Drug-resistant Leukemia Cells Research Articles

Antioxidant activity and cytotoxicity of Cyrtosperma johnstonii extracts on drug sensitive and resistant leukemia and small cell lung carcinoma cells

Context: The number of patients with cancer is increasing. New therapeutic agents to overcome drug-resistant tumors are urgently needed. Cyrtosperma johnstonii N.E. Br. (Araceae) is used for treatment of cancer in Thai traditional medicine. This study aimed to evaluate antioxidant activity and cytotoxicity of C. johnstonii extracts on human cancer cells.Materials and methods: Dried powder of C. johnstonii rhizomes was extracted with several solvents. The 0.1 mg/ml extract solution was tested for antioxidant activity by 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and ferric reducing antioxidant power (FRAP) assays. Color formation from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to determine cell viability. Standardization of the extract was performed by high-performance liquid chromatography (HPLC) with photodiode array detector at 254 and 360 nm. Cell cycle arrest was evaluated by flow cytometry after 5 min, 12 h and 24 h treated with 20 µg/ml of the acetone extract.Results: The acetone extract exhibited the highest phenolic content and antioxidant activity (TEAC and EC values = 19.2 ± 0.14 and 19.2 ± 0.31 mM/mg, respectively). The IC50 values for leukemia ranged from 11 ± 1 to 29 ± 3 µg/ml and from 5 ± 2 to 6 ± 0 µg/ml for small cell lung carcinoma cells. Cell cycle arrest occurred at the G2/M phase followed by apoptosis. HPLC analysis revealed that rutin is the major constituents of the extract.Discussion and conclusion: The acetone extract of C. johnstoni is a promising source of natural antioxidants and anticancer. The extract inhibits cancer cells effectively with less effect on normal cells.

Glutathionylation of UCP2 sensitizes drug resistant leukemia cells to chemotherapeutics

Uncoupling protein-2 (UCP2) is used by cells to control reactive oxygen species (ROS) production by mitochondria. This ability depends on the glutathionylation state of UCP2. UCP2 is often overexpressed in drug resistant cancer cells and therein controls cell ROS levels and limits drug toxicity. With our recent observation that glutathionylation deactivates proton leak through UCP2, we decided to test if diamide, a glutathionylation catalyst, can sensitize drug resistant cells to chemotherapeutic agents. Using drug sensitive HL-60 cells and the drug resistant HL-60 subline, Mx2, we show that chemical induction of glutathionylation selectively deactivates proton leak through UCP2 in Mx2 cells. Chemical glutathionylation of UCP2 disables chemoresistance in the Mx2 cells. Exposure to 200μM diamide led to a significant increase in Mx2 cell death that was augmented when cells were exposed to either menadione or the anthracycline doxorubicin. Diamide also sensitized Mx2 cells to a number of other chemotherapeutics. Proton leak through UCP2 contributed significantly to the energetics of the Mx2 cells. Knockdown of UCP2 led to a significant decrease in both resting and state 4 (i.e., proton leak-dependent) respiration (~43% and 62%, respectively) in Mx2 cells. Similarly diamide inhibited proton leak-dependent respiration by ~64%. In contrast, diamide had very little effect on proton leak in HL-60 cells. Collectively, our observations indicate that manipulation of UCP2 glutathionylation status can serve as a therapeutic strategy for cancer treatment.

Combined treatment of 3-hydroxyflavone and imatinib mesylate increases apoptotic cell death of imatinib mesylate-resistant leukemia cells

Imatinib mesylate, a Bcr/Abl tyrosine kinase inhibitor, is widely used in treating chronic myeloid leukemia. However, drug-resistance of leukemia cells becomes an emergent problem. Herein, various flavonoids were screened for applicability in leukemia treatment, and 3-hydroxyflavone (3-HF) was found to be most effective in reducing cancer cell viability. The combination of 3-HF and imatinib mesylate resulted in significant apoptotic cell death in imatinib mesylate-resistant leukemia cells. Combined treatment resulted in apparent activation of caspases and decrease of the oncoprotein phosphor-Bcr/Abl in leukemia cells. Our results suggest that this combined treatment is beneficial in imatinib mesylate-resistant chronic myelogenous leukemia.

Androgen Receptor Signaling Regulates Cell Growth and Vulnerability to Doxorubicin in Bladder Cancer

There are several reports of androgen receptor in bladder cancer cases but androgen receptor expression and the function of androgen/androgen receptor signaling in bladder cancer remain unclear. We investigated androgen receptor expression and the role of androgen/androgen receptor signaling in bladder cancer. We evaluated AR mRNA expression in bladder cancer tissue by quantitative real-time polymerase chain reaction. The role of androgen receptor in cell growth and drug sensitivity was also evaluated in vitro and in vivo in several bladder cancer cell lines. AR mRNA expression inversely correlated with bladder cancer grade, stage and spread. Of several bladder cancer cell lines UMUC3 and MBT-2 markedly expressed androgen receptor transcript and protein. In each cell line androgen/androgen receptor signaling blockade using androgen deprivation, blockade knockdown and antiandrogen agents decreased cell growth, colony formation and cell viability. Androgen receptor expression was implicated in doxorubicin resistance. Inversely androgen receptor deprivation and knockdown made UMUC3 cells sensitive to doxorubicin. Finally, castration slightly suppressed UMUC3 tumor growth in vivo, although this did not attain statistical significance. AR transcript expression inversely correlates with bladder cancer clinicopathological characteristics. Androgen/androgen receptor signaling has an important role in the growth and vulnerability to doxorubicin of bladder cancer cells that express androgen receptor. Androgen/androgen receptor signaling might be a possible prophylactic and therapeutic target for bladder cancer that shows androgen receptor expression.

Abstract IA1: Keynote Presentation: BRAF and RAS signaling in melanoma from basic to translational and clinical research

Abstract BRAF is a protein kinase that is mutated in about half of human melanomas. Its activator, the small G-protein NRAS, is mutated in another 20% of cases. Drugs that inhibit BRAF mediate impressive responses in melanoma patients whose tumors carry mutations in BRAF, but are not effective in patients whose tumors express wild-type BRAF. Unexpectedly however, whereas BRAF drugs inhibit MEK/ERK signaling in melanoma cells that express oncogenic BRAF, they activate MEK/ERK signaling in cells that express oncogenic RAS. This paradoxical activation of MEK/ERK signaling in melanoma cells that express oncogenic RAS occurs because BRAF drugs drive the formation of dimers between BRAF and a closely related family member, called CRAF. These hetero- and homo-dimers contain one partner that is drug-bound and one partner that is drug-free, and the drug-bound partner activates the drug-free partner through a conformational change, or by acting as a scaffold protein. Notably, this paradoxical activation drives the formation of non-melanoma skin lesions (keratoacanthomas, squamous cell carcinomas) in about a third of patients. Using a two-stage skin carcinogenesis model, we find that this is not because BRAF inhibitors are tumor promoters per se; rather they act by accelerating the inevitable in susceptible patients by driving the growth of pre-existing lesions through paradoxical RAF activation. We also find that nilotinib is a weak RAF inhibitor that activates the RAF paradox and thereby drives survival in drug-resistant chronic mylogenous leukemia (CML) cells. Our data show how improved understanding of signaling responses to BRAF inhibitors can lead to improved patient care.

Abstract B50: Per oral nanogel conjugates of activated nucleoside analogs are effective in the treatment of drug-resistant tumors

Abstract Purpose: Chemotherapeutic application of cytotoxic nucleoside analogs and bases resulted in the development of specific mechanisms of drug resistance. Numerous attempts to develop phosphorylated prodrugs of nucleoside analogs effective in vivo have not yet yielded clinical drugs. On the other hand, there exist a very limited list of oral anticancer drugs despite of the fact that per oral administration remains a preferred route of administration due to its convenience and better patient compliancy. Recently, we developed novel conjugates of biocompatible polymers and phosphorylated nucleoside analogs, which fold into small nanogel particles in aqueous media and show an enhanced activity against aggressive and drug-resistant tumor cells. These nanogel-drug conjugates are stable in conditions existing in the gastrointestinal tract, but can release activated drug molecules after internalization and enzymatic hydrolysis in cancer cells. Method: Poly(vinyl alcohol), a nontoxic biocompatible polymer, having excellent mucoadhesive properties that are important for gastrointestinal (GI) absorption, was decorated with cholesterol moieties(CPVA) and conjugated with floxuridine(FU) and N-acylated gemcitabine(Gem) via a tetraphosphorylated linker. These CPVA-drug conjugates formed compact nanogels (20-60 nm in diameter) in aqueous media. Drug content in the conjugates was usually 8–12%. We examined cytotoxicity of the nanogel-drug conjugates in human breast, prostate and drug-resistant pancreatic cancer, leukemic and lymphoma cells. Tumor growth inhibition was evaluated in s.c. xenograft animal models following the administration of nanogel-drug conjugates peritumorally or orally, for example, by liquid gavage or with drinking water ad libitum. Results: The hydrophobic modification by cholesterol moieties played a significant part in the intracellular drug accumulation due to higher cytotoxic activity. In most cases, the nanogel-drug conjugates demonstrated a 3- to 30-fold higher efficacy than nucleoside analogs showing both cytotoxic and cytostatic activities. We found selective drug release patterns depending on the environmental pH and tissue-related enzymatic activities. Importantly, these conjugates were stable at low pH and, therefore, could be administered orally. High viscosity of nanogel-drug conjugate solutions is also a positive factor increasing their absorption in GI tract and providing a depot-like pattern of drug release after s.c. injections. Due to the molecular mass of nanogel-drug conjugates (>50 kDa), absorbed drug nanoformulations can potentially be retained in tumors via leaking neovasculature due to the enhanced permeability and retention effect. In all experiments, we observed sustained antitumor effects at relatively low drug doses compared to control groups and no chronic toxicity or weight loss was detected. Conclusion: Nanogel drug conjugates demonstrated advanced accumulation and drug release profiles during the treatment of cancer cells resistant to nucleoside analogs and displayed a strong growth inhibition of drug-resistant tumors in xenograft animal models following per oral administration. Therefore, nanogel drug conjugates constitute a novel efficient formulation for the treatment of drug resistant aggressive cancers. Acknowledgements: The National Cancer Institute's support CA136921 (for S.V.V.) is greatly appreciated.

731. Specific RNA Cleavage by Oligodeoxyribozyme Suppresses Proliferation of Drug-Resistant Leukemia Cells

731. Specific RNA Cleavage by Oligodeoxyribozyme Suppresses Proliferation of Drug-Resistant Leukemia Cells

Abstract 796: Novel mechanisms of drug resistance in leukemia cells associated with changes in HoxA9 and microRNA expression

Abstract The acquisition of resistance to anticancer drugs is a key obstacle to successful cancer therapy. An increasing number of studies have investigated the roles of microRNAs in drug resistance. We previously reported that the upregulation of miR-135b and miR-196b correlated positively with acquired drug resistance in the human T-cell leukemia cell line, CCRF-CEM (CEM), and that the elevation of expression of these two microRNAs in response to the DNA damaging agent etoposide appears to be histiotype-specific (T-T Ho et al, Proceedings of the 102nd Annual Meeting of the AACR 2011). To develop a mechanistic understanding of why these microRNAs are differentially expressed, we have studied miR-196b, which maps between the HoxA9 and HoxA10 genes on chromosome 7p15.2, suggesting that miR-196b and HoxA genes might be co-activated. Indeed, we found upregulation of HoxA9 mRNA after short-term (48 h) exposure of CEM cells to the topoisomerase II inhibitors etoposide and doxorubicin. Of note, we also found that HoxA9 is constitutively overexpressed in multidrug-resistant CEM/VM-1-5 cells. Of interest, we found that HoxA9 is clearly associated with drug resistance, as knockdown of HoxA9 using esiRNA (a heterogeneous mixture of siRNAs that all target the same mRNA) partially sensitized multidrug-resistant CEM/VM-1-5 cells to etoposide. This indicates that the expression of miR-196b is linked to HoxA gene transcription and to drug responsiveness. We subsequently confirmed that the expression of HoxA9 mRNA follows the same pattern in other leukemia cells. For example, both miR-196b and HoxA9 are upregulated in response to short-term etoposide exposure in HL-60 (acute promyelocytic leukemia), but not in solid tumor-derived MCF7 (breast cancer cell line) and its etoposide-resistant subline MCF7/VP, or Rh30 (rhabdomyosarcoma cell line) and its etoposide-resistant subline, Rh30/V1. We are presently assessing the role of HoxA9 in drug resistance in other leukemia cell lines and investigating the mechanistic basis for this effect. In summary, we report here a novel mechanism of anticancer drug resistance in leukemia cells that is associated with HoxA9 and specific changes in microRNA expression. This is the first report that HoxA9 may impact on anticancer drug resistance. (Supported in part by R01 CA40570 from NCI to WTB, and in part by UIC.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 796. doi:1538-7445.AM2012-796

Clinical impact of in vitro cellular drug resistance on childhood acute lymphoblastic leukemia in Taiwan

Despite the successful treatment of childhood acute lymphoblastic leukemia (ALL), the resistance to chemotherapy in ALL cells continues to play an important role in treatment failure. In vitro drug resistance determined using an MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was carried out in 16 children with newly diagnosed ALL between November 2009 and December 2010. The in vitro therapeutic effects of asparaginase, vincristine, prednisolone, dexamethasone, epirubicin and cytarabine were examined. Although there was no significant association between in vitro drug resistance of leukemic cells and ALL subtypes, ETV6–RUNX1 ALL tended to be more sensitive to asparaginase, vincristine and prednisolone. Leukemic cells from girls were significantly more sensitive to epirubicin compared with boys (p = 0.008). Higher leukocyte count at diagnosis was correlated with in vitro resistance to asparaginase and prednisolone (p = 0.03 and 0.05, respectively). Relapse or death occurred in five patients. The leukemic cells from these five patients demonstrated increased in vitro resistance to asparaginase compared to those from the other 11 patients (p = 0.009). From the present case series, the demonstrated in vitro resistance to chemotherapeutic agents may have a prognostic value in children with ALL before comprehensive minimal residual disease measurement is available.

Self-assembled squalenoyl-cytarabine nanostructures as a potent nanomedicine for treatment of leukemic diseases

BackgroundIn this investigation, the antileukemic activity of a new nanomedicine based on the conjugation of 1,1′,2-tris-nor-squalenic acid with cytarabine (Ara-C) was evaluated.MethodsSqualenoyl-Ara-C conjugate (Sq-Ara-C) self-assembled nanosystems were obtained by the nanoprecipitation method and characterized in vitro and in vivo.ResultsThis new nanomedicine, which had a mean diameter of approximately 150 nm, improved the in vitro antitumoral activity of Ara-C in different cancer cell lines (L1210, K562, and MCF-7). Sq-Ara-C nanomedicine allowed reduction of the IC50 value with respect to the free drug and was also active against drug-resistant leukemic cells (L1210R). A noticeable increase in the survival rate of mice with aggressive metastatic L1210R leukemia was observed after treatment with Sq-Ara-C (50 mg/kg) as compared with the free active compound (100 mg/kg). Finally, evaluation of the biodistribution and pharmacokinetic profiles of the drug demonstrated that these nanoaggregates preferentially localized to the liver and spleen, and protected the drug from physiological metabolism.ConclusionSqualenoylation of cytarabine offers several pharmacological benefits both in vitro and in vivo.

Proteasome inhibitor bortezomib overcomes P‐gp‐mediated multidrug resistance in resistant leukemic cell lines

To study the effect of bortezomib alone or in combination with daunorubicin (DNR) on an mdr1 single-factor drug-resistant leukemia cell line K562/MDR1, a multifactor-resistant cell line K562/A02, a drug-sensitive cell line K562, and primary cells from acute myeloid leukemia patients. The cell lines were exposed to bortezomib, DNR, and bortezomib plus DNR, and cell proliferation, cell cycle, apoptosis rate, and expression of MDR1/BCL2 were analyzed. Bortezomib potently inhibited growth and increased the apoptosis rate in the cell lines. In K562/MDR1 and K562/A02, the calcium channel blocker verapamil reduced the 50% inhibitory concentration and apoptosis rate of DNR, a P-gp protein substrate, but not of bortezomib. Bortezomib plus DNR had synergistic effect on antiproliferation (synergistic ratio > 1). Apoptosis was substantially more increased by the combination of two drugs than by bortezomib alone. Bortezomib arrested the cell cycles of three cell lines at the G2/M stage, decreased BCL2 mRNA expression, but did not affect MDR1 mRNA levels. The antiproliferative role of bortezomib was also confirmed in primary leukemia cells. Bortezomib is a promising potential therapy for acute leukemia, especially mdr1 drug-resistant leukemia.

Angiopoietin1 contributes to the maintenance of cell quiescence in EVI1high leukemia cells

Ecotropic viral integration site-1 (EVI1) is an oncogenic transcription factor in human acute myeloid leukemia (AML) associated with poor prognosis. Because the drug-resistance of leukemia cells is partly dependent on cell quiescence in the bone marrow niche, EVI1 may be involved in cell cycle regulation in leukemia cells. As a candidate regulator of the cell cycle in leukemia cells with high EVI1 expression (EVI1high), we analyzed angiopoietin1 (Ang1), which is a down-regulated gene in EVI1-deficient mice and is involved in the quiescence of hematopoietic stem cells. The results of real-time PCR analyses showed that Ang1 is highly expressed in leukemia cell lines and primary AML cells with EVI1high expression. Introduction of shRNA against EVI1 into EVI1high leukemia cells down-regulated Ang1 expression. Moreover, knockdown of Ang1 in EVI1high leukemia cells promoted cell cycle progression and down-regulated the CDK inhibitor p18 (INK4c). Treatment with a decoy Tie2/Fc protein also down-regulated the expression of p18. These results suggest that Ang1/Tie2 signaling may suppress cell cycle progression via maintenance of G0/G1 phase through up-regulation of p18 expression. This mechanism may help to maintain EVI1high leukemia cells in the bone marrow niche and promote resistance to anti-cancer drugs.

Ultrasound Mediated Drug-Loaded Nanoparticles Crossing Cell Membranes as a New Strategy to Reverse Cancer Multidrug Resistance

The emergence of the multidrug resistance (MDR) phenomenon in tumor has rendered many currently available chemotherapeutic drugs ineffective. Although many strategies have been explored to overcome MDR, the results have been disappointing to the obstacle. The aim of this study was to investigate whether the new strategy of combining drug-loaded nanoparticles (Nps) and ultrasound (US) would show useful effects on the reversal of MDR in tumor. The MDR leukemia K562/A02 cells were treated with the daunorubicin (DNR)-loaded TiO2 Nps drug carrier and US exposure. We observed good biocompatibility of the therapeutic approach, and the fresh evidence from the electrochemical studies, MTT assays, and caspase-3 immunocytochemistry demonstrated that the strategy could significantly increase the uptake of DNR by drug-resistant leukemia cells, and enhance the sensitivity of the MDR cells to the chemotherapeutic agents after released in the cells. The resisting fold became obviously lower and the apoptosis was induced in the cells as well. It was therefore concluded that the strategy could have good reversal ability of MDR in tumor. These findings reveal that the reversal of MDR in tumor by US mediated drug-loaded Nps crossing cell membranes could represent promising approach in cancer therapy.

The effect of glucosylceramide synthase on P-glycoprotein function in K562/AO2 leukemia drug-resistance cell line

Previous work from our laboratory demonstrated that glucosylceramide synthase (GCS) and multidrug resistance 1 gene (MDR1) are co-overexpressed in drug-resistant leukemia cells. We hypothesized that GCS and MDR1 may interact. In this study, we used RNA interference (RNAi) to silence the GCS or MDR1 gene in K562/AO2 drug-resistant cells. The sensitivity of cells to different treatments with doxorubicin was evaluated. We used Taqman probe fluorescence real-time quantitative PCR, and detected expression of GCS and MDR1 mRNAs in different interfering groups. Intracellular mean fluorescence intensity (MFI), which represents rhodamine123 (rh123) retention, was determined by flow cytometry (FCM). An MTT cytotoxicity assay showed that the 50% inhibition concentration (IC50) of doxorubicin of K562/AO2 cells (138.25 ± 3.75 µg/ml) was significantly higher than that of K562 drug-sensitive cells (2.125 ± 0.125 µg/ml), and that IC50 was evidently lower in K562/AO2 cells, whether it was transfected with a small interfering RNA (siRNA) targeting GCS (GCSsiRNA) or one targeting MDR1 (MDR1siRNA). Compared with untreated K562/AO2 cells, the inhibition rates of GCS mRNA in the cells transfected with GCSsiRNA for 9 and 36 h were 56.67 ± 9.29% (p < 0.05) and 74 ± 6.38% (p < 0.05), respectively. Interestingly, the expression of MDR1 mRNA was also inhibited to 51.7 ± 4.5% (p < 0.05) 36 h after transfection with GCSsiRNA, but there was no significant difference in MDR1 expression at 9 h post-transfection in cells treated with GCSsiRNA and a negative control. It is well known that rh123 retention in cells results from an efflux function of P-glycoprotein (P-gp). In K562 cells, rh123 retention was much higher than in K562/AO2 cells (p < 0.01). We also noted that rh123 retention in the K562/AO2 cells transfected with GCSsiRNA for 48 h was significantly higher than in the negative control group. In conclusion, we show in the present study that inhibition of the GCS gene affects the expression of MDR1 mRNA and P-gp function.

Reversion of multidrug-resistance by proteasome inhibitor bortezomib in K562/DNR cell line

To observe the reversion of multi-drug resistance by proteasome inhibitor bortezomib in K562/DNR cell line and to analyze the possible mechanism of reversion of multidrug-resistance. MTT method was used to determine the drug resistance of K562/DNR cells and the cellular toxicity of bortezomib. K562/DNR cells were cultured for 12 hours, 24 hours and 36 hours with 100 μg/ml DNR only or plus 4 μg/L bortezomib. The expressions of NF-κB, IκB and P-gp of K562/DNR were detected with Western blot method, the activity of NF-κB was tested by ELISA method and the apoptosis rate was observed in each group respectively. The IC50 of DNR on cells of K562/S and K562/DNR groups were 1.16 μg/ml and 50.43 μg/mL, respectively. The drug-resistant fold was 43.47. The IC10 of PS-341 on Cell strain K562/DNR was 4 μg/L. Therefore, 4 μg/L was selected as the concentration for PS-341 to reverse drug-resistance in this study. DNR induced down-regulation of IκB expression, up-regulation of NF-κB and P-gp expression. After treatment with PS-341, a proteasome inhibitor, the IκB degradation was inhibited, IκB expression increased, NF-κB and P-gp expression decreased in a time dependent manner. Compared to DNR group, the NF-κB p65 activity of DNR+PS-341 group was decreased. Compared to corresponding DNR group, DNR induced apoptosis rate increases after addition of PS-341 in a time dependent manner. Proteasome inhibitor bortezomib can convert the leukemia cell drug resistance. The mechanism may be that bortezomib decreases the degradation of IκB and the expression of NF-κB and P-gp, therefore induces the apoptosis of multi-drug resistant cells.

Redox active copper chelate overcomes multidrug resistance in T-lymphoblastic leukemia cell by triggering apoptosis

Multidrug resistance (MDR) mediated by the over expression of drug efflux protein P-glycoprotein (P-gp) is one of the major impediments to successful treatment of cancer. P-gp acts as an energy-dependent drug efflux pump and reduces the intracellular concentration of structurally unrelated drugs inside the cells. Therefore, there is an urgent need for development of new molecules that are less toxic to normal cell and preferentially effective against drug resistant malignant cells. In this preclinical study we report the apoptotic potential of copper N-(2-hydroxyacetophenone) glycinate (CuNG) on doxorubicin resistant T lymphoblastic leukaemia cells (CEM/ADR5000). To evaluate the cytotoxic effect of CuNG, we used different normal cell lines (NIH 3T3, Chang liver and human PBMC) and cancerous cell lines (CEM/ADR5000, parental sensitive CCRF-CEM, SiHa and 3LL) and conclude that CuNG preferentially kills cancerous cells, especially both leukemic cell types irrespective of their MDR status, while leaving normal cell totally unaffected. Moreover, CuNG involves reactive oxygen species (ROS) for induction of apoptosis in CEM/ADR5000 cells through the intrinsic apoptotic pathway. This is substantiated by our observation that antioxidant N-acetyle-cysteine (NAC) and PEG catalase could completely block ROS generation and, subsequently, abrogates CuNG induced apoptosis. On the other hand, uncomplexed ligand N-(2-hydroxyacetophenone) glycinate (NG) fails to generate a significant amount of ROS and concomitant induction of apoptosis in CEM/ADR5000 cells. Therefore, CuNG induces drug resistant leukemia cells to undergo apoptosis and proves to be a molecule having therapeutic potential to overcome MDR in cancer.

The rare-earth yttrium complex [YR(mtbmp)(thf)] triggers apoptosis via the extrinsic pathway and overcomes multiple drug resistance in leukemic cells

A major problem in treating patients with acute lymphoblastic leukemia is the development of drug resistance. In the current study, we investigated the anticancer properties of the novel rare-earth yttrium complex [YR(mtbmp)(thf)] in various established cell lines, and moreover, we identified the involved apoptotic pathway. Further aim was to investigate whether synergistic effects could be reached in combination with the conventional drug vincristine. We used the yttrium complex [YR(mtbmp)(thf)] in cells of leukemia (Nalm-6) and lymphoma (BJAB) and identified the main mechanism of the apoptosis induction by measuring the amount of hypodiploid DNA via FACS Scan analysis. Exposure of BJAB cells to [YR(mtbmp)(thf)] led to a death receptor-mediated reduction of cell viability and induction of apoptosis. The independence of Bcl-2 expression supports the suggestion that the [YR(mtbmp)(thf)]-induced apoptosis is mainly mediated via the extrinsic pathway. The extensive anti-tumor activity of [YR(mtbmp)(thf)] could be underlined by its capability to overcome multiple drug resistance in leukemic cells (Nalm-6) that are characterized by an overexpression of P-glycoprotein. [YR(mtbmp)(thf)] in combination with the conventional drug vincristine displayed impressive synergistic effects. We demonstrate in vitro efficiency of [YR(mtbmp)(thf)] in cells of hematological malignancies and reveal its ability to be a possible agent for polychemotherapy.

Adjuvant CD49d Blockade Eradicates Chemoresistant ALL

AbstractAbstract 869Despite the recent advances in chemotherapy for acute lymphoblastic leukemia (ALL), drug resistance resulting in relapse and long-term side effects of current treatments warrant new treatment modalities. Integrin α4β1 (VLA4/ITGA4/CD49d) mediates adhesion of hematopoietic cells onto bone marrow cells and has been implicated in cell adhesion-mediated drug resistance of leukemia cells. Gene expression analyses indicate that VLA4 is upregulated in B-lineage Acute Lymphocytic Leukemia (ALL). Therefore, we hypothesize that VLA4 might be a potential target for treatment of drug resistant ALL To test our hypothesis, we determined the effect of VLA4 inhibition on engraftment of primary pre-B ALLs using a humanized CD49d antibody, Tysabri, as a single agent in our NOD/SCID xenograft model of primary pre-B ALL. Tysabri is known to mobilize normal hematopoietic progenitor cells into the circulation. It blocks binding of VLA-4 to its counter receptors VCAM-1 and osteopontin and we have shown previously in a small pilot study that adjuvant administration with chemotherapy sensitizes one drug resistant primary ALL in vivo to drug treatment. In this study, we injected primary ALL cells from eight different donors into NOD/SCID mice. The samples encompass various cytogenetic aberrations (BCR-ABL, E2A-PBX, MLL-AF, normal karyotype). Cells were luciferase-transduced for in vivo cell tracking and pretreated in vitro with either Tysabri (n=3 per leukemia, n=24 total) or human Ig as a control (n=3 per leukemia, n=24 total). Recipients of Tysabri treated leukemias showed significantly prolonged median survival time (BCR-ABL: MST=112days, E2A-PBX: MST=83days, MLL-AF4: MST=51days; Normal karyotype: MST=48days) compared to control groups (BCR-ABL: MST=84days, E2A-PBX: MST=54days, MLL-AF4: MST=35days; Normal: MST=39days) (p<0.05). Therefore, engraftment of leukemia was significantly delayed in the Tysabri-treated groups as determined by bioluminescent imaging (p<0.05) and survival analysis (p<0.05). Next, we injected two luciferase-labeled pre-B ALLs (US7R, RS4;11) into NOD/SCID mice, which were then treated intraperitoneally with saline (US7R: n=4; RS4;11: n=3), Tysabri (US7R: n=4; RS4;11: n=3), VDL (Vincristine, Dexamethasone and L-Asparaginase) (US7R: n=9; RS4;11: n=5), or VDL+Tysabri (US7R: n=9; RS4;11: n=5), for 4 weeks. Tysabri-treated groups showed prolonged survival time (US7R: MST=52days; RS4;11: MST=83) compared with saline-treated groups (US7R: MST=38days; RS4;11: MST=60 days) (p=0.007). VDL-only treated animals died rapidly (US7R: MST=74days; RS4;11: MST=109 days), however, the animals treated with the combination VDL+ Tysabri, survived disease-free until the end of follow-up (US7R: MST=151days; RS4;11: MST=141 days) (p<0.0001). The sacrificed animals showed absence of human CD45 in spleen, liver, bone marrow and lung by immunohistochemistry and flow cytometry indicating eradication of recalcitrant leukemia cells. We have also shown in vivo using an immunocompetent mouse model that VLA4 ablation does not result in dose-limiting toxicity to normal hematopoietic cells after VDL or 5-FU treatment. To understand further the role of VLA4 deletion in ALL, we established a model of murine leukemia using bone marrow cells from VLA4 floxed mice, retrovirally transformed with BCR-ABL1 p210 and cmyc. Subsequent to leukemic outgrowth, cells were transduced with either Empty GFP control, or Cre-GFP vector to delete VLA4. Knockout of VLA4 in transduced cells was detected by PCR on genomic DNA and by flow cytometry (Empty GFP control: 97% CD49+; Cre-GFP vector: 0.8% CD49+). Upon in vitro culturing of the cells 4-fold more VLA4 deleted cells were found in the supernatant compared to the control cells (p<0.05) determined by Trypan blue exclusion counts of dead cells, indicating that CD49d in murine leukemia is required for cell adhesion. Further functional studies addressing engraftment and gene expression upon induced VLA4 deletion are ongoing. Taken together, our data demonstrate that CD49d-blockade with adjuvant chemotherapy can eradicate chemotherapy-resistant leukemia. Further studies are warranted to understand and evaluate preclinically adjuvant inhibition of integrins to overcome relapse of leukemia. Disclosures:No relevant conflicts of interest to declare.

Drug resistance in mutant FLT3-positive AML

Mutant Fms-Like Tyrosine kinase-3 (FLT3), which is expressed in the leukemic cells of a subpopulation of acute myeloid leukemia (AML) patients, represents an attractive target for the therapy of AML. There are several FLT3 inhibitors presently in clinical trials with sufficient efficacy and toxicity features to warrant further testing in combination with standard therapies. However, the transient and partial responses observed in AML patients treated with FLT3 inhibitors, coupled with the discovery of drug-resistant leukemic blast cells in AML patients, have made resistance to FLT3 inhibitors a growing concern. In this study, we provide an overview of the role of mutant FLT3 in AML, FLT3 inhibitors under clinical and preclinical investigation, mechanisms of resistance to FLT3 inhibitors, and possible therapeutic approaches to overcoming this resistance.

3-Deazauridine enhances the antileukemic action of 5-aza-2′-deoxycytidine and targets drug-resistance due to deficiency in deoxycytidine kinase

New approaches should be sought to treat high-risk acute lymphoblastic leukemia (ALL). Since aberrant DNA methylation plays an important role in leukemogenesis of ALL, it can be targeted by 5-aza-2′-deoxycytidine (5-AZA-CdR), a potent inhibitor of DNA methylation. 5-AZA-CdR is a prodrug that is activated by deoxycytidine kinase (DCK). Leukemic cells lacking DCK are drug-resistant. In a previous phase I study, we reported that 5-AZA-CdR could induce remissions in ALL. However, some patients developed drug-resistance due to deficiency in DCK. These observations aroused our interest in 3-deazauridine (3-DU), a CTP synthetase inhibitor that is effective against leukemic cells deficient in DCK. In this report, we observed that 3-DU enhanced the in vitro antineoplastic action of 5-AZA-CdR on human leukemic cells by increasing its incorporation into DNA. Using an optimized dose-schedule we showed that this combination could cure some mice bearing L1210 leukemia, even in the presence of a subpopulation of drug-resistant (L1210/ARA-C) leukemic cells lacking DCK. 3-DU alone also cured some mice with L1210/ARA-C leukemia. In a pilot study on 3 relapsed patients with advanced ALL, the combination of 5-AZA-CdR and 3-DU produced a marked reduction in leukemic blasts, confirming our preclinical observations. Furthermore, after several treatments with these agents all three patients developed drug-resistance to 5-AZA-CdR as determined by an in vitro drug sensitivity test. In two patients we showed by enzymatic analysis that the drug-resistance was due to deficiency in DCK. Our preclinical and clinical results provide a strong rationale to further investigate the combination of 5-AZA-CdR and 3-DU for the treatment of advanced ALL.