The (un)SAFE and RISK(y) sides of doxorubicin-induced cardiotoxicity

[摘要] ENGLISH ABSTRACT: IntroductionThe discovery of Doxorubicin in the 1960s has drastically improved the survival rates of cancerpatients, however, its success is limited by dose-dependent cardiotoxicity. While much of the literaturehas focused on acute cardiotoxicity which is minor and generally reversible, chronic cardiotoxicityposes a serious threat to cancer survivors since it can lead to dilative cardiomyopathy, congestive heartfailure and even death. The mechanisms that contribute to cardiotoxicity are still a matter ofcontroversy, however, oxidative stress-induced myocardial damage and apoptosis are thought to be themajor role players. Reperfusion injury, also characterized by oxidative stress and apoptosis, occurs as aresult of restoring blood flow to an ischemic heart. Fortunately, pre- and post-conditioning aretechniques employed to minimize this damage and are thought to do so by activating the reperfusioninjury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways. The RISKpathway involves the pro-survival kinases, Erk1/2 and Akt, while the SAFE pathway, triggered byTNF-α, involves Jak2 and STAT3. Since both reperfusion injury and Doxorubicin-inducedcardiotoxicity share similar characteristics, this study aimed to determine whether the RISK and SAFEpathways are activated in response to long-term Doxorubicin treatment. Furthermore, this study aimedto determine whether TNF-α is produced during treatment, since its role in Doxorubicin-inducedcardiotoxicity is still relatively unknown.Methods H9c2 cardiomyocytes and differentiated C2C12 myotubes were treated daily with increasingconcentrations of Doxorubicin for a total of 120 hours. Cell viability, apoptosis and necrosis wereassessed using the MTT, Caspase-Glo® 3/7 and lactate dehydrogenase assays respectively. TNF-αproduction was measured using Quantikine® ELISA kits and various assays were used to assessoxidative stress, anti-oxidant capacity and anti-oxidant status. The protein expression of the RISK andSAFE pathways were analysed by western blotting using both phospho-specific and total antibodies.Results and DiscussionTreatment with Doxorubicin caused a time- and dose-dependent decrease in cell viability in both celllines and this was accompanied by an increase in apoptosis. In the H9c2 cardiomyocytes, treatmentwith 0.2 μM Doxorubicin yielded significant levels of TNF-α after 120 hours and we can speculate thatthese low levels partially protected the cells from the toxic effects of Doxorubicin by activating the SAFE pathway, since both Jak2 and STAT3 were phosphorylated at this concentration. Treatment with1 μM Doxorubicin caused a larger and biphasic pattern of TNF-α release, which may have thencontributed to the decrease in cell viability, since the SAFE pathway was not activated at thisconcentration. Akt was phosphorylated during the first 72 hours of treatment with the low dose ofDoxorubicin, but chronic treatment prevented this phosphorylation. While Erk1/2 was notphosphorylated at all at the low dose of Doxorubicin, neither Akt nor Erk1/2 was phosphorylated at thehigh dose and their inhibition may contribute to the cardiotoxic effects of Doxorubicin. In the C2C12myotubes, a significant amount of TNF-α was produced after 120 hours of treatment with the low doseof Doxorubicin. Treatment with the high dose of Doxorubicin induced significant TNF-α production atevery time point. While STAT3 was phosphorylated at the serine residue after treatment with the lowdose of Doxorubicin, treatment with the high dose induced phosphorylation at the tyrosine residue in atime-dependent manner. p-Jak2 expression was significantly down-regulated at both concentrations ofDoxorubicin, suggesting that STAT3 proteins can by-pass activation by Jak2. The Erk1/2 leg of theRISK pathway was also not activated for the majority of the treatment period, however, p-Aktexpression was increased at the low concentration of Doxorubicin relative to total Akt expression.ConclusionThese observations indicate that treatment with Doxorubicin causes a severe, dose-dependent loss inviability which is likely to mediated by high concentrations of TNF-α (induced by high concentrationsof Doxorubicin) and down-regulation of protective signaling pathways. TNF-α may confer partialprotection at low concentrations by activating the SAFE pathway. However, activation of the SAFEpathway could not provide sufficient protection from Doxorubicin, most probably because the RISKpathway was not simultaneously activated. Our results also clearly highlight the differences betweenacute and chronic treatment since a single high dose of Doxorubicin produced vastly differentresponses to cumulative treatment with a low dose. Before one can extrapolate these results into the clinical setting, further research is required to provide a better understanding of the RISK and SAFEpathways and whether stimulation thereof will provide a protective effect. In addition, although ourstudy has shown that TNF-α is produced in response to Doxorubicin treatment, its true role, whetherbeneficial or detrimental, remains to be determined.

[发布机构] Stellenbosch University