flank of the same mice, respectively. (H) Comparison of tumor growth in mice bearing T-Rex/K-ras cells with and without long-term pre-induction of K-rasG12V expression. After cell inoculation, all mice received doxycycline during the 60-day observation period.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Warburg O. On the origin of cancer cells. Science. 1956;123:309–314. - PubMed
    1. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009;324:1029–1033. - PMC - PubMed
    1. Kaira K, Endo M, Abe M, et al. Biologic correlates of (1)F-FDG uptake on PET in pulmonary pleomorphic carcinoma. Lung Cancer. 2011;71:144–150. - PubMed
    1. Vergez S, Delord JP, Thomas F, et al. Preclinical and clinical evidence that Deoxy-2-[18F]fluoro-D-glucose positron emission tomography with computed tomography is a reliable tool for the detection of early molecular responses to erlotinib in head and neck cancer. Clin Cancer Res. 2010;16:4434–4445. - PubMed
    1. Vafa O, Wade M, Kern S, et al. c-Myc can induce DNA damage, increase reactive oxygen species, and mitigate p53 function: a mechanism for oncogene-induced genetic instability. Mol Cell. 2002;9:1031–1044. - PubMed

Publication types

LinkOut - more resources