Colon-26 Research Articles

Effects of Neuromuscular Electrical Stimulation in Combination with Glutamine Administration on Skeletal Muscle Atrophy in Colon-26 Tumor-Bearing Mice

The depressed protein synthetic response, a phenomenon termed anabolic resistance, has been shown to be involved in muscle wasting induced by cancer cachexia. Moreover, a positive relationship between the protein synthetic rate and intracellular glutamine (GLN) concentration has been found in skeletal muscles. This study investigated the effects of neuromuscular electrical stimulation (ES) and GLN administration on muscle wasting and GLN metabolism in colon-26 (C-26) tumor-bearing mice. CD2F1 mice were divided into 8 groups: control (CNT), CNT+ES, CNT+GLN, CNT+ES+GLN, C-26, C-26+ES, C-26+GLN, C-26+ES+GLN. Cancer cachexia was induced by subcutaneous injection of C-26 cells and developed for four weeks. ES was performed on the left plantar flexor muscles every other day, and GLN (1 g/kg) was administered daily intraperitoneally starting one day after the C-26 injection. Tumor-free body mass and fast-twitch gastrocnemius (Gas) muscle weight were lower in the C-26 group than in the CNT group (-19% and -17%, respectively). Neither ES training nor GLN administration, alone or in combination, ameliorated the loss of Gas muscle weight in the C-26 mice. However, ES training in combination with GLN administration inhibited the increased expression of GLN synthetase (GS) in the C-26 muscles. Thus, it is likely that GLN plays a critical role in muscle protein metabolism and, therefore, can be targeted as a tentative treatment of cancer cachexia.

Sulforaphane attenuates cancer cell-induced atrophy of C2C12 myotubes

Cancer cachexia is common in many cancers and the loss of skeletal muscle mass compromises the response to therapies and quality of life. A contributing mechanism is oxidative stress and compounds able to attenuate it may be protective. Sulforaphane (SFN), a natural antioxidant in cruciferous vegetables, activates nuclear factor erythroid 2-related factor 2 (Nrf2) signaling to decrease oxidative stress. Although SFN has potential as a cancer therapeutic, whether it can attenuate muscle wasting in the absence or presence of chemotherapy is unknown. In healthy C2C12 myotubes, SFN administration for 48 h induced hypertrophy through increased myoblast fusion via Nrf2 and ERK signaling. To determine whether SFN could attenuate wasting induced by cancer cells, myotubes were cocultured with or without Colon-26 (C-26) cancer cells for 48 h and treated with 5-fluorouracil (5-FU, 5 µM) or vehicle (DMSO). SFN (10 µM) or DMSO was added for the final 24 h. Coculture with cancer cells in the absence and presence of 5-FU reduced myotube width by ∼30% (P < 0.001) and ∼20% (P < 0.01), respectively, which was attenuated by SFN (P < 0.05). Exposure to C-26 conditioned media reduced myotube width by 15% (P < 0.001), which was attenuated by SFN. Western immunoblotting and qRT-PCR confirmed activation of Nrf2 signaling and antioxidant genes. Coadministration of Nrf2 inhibitors (ML-385) or MEK inhibitors (PD184352) revealed that SFN's attenuation of atrophy was blocked by ERK inhibition. These data support the chemoprotective and antioxidative function of SFN in myotubes, highlighting its therapeutic potential for cancer-related muscle wasting.

Diet-Induced Gene Expression Changes of Cachectic Muscle, Adipose, and Liver

ObjectivesCancer cachexia is a systemic disease characterized by muscle and adipose loss that cannot be reversed by increasing caloric intake. Our previous research has shown insulin resistance precedes cancer cachexia in the C26 mouse model of cachexia, and a diet high in linoleic acid, the essential omega-6 polyunsaturated fatty acid, attenuates the C26-induced insulin resistance. Therefore, to better understand how dietary linoleic acid is improving insulin sensitivity, we characterized gene expression changes in three major tissues responsible for controlling insulin sensitivity: skeletal muscle, adipose, and liver. MethodsMale CD2F1 (Charles River, MA) were randomized to semi-purified diet (24% fat by weight) containing fat prominently from lard, or containing fat prominently from safflower oil (a linoleic acid-rich oil). One week after diet randomization, mice were inoculated with colon-26 (C26) adenocarcinoma cells (1.0E6 cells). 13 days after inoculation mice were euthanized and gastrocnemius skeletal muscle, epididymal white adipose tissue, and liver tissue were collected for total transcriptome analysis using poly-A enriched next generation RNA-sequencing. Differentially expressed genes were selected based on p-values < 0.05. ResultsThere were no detectable differences in body weight or food intake between the two diets in mice with C26 tumors. Between the two diets 12 genes were differentially expressed in the muscle, while 57 genes were differentially expressed in the liver, and 314 genes were differentially expressed in adipose. ConclusionsA linoleic acid enriched diet had little effect on the skeletal muscle transcriptome but induced larger transcriptome changes in liver and adipose. This could suggest dietary linoleic acid increases insulin sensitivity through affecting metabolism in adipose and liver, rather than skeletal muscle. Determining these diet-induced transcriptome changes allows us to better target tissue-specific molecular mechanisms of linoleic acid in future research. Funding SourcesThis work was supported by the Pelotonia Fellowship Program, the Ohio State University Comprehensive Cancer Center, and the National Institutes of Health.

The Mitochondria-Targeting Agent MitoQ Improves Muscle Atrophy, Weakness and Oxidative Metabolism in C26 Tumor-Bearing Mice.

Cancer cachexia is a debilitating syndrome characterized by skeletal muscle wasting, weakness and fatigue. Several pathogenetic mechanisms can contribute to these muscle derangements. Mitochondrial alterations, altered metabolism and increased oxidative stress are known to promote muscle weakness and muscle catabolism. To the extent of improving cachexia, several drugs have been tested to stimulate mitochondrial function and normalize the redox balance. The aim of this study was to test the potential beneficial anti-cachectic effects of Mitoquinone Q (MitoQ), one of the most widely-used mitochondria-targeting antioxidant. Here we show that MitoQ administration (25 mg/kg in drinking water, daily) in vivo was able to improve body weight loss in Colon-26 (C26) bearers, without affecting tumor size. Consistently, the C26 hosts displayed ameliorated skeletal muscle and strength upon treatment with MitoQ. In line with improved skeletal muscle mass, the treatment with MitoQ was able to partially correct the expression of the E3 ubiquitin ligases Atrogin-1 and Murf1. Contrarily, the anabolic signaling was not improved by the treatment, as showed by unchanged AKT, mTOR and 4EBP1 phosphorylation. Assessment of gene expression showed altered levels of markers of mitochondrial biogenesis and homeostasis in the tumor hosts, although only Mitofusin-2 levels were significantly affected by the treatment. Interestingly, the levels of Pdk4 and CytB, genes involved in the regulation of mitochondrial function and metabolism, were also partially increased by MitoQ, in line with the modulation of hexokinase (HK), pyruvate dehydrogenase (PDH) and succinate dehydrogenase (SDH) enzymatic activities. The improvement of the oxidative metabolism was associated with reduced myosteatosis (i.e., intramuscular fat infiltration) in the C26 bearers receiving MitoQ, despite unchanged muscle LDL receptor expression, therefore suggesting that MitoQ could boost β-oxidation in the muscle tissue and promote a glycolytic-to-oxidative shift in muscle metabolism and fiber composition. Overall, our data identify MitoQ as an effective treatment to improve skeletal muscle mass and function in tumor hosts and further support studies aimed at testing the anti-cachectic properties of mitochondria-targeting antioxidants also in combination with routinely administered chemotherapy agents.

Metabolic Biomarkers for the Early Detection of Cancer Cachexia.

Background: Cancer cachexia is a severe metabolic disorder characterized by progressive weight loss along with a dramatic loss in skeletal muscle and adipose tissue. Like cancer, cachexia progresses in stages starting with pre-cachexia to cachexia and finally to refractory cachexia. In the refractory stage, patients are no longer responsive to therapy and management of weight loss is no longer possible. It is therefore critical to detect cachexia as early as possible. In this study we applied a metabolomics approach to search for early biomarkers of cachexia.Methods: Multi-platform metabolomics analyses were applied to the murine Colon-26 (C26) model of cachexia. Tumor bearing mice (n = 5) were sacrificed every other day over the 14-day time course and control mice (n = 5) were sacrificed every fourth day starting at day 2. Linear regression modeling of the data yielded metabolic trajectories that were compared with the trajectories of body weight and skeletal muscle loss to look for early biomarkers of cachexia.Results: Weight loss in the tumor-bearing mice became significant at day 9 as did the loss of tibialis muscle. The loss of muscle in the gastrocnemius and quadriceps was significant at day 7. Reductions in amino acids were among the earliest metabolic biomarkers of cachexia. The earliest change was in methionine at day 4. Significant alterations in acylcarnitines and lipoproteins were also detected several days prior to weight loss.Conclusion: The results of this study demonstrate that metabolic alterations appear well in advance of observable weight loss. The earliest and most significant alterations were found in amino acids and lipoproteins. Validation of these results in other models of cachexia and in clinical studies will pave the way for a clinical diagnostic panel for the early detection of cachexia. Such a panel would provide a tremendous advance in cachectic patient management and in the design of clinical trials for new therapeutic interventions.

Mechanisms of chemotherapy‐induced muscle wasting in mice with cancer cachexia

AbstractBackgroundCachexia is a debilitating complication of cancer characterized by progressive wasting and weakness of skeletal muscles that reduces quality of life and can compromise survival. Many anticancer treatments, such as chemotherapy, also cause muscle wasting, which impairs the response to treatment. Given that many cancer patients present with cachexia at the initiation of treatment, we investigated whether cachectic mice were susceptible to chemotherapy‐induced muscle wasting and to investigate contributing mechanisms, including the dysregulation of microRNAs (miRs).MethodsCachectic colon‐26 (C‐26) tumour‐bearing mice were given 5‐fluourouracil (5‐FU) chemotherapy or vehicle treatment and analysed for muscle mass, fibre size and composition, and miR expression. Mechanisms were validated in vitro using C2C12 cell culture and miR mimics and inhibitors and were confirmed in vivo by injecting muscles of 5‐FU‐treated cachectic mice with recombinant adeno‐associated viral (rAAV) vectors encoding a miR sponge.ResultsIn cachectic tumour‐bearing mice, 5‐FU chemotherapy exacerbated the loss of skeletal muscle mass compared with vehicle treatment (by −12% to −20%, P &lt; 0.05). miR expression profiling, quantitative real‐time PCR, and in vitro analyses revealed contributing mechanisms including miR‐351‐3p‐dependent ERK2 inhibition. Intramuscular injection of rAAV vectors encoding a sponge to reduce miR‐351‐3p expression in 5‐FU‐treated cachectic mice enhanced ERK phosphorylation (+18%, P &lt; 0.05) and increased muscle fibre size (+15%, P &lt; 0.01). Hsa‐miR‐125a‐3p shares similar predicted gene targets as mmu‐miR‐351‐3p, and its inhibition in human muscle cells in vitro prevented 5‐FU‐induced atrophy (P &lt; 0.001) and increased ERK phosphorylation (P &lt; 0.001).ConclusionsThe findings implicate miR‐351‐3p‐mediated ERK2 inhibition as a contributing mechanism in chemotherapy‐induced muscle wasting in mice with cancer cachexia and that its inhibition is a promising adjunct therapy for preserving muscles during cancer treatment.

The Effect of Mechanical Stretch on Myotube Growth Suppression by Colon-26 Tumor-Derived Factors.

Preclinical models and in vitro experiments have provided valuable insight into the regulation of cancer-induced muscle wasting. Colon-26 (C26) tumor cells induce cachexia in mice, and conditioned media (CM) from these cells promotes myotube atrophy and catabolic signaling. While mechanical stimuli can prevent some effects of tumor-derived factors on myotubes, the impact of mechanical signaling on tumor-derived factor regulation of myosin heavy chain (MyHC) expression is not well understood. Therefore, we examined the effects of stretch-induced mechanical signaling on C2C12 myotube growth and MyHC expression after C26 CM exposure. C26 CM was administered to myotubes on day 5 of differentiation for 48 h. During the last 4 or 24 h of C26 CM exposure, 5% static uniaxial stretch was administered. C26 CM suppressed myotube growth and MyHC protein and mRNA expression. Stretch for 24 h increased myotube size and prevented the C26 CM suppression of MyHC-Fast protein expression. Stretch did not change suppressed MyHC mRNA expression. Stretch for 24 h reduced Atrogin-1/MAFbx, MuRF-1, and LC3B II/I ratio and increased integrin β1D protein expression and the myogenin-to-MyoD protein ratio. Stretch in the last 4 h of CM increased ERK1/2 phosphorylation but did not alter the CM induction of STAT3 or p38 phosphorylation. These results provide evidence that in myotubes pre-incubated with CM, the induction of mechanical signaling can still provide a growth stimulus and preserve MyHC-Fast protein expression independent of changes in mRNA expression.

The Effect of Mechanical Stretch on Myotube Growth Suppression by Tumor‐derived Factors

The mechanisms of cancer-induced muscle wasting are widely investigated with in vivo preclinical models and in vitro experiments examining effects of tumor-derived factors. Myogenic differentiation is associated with rapid increases in myotube size and contractile properties, which tumor-derived factors can suppress. Specifically, colon-26 (C26) conditioned media induces catabolic signaling in cultured myotubes. While mechanical stimuli can induce anabolic signaling and myotube growth, the effect of mechanical stretch on myotubes treated with C26 conditioned media (CM) is not known. Therefore, we examined if uniaxial mechanical stretch could reverse myotube growth suppression caused by C26 conditioned media. Cachectic C26 or non-cachectic EL4 lymphoma CM was collected after 48h (~90% confluence). C2C12 myoblasts were seeded on type I collagen-coated polystyrene or Flexcell uniaxial stretch plates, proliferated, then placed in differentiation media for 5-7 days. On day 5 of differentiation, myotubes were treated with 50% control, C26, or EL4 CM for 48h. An additional experiment stretched myotubes 5% for the last 24h of the C26 incubation, and myotubes were collected immediately post stretch. Myotube diameter quantification used digital images. For all comparisons, at least three replicates from 2 independent experiments were used. Myotubes increased diameter (28%, p<0.0001) and myosin heavy chain-fast (MHC-F) protein expression (IOD/ug protein; 127%, p<0.0001) between days 5 and 7 of differentiation. Myotube growth was associated with increased AktT308 phosphorylation and suppressed E3 ligase expression. C26, but not EL4 suppressed myotube diameter (C26: -18%, p<0.0001; EL4: 1%, p=0.708) and MHC-F protein expression (C26: -62%, p<0.0001; EL4: 0%, p=0.999). C26 suppressed AktS473, ERK1/2, and 4E-BP1phosphorylation and increased MuRF1 and Atrogin1 expression. The C26 decrease in myotube diameter was not rescued by 24h of stretch (C26 control: -18%, p<0.0001; C26 stretch: -12%, p=0.0005). Stretch prevented C26 suppression of MHC-F protein expression (C26 control: -18%, p=0.001; C26 stretch: 11%, p=0.389). In summary, MHC-F protein concentration was rescued by mechanical stretch in C26 treated myotubes independent of myotube size. Additional research is warranted to determine how mechanical stretch prevents myosin's catabolic regulation in myotubes by tumor-derived factors.

Mitochondrial dynamics and quality control are altered in a hepatic cell culture model of cancer cachexia.

Hepatic mitochondrial function loss is associated with cancer cachexia pathology in vivo. Here, we examined if hepatic mitochondrial defects observed in vivo in the cachexic liver also recapitulate during the in vitro treatment of mouse hepatocytes with tumor conditioned media. In vitro experiments were combined with proteome-wide expression analysis of cachexic liver tissue curated for mitochondrial dynamics and quality control proteins, to determine the fidelity of hepatic mitochondrial maladaptation in cancer cachexia pathology. AML12 hepatocytes were exposed to colon-26 (C26) and Lewis lung carcinoma (LLC) conditioned media for 6-72h and assayed for cell viability, membrane potential, respiratory function, H2O2 production, total ROS/RNS, and mitochondrial dynamics and quality control proteins by immunoblotting. Liver tissue from cachexic C26 mice was analyzed by TMT-based quantitative proteomics for in vivo comparison. Cell viability, membrane potential, H2O2 production, total ROS/RNS, and respiration were decreased 48-72h after exposure to C26 and/or LLC. Protein expression of treated hepatocytes and cachexic liver tissue showed altered mitochondrial dynamics and quality control, in a manner that suggests limited fusion and content mixing, but also impaired ability to fragment and clear damaged mitochondria. Two strategies to maintain mitochondrial health, therefore, may not be functioning sufficiently in the cachexic liver. Together these findings imply adverse effects of C26 and LLC exposure on hepatocyte health, due to impaired mitochondrial function and remodeling. Exposure of mouse hepatocytes to tumor conditioned media models aspects of cachexic liver mitochondria dysfunction in vivo and validates the importance of hepatic mitochondrial maladaptation in cancer cachexia pathology.

Improved Antitumor Outcomes for Colon Cancer Using Nanomicelles Loaded with the Novel Antitumor Agent LA67.

BackgroundLA67 is a derivative of triptolide that exhibits strong antitumor activity. This derivative has a better safety profile than triptolide, but is limited by poor aqueous solubility.Aim and MethodsTo improve solubility and further increase therapeutic efficacy, we prepared LA67-loaded polymeric micelles (LA67-PMs) using a film hydration method. The physicochemical properties of LA67-PMs were investigated, and the antitumor activity of this formulation against Colon26 (C26) cancer cell line was evaluated in vitro and in vivo with LA67 as a control.ResultsPolymeric micelles containing LA67 had a particle size of 17.88 nm and a drug entrapment efficiency of 94.84%. This formulation dispersed completely in aqueous solution and exhibited slow, sustained release of LA67. Cellular uptake assay showed that LA67-PMs delivered LA67 to cancer cells with greater efficiency than free LA67, which resulted in increased LA67 accumulation in cancer cells. Cell counting kit 8 (CCK-8) assay showed that blank polymeric micelles (PMs) exhibited low toxicity and LA67-PMs exerted pronounced anti-proliferation effects against C26 cells. Furthermore, LA67-PMs induced apoptosis and repressed migration more effectively than free LA67. In vivo evaluation of antitumor activity showed that LA67-PMs inhibited tumor growth and distant organ metastasis to a greater extent than LA67, which resulted in improved survival rate. The potential mechanisms of these effects may have been induction of apoptosis, inhibition of cell proliferation, and neovascularization.ConclusionOur study showed that LA67-PMs may be a promising formulation for treatment of colon cancer.

Tissue-specific dysregulation of mitochondrial respiratory capacity and coupling control in colon-26 tumor-induced cachexia.

In addition to skeletal muscle dysfunction, cancer cachexia is a systemic disease involving remodeling of nonmuscle organs such as adipose and liver. Impairment of mitochondrial function is associated with multiple chronic diseases. The tissue-specific control of mitochondrial function in cancer cachexia is not well defined. This study determined mitochondrial respiratory capacity and coupling control of skeletal muscle, white adipose tissue (WAT), and liver in colon-26 (C26) tumor-induced cachexia. Tissues were collected from PBS-injected weight-stable mice, C26 weight-stable mice and C26 mice with moderate (10% weight loss) and severe cachexia (20% weight loss). The respiratory control ratio [(RCR) an index of oxidative phosphorylation (OXPHOS) coupling efficiency] was low in WAT during the induction of cachexia because of high nonphosphorylating LEAK respiration. Liver RCR was low in C26 weight-stable and moderately cachexic mice because of reduced OXPHOS. Liver RCR was further reduced with severe cachexia, where Ant2 but not Ucp2 expression was increased. Ant2 was inversely correlated with RCR in the liver (r = -0.547, P < 0.01). Liver cardiolipin increased in moderate and severe cachexia, suggesting this early event may also contribute to mitochondrial uncoupling. Impaired skeletal muscle mitochondrial respiration occurred predominantly in severe cachexia, at complex I. These findings suggest that mitochondrial function is subject to tissue-specific control during cancer cachexia, whereby remodeling in WAT and liver arise early and may contribute to altered energy balance, followed by impaired skeletal muscle respiration. We highlight an under-recognized role of liver and WAT mitochondrial function in cancer cachexia and suggest mitochondrial function of multiple tissues to be therapeutic targets.

Muscle and serum metabolomes are dysregulated in colon-26 tumor-bearing mice despite amelioration of cachexia with activin receptor type 2B ligand blockade.

Cancer-associated cachexia reduces survival, which has been attenuated by blocking the activin receptor type 2B (ACVR2B) ligands in mice. The purpose of this study was to unravel the underlying physiology and novel cachexia biomarkers by use of the colon-26 (C26) carcinoma model of cancer cachexia. Male BALB/c mice were subcutaneously inoculated with C26 cancer cells or vehicle control. Tumor-bearing mice were treated with vehicle (C26+PBS) or soluble ACVR2B either before (C26+sACVR/b) or before and after (C26+sACVR/c) tumor formation. Skeletal muscle and serum metabolomics analysis was conducted by gas chromatography-mass spectrometry. Cancer altered various biologically functional groups representing 1) amino acids, 2) energy sources, and 3) nucleotide-related intermediates. Muscle metabolomics revealed increased content of free phenylalanine in cancer that strongly correlated with the loss of body mass within the last 2 days of the experiment. This correlation was also detected in serum. Decreased ribosomal RNA content and phosphorylation of a marker of pyrimidine synthesis revealed changes in nucleotide metabolism in cancer. Overall, the effect of the experimental C26 cancer predominated over blocking ACVR2B ligands in both muscle and serum. However, the level of methyl phosphate, which was decreased in muscle in cancer, was restored by sACVR2B-Fc treatment. In conclusion, experimental cancer affected muscle and blood metabolomes mostly independently of blocking ACVR2B ligands. Of the affected metabolites, we have identified free phenylalanine as a promising biomarker of muscle atrophy or cachexia. Finally, the decreased capacity for pyrimidine nucleotide and protein synthesis in tumor-bearing mice opens up new avenues in cachexia research.

Cachexia induced by cancer and chemotherapy yield distinct perturbations to energy metabolism.

BackgroundCancer cachexia is a metabolic disorder involving perturbed energy balance and altered mitochondrial function. Chemotherapy is a primary treatment option for many types of cancer, but there is substantial evidence that some chemotherapeutic agents can also lead to the development and progression of cachexia. In this study, we apply a comprehensive and systems level metabolomics approach to characterize the metabolic perturbations in murine models of cancer‐induced and chemotherapy‐induced cachexia. Knowledge of the unique pathways through which cancer and chemotherapy drive cachexia is necessary in order to develop effective treatments.MethodsThe murine Colon26 (C26) adenocarcinoma xenograft model was used to study the metabolic derangements associated with cancer‐induced cachexia. In vivo administration of Folfiri (5‐fluorouracil, irinotecan, and leucovorin) was used to model chemotherapy‐induced cachexia. Comprehensive metabolic profiling was carried out using both nuclear magnetic resonance‐based and mass spectrometry‐based platforms. Analyses included plasma, muscle, and liver tissue to provide a systems level profiling.ResultsThe study involved four groups of CD2F1 male mice (n = 4–5), including vehicle treated (V), C26 tumour hosts (CC), Folfiri treated (F), and C26 tumour hosts treated with Folfiri (CCF). Significant weight loss including skeletal muscle was observed for each of the experimental groups with the tumour hosts showing the most dramatic change (−3.74 g vs. initial body weight in the CC group). Skeletal muscle loss was evident in all experimental groups compared with V, with the CCF combination resulting in the most severe depletion of quadriceps mass (−38% vs. V; P < 0.001). All experimental groups were characterized by an increased systemic glucose demand as evidenced by decreased levels of circulating glucose (−47% in CC vs. V; P < 0.001) and depletion of liver glucose (−51% in CC vs. V; P < 0.001) and glycogen (−74% in CC vs. V; P < 0.001). The cancer‐induced and chemotherapy‐induced cachexia models displayed unique alterations in flux through the tricarboxylic acid cycle and β‐oxidation pathways. Cancer‐induced cachexia was uniquely characterized by a dramatic elevation in low‐density lipoprotein particles (+6.9‐fold vs. V; P < 0.001) and a significant increase in the inflammatory marker, GlycA (+33% vs. V; P < 0.001).ConclusionsThe results of this study demonstrated for the first time that cancer‐induced and chemotherapy‐induced cachexia is characterized by a number of distinct metabolic derangements. Effective therapeutic interventions for cancer‐induced and chemotherapy‐induced cachexia must take into account the specific metabolic defects imposed by the pathological or pharmacological drivers of cachexia.

Muscle regeneration is disrupted by cancer cachexia without loss of muscle stem cell potential.

Cancer cachexia is a severe, debilitating condition characterized by progressive body wasting associated with remarkable loss of skeletal muscle weight. It has been reported that cancer cachexia disturbs the regenerative ability of skeletal muscle, but the cellular mechanisms are still unknown. Here, we investigated the skeletal muscle regenerative process in mouse colon-26 (C26) tumor cell-bearing mice as a C26 cancer cachexia model. Although the proliferation and differentiation abilities of muscle stem cells derived from the C26 tumor cell-bearing mice were sustained in vitro, the proliferation and differentiation were severely impaired in the cachexic mice. The numbers of both macrophages and mesenchymal progenitors, which are critical players in muscle regeneration, were reduced in the cancer cachexic mice, indicating that the skeletal muscle regeneration process was disrupted by cancer cachexia. Furthermore, the number of infiltrated neutrophils was also reduced in cancer cachexia mice 24 hours after muscle injury, and the expression of critical chemokines for muscle regeneration was reduced in cancer cachexia model mice compared to control mice. Collectively, although the ability to regeneration of MuSCs was retained, cancer cachexia disturbed skeletal muscle regenerative ability by inhibiting the orchestrated muscle regeneration processes.

Transgenic mice that accept Luciferase- or GFP-expressing syngeneic tumor cells at high efficiencies.

Jellyfish green fluorescent protein (GFP) and firefly luciferase can serve as versatile tracking markers for identification and quantification of transplanted cancer cells in vivo. However, immune reactions against these markers can hamper the formation of syngraft tumors and metastasis that follows. Here, we report two transgenic (Tg) mouse lines that express nonfunctional mutant marker proteins, namely modified firefly luciferase (Luc2) or enhanced GFP (EGFP). These mice, named as Tg-mLuc2 and Tg-mEGFP, turned out to be immunologically tolerant to the respective tracking markers and thus efficiently accepted syngeneic cancer cells expressing the active forms of the markers. We then injected intrarectally the F1 hybrid Tg mice (BALB/c×C57BL/6J) with Colon-26 (C26) colon cancer cells that originated from a BALB/c mouse. Even when C26 cells expressed active Luc2 or EGFP, they formed primary tumors in the Tg mice with only 104 cells per mouse compared with more than 106 cells required in the nontransgenic BALB/c hosts. Furthermore, we detected metastatic foci of C26 cells in the liver and lungs of the Tg mice by tracking the specific reporter activities. These results show the usefulness of the Tg mouse lines as recipients for transplantation experiments with the non-self tracking marker-expressing cells.

Mitochondrial Respiratory Capacity and Coupling Control of Skeletal Muscle in Colon-26 Tumor-Induced Cachexia

Cancer cachexia is a life-threatening, paraneoplastic syndrome featuring unintended weight loss and skeletal muscle atrophy. Mitochondria, the major providers of cellular energy, couple oxygen consumption to ATP synthesis (i.e. oxidative phosphorylation, OXPHOS). Impaired mitochondrial bioenergetics (e.g. respiration) is associated with the pathophysiology of multiple diseases. The control of mitochondrial respiration in skeletal muscle during the induction and progression of cancer cachexia is not well understood. PURPOSE: To investigate mitochondrial respiratory capacity and coupling control of skeletal muscle in the colon-26 model of cancer cachexia. METHODS: Balb/c males (10 wks) were assigned to control or colon-26 (C26). C26 mice were injected with 1x106 tumor cells, and tissue collected on days 7, 14, and 21 post-injection. In this model, mice develop palpable tumors at day 7, and cachexia by day 21. Controls were injected with PBS and tissue collected on day 0. Respiration was measured in permeabilized fibers from the medial gastrocnemius via high-resolution respirometry. A substrate-uncoupler-inhibitor titration protocol was used to evaluate Complex I OXPHOS (CIP), Complex I+II OXPHOS (CI+IIP), and electron transfer system capacity (ETS). Efficiency of the OXPHOS system was determined from the ratio CI+IIP/ETS (P/E). RESULTS: CIP was significantly lower (p<0.05) at day 21 (4.8±1.6 pmol/s/mg) in comparison to day 0 (53.4±7.0), day 7 (57.4±6.5), and day 14 (60.0±2.9). CI+IIP was significantly lower (p<0.05) at day 21 (22.3±2.3 pmol/s/mg) in comparison to day 0 (65.5±7.8), day 7 (69.3±8.5), and day 14 (73.8±4.8). Maximal ETS was significantly lower (p<0.05) at day 21 (24.1 pmol/s/mg) in comparison to day 0 (83.7±13.8), day 7 (84.4±12.3), and day 14 (105.1±7.3). P/E was not significantly different across timepoints (p>0.05). CONCLUSION: Phosphorylating respiration with electron input from Complex I and I+II, and maximal electron transfer system capacity (i.e. non-coupled respiration) was significantly reduced at day 21 concomitant with cachexia, but not at earlier timepoints, suggesting that changes in oxidative metabolism occur as a consequence of cachexia rather than having a causative role. Loss of respiratory capacity may compromise muscle function and physical independence.

Bone mineral density and content are differentially impacted by aerobic and resistance training in the colon-26 mouse model of cancer cachexia

BackgroundCancer cachexia is a debilitating paraneoplastic syndrome featuring unintended weight loss and skeletal muscle atrophy. Evidence suggests that bone health may also be compromised, further limiting mobility and quality of life. Aerobic and resistance training was recently reported to differentially affect skeletal muscle adaptations in cancer cachectic mice. The purpose of this investigation was to assess the effects of aerobic and resistance training on bone mineral density (BMD) and bone mineral content (BMC) in mice with colon-26 (C26) tumor-induced cachexia.MethodsTwelve-month old Balb/c mice were aerobic-trained (wheel running 5 days/week) or resistance-trained (weighted ladder climbing 3days/week) for 8 weeks prior to C26 cell injection, followed by an additional three weeks of exercise. BMD and BMC were assessed pre- and post-training by dual-energy x-ray absorptiometry.ResultsResistance-trained C26 mice lost total BMD by 7% (p = 0.06), which did not occur in aerobic-trained C26 mice. In terms of pelvic bone, both resistance- and aerobic-trained C26 mice had significantly lower BMD values (−12%, p = 0.01 and −6%, p = 0.04, respectively), albeit to a lesser degree in aerobic-trained C26 mice. Furthermore, resistance-trained C26 mice tended to lose total BMC (−12%), whereas aerobic-trained C26 mice maintained total BMC. In mice without C26 tumors, resistance training significantly increased total BMD (+13%, p = 0.001).ConclusionsAerobic and resistance training may differentially affect bone status in C26 cancer cachexia, with high resistance loading possibly being detrimental to total and pelvis BMD, a region expected to bear significant loading stress and contribute substantially to overall mobility. Because resistance training improved BMD in tumor-free mice, the C26 tumor burden appeared to impair the beneficial effect of resistance training on bone mass.

Oral Treatment with the Ghrelin Receptor Agonist HM01 Attenuates Cachexia in Mice Bearing Colon-26 (C26) Tumors.

The gastrointestinal hormone ghrelin reduces energy expenditure and stimulates food intake. Ghrelin analogs are a possible treatment against cancer anorexia-cachexia syndrome (CACS). This study aimed to investigate whether oral treatment with the non-peptidergic ghrelin receptor agonist HM01 counteracts CACS in colon-26 (C26) tumor-bearing mice. The C26 tumor model is characterized by pronounced body weight (BW) loss and muscle wasting in the absence of severe anorexia. We analyzed the time course of BW loss, body composition, muscle mass, bone mineral density, and the cytokines interleukin-6 (IL-6) and macrophage-inhibitory cytokine-1 (MIC-1). Moreover, we measured the expression of the muscle degradation markers muscle RING-finger-protein-1 (MuRF-1) and muscle atrophy F-box (MAFbx). After tumor inoculation, MIC-1 levels increased earlier than IL-6 and both cytokines were elevated before MuRF-1/MAFbx expression increased. Oral HM01 treatment increased BW, fat mass, and neuronal hypothalamic activity in healthy mice. In tumor-bearing mice, HM01 increased food intake, BW, fat mass, muscle mass, and bone mineral density while it decreased energy expenditure. These effects appeared to be independent of IL-6, MIC-1, MuRF-1 or MAFbx, which were not affected by HM01. Therefore, HM01 counteracts cachectic body weight loss under inflammatory conditions and is a promising compound for the treatment of cancer cachexia in the absence of severe anorexia.

Increased hypoxia-inducible factor-1α in striated muscle of tumor-bearing mice.

Cancer cachexia is a progressive wasting disease resulting in significant effects on the quality of life and high mortality. Most studies on cancer cachexia have focused on skeletal muscle; however, the heart is now recognized as a major site of cachexia-related effects. To elucidate possible mechanisms, a proteomic study was performed on the left ventricles of colon-26 (C26) adenocarcinoma tumor-bearing mice. The results revealed several changes in proteins involved in metabolism. An integrated pathway analysis of the results revealed a common mediator in hypoxia-inducible factor-1α (HIF-1α). Work by other laboratories has shown that extensive metabolic restructuring in the C26 mouse model causes changes in gene expression that may be affected directly by HIF-1α, such as glucose metabolic genes. M-mode echocardiography showed progressive decline in heart function by day 19, exhibited by significantly decreased ejection fraction and fractional shortening, along with posterior wall thickness. Using Western blot analysis, we confirmed that HIF-1α is significantly upregulated in the heart, whereas there were no changes in its regulatory proteins, prolyl hydroxylase domain-containing protein 2 (PHD2) and von Hippel-Lindau protein (VHL). PHD2 requires both oxygen and iron as cofactors for the hydroxylation of HIF-1α, marking it for ubiquination via VHL and subsequent destruction by the proteasome complex. We examined venous blood gas values in the tumor-bearing mice and found significantly lower oxygen concentration compared with control animals in the third week after tumor inoculation. We also examined select skeletal muscles to determine whether they are similarly affected. In the diaphragm, extensor digitorum longus, and soleus, we found significantly increased HIF-1α in tumor-bearing mice, indicating a hypoxic response, not only in the heart, but also in skeletal muscle. These results indicate that HIF-1α may contribute, in part, to the metabolic changes that occur during cancer cachexia.NEW & NOTEWORTHY We used proteomics and metadata analysis software to identify contributors to metabolic changes in striated muscle during cancer cachexia. We found increased expression of hypoxia-inducible factor-1α in the heart and skeletal muscle, suggesting a potential target for the therapeutic treatment of cancer cachexia.

Cancer and Chemotherapy Contribute to Muscle Loss by Activating Common Signaling Pathways.

Cachexia represents one of the primary complications of colorectal cancer due to its effects on depletion of muscle and fat. Evidence suggests that chemotherapeutic regimens, such as Folfiri, contribute to cachexia-related symptoms. The purpose of the present study was to investigate the cachexia signature in different conditions associated with severe muscle wasting, namely Colon-26 (C26) and Folfiri-associated cachexia. Using a quantitative LC-MS/MS approach, we identified significant changes in 386 proteins in the quadriceps muscle of Folfiri-treated mice, and 269 proteins differentially expressed in the C26 hosts (p < 0.05; −1.5 ≥ fold change ≥ +1.5). Comparative analysis isolated 240 proteins that were modulated in common, with a large majority (218) that were down-regulated in both experimental settings. Interestingly, metabolic (47.08%) and structural (21.25%) proteins were the most represented. Pathway analysis revealed mitochondrial dysfunctions in both experimental conditions, also consistent with reduced expression of mediators of mitochondrial fusion (OPA-1, mitofusin-2), fission (DRP-1) and biogenesis (Cytochrome C, PGC-1α). Alterations of oxidative phosphorylation within the TCA cycle, fatty acid metabolism, and Ca2+ signaling were also detected. Overall, the proteomic signature in the presence of both chemotherapy and cancer suggests the activation of mechanisms associated with movement disorders, necrosis, muscle cell death, muscle weakness and muscle damage. Conversely, this is consistent with the inhibition of pathways that regulate nucleotide and fatty acid metabolism, synthesis of ATP, muscle and heart function, as well as ROS scavenging. Interestingly, strong up-regulation of pro-inflammatory acute-phase proteins and a more coordinated modulation of mitochondrial and lipidic metabolisms were observed in the muscle of the C26 hosts that were different from the Folfiri-treated animals. In conclusion, our results suggest that both cancer and chemotherapy contribute to muscle loss by activating common signaling pathways. These data support the undertaking of combination strategies that aim to both counteract tumor growth and reduce chemotherapy side effects.