Chemotherapeutic agents induce mitochondrial superoxide production and toxicity but do not alter respiration in skeletal muscle in vitro
Introduction
Characterised by abnormal cell growth, cancer constitutes a group of diseases which is rising in incidence and is a primary contributor to worldwide mortality rates and disease burden (Stewart and Wild, 2014). Alongside surgical resection (for solid tumours) and radiotherapy, chemotherapy is a staple of anti-cancer treatment. Chemotherapeutic agents are cytotoxic drugs that impart anti-cancer efficacy either at the DNA or RNA level to arrest the cell cycle and inhibit the rapid cellular proliferation characteristic of cancerous tissues (Ad et al., 2000, Ogston et al., 2003). Chemotherapeutic agents can be classified according to their chemical structure and specific mode of action (Sorensen et al., 2016) and administered either alone or in combination regimens at varying dosages and for varying duration.
Whilst chemotherapy is an effective first-line treatment against cancer, it notoriously induces a myriad of serious clinical complications that range in severity from alopecia to cardiomyopathy, but which in all cases, severely impact patient quality of life (QoL) (Zitvogel et al., 2008, Gilliam and St Clair, 2011, Greene et al., 1993). This is because chemotherapy is delivered systemically and can both penetrate and mediate effects in healthy tissues (Fojo, 2001, Lind, 2008) – albeit side-effects are most obvious in highly mitotic tissues such as hair, skin and gastrointestinal epithelium (Greene et al., 1993, Coates et al., 1983, Love et al., 1989). One side-effect of chemotherapy that has emerged as a significant predictor of post-treatment morbidity, mortality and/or reduced QoL is skeletal muscle wasting and dysfunction (Zitvogel et al., 2008, Järvelä et al., 2012, van Brussel et al., 2006, Scheede-Bergdahl and Jagoe, 2013, Miyamoto et al., 2015, Blauwhoff-Buskermolen et al., 2016). To this effect, the maintenance of skeletal muscle mass – and therefore body mass – is required to maintain optimal dosage and tolerability of chemotherapy (Ali et al., 2016) and other anti-neoplastic treatment options (Prado et al., 2009, Antoun et al., 2010). It is only in recent years that data has emerged highlighting chemotherapeutic drugs are capable of independently promoting skeletal muscle dysfunction and wasting (Bredahl et al., 2016, Dirks-Naylor et al., 2013, Gilliam et al., 2009, Gilliam et al., 2011, Gilliam et al., 2012, Smuder et al., 2011). This is particularly true for the anthracycline doxorubicin (DOX), which induces an array of deleterious effects in skeletal muscle including atrophy signaling, insulin resistance and glucose dysregulation, weakness and fatigue (Bredahl et al., 2016, Dirks-Naylor et al., 2013, Gilliam et al., 2009, Gilliam et al., 2011, Gilliam et al., 2012, Smuder et al., 2011, Ariaans et al., 2015). These effects are secondary to its potent capacity to induce oxidative stress as a consequence of drug metabolism by oxidases (Davies and Doroshow, 1986, Doroshow and Davies, 1986), which are abundant in skeletal muscle. However, novel data also demonstrates that clinically-relevant combination chemotherapies including FOLFOX (folinic acid (leucovorin), 5-fluorouracil (5FU) and oxaliplatin (OX)) and FOLFIRI (leucovorin, 5FU and irinotecan (IR)) also promote skeletal muscle wasting and dysfunction in mice (Barreto et al., 2016a). Notably, each of these drugs derives from a different class of drugs with unique mechanistic action. For example, DOX is an anti-tumor antibiotic and topoisomerase II inhibitor that blocks all stages of the cell cycle; IR is a specific topoisomerase II inhibitor that blocks DNA replication; OX is an alkylating agent that generates DNA lesions and arrests DNA and RNA synthesis; and 5FU is an antimetabolite, which inhibits DNA and RNA formation (reviewed in (Sorensen et al., 2016)). This suggests that skeletal muscle can be deleteriously affected by chemotherapeutic agents from a variety of drug classes, potentially via a common pathogenesis. Furthermore, chemotherapy-induced myopathy appears to be most detrimental and persistent when drugs are administered during early childhood, presumably when skeletal muscle growth is hyperplastic and skeletal muscle stem (satellite) cells are highly proliferative (Scheede-Bergdahl and Jagoe, 2013, Ness et al., 2007, Ness et al., 2012). These data suggest that mechanisms controlling growth rather than maintenance are more severely impacted.
While the mechanisms underlying chemotherapy-induced skeletal muscle wasting and dysfunction remain mostly uncharacterised, the general consensus is that the induction of skeletal muscle atrophy is almost always preceded in the first instance by elevated mitochondrial reactive oxygen species (mtROS) production (Gilliam and St Clair, 2011, Dirks-Naylor et al., 2013, Gilliam et al., 2012, Davies and Doroshow, 1986, Doroshow and Davies, 1986, Chen et al., 2003, Chen et al., 2007). This suggests that the mitochondria might be inadvertent targets of chemotherapeutic agents such that mitochondrial damage and/or dysfunction drives ROS production or vice versa, ultimately reducing cellular energy production capacity and inducing skeletal muscle wasting and dysfunction (Sorensen et al., 2016, Gilliam and St Clair, 2011, Gilliam et al., 2009, Gilliam et al., 2011, Gilliam et al., 2012, Gouspillou et al., 2015). In the past decade mitochondria have emerged as key players in the pathogenesis of a variety of diseases, with mitochondrial dysfunction and toxicity manifesting as skeletal muscle-specific symptomology (Scheede-Bergdahl and Jagoe, 2013, Frohnert and Bernlohr, 2013, Gilliam et al., 2013, Holzerová and Prokisch, 2015, Hsin-Chen et al., 2000, Ide et al., 2001, Kerr, n.d, Lenaz, 2012, Powers et al., 2012, Talvensaari et al., 1996). As such, we have recently proposed a hypothetical model via which chemotherapeutic agents might target the mitochondria to promote skeletal muscle wasting (Sorensen et al., 2016). Indeed, our hypothesis has been given credence by recent data highlighting that the overexpression of the mitochondrial antioxidant enzyme catalase can reduce DOX-induced skeletal muscle wasting and dysfunction in mice (Gilliam et al., 2016).
Although the anti-neoplastic effects of chemotherapeutic agents are well established, there is little data describing their effects on the skeletal muscular system with the exception of DOX (Bredahl et al., 2016, Gilliam et al., 2013). Therefore, the aim of this study was to characterise the effects of a variety of chemotherapeutic agents on mitotically active skeletal muscle myoblasts and differentiated skeletal muscle myotubes. Specifically, we aimed to determine whether a selection of chemotherapeutic agents typically used in the treatment of colorectal cancer (DOX, OX, IR, 5FU and OX + 5FU) and from different drug classes, induce deleterious effects on mitochondrial function, viability and ROS production and whether this results in the compromise of skeletal muscle size.
Section snippets
Establishment and maintenance of C2C12 myoblast and myotube cultures
A commercially available C2C12 mouse myoblast cell line (ATCC via Sigma, Australia) was obtained and cryogenically preserved in liquid nitrogen at 300,000 cells/vial. C2C12 cells were grown and maintained in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with 10% foetal bovine serum (Fisher Biotec) and 1% penicillin streptomycin (Gibco) (referred to from this point onward as growth media (GM)) at 37 °C with 5% carbon dioxide. Cells were seeded at 100,000 cells/well in 6 well
Dose-response relationships
In order to derive a treatment dosage that was sufficiently toxic to enable the quantification of potential mechanisms, but which was not so toxic that there were no cells available to assay, dose-response curves and the LD50 were determined for each chemotherapeutic agent. The effect of in vitro chemotherapeutic agent treatment on C2C12 myoblasts was established for DOX, IR, OX, 5FU and an OX + 5FU combination (Fig. 1), and the 5FU VEH (data not shown). For DOX, concentrations spanning 10 pM to 1
Discussion
Mitochondrial damage, dysfunction and ROS production have been implicated in the skeletal muscle wasting and dysfunction induced by systemic chemotherapy treatment in human patients and animal models (Sorensen et al., 2016, Scheede-Bergdahl and Jagoe, 2013). We have thus investigated the effects of chemotherapeutic agents from various drug classes that are typically used in the treatment of colorectal cancer, on mitochondrial function in skeletal muscle. Using in vitro C2C12 myoblast and
Limitations
One notable limitation of our study was that a complete dose-response curve could not be obtained for 5FU or the OX + 5FU combination treatment due to a > 10% DMSO concentration being required as a solvent, which itself induced cell death. Indeed, this is a limitation that we have also observed in our animals studies in which the 10% DMSO vehicle has a considerable negative impact upon body weights and lean mass compared to untreated control animals (Campelj, D. & Rybalka, E., personal
Conclusions
In summary, we have demonstrated in the present study that chemotherapeutic agents, regardless of chemotype, reduce the viability of the mitochondrial pool in in vitro myoblast and myotube cultures. Concomitant with the loss of mitochondrial viability was a reduction in myotube diameter, which was not associated with the loss of mitochondrial function and therefore the bioenergetical status of the cells. In myoblasts, loss of mitochondrial viability was also associated with a drastically
Competing interests
The authors declare no conflicts of interest.
Funding
This work was supported by grants from the College of Health and Biomedicine Research Interruption Enhancement Program (ER) and the Institute of Sport, Exercise and Active Living (ISEAL) Clinical Exercise Program funding schemes (both Victoria University).
Authors' contributions
ER, AH and KN conceived the study, obtained funding and designed the experiments. BC, CT and JS contributed to the acquisition of the data. ER, CT, BC and JS and contributed to the analysis of the data. ER wrote the manuscript. All authors contributed to the interpretation of the data and the editing of the manuscript. All authors approve of the final manuscript submission and agree to be accountable for all aspects of the work.
Acknowledgements
The authors wish to thank Dr. Erik Hanson for assistance with the establishment of the C2C12 cell cultures.
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