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- Material and Methods
- Supporting Information
Cancer cachexia describes the progressive skeletal muscle wasting and weakness associated with many cancers. Cachexia reduces mobility and quality of life and accounts for 20–30% of all cancer-related deaths. Activation of the renin–angiotensin system causes skeletal muscle wasting and weakness. We tested the hypothesis that treatment with the angiotensin converting enzyme (ACE) inhibitor, perindopril, would enhance whole body and skeletal muscle function in cachectic mice bearing Colon-26 (C-26) tumors. CD2F1 mice received a subcutaneous injection of phosphate buffered saline or C-26 tumor cells inducing either a mild or severe cachexia. The following day, one cohort of C-26 mice began receiving perindopril in their drinking water (4 mg kg−1 day−1) for 21 days. In mild and severe cachexia, perindopril increased measures of whole body function (grip strength and rotarod) and reduced fatigue in isolated contracting diaphragm muscle strips (p < 0.05). In severely cachectic mice, perindopril reduced tumor growth, improved locomotor activity and reduced fatigue of tibialis anterior muscles in situ (p < 0.05), which was associated with increased oxidative enzyme capacity (succinate deyhydrogenase, p < 0.05). Perindopril attenuated the increase in MuRF-1 and IL-6 mRNA expression and enhanced Akt phosphorylation in severely cachectic mice but neither body nor muscle mass was increased. These findings support the therapeutic potential of ACE inhibition for enhancing whole body function and reducing fatigue of respiratory muscles in early and late stage cancer cachexia and should be confirmed in future clinical trials. Since ACE inhibition alone did not enhance body or muscle mass, co-treatment with an anabolic agent may be required to address these aspects of cancer cachexia.
Cancer cachexia is a complex multifactorial syndrome characterized by a progressive loss of skeletal muscle mass with or without loss of fat mass and is associated with significant functional impairments. Cachexia is present in up to 80% of patients with advanced cancer and in 60–80% of those diagnosed with gastrointestinal, pancreatic and lung cancer. It can develop in stages from pre-cachexia to cachexia to refractory cachexia. Cachexia causes severe fatigue and reduces mobility, leading to a loss of functional independence and a reduction in overall quality of life. It can also impair the response to chemotherapy, and the eventual failure of respiratory and cardiac muscle function is responsible for 20–30% of all cancer-related deaths. Treatments are therefore needed to improve patient quality of life and reduce mortality.
The renin–angiotensin system (RAS) is typically associated with the regulation of blood pressure and water balance and has well-characterized effects including vasoconstriction. RAS inhibition is used widely for treating hypertension. Local RAS exist in multiple tissues and have diverse effects. Angiotensin peptides such as angiotensin I (Ang I) and Ang II are produced within skeletal muscle[5, 6] and stimulation of these peptides may contribute to muscle breakdown, either directly or indirectly by numerous mechanisms including enhanced protein degradation, reduced protein synthesis, inflammation, and apoptosis. RAS expression is up-regulated in several conditions associated with muscle wasting and weakness, including sarcopenia, muscular dystrophies, chronic heart failure and chronic renal failure; and may contribute to their pathophysiology. RAS upregulation occurs in patients with colorectal liver metastases and laryngeal cancer which are both associated with severe cachexia. RAS inhibition therefore represents a potential therapeutic strategy for reducing cancer cachexia. Supporting this contention is our recent finding that mice lacking the angiotensin type 1A receptor (AT1A−/−) have increased whole body and skeletal muscle function compared with wild type mice.
AT1 antagonism and inhibition of the angiotensin converting enzyme (ACE), which mediates conversion of Ang I to Ang II, have been shown to improve skeletal muscle pathophysiology in conditions associated with muscle wasting including laceration injury, sarcopenia and the muscular dystrophies. Only one preclinical study has investigated the therapeutic potential of RAS inhibition for attenuating cancer cachexia and found that treatment with a high dose (30 mg kg−1) of the ACE inhibitor, imidapril, attenuated both the loss of body mass and tumor growth in a murine model of cancer cachexia. However, this study did not test whether imidapril also improved skeletal muscle function, which is an important unresolved question since this is the main outcome affecting the quality of life and mortality of patients with cancer cachexia. Although there are no listed clinical studies (on PubMed), a nonpeer-reviewed report of a Phase III clinical trial by Ark Therapeutics found that imidapril significantly attenuated the loss of body mass and reduction in grip strength in cachectic patients with nonsmall cell lung cancer (NSCLC) and colorectal cancer, but not pancreatic cancer. However, when the results for all patients were combined, the improvements were not statistically significant. Taken together, the limited data available support the therapeutic potential of RAS inhibition for cancer cachexia, but there is an unmet need to more comprehensively investigate the efficacy of RAS inhibition for attenuating functional deficits in cancer cachexia. We examined the efficacy of the long-acting ACE inhibitor, perindopril, for enhancing whole body and skeletal muscle function in mildly cachectic and severely cachectic mice bearing Colon-26 (C-26) tumors. As treatments are initiated at different stages of the cachexia spectrum, it is important to test the efficacy of potential therapies in models with varying degrees of cachexia to maximize translation of preclinical findings. We therefore tested the hypothesis that perindopril would increase whole body and skeletal muscle function in both mildly cachectic and severely cachectic C-26 tumor-bearing mice.
- Top of page
- Material and Methods
- Supporting Information
The most important finding of clinical relevance from this study was the demonstrated efficacy of the long-acting ACE inhibitor, perindopril, to enhance whole body and skeletal muscle function in tumor-bearing mice at different stages of the cachexia spectrum. In both mildly and severely cachectic mice, perindopril improved whole body function and reduced fatigue of diaphragm muscle strips. In severely cachectic mice, perindopril also improved locomotor activity and attenuated the deleterious metabolic aberrations in untreated cachectic mice. Taken together, these findings highlight the therapeutic potential of ACE inhibition for enhancing whole body function and reducing fatigue of respiratory muscles in early and late stage cancer cachexia and should be confirmed in future clinical studies.
Patients with cancer cachexia have a 25% reduction in grip strength which affects their ability to perform everyday tasks such as rising from a chair or bed, performing home duties and maintaining personal hygiene. The reduction in grip strength in cachectic patients is also correlated strongly with postoperative complications. Perindopril prevented completely the 22% decrease in grip strength in severely cachectic mice and increased grip strength in mildly cachectic mice. These findings are consistent with a Phase III clinical trial showing improved grip strength with imidapril in cachectic NSCLC patients. They also indicate that ACE inhibition could improve the ability of affected patients to perform even the simplest tasks and may reduce their risk of postoperative complications.
Cachectic patients have impaired mobility and lower levels of physical activity that reduces their functional independence. Perindopril prevented the decrease in mobility as assessed by rotarod performance and attenuated the reduction in locomotor activity in severely cachectic mice, and enhanced mobility in mildly cachectic mice. The reduction in physical activity levels in cachectic patients is due to a combination of pain, fatigue and depression, and a vicious cycle ensues whereby reduced physical activity levels compound the depression. Physical activity levels in our severely cachectic mice were monitored over 24 hr, but it would be of interest to examine whether perindopril increased the performance of voluntary exercise in tumor-bearing mice. The maintenance of physical activity levels might not only attenuate cachexia, but also reduce depression.
Respiratory failure is one of the major causes of death in cancer cachexia. Remarkably, perindopril was able to reduce fatigue of isolated diaphragm muscle strips from mice with mild and severe cachexia. These findings support those of a recent study reporting diaphragm muscle wasting after Ang II infusion in mice, and are consistent with perindopril attenuating the reduction in and in severely cachectic mice. The improved fatigue resistance with perindopril is clinically significant since respiratory muscles work continuously during life and cachectic patients often die from respiratory failure. By reducing fatigue of respiratory muscles, ACE inhibition could potentially prolong survival of cachectic patients.
Limb muscle strength is impaired in cancer cachexia, causing affected patients to reduce their functional independence. Perindopril attenuated the reduction in submaximal force and reduced fatigue in TA muscles of severely cachectic mice. Given that the force required of muscles for normal daily activities is never close to maximal, the improved submaximal forces with perindopril would be beneficial for performing normal daily tasks. The fatigue resistance with perindopril was associated with increased fiber oxidative enzyme capacity (SDH) in both the fast oxidative type IIa fibers and the fast glycolytic type IIx/b fibers. As severe fatigue is a devastating consequence of cachexia that affects nearly all cancer patients, attenuating fatigue would dramatically improve the quality of life of affected patients. The improvements in whole body and skeletal muscle function with perindopril occurred without affecting muscle mass or fiber size; findings consistent with our previous observation that AT1A−/− mice have increased whole body and skeletal muscle function despite having smaller muscles. The improvements in these mice were attributed to a fiber-type shift toward a greater proportion of fast glycolytic type IIx/b fibers and a smaller proportion of fast oxidative type IIa fibers. However, a similar fiber-type shift was not found in the current study so the mechanisms contributing to the improvement in muscle function with perindopril remain uncertain and should be investigated in future studies. Although some previous studies have found no effect of acute RAS inhibition on muscle mass in dystrophic mdx mice, other studies have reported increased muscle mass. Real-Time RT-PCR analyses confirmed that perindopril prevented the increase in ACE mRNA in severely cachectic mice. Perindopril also attenuated expression of genes involved in protein degradation (MuRF-1, atrogin-1) and inflammation (IL-6), and proteins involved in apoptosis (caspase3), as well as increased expression of proteins involved in protein synthesis (phosphorylated Akt). These findings are consistent with previous reports showing that stimulation of Ang I and Ang II induce skeletal muscle breakdown by enhancing protein degradation, inflammation and muscle apoptosis, and by reducing protein synthesis.
Metabolic abnormalities including increased fat oxidation and reduced CHO oxidation are prevalent in patients with cancer cachexia and are thought to contribute to the pathogenesis. Remarkably, perindopril prevented the metabolic alterations in severely cachectic tumor-bearing mice, returning fat and CHO oxidation and RER to levels similar to those of PBS controls. The increase in fat oxidation and the reduction in CHO oxidation in cachectic patients has been linked to insulin resistance and correction of these abnormalities is consistent with the well characterized improvement in whole body sensitivity with ACE inhibition.
Experimental studies in rodents have reported that ACE inhibition reduced the size of various tumors including colorectal liver tumors and MAC16 colon tumors. Retrospective studies have also shown that ACE inhibition is associated with attenuation of tumor growth of the prostate, lung, colon and breast. To our knowledge, this is the first study showing that ACE inhibition reduced the size of C-26 tumors from severely cachectic mice. Interestingly, perindopril had no effect on C-26 tumor size in mildly cachectic mice. ACE inhibition has been proposed to inhibit tumor angiogenesis and growth, and to induce apoptosis. Although beyond the scope of this study, future studies should compare the effects of perindopril on the angiogenesis, growth and apoptosis of tumors induced with the two different C-26 cell lines. Importantly, the differing effects of perindopril on C-26 tumor size indicate that the improvements in whole body and skeletal muscle function in the severely cachectic mice was not simply due to a smaller tumor burden.
Cardiac atrophy leading to cardiac failure is one of the major causes of death in cancer cachexia and is the cause of >7% of all cancer-related deaths. Anti-cancer therapies can cause cardiotoxicity and exacerbate the cardiac dysfunction in cancer patients. Consistent with our previous study, cardiac atrophy was apparent in mice with severe but not mild cachexia. Heart failure has been linked to reduced expression of α-MHC due to slowed rates of rise of force and reduced ejection time and functional cardiac impairments in C-26 tumor-bearing mice have been associated with reduced α-MHC expression. Although assessment of cardiac function was beyond the scope of the current study, the mRNA expression of α-MHC was reduced in C-26 tumor-bearing mice indicating impaired cardiac function. ACE inhibitors have well known cardioprotective effects and can prevent cardiotoxicity and improve cardiac function in patients receiving chemotherapy. ACE inhibitors also reduce heart mass by lowering blood pressure, decreasing cardiac load and increasing cardiac α-MHC expression. Consistent with these effects, perindopril reduced heart mass and attenuated the decrease in α-MHC mRNA in tumor-bearing mice, indicating functional improvement with perindopril and that the reduction in heart mass was physiological rather than pathological.