The chemotherapy drug doxorubicin (Dox) is commonly used for treating a variety of human cancers; however, it is highly cardiotoxic and induces heart failure. We previously reported that the Bcl-2 mitochondrial death protein Bcl-2/19kDa interaction protein 3 (Bnip3), is critical for provoking mitochondrial perturbations and necrotic cell death in response to Dox; however, the underlying mechanisms had not been elucidated. Herein, we investigated mechanism that drives Bnip3 gene activation and downstream effectors of Bnip3-mediated mitochondrial perturbations and cell death in cardiac myocytes treated with Dox.Nuclear factor-κB (NF-κB) signalling, which transcriptionally silences Bnip3 activation under basal states in cardiac myocytes was dramatically reduced following Dox treatment. This was accompanied by Bnip3 gene activation, mitochondrial injury including calcium influx, permeability transition pore (mPTP) opening, loss of nuclear high mobility group protein 1, reactive oxygen species production, and cell death. Interestingly, impaired NF-κB signalling in cells treated with Dox was accompanied by protein complexes between Bnip3 and cyclophilin D (CypD). Notably, Bnip3-mediated mPTP opening was suppressed by inhibition of CypD-demonstrating that CypD functionally operates downstream of Bnip3. Moreover, restoring IKKβ-NF-κB activity in cardiac myocytes treated with Dox suppressed Bnip3 expression, mitochondrial perturbations, and necrotic cell death.The findings of the present study reveal a novel signalling pathway that functionally couples NF-κB and Dox cardiomyopathy to a mechanism that is mutually dependent upon and obligatorily linked to the transcriptional control of Bnip3. Our findings further demonstrate that mitochondrial injury and necrotic cell death induced by Bnip3 is contingent upon CypD. Hence, maintaining NF-κB signalling may prove beneficial in reducing mitochondrial dysfunction and heart failure in cancer patients undergoing Dox chemotherapy.
Herein we describe a novel survival pathway that operationally links alternative pre‐mRNA splicing of the hypoxia‐inducible death protein B cl‐2 19kD I nteracting P rotein 3 (Bnip3) to the unique glycolytic phenotype in cancer cells. While a full length Bnip3 protein (Bnip3FL) encoded by exons 1–6 was expressed isoform in normal cells and promoted cell death, a truncated spliced variant of Bnip3 mRNA deleted for exon 3 (Bnip3Dex3) was preferentially expressed in several human adenocarcinomas promoted survival. Reciprocal inhibition of the Bnip3Dex3/Bnip3FL isoform ratio by inhibiting pyruvate dehydrogenase kinase isoform 2 (PDK2) in Panc‐1 cells rapidly induced mitochondrial perturbations and cell death. The findings of the present study reveal a novel survival pathway that functionally couples the unique glycolytic phenotype in cancer cells to hypoxia resistance via a PDK2‐ dependent mechanism that switches Bnip3 from cell death to survival. Discovery of the survival Bnip3Dex3 isoform may fundamentally explain how certain cells resist Bnip3 and avert death during hypoxia.
Autophagy is a homeostatic process by which damaged organelles such as mitochondria are degraded by an autophagosomal regulated pathway. Accordingly, excessive autophagy can be detrimental and prom...
Major tool of apoptosis detection, even today is the routine morphology based on already established histological techniques. Therefore, validation of new techniques becomes critical. Various aspects of apoptotic events have been extensively researched to elucidate the common biochemical pathways leading to this critical and unique phenomenon. Till now, apart from prototypical apoptotic morphology , techniques like DNA fragmentation estimation, specific sera against apoptotic components have been established. With each of these techniques, the essential requirements for apoptotic detection have been tried to establish like differentiating the apoptotic cells from non apoptotic cells, stages of apoptotic events and sensitivity of these techniques for apoptotic cells. In this review, we have not only tried to encompass various recent technique advancements in apoptosis detection, but also elaborate the pitfalls of all the techniques that shake the interpretation of results starting from the routine to the latest ones.
Nuclear Factor-κB (NF-κB) is ubiquitously present transcription factor that regulates a variety of cellular functions including cell survival. Herein, we show a critical role for NF-κB signaling in regulation of mitochondrial permeability transition pore opening (mPTP) of cardiac myocytes that involves cyclophilin D (CypD). Cardiac myocytes expressing a kinase defective form of IKKβ (IKK K-M ), the principle IKK required for NF-κB activation, displayed impaired NF-κB gene activity. Defects in NF-κB signaling coincided with an increase in mPTP opening and cell death. Interestingly, mPTP opening and cell death observed in the NF-κB defective cardiomyocytes was supressed by inhibition of mPTP modulator CypD, with cyclosporin A (CSA) or by siRNA knock down (CypDsiRNA), suggesting a link between mPTP regulation and NF-κB signaling. Earlier, we reported that doxorubicin (Dox) treatment resulted in severe ultra-structural defects including disrupted mitochondrial cristae and impaired respiration, increased mitochondrial calcium overload, mPTP opening and a widespread cell death. Interestingly, we observed a dramatic reduction in NF-κB signaling in cardiac myocytes treated with doxorubicin (18 Hrs), coupled with mitochondrial dysfunction including impaired respiration. Inhibition of CyPD suppressed doxorubicin induced cell death of cardiac myocytes. Finally restoration of NF-κB signaling in cardiac myocytes treated with doxorubicin by IKKβ, active kinase, suppressed mitochondrial calcium overload, mitochondrial perturbations, respiration and cell death. The data herein, provides the first direct evidence that impaired NF-κB signaling predispose Dox treated cardiac myocytes to cell death. Hence, interventions that preserve NF-κB survival signaling pathways in the heart may prove beneficial in reducing cardiac dysfunction and heart failure in cancer patients undergoing doxorubicin chemotherapy.
The advent of new chemotherapeutic agents has tremendously increased the survival rate of cancer patients. However, the majority, if not all the anticancer therapies in clinical use, is associated with adverse cardiotoxicity, which affects the quality of life of cancer patient survivors. Cardiac arrhythmia, ischemia, pericarditis, and systolic and diastolic dysfunction are the common abnormalities most cancer patient survivors experience for rest of their lives. Chemotherapy is the most common cancer treatment and mainly includes anthracyclines, alkylating agents, antimetabolites, and mitotic and topoisomerase inhibitors (Minami M, Matsumoto S, Horiuchi H. Cardiovascular side-effects of modern cancer therapy. Circ J 2010;74:1779–86). Over the past 3 decades, anthracyclines have been proven as an efficient oncological medication and are among the most widely prescribed drugs used to treat human malignancies (Torti FM, Bristow MM, Lum BL, Carter SK, Howes AE, Aston DA, Brown Jr BW, Hannigan Jr JF, Meyers FJ, Mitchell EP. Cardiotoxicity of epirubicin and doxorubicin: assessment by endomyocardial biopsy. Cancer Res 1986;46:3722–27), however, a major consideration includes the associated cardiotoxic effects that limit their usage. Of note, approximately two million people in the USA alone may be at risk of delayed anthracycline toxicity (Eschenhagen T, Force T, Ewer MS, de Keulenaer GW, Suter TM, Anker SD, Avkiran M, de AE, Balligand JL, Brutsaert DL, Condorelli G, Hansen A, Heymans S, Hill JA, Hirsch E, Hilfiker-Kleiner D, Janssens S, de JS, Neubauer G, Pieske B, Ponikowski P, Pirmohamed M, Rauchhaus M, Sawyer D, Sugden PH, Wojta J, Zannad F, Shah AM. Cardiovascular side effects of cancer therapies: a position statement from the Heart Failure Association of the European Society of Cardiology. Eur J Heart Fail 2011;13:1–10). For the effective management of cancer patients it is imperative to reduce or minimize the side effects of anthracyclines on the heart. Therefore, understanding the molecular mechanisms that underlie the cardiotoxic effects of anthracycline and related compounds is of paramount importance in reducing morbidity and mortality in cancer patients treated with these agents. The current literature describing the molecular mechanisms underlying anthracycline cardiomyopathy will be discussed in this chapter.
Hypoxia exerts broad effects on cardiomyocyte function and viability, ranging from altered metabolism and mitochondrial physiology to apoptotic or necrotic cell death. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a key regulator of cardiomyocyte metabolism and mitochondrial function and is down-regulated in hypoxia; however, the underlying mechanism is incompletely resolved. Using primary rat cardiomyocytes coupled with electrophoretic mobility shift and luciferase assays, we report that hypoxia impaired mitochondrial energetics and resulted in an increase in nuclear localization of the Nuclear Factor-κB (NF-κB) p65 subunit, and the association of p65 with the PGC-1α proximal promoter. Tumor necrosis factor α (TNFα), an activator of NF-κB signaling, similarly reduced PGC-1α expression and p65 binding to the PGC-1α promoter in a dose-dependent manner, and TNFα-mediated down-regulation of PGC-1α expression could be reversed by the NF-κB inhibitor parthenolide. RNA-seq analysis revealed that cardiomyocytes isolated from p65 knockout mice exhibited alterations in genes associated with chromatin remodeling. Decreased PGC-1α promoter transactivation by p65 could be partially reversed by the histone deacetylase inhibitor trichostatin A. These results implicate NF-κB signaling, and specifically p65, as a potent inhibitor of PGC-1α expression in cardiac myocyte hypoxia.
