Mitochondria dysfunction in lung cancer-induced muscle wasting in C2C12 myotubes

Abstract

Aims: Cancer cachexia is a syndrome which results in severe loss of muscle mass and marked fatigue. Conditioned media from cachexia-inducing cancer cells triggers metabolic dysfunction in skeletal muscle, including decreased mitochondrial respiration, which may contribute to fatigue. We hypothesized that Lewis lung carcinoma conditioned medium (LCM) would impair the mitochondrial electron transport chain (ETC) and increase production of reactive oxygen species, ultimately leading to decreased mitochondrial respiration. We incubated C2C12 myotubes with LCM for 30 min, 2, 4, 24 or 48 h. We measured protein content by western blot; oxidant production by 2',7'-dichlorofluorescin diacetate (DCF), 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF), and MitoSox; cytochrome c oxidase activity by oxidation of cytochrome c substrate; and oxygen consumption rate (OCR) of intact myotubes by Seahorse XF Analyzer.

Results: LCM treatment for 2 or 24 h decreased basal OCR and ATP-related OCR, but did not alter the content of mitochondrial complexes I, III, IV and V. LCM treatment caused a transient rise in reactive oxygen species (ROS). In particular, mitochondrial superoxide (MitoSOX) was elevated at 2 h. 4-Hydroxynonenal, a marker of oxidative stress, was elevated in both cytosolic and mitochondrial fractions of cell lysates after LCM treatment.

Conclusion: These data show that lung cancer-conditioned media alters electron flow in the ETC and increases mitochondrial ROS production, both of which may ultimately impair aerobic metabolism and decrease muscle endurance.

Keywords: cachexia; electron transport chain; mitochondria; oxidants; skeletal muscle.

Figure 1

Oxygen consumption rate (OCR) in C2C12 myotubes. Eleven total OCR measurements were taken over 2 h: 3 basal respiration, 3 Oligomycin-sensitive respiration, 3 maximal respiratory capacity, and 2 non-mitochondrial respiration. The x-axis in (A) describes the measurement number. (A) OCR normalized to VDAC content presented at % basal control in control myotubes vs. myotubes treated for 30 min, 2 or 24 h with LCM (n = 12, ^*P < 0.01 for treatment effect, repeated measures ANOVA). (B) VDAC content in myotubes treated with LCM, represented as percent control. (C) Change basal respiration in myotubes treated with LCM, expressed as percent of control respiration (n = 12, ^*P < 0.01, ANOVA, Bonferroni post-hoc). (D) ATP-related OCR/VDAC in myotubes treated with LCM expressed as percent basal control (n = 12, ^*P < 0.05, ANOVA, Bonferroni post-hoc test).

Figure 2

Protein content of electron transport chain complexes. Samples were equal protein loaded, which was determined via SDS-PAGE and staining with Simply Blue. Western blot membranes showed no changes in protein content for mitochondrial complex I NDUFA9, complex III subunit core I, complex IV subunit IV, and complex V, subunit α (n = 6).

Figure 3

The effect of LCM treatment on cytochrome c oxidase activity. (A) Cytochrome C absorbance decreases after a 2 h exposure to LCM, indicating an increase in cytochrome c oxidase activity. (B) Cytochrome oxidase activity defined by the rate of change in the linear range per mg protein. LCM treatment for 2 h increased activity to 144% of control (n = 8, ^*P < 0.01, t-test).

Figure 4

LCM treatment alters oxidant production. (A) DCFH fluorescence, a measurement of cytosolic oxidant levels, was increased by LCM treatment for 30 min and 4 h, but reduced by 24 h of treatment, represented as percent change (n = 10, ^*P < 0.05, ANOVA, Bonferroni post-hoc). (B) Unaltered DAF fluorescence in myotubes treated with LCM for 30 min, 2, and 24 h, represented as percent change (n = 20, non-significant, ANOVA, Bonferroni post-hoc). (C) Fluorescence images of control myotubes (top panel) and LCM-treated myotubes (bottom panel) treated with MitoSox. (D) 2 h of LCM treatment increased MitoSox fluorescence compared to control; 2 h of LCM treatment combined with SS31 decreased Mitosox fluorescence compared to LCM-treated myotubes; and 2 h of LCM treatment combined with NAC did not alter MitoSox fluorescence significantly (n = 4, ^*P < 0.05, Bonferroni post-hoc).

Figure 5

The effect of LCM treatment on UCP3 content. The left panel shows a representative western blot with anti-UCP3 in control and LCM-treated myotubes. The right panel shows the quantification of western blot represented as percent control (n = 6, t-test). 24 h of LCM treatment does not alter UCP3 content.

Figure 6

LCM treatment oxidizes proteins. The upper panel shows western blot for anti-4HNE in cytosolic and mitochondrial myotube fractions treated with LCM for 2 or 48 h, vs. control myotubes. The bottom panel shows the quantification anti-4HNE western blots (n = 4, ^*P < 0.05, ANOVA, Bonferroni post-hoc). LCM treatment for 2 and 48 h increased HNE content in both cytosolic and mitochondrial portions of cell lysates.