Single Injection Of Doxorubicin Research Articles

Entorhinal Cortex Circuit Dysfunction in Mouse Models of APOE4 and Chemotherapy‐Induced Cognitive Impairment

AbstractBackground APOE4 has broad effects, one of which is worsened cognitive outcomes in cancer survivors following chemotherapy treatment compared to APOE3.Method1‐2 month old female APOE3 and APOE4‐Targeted Replacement (TR) mice were used for whole cell patch‐clamp experiments in the entorhinal cortex. Chemotherapy was introduced via a single injection of doxorubicin (10 mg/kg) or saline (control) and treated APOE‐TR mice were euthanized 1 week post‐injection. 0.5% biocytin internal solution was used to visualize cell morphology post hoc under a confocal microscope.ResultsPyramidal cells in the entorhinal cortex of APOE4‐TR mice (4 mice, n = 26 cells) exhibit significantly decreased spontaneous excitatory and inhibitory postsynaptic current frequencies, thereby contributing to an elevated excitatory‐inhibitory balance compared to APOE3‐TR mice (4 mice, n = 23 cells). Doxorubicin‐treated APOE3‐TR mice (4 mice, n = 33 cells) show a significant increase in spontaneous excitatory and inhibitory postsynaptic current amplitudes compared to controls (2 mice, n = 16 cells), while doxorubicin‐treated APOE4‐TR mice (5 mice, n = 20 cells) display no such synaptic changes compared to controls (4 mice, n = 19 cells).Conclusion APOE4 has baseline effects on entorhinal cortex circuit inhibition and the lack of response to chemotherapy may reflect reduced resilience to stressors.

FGF10 mitigates doxorubicin-induced myocardial toxicity in mice via activation of FGFR2b/PHLDA1/AKT axis.

Doxorubicin is a common chemotherapeutic agent in clinic, but myocardial toxicity limits its use. Fibroblast growth factor (FGF) 10, a multifunctional paracrine growth factor, plays diverse roles in embryonic and postnatal heart development as well as in cardiac regeneration and repair. In this study we investigated the role of FGF10 as a potential modulator of doxorubicin-induced cardiac cytotoxicity and the underlying molecular mechanisms. Fgf10+/- mice and an inducible dominant negative FGFR2b transgenic mouse model (Rosa26rtTA; tet(O)sFgfr2b) were used to determine the effect of Fgf10 hypomorph or blocking of endogenous FGFR2b ligands activity on doxorubicin-induced myocardial injury. Acute myocardial injury was induced by a single injection of doxorubicin (25 mg/kg, i.p.). Then cardiac function was evaluated using echocardiography, and DNA damage, oxidative stress and apoptosis in cardiac tissue were assessed. We showed that doxorubicin treatment markedly decreased the expression of FGFR2b ligands including FGF10 in cardiac tissue of wild type mice, whereas Fgf10+/- mice exhibited a greater degree of oxidative stress, DNA damage and apoptosis as compared with the Fgf10+/+ control. Pre-treatment with recombinant FGF10 protein significantly attenuated doxorubicin-induced oxidative stress, DNA damage and apoptosis both in doxorubicin-treated mice and in doxorubicin-treated HL-1 cells and NRCMs. We demonstrated that FGF10 protected against doxorubicin-induced myocardial toxicity via activation of FGFR2/Pleckstrin homology-like domain family A member 1 (PHLDA1)/Akt axis. Overall, our results unveil a potent protective effect of FGF10 against doxorubicin-induced myocardial injury and identify FGFR2b/PHLDA1/Akt axis as a potential therapeutic target for patients receiving doxorubicin treatment.

EFFECTS OF 5-HYDROXYPYRIMIDINE DERIVATIVE ON GROWTH AND METASTASIS OF MELANOMA B16 IN C57BL/6 MICE

Introduction. Suppression of activation of an alternative immune response is promising approach of tumor immunotherapy. In this study we evaluated antitumor and antimetastatic activity of SNK-411.Objective. Evaluation of antitumor and antimetastatic activity of 5-hydroxypyrimidine derivative SNK-411 in mouse melanoma B16 model.Materials and methods. Antitumor and antimetastatic activity of the SNK-411 were studied in tests on male C57BL/6 mice using the B16-F10 melanoma model. SNK-411 was injected intraperitoneally at doses of 10 and 25 mg/kg from day 2 to day 15 of melanoma development. Doxorubicin was injected at dose of 4 mg/kg on day 2 of tumor development to act as positive control. Antitumor and antimetastatic activity were studied by calculation of tumor growth inhibition and metastasis inhibition index (MII).Results. SNK-411 at doses of 10 and 25 mg/kg and in combination with single injection of doxorubicin in dose of 4 mg/kg showed antimetastatic activity. MII in SNK-411 at 10 mg/kg dose was 72 %, at dose of 25 mg/kg was 82,9 %. The combination of 14-day course of intraperitoneal injections of SNK-411 at dose of 10 mg/kg and injection of doxorubicin 4 mg/kg revealed MII 97,1 %, in half of mice in this group metastasis were not observed on 21st day of melanoma development. All results are statistically significant. There was no significant inhibition of tumor growth in all groups.Conclusion. SNK-411 has antimetastatic activity in tests on melanoma B16 model. Further investigation is required.

Exercise, but Not Metformin Prevents Loss of Muscle Function Due to Doxorubicin in Mice Using an In Situ Method.

Though effective in treating various types of cancer, the chemotherapeutic doxorubicin (DOX) is associated with skeletal muscle wasting and fatigue. The purpose of this study was to assess muscle function in situ following DOX administration in mice. Furthermore, pre-treatments with exercise (EX) or metformin (MET) were used in an attempt to preserve muscle function following DOX. Mice were assigned to the following groups: control, DOX, DOX + EX, or DOX + MET, and were given a single injection of DOX (15 mg/kg) or saline 3 days prior to sacrifice. Preceding the DOX injection, DOX + EX mice performed 60 min/day of running for 5 days, while DOX + MET mice received 5 daily oral doses of 500 mg/kg MET. Gastrocnemius–plantaris–soleus complex function was assessed in situ via direct stimulation of the sciatic nerve. DOX treatment increased time to half-relaxation following contractions, indicating impaired recovery (p < 0.05). Interestingly, EX prevented any increase in half-relaxation time, while MET did not. An impaired relaxation rate was associated with a reduction in SERCA1 protein content (p = 0.07) and AMPK phosphorylation (p < 0.05). There were no differences between groups in force production or mitochondrial respiration. These results suggest that EX, but not MET may be an effective strategy for the prevention of muscle fatigue following DOX administration in mice.

Coronary microcirculation damage in anthracycline cardiotoxicity.

AimsThe aim of this study was to study changes in coronary microcirculation status during and after several cycles of anthracycline treatment.Methods and resultsLarge-white male pigs (n=40) were included in different experimental protocols (ExPr.) according to anthracycline cumulative exposure [0.45 mg/kg intracoronary (IC) doxorubicin per injection] and follow-up: control (no doxorubicin); single injection and sacrifice either at 48 h (ExPr. 1) or 2 weeks (ExPr. 2); 3 injections 2 weeks apart (low cumulative dose) and sacrifice either 2 weeks (ExPr. 3) or 12 weeks (ExPr. 4) after third injection; five injections 2 weeks apart (high cumulative dose) and sacrifice 8 weeks after fifth injection (ExPr. 5). All groups were assessed by serial cardiac magnetic resonance (CMR) to quantify perfusion and invasive measurement of coronary flow reserve (CFR). At the end of each protocol, animals were sacrificed for ex vivo analyses. Vascular function was further evaluated by myography in explanted coronary arteries of pigs undergoing ExPr. 3 and controls. A single doxorubicin injection had no impact on microcirculation status, excluding a direct chemical toxicity. A series of five fortnightly doxorubicin injections (high cumulative dose) triggered a progressive decline in microcirculation status, evidenced by reduced CMR-based myocardial perfusion and CFR-measured impaired functional microcirculation. In the high cumulative dose regime (ExPr. 5), microcirculation changes appeared long before any contractile defect became apparent. Low cumulative doxorubicin dose (three bi-weekly injections) was not associated with any contractile defect across long-term follow-up, but provoked persistent microcirculation damage, evident soon after third dose injection. Histological and myograph evaluations confirmed structural damage to arteries of all calibres even in animals undergoing low cumulative dose regimes. Conversely, arteriole damage and capillary bed alteration occurred only after high cumulative dose regime.ConclusionSerial in vivo evaluations of microcirculation status using state-of-the-art CMR and invasive CFR show that anthracyclines treatment is associated with progressive and irreversible damage to the microcirculation. This long-persisting damage is present even in low cumulative dose regimes, which are not associated with cardiac contractile deficits. Microcirculation damage might explain some of the increased incidence of cardiovascular events in cancer survivors who received anthracyclines without showing cardiac contractile defects.

Anti-oxidant impact of Lisinopril and Enalapril against acute kidney injury induced by doxorubicin in male Wistar rats: involvement of kidney injury molecule-1

Doxorubicin (DOX) is a standard anticancer agent exerting devastating effects as nephrotoxicity, hepatotoxicity and cardiotoxicity. The purpose of this study was to increase the clinical use of DOX through decreasing its detrimental effects via combination with ACE inhibitors to ameliorate the induced acute kidney injury (AKI). AKI was induced by a single injection of DOX (7.5 mg/kg; i.p.) as Group 1; control (vehicle), Group 2; DOX (7.5 mg/kg; i.p.) single dose, Group 3 and 4; Lisinopril (Lis, 20 mg/kg) and Enalapril (Enal, 40 mg/kg) orally administration for 15 consecutive days after DOX injection, respectively. Serum samples were used to measure creatinine and BUN, tissue samples were extracted to determine myeloperoxidase (MPO), malondialdehyde (MDA), total antioxidant capacity (TAC) and kidney injury molecule (KIM-1) using ELISA technique. Heme oxygenase (HO-1) RNA expression was quantified in tissue using real time polymerase chain reaction (PCR). Parts of the kidney tissue were kept in formalin for immunohistochemical demonstration of Cleaved Caspase-3 and NF-κβ immune staining and the other part was used for pathological examination. Oral treatment with Lis (20 mg/kg) and Enal (40 mg/kg) for 15 consecutive days reversed DOX effects as they reduced the serum creatinine and BUN, kidney levels of MPO and MDA, whereas the drugs increased tissue TAC. The administration of Lis and Enal with DOX also reduced KIM-1and HO-1 RNA expression. A significant decrease in cleaved caspase-3 and NF-κβ immunostainings in conjunction with pronounced amelioration in pathologies in the rat kidney were observed. We concluded that DOX adverse effects can be controlled by Lis and Enal.

Synthesis and Antitumor and Antimetastatic Activity of 5-hydroxypyrimidine Derivatives

2-Isobutyl-4,6-dimethyl-5-hydroxypyrimidine (SNK-411) and its salt (SNK-578) were synthesized. Antitumor and antimetastatic activity of the new derivative 5-hydroxypyrimidine SNK-578 were studied in tests on male C57BL/6 mice using the B16 melanoma model. The standard grafted dose was 5 × 106 tumor cells mouse. SNK-578 was injected i.p. at doses of 10 and 25 mg/kg for two weeks from day 2 to day 15 of B16 melanoma development. Doxorubicin was injected to mice at a dose of 4 mg/kg on day 2 of tumor development to act as a positive control and to reveal an additive effect from combined single use of doxorubicin and injection of a course of SNK-578. The combination of i.p. injection of a course of SNK-578 at a dose of 10 mg/kg and a single i.p. injection of doxorubicin at a dose of 4 mg/kg revealed statistically significant tumor growth inhibition that was expressed as a decrease of tumor volume by 2.4, 1.9, and 1.5 times on day 11, 15, and 21 of the test as compared with the active control group that did not receive SNK-578. SNK-578 possessed pronounced antimetastatic activity at doses of 10 and 25 mg/kg for monotherapy and coadministration with doxorubicin at a dose of 4 mg/kg. The metastasis inhibition index (MII) after SNK-578 injection at a dose of 10 mg/kg was 75.8%; at 25 mg/kg, 92.3%. The most pronounced antimetastatic effect was observed after combined i.p. injection of SNK-578 at a dose of 10 mg/kg for 14 d and a single injection of doxorubicin at a dose of 4 mg/kg. Metastases were not observed in six of nine mice on day 21 of B16 melanoma development; the other three had 1, 2, and 3 very small metastases so that the MII was 98.9%.

Structural Analysis of the Myocardium in Experimental Anthracycline-Induced Cardiomyopathy Combined with Adrenergic Stimulation.

We studied dynamic changes in the total number of cardiomyocytes and the character of structural lesions in the myocardium in rats with modeled anthracycline-induced cardiomyopathy provoked by a single injection of doxorubicin in a dose of 10 mg/kg alone or in combination with subsequent adrenergic stimulation. The injections of epinephrine during the development of anthracycline-induced cardiomyopathy resulted in more pronounced loss of body weight, stronger decrement of the heart weight, and more severe decrease of the cardiomyocyte count in comparison with the corresponding changes induced by doxorubicin alone. The basic lesions of cardiomyocytes in anthracycline-induced cardiomyopathy are the lytic alterations and subsegmental contractures; in contrast, combined use of doxorubicin and epinephrine provoked degree II and III contractures. The revealed necrobiotic changes of cardiomyocytes resulted in their death and pronounced decrease of their number at the initial terms of the study. Hypertrophy observed at later terms of the experiments in parallel with partial recovery of cardiomyocyte number reflected the development of regenerative and adaptivecompensatory processes induced by massive death and elimination of the parenchymatous cells (up to 36-37% of population).

Disruption of ROCK1 gene restores autophagic flux and mitigates doxorubicin-induced cardiotoxicity.

Doxorubicin is among the essential medicines with a wide antitumor spectrum, but its clinical application is limited by its cardiotoxicity. We recently discovered that ROCK1 is a key molecule in mediating cardiac remodeling in response to various stresses. To determine the roles of ROCK1 in doxorubicin cardiotoxicity, we gave three doses of doxorubicin injections to wild type (WT) and ROCK1−/− mice with one week intervals between treatments, the cumulative dose being 24 mg/kg. ROCK1−/− mice exhibited preserved cardiac function, reduced apoptosis, autophagy and fibrosis compared to the WT mice. To further determine the cellular mechanisms, we have examined the role of ROCK1 in cardiomyocytes using cardiomyocyte-specific knockout mice, MHC-Cre/ROCK1fl/fl, which partially reproduced the cardioprotective characteristics of ROCK1−/− mice, indicating that ROCK1 in both cardiomyocytes and non-cardiomyocytes mediates doxorubicin cardiotoxicity. To elucidate the molecular mechanisms, a detailed time course study after a single doxorubicin injection at 10 mg/kg was performed in ROCK1−/− and MHC-Cre/ROCK1fl/fl mice. The molecular analysis revealed that both ROCK1−/− and MHC-Cre/ROCK1fl/fl hearts exhibited significant reduction of doxorubicin-induced early responses including increased apoptotic (Bax) and autophagic (p62/SQSTM1 and LC3-II) markers, associated with reduced Beclin 1 phosphorylation on Thr119, supporting reduced Beclin 1-mediated autophagy initiation due to increased association of Beclin 1 with Bcl 2 or Bcl-XL in these hearts compared to the WT or ROCK1fl/fl mice. These results support that ROCK1 deficiency is cardioprotective against doxorubicin-induced cardiotoxicity at least in part through reducing Beclin 1-mediated autophagy initiation in cardiomyocytes and restoring autophagic flux to ameliorate doxorubicin cardiotoxicity.

Inflammatory mediators in a short-time mouse model of doxorubicin-induced cardiotoxicity

Doxorubicin (DOXO) is commonly used to treat a wide range of malignant tumors, but its clinical use is limited by acute and chronic cardiotoxicity. The precise mechanism underlying DOXO-induced cardiotoxicity is still not completely elucidated, but cardiac inflammation seems to be involved. Effects of DOXO on proinflammatory cytokines, inflammatory cell infiltration, and necrosis have been proven only when a functional impairment has already occurred, so this study aimed to investigate the acute effect of DOXO administration in mouse heart. The results of our study demonstrated alterations in cardiac function parameters assessed by ultrasound within 24h after a single injection of DOXO, with a cumulative effect along the increase of the dose and the number of DOXO administrations. At the same time, DOXO causes a significant production of proinflammatory cytokines (such as TNF-α and IL-6) with a concomitant reduction of IL-10, a well-known antiinflammatory cytokine. Furthermore, overexpression of inducible nitric oxide synthase (iNOS) in heart tissue and increased levels of serum nitrite in DOXO-treated mice were detected. Notably, DOXO administration significantly increased nitrotyrosine expression in mouse heart. Our data support the hypothesis that these early events, could be responsible for the later onset of more severe deleterious remodeling leading to DOXO induced cardiomyopathy.

Oxidative Stress Induced Ventricular Arrhythmia and Impairment of Cardiac Function in &lt;i&gt;Nos1ap&lt;/i&gt; Deleted Mice

Genome-wide association study has identified that the genetic variations at NOS1AP (neuronal nitric oxide synthase-1 adaptor protein) were associated with QT interval and sudden cardiac death (SCD). However, the mechanism linking a genetic variant of NOS1AP and SCD is poorly understood. We used Nos1ap knockout mice (Nos1ap(-/-)) to determine the involvement of Nos1ap in SCD, paying special attention to oxidative stress.At baseline, a surface electrocardiogram (ECG) and ultrasound echocardiography (UCG) showed no difference between Nos1ap(-/-) and wild-type (WT) mice. Oxidative stress was induced by a single injection of doxorubicin (Dox, 25 mg/kg). After Dox injection, Nos1ap(-/-) showed significantly higher mortality than WT (93.3 versus 16.0% at day 14, P < 0.01). ECG showed significantly longer QTc in Nos1ap(-/-) than WT, and UCG revealed significant reduction of fractional shortening (%FS) only in Nos1ap(-/-) after Dox injection. Spontaneous ventricular tachyarrhythmias were documented by telemetry recording after Dox injection only in Nos1ap(-/-). Ex vivo optical mapping revealed that the action potential duration (APD)90 was prolonged at baseline in Nos1ap(-/-), and administration of Dox lengthened APD90 more in Nos1ap(-/-) than in WT. The expression of Bnp and the H2O2 level were higher in Nos1ap(-/-) after Dox injection. Nos1ap(-/-) showed a reduced amplitude of calcium transient in isolated cardiomyocytes after Dox injection. Administration of the antioxidant N-acetyl-L-cysteine significantly reduced mortality of Nos1ap(-/-) by Dox injection, accompanied by prevention of QT prolongation and a reduction in %FS.Although Nos1ap(-/-) mice have apparently normal hearts, oxidative stress evokes ventricular tachyarrhythmia and heart failure, which may cause sudden cardiac death.

Quercetin attenuates doxorubicin cardiotoxicity by modulating Bmi‐1 expression

Doxorubicin-based chemotherapy induces cardiotoxicity, which limits its clinical application. We previously reported the protective effects of quercetin against doxorubicin-induced hepatotoxicity. In this study, we tested the effects of quercetin on the expression of Bmi-1, a protein regulating mitochondrial function and ROS generation, as a mechanism underlying quercetin-mediated protection against doxorubicin-induced cardiotoxicity. Effects of quercetin on doxorubicin-induced cardiotoxicity was evaluated using H9c2 cardiomyocytes and C57BL/6 mice. Changes in apoptosis, mitochondrial function, oxidative stress and related signalling were evaluated in H9c2 cells. Cardiac function, serum enzyme activity and reactive oxygen species (ROS) generation were measured in mice after a single injection of doxorubicin with or without quercetin pre-treatment. In H9c2 cells, quercetin reduced doxorubicin-induced apoptosis, mitochondrial dysfunction, ROS generation and DNA double-strand breaks. The quercetin-mediated protection against doxorubicin toxicity was characterized by decreased expression of Bid, p53 and oxidase (p47 and Nox1) and by increased expression of Bcl-2 and Bmi-1. Bmi-1 siRNA abolished the protective effect of quercetin against doxorubicin-induced toxicity in H9c2 cells. Furthermore, quercetin protected mice from doxorubicin-induced cardiac dysfunction that was accompanied by reduced ROS levels and lipid peroxidation, but enhanced the expression of Bmi-1 and anti-oxidative superoxide dismutase. Our results demonstrate that quercetin decreased doxorubicin-induced cardiotoxicity in vitro and in vivo by reducing oxidative stress by up-regulation of Bmi-1 expression. The findings presented in this study have potential applications in preventing doxorubicin-induced cardiomyopathy.

P253Oxidative stress causes the release of a specific subset of proteins from viable cardiac myocytes

Purpose: It is widely accepted that oxidative stress plays a major role in cardiovascular diseases. The aim of this study is to identify novel biomarkers that are released from cardiomyocytes upon oxidative stress before the onset of necrosis. Methods and Results: Firstly, isolated mouse hearts were subjected to Langendorff perfusion with 1 mM H2O2 for 15 min to induce maximal oxidative stress in the absence of cell death, as detected by lactate dehydrogenase release. Notably, SDS/PAGE of tissue samples displayed the disappearance of a band migrating at 72 kDa in the H2O2 group as compared to the normoxic control. Analysis by mass spectrometry identified two proteins, namely EHD4 and HSPA8. Concomitantly, these proteins were found in the coronary effluent. We confirmed this result by both Western blot and immunohistochemistry. In particular, H2O2-perfused heart cryosections displayed 77% and 70% decrease in immunofluorescence as compared to normoxic-perfused hearts for EDH4 and HSPA8, respectively. EHD4 belongs to the EHD protein family that are involved in intracellular trafficking, while HSPA8 is a chaperonin. Then, we evaluated another model of oxidative stress caused by single injection of doxorubicin (15 mg/kg). Mice were sacrificed after 3 days. The EHD4 content was reduced in hearts of doxorubicin-treated mice, as compared to hearts of vehicle-treated animals. To confirm that protein release can occur the absence of cell death, we treated an atriomal murine cell line (HL1) with: H2O2 (10-100 μM), doxorubicin (1-6 μM), or H2O2 (10 μM) + doxorubicin (6 μM). We observed that the intracellular EHD4 content was significantly reduced upon the different treatments, as detected by Western blot and immunocytochemistry in the absence of any loss of cell viability, as detected by Hoecst 22358/Propidium Iodide staining. The release of HSPA8 in the tested protocols was less evident. Conclusions: EHD4 appears as the most prominent protein released by cardiac myocytes undergoing oxidative stress in the absence of cell death. Therefore, EHD4 release should be exploited as a novel plasma biomarker of early oxidative stress, occurring before the onset of necrosis. Further studies will elucidate the mechanisms underlying protein release in viable cardiac myocytes and the effects of specific protein loss on the susceptibility to cell injury.

Paneth cells expand from newly created and preexisting cells during repair after doxorubicin-induced damage

Paneth cell numbers increase following intestinal damage, but mechanisms driving this process are not understood. We hypothesized that the increase in Paneth cell numbers is due to recruitment of cells from a preexisting pool of secretory progenitors. Mice were given a single injection of doxorubicin (Dox), and intestinal tissue was collected 0-168 h after treatment. Paneth, goblet, and intermediate cells were counted and evaluated for cell morphology. Quantitative RT-PCR was used to measure expression of various genes associated with Paneth cell allocation and maturation. Paneth cells were birth dated using incorporation of thymidine analogs given before or after Dox. Staining revealed "intermediate" cells, which were rarely observed in control crypts but increased significantly in number 96 and 120 h after Dox treatment. Birth dating of intermediate cells 5 days after Dox treatment revealed that 24% of these cells took up thymidine analog given prior to Dox treatment and 36% took up thymidine analog given after Dox treatment. Quantitative RT-PCR demonstrated a significant increase in Spdef, Atoh1, Sox9, EphB3, Mist, Wnt5a, FGF-9, and FGF-18 mRNAs and a significant decrease in Indian hedgehog mRNA. Expansion of the Paneth cell compartment after Dox treatment is due to generation of new cells and recruitment of cells from an existing pool. These cells express Paneth and goblet biomarkers and are found only during repair. Expansion of these cells correlates temporally with reduced Indian hedgehog and increased FGF and Wnt mRNA. These findings are significant, as they provide a first step in understanding mechanisms of Paneth cell expansion during mucosal repair.

Abstract B38: Iodine supplement exerts antineoplastic adjuvancy, chemoresistance inhibition and cardioprotection in mammary cancer treatment with anthracyclines

Abstract Mortality for mammary cancer (MC) has decreased in the past decade, owing principally to early detection and the use of neoadjuvant chemotherapy. Of several preparations that cause regression of MC, Doxorubicin (DOX) is the most active, first-line monotherapeutic. Nevertheless, its use has been limited due to the rapid development of chemoresistance and cardiotoxicity caused by free radicals. In previous studies we have shown that supplementation with moderately high doses of molecular iodine (I2) has a powerful antineoplastic effect in methylnitrosourea (MNU)-induced MC models. These studies also showed a consistent antioxidant effect of I2 in normal and tumoral tissues. In the present study we analyzed the effect of I2 in combination with DOX treatment. Female Sprague Dawley rats with MNU-induced MC were treated with the therapeutic DOX dose (16 mg/kg) and with two lower doses (8 and 4 mg/kg) with and without 0.05% I2 in drinking water. In a first, short-term experiment, iodine treatment began 2 days before a single DOX injection (ip), and the animals were analyzed after 7 days. In a second experiment, animals received two injections (days zero and 14) of 4 mg/kg DOX without or with long-term I2 treatment throughout the 56 days of observation. The results showed that at every DOX dose, short-term I2 treatment induced synergic antineoplastic effects (decreasing tumor size and PCNA signal) with significant protection against body weight loss and cardiotoxicity (CK-MB enzyme and cardiac lipoperoxidation). With long-term I2 treatment, mammary tumors became more sensitive to 4 mg/kg DOX, decreasing chemoresistance markers (Bcl2 and survivin) and increasing peroxisome proliferator-activated receptor type gamma expression, indicating a specific interaction of I2 with these receptors. The authors thank Felipe Ortóz for his technical assistance and Leonor Casanova for academic supports. Supported in part by PAPIIT/UNAM 201210 and CONACYT 78955. Citation Format: Carmen Aceves, Yunuen Alfaro-Hernandez, Guadalupe Delgado. Iodine supplement exerts antineoplastic adjuvancy, chemoresistance inhibition and cardioprotection in mammary cancer treatment with anthracyclines. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B38.

Doxorubicin impairs skeletal muscle mitochondrial respiratory capacity in skeletal muscle

Debilitating muscle weakness and fatigue are common side effects of chemotherapy in cancer patients, limiting recovery and increasing morbidity. Our previous work shows that doxorubicin, a common chemotherapy drug, elevates reactive oxygen species (ROS) and decreases contractile force in skeletal muscle. As mitochondria represent a primary source of oxidant generation in muscle, we hypothesized that doxorubicin may compromise mitochondrial respiratory control and increase ROS production. 72 hrs following a single doxorubicin injection (20 mg/kg), maximal ADP‐stimulated O2 consumption was decreased during respiration supported by palmitoylcarnitine (PC) (−46 ± 26 %, p&lt;0.05) and pyruvate/malate (−43 ± 21 %, p&lt;0.05, n=6/group) in permeabilized fiber bundles (PmFB) from red gastrocnemius muscle. PmFB from doxorubicin treated animals also display increased rates of mitochondrial H2O2 emission with PC (23 ± 2 %) and 3‐phosphoglycerate (49 ± 2 %, p=0.1). Our data indicate that doxorubicin impairs both maximal State 3, and fatty‐acid‐supported respiration. In addition, doxorubicin promotes mitochondrial ROS generation, a potential underlying cause of doxorubicin‐induced muscle dysfunction.Supported by DK073488

Doxorubicin-induced glomerulosclerosis with proteinuria in GFP-GABARAP transgenic mice

Autophagy is a process of cellular degradation, and its dysfunction elicits many pathological symptoms. However, the contribution of autophagy to kidney glomerular function has not been fully clarified. We previously reported that LC3, a promising executor of autophagy, played an important role in recovery from podocyte damage in an experimental nephrosis model (Asanuma K, Tanida I, Shirato I, Ueno T, Takahara H, Nishitani T, Kominami E, Tomino Y. FASEB J 17: 1165-1167, 2003). γ-Aminobutyric acid A receptor-associated protein (GABARAP), has recently been characterized as another homolog of LC3, although its precise role in autophagy remains unclear. We recently generated green fluorescent protein (GFP)-GABARAP transgenic mice, in which GFP-GABARAP is abundantly expressed in glomerular podocytes. We found that the transgenic mice showed no obvious phenotype, and podocytes isolated from these mice manifested autophagic activity almost equivalent to that of wild-type mice when measured in vitro. Surprisingly, a single injection of doxorubicin caused a greater increase in proteinuria and sclerotic glomeruli in transgenic mice compared with wild-type mice. Under these conditions, neither GFP-GABARAP nor endogenous GABARAP appeared to be recruited to autophagosomes, and both remained in the cytosol. Moreover, the cytosolic GFP-GABARAP was significantly colocalized with p62 to form aggregates. These results indicate that the GFP-GABARAP/p62 complex is responsible for impairment of glomerular function and that it retards recovery from the effects of doxorubicin.

Overexpression of Nrdp1 in the Heart Exacerbates Doxorubicin-Induced Cardiac Dysfunction in Mice

BackgroundCardiac cell death and generation of oxidative stress contribute to doxorubicin (DOX)-induced cardiac dysfunction. E3 ligase Nrdp1 plays a critical role in the regulation of cell apoptosis, inflammation and production of reactive oxygen species (ROS), which may contribute to heart failure. However, the role of Nrdp1 in DOX-induced cardiac injury remains to be determined.Methods and ResultsWe examined the effect of Nrdp1 overexpression with DOX treatment in rat neonatal cardiomyocytes and mouse heart tissue. Cardiomyocytes were infected with adenovirus containing GFP (Ad-GFP), Nrdp1 wild-type (Ad-Nrdp1) or the dominant-negative form of Nrdp1 (Ad-Dn-Nrdp1), then treated with DOX for 24 hr. DOX treatment increased cell death and apoptosis, with Ad-Nrdp1 infection enhancing these actions but Ad-Dn-Nrdp1 infection attenuating these effects. Furthermore, 5 days after a single injection of DOX (20 mg/kg, intraperitoneally), Nrdp1 transgenic mice (TG) showed decreased cardiac function and increased apoptosis, autophagy and oxidative stress as compared with wild-type (WT) mice (P<0.01). Survival rate was significantly lower in Nrdp1 TG mice than in WT mice 10 days after DOX injection (P<0.01).Conclusions/SignificanceThese results were associated with decreased activation of Akt, extracellular signal-regulated kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3) signaling pathways. Nrdp1 may be a key mediator in the development of cardiac dysfunction after DOX treatment and associated with inhibition of Akt, ERK1/2 and STAT3. Nrdp1 may be a new therapeutic target in protecting against the cardiotoxic effects of DOX.

Pretreatment with Statin Attenuates the Cardiotoxicity of Doxorubicin in Mice

Cardiotoxicity, which may result from intense cardiac oxidative stress and inflammation, is the main limiting factor of the anticancer therapy using doxorubicin. Because statins might exert beneficial pleiotropic cardiovascular effects, among other things, by anti-inflammatory and antioxidative mechanisms, we investigated whether or not fluvastatin pretreatment can attenuate doxorubicin-induced cardiotoxicity. Five days after a single injection of doxorubicin (20 mg/kg; i.p.), left ventricular (LV) function was measured in fluvastatin-treated (DoxStatin; 100 mg/kg/day, p.o.) and saline-treated (doxorubicin) mice (n = 8 per group) by a micro conductance catheter. Untreated mice served as controls (placebo; n = 8 per group). After measurement of cardiac function, LV tissues were analyzed by molecular biological and immunohistologic methods. Injection resulted in significantly impaired LV function (LV pressure, -29%; dp/dtmax, -45%; cardiac output, -68%; P < 0.05) when compared with placebo. This was associated with a significant increase in cardiac oxidative stress, inflammation and apoptotic mechanisms, as indicated by significant increased cardiac lipid peroxidation activity, protein expression of nitrotyrosine, tumor necrosis factor alpha and Bax (P < 0.05). In contrast, DoxStatin mice showed improved LV function (LV pressure, +24%; dp/dtmax, +87%; cardiac output, +87%; P < 0.05) when compared with untreated doxorubicin mice. This was associated with reduced cardiac expression of nitrotyrosine, enhanced expression of the mitochondrial located antioxidative SOD 2, attenuated mitochondrial apoptotic pathways, and reduced cardiac inflammatory response. Statin pretreatment attenuates doxorubicin-induced cardiotoxicity via antioxidative and anti-inflammatory effects.

Abstract 5355: An Alpha-1A-Adrenergic Receptor Subtype Agonist Prevents Cardiomyopathy Without Increasing Blood Pressure

Background: Alpha-1-adrenergic receptor (AR) agonists classically increase blood pressure (BP). Among the 3 alpha-1-AR subtypes, A, B, and D, the alpha-1A is required for cardiac protection in a knockout (KO) mouse, and is sufficient for protection of cultured cardiac myocytes, via ERK activation. However, it is unknown if activation of the alpha-1A-subtype by a drug can protect the heart in vivo. Hypothesis: At a dose that does not increase BP, an agonist selective for the alpha-1A-AR subtype can prevent cardiomyopathy. Methods: We gave the alpha-1A agonist A61603 (A6) to 11 week-old wild type (WT) male C57Bl6J mice by osmotic minipump. We induced cardiomyopathy with a single dose of doxorubicin (DOX) (25 mg/kg IP), a cardiotoxic cancer drug. We measured BP by tail cuff, activated (phosphorylated, P)-ERK by immunoblot, heart mRNAs by RT-qPCR, fractional shortening (FS) by echocardiog-raphy (ECHO), myocyte necrosis by serum creatine kinase (CK), apoptosis by TUNEL stain, and fibrosis by sirius red stain. Results : In dose-finding experiments (0.01–100 ug/kg/d), A6 at 10 ng/kg/d over 7 days had no effect on daily tail cuff BP (average mmHg Vehicle 115 ± 4; A6 119 ± 4), but increased heart P-ERK (1.7-fold) and the mRNAs for beta-MyHC and ANF (5-fold). A6 at higher doses increased BP. Next, A6 at the non-hypertensive dose (10 ng/kg/d) or vehicle was infused over 7 days after a single DOX injection. The TABLE shows that DOX caused cardiomyopathy, with reduced survival and FS, and increased necrosis, apoptosis, and fibrosis (data are mean ± SE). A6 prevented all of these abnormalities. In alpha-1A-subtype KO mice, DOX caused increased apoptosis and mortality compared with WT mice, and A6 had no beneficial effect (not shown), indicating specificity of A6. Conclusions: A very low dose of an agonist selective for the alpha-1A-AR subtype can activate cardiac survival signaling (P-ERK), induce cardiac fetal genes, and prevent DOX-induced cardiomyopathy, all without increasing BP.