Preventing loss of sirt1 lowers mitochondrial oxidative stress and preserves C2C12 myotube diameter in an in vitro model of cancer cachexia

Abstract

Cancer cachexia is a multifactorial syndrome associated with advanced cancer that contributes to mortality. Cachexia is characterized by loss of body weight and muscle atrophy. Increased skeletal muscle mitochondrial reactive oxygen species (ROS) is a contributing factor to loss of muscle mass in cachectic patients. Mice inoculated with Lewis lung carcinoma (LLC) cells lose weight, muscle mass, and have lower muscle sirtuin-1 (sirt1) expression. Nicotinic acid (NA) is a precursor to nicotinamide dinucleotide (NAD+) which is exhausted in cachectic muscle and is a direct activator of sirt1. Mice lost body and muscle weight and exhibited reduced skeletal muscle sirt1 expression after inoculation with LLC cells. C2C12 myotubes treated with LLC-conditioned media (LCM) had lower myotube diameter. We treated C2C12 myotubes with LCM for 24 h with or without NA for 24 h. C2C12 myotubes treated with NA maintained myotube diameter, sirt1 expression, and had lower mitochondrial superoxide. We then used a sirt1-specific small molecule activator SRT1720 to increase sirt1 activity. C2C12 myotubes treated with SRT1720 maintained myotube diameter, prevented loss of sirt1 expression, and attenuated mitochondrial superoxide production. Our data provides evidence that NA may be beneficial in combating cancer cachexia by maintaining sirt1 expression and decreasing mitochondrial superoxide production.

Keywords: cancer cachexia; mitochondria; nicotinic acid; oxidative stress; sirtuin‐1.

FIGURE 1

LLC inoculation causes loss of body weight, muscle mass, and decreases muscle sirt1 expression. (a) Whole animal tumor‐free body weight. (b) Wet muscle weights for gastrocnemius and soleus. (c) Sirt1 and total protein western blots and quantification of sirt1 relative to total protein. (d) Correlation of body weight and gastrocnemius weight to Sirt1 expression in the gastrocnemius muscle. (e) PGC‐1α and Nox4 protein expression normalized to GAPDH; western blots and quantification. (a–c, e) Student's t‐test. (d) Linear regression analysis. Groups: N = non‐tumor; T = tumor. n = 6 for all groups.

FIGURE 2

NA attenuates LCM‐mediated loss of C2C12 myotube diameter and prevents loss of sirt1 protein expression and total protein acetylation. C2C12 myotubes were incubated with LCM with and without 0.75 mM NA for 24 h. (a) Representative images of C2C12 myotubes treated with NCM, LCM +/− NA. Scale bar = 100 μm. (b) Sirt1 protein expression was significantly lower in LCM‐treated C2C12 myotubes which was rectified with NA treatment while both PGC‐1α and Nox4 protein expression were unchanged. All blots normalized to total protein expression. (c) LCM increased total protein lysine acetylation which was prevented with NA. (a) Two‐way ANOVA with Tukey's post hoc test for multiple comparisons. (b, c) One‐way ANOVA with multiple comparisons. Groups: N=NCM, L = LCM, NA = LCM + NA. n = 3 for all groups.

FIGURE 3

SRT1720 attenuates LCM‐mediated loss of C2C12 myotube diameter and prevents loss of sirt1 protein expression and maintains sirt1 activity. C2C12 myotubes were incubated with LCM with and without 2 μM SRT1720 for 24 h. (a) C2C12 myotube diameter was significantly decreased 24 h after incubation with LLC‐conditioned media which was prevented with supplementation of SRT1720. Scale bar = 100 μm. (b) Sirt1 protein expression was significantly lower in LCM‐treated C2C12 myotubes which was partially improved with SRT1720 administration. (a) Two‐way ANOVA with Tukey's post hoc test for multiple comparisons (b) One‐way ANOVA with Tukey's post hoc test for multiple comparisons. Groups: N = NCM, L = LCM, S = LCM + SRT1720. n = 3 for all groups.

FIGURE 4

LCM causes increased mitochondrial superoxide in C2C12 myotubes which is ameliorated by either NA or SRT1720 administration. C2C12 myotubes were incubated with LCM with and without NA or SRT1720 for 24 h. Myotubes were treated with Mitosox Red and MitoTracker Green in order to measure mitochondrial superoxide. (a) Representative images of mitochondria (green) and mitochondrial superoxide (red). Images taken at 40× magnification using confocal microscopy. (b) Quantification of the ratio of mitochondrial superoxide (red) to mitochondria (green). (b) One‐way ANOVA with Tukey's post hoc test for multiple comparisons; Scale bar = 50 μm. Approximately 50 fibers/well measured. N = 3 in each group.