Idarubicin Treatment Research Articles

Idarubicin combats abiraterone and enzalutamide resistance in prostate cells via targeting XPA protein

Although second-generation therapies like abiraterone (ABI) and enzalutamide (ENZ) benefit patients with castration-resistant prostate cancer (CRPC), drug resistance frequently occurs, eventually resulting in therapy failure. In this study, we used two libraries, FDA-approved drug library and CRISP/Cas9 knockout (GeCKO) library to screen for drugs that overcome treatment resistance and to identify the potential drug-resistant genes involved in treatment resistance. Our screening results showed that the DNA-damaging agent idarubicin (IDA) overcame abiraterone and enzalutamide resistance in prostate cancer cells. IDA treatment inhibited the DNA repair protein XPA expression in a transcription-independent manner. Consistently, XPA knockout sensitized prostate cancer cells to abiraterone and enzalutamide treatment. In conclusion, IDA combats abiraterone and enzalutamide resistance by reducing XPA protein level in prostate cancer.

Chemotherapy-Induced Survivin Regulation in Acute Myeloid Leukemia Cells

Survivin is a 16.5 kDa protein highly expressed in centrosomes, where it controls proper sister chromatid separation. In addition to its function in mitosis, survivin is also involved in apoptosis. Overexpression of survivin in many cancer types makes it a suitable target for cancer therapy. Western blotting and confocal microscopy were used to characterize the effect of chemotherapy on acute myeloid leukemia (AML) cells. We found enhanced survivin expression in a panel of AML cell lines treated with cytarabine (Ara-C), which is part of a first-line induction regimen for AML therapy. Simultaneously, Ara-C caused growth arrest and depletion of the mitotic cell fraction. Subsequently, the effect of a second component of standard therapy protocol, idarubicin, and of a known survivin inhibitor, YM-155, on cell viability and survivin expression and localization in AML cells was investigated. Idarubicin reversed Ara-C-induced survivin upregulation in the majority of AML cell lines. YM-155 caused survivin deregulation together with a viability decrease in cells resistant to idarubicin treatment, suggesting that YM-155 might be efficient in a specific subset of AML patients. Expression levels of other apoptosis-related proteins, in particular X-linked inhibitor of apoptosis (XIAP), Mcl-1, and p53, and of the cell-cycle inhibitor p21 considerably changed in almost all cases, confirming the off-target effects of YM-155.

Measurable residual disease after the first consolidation predicts the outcomes of patients with acute promyelocytic leukemia treated with all-trans retinoic acid and chemotherapy.

We stratified patients with newly diagnosed acute promyelocytic leukemia (APL) according to a white blood cell (WBC) count of ≥ 3 × 109/L (high risk) or < 3 × 109/L (low risk) before administering risk-adapted chemotherapy in combination with all-trans retinoic acid (ATRA). In total, 27 low-risk and 23 high-risk patients were assigned to receive induction and three courses of consolidation with ATRA and anthracycline, followed by 2-year maintenance regimen. High-risk group additionally received cytarabine during 1st consolidation and another one-shot idarubicin treatment during 3rd consolidation. We prospectively monitored measurable residual disease (MRD) after induction and each consolidation. In the low-risk and high-risk groups, 5-year disease-free survival (DFS) rates were 86.5% and 81.2% (p = 0.862), and 5-year overall survival rates were 100% and 84.8% (p = 0.062), respectively. In the MRD-negative and MRD-positive groups, 5-year DFS rates were 91.7% and 78.4% (p = 0.402) and 84.7% and 60.0% (p = 0.102) after induction and 1st consolidation, respectively. Relapse rates were 8.3% and 13.3% (p = 0.570) and 9.0% and 40.0% (p = 0.076) after induction and 1st consolidation, respectively. Achieving MRD-negativity after 1st consolidation, rather than after induction, was a potential predictor of relapse and DFS in patients with APL treated with ATRA + chemotherapy.

DNA Repair Genes' Expression Abnormalities in De Novo Acute Myelogenous Leukemia (AML): Implications for Targeted Treatment

INTRODUCTION AML is the most common malignant myeloid disorder in adults. Relapses are initiated by chemoresistant leukemic cells. DNA damage and repair mechanisms influence not only the genetic predisposition to leukemia but are also very important for refractoriness to treatment. The aim of this study was to investigate the possible alterations in the gene expression profile in DNA damage signaling pathways in two leukemic cell lines following their exposure to chemotherapeutic agents and verify the findings in AML patients. METHODS Kasumi-1 and MV4-11 AML cells were treated with either idarubicin (0.1μΜ) for 6h or cytarabine (1μΜ) for 48h. Dead cells were eliminated from drug-treated cells using the appropriate commercial kit. Gene expression profiling through PCR arrays analysis (RT2Profiler, Qiagen) was performed after RNA extraction from untreated, drug-treated and chemoresistant (live) cells following their exposure to cytotoxic agents. Human DNA Damage Signaling pathway related genes' expression was evaluated and analyzed through RT2Profiler PCR Array data analysis tool. Following our initial results, two genes were selected for further analysis: PPP1R15A and HUS-1 genes' relative expression was evaluated by qRT-PCR analysis with QuantiTect Primer Assays kit (Qiagen) using the 2^-∆∆Ct method. The analysis included 28 de novo AML patients before the onset of the 7+3 combination chemotherapy and 16 healthy donors. Eighteen cases had normal karyotype including 7 with flt3 mutation, 1 case had inv(16) and 9 cases intermediate risk karyotype. Statistics were performed through One Way Anova analysis. RESULTS PCR Array analysis after idarubicin and cytarabine treatment of Kasumi-1 cells revealed a significant up-regulation of genes involved in apoptosis, cell cycle, DNA damage and repair, and ATM/ATR signaling. Significant differences in their gene expression patterns were observed between cytarabine-treated Kasumi-1 cells and chemoresistant ones. HUS-1 gene (DSB) was 3x fold up-regulated in cytarabine-treated cells and 0.7x fold down-regulated in chemoresistant cells compared to untreated cells. Cytarabine and idarubicin treatment of MV4-11 cells led to an up-regulation of genes involved in cell cycle, DNA damage repair, including DSB repair and NER mechanisms. Most importantly, PPP1R15A gene's expression in both cytarabine and idarubicin chemoresistant MV4-11 cells was significantly 4.2x and 2.7x fold up-regulated compared to drug treated cells. Following these results the expression level of genes PPP1R15A and HUS1 was examined in the bone marrow cells of AML patients in order to verify their association with chemoresistance. PPP1R15A gene's relative expression was significantly up-regulated in non-responding to induction chemotherapy AML patients compared to responding (median: 2.705 vs. 0.73, p&lt;0.05) and in non-responding to chemotherapy AML patients compared to controls (median: 2.705 vs. 0.577, p&lt;0.01). HUS1 gene's relative expression was remarkably down-regulated in AML patients compared to controls (median: 1.585 vs. 7.74, p&lt;0.001). This was also observed comparing responding and refractory to chemotherapy AML patients to controls (median: 1.09 vs. 7.74, p&lt;0.001 and 1.585 vs. 7.74, p&lt;0.05, respectively). CONCLUSIONS The up-regulation of PPP1R15A gene in chemoresistant MV4-11 cells after treatment with cytotoxic agents is justified since this gene participates in growth arrest and apoptosis in response to DNA damage, negative growth signals and protein malfolding by binding to protein phosphatase PP1, and attenuating the translational elongation of key transcription factors through dephoshorylation of eukaryotic initiation factor 2a(eIF2a). Most importantly the significant increased baseline expression of PPP1R15A in AML chemoresistant patients indicates its involvement in chemoresistance mechanisms and paves the way for targeted treatment. HUS1 gene's expression was remarkably depressed in de novo AML samples. This gene is required for the optimal ATM/ATR signaling response to DSBs and replication stress forming part of the RAD9A-RAD1-HUS1 (9-1-1) complex functioning as a damage sensor in checkpoint pathway. Therefore the described above reduced expression in AML samples indicates reduced ATM-ATR response to DSBs associated with genetic instability and offers new options for synthetic lethality treatment strategies Disclosures Symeonidis: Tekeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding. Pappa:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding; Celgene / GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

PB1703 PPP1R15A GENE IS OVEREXPRESSED IN CHEMORESISTANT ACUTE MYELOID LEUKEMIA (AML)

Background: AML is the most common malignant myeloid disorder in adults. Relapses are initiated by chemoresistant leukemic cells. DNA damage and repair mechanisms influence not only the genetic predisposition to leukemia but are also very important for refractoriness to treatment. Aims: The aim of this study was to investigate the possible alterations in the gene expression profile in DNA damage signaling pathways and apoptosis in two leukemic cell lines following their exposure to chemotherapeutic agents, idarubicin and cytarabine and to confirm the results in AML patients’ cells. Methods: Kasumi-1 and MV4-11 AML cells were treated with either idarubicin (0.1 μM) for 6 h or cytarabine (1 μM) for 48 h. Dead cells were eliminated after exposure to the drug using the appropriate commercial kit. Gene expression profiling through PCR arrays analysis (RT2Profiler, Qiagen) was performed after RNA extraction from untreated, drug-treated and chemoresistant (live) cells. Human DNA Damage Signaling pathway related genes’ expression was evaluated and analyzed through RT2Profiler PCR Array data analysis tool. Following our initial results, PPP1R15A gene's relative expression was evaluated by qRT-PCR analysis with QuantiTect Primer Assays kit (Qiagen) in 12 de novo AML patients before the onset of the 7 + 3 combination chemotherapy and 17 healthy donors using the 2^-ΔΔCt method. Results: PCR Array analysis after idarubicin treatment of Kasumi-1 cells revealed a significant up-regulation of genes involved in apoptosis (BBC3), cell cycle (CDKN1A, PPP1R15A), DNA damage and repair (PNP), and ATM/ATR signaling (RBBP8) while cytarabine treatment led to the up-regulation of genes involved mostly in the DSB repair pathway i.e. HUS1,MLH1,NBN,XRCC1,XRCC2). Interestingly, significant differences in their gene expression patterns were observed between cytarabine-treated Kasumi-1 cells and chemoresistant ones. HUS-1 gene (DSB) was up-regulated in cytarabine-treated cells and down-regulated in chemoresistant cells, while MLH1 and NBN genes presented the opposite pattern. Most importantly, PPP1R15A gene's expression in both cytarabine and idarubicin chemoresistant MV4-11 cells was significantly up-regulated compared to drug treated cells. PPP1R15A gene's relative expression was significantly up-regulated in AML patients’ cells compared to controls (median: 0.72 vs 1.72, p < 0.05) and in non-responding to chemotherapy AML patients compared to controls (median: 0.72 vs 1.21, p < 0.05). Summary/Conclusion: Our data suggest differences in the gene expression pattern between chemoresistant cells and drug-treated cells, indicating the significance of DNA damage and repair pathways involved in chemoresistance. Specifically, the up-regulation of PPP1R15A gene in chemoresistant MV4-11 cells after treatment with both agents is of great importance since this gene participates in cell cycle and its transcript levels are increased following stressful growth arrest and treatment with DNA-damaging agents. Considering that MV4-11 is an AML chemoresistant cell line, up-regulation of the expression of PPP1R15A gene which facilitates recovery of cells from stress is totally compatible. These findings were further verified by qRT-PCR demonstrating overexpression of PPP1R15A gene in AML chemoresistant patients indicating the involvement of this gene in chemoresistance mechanisms.

Dexrazoxane Averts Idarubicin-Evoked Genomic Damage by Regulating Gene Expression Profiling Associated With the DNA Damage-Signaling Pathway in BALB/c Mice.

Idarubicin is an anthracycline antileukemic agent widely used in the treatment of hematological malignancies. However, cardiotoxicity and secondary leukemia have been reported after idarubicin treatment. Dexrazoxane is the only medication approved by FDA to prevent anthracycline-evoked cardiotoxicity. However, lack of information on the genomic damage caused by the combination of these 2 drugs prompted us to conduct the current study. We treated mice with different doses of idarubicin and/or dexrazoxane. Genomic DNA damage, apoptosis, and reactive oxygen species (ROS) were evaluated in the bone marrow cells. Our results demonstrate that mice pretreated of with dexrazoxane had significant lower genomic damage, apoptosis and ROS generation compared with that of mice treated with idarubicin alone, an effect that was dependent on dexrazoxane dose. The expression of 84 genes implicated in DNA damage-signaling pathways was quantified using an RT2 Profiler PCR Array. Idarubicin treatments altered the expression of 58 genes, and 16 of those were expressed at significantly different level. In treatments combining idarubicin and dexrazoxane, substantial restorations of mRNA expression of these genes were observed. RT-qPCR was performed for selected genes and the alteration of these genes was confirmed. Alterations in mRNA expression of a subset of genes were further proved by Western blotting analysis of protein levels, which nearly showed similar alterations. Conclusively, dexrazoxane can be safely co-administered with idarubicin. Moreover, dexrazoxane minimizes idarubicin-evoked genomic damage via its radical scavenging and DNA repair-enhancing activities. Thus, dexrazoxane may help avert secondary malignancies in cancer patients in remission who are exposed to idarubicin.

Abstract A039: Caspase-2 and oxidative-ER stress crosstalk regulates the exposure of “eat me” signal calreticulin by high hydrostatic pressure treated cancer cells

Abstract Immunogenic cell death (ICD) is a type of cell death induced by various chemotherapeutics, physical modalities such as photodynamic therapy, radiotherapy and high hydrostatic pressure (HHP). HHP induces immunogenic cell death of cancer cell is currently tested in running II. and III. phase of clinical trial for preparation of active cellular cancer immunotherapy (DCVAC). Previous studies have shown that the immunogenicity of ICD mainly relies on surface exposed calreticulin (ecto-CRT); CRT is normally located in the lumen of the endoplasmic reticulum (ER) and may translocate to the cell surface within hours after treatment. Although ICD can be triggers by HHP, underlying danger signaling pathways remain unclear. Therefore ovarian (OV90) cancer cell line was treated by HHP, UV-B and anthracycline (Idarubicin). The kinetics of ICD markers and key components of ER stress-mediated apoptotic pathway were analyzed by flow cytometry, western blotting and confocal microscopy. In this work we described the ability of HHP triggers oxidative ER-stress (the production of reactive oxygen species (ROS) together with ER stress response) which results in phosphorylation of PERK and eIF2α. We also observed a rapid caspase-8 activation subsequently followed by BAP31 cleavage, Bid activation and caspase-3 activation. While caspase-8 activation was necessary for cell surface exposure of calreticulin (ecto-CRT) after HHP treatment, proapoptotic proteins Bax and Bak were dispensable for this process. Moreover, we identified that HHP treatment leads to caspase-2 activation. Whereas Idarubicin treatment has no effect on ecto-CRT exposure in Caspase-2 knockdown tumor cells, ecto-CRT was significantly decreased in these cells after HHP treatment. In addition, phagocytosis of HHP-treated Caspase-2 knockdown tumor cells was compromised in contrast to Idarubicin-treated knockout tumor cells. In conclusion, we characterized the molecular mechanism of ecto-CRT exposure pathway induced by HHP which is analogous to that induced by anthracycline treatment. For the first time we provide evidence that caspase-2 might play an important role in the HHP-mediated ecto-CRT exposure. Citation Format: Irena Kusova Moserova, Iva Truxova, Abhishek G, Garg, Patrizia Agostinis, Piere Francois Cartron, Sarka Vosahlikova, Radek Spisek, Jitka Fucikova. Caspase-2 and oxidative-ER stress crosstalk regulates the exposure of “eat me” signal calreticulin by high hydrostatic pressure treated cancer cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A039.

Cost-Effectiveness Analysis of Treating Acute Promyelocytic Leukemia Patients With Arsenic Trioxide and Retinoic Acid in the United States.

This study estimated the cost-effectiveness of arsenic trioxide (ATO) added to all-trans retinoic acid (ATRA) when used in first-line acute promyelocytic leukemia (APL) treatment. A Markov cohort model was developed with 3 states: stable disease (during first- or second-line treatment), disease event, and death. Newly diagnosed patients with low- to intermediate-risk APL were included and each month could remain in their current health state or move to another. Treatment consisted of ATO+ ATRA, ATRA+ idarubicin (IDA), or ATRA+ cytarabine (AraC)+ additional chemotherapy. After an initial disease event, patients discontinued first-line therapy and switched to a second-line ATO regimen. Efficacy and safety data were obtained from published trials; quality of life/utility estimates were obtained from the literature; costs were obtained from US data sources. Costs and outcomes over time were used to calculate incremental cost-effectiveness ratios (ICERs). Deterministic and probabilistic sensitivity analyses were conducted. Compared to ATRA+ AraC+ additional chemotherapy, ATRA+ IDA treatment had ICERs of $2933 per life-year (LY) saved and $3122 per quality-adjusted life-year (QALY) gained. Compared to the ATRA+ IDA regimen, first-line ATO+ ATRA treatment had ICERs of $4512 per LY saved and $5614 per QALY gained. Results were sensitive to changes in pharmacy costs of the ATO+ ATRA regimen during consolidation. The ATO+ ATRA regimen is highly cost-effective compared to ATRA+ AraC+ additional chemotherapy or ATRA+ IDA in the treatment of newly diagnosed low- to intermediate-risk APL patients.

Identifying genes that mediate anthracyline toxicity in immune cells

The role of the immune system in response to chemotherapeutic agents remains elusive. The interpatient variability observed in immune and chemotherapeutic cytotoxic responses is likely, at least in part, due to complex genetic differences. Through the use of a panel of genetically diverse mouse inbred strains, we developed a drug screening platform aimed at identifying genes underlying these chemotherapeutic cytotoxic effects on immune cells. Using genome-wide association studies (GWAS), we identified four genome-wide significant quantitative trait loci (QTL) that contributed to the sensitivity of doxorubicin and idarubicin in immune cells. Of particular interest, a locus on chromosome 16 was significantly associated with cell viability following idarubicin administration (p = 5.01 × 10−8). Within this QTL lies App, which encodes amyloid beta precursor protein. Comparison of dose-response curves verified that T-cells in App knockout mice were more sensitive to idarubicin than those of C57BL/6J control mice (p < 0.05). In conclusion, the cellular screening approach coupled with GWAS led to the identification and subsequent validation of a gene involved in T-cell viability after idarubicin treatment. Previous studies have suggested a role for App in in vitro and in vivo cytotoxicity to anticancer agents; the overexpression of App enhances resistance, while the knockdown of this gene is deleterious to cell viability. Further investigations should include performing mechanistic studies, validating additional genes from the GWAS, including Ppfia1 and Ppfibp1, and ultimately translating the findings to in vivo and human studies.

Safety and pharmacokinetics of the antisense oligonucleotide (ASO) LY2181308 as a single-agent or in combination with idarubicin and cytarabine in patients with refractory or relapsed acute myeloid leukemia (AML)

Survivin is expressed in tumor cells, including acute myeloid leukemia (AML), regulates mitosis, and prevents tumor cell death. The antisense oligonucleotide sodium LY2181308 (LY2181308) inhibits survivin expression and may cause cell cycle arrest and restore apoptosis in AML. In this study, the safety, pharmacokinetics, and pharmacodynamics/efficacy of LY2181308 was examined in AML patients, first in a cohort with monotherapy (n = 8) and then post-amendment in a cohort with the combination of cytarabine and idarubicin treatment (n = 16). LY2181308 was administered with a loading dosage of three consecutive daily infusions of 750 mg followed by weekly intravenous (IV) maintenance doses of 750 mg. Cytarabine 1.5 g/m(2) was administered as a 4-hour IV infusion on Days 3, 4, and 5 of Cycle 1, and idarubicin 12 mg/m(2) was administered as a 30-minute IV infusion on Days 3, 4, and 5 of Cycle 1. Cytarabine and idarubicin were administered on Days 1, 2, and 3 of each subsequent 28-day cycle. Reduction of survivin was evaluated in peripheral blasts and bone marrow. Single-agent LY2181308 was well tolerated and survivin was reduced only in patients with a high survivin expression. In combination with chemotherapy, 4/16 patients had complete responses, 1/16 patients had incomplete responses, and 4/16 patients had cytoreduction. Nine patients died on study: 6 (monotherapy), 3 (combination). LY2181308 alone is well tolerated in patients with AML. In combination with cytarabine and idarubicin, LY2181308 does not appear to cause additional toxicity, and has shown some clinical benefit needing confirmation in future clinical trials.

Superior Long-Term Outcome With Idarubicin Compared With High-Dose Daunorubicin in Patients With Acute Myeloid Leukemia Age 50 Years and Older

Although standard chemotherapy remains associated with a poor outcome in older patients with acute myeloid leukemia (AML), it is unclear which patients can survive long enough to be considered as cured. This study aimed to identify factors influencing the long-term outcome in these patients. The study included 727 older patients with AML (median age, 67 years) treated in two idarubicin (IDA) versus daunorubicin (DNR) Acute Leukemia French Association trials. Prognostic analysis was based on standard univariate and multivariate models and also included a cure fraction model to focus on long-term outcome. Age, WBC count, secondary AML, Eastern Cooperative Oncology Group (ECOG) performance status (PS), and adverse-risk and favorable-risk AML subsets (European LeukemiaNet classification) all influenced complete remission (CR) rate and overall survival (OS). IDA random assignment was associated with higher CR rate, but not with longer OS (P = .13). The overall cure rate was 13.3%. Older age and ECOG-PS more than 1 negatively influenced cure rate, which was higher in patients with favorable-risk AML (39.1% v 8.0% in adverse-risk AML; P < .001) and those treated with IDA (16.6% v 9.8% with DNR; P = .018). The long-term impact of IDA was still observed in patients younger than age 65 years, although all of the younger patients in the DNR control arm received high DNR doses (cure rate, 27.4% for IDA v 15.9% for DNR; P = .049). In multivariate analysis, IDA random assignment remained associated with a higher cure rate (P = .04), together with younger age and favorable-risk AML, despite not influencing OS (P = .11). In older patients with AML, younger age, favorable-risk AML, and IDA treatment predict a better long-term outcome.

Bone Marrow Mesenchymal Stromal Cells Protect Acute Lymphocytic Leukemia Cells From Cytotoxic Agents Via MAPK/Erk and Wnt/β-Catenin Signal Pathways

Abstract 4736Acute lymphoblastic leukemia (ALL) remains one of the greatest challenges in oncology. Relapsed ALL is a leading cause of death in young people, and further improvements in outcome will required the development of therapeutic approaches directed against rational therapeutic targets. Increasing evidence indicated that the bone marrow microenvironment plays a crucial role in the pathogenesis of leukemia by promoting tumor cell growth and survival as well as drug resistance. As a pivotal component of the bone marrow microenvironment, how bone marrow derived mesenchymal stromal cells (MSCs) affect ALL cells and the underlying mechanisms remain unclear.In this study, bone marrow was obtained from healthy adult donors and MSCs were cultured and identified as previous report. Our study applied the co-culture system to mimic the bone marrow microenvironment. To study the effect of bone marrow stroma on ALL cells, three different sub-types of ALL cell lines including Sup-B15, Reh, and MOTL-4 were cultured with and without an underlying confluent layer of MSC. Survival of ALL cells was detected by CCK-8 kit and apoptosis was tested by PI/Annexin V assay. Cell cycle was evaluated by flow cytometry. In addition, Western-bloting was used in mechanism study.The results demonstrated that MSCs improved the survival of ALL cells induced by the cytotoxic agents Idarubicin (IDA) and Etoposide (Vp-16). Number of viable ALL cells were increased remarkably in presence of MSCs. In Vp-16 treatment group, the ratios of viable ALL cell were 1:1.17±0.05 (Sup-B15: Sup-B15+MSC, P<0.05), 1:1.20±0.02 (Reh: Reh+MSC, P<0.05) and 1:1.74±0.16 (MOLT-4: MOLT-4+MSC, P<0.05) respectively. In IDA treatment group, the ratios were 1:1.39±0.11 (Sup-B15: Sup-B15+MSC P<0.05), 1:1.67±0.05 (Reh: Reh+MSC, P<0.01) and 1:1.06±0.07 (MOLT-4: MOLT-4+MSC) respectively. Our data also indicated that coculture with normal MSCs protected ALL cells from apoptosis induced by IDA and Vp-16. The propotion of apoptotic cells was reduced in presence of MSCs both in Vp-16 treatment (Sup-B15: 26.46 ±0.70 vs. 22.00±5.35%, Reh: 18.16±2.09 vs. 16.94±4.34%, MOLT-4: 71.54 ±19.15 vs.62.59±35.63%) and IDA treatment (Sup-B15: 46.57±3.74 vs. 28.86±5.99, Reh: 48.58±6.73 vs. 36.49±5.33%, MOLT-4: 71.54 ±43.72±6.76 vs. 25.04±6.84, P<0.05 respectively).To clarify the mechanisms of the protective role of MSCs, we compared the expression levels of apoptosis and survival related proteins in ALL cells cultured without or with MSCs. The data indicated that MAPK/Erk,Wnt/β-catenin signal pathways were activated in ALL cells co-cultured with MSC. ALL cells showed significantly higher expression of p-Erk and β-catenin in presence of MSCs. At the same time, the cycle inhibition protein p21, mitochondria mediated apoptosis proteins, and the oncogene c-myc were down-regulated.Our study provides experimental basis for the exploring of novel ALL therapy that targets the microenvironment. Disclosures:No relevant conflicts of interest to declare.

Potent antitumor activities of recombinant human PDCD5 protein in combination with chemotherapy drugs in K562 cells

Conventional chemotherapy is still frequently used. Programmed cell death 5 (PDCD5) enhances apoptosis of various tumor cells triggered by certain stimuli and is lowly expressed in leukemic cells from chronic myelogenous leukemia patients. Here, we describe for the first time that recombinant human PDCD5 protein (rhPDCD5) in combination with chemotherapy drugs has potent antitumor effects on chronic myelogenous leukemia K562 cells in vitro and in vivo. The antitumor efficacy of rhPDCD5 protein with chemotherapy drugs, idarubicin (IDR) or cytarabine (Ara-C), was examined in K562 cells in vitro and K562 xenograft tumor models in vivo. rhPDCD5 protein markedly increased the apoptosis rates and decreased the colony-forming capability of K562 cells after the combined treatment with IDR or Ara-C. rhPDCD5 protein by intraperitoneal administration dramatically improved the antitumor effects of IDR treatment in the K562 xenograft model. The tumor sizes and cell proliferation were significantly decreased; and TUNEL positive cells were significantly increased in the combined group with rhPDCD5 protein and IDR treatment compared with single IDR treatment groups. rhPDCD5 protein, in combination with IDR, has potent antitumor effects on chronic myelogenous leukemia K562 cells and may be a novel and promising agent for the treatment of chronic myelogenous leukemia.

Thrombosis of the cerebral veins and sinuses in acute promyelocytic leukemia after all-trans retinoic acid treatment: a case report

Thrombosis of the cerebral veins or sinuses is a rare cerebrovascular disorder, which seldom represents a complication of acute promyelocytic leukemia. To the best of our knowledge, it never occurred during treatment with all-trans retinoic acid. We report a case of a 35-year-old woman affected by acute promyelocytic leukemia, who developed massive thrombosis of the cerebral sinuses and veins when she was in complete morphological and molecular remission after all-trans retinoic acid and idarubicin treatment. Anticoagulant therapy contributed to progressive dissolution of the thrombosis as documented by magnetic resonance imaging with complete disappearance of neurological signs without sequelae.

Adenovirus-mediated PDCD5 gene transfer sensitizes K562 cells to apoptosis induced by idarubicin in vitro and in vivo

PDCD5 (programmed cell death 5) accelerates apoptosis of certain tumor cells and is expressed at low levels in marrow-nucleated cells of AML and CML patients. In the present study, we evaluated the effects of PDCD5 overexpression on drug sensitivity of leukemia cells. K562 cells were treated with idarubicin (IDR) alone or in combination with adenoviral vectors expressing PDCD5 (Ad-PDCD5). As shown by annexin-V-FITC/PI dual labeling, apoptosis rates were markedly increased after combined treatment with Ad-PDCD5 compared to IDR treatment alone. We observed that PDCD5 overexpression significantly improves the antitumor effects of low dose IDR treatment in vivo. Tumor sizes were significantly decreased in combined Ad-PDCD5 and low dose IDR treatment groups compared with single IDR treatment groups. Similar results were obtained with combined systemic treatment of Ad-PDCD5 and low dose IDR, and combined treatment with Ad-PDCD5 local injection and low dose IDR i.p. injection. These results indicate that Ad-PDCD5 may be a promising agent for enhancing chemosensitivity.

Sensitivity of Childhood Acute Lymphoblastic Leukemia (ALL) to Idarubicin Is Significantly Higher Than to Daunorubicin Treatment Based on Ex Vivo Apoptosis Induction.

Anthracyclines, especially daunorubicin, play a very important role in the treatment of acute lymphoblastic leukemia (ALL) and the relapsed ALL in childhood.In the present study, primary lymphoblasts isolated from 65 children with de novo ALL (median: 5.8 years; range: 1.9 – 16.9 years) and relapsed ALL (median: 12.7 years; range: 1.3 – 17.9 years) were treated with daunorubicin (10 mmol/l) or idarubicin (2 mmol/l) in vitro. We could show that both anthracylines induce apoptosis, as evidenced by measurement of genomic DNA fragmentation. Interestingly, daunorubicin only induced modest apoptosis, whereas idarubicin displayed a significantly stronger apoptosis inducing effect. Furthermore the treatment of daunorubicin-resistant lymphoblasts with idarubicin resulted in good response in most of the resistant cell populations.Out of the 65 patients analysed in this study 23 were female (13 de novo ALL, 10 relapsed ALL) and 42 were male (29 de novo ALL, 13 relapsed ALL). Primary lymphoblasts were obtained by bone marrow aspiration and separated by centrifugation over Ficoll. Within these cell populations following immunologic subgroups were found: 35 c-ALL, 10 pre-B-ALL, 7 pro-B-ALL, 10 T-ALL and 3 pre-T-ALL.Daunorubicin induced apoptosis in 33 out of 65 lymphoblast populations (response rate 50.8 %). Nevertheless, a far higher response rate was observed for idarubicin with 59/65 (90,8 %) (p < 0.008), if response is defined as apoptosis induction higher than 1 %. Daunorubicin-resistance was found in 32/65 (49,2 %), resistance to both was observed in 6/65 (9,2 %). Treatment of daunorubicin-resistant lymphoblasts with idarubicin resulted in significant apoptosis induction in 26 out of 32 cell populations (81,3 %).We clearly demonstrated here that the in vitro treatment of lymphoblasts from children with de novo or relapsed ALL with idarubicin induces significantly higher response rates than daunorubicin treatment. The ex vivo sensitivity of daunorubicin-resistant lymphoblasts of childhood ALL to idarubicin treatment reflects the better potency of idarubicin to induce apoptosis and to overcome daunorubicin resistance. These data prompted us to study the clinical relevance of idarubicin in ongoing clinical trials to improve existing therapeutic regiments. First clinical data point to a good tolerability of idarubicin in the treatment of relapsed ALL in childhood.

Effect of imatinib on haematopoietic recovery following idarubicin exposure.

SCF is a potent pro-proliferative cytokine crucial for haematopoiesis, which binds to c-kit and activates its tyrosine kinase activity. Inactivating mutations of either SCF or c-kit have been described in mice and lead to increased sensitivity to treatment with ionising radiation. Imatinib is a tyrosine kinase inhibitor with high affinity for c-Abl, PDGFR and c-kit. In this study we investigated the effect of concomitant administration of imatinib and idarubicin, an anthracycline with haematosuppressive activity, in nu/nu mice and murine bone marrow cells. Double-treated animals showed significantly increased mortality compared to mice that received imatinib or idarubicin alone only when idarubicin and imatinib were given simultaneously. The combined treatment induced a more severe neutropenia with a slower recovery when compared to mice treated with idarubicin alone. The myeloid metaplasia usually observed in the spleen after idarubicin treatment was absent in mice co-treated with imatinib. Bone marrow from double-treated animals also showed decreased numbers of megakaryocytes and myeloid precursor cells. In vitro culture of murine bone marrow cells in the presence of imatinib inhibited SCF-induced proliferation and recovery from treatment with idarubicin. Our results indicate that the simultaneous administration of imatinib enhances idarubicin-induced haematopoietic toxicity in vivo and in vitro.

Arsenic trioxide- and idarubicin-induced remissions in relapsed acute promyelocytic leukaemia: clinicopathological and molecular features of a pilot study.

Arsenic trioxide (As2O3) effectively induces remissions in relapsed acute promyelocytic leukaemia (APL), but the safety of its long-term administration is unknown. The anthracycline idarubicin is highly active alone or in combination chemotherapy for the treatment of APL. To minimize arsenic exposure and based on the high sensitivity of APL cells to anthracyclines, we conducted a prospective study to evaluate induction with As2O3 followed by consolidation with idarubicin in the treatment of APL in relapse. Eight patients were treated with As2O3 at a daily dose of 10 mg until remission, followed by three monthly courses of idarubicin, at 6 mg/m(2)/day for 5 days in the first course and 6 mg/m(2)/day for 2 days in the subsequent two courses. All patients achieved morphological but not molecular remission after As2O3 treatment. During As2O3 therapy, an increase in white cell count peaking at a median of 17 days occurred in all the cases. Serial flow cytometric analysis of apoptosis, with mitochondrial APO2.7 antigen expression and the sub-G1 cell fraction on DNA histogram as markers, showed induction of apoptosis of APL cells in vivo. With both qualitative and real-time quantitative polymerase chain reaction, all patients were shown to attain molecular remission after subsequent idarubicin treatment. With a median follow up of 13 months, seven of eight patients have remained in complete clinical remission, with six patients in molecular remission as well. One patient who was in third remission became PCR-positive after being transiently negative. One patient died from an intracranial extramedullary relapse after achieving marrow molecular remission. We conclude that As2O3 induction followed by idarubicin consolidation is an effective therapy for APL in relapse. This regimen avoids the possible long-term toxicities of As2O3 and mutagenicity of combination chemotherapy, a strategy that might be suitable for this potentially curable leukaemia.

Flow cytometry detection of caspase 3 activation in preapoptotic leukemic cells.

Procaspase 3 is a constitutive proenzyme that is activated by cleavage during apoptosis. The resulting enzyme is able to cleave several target proteins after the second aspartate of a DEVD sequence common to all the substrates of caspases 3 and 7 (DEVDase). Because active caspase 3 is a common effector in several apoptotic pathways, it may be a good marker to detect (pre-)apoptotic cells by flow cytometry (FCM). Materials and Methods Apoptosis was induced in U937 or bone marrow mononuclear cells by daunorubicin (DNR), idarubicin (IDA), or camptothecin (CAM). Viable and apoptotic cells were sorted by FCM on the basis of either fluorescein isothiocyante (FITC)-annexin V binding or DiOC6(3) accumulation. DEVDase activity was measured in sorted populations by spectrofluorometry. Cleaved caspase 3 was labeled in situ with phycoerythrin (PE)-conjugated anti-activated caspase 3 antibodies and analyzed by FCM. DEVDase activity was detected in sorted viable CAM- and DNR-treated U937 cells, demonstrating that a partial caspase activation occurred earlier than phosphatidyl-serine exposure and mitochondrial membrane potential dissipation. The presence of a low amount of active caspase 3 in the treated viable cells was confirmed in situ with PE-conjugated anti-active caspase 3 antibodies. The same antibody was used in combination with FITC-annexin V and CD45-PC5 to study caspase 3 activation in acute leukemia blast cells after in vitro DNR and IDA treatment. Both anthracyclines induced a caspase 3-dependent apoptosis that was more efficient in blast cells than in contaminating lymphocytes. These results demonstrate that anti-active caspase 3 labeling can be an alternative to fluorogenic substrates to efficiently detect early apoptosis by FCM in heterogeneous samples. They also confirm that anthracyclines induce blast cell apoptosis by a caspase 3-dependent pathway.

Effect of the cytostatic agent idarubicin on fibroblasts of the human Tenon’s capsule compared with mitomycin C

BACKGROUND/AIMSTo investigate the in vitro effect of a short time exposure to the anthracycline idarubicin on proliferation, protein synthesis, and motility of human Tenon’s capsule fibroblasts in comparison with the...