Doxorubicin-induced Cardiomyocyte Apoptosis Research Articles

Genistein alleviates doxorubicin-induced cardiomyocyte autophagy and apoptosis via ERK/STAT3/c-Myc signaling pathway in rat model.

Doxorubicin (Dox) is a highly effective anti-neoplastic agent. Still, its utility in the clinic has been hindered by toxicities, including vomiting, hematopoietic suppression and nausea, with cardiotoxicity being the most serious side effect. Genistein (Gen) is a natural product with extensive biological effects, including anti-oxidation, anti-tumor, and cardiovascular protection. This study evaluated whether Gen protected the heart from Dox-induced cardiotoxicity and explored the underlying mechanisms. Male Sprague-Dawley (SD) rats were categorized into control (Ctrl), genistein (Gen), doxorubicin (Dox), genistein 20 mg/kg/day + doxorubicin (Gen20 + Dox) and genistein 40 mg/kg/day + doxorubicin (Gen40 + Dox) groups. Six weeks after injection, immunohistochemistry (IHC), transmission electron microscopy (TEM), and clinical cardiac function analyses were performed to evaluate the effects of Dox on cardiac function and structural alterations. Furthermore, each heart histopathological lesions were given a score of 0-3 in compliance with the articles for statistical analysis. In addition, molecular and cellular response of H9c2 cells toward Dox were evaluated through western blotting, Cell Counting Kit-8 (CCK8), AO staining and calcein AM/PI assay. Dox (5 μM in vitro and 18 mg/kg in vivo) was used in this study. In vivo, low-dose Gen pretreatment protected the rat against Dox-induced cardiac dysfunction and pathological remodeling. Gen inhibited extracellular signal-regulated kinase1/2 (ERK1/2)'s phosphorylation, increased the protein levels of STAT3 and c-Myc, and decreased the autophagy and apoptosis of cardiomyocytes. U0126, a MEK1/2 inhibitor, can mimic the effect of Gen in protecting against Dox-induced cytotoxicity both in vivo and in vitro. Molecular docking analysis showed that Gen forms a stable complex with ERK1/2. Gen protected the heart against Dox-induced cardiomyocyte autophagy and apoptosis through the ERK/STAT3/c-Myc signaling pathway.

Aging-Associated ALKBH5-m6A Modification Exacerbates Doxorubicin-Induced Cardiomyocyte Apoptosis Via AT-Rich Interaction Domain 2.

Chemotherapy-induced cardiovascular disease is a growing concern in the elderly population who have survived cancer, yet the underlying mechanism remains poorly understood. We investigated the role of ALKBH5 (AlkB homolog 5), a primary N6-methyladenosine (m6A) demethylase, and its involvement in m6A methylation-mediated regulation of targets in aging-associated doxorubicin-induced cardiotoxicity. To validate the relationship between doxorubicin-induced cardiotoxicity and aging, we established young and old male mouse models. ALKBH5 expression was modulated through adeno-associated virus 9 (in vivo), Lentivirus, and siRNAs (in vitro) to examine its impact on cardiomyocyte m6A modification, doxorubicin-induced cardiac dysfunction, and remodeling. We performed mRNA sequencing, methylated RNA immunoprecipitation sequencing, and molecular assays to unravel the mechanism of ALKBH5-m6A modification in doxorubicin-induced cardiotoxicity. Our data revealed an age-dependent increase in doxorubicin-induced cardiac dysfunction, remodeling, and injury. ALKBH5 expression was elevated in aging mouse hearts, leading to reduced global m6A modification levels. Through mRNA sequencing and methylated RNA immunoprecipitation sequencing analyses, we identified ARID2 (AT-rich interaction domain 2) as the downstream effector of ALKBH5-m6A modulation in cardiomyocytes. Further investigations revealed that ARID2 modulates DNA damage response and enhances doxorubicin-induced cardiomyocyte apoptosis. Our findings provide insights into the role of ALKBH5-m6A modification in modulating doxorubicin-induced cardiac dysfunction, remodeling, and cardiomyocyte apoptosis in male mice. These results highlight the potential of ALKBH5-targeted treatments for elderly patients with cancer in clinical settings.

Recent Advances in the Mechanisms of Cell Death and Dysfunction in Doxorubicin Cardiotoxicity.

Despite recent advances in cancer therapy, anthracycline-based combination therapy remains the standardized first-line strategy and has been found to have effective antitumor actions. Anthracyclines are extremely cardiotoxic, which limits the use of these powerful chemotherapeutic agents. Although numerous studies have been conducted on the cardiotoxicity of anthracyclines, the precise mechanisms by which doxorubicin causes cardiomyocyte death and myocardial dysfunction remain incompletely understood. This review highlights recent updates in mechanisms and therapies involved in doxorubicin-induced cardiomyocyte death, including autophagy, ferroptosis, necroptosis, pyroptosis, and apoptosis, as well as mechanisms of cardiovascular dysfunction resulting in myocardial atrophy, defects in calcium handling, thrombosis, and cell senescence. We sought to uncover potential therapeutic approaches to manage anthracycline cardiotoxicity via manipulation of crucial targets involved in doxorubicin-induced cardiomyocyte death and dysfunction.

Everolimus prevents doxorubicin-induced apoptosis in H9c2 cardiomyocytes but not in MCF-7 cancer cells: Cardioprotective roles of autophagy, mitophagy, and AKT

Cardiotoxicity is a severe side effect of the chemotherapeutic agent doxorubicin (DOX). We recently showed that DOX-induced cardiomyocyte apoptosis and death were attenuated through autophagy pre-induction. Herein, we assessed how the autophagy/mitophagy-inducing antitumor drug everolimus (EVL) affected DOX-induced cytotoxicity in the rat cardiomyocyte cell line H9c2 and human breast cancer cell line MCF-7. Apoptosis was assessed using annexin V assay. Autophagy and mitophagy were assessed using fluorescence assays. Cellular protein levels were determined using western blotting. Pretreatment with EVL (1 nM) before DOX exposure inhibited mammalian target of rapamycin (mTOR) activity, induced autophagy and mitophagy, and activated protein kinase B (AKT) in H9c2 cells. In mitochondria, DOX (1 μM) induced structural damage (decreased membrane potential and release of cytochrome c), increased superoxide levels, decreased apoptosis inhibitor Bcl-2, and increased apoptosis inducer Bax, leading to apoptosis and reduced viability in H9c2 cells. EVL pretreatment suppressed DOX-induced changes. EVL anti-apoptotic effects were inhibited by treatment with MK-2206, a selective AKT inhibitor. Furthermore, EVL suppressed DOX-induced cardiotoxicity through autophagy/mitophagy and AKT activation but did not attenuate DOX-induced apoptosis or reduction in viability in MCF-7 cells. Altogether, EVL can protect cardiomyocytes from DOX-induced apoptosis and toxicity without reducing DOX antitumor effects, allowing safer chemotherapy.

Secreted APE1/Ref-1 inhibits doxorubicin-induced cardiotoxicity via suppressing ROS generation and p53 pathway

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This research is supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare and Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Republic of Korea. Background Doxorubicin is an anthracycline anticancer drug with a potent therapeutic effect against various malignancies. However, its use is limited due to cardiotoxicity, which can lead to heart failure. Oxidative stress is a key player in the development of anthracycline cardiotoxicity. Apurinic/apyrimidinic endonuclease 1/redox factor-1(APE1/Ref-1) is a multifunctional protein that regulates oxidative stress and anti-inflammatory function. Secreted APE1/Ref-1 has shown anti-oxidant and anti-inflammatory properties, but its therapeutic potential in various diseases is largely unknown. Purpose This study aims to test the protective effect of secreted APE1/Ref-1 in the doxorubicin-induced cardiomyopathy model in vivo and in vitro. Methods H9C2 cells were treated with doxorubicin(2uM) to induce cardiotoxicity. Adenovirus preprotrypsin leading sequence APE1/Ref-1(AdPPT-LS-APE1/Ref-1), a secretory APE1/Ref-1 adenovirus encoding human APE1/Ref-1 gene, was amplified in HEK293T cells. H9C2 cells were then treated with the supernatant of the amplified AdPPT-LS-APE1/Ref-1. In vivo model of doxorubicin-induced cardiotoxicity was established by injecting doxorubicin(15mg/kg) into C57BL/6 mice via the peritoneum. The conditioned media of HEK293T cells with AdPPT-LS-APE1/Ref-1 transfection was injected i.p. after 24h. The severity of cardiotoxicity was evaluated by echocardiography and plasma NT-proBNP. Cell survival, morphology, and ROS level were examined. The expression of cellular APE1/Ref-1, p53, Bax/Bcl-2, and Caspase-3 were analyzed by Western blot and ELISA. Redox activity was measured using the DTNB reduction assay. Results Doxorubicin caused apoptosis and oxidative stress in H9C2 cells and cardiomyopathy in mice. NT-proBNP level increased both in vitro(p<0.001) and in vivo(p<0.05) upon administering doxorubicin. Interestingly, doxorubicin increased p53 level in a dose-dependent manner in 48 hr, but the cellular APE1/Ref-1 level did not change. Secreted APE1/Ref-1 in the conditional media showed redox activity and decreased NT-proBNP in doxorubicin-treated H9C2 cells and mice(p<0.001 and p<0.05, respectively). Doxorubicin aggravated cell death(p<0.001) and oxidative tissue damage(p<0.001) in H9C2 cells, while pre-treatment of secreted APE1/Ref-1 inhibited cell death in H9C2 cells(p<0.05). Secreted APE1/Ref-1 significantly reduced the expression of p53(p<0.001) and its effectors, Bax/Bcl-2(p<0.05) and Caspase-3(p<0.05) in doxorubicin-treated H9C2 cells. It also reduced p53(p<0.05) along with Bax/Bcl-2(p<0.05) and Caspase-3(p<0.05) in doxorubicin-treated mice(n=4 in each group). Finally, secreted APE1/Ref-1 significantly decreased intracellular ROS production(p<0.001). Conclusion Secreted APE1/Ref-1 is protective against doxorubicin-induced cardiomyocyte apoptosis and oxidative stress by suppressing ROS generation and p53 pathway. APE1/Ref-1 showed therapeutic potential for the prevention and treatment of doxorubicin-induced cardiotoxicity.

Abstract P2082: Igfbp-3 Activates Pro-survival Pathways To Mitigate Anthracycline-induced Cardiomyocyte Injury

Rationale: Anthracyclines such as doxorubicin are among the most effective chemotherapeutic agents; however, their use leads to frequent cardiovascular complications, including heart failure, due to the activation of multiple cardiomyocyte injury pathways. Insulin-like growth factor binding protein-3 (IGFBP3) is strongly upregulated in cardiomyocytes after administration of doxorubicin. However, its role in doxorubicin-induced cardiomyocyte apoptosis and survival remains controversial. Methods and Results: We demonstrate that IGFBP3 is strongly induced by doxorubicin in neonatal ventricular myocytes (NRVMs) 12 hours after doxorubicin treatment by RT-PCR (4.2 fold vs. control, n=3, P<0.05) and Western blot. Adenoviral overexpression of IGFBP3 in NRVMs leads to a significant reduction of phosphorylated H2A histone (γH2AX) after doxorubicin treatment, a hallmark of DNA double-strand breaks (DSBs), as well as topoisomerase IIβ, a key mediator of doxorubicin induced DNA DSBs. Conversely, silencing of IGFBP3 with siRNA leads to a significant increase of γH2AX expression in NRVMs. Adenoviral shRNA silencing of IGFBP3 in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) further demonstrates a 44% and 52% decrease in contraction magnitude and velocity in response to doxorubicin compared with scramble shRNA (scramble shRNA, n=5, shIGFBP3, n=3, P<0.05). Immunofluorescent imaging and cell fractionation assays conducted in NRVMs indicate that doxorubicin initiates IGFBP3 nuclear translocation, suggesting an active role in the cardiomyocyte response to doxorubicin-induced cell injury. Next generation sequencing of hiPSC-CMs treated with doxorubicin reveals that overexpression of IGFBP3 leads to upregulation of cardiomyocyte survival pathways, including TNF receptors without cytoplasmic death domain (TNFRSF10C and D), BCL2 family gene BCL2L1, the p53 target ACER2, the p53 co-factor FBXW7, the β-catenin mediator TBX3 and the ataxia telangiectasia mutated (ATM) activator ZNF423. Conclusion: Our findings indicate that IGFBP3 is strongly induced in cardiomyocytes by doxorubicin treatment. Overexpression of IGFBP3 attenuates doxorubicin induced DNA DSBs and promotes pro-survival signaling pathways.

Hydroxytyrosol Prevents Doxorubicin-Induced Oxidative Stress and Apoptosis in Cardiomyocytes.

Doxorubicin (Dox) is a highly effective chemotherapeutic agent employed in the handling of hematological and solid tumors. The effective use of Dox in cancer therapy has been seriously limited due to its well-known cardiotoxic side effects, mainly mediated by oxidative damage. Therefore, the identification of an effective and safe antagonist against Dox-induced cardiotoxicity remains a challenge. In this respect, as plant polyphenols have attracted considerable interest due to their antioxidant properties and good safety profile, hydroxytyrosol (HT), the major phenolic compound in olive oil, could be a potential candidate due to its remarkable antioxidant and anticancer powers. In this study, the effect of HT was tested on Dox-induced cardiotoxicity by using a combination of biochemical and cellular biology techniques. Interestingly, HT was able to counteract Dox-induced cytotoxicity in cardiomyocytes by acting on the SOD2 level and the oxidative response, as well as on apoptotic mechanisms mediated by Bcl-2/Bax. At the same time, HT did not to interfere with the antitumorigenic properties of Dox in osteosarcoma cells. This study identifies new, beneficial properties for HT and suggests that it might be a promising molecule for the development of additional therapeutic approaches aimed at preventing anthracycline-related cardiotoxicity and improving long-term outcomes in antineoplastic treatments.

Ginsenoside Rg2 Attenuates Doxorubicin-induced Cardiomyocyte Apoptosis via the PI3K/Akt Pathway

Doxorubicin, sold under the brand name Adriamycin among others, is an important drug for cancer therapy; however, its use is limited by its cardiotoxicity. Ginsenoside Rg2 is extracted from Panax ginseng C.A.Mey., Araliaceae, which is believed to have cardioprotective properties. However, to date, there have been no reports on whether ginsenoside Rg2 could protect cardiomyocytes against doxorubicin. In this study, we investigated the action and the underlying mechanisms of cardioprotection of ginsenoside Rg2 upon doxorubicin treatment. Cell counting kit-8 was used to determine cell viability; in addition, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling staining was used to detect apoptotic cells. Western blotting was used to investigate the relevant pathways. LY294002, a phosphatidylinositol 3-kinase inhibitor, was also used in this study. Ginsenoside Rg2 significantly (p < 0.01) neutralized cardiomyocyte apoptosis induced by doxorubicin in a dose-dependent manner, but this effect was blocked by LY294002. Furthermore, ginsenoside Rg2 upregulated protein kinase B phosphorylation through the phosphatidylinositol 3-kinase/protein kinase B pathway and inhibited p53 expression. These results suggest that ginsenoside Rg2 could attenuate doxorubicin-induced cardiomyocyte apoptosis via the phosphatidylinositol 3-kinase/protein kinase B pathway.Graphical abstract

The circular RNA circNlgnmediates doxorubicin-inducedcardiac remodeling and fibrosis

Doxorubicin is a chemotherapeutic medication commonly used to treat many types of cancers, but it has side effects including vomiting, rash, hair loss, and bone marrow suppression. The most dangerous side effects are cardiomyopathy, cardiofibrosis, and heart failure, as doxorubicin generates cytotoxicity and stops DNA replication. There is no treatment to block these side effects. We have developed a transgenic mouse line overexpressing the circular RNA circNlgn and shown that circNlgn is a mediator of doxorubicin-induced cardiofibrosis. Increased expression of circNlgn decreased cardiac function and induced cardiofibrosis by upregulating Gadd45b, Sema4C, and RAD50 and activating p38 and pJNK in circNlgn transgenic heart. Silencing circNlgn decreased the effects of doxorubicin on cardiac cell activities and prevented doxorubicin-induced expression of fibrosis-associated molecules. The protein (Nlgn173) translated by circNlgn could bind and activate H2AX, producing γH2AX, resulting in upregulation of IL-1b, IL-2Rb, IL-6, EGR1, and EGR3. We showed that silencing these molecules in the signaling pathway prevented doxorubicin-induced cardiomyocyte apoptosis, increased cardiomyocyte viability, decreased cardiac fibroblast proliferation, and inhibited collagen production. This mechanism may hold therapeutic implications for mitigating the side effects of doxorubicin therapy in cancer patients.

MicroRNA-194-5p Attenuates Doxorubicin-Induced Cardiomyocyte Apoptosis and Endoplasmic Reticulum Stress by Targeting P21-Activated Kinase 2.

ObjectiveMany studies have reported that microRNAs (miRs) are involved in the regulation of doxorubicin (DOX)-induced cardiotoxicity. MiR-194-5p has been reported significantly upregulated in patients with myocardial infarction; however, its role in myocardial diseases is still unclear. Various stimuluses can trigger the endoplasmic reticulum (ER) stress and it may activate the apoptosis signals eventually. This study aims to explore the regulatory role of miR-194-5p in DOX-induced ER stress and cardiomyocyte apoptosis.MethodsH9c2 was treated with 2 μM DOX to induce apoptosis, which is to stimulate the DOX-induced cardiotoxicity model. The expression of miR-194-5p was detected by quantitative real-time PCR (qRT-PCR); the interaction between miR-194-5p and P21-activated kinase 2 (PAK2) was tested by dual luciferase reporter assay; terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and caspase-3/7 activity were used to assess apoptosis; trypan blue staining was applied to measure cell death; Western blotting was performed to detect protein expressions; and ER-related factors splicing X-box binding protein 1 (XBP1s) was detected by polyacrylamide gel electrophoresis and immunofluorescence to verify the activation of ER stress.ResultsMiR-194-5p was upregulated in cardiomyocytes and mouse heart tissue with DOX treatment, while the protein level of PAK2 was downregulated. PAK2 was predicted as the target of miR-194-5p; hence, dual luciferase reporter assay indicated that miR-194-5p directly interacted with PAK2 and inhibited its expression. TUNEL assay, caspase-3/7 activity test, and trypan blue stain results showed that either inhibition of miR-194-5p or overexpression of PAK2 reduced DOX-induced cardiomyocyte apoptosis. Silencing of miR-194-5p also improved DOX-induced cardiac dysfunction. In addition, DOX could induce ER stress in H9c2, which led to XBP1 and caspase-12 activation. The expression level of XBP1s with DOX treatment increased first then decreased. Overexpression of XBP1s suppressed DOX-induced caspase-3/7 activity elevation as well as the expression of cleaved caspase-12, which protected cardiomyocyte from apoptosis. Additionally, the activation of XBP1s was regulated by miR-194-5p and PAK2.ConclusionOur findings revealed that silencing miR-194-5p could alleviate DOX-induced cardiotoxicity via PAK2 and XBP1s in vitro and in vivo. Thus, the novel miR-194-5p/PAK2/XBP1s axis might be the potential prevention/treatment targets for cancer patients receiving DOX treatment.

MiR-488-3p Protects Cardiomyocytes against Doxorubicin-Induced Cardiotoxicity by Inhibiting CyclinG1.

Objective To investigate the protective effects and regulatory mechanism of miR-488-3p on doxorubicin-induced cardiotoxicity. Methods The C57BL/6 mice and primary cardiomyocytes were used to construct doxorubicin-induced cardiomyocyte injury models in vivo and in vitro. The levels of miR-488-3p and its downstream target genes were analyzed by quantitative real-time PCR. Mouse cardiac function, cell survival, cellular injury-related proteins, and the apoptosis level of cardiomyocytes were analyzed by echocardiography, MTT analysis, Western blotting, and DNA laddering separately. Results Cardiomyocyte injury caused by a variety of stimuli can lead to the reduction of miR-488-3p level, especially when stimulated with doxorubicin. Doxorubicin led to significant decrease in cardiac function, cell autophagic flux blockage, and apoptosis in vivo and in vitro. The expression of miR-488-3p's target gene, CyclinG1, increased remarkably in the doxorubicin-treated neonatal mouse cardiomyocytes. Overexpression of miR-488-3p inhibited CyclinG1 expression, increased cardiomyocyte viability, and attenuated doxorubicin-induced cardiomyocyte autophagic flux blockage and apoptosis. Conclusions miR-488-3p is one of the important protective miRNAs in doxorubicin-induced cardiotoxicity by inhibiting the expression of CyclinG1, which provides insight into the possible clinical application of miR-488-3p/CyclinG1 as therapeutic targets in doxorubicin-induced cardiovascular diseases.

A novel β2-AR agonist, Higenamine, induces β-arrestin-biased signaling.

The biased ligands in G protein-coupled receptors (GPCRs) have opened new avenues for developing safer and more effective drugs. However, the identification of such biased ligands as drug candidates is highly desirable. Here, we report that Higenamine, a compound isolated from a Chinese herb, functions as a novel β-arrestin-biased ligand of the β2-adrenergic receptor (β2-AR). The radioligand binding assays demonstrated that Higenamine was the ligand of β2-AR. Higenamine induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), which can be blocked by propranolol, an inhibitor of β2-AR. The Gi protein inhibitor, pertussis toxin, had no effect on the phosphorylation of ERK1/2 induced by Higenamine. Furthermore, Higenamine induced ERK1/2 phosphorylation through transactivation of Epithelial growth factor receptor (EGFR). We also found that Higenamine-induced-ERK1/2 phosphorylation is dependent on β-arrestin1/2, and HG inhibits Doxorubicin-induced cardiomyocyte apoptosis. Our results identify Higenamine as a novel biased ligand via the β-arrestin-dependent pathway. These findings give us a better understanding of Higenamine's potential role in designing diagnostic and therapeutic strategies.

Long Noncoding RNA SOX2-OT Aggravates Doxorubicin-Induced Apoptosis of Cardiomyocyte by Targeting miR-942-5p/DP5.

BackgroundLong non-coding RNAs (LncRNAs) play important roles in doxorubicin (DOX)-induced apoptosis of cardiomyocytes. However, the function of lncRNA SOX2-OT is unclear. This study was carried out to investigate the function of SOX2-OT in doxorubicin-induced cardiomyocyte apoptosis.MethodsqRT-PCR and immunoblotting were used to detect the expression levels of SOX2-OT, miR-942-5p and death protein-5 (DP5) in DOX-treated primary cardiomyocytes and rat models. The relationship among miR-942-5p, SOX2-OT, and DP5 was explored by luciferase reporter assay. The effects of SOX2-OT, miR-942-5p and DP5 on doxorubicin-induced cardiomyocyte apoptosis were evaluated by Annexin V-FITC/PI method and caspase-3 activity assay. The effect of SOX2-OT on cardiomyocyte apoptosis was analyzed by TUNEL staining and echocardiography.ResultsSOX2-OT and DP5 were highly expressed, while miR-942-5p was down-regulated in DOX-treated primary cardiomyocytes and rat model. SOX2-OT can upregulate DP5 as a sponge of miR-942-5p, which was a direct target of miR-942-5p. In addition, miR-942-5p reversed the protective effect of knockdown of SOX2-OT on cardiomyocytes by inhibiting the expression of DP5 in vitro and in vivo.ConclusionKnockdown of SOX2-OT down-regulated DP5 via sponging miR-942-5p and inhibiting DOX-induced apoptosis of primary cardiomyocytes.

Orosomucoid 1 Attenuates Doxorubicin-Induced Oxidative Stress and Apoptosis in Cardiomyocytes via Nrf2 Signaling.

Doxorubicin (DOX) is an effective anticancer drug, but its therapeutic use is limited by its cardiotoxicity. The principal mechanisms of DOX-induced cardiotoxicity are oxidative stress and apoptosis in cardiomyocytes. Orosomucoid 1 (ORM1), an acute-phase protein, plays important roles in inflammation and ischemic stroke; however, the roles and mechanisms of ORM1 in DOX-induced cardiotoxicity remain unknown. Therefore, in the present study, we aimed to investigate the function of ORM1 in cardiomyocytes experiencing DOX-induced oxidative stress and apoptosis. A DOX-induced cardiotoxicity animal model was established in C57BL/6 mice by administering an intraperitoneal injection of DOX (20 mg/kg), and the control group was intraperitoneally injected with the same volume of sterilized saline. The effects were assessed after 7 d. Additionally, H9c2 cells were stimulated with DOX (10 μM) for 24 h. The results showed decreased ORM1 and increased oxidative stress and apoptosis after DOX stimulation in vivo and in vitro. ORM1 overexpression significantly reduced DOX-induced oxidative stress and apoptosis in H9c2 cells. ORM1 significantly increased the expression of nuclear factor-like 2 (Nrf2) and its downstream protein heme oxygenase 1 (HO-1) and reduced the expression of the lipid peroxidation end product 4-hydroxynonenal (4-HNE) and the level of cleaved caspase-3. In addition, Nrf2 silencing reversed the effects of ORM1 on DOX-induced oxidative stress and apoptosis in cardiomyocytes. In conclusion, ORM1 inhibited DOX-induced oxidative stress and apoptosis in cardiomyocytes by regulating the Nrf2/HO-1 pathway, which might provide a new treatment strategy for DOX-induced cardiotoxicity.

Vanillin Prevents Doxorubicin-Induced Apoptosis and Oxidative Stress in Rat H9c2 Cardiomyocytes

Doxorubicin (doxo) is an effective anticancer compound in several tumor types. However, as a consequence of oxidative stress induction and ROS overproduction, its high cardiotoxicity demands urgent attention. Vanillin possesses antioxidant, antiproliferative, antidepressant and anti-glycating properties. Therefore, we investigated the potential vanillin protective effects against doxo-induced cardiotoxicity in H9c2 cells. Using multiparametric approach, we demonstrated that vanillin restored both cell viability and damage in response to doxo exposure. Contextually, vanillin decreased sub-G1 appearance and caspase-3 and PARP1 activation, reducing the doxo-related apoptosis induction. From a mechanistic point of view, vanillin hindered doxo-induced ROS accumulation and impaired the ERK phosphorylation. Notably, besides the cardioprotective effects, vanillin did not counteract the doxo effectiveness in osteosarcoma cells. Taken together, our results suggest that vanillin ameliorates doxo-induced toxicity in H9c2 cells, opening new avenues for developing alternative therapeutic approaches to prevent the anthracycline-related cardiotoxicity and to improve the long-term outcome of antineoplastic treatment.

Abstract MP131: The CDK2-FOXO1 Axis Mediates Doxorubicin-induced Cardiomyocyte Apoptosis and Atrophy

Recent clinical investigations indicate that anthracycline-based chemotherapy induces early declines in heart mass in cancer patients. A decline in heart mass can be caused by a decrease in cardiac cell number due to exaggerated cell death, and/or a reduction in cell size via atrophy. Our previous study reveals that the anthracycline doxorubicin (DOX) induces apoptotic death of cardiomyocytes through activation of cyclin-dependent kinase 2 (CDK2). However, the signaling pathway downstream of CDK2 remains to be characterized. In addition, it is unclear whether the same pathway also mediates cardiac atrophy. Here we demonstrated that DOX exposure induced CDK2-dependent phosphorylation of forkhead box O1 (FOXO1) at S249, leading to transcription of its pro-apoptotic target gene Bim. In cultured cardiomyocytes, treatment with the small molecule FOXO1 inhibitor AS1842856, or transfection with FOXO1-specific siRNAs protected against DOX-induced apoptosis and mitochondrial damage. Oral administration of AS1842856 in mice hindered apoptosis and prevented DOX-induced decline in left ventricular systolic function. Intriguingly, pharmacologic inhibition of FOXO1 also attenuated DOX-induced cardiac atrophy, likely due to repression of MuRF1, a pro-atrophic FOXO1 target gene. In conclusion, DOX exposure induces CDK2-dependent FOXO1 activation, resulting in cardiomyocyte apoptosis and atrophy. Our results identify FOXO1 as a promising drug target for DOX-induced cardiotoxicity. FOXO1 inhibitors may represent a novel class of therapeutics for cardioprotection during chemotherapy.

Sodium thiosulfate prevents doxorubicin-induced DNA damage and apoptosis in cardiomyocytes in mice

AimDoxorubicin (DOX) induces dose-dependent cardiotoxicity due to reactive oxygen species (ROS)-mediated oxidative stress and subsequent apoptosis of cardiomyocytes. We aimed to assess whether sodium thiosulfate (STS), which has antioxidant and antiapoptotic properties, exerts cardioprotective effects on DOX-induced cardiomyopathy. Main methodsMale C57BL/6N mice were divided into four groups, control, DOX, STS, and DOX + STS, and administered DOX (20 or 30 mg/kg) or normal saline intraperitoneally, followed by an injection of STS (2 g/kg) or normal saline 4 h later. Key findingsThe DOX group showed a poorer 6-day survival and decreased cardiac function than the DOX + STS group. The DOX group showed a marked increase in the plasma creatine kinase isoenzyme myocardial band (CK-MB) and lactate dehydrogenase (LDH) levels 10 h after DOX injection, while the DOX + STS group showed suppression of DOX-induced elevation of CK-MB and LDH levels. The DOX group showed increased 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels in the heart, whereas the DOX + STS group showed increased catalase and superoxide dismutase (SOD) activities and decreased 8-OHdG levels in the heart compared with DOX group, suggesting that STS reduces DOX-induced DNA damage by improving antioxidant enzymes activities in cardiomyocytes. Additionally, the DOX + STS group showed attenuation of cleaved caspase-3 and DNA fragmentation in cardiomyocytes compared with the DOX group, suggesting that STS suppresses DOX-induced apoptosis in cardiomyocytes. SignificanceSTS exerts cardioprotective effects against DOX-induced cardiac dysfunction partly by improving antioxidant defense and suppressing apoptosis, indicating the therapeutic potential of STS against DOX-induced cardiomyopathy.

Dexrazoxane Protects Cardiomyocyte from Doxorubicin-Induced Apoptosis by Modulating miR-17-5p.

The usage of doxorubicin is hampered by its life-threatening cardiotoxicity in clinical practice. Dexrazoxane is the only cardioprotective medicine approved by the FDA for preventing doxorubicin-induced cardiac toxicity. Nevertheless, the mechanism of dexrazoxane is incompletely understood. The aim of our study is to investigate the possible molecular mechanism of dexrazoxane against doxorubicin-induced cardiotoxicity. We established a doxorubicin-induced mouse and cardiomyocyte injury model. Male C57BL/6J mice were randomly distributed into a control group (Con), a doxorubicin treatment group (DOX), a doxorubicin plus dexrazoxane treatment group (DOX+DEX), and a dexrazoxane treatment group (DEX). Echocardiography and histology analyses were performed to evaluate heart function and structure. DNA laddering, qRT-PCR, and Western blot were performed on DOX-treated cardiomyocytes with/without DEX treatment in vitro. Cardiomyocytes were then transfected with miR-17-5p mimics or inhibitors in order to analyze its downstream target. Our results demonstrated that dexrazoxane has a potent effect on preventing cardiac injury induced by doxorubicin in vivo and in vitro by reducing cardiomyocyte apoptosis. MicroRNA plays an important role in cardiovascular diseases. Our data revealed that dexrazoxane could upregulate the expression of miR-17-5p, which plays a cytoprotective role in response to hypoxia by regulating cell apoptosis. Furthermore, the miRNA and protein analysis revealed that miR-17-5p significantly attenuated phosphatase and tensin homolog (PTEN) expression in cardiomyocytes exposed to doxorubicin. Taken together, dexrazoxane might exert a cardioprotective effect against doxorubicin-induced cardiomyocyte apoptosis by regulating the expression of miR-17-5p/PTEN cascade.

Repeated Remote Ischemic Conditioning Reduces Doxorubicin-Induced Cardiotoxicity

ObjectivesThis study investigated the cardioprotective effect of repeated remote ischemic preconditioning (rRIC) on doxorubicin-induced cardiotoxicity in mice. BackgroundDoxorubicin is an effective chemotherapeutic agent for a wide range of tumor types but its use and dosing are limited by acute and chronic cardiotoxicity. Remote ischemic conditioning (RIC) is cardioprotective in multiple cardiovascular injury models, but the effectiveness of rRIC in doxorubicin-induced cardiotoxicity has not been fully elucidated. MethodsrRIC was performed on mice before and after doxorubicin administration. Cardiac function was assessed by echocardiography and myocardial biology was tested by molecular approaches. ResultsDoxorubicin administration induced acute cardiotoxicity, as indicated by reduced cardiac function, reduced myocyte cross-section area and increased extracellular collagen deposition, increased circulating cardiac muscle damage markers, and decreased heart weight. Doxorubicin also adversely affected other organs, including the kidney, liver, and spleen, as evaluated by circulating markers or organ weight loss. rRIC not only abrogated doxorubicin-induced cardiotoxicity (left ventricular ejection fraction, doxorubicin 47.5 ± 1.1%, doxorubicin + rRIC 51.6 ± 0.7%, p = 0.017), but also was associated with multiorgan protection. Within the myocardium, rRIC attenuated doxorubicin-induced cardiomyocyte apoptosis, reduced inflammation, and increased autophagy signaling. ConclusionsrRIC may be a promising approach to reduce doxorubicin-induced cardiotoxicity.

Human Amnion Membrane Proteins Prevent Doxorubicin-Induced Oxidative Stress Injury and Apoptosis in Rat H9c2 Cardiomyocytes.

Doxorubicin (DOX) is widely used as an effective chemotherapy agent in cancer treatment. Cardiac toxicity in cancer treatment with DOX demand urgent attention and no effective treatment has been established for DOX-induced cardiomyopathy. It has been well documented that human amniotic membrane proteins (AMPs), extracted from amnion membrane (AM), have antioxidant, anti-apoptotic, and cytoprotective properties. Therefore, in this study, we aimed to investigate the protective effects of AMPs against cardiotoxicity induced by DOX in cultured rat cardiomyocyte cells (H9c2). DOX-induced cell injury was evaluated using multi-parametric assay including thiazolyl blue tetrazolium bromide (MTT), the release of lactic dehydrogenase (LDH), intracellular Ca2+ , reactive oxygen species (ROS) levels, cellular antioxidant status, mitochondrial membrane potential (ΔΨm), malondialdehyde (MDA), and NF-κB p65 DNA-binding activity. Moreover, expression profiling of apoptosis-related genes (P53, Bcl-2, and Bax) and Annexin V by flow cytometry were used for cell apoptosis detection. It was shown that AMPs pretreatment inhibited the cell toxicity induced by DOX. AMPs effectively attenuated the increased levels of LDH, Ca2+ , ROS, and MDA and also simultaneously elevated the ΔΨm and antioxidant status such as superoxide dismutase (SOD) and Catalase (CAT) in pretreated H9c2 cardiomyocytes. Besides, the activity of NF-kB p65 was reduced and the p53 and Bax protein levels were inhibited in these myocardial cells subjected to DOX. These findings provide the first evidence that AMPs potently suppressed DOX-induced toxicity in cardiomyocytes through inhibition of oxidative stress and apoptosis. Thus, AMPs can be a potential therapeutic agent against DOX cardiotoxicity.