Involvement of Yes‐associated protein 1 (YAP1) in doxorubicin‐induced cytotoxicity in H9c2 cardiac cells

Cardiac cell death is one of the major events implicated in doxorubicin‐induced cardiotoxicity, which leads to heart failure. We recently reported that Yes‐associated protein 1 (YAP1) regulates cell survival and apoptosis. However, it is unclear whether YAP1 regulates doxorubicin‐induced cell death in cardiomyocytes. We investigated whether YAP1 is involved in doxorubicin‐induced cell death using H9c2 cardiac cells and mouse heart. In an in vivo study, YAP1 protein expression was significantly decreased in hearts of doxorubicin‐treated mice with increased caspase‐3 activation. Doxorubicin also caused cell death by increasing caspase‐3 activation in H9c2 cells. Doxorubicin reduced YAP1 protein expression and messenger RNA expression accompanied by increased phosphorylation of YAP1 at Ser127. Doxorubicin further increased cell death with increased caspase‐3/7 activation in the absence of YAP1 when compared with doxorubicin or siYAP1 treatment alone. Overexpression of constitutively active YAP1 (YAP1–5SA) using an adenovirus gene transfer technique significantly reversed doxorubicin‐induced cell death by decreasing caspase‐3/7 activation in H9c2 cells. Akt, a potential prosurvival factor, decreased in doxorubicin‐ and YAP1 short interfering RNA (siRNA)‐treated cells. Doxorubicin further significantly decreased Akt protein expression when YAP1 was silenced. Overexpression of YAP1 canceled decreased Akt protein expression induced by doxorubicin treatment in H9c2 cells. In conclusion, these results suggest that doxorubicin‐induced cardiac cell death is mediated in part by down‐regulation of YAP1 and YAP1‐targeted gene, Akt. Modulating YAP1 and its related Hippo pathway on local cardiomyocytes may be a promising therapeutic approach for doxorubicin‐induced cardiotoxicity.     

Tricyclic isoxazoles are novel inhibitors of the multidrug resistance protein (MRP1)

Tricyclic isoxazoles were identified from a screen as a novel class of selective multidrug resistance protein (MRP1) inhibitors. From a screen lead, SAR efforts resulted in the preparation of LY 402913 (9h), which inhibits MRP1 and reverses drug resistance to MRP1 substrates, such as doxorubicin, in HeLa-T5 cells (EC(50)=0.90 microM), while showing no inherent cytotoxicity. Additionally, LY 402913 inhibits ATP-dependent, MRP1-mediated LTC(4) uptake into membrane vesicles prepared from the MRP1-overexpressing HeLa-T5 cells (EC(50)=1.8 microM). LY 402913 also shows selectivity ( approximately 22-fold) against the related transporter, P-glycoprotein, in HL60/Adr and HL60/Vinc cells. Finally, when dosed in combination with the oncolytic MRP1 substrate vincristine, LY 402913 delays the growth of MRP1-overexpressing tumors in vivo.     

RLIP76 is the major ATP-dependent transporter of glutathione-conjugates and doxorubicin in human erythrocytes.

We have recently demonstrated that RLIP76, a Ral-binding GTPase activating protein mediates ATP-dependent transport of glutathione (GSH) conjugates of electrophiles (GS-E) as well as doxorubicin (DOX), and that it is identical with DNP-SG ATPase, a GS-E transporter previously characterized by us in erythrocyte membranes (Awasthi et al. Biochemistry 39, 9327-9334). Multidrug resistance-associated protein (MRP1) belonging to the family of the ABC-transporters has also been suggested to be a GS-E transporter in human erythrocytes. Using immunological approaches, the present studies were designed to elucidate the relative contributions of RLIP76, MRP1, and P-glycoprotein (Pgp), in the ATP-dependent transport of GS-E and DOX in human erythrocytes. In Western blot analyses using antibodies against RLIP76, a strong expression of RLIP76 was observed in erythrocytes. Immunohistochemical studies using a fluorescent probe showed association of RLIP76 with erythrocyte membrane, which was consistent with its transport function. Neither MRP1 nor Pgp were detected in erythrocytes when the antibodies against MRP1 or Pgp were used. In erythrocyte inside-out vesicles (IOVs) coated with antibodies against RLIP76, a dose-dependent inhibition of the ATP-dependent transport of DOX and GS-E, including S-(dinitrophenyl)glutathione (DNP-SG), leukotriene C(4), and the GSH conjugate of 4-hydroxynonenal, was observed with a maximal inhibition of about 70%. On the contrary, in the IOVs coated with the antibodies against MRP1 or Pgp no significant inhibition of the ATP-dependent transport of these compounds was observed. These findings suggest that RLIP76 is the major ATP-dependent transporter of GS-E and DOX in human erythrocytes.     

Overexpression of Nd1, a novel Kelch family protein, in the heart of transgenic mice protects against doxorubicin-induced cardiomyopathy

Doxorubicin is one of the most effective drugs available for cancer chemotherapy. However, the clinical use of doxorubicin has been greatly limited because of severe side effects on cardiomyocytes. Since Nd1-L, a novel actin-binding protein, is expressed most abundantly in the heart of adult mice, we examined a role of Nd1-L in doxorubicin-induced cardiomyopathy. When doxorubicin (5 mg/kg × 4 times) was injected into adult mice at a 3-day-interval, approximately 50% of injected mice died within 4 weeks of the first injection. Nd1-L mRNA expression in the heart decreased within 3 weeks after the first injection and many cardiomyocytes of injected mice died by apoptosis. Overexpression of Nd1-L in the heart of transgenic mice protected the cardiomyocytes from apoptosis and improved survival rate after doxorubicin injection. Furthermore, activation of Erk1/2 was observed in cultured cells overexpressing Nd1-L. Thus, Nd1-L plays a critical role in protecting the heart from doxorubicin-induced cardiomyopathy.     

Bicarbonate exacerbates oxidative injury induced by antitumor antibiotic doxorubicin in cardiomyocytes

Doxorubicin, a broad-spectrum antitumor antibiotic, causes dose-dependent cardiomyopathy and heart failure. Although the exact molecular mechanisms of cardiotoxicity are not well established, oxidative mechanisms involving doxorubicin-induced superoxide anion production have been proposed. In this study, we show that bicarbonate, a physiologically relevant tissue component, greatly amplified doxorubicin-induced cardiomyocyte injury. Bicarbonate also enhanced inactivation of aconitase, a crucial tricarboxylic acid cycle enzyme, in cardiomyocytes exposed to doxorubicin. The cell-permeable superoxide dismutase mimetic, Mn(III)tetrakis (4-benzoic acid) porphyrin, reversed doxorubicin-induced cardiomyocyte injury. Bicarbonate enhanced the inactivation of purified mitochondrial aconitase in the xanthine/xanthine oxidase system, generating superoxide. The results suggest that bicarbonate amplifies the prooxidant effect of superoxide. Bicarbonate also caused an increased loading of cardiomyocytes with doxorubicin. We conclude that the bicarbonate-mediated increase in doxorubicin toxicity is due to increased intracellular loading of doxorubicin in cardiomyocytes and subsequent exacerbation of superoxide-mediated cardiomyocyte injury.     

Doxorubicin Impairs the Insulin-Like Growth Factor-1 System and Causes Insulin-Like Growth Factor-1 Resistance in Cardiomyocytes

BackgroundInsulin-like growth factor-1 (IGF-1) promotes the survival of cardiomyocytes by activating type 1 IGF receptor (IGF-1R). Within the myocardium, IGF-1 action is modulated by IGF binding protein-3 (IGFBP-3), which sequesters IGF-1 away from IGF-1R. Since cardiomyocyte apoptosis is implicated in anthracycline cardiotoxicity, we investigated the effects of the anthracycline, doxorubicin, on the IGF-1 system in H9c2 cardiomyocytes.ConclusionsDoxorubicin down-regulates IGF-1R and up-regulates IGFBP-3 via p53 and oxidative stress in H9c2 cells. This leads to resistance to IGF-1 that may contribute to doxorubicin-initiated apoptosis. Further studies are needed to confirm these findings in human cardiomyocytes and explore the possibility of manipulating the IGF-1 axis to protect against anthracycline cardiotoxicity.     

Selective potentiation of drug cytotoxicity by NSAID in human glioma cells: the role of COX-1 and MRP.

Here, we report that nonsteroidal anti-inflammatory drugs (NSAID) enhance the cytotoxic effects of doxorubicin and vincristine in T98G human malignant glioma cells. The cytotoxicity of BCNU, cisplatin, VM26, camptothecin, and cytarabine is unaffected by NSAID. No free radical formation is induced by doxorubicin or vincristine in the absence or presence of NSAID. Doxorubicin and vincristine cytotoxicity in the absence or presence of NSAID are unaffected by free radical scavengers. Functional inhibitors of phospholipase A2 (PLA2), such as dexamethasone and quinacrine, do not mimick the effects of NSAID. T98G cells, but not LN-18, LN-229, LN-308, or A172 glioma cells, express cyclooxygenase (COX-1) and NSAID do not modulate drug cytotoxicity in the other cell lines, except T98G. Thus, augmentation of doxorubicin and vincristine cytotoxicity by NSAID correlates with COX-1 expression. However, ectopic expression of COX-1 in LN-229 cells does not induce the phenotype of T98G cells, indicating that COX-1 inhibition does not mediate the effects of NSAID on drug cytotoxicity. In contrast, a multidrug resistance (MDR) phenotype due to expression of the multidrug resistance-associated protein (MRP) is most prominent in T98G cells and is amenable to modulation by indomethacin, suggesting that inhibition of MRP is at least in partly responsible for the potentiation of doxorubicin and vincristine cytotoxicity by NSAID.     

Metformin protects against doxorubicin-induced cardiotoxicity: involvement of the adiponectin cardiac system

Doxorubicin has cardiotoxic effects that limit its clinical benefit in cancer patients. Metformin exerts cardioprotective actions via AMP-activated protein kinase (AMPK) and increases the expression of adiponectin and its receptors (adipoR1 and adipoR2) in skeletal muscle and adipose tissue, but its effect on cardiac tissue is still unknown. This work aimed to study whether metformin exerts any protective action against the cardiotoxicity of doxorubicin and whether the cardiac system of adiponectin is involved in any such action. The addition of doxorubicin (5μM) to adult mouse cardiomyocytes (HL-1 cell line) induced apoptosis, which was characterized by a loss of cell viability, activation of caspases, and fragmentation of the genetic material. Doxorubicin treatment also caused a decrease in the activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase. Pretreatment with metformin (4mM, 24h) provided protection against doxorubicin-induced damage. This pretreatment significantly increased cell viability, attenuated the activation of caspases and the fragmentation of genetic material, and restored the antioxidant activity. In addition, metformin up-regulated the expression of adiponectin and its receptors, adipoR1 and adipoR2, in cardiomyocytes. In contrast, silencing either adipoR1 or adipoR2 with siRNA inhibited the AMPK activation and the protective effects of metformin. Taken together, these results demonstrate that metformin protects cardiomyocytes from doxorubicin-induced damage and that the cardiac adiponectin system plays an important role in this protective action.     

Attenuation of Doxorubicin-Induced Cardiotoxicity by mdivi-1: A Mitochondrial Division/Mitophagy Inhibitor

Doxorubicin is one of the most effective anti-cancer agents. However, its use is associated with adverse cardiac effects, including cardiomyopathy and progressive heart failure. Given the multiple beneficial effects of the mitochondrial division inhibitor (mdivi-1) in a variety of pathological conditions including heart failure and ischaemia and reperfusion injury, we investigated the effects of mdivi-1 on doxorubicin-induced cardiac dysfunction in naïve and stressed conditions using Langendorff perfused heart models and a model of oxidative stress was used to assess the effects of drug treatments on the mitochondrial depolarisation and hypercontracture of cardiac myocytes. Western blot analysis was used to measure the levels of p-Akt and p-Erk 1/2 and flow cytometry analysis was used to measure the levels p-Drp1 and p-p53 upon drug treatment. The HL60 leukaemia cell line was used to evaluate the effects of pharmacological inhibition of mitochondrial division on the cytotoxicity of doxorubicin in a cancer cell line. Doxorubicin caused a significant impairment of cardiac function and increased the infarct size to risk ratio in both naïve conditions and during ischaemia/reperfusion injury. Interestingly, co-treatment of doxorubicin with mdivi-1 attenuated these detrimental effects of doxorubicin. Doxorubicin also caused a reduction in the time taken to depolarisation and hypercontracture of cardiac myocytes, which were reversed with mdivi-1. Finally, doxorubicin caused a significant elevation in the levels of signalling proteins p-Akt, p-Erk 1/2, p-Drp1 and p-p53. Co-incubation of mdivi-1 with doxorubicin did not reduce the cytotoxicity of doxorubicin against HL-60 cells. These data suggest that the inhibition of mitochondrial fission protects the heart against doxorubicin-induced cardiac injury and identify mitochondrial fission as a new therapeutic target in ameliorating doxorubicin-induced cardiotoxicity without affecting its anti-cancer properties.     

Impact of Doxorubicin Treatment on the Physiological Functions of White Adipose Tissue

White adipose tissue (WAT) plays a fundamental role in maintaining energy balance and important endocrine functions. The loss of WAT modifies adipokine secretion and disrupts homeostasis, potentially leading to severe metabolic effects and a reduced quality of life. Doxorubicin is a chemotherapeutic agent used clinically because of its good effectiveness against various types of cancer. However, doxorubicin has deleterious effects in many healthy tissues, including WAT, liver, and skeletal and cardiac muscles. Our objective was to investigate the effects of doxorubicin on white adipocytes through in vivo and in vitro experiments. Doxorubicin reduced the uptake of glucose by retroperitoneal adipocytes and 3T3-L1 cells via the inhibition of AMP-activated protein kinase Thr172 phosphorylation and glucose transporter 4 content. Doxorubicin also reduced the serum level of adiponectin and, to a greater extent, the expression of genes encoding lipogenic (Fas and Acc) and adipogenic factors (Pparg, C/ebpa, and Srebp1c) in retroperitoneal adipose tissue. In addition, doxorubicin inhibited both lipogenesis and lipolysis and reduced the hormone-sensitive lipase and adipose tissue triacylglycerol lipase protein levels. Therefore, our results demonstrate the impact of doxorubicin on WAT. These results are important to understand some side effects observed in patients receiving chemotherapy and should encourage new adjuvant treatments that aim to inhibit these side effects.     

Subcellular localization and activity of multidrug resistance proteins

The multidrug resistance (MDR) phenotype is associated with the overexpression of members of the ATP-binding cassette family of proteins. These MDR transporters are expressed at the plasma membrane, where they are thought to reduce the cellular accumulation of toxins over time. Our data demonstrate that members of this family are also expressed in subcellular compartments where they actively sequester drugs away from their cellular targets. The multidrug resistance protein 1 (MRP1), P-glycoprotein, and the breast cancer resistance protein are each present in a perinuclear region positive for lysosomal markers. Fluorescence-activated cell sorting analysis suggests that these three drug transporters do little to reduce the cellular accumulation of the anthracycline doxorubicin. However, whereas doxorubicin enters cells expressing MDR transporters, this drug is sequestered away from the nucleus, its subcellular target, in vesicles expressing each of the three drug resistance proteins. Using a cell-impermeable inhibitor of MRP1 activity, we demonstrate that MRP1 activity on intracellular vesicles is sufficient to confer a drug resistance phenotype, whereas disruption of lysosomal pH is not. Intracellular localization and activity for MRP1 and other members of the MDR transporter family may suggest different strategies for chemotherapeutic regimens in a clinical setting.     

Up-regulation and cytoprotective role of epithelial multidrug resistance-associated protein 1 in inflammatory bowel disease

MRP1 (multidrug resistance-associated protein 1) is well known for its role in providing multidrug resistance to cancer cells. In addition, MRP1 has been associated with both pro- and anti-inflammatory functions in nonmalignant cells. The pro-inflammatory function is evident from the fact that MRP1 is a high affinity transporter for cysteinyl-leukotriene C4 (LTC4), a lipid mediator of inflammation. It remains unexplained, however, why the absence of Mrp1 leads to increased intestinal epithelial damage in mice treated with dextran-sodium sulfate, a model for inflammatory bowel disease (IBD). We found that MRP1 expression is induced in the inflamed intestine of IBD patients, e.g. Crohn disease and ulcerative colitis. Increased MRP1 expression was detected at the basolateral membrane of intestinal epithelial cells. To study a putative role for MRP1 in protecting epithelial cells against inflammatory cues, we manipulated MRP1 levels in human epithelial DLD-1 cells and exposed these cells to cytokines and anti-Fas. Inhibition of MRP1 (by MK571 or RNA interference) resulted in increased cytokine- and anti-Fas-induced apoptosis of DLD-1 cells. Opposite effects, e.g. protection of DLD-1 cells against cytokine- and anti-Fas-induced apoptosis, were observed after recombinant MRP1 overexpression. Inhibition of LTC4 synthesis reduced anti-Fas-induced apoptosis when MRP1 function was blocked, suggesting that LTC4 is the pro-apoptotic compound exported by epithelial MRP1 during inflammation. These data show that MRP1 protects intestinal epithelial cells against inflammation-induced apoptotic cell death and provides a functional role for MRP1 in the inflamed intestinal epithelium of IBD patients.     

Cyclohexyl-linked tricyclic isoxazoles are potent and selective modulators of the multidrug resistance protein (MRP1)

Structure–activity relationship (SAR) studies on the tricyclic isoxazole series of MRP1 modulators have resulted in the identification of potent and selective inhibitors containing cyclohexyl-based linkers. These studies ultimately identified compound 21b, which reverses drug resistance to MRP1 substrates, such as doxorubicin, in HeLa-T5 cells (EC₅₀ = 0.093 μM), while showing no inherent cytotoxicity. Additionally, 21b inhibits ATP-dependent, MRP1-mediated LTC₄ uptake into membrane vesicles prepared from the MRP1-overexpressing HeLa-T5 cells (EC₅₀ = 0.064 μM) and shows selectivity (1115-fold) against the related transporter, P-glycoprotein, in HL60/Adr and HL60/Vinc cells. Finally, when dosed in combination with the oncolytic MRP1 substrate vincristine, 21b showed tumor regression and growth delay in MRP1-overexpressing tumors in vivo.     

Exposing cardiomyocytes to subclinical concentrations of doxorubicin rapidly reduces their creatine transport

Doxorubicin is commonly used to treat leukemia, lymphomas, and solid tumors, such as soft tissue sarcomas or breast cancer. A major side effect of doxorubicin therapy is dose-dependent cardiotoxicity. Doxorubicin's effects on cardiac energy metabolism are emerging as key elements mediating its toxicity. We evaluated the effect of doxorubicin on [(14)C]creatine uptake in rat neonatal cardiac myocytes and HL-1 murine cardiac cells expressing the human creatine transporter protein. A significant and irreversible decrease in creatine transport was detected after an incubation with 50-100 nmol/l doxorubicin. These concentrations are well below peak plasma levels (5 μmol/l) and within the ranges (25-250 nmol/l) for steady-state plasma concentrations reported after the administration of 15-90 mg/m(2) doxorubicin for chemotherapy. The decrease in creatine transport was not solely because of increased cell death due to doxorubicin's cytotoxic effects. Kinetic analysis showed that doxorubicin decreased V(max), K(m), and creatine transporter protein content. Cell surface biotinylation experiments confirmed that the amount of creatine transporter protein present at the cell surface was reduced. Cardiomyocytes rely on uptake by a dedicated creatine transporter to meet their intracellular creatine needs. Our findings show that the cardiomyocellular transport capacity for creatine is substantially decreased by doxorubicin administration and suggest that this effect may be an important early event in the pathogenesis of doxorubicin-mediated cardiotoxicity.     

Increase in multidrug transport activity is associated with oocyte maturation in sea stars

In this study, we report on the presence of efflux transporter activity before oocyte maturation in sea stars and its upregulation after maturation. This activity is similar to the multidrug resistance (MDR) activity mediated by ATP binding cassette (ABC) efflux transporters. In sea star oocytes the efflux activity, as measured by exclusion of calcein-am, increased two-fold 3 h post-maturation. Experiments using specific and non-specific dyes and inhibitors demonstrated that the increase in transporter activity involves an ABCB protein, P-glycoprotein (P-gp), and an ABCC protein similar to the MDR-associated protein (MRP)-like transporters. Western blots using an antibody directed against mammalian P-gp recognized a 45 kDa protein in sea star oocytes that increased in abundance during maturation. An antibody directed against sea urchin ABCC proteins (MRP) recognized three proteins in immature oocytes and two in mature oocytes. Experiments using inhibitors suggest that translation and microtubule function are both required for post-maturation increases in transporter activity. Immunolabeling revealed translocation of stored ABCB proteins to the plasma cell membrane during maturation, and this translocation coincided with increased transport activity. These MDR transporters serve protective roles in oocytes and eggs, as demonstrated by sensitization of the oocytes to the maturation inhibitor, vinblastine, by MRP and PGP-specific transporter inhibitors.     

Protective effect of miR378* on doxorubicin‐induced cardiomyocyte injury via calumenin

Doxorubicin (Dox) is a highly effective antitumor antibiotic, however myocardial toxicity severely limits its use clinically. The pathogenesis of doxorubicin‐induced cardiomyopathy is unclear. In Dox cardiomyopathy mice, there is a decline in cardiac function, a change in myocardial pathology and a reduction in miR378* expression. Expression changes in calumenin, an endoplasmic reticulum stress (ERS) chaperone protein and pathway factor, as well as apoptosis, were observed in cardiomyocytes after doxorubicin‐induced injury. However, miR378* increased calumenin expression, eased ERS, and reduced cardiomyocyte apoptosis, while, silencing miR378* reduced calumenin expression, aggravated ERS, and increased cardiomyocyte apoptosis. The above results indicate that miR378* alleviates ERS and inhibits the activation of the ERS‐mediated apoptosis signaling pathway in cardiomyocytes via regulating calumenin expression, thereby reducing cardiomyocyte apoptosis after doxorubicin‐induced injury. Increasing miR378* expression may be a new way to improve cardiac function and quality of life in patients with Dox cardiomyopathy.     

Human pluripotent stem cell-derived cardiomyocytes for studying energy metabolism

Cardiomyocyte energy metabolism is altered in heart failure, and primary defects of metabolic pathways can cause heart failure. Studying cardiac energetics in rodent models has principal shortcomings, raising the question to which extent human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) can provide an alternative. As metabolic maturation of CM occurs mostly after birth during developmental hypertrophy, the immaturity of hiPSC-CM is an important limitation. Here we shortly review the physiological drivers of metabolic maturation and concentrate on methods to mature hiPSC-CM with the goal to benchmark the metabolic state of hiPSC-CM against in vivo data and to see how far known abnormalities in inherited metabolic disorders can be modeled in hiPSC-CM. The current data indicate that hiPSC-CM, despite their immature, approximately mid-fetal state of energy metabolism, faithfully recapitulate some basic metabolic disease mechanisms. Efforts to improve their metabolic maturity are underway and shall improve the validity of this model.