Murine C26 colon adenocarcinoma cells were provided by Donna McCarthy (Ohio State University, Columbus, OH, United States) and cultured in high-glucose (4.5 g/L) DMEM supplied with 10% fetal bovine serum, 1% glutamine, 1% sodium pyruvate, and 1% penicillin and streptomycin. Cells were maintained in a 5% CO₂, 37°C humidified incubator. Murine C2C12 skeletal myoblasts (ATCC, Manassas, VA) were grown in high glucose DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 100 mg/ml sodium pyruvate, 2 mM L-glutamine, and maintained at 37°C in 5% CO₂. Myotubes were generated by exposing the myoblasts to DMEM containing 2% horse serum (i.e., differentiation medium, DM), and replacing the medium every other day for 5 days. To determine the effect on myotube size dependent on MitoQ (MitoQ Limited, Auckland, New Zealand), myotubes were exposed to 20% C26 conditioned medium (CM) in combination with 75 µM MitoQ (C26 + MitoQ) or triphenylphosphonium cation (TPP, used as non-specific, inactive compound; C26 + TPP) for up to 48 h. Control myotubes were exposed to the same percentage of unconditioned media (UM).

All animal studies were approved by the Institutional Animal Care and Use Committee at Indiana University School of Medicine and complied with the National Institutes of Health Guidelines for Use and care of Laboratory Animals and with the 1964 Declaration of Helsinki and its later amendments. All animals were maintained on a regular dark-light cycle (light from 8 a.m. to 8 p.m.), with free access to food and water during the whole experimental period. For the experiments, CD2F1 male mice (Envigo, Indianapolis, IN) were used and housed in a pathogen-free facility at Indiana University Laboratory Animal Resource Center (up to 4 per cage). When the mice were 11 weeks of age, 1 × 10⁶ C26 cells were inoculated subcutaneously (s.c.) in sterile saline. Mice were randomized into four groups: mice receiving TPP (TPP; n = 4), mice treated with MitoQ (MitoQ; n = 4), C26 tumor-bearing mice receiving TPP (C26 + TPP; n = 6) and C26 tumor-bearing mice treated with MitoQ (C26 + MitoQ; n = 7). MitoQ was administered in drinking water at 200 μM concentration for the entire duration of the study, starting 2 weeks before tumor inoculation (i.e., at 9 weeks of age). Keeping in mind that each animal generally drinks ∼5 ml of solution per day (data not shown), this dosing equals approximately a 25 mg/kg dosing, in line with previous reports (Ghosh et al., 2010). The mice were monitored for the entire duration of the experiments. After 13 days from tumor injection the mice were sacrificed under light anesthesia (5% isoflurane in oxygen). Several tissues were collected, weighed, snap frozen in liquid nitrogen and stored at −80°C for further analyses. The tibialis anterior muscle was frozen in liquid nitrogen-cooled isopentane, mounted in OCT and stored for morphological analyses.

The evaluation of the whole-body strength in mice was assessed as previously described (Bonetto et al., 2015). The absolute grip strength (peak force, expressed in grams) was recorded by means of a grip strength meter (Columbus Instruments, Columbus, OH). Five measurements were completed, and the top three measurements were included in the analysis. In order to avoid habituation, the animals were tested for grip strength at baseline (day 0) and at time of sacrifice (day 13). The investigators were not blinded during the testing.

To assess skeletal muscle atrophy, 10-μm-thick cryosections of tibialis anterior muscles taken at the mid-belly were processed for immunostaining as described previously (Huot et al., 2020b). Briefly, sections were blocked for 1 h at room temperature and incubated overnight at 4°C with a dystrophin primary antibody [1:50, #MANDRA1 (7A10), Developmental Studies Hybridoma Bank, Iowa City, Iowa, United States], followed by a 1 h secondary antibody (AlexaFluor 555, 1:1,000, A21127, Thermo Fisher Scientific) incubation at room temperature. Entire dystrophin-stained sections were analyzed for CSA using a Lionheart LX automated microscope (BioTek Instruments).

C2C12 cell layers were fixed in ice-cold acetone-methanol and incubated with anti-myosin heavy chain antibodies (MF-20, diluted 1:200; Developmental Studies Hybridoma Bank, Iowa City, IA) and an AlexaFluor 488-labeled secondary antibody (Invitrogen, Grand Island, NY). Analysis of myotube size was performed by measuring the minimum diameter of long, multi-nucleate fibers avoiding regions of clustered nuclei on a calibrated image using the ImageJ 1.43 software. Three biological replicates were used for each experimental condition. The results of each replicate were then averaged to obtain the final myotube size.

Total protein extracts were obtained by lysing cell layers or homogenizing 100 mg gastrocnemius muscle tissue in radioimmunoprecipitation assay (RIPA) buffer [150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 50 mM Tris, pH 8.0] completed with protease (Roche, Indianapolis, IN, United States) and phosphatase (Thermo Scientific, Rockford, IL, United States) inhibitor cocktails. Cell debris were removed by centrifugation (15 min, 14,000 g), and the supernatant was collected and stored at −80°C. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay method (Thermo Scientific). Protein extracts (30 μg) were then electrophoresed in 4–15% gradient SDS Criterion TGX precast gels (Bio-Rad, Hercules, CA, United States). Gels were transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked with SEA BLOCK blocking reagent (Thermo Scientific) at room temperature for 1 h, followed by an overnight incubation with diluted antibody in SEA BLOCK buffer containing 0.2% Tween-20 at 4°C with gentle shaking. After washing with PBS containing 0.2% Tween-20, the membrane was incubated at room temperature for 1 h with either anti-rabbit IgG (H + L) DyLight 800 or anti-mouse IgG (H + L) DyLight 600 (Cell Signaling Technologies, Danvers, MA, United States). Blots were then visualized with Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, United States). Optical density measurements were taken using the Gel-Pro Analyzer software. Antibodies used were: p-Akt-S473 (#4060), Akt (#9272), p-mTOR (#5536S), mTOR (7C10), p-4E-BP1 (#236B4), 4E-BP1 (#53H11), OPA1 (#80471) from Cell Signaling Technologies, PDK4 (#ab214938), p-PDH (#ab177461), PDH (#ab168379), PGC1α (#ab3242) from Abcam, and α-Tubulin (#12G10) from Developmental Studies Hybridoma Bank (Iowa City, IA, United States).

RNA from gastrocnemius muscle was isolated using the miRNeasy Mini kit (Qiagen, Valencia, CA, United States), following the protocol provided by the manufacturer. RNA was quantified by using a Synergy H1 spectrophotometer (Biotek, Winooski, VT, United States). Total RNA was reverse transcribed to cDNA using the Verso cDNA kit (Thermo Fisher Scientific, Waltham, MA, United States). Transcript levels were measured by Real-Time PCR (Light Cycler 96, Roche, Indianapolis, IN, United States) taking advantage of the TaqMan gene expression assay system (Life Technologies, Carlsbad, CA). Expression levels for Atrogin-1 (Mm00499523_m1), Murf-1 (Mm01185221_m1), Fis1 (Mm00481580), Mfn (Mm00500120), PDK4 (Mm01166879), Pink1 (Mm00550827), Parkin (Mm00450187), Opa1 (Mm01349707), Pgc1α (Mm01208835), CytB (Mm04225271_g1), Lipe (Mm00495359), Pnpla2 (Mm00503040), Fasn (Mm00662319), Srebf1 (Mm00550338), Srebf2 (Mm01306292), Plin1 (Mm00558672_m1), Cpt1b (Mm.PT.6753512a1), Acaa1a (Mm.PT.18700004a1), Hadha (Mm.PT.33859811a1) were detected. Gene expression was normalized to TBP (Mm01277042_m1) levels using the standard 2^−ΔCT methods. The expression of TBP was not significantly altered in any of the experimental groups.

The levels of LDL receptor were measured in gastrocnemius skeletal muscle homogenates from all groups by using a specific ELISA kit (#MLDLR0; Bio-Techne Corporation, Minneapolis, MN, United States), according to the manufacturers protocol.

For ORO staining, tibialis anterior muscles were sectioned (10 μm) and immediately fixed in ice cold formaldehyde (3.7%; Thermo Fisher Scientific) for 1 h. Sections were serially washed in Milli-Q water (MilliporeSigma) and stained in ORO working solution prepared as previously described (Huot et al., 2020a); for 45 min at room temperature in the dark. Following ORO staining, sections were again serially washed in Milli-Q water and then rinsed in running tap water for 10 min. Sections were mounted in 50% glycerol in PBS and photographed using an Axio Observer.Z1 motorized microscope (Zeiss). Entire ORO-stained sections were analyzed for signaling intensity and area of positive staining using ImageJ software.

The enzymatic activities of hexokinase (HK), pyruvate dehydrogenase (PDH) and succinate dehydrogenase (SDH) were measured in gastrocnemius muscle homogenates using Colorimetric Assay Kits (#MAK091, MAK183 and #MAK197, respectively) from Sigma-Aldrich according to the manufacturer’s instruction. Briefly, 10 mg of skeletal muscle tissue was homogenized in 100 μL of ice-cold assay buffer and then centrifuged, and 10 μL of homogenate was added to 96-well plates. Appropriate reaction mix was added to each of the wells and the product of enzyme reaction, which results in a colorimetric (600 nm for SDH and 450 nm for HK and PDH) product proportional to the enzymatic activity. The absorbance was recorded by incubating the plate at 37°C taking measurements (600 or 450 nm) every 5 for 30 min.

Two-way analysis of variance (ANOVA) tests were performed to determine differences between experimental groups. Post hoc comparisons were accomplished via a Tukey’s test, with statistical significance set a priori at p ≤ 0.05. All statistics were performed using GraphPad Prism 8.4.1. Data are presented as means ± standard deviation.

To clarify the in vitro effects of MitoQ in C2C12 myotube cultures exposed to tumor-derived conditioned medium (CM), fully differentiated C2C12 myotubes were administered 20% C26 CM and treated with MitoQ 75 μM, C26 + MitoQ) for 48 h. In line with our previous observations (Pin et al., 2019b), myotubes incubated with CM generated from C26 cells in combination with TPP showed reduced size when compared to TPP-treated controls (−23%, p < 0.01; Figure 1A). Interestingly, MitoQ protected the myotubes from atrophy induced by tumor-derived factors (Figures 1A,B). The muscle-specific ubiquitin ligase Murf1 was increased after exposure to C26 CM (+98%, p < 0.001; Figure 1C), although the treatment was only able to prevent the overexpression of Atrogin-1 (−30%, p < 0.001, Figure 1C). We then checked the expression of PDK4, involved in the regulation of the pyruvate dehydrogenase complex (PDH) and a key enzyme in energy metabolisms that we showed upregulated in the muscle of cachectic mice (Pin et al., 2019b). The myotubes exposed to C26 showed PDK4 increased at both mRNA and protein levels (+54% p < 0.01, and +213%, p < 0.001, respectively; Figures 1C–E), consistent with the increased phosphorylation of PDH (+56%, p < 0.01, Figures 1D,E). Administration of MitoQ reduced the levels of PDK4 gene expression, as well as the phosphorylation of PDH (−51% and −26%, p < 0.01, respectively; Figures 1C–E) in the C26 CM-treated myotubes. Since intramuscular lipid metabolism can be compromised during cachexia, we investigated the expression of Lipase E hormone sensitive (LIPE), known to be involved in intramuscular lipid accumulation (Haemmerle et al., 2002). Lipe was unchanged in the C26+TPP-treated myotubes, although MitoQ administration enhanced its expression (+53%, p < 0.05 vs. C26 + TPP; Figure 1C). To verify if the anabolic signaling was equally affected by the treatment, we assessed the phosphorylation of AKT and found it drastically increased by MitoQ administration (MitoQ: +2.5-fold, p < 0.001; C26 + MitoQ: +7-fold, p < 0.001 vs. TPP; Figures 1D,E). Similarly, the reduced anabolic signaling supported by the downregulation of p-4EBP1 (−33%, p < 0.01 vs. TPP) was corrected by the administration of MitoQ (+33%, p < 0.05 vs. C26 + TPP; Figures 1D,E).

To determine if MitoQ was sufficient to improve cancer cachexia, CD2F1 male mice were treated with MitoQ starting 2 weeks before the inoculum of C26 tumor cells and until the day of sacrifice. As shown in Figure 1A, body weights at time of tumor injection (i.e., 11 weeks of age) were comparable across all experimental groups. The C26 + TPP hosts showed significant loss of body weight (BW) starting 9 days after tumor implantation (Figure 2B), whereas the administration of MitoQ was able to partially correct body wasting without interfering with tumor growth (Figures 2B–D). The C26 + TPP hosts lost skeletal muscle mass, as suggested by the reduced tibialis anterior (−17%, p < 0.001), gastrocnemius (−21%, p < 0.001) and quadriceps (−29%, p < 0.001) muscle mass (Figure 2E, Supplementary Figure S1A), along with the reduction of muscle strength (−45%, p < 0.001; Figure 2F, Supplementary Figure S1B) and muscle cross-sectional area (−9%, p < 0.05; Supplementary Figure S1C). Notably, the C26 + MitoQ group was partially protected from muscle mass loss, as supported by the increased size of the tibialis (+8%, p < 0.05) and gastrocnemius (+12%, p < 0.05) muscles (Figure 2E, Supplementary Figure S1A). Consistently, also muscle strength in the C26 hosts was improved by the treatment with MitoQ (+40%, p < 0.05; Figure 2F, Supplementary Figure S1B). Moreover, the spleen mass was severely increased in the C26 bearers independent of the treatment (+120%, p < 0.001 vs. TPP and +162%, p < 0.001 vs. MitoQ), whereas heart and liver were unchanged (Supplementary Figure S1D). Interestingly, the white adipose tissue (WAT) was severely depleted in the C26 + TPP hosts (−58%, p < 0.001; Supplementary Figure S1D), whereas it was 50% increased, though not significantly, in the tumor-bearing mice treated with MitoQ (p = 0.0675; Supplementary Figure S1D). For this reason, we decided to study the expression of the mitochondrial uncoupling protein-2 (UCP2), the most abundant UCP isoform in WAT, involved in the regulation of energy homeostasis (Pinkney et al., 2000). We found that MitoQ was able to reduce the expression of UCP2 in WAT in the tumor-bearing mice (−59%, p < 0.05; Supplementary Figure S1E).

As previously described (Huot et al., 2020a) and consistent with the reduced skeletal muscle mass, the mRNA expression of markers of protein catabolism such as Atrogin-1 and MuRF-1 was markedly increased in the muscle of C26 tumor hosts (+289%, p < 0.001; +912%, p < 0.001, respectively; Figure 3A) vs. TPP. Interestingly, the administration of MitoQ was able to partially revert the hypercatabolic state in the muscle of the C26 bearers, as shown by the reduced mRNA expression of Atrogin-1 (−64%, p < 0.01), MuRF-1 (−62%, p < 0.01), and Foxo1 (−53%, p < 0.05; Figure 3A). Since skeletal muscle atrophy can be driven by an unbalance between protein degradation and synthesis (Bonaldo and Sandri, 2013), we investigated the expression of markers of anabolic signaling previously found altered in cancer cachexia (Huot et al., 2020a). As shown in Figures 3B,C, the p-AKT/AKT and p-mTOR/mTOR ratios were unchanged in all groups, whereas the p-4EBP1/4EBP1 ratio was reduced in the cachectic muscles (−70%, p < 0.001, respectively) with respect to the TPP-treated mice. These data suggest that, unlike our in vitro data (Figure 1), the treatment with MitoQ was unable to improve the anabolic signal, in line with unchanged AKT, mTOR or 4EBP1 phosphorylation levels (Figures 3B,C).

Since MitoQ acts directly within the mitochondria, we investigated markers of mitochondrial biogenesis, turnover and function. In line with our previous findings (Pin et al., 2019b; Huot et al., 2020a), the muscle of C26 bearers showed reduced protein and gene expression for PGC1α, one of the main regulators of mitochondrial biogenesis (−30%, p < 0.05 and −54%, p < 0.01, respectively; Figures 4A–C). The administration of MitoQ contributed to preserve PGC1α protein expression in the C26 hosts when compared to the TPP-treated hosts (−47% p < 0.05 and Figures 4A,B). With regards to the markers of mitochondrial dynamics and mitophagy, the mRNA expression levels of Fis1 and Parkin were unchanged across all groups (Figure 4C), whereas the regulators of mitochondrial fusion and fission OPA1, Mitofusin-2 and Pink1 were downregulated in the muscle of C26 + TPP hosts. Interestingly, only the expression of Mitofusin-2 was significantly improved by the treatment (+70%, p < 0.05; Figure 4C). As shown in Figures 4A,B, OPA1 protein levels were not significantly affected by the growth of the C26 tumor, whereas MitoQ was able to upregulate the expression of OPA1 in the muscle of both healthy and tumor-bearing mice (Figures 4A,B). To clarify if the oxidative function was improved using mitochondria-targeting agents we analyzed the gene expression of the mitochondria-specific enzymes Cytochrome C (CytC) and Cytochrome B (CytB) and found that only the former was significantly reduced in the muscle of the tumor-bearing mice, whereas MitoQ did not provide any protective effect (Figure 4C). In line with our in vitro data, PDK4 was increased at both gene and protein levels in the tumor hosts, whereas MitoQ partially prevented PDK4 overexpression (Figures 4A–C). Moreover, PDH phosphorylation was not increased in the C26 model and MitoQ treatment had no effect in any of the groups (Figures 4A,B). To evaluate if the changes observed at molecular levels were also followed by modulation of the energetic metabolism, we assessed the activity of key enzymes, including HK, PDH and SDH. As shown in Figure 4D, HK and SDH enzymatic activities were unchanged in the muscle of C26 + TPP mice, whereas the treatment with MitoQ reduced HK (−27%, p < 0.001) and increased SDH (+80%, p < 0.01; Figure 4D) in the presence of C26 tumors. Finally, consistent with the expression of PDK4, the activity of PDH was markedly reduced in the cachectic muscles (−17%, p < 0.05) and corrected by administration of MitoQ (+24%, p < 0.001; Figure 4D).

Abnormal lipid metabolism, as suggested by hyperlipidemia, elevated levels of LDL-R (Pin et al., 2019a) and increased myosteatosis (Huot et al., 2020a) characterize the C26 models and could reflect altered utilization of fat as energy substrate. Hence, we investigated whether the use of MitoQ could improve these alterations. Similar to our previous study (Pin et al., 2019a), the levels of LDL-receptor (LDL-R) were increased in the muscle of cachectic hosts (+10%, p < 0.05; Figure 5A), in line with the elevated intramuscular fat accumulation, as measured by Oil Red-O in the tibialis anterior transversal sections from C26 tumor-bearing mice (Figure 5B). Interestingly, MitoQ treatment did not modulate the expression of LDL-R (Figure 5A), although it was able to decrease fat accumulation in the skeletal muscle (Figure 5B). To investigate the molecular mechanisms subtending to the changes observed in fat accumulation we assessed the expression of markers of lipogenesis and lipolysis in the skeletal muscle of control and tumor hosts. The levels of Lipe, a regulator of lipolysis, as well as of Plin1 and Srebf1, involved in the control of lipid metabolism, were unchanged in the tumor hosts (Figure 5C). Conversely, the regulators of lipogenesis Fasn and Srebf2 were markedly reduced (−87% and −68%, p < 0.001, respectively) and Pnpla2 was increased (+273%, p < 0.001; Figure 5C). Upon MitoQ administration, Lipe and Srebf2 were found significantly increased in the skeletal muscle of C26 bearers (+63% and +40%, p < 0.05, respectively; Figure 5C). Since the changes in intramuscular fat accumulation could be associated with altered utilization of fat as energy substrate, markers of β-oxidation were also measured. The gene expression for Cpt1b, Acaa1a and Hadha was reduced in the cachectic muscle (−57%, −45% and −53%, p < 0.01, respectively; Figure 5D), whereas only Cpt1b was positively regulated by the treatment (+64%, p < 0.05; Figure 5D).