Epirubicin Exposure Research Articles

Abstract P2179: Endothelial-derived Extracellular Vesicles Communicate With Cardiomyocytes To Elicit Cancer Therapy-related Cardiac Dysfunction

Background: Treatment with the anthracycline, epirubicin, can result in cancer-therapy related cardiac dysfunction (CTRCD), a leading cause of non-malignant mortality in cancer survivors. Endothelial cells (ECs) lining the blood vessels are the first point of contact of epirubicin and secrete factors that are pivotal in regulating cardiac function. Of these factors, extracellular vesicles (EVs) are known to mediate intercellular communication. It remains unknown whether and how EC-EVs affect cardiomyocyte function in the context of CTRCD. Hypothesis: We hypothesize that epirubicin exposure leads to altered EC-EV cargo which mediates cardiomyocyte dysfunction. Methods: Human umbilical vein endothelial cells (HUVEC) were exposed to the peak and trough concentrations of epirubicin and endothelial function was assessed. EC-EVs were isolated via ultracentrifugation and EV size and concentration were measured through nanoparticle tracking analysis. The protein content of EC-EVs was determined using liquid chromatography with tandem mass spectrometry. Pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were exposed to physiological concentrations of EVs (1x1010 particles/mL) and contractility was assessed in real-time. Results: Epirubicin exposure increased endothelial leak, apoptosis, and adhesion molecule expression. We observed an increase in size and concentration of EC-EVs and proteomic analysis revealed altered EV cargo including reduced cardiomyocyte protective factors, CNP and eNOS in EVs from epirubicin treated HUVEC in comparison to control. Treatment of iPSC-cardiomyocytes with EVs from epirubicin treated HUVECs resulted in reduced contractility compared to EVs from healthy ECs. Cleavage of EC-EV surface proteins via proteinase K abrogated the effects of EC-EVs on iPSC-cardiomyocytes. Cardiomyocyte-specific pathways targeted by EC-EVs from epirubicin- compared to control-treated cells are currently on-going. Conclusion: We demonstrate that epirubicin treatment leads to endothelial dysfunction and altered release of EVs capable of mediating cardiomyocyte dysfunction. Our results show the potential role of EC-EVs in CTRCD pathogenesis and highlight their influence on cardiomyocyte function.

A Physiologically Based Pharmacokinetic Model to Predict Determinants of Variability in Epirubicin Exposure and Tissue Distribution.

Epirubicin is an anthracycline antineoplastic drug that is primarily used in combination therapies for the treatment of breast, gastric, lung and ovarian cancers and lymphomas. Epirubicin is administered intravenously (IV) over 3 to 5 min once every 21 days with dosing based on body surface area (BSA; mg/m2). Despite accounting for BSA, marked inter-subject variability in circulating epirubicin plasma concentration has been reported. In vitro experiments were conducted to determine the kinetics of epirubicin glucuronidation by human liver microsomes in the presence and absence of validated UGT2B7 inhibitors. A full physiologically based pharmacokinetic model was built and validated using Simcyp® (version 19.1, Certara, Princeton, NJ, USA). The model was used to simulate epirubicin exposure in 2000 Sim-Cancer subjects over 158 h following a single intravenous dose of epirubicin. A multivariable linear regression model was built using simulated demographic and enzyme abundance data to determine the key drivers of variability in systemic epirubicin exposure. Multivariable linear regression modelling demonstrated that variability in simulated systemic epirubicin exposure following intravenous injection was primarily driven by differences in hepatic and renal UGT2B7 expression, plasma albumin concentration, age, BSA, GFR, haematocrit and sex. By accounting for these factors, it was possible to explain 87% of the variability in epirubicin in a simulated cohort of 2000 oncology patients. The present study describes the development and evaluation of a full-body PBPK model to assess systemic and individual organ exposure to epirubicin. Variability in epirubicin exposure was primarily driven by hepatic and renal UGT2B7 expression, plasma albumin concentration, age, BSA, GFR, haematocrit and sex.

Circulating Levels of Epirubicin Cause Endothelial Senescence While Compromising Metabolic Activity and Vascular Function

Anthracycline-based chemotherapy is a common treatment for cancer patients. Because it is delivered intravenously, endothelial cells are exposed first and to the highest concentrations, prior to diffusion to target cells. Not surprisingly, vascular dysfunction is a consequence of anthracycline therapy. While chemotherapy-induced endothelial damage at administration sites has been investigated, the effects of lower doses encountered by distant microvascular networks has not. The aim of this study was to investigate the impact of epirubicin, a widely used anthracycline, on healthy endothelial cells to elucidate its effects on microvascular physiology. Here, endothelial cells were briefly exposed to low doses of epirubicin to recapitulate levels in circulation following dilution in the blood and compound half-life in circulation. Both immediate and prolonged responses to treatment were assessed to determine changes in endothelial function. Epirubicin caused a decrease in proliferation and viability in hUVEC, with lower doses resulting in a senescent phenotype in a large proportion of cells, accompanied by a significant increase in pro-inflammatory cytokines and a significant decrease in metabolic activity. Epirubicin exposure also impaired endothelial function with delayed wound closure, reduced angiogenic potential and increased monolayer permeability downstream of VE-cadherin internalization. Primary lung endothelial cells obtained from epirubicin-treated mice similarly demonstrated reduced viability and functional impairment. In vivo, epirubicin treatment resulted in persistent reduction in lung vascular density and significantly increased infiltration of myeloid cells. Modulation of endothelial status and inflammatory tissue microenvironment observed in response to low doses of epirubicin may predict risk for long-term secondary pathologies associated with chemotherapy.

Application of a combination of echocardiographic techniques in an experimental model of epirubicin-induced cardiotoxicity.

This study compared the potential ability of multinomial echocardiographic parameters in early detection, prediction and combined diagnosis of antineoplastic-related cardiotoxicity. Male Balb/c mice were repeatedly administered with low doses of epirubicin (6 × 3mg/kg; n = 20) to induce cardiac injury or with placebo as control (n = 10). Conventional and strain parameters as well as myocardial performance index (MPI) were analyzed at baseline, 1day after the second, fourth and sixth cycle, and 12days after completion of chemotherapy (as follow-up) by a high-resolution rodent ultrasound machine. After the experiment, serum cTnI levels were measured, and myocardialinjury was evaluated by histological analyses. Thirteen mice developed cardiotoxicity after epirubicin exposure. Global longitudinal (GLS), radial strain (GRS) and longitudinal strain rate (LSR) were markedly decreased (all P ≤ 0.01) and MPI was increased (P ≤ 0.05) at the completion of treatment compared with baseline values. GLS expressed the best correlations with myocardial pathological injury, especially with collagen content (ρ = -0.68, P < 0.01). Additionally, GLS and MPI were associated with serum cTnI levels. A > 9.5% decrease in GLS from baseline to the fourth cycle of chemotherapy could predict future cardiotoxicity (odds ratio = 0.331, P < 0.05). GLS (cutoff value, -15.16%) combined with MPI (cutoff value, 0.64) could improve the accuracy of diagnosing cardiotoxicity (sensitivity, 92%; specificity, 87%). GLS was the only predictor of cardiotoxicity. GLS combined with MPI may provide a noninvasive and accurate method for the early detection of cardiotoxicity.

Distinct effects of epirubicin, cisplatin and cyclophosphamide on ovarian somatic cells of prepuberal ovaries.

In vitro culture models were used to characterize the effects of chemotherapeutic drugs and of LH on somatic cells from prepuberal mouse ovaries. All cell types (pre- and granulosa cells, pre-thecal and OSE cells) underwent apoptosis following Epirubicin (0.5μM) exposure for 24hrs (about 60%) and 48hrs (>80%). Cisplatin (10μM) and the Cyclophosphamide active metabolite, Phosphoramide Mustard (10μM), didn’t cause apoptosis in 90% of pre-thecal and pre-granulosa cells up to 72hrs of exposure, although they suffered extensive DNA damage and cell cycle arrest, and acquired stress induced premature senescence (SIPS) features. Cultured granulosa cells didn’t show evident DNA damage and remained viable without acquiring SIPS features; OSE cells were resistant to apoptosis and SIPS but not to DNA damage. These latter, like pre-thecal and pre-granulosa cells, were able of efficient DNA repair involving MLH1-dependent MMR pathways. SIPS features were also observed in ovary after in vivo treatment with Cisplatin. LH (200mIU/mL) didn’t significantly influence apoptosis, SIPS and DNA damage but favoured DNA repair. These results show that somatic cells of prepuberal ovary response to drugs in different ways, either undergoing apoptosis or SIPS, either showing resistance to Cisplatin and Phosphoramide Mustard. Moreover, a new role of LH in promoting DNA repair was shown.

Mutagenicity assessment of environmental contaminations in a hospital centralized reconstitution unit

IntroductionCytotoxic drug exposure of hospital staff preparing intravenous chemotherapy is a major issue and related mutagenic risks should be more explored. The aim of this study was to assess the mutagenicity of several cytotoxic mixtures prepared at fixed concentrations, and the mutagenicity of environmental samples collected in a hospital centralized reconstitution unit. In parallel cytotoxic exposure in environmental samples was quantified. MethodsEnvironmental samples were performed by wiping method using swabs in five critical production unit areas. Mutagenicity was assessed with a liquid microplate AMES test using two salmonella typhimurium strains (TA98 and TA100), in prepared cytotoxic mixtures containing 14 cytotoxic drugs (cyclophosphamide, cytarabine, dacarbazine, docetaxel, doxorubicin, epirubicin, etoposide, 5-fluorouracil, gemcitabine, ifosfamide, irinotecan, methotrexate, paclitaxel and pemetrexed) according a dichotomous strategy and in environmental samples. Cytotoxic drugs were quantified in samples using liquid chromatography coupled to mass tandem spectrometry. ResultsMutagenesis was observed for the mix of 14 cytotoxic drugs with TA98 strain ± S9 fraction but not TA100 strain. After dichotomous approach, only doxorubicin and epirubicin exposure were associated to mutagenesis. The mutagenesis observed was expressed at lower concentrations with the mix of the 14 drugs than with anthracyclins alone, assuming a synergistic effect. Despite measurable level of cytotoxic contamination in environmental samples, no mutagenesis was highlighted in Ames tests performed on these environmental samples. ConclusionsThe analyses carried out show the conservation of the mutagenicity of cytotoxic drugs found in very low quantities in the environment. The traces of cytotoxic drugs found in our unit regularly exceed the limits given by some authors. This approach may be considered as a new tool to monitor environmental contamination by cytotoxic drugs.

Exposition du personnel des établissements de soin aux médicaments anticancéreux : de l’évaluation à la prévention

Cytotoxic drug exposure of hospital staff preparing intravenous chemotherapy is a major issue and related mutagenic risks should be more explored. The aim of this study was to assess the mutagenicity of several cytotoxic mixtures prepared at fixed concentrations, and the mutagenicity of environmental samples collected in a hospital centralized reconstitution unit. In parallel cytotoxic exposure in environmental samples was quantified.Environmental samples were performed by wiping method using swabs in five critical production unit areas. Mutagenicity was assessed with a liquid microplate AMES test using two salmonella typhimurium strains (TA98 and TA100), in prepared cytotoxic mixtures containing 14 cytotoxic drugs (cyclophosphamide, cytarabine, dacarbazine, docetaxel, doxorubicin, epirubicin, etoposide, 5-fluorouracil, gemcitabine, ifosfamide, irinotecan, methotrexate, paclitaxel and pemetrexed) according a dichotomous strategy and in environmental samples. Cytotoxic drugs were quantified in samples using liquid chromatography coupled to mass tandem spectrometry.Mutagenesis was observed for the mix of 14 cytotoxic drugs with TA98 strain ± S9 fraction but not TA100 strain. After dichotomous approach, only doxorubicin and epirubicin exposure were associated to mutagenesis. The mutagenesis observed was expressed at lower concentrations with the mix of the 14 drugs than with anthracyclins alone, assuming a synergistic effect. Despite measurable level of cytotoxic contamination in environmental samples, no mutagenesis was highlighted in Ames tests performed on these environmental samples.The analyses carried out show the conservation of the mutagenicity of cytotoxic drugs found in very low quantities in the environment. The traces of cytotoxic drugs found in our unit regularly exceed the limits given by some authors. This approach may be considered as a new tool to monitor environmental contamination by cytotoxic drugs.

Activation of Akt characterizes estrogen receptor positive human breast cancers which respond to anthracyclines.

Anthracyclines are key components of human breast cancer chemotherapy. Here, we explored the role of Akt signaling in anthracycline resistance.The antitumor activity of doxorubicin and Akt inhibitor A-443654 alone or combined was examined in estrogen receptor (ER) positive and negative human breast cancer cell lines. Further, we examined mRNA changes induced by anthracyclines in locally advanced breast cancers biopsied before and after treatment in two clinical trials.Doxorubicin increased Akt phosphorylation in ER positive MCF7 and T47D cell lines, with no effect in ER negative MDA-MB231 breast cancer cells. A-443654 was significantly more cytotoxic in doxorubicin-resistant compared to doxorubicin-naïve MCF7. This difference was not observed in MDA-MB231. Among 24 patients, AKT1 gene expression increased 24 hrs after the initial epirubicin exposure in ER positive tumors responding to therapy (n=6), as compared to ER positive non-responders (n=7) or ER negative tumors (n=11). In contrast, AKT1 mRNA changes after 16 weeks of doxorubicin were unrelated to clinical response and ER status (n=30).In conclusion, rapid Akt activation was observed in ER positive breast cancers which responded to anthracyclines. Increased cytotoxicity of A-443654 in doxorubicin-resistant MCF7 cells indicates a possible role for Akt inhibitors in ER positive breast cancers where chemoresistance evolves.

MiR-671-5p inhibits epithelial-to-mesenchymal transition by downregulating FOXM1 expression in breast cancer.

MicroRNA (miRNA) dysfunction is associated with a variety of human diseases, including cancer. Our previous study showed that miR-671-5p was deregulated throughout breast cancer progression. Here, we report for the first time that miR-671-5p is a tumor-suppressor miRNA in breast tumorigenesis. We found that expression of miR-671-5p was decreased significantly in invasive ductal carcinoma (IDC) compared to normal in microdissected formalin-fixed, paraffin-embedded (FFPE) tissues. Forkhead Box M1 (FOXM1), an oncogenic transcription factor, was predicted as one of the direct targets of miR-671-5p, which was subsequently confirmed by luciferase assays. Forced expression of miR-671-5p in breast cancer cell lines downregulated FOXM1 expression, and attenuated the proliferation and invasion in breast cancer cell lines. Notably, overexpression of miR-671-5p resulted in a shift from epithelial-to-mesenchymal transition (EMT) to mesenchymal-to-epithelial transition (MET) phenotypes in MDA-MB-231 breast cancer cells and induced S-phase arrest. Moreover, miR-671-5p sensitized breast cancer cells to cisplatin, 5-fluorouracil (5-FU) and epirubicin exposure. Host cell reactivation (HCR) assays showed that miR-671-5p reduces DNA repair capability in post-drug exposed breast cancer cells. cDNA microarray data revealed that differentially expressed genes when miR-671-5p was transfected are associated with cell proliferation, invasion, cell cycle, and EMT. These data indicate that miR-671-5p functions as a tumor suppressor miRNA in breast cancer by directly targeting FOXM1. Hence, miR-671-5p may serve as a novel therapeutic target for breast cancer management.

Abstract 3062: miR-671-5p promotes epithelial-to-mesenchymal transition by downregulating FOXM1 expression in breast cancer

Abstract microRNA (miRNA) dysfunction is associated with a variety of human diseases including cancer. Our previous study showed that miR-671-5p was deregulated during breast cancer progression. We aim to decipher the functional mechanism of miR-671-5p in breast cancer. We found that expression of miR-671-5p was decreased significantly in ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) compared to normal and atypical ductal hyperplasia (ADH) in the microdissected formalin-fixed, paraffin-embedded (FFPE) tissues. Forkhead Box M1 (FOXM1), as an oncogenic transcription factor, was predicted as one of the direct targets of miR-671-5p, which was subsequently confirmed by luciferase assays. Forced expression of miR-671-5p in breast cancer cell lines downregulated FOXM1 expression, and attenuated the proliferation and invasion in breast cancer cell lines. Notably, overexpression of miR-671-5p resulted in a shift from epithelial-to-mesenchymal transition (EMT) to mesenchymal-to-epithelial transition (MET) phenotypes in MDA-MB-231 breast cancer cells and induced S-phase arrest. Moreover, miR-671-5p sensitized breast cancer cells to cisplatin, 5-fluorouracil (5-FU) and epirubicin exposure. Host cell reactivation (HCR) assays showed that miR-671-5p reduces DNA repair capability in post-drug exposed breast cancer cells. These data indicates that miR-671-5p functions as a tumor suppressor miRNA by directly targeting FOXM1 in breast cancer. Hence, miR-671-5p may serve as a novel therapeutic target for breast cancer management. Citation Format: Xiaohui Tan, Yebo Fu, Liang Chen, Shejuan An, Woojin Lee, Yinglei Lai, Katayoon Rezaei, Sana Tabbara, Christine B. Teal, Yan-gao Man, Robert Siegel, Rachel F. Brem, Sidney W. Fu. miR-671-5p promotes epithelial-to-mesenchymal transition by downregulating FOXM1 expression in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3062. doi:10.1158/1538-7445.AM2015-3062

MiR-638 mediated regulation of BRCA1 affects DNA repair and sensitivity to UV and cisplatin in triple-negative breast cancer.

IntroductionTriple-negative breast cancer (TNBC) represents 15 to 20% of all types of breast cancer; however, it accounts for a large number of metastatic cases and deaths, and there is still no effective treatment. The deregulation of microRNAs (miRNAs) in breast cancer has been widely reported. We previously identified that miR-638 was one of the most deregulated miRNAs in breast cancer progression. Bioinformatics analysis revealed that miR-638 directly targets BRCA1. The aim of this study was to investigate the role of miR-638 in breast cancer prognosis and treatment.MethodsFormalin-fixed, paraffin-embedded (FFPE) breast cancer samples were microdissected into normal epithelial and invasive ductal carcinoma (IDC) cells, and total RNA was isolated. Several breast cancer cell lines were used for the functional analysis. miR-638 target genes were identified by TARGETSCAN-VERT 6.2 and miRanda. The expression of miR-638 and its target genes was analyzed by real-time qRT-PCR and Western blotting. Dual-luciferase reporter assay was employed to confirm the specificity of miR-638 target genes. The biological function of miR-638 was analyzed by MTT chemosensitivity, matrigel invasion and host cell reactivation assays.ResultsThe expression of miR-638 was decreased in IDC tissue samples compared to their adjacent normal controls. The decreased miR-638 expression was more prevalent in non-TNBC compared with TNBC cases. miR-638 expression was significantly downregulated in breast cancer cell lines compared to the immortalized MCF-10A epithelial cells. BRCA1 was predicted as one of the direct targets of miR-638, which was subsequently confirmed by dual-luciferase reporter assay. Forced expression of miR-638 resulted in a significantly reduced proliferation rate as well as decreased invasive ability in TNBC cells. Furthermore, miR-638 overexpression increased sensitivity to DNA-damaging agents, ultraviolet (UV) and cisplatin, but not to 5-fluorouracil (5-FU) and epirubicin exposure in TNBC cells. Host cell reactivation assays showed that miR-638 reduced DNA repair capability in post UV/cisplatin-exposed TNBC cells. The reduced proliferation, invasive ability, and DNA repair capabilities are associated with downregulated BRCA1 expression.ConclusionsOur findings suggest that miR-638 plays an important role in TNBC progression via BRCA1 deregulation. Therefore, miR-638 might serve as a potential prognostic biomarker and therapeutic target for breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0435-5) contains supplementary material, which is available to authorized users.

Two‐dimensional speckle tracking echocardiography combined with high‐sensitive cardiac troponin T in early detection and prediction of cardiotoxicity during epirubicine‐based chemotherapy

To investigate whether alterations of myocardial strain and high-sensitive cardiac troponin T (cTnT) could predict future cardiac dysfunction in patients after epirubicin exposure. Seventy-five patients with non-Hodgkin lymphoma treated with epirubicin were studied. Blood collection and echocardiography were performed at baseline, 1 day after the third cycle, and 1 day after completion of chemotherapy. Patients were studied using echocardiography during follow-up. Global longitudinal (GLS), circumferential (GCS), and radial strain (GRS) were calculated using speckle tracking echocardiography. Left ventricular ejection fraction was analysed by real-time 3D echocardiography. Cardiotoxicity was defined as a reduction of the LVEF of ≥5% to <55% with symptoms of heart failure or an asymptomatic reduction of the LVEF of ≥10% to <55%. Fourteen patients (18.67%) developed cardiotoxicity after treatment. GLS (-18.48 ± 1.72% vs. -15.96 ± 1.6%), GCS (-20.93 ± 2.86% vs. -19.20 ± 3.21%), and GRS (39.23 ± 6.44% vs. 34.98 ± 6.2%) were markedly reduced and cTnT was elevated from 0.0010 ± 0.0020 to 0.0073 ± 0.0038 ng/mL (P all < 0.01) at the completion of chemotherapy compared with baseline values. A >15.9% decrease in GLS [sensitivity, 86%; specificity, 75%; area under the curve (AUC) = 0.815; P = 0.001] and a >0.004 ng/mL elevation in cTnT (sensitivity, 79%; specificity, 64%; AUC = 0.757; P = 0.005) from baseline to the third cycle of chemotherapy predicted later cardiotoxicity. The decrease in GLS remained the only independent predictor of cardiotoxicity (P = 0.000). GLS combined with cTnT may provide a reliable and non-invasive method to predict cardiac dysfunction in patients receiving anthracycline-based chemotherapy.

Early detection of anthracycline-induced cardiotoxicity using two-dimensional speckle tracking echocardiography

To examine the subclinical myocardial injury shortly after epirubicin exposure in asymptomatic patients with large B-cell non-Hodgkin lymphoma using 2-dimensional (2D) speckle tracking echocardiography. Sixty-seven patients aged 52.58 ± 13.86 years with large B-cell non-Hodgkin lymphoma treated with epirubicin were studied. Echocardiography was performed at baseline and 1 day after completion of the therapy. Global and regional longitudinal (LS), circumferential (CS) and radial strain (RS), standard deviation of time to peak LS (TLS-SD), CS (TCS-SD), RS(TRS-SD) were calculated using 2D speckle tracking echocardiography. Despite normal left ventricular ejection fraction, global LS (-18.30 ± 1.87% vs.-16.18 ± 1.92%; p < 0.01), CS (-20.37 ± 2.89% vs. -18.25 ± 2.40%, p < 0.01) and RS (39.95 ± 5.79% vs. 36.15 ± 5.79%, p < 0.01), were remarkably reduced compared to baseline values. However, TLS-SD, TCS-SD and TRS-SD showed no significant difference after epirubicin exposure. Hypertension was an independent predictor of reduction of global LS, CS and RS. Subtle abnormalities in myocardial systolic function were present in asymptomatic patients shortly after anthracycline exposure, which could be detected by 2D speckle tracking echocardiography.

Two-dimensional speckle tracking echocardiography in early detection and prediction of cardiotoxocity during epirubicine based chemotherapy

Objective: To investigate whether alterations of myocardial strain and high sensitive cardiac Troponin T (cTnT) could predict future cardiac dysfunction in patients after epirubicin exposure. Methods: Sixty-five patients aged 52.46±13.58 years with newly-diagnosed large B-cell non-Hodgkin lymphoma treated with epirubicin were studied. Blood collection and echocardiography were performed at baseline, 1 day after the third cycle, and 1 day after chemotherapy completion. After 4-6 months of chemotherapy, patients were studies using echocardiography. cTnT was detected with a highly sensitive assay. Longitudinal (LS), Circumferential (CS) and Radial Strain (RS) were calculated using two-dimensional speckle tracking echocardiography. Left Ventricular Ejection Fraction (LVEF) was analyzed by real-time 3D echocardiography. Cardiotoxicity was defined as a reduction of the Left Ventricular Ejection Fraction (LVEF) of ≥5% to <55% with symptoms of heart failure or an asymptomatic reduction of the LVEF of ≥10% to <55%. Results: LVEF remained stable and within normal limits in the whole course of chemotherapy, however decreased from 65.13±3.65% at baseline to 60.58±4.56% during follow-up (p=0.000). Twelve patients (18.46%) developed cardiotoxicity 4-6 months after treatment. Global LS (-18.56±1.69% vs -15.79±1.53%), CS (-20.88±2.67% vs -19.23±3.21%), RS (39.32±6.36% vs 34.79±6.15%) were markedly reduced and cTnT elevated from 0.0010±0.0020ng/ml to 0.0072±0.0035ng/ml (p all 15.9% decrease in longitudinal strain (sensitivity: 86%, specificity: 75%) and a >0.004ng/ml elevation in cTnT levels (sensitivity: 79%, specificity: 64%) from baseline to the third cycle of chemotherapy predicted later cardiotoxicity. Conclusions: Longitudinal strain combined with high sensitive cTnT may provide a reliable and non-invasive method to predict cardiac dysfunction in patients receiving anthracycline-based chemotherapy.

GW24-e1764 Speckle Tracking Imaging Combined with High Sensitive Cardiac Troponin T in Early Detection and Prediction of Cardiotoxicity during Anthracycline-based Chemotherapy

ObjectivesTo detect the early subclinical myocardial injury during and shortly after epirubicin exposure in asymptomatic patients with large B-cell non-Hodgkin lymphoma by two-dimensional speckle tracking imaging and high sensitive cardiac...

Epirubicin-mediated expression of miR-302b is involved in osteosarcoma apoptosis and cell cycle regulation

Epirubicin is widely used in osteosarcoma chemotherapy. Growing evidence indicates that the microRNA (miRNA) expression levels which are induced by chemotherapeutic agents play an important role in osteosarcoma development and progression. In this study we investigate the alterations of miRNA expression in the osteosarcoma cells after epirubicin treatment and whether miRNAs can enhance its anti-osteosarcoma effect. After epirubicin exposure, microarray shows 40 miRNAs are differentially expressed in osteosarcoma cells including 24 down-regulated miRNAs. Notably, miR-302b, which is stably low-expressed in osteosarcoma, could be induced by the epirubicin. Furthermore, we find that miR-302b can inhibit the osteosarcoma cell proliferation, promote cell apoptosis and cell cycle arrest MiR-302b can activate caspase-3 and regulate the Akt/pAkt, Bcl-2, Bim expression to increase the cell apoptosis. Meanwhile, miR-302b also attenuates cyclin D1 and CDKs expression to induce cell cycle arrest. Therefore, our results suggest miR-302b can play an essential role in osteosarcoma treatment as a potential tumor suppressor.

Altered chemotherapeutic response of primary human breast cancer epithelial cells (HBCEC) and breast cancer cell lines

11506 Background: A novel technique to obtain individual primary cultures of human breast cancer biopsies was filed for patent (PCT/DE 2006/000608). The different individualized HBCEC (human breast cancer epithelial cell) cultures will be characterized and chemotherapeutic effects will be compared to established breast cancer cell lines. Methods: Primary HBCEC from 20 different breast cancer patients were characterized for epithelial cell and tumor markers by immunofluorescence and PCR. Following treatment with 1μM epirubicin for 1h up to 72h differences in protein expression patterns were compared to the similarly treated MCF-7 cell line by 2D gel electrophoresis. Differentially expressed protein spots were identified by mass spectrometry and confirmed by appropriate Western blot analysis. Results: Characterization of primary HBCEC revealed continuous mitosis and cell cycle progression for more than one year in culture with no significant contamination by fibroblasts or other cell types. Whereby HBCEC underwent cell death within 72h of epirubicin treatment analysis by 2D gel and subsequent protein identification by MALDI-TOF/TOF mass spectrometry exhibited a variety of differences compared to MCF-7 cells including HSP27 and prohibitin. Appropriate Western blots confirmed these differences and revealed altered expression levels for HSP27 and prohibitin in the course of epirubicin exposure in HBCEC and MCF-7 cells, respectively, suggesting altered signalling pathways in either primary breast cancer cells or the tumor cell line. Conclusions: Individualized primary HBCEC from various patients could provide a cellular platform beyond breast cancer cell lines, which eventually meet the requirements for an appropriate breast cancer testing system including the characterization of biomarkers and the identification of potential molecular targets. No significant financial relationships to disclose.

Plasma and Tissue Pharmacokinetics of Epirubicin and Paclitaxel in Patients Receiving Neoadjuvant Chemotherapy for Locally Advanced Primary Breast Cancer

The objective of the study was to assess individual distribution of antineoplastic drugs into the tumor. Twelve advanced-stage primary breast cancer patients with neoadjuvant epirubicin+paclitaxel chemotherapy were studied. Plasma concentrations of epirubicin and paclitaxel were monitored for 24 h. Epirubicin concentrations in subcutaneous and tumor tissues were measured using microdialysis up to 12 h postdose. Epirubicin concentrations were described by a compartmental population pharmacokinetic model (NONMEM). Noncompartmental analysis was used for paclitaxel. Plasma pharmacokinetics corresponded to published data. Mean epirubicin exposure in the tumor and in subcutaneous tissue was very similar, but tissue Cmax and area under the curve values reached only (means) 1% and 11%, respectively, of plasma values. Epirubicin doses were significantly correlated to tumor exposure irrespective of body surface area. There is no specific barrier for epirubicin to reach primary breast cancer tumors.

The nuclear membrane in multidrug resistance: microinjection of epirubicin into bladder cancer cell lines

To assess whether microinjecting epirubicin into cells showing multidrug resistance (MDR, common to many cancers, including bladder cancer, with resistance to, e.g. anthracyclines and mitomycin C) spares the nucleus, as when these drugs accumulate, distribution in MDR cells characteristically spares the nucleus, suggesting that the nuclear membrane is responsible for excluding cytotoxic drugs from MDR nuclei. Nuclear exclusion of drugs is an important feature of resistance in MDR cells, as many MDR-susceptible drugs have cytotoxic actions within the nucleus. Drug accumulation in 'classical' P-glycoprotein-mediated MDR cells is greatly reduced by efflux. Microinjection of epirubicin into the cytoplasm of MDR cells bypasses the P-glycoprotein efflux pump on the plasma membrane. Nuclear sparing would directly implicate the nuclear membrane in this phenomenon. Because of their fluorescence properties, which allow study by confocal microscopy and flow cytometry, anthracyclines have also been used extensively to investigate MDR. Thus sensitive (MGH-U1 and RT112) and MDR (MGH-U1R and MGH-U1-MMC) bladder cancer cell lines were used. Adherent cells from each cell line were individually microinjected with epirubicin (0.5 mg/mL) and a 77 kDa fluorescein isothiocyanate (FITC)-dextran (0.5 mg/mL). The pattern of nuclear epirubicin uptake in injected cells was then evaluated by confocal microscopy. The 77 kDa FITC-dextran allowed easier identification of injected cells and was also excluded from their nuclei. Sensitive bladder cancer cell lines all showed a nuclear accumulation pattern of epirubicin, consistent with their normal uptake after exposure to epirubicin. The MDR cell lines showed the characteristic nuclear-sparing pattern of epirubicin uptake, similar to the normal uptake pattern after epirubicin exposure. The 77 kDa FITC-dextran showed clearly which cells had been microinjected, and was excluded from the nuclei of all injected cells. Cell viability was confirmed by acridine-orange staining after initial visualization of injected cells. The nuclear membrane is responsible for the nuclear exclusion of epirubicin in MDR cells. Further work is necessary to determine the mechanisms involved.

Alteration in Epirubicin Pharmacokinetics and Metabolism by Dexverapamil: Results from a Phase II Study in Patients with Metastatic Breast Cancer

Background: To study the effect of the resistance-modifying agent (RMA) dexverapamil on the pharmacokinetics and metabolism of epirubicin. Methods: Nine women with metastatic breast cancer who received epirubicin (120 mg/m 2 ) as a 0.25-hour infusion without dexverapamil during the first treatment cycle and with dexverapamil (12 × 300 mg p. o. every 6 h) during the third treatment cycle were monitored with frequent plasma samples up to 48 h postinfusion. Epirubicin and its metabolites and dexverapamil and its main metabolite nordexverapamil were measured using a reverse-phase high-pressure liquid chromatography assay. Results: The concomitant administration of dexverapamil resulted in an increase in mean (±SD) epirubicin clearance from 1,408 (610) ml/min/m 2 to 2,004 (1,696) ml/min/m 2 , a decrease in epirubicin exposure (area under the curve (AUC)) from 2,968 (1,219) mg × h/ml to 1,901 (494) mg × h/ml, and an increase of the volume of distribution at steady state from 1,436 (1,231) l/m 2 to 2,425 (1,340) l/m 2 . The mean residence time and the elimination half-life were not different. There was a significant increase of epiglucuronide and the 7-deoxy-aglycones. At the time of epirubicin administration mean dexverapamil and nordexverapamil plasma levels were 790±409 and 755 ± 274 ng/ml, respectively. Conclusion: Dexverapamil had a significant effect on the pharmacokinetics and metabolism of epirubicin. However, other than observed in previous studies of RMAs and cytotoxic drugs, dexverapamil did not increase but reduce the AUC of epirubicin and had no effect on its toxicity. These data suggest that the type of pharmacologic interaction between RMAs and cytotoxic drugs can differ depending on the particular compounds used in combination.