Department of Cardiovascular Medicine, University of Kansas School of Medicine, Kansas City, KS, United States of America.
PLoS One. 2023 Oct 19;18(10):e0277747. doi: 10.1371/journal.pone.0277747. eCollection 2023.
Doxorubicin, an anthracycline chemotherapeutic known to incur heart damage, decreases heart function in up to 11% of patients. Recent investigations have implicated the Wnt signaling cascade as a key modulator of cardiac tissue repair after myocardial infarction. Wnt upregulation in murine models resulted in stimulation of angiogenesis and suppression of fibrosis after ischemic insult. However, the molecular mechanisms of Wnt in mitigating doxorubicin-induced cardiac insult require further investigation. Identifying cardioprotective mechanisms of Wnt is imperative to reducing debilitating cardiovascular adverse events in oncologic patients undergoing treatment.
Exposing human cardiomyocyte AC16 cells to varying concentrations of Wnt10b and DOX, we observed key metrics of cell viability. To assess the viability and apoptotic rates, we utilized MTT and TUNEL assays. We quantified cell and mitochondrial membrane stability via LDH release and JC-1 staining. To investigate how Wnt10b mitigates doxorubicin-induced apoptosis, we introduced pharmacologic inhibitors of key enzymes involved in apoptosis: FR180204 and SB203580, ERK1/2 and p38 inhibitors. Further, we quantified apoptotic executor enzymes, caspase 3/7, via immunofluorescence.
AC16 cells exposed solely to doxorubicin were shrunken with distorted morphology. Cardioprotective effects of Wnt10b were demonstrated via a reduction in apoptosis, from 70.1% to 50.1%. LDH release was also reduced between doxorubicin and combination groups from 2.27-fold to 1.56-fold relative to the healthy AC16 control group. Mitochondrial membrane stability was increased from 0.67-fold in the doxorubicin group to 5.73 in co-treated groups relative to control. Apoptotic protein expression was stifled by Wnt10b, with caspase3/7 expression reduced from 2.4- to 1.3-fold, and both a 20% decrease in p38 and 40% increase in ERK1/2 activity.
Our data with the AC16 cell model demonstrates that Wnt10b provides defense mechanisms against doxorubicin-induced cardiotoxicity and apoptosis. Further, we explain a mechanism of this beneficial effect involving the mitochondria through simultaneous suppression of pro-apoptotic p38 and anti-apoptotic ERK1/2 activities.
多柔比星是一种蒽环类化疗药物，已知会导致心脏损伤，多达 11%的患者会出现心脏功能下降。最近的研究表明，Wnt 信号级联反应是心肌梗死后心脏组织修复的关键调节剂。在小鼠模型中上调 Wnt 会导致缺血性损伤后血管生成的刺激和纤维化的抑制。然而，Wnt 在减轻多柔比星引起的心脏损伤中的分子机制仍需要进一步研究。确定 Wnt 的心脏保护机制对于减少接受治疗的肿瘤患者的衰弱性心血管不良事件至关重要。
我们将人心肌细胞 AC16 暴露于不同浓度的 Wnt10b 和 DOX 中，观察细胞活力的关键指标。为了评估细胞活力和凋亡率，我们使用了 MTT 和 TUNEL 测定法。通过 LDH 释放和 JC-1 染色来量化细胞和线粒体膜的稳定性。为了研究 Wnt10b 如何减轻多柔比星诱导的凋亡，我们引入了参与凋亡的关键酶的药理抑制剂：FR180204 和 SB203580，ERK1/2 和 p38 抑制剂。此外，我们通过免疫荧光定量分析了凋亡执行器酶 caspase 3/7。
仅用多柔比星处理的 AC16 细胞体积缩小，形态扭曲。Wnt10b 的心脏保护作用表现为凋亡减少，从 70.1%降至 50.1%。LDH 释放也从多柔比星和联合组相对于健康的 AC16 对照组的 2.27 倍减少至 1.56 倍。线粒体膜稳定性从多柔比星组的 0.67 倍增加到联合治疗组的 5.73 倍。Wnt10b 抑制了凋亡蛋白的表达，caspase3/7 的表达从 2.4 倍降低至 1.3 倍，同时 p38 降低 20%，ERK1/2 活性增加 40%。
我们使用 AC16 细胞模型的数据表明，Wnt10b 提供了防御机制，可抵抗多柔比星引起的心脏毒性和凋亡。此外，我们通过同时抑制促凋亡 p38 和抗凋亡 ERK1/2 活性，解释了这种有益作用的机制涉及线粒体。
