Multidrug-resistant Leukemia Cells Research Articles

Methylation of SFRP5 is related to multidrug resistance in leukemia cells

Methylation of secreted frizzle-related protein (SFRP) genes activates Wnt/ß-catenin signaling and promotes tumor development. This study investigated whether SFRP5 gene methylation causes multidrug resistance (MDR) in leukemia through the Wnt/ß-catenin signaling, leading to the upregulation of the mdr1 gene and its product, P-glycoprotein (P-gp). Methylation-specific PCR identified SFRP5 gene methylation in cultured bone mononuclear cells from 7/12 patients with acute leukemia and in four human leukemia cell lines (HL-60, Raji, U937 and KG1a). Western blotting revealed absent SFRP5 protein expression in cells from 5/7 patients with SFRP5 gene methylation and in all cell lines. Treatment with a demethylation agent (DAC) rescued SFRP5 expression. mdr1 mRNA and P-gp protein were detected in cells from 3/5 patients with absent SFRP5, and in the KG1a cell line; these cells also had the highest levels of activated ß-catenin. In cells from these three patients, DAC rescued SFRP5 expression and downregulated mdr1 and P-gp. SFRP5 protein expression was rescued in transgenic KG1a/SFRP5 cells, compared with KG1a/eGFP or untransfected KG1a cells. mdr1 and P-gp in KG1a/SFRP5 cells were downregulated. Doxorubicin IC50 values were significantly lower in KG1a/SFRP5 (0.573±0.131 μM) than in KG1a (0.963±0.115) or KG1a/eGFP (0.917±0.138) cells (P<0.05). We conclude that SFRP5 gene methylation in leukemia cells activates Wnt/ß-catenin signaling to upregulate mdr1/P-gp expression and cause MDR. Recovery of SFRP5 expression reversed MDR in the KG1a leukemia cell line. Our results suggest that modulating SFRP5 methylation could decrease MDR in leukemia patients.

Assessment of effects of multi drug resistance on dielectric properties of K562 leukemic cells using electrorotation

In this study, dielectric characterization of multidrug resistant (MDR) K562 human leukemia cells was carried out using a MEMS based electrorotation (ER) device with 3D electrodes.

Increased 14-3-3ζ Expression In The Multidrug-Resistant Leukemia Cell Line HL-60/Vcr As Compared To HL-60 Cell Line Mediates Cell Growth and Apoptosis

ObjectivesAcute myeloid leukemia (AML) recurrence is largely a result of multidrug resistance (MDR). Because vincristine-resistant HL-60 cells (HL-60/VCR) express greater levels of 14-3-3¦Æ, which is associated with apoptosis and chemosensitivity in other cancers, this study sought to examine its role in AML chemosensitivity using HL-60 and HL-60/VCR cells. MethodsThe mRNA and protein expression of 14-3-3¦Æ, mdr1, Pgp, BCL-2 and MCL-1 were examined using semi-quantitative RT-PCR and Western blot analyses, respectively. The subcellular location of 14-3-3¦Æ protein in HL-60 and HL-60/VCR cells was determined using immunofluorescence and confocal microscopy. After siRNA-mediated silencing of 14-3-3¦Æ in HL-60 AND HL-60/VCR cells, cell growth and cell cycle progression were determined by direct counting and flow cytometry, respectively. The effect of 14-3-3¦Æ siRNA on topotecan (TPT)-induced apoptosis was evaluated using acridine orange/ethidium bromide and Annexin V staining as well as TUNEL analysis. NF-¦ÊB-DNA biding was also assessed using electrophoretic mobility shift assay. ResultsAs compared to HL-60 cells, increased 14-3-3¦Æ mRNA and protein expression was observed in HL-60/VCR cells. In addition, increased mdr-1 mRNA as well as Pgp, Bcl-2, and Mcl-1 protein expression were observed in HL-60/VCR cells. In both HL-60 and HL-60/VCR cells, 14-3-3¦Æ was observed in the cytoplasm and nuclear compartments. 14-3-3¦Æ siRNA significantly reduced HL-60 and HL-60/VCR cell growth after 48 h and increased the proportion of cells in the G0/G1 phase. Moreover, 14-3-3¦Æ siRNA significantly increased the sensitivity of both HL-60 and HL-60/VCR cells to TPT possibly through inhibition of Bcl-2, Mcl-1 and Pgp protein expression. Conversely, increased Bad and Noxa protein expression was observed with 14-3-3¦Æ siRNA. NF-ĸB DNA binding was reduced with 14-3-3¦Æ siRNA. ConclusionsSilencing of 14-3-3¦Æ increased the sensitivity of both sensitive and resistant HL-60 cells to TPT-induced apoptosis possibly through altering the expression of apoptosis-associated proteins, suggesting that it may be a potential target for MDR AML. Disclosures:No relevant conflicts of interest to declare.

Bioreductive activation of mitoxantrone by NADPH cytochrome P450 reductase does not change its apoptotic stimuli properties in regard to sensitive and multidrug resistant leukaemia HL60 cells

The objective of this study was to examine the effect of bioreductive activation of antitumour drug, mitoxantrone (MX), by liver NADPH cytochrome P450 reductase (CPR) on inducing apoptosis of human promyelocytic sensitive leukaemia HL60 cell line and its multidrug resistance (MDR) sublines exhibiting two different phenotypes of MDR related to the overexpression of P-glycoprotein (HL60/VINC) or MRP1 (HL60/DOX). It was found that non-activated as well as CPR-activated form of MX used at IC90 were able to influence cell cycle of sensitive HL60 as well as resistant cells and induce apoptosis. Interestingly, it was evidenced that HL60/VINC cells were more susceptible to undergo caspase-3/caspase-8-dependent apoptosis induced by both studied forms of MX compared to HL60 and HL60/DOX cells. However, the examined agent did not change the expression of Fas receptors on the surface of HL60 sensitive as well as resistant cells regardless of its form used in the study. Obtained results suggest that CPR-dependent reductive activation of MX does not change its apoptotic stimuli properties in regard to sensitive HL60 and multidrug resistant (HL60/VINC and HL60/DOX) leukaemia cells. Nevertheless, taking into account that side toxic effects observed in course of patient treatment with antitumour drugs are dose-dependent, it seems that the reported increase in antiproliferative activity and ability to induce apoptosis of MX after its reductive activation by exogenous CPR against the MDR cells overexpressing both P-glycoprotein and MRP1 at much more lower concentrations of this drug could be of clinical importance for the treatment of tumours resistant to classical chemotherapy.

Hypoxia influences stem cell-like properties in multidrug resistant K562 leukemic cells

The present study investigates the potential role of hypoxia in maintaining stem cell-like properties and therapeutic resistance in K562 leukemic cell. Western blot, flow cytometry and cell viability assays were used to investigate the effects of hypoxia (1% O2) on cell proliferation, drug resistance and expression of the hypoxia inducible factor-2α (HIF-2α), the octamer-binding transcription factor 4 (Oct4), CD133, CD34 and the ATP-binding cassette sub-family G member 2 (ABCG2) as well as Smad2 phosphorylation in the drug resistant cell line K562/DOX and its parental cell line. Hypoxia induced growth inhibition and significantly upregulated HIF-2α, CD133, Oct4, CD34 and ABCG2 expression in the wild type K562 cells (p<0.05). The IC50 of doxorubicin was also enhanced about 2.5-fold in hypoxia. In contrast, the K562/DOX cells, which showed significantly higher ABCG2 expression and IC50 for various drugs, no significant difference in cell proliferation was observed between hypoxia and normoxia. The hypoxia-induced upregulation of HIF-2α, CD133, Oct4, CD34 and ABCG2 expression was significantly lower than in the wild type cells (p<0.05). Moreover, hypoxia induced the phosphorylation of Smad2 and additional treatment with SD-208, an inhibitor of the TGF-β receptor I kinase, resulted in a dose-dependent downregulation of CD133 and Oct4 in the K562/DOX cells. Hypoxia plays an important role in enhancing the stem cell-like properties and to induce multidrug resistance of leukemia cells. The activation of the TGF-β/Smad2 signaling pathway may be involved in the regulation of this pathophysiological process.

Abstract 3294: Apoptotic effect of thymus extract plus Lactobacillus kefiri P-IF on human myeloid leukemia (HL60/AR) cancer cells.

Abstract Apoptosis involves a cascade of biochemical events that is tightly regulated. Induction of apoptosis in tumor cells is a promising approach to curing cancer. Therefore, new and safe treatment methods that maximize cancer cells apoptosis are therefore highly sought after. In this study, we examined the effect of gross thymus extract (GTE) combined with freeze dried, heat-killed Lactobacillus kefiri P-IF in the induction of apoptosis in human multidrug-resistant (MDR) myeloid leukemia (HL60/AR) cells in vitro and evaluated the mechanism underlying its effect. L. kefiri P-IF is a specific strain of lactic acid bacteria that has a unique DNA sequence and PET scans show a 99.6% homology with regular kefiris. To identify any synergistic effect of these two products, tumor cells were cultured for three days with 1) GTE at a concentration of 1.25 mg/ml or 2) L.kefiri P-IF at a concentration of 1.25 mg/ml or 3) a combination of both products at the same concentrations. The apoptotic response was assessed by propidium iodide assay and expression of Bcl2, anti-apoptotic protein, was determined by flow cytometry. Results showed that GTE and L.kefiri P-IF induced apoptosis in HL60/AR cells; GTE caused 37% apoptosis while L.kefiri P-IF caused 17% apoptosis, as compared to 7% for control cancer cells without treatment. When cancer cells were cultured in the presence of both GTE and L.kefiri P-IF, 60% apoptosis of cancer cells was detected. This data was confirmed by giemsa stained cytospin preparation. The synergistic apoptotic effect was associated with decreased expression of Bcl2. MFC: 74, 68, 61 and 50 for control untreated cells, GTE, L.kefiri P-IF and GTE + kefiri P-IF respectively. Atomic Force Microscopy studies were performed and showed clear signs of apoptotic cancer cells including membrane blebbing. These results demonstrate that GTE + L.kefiri P-IF induces apoptosis on cancer cells that exceed the effect of either agent alone, and this agent may represent a new class of adjuvants for the treatment of leukemia. GTE (Thymax) was provided by the YS Nature Company, Tokyo, Japan; L.kefiri P-IF was provided by Paitos Co., Ltd. Yokohama, Kanagawa, Japan. Citation Format: Mamdooh H. Ghoneum, Mark Henary, Yoshikazu Seto. Apoptotic effect of thymus extract plus Lactobacillus kefiri P-IF on human myeloid leukemia (HL60/AR) cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3294. doi:10.1158/1538-7445.AM2013-3294 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

P05.06 - Targeting Tumor Cell Metabolism: Restriction of Glucose Consumption Sensitizes Multidrug Resistant (MDR) Leukemia Cells to the Effects of Imatinib Mesylate

P05.06 - Targeting Tumor Cell Metabolism: Restriction of Glucose Consumption Sensitizes Multidrug Resistant (MDR) Leukemia Cells to the Effects of Imatinib Mesylate

Phytochemical Analysis and Cytotoxicity Towards Multidrug-Resistant Leukemia Cells of Essential Oils Derived from Lebanese Medicinal Plants

Juniperus excelsa fruit essential oil as well as J. oxycedrus, Cedrus libani, and Pinus pinea wood essential oils have been obtained with yields between 2.2 ± 0.3 % to 3.4 ± 0.5 % and analyzed by gas chromatography. Sesquiterpenes mainly characterized C. libani and J. oxycedrus essential oils, while in P. pinea and J. excelsa, monoterpenes were the most abundant compounds. In J. oxycedrus, cis-calamenene (7.8 %), cuparene (3.8 %), and cis-thujopsenal (2.0 %) have been detected for the first time. The cytotoxic activity of these essential oils against drug-sensitive CCRF-CEM and multidrug-resistant P-glycoprotein-expressing CEM/ADR5000 leukemia cells has been investigated (IC₅₀ values: 29.46 to 61.54 µg/mL). Remarkably, multidrug-resistant CEM/ADR5000 cells did not reveal cross-resistance, indicating that these essential oils might be useful to treat otherwise drug-resistant and refractory tumors.

An electrochemical method to assay the reversal effect on multi-drug resistance in tumor cells

The occurrence of multi-drug resistance (MDR) has become a major obstacle to the successful performance of chemotherapy for cancer patients, so it is highly required to develop methods to assay the reversal effect on MDR in tumor cells. In this paper, we report a simple but efficient electrochemical method to assay the reversal effect on MDR by using limonin as reversal agent and multi-drug resistant leukemia cells K562/ADM as target cells. The strategy is based on the over-expression of P-glycoprotein (P-gp) in K562/ADM cells that may promote the efflux of the intracellular anticancer drug kaempferol. Consequently, kaempferol molecules accumulate outside the cells. They then adsorb onto the surface of the working electrode, which is modified by β-cyclodextrin/multi-walled carbon nanotubes (β-CD/MWCNTs), to give a well-defined electrochemical response. However, after K562/ADM cells have been incubated with the P-gp inhibitor limonin, efflux of the intracellular anticancer drug molecules will be prohibited, so the peak currents can be observed to decrease in a limonin concentration-dependent manner. Therefore, the proposed method in this work can efficiently characterize the reversal effect of limonin on MDR, which might have potential use in screening the reversal agents for cancer treatments in the future.

171 Bcl-2 Family Members and Survival Under Stress Conditions in Multidrug Resistant Leukemic Cells

171 Bcl-2 Family Members and Survival Under Stress Conditions in Multidrug Resistant Leukemic Cells

Cryptotanshinone induces cell cycle arrest and apoptosis of multidrug resistant human chronic myeloid leukemia cells by inhibiting the activity of eukaryotic initiation factor 4E

Cryptotanshinone (CPT), a diterpene quinone isolated from Salvia miltiorrhiza, is recently reported to have obvious anticancer activities against diverse cancer cells. However, the effect and regulatory mechanism of CPT remain unclear in human chronic myeloid leukemia (CML) cells. In this study, we investigated the antiproliferative activity of CPT on the multidrug resistant CML cells K562/ADM. Our results demonstrated that CPT decreased the cell viability of K562/ADM cells by inducing cell cycle arrest and apoptosis through suppressing the expression of cyclin D1 and Bcl-2. Further studies indicated that CPT mainly functions at post-transcriptional levels, suggesting the involvement of eukaryotic initiation factor 4E (eIF4E). CPT significantly reduced the expression and activity of eIF4E in K562/ADM cells. Overexpression of eIF4E obvious conferred resistance to the CPT antiproliferation and proapoptotic activity as well as the cyclin D1 and Bcl-2 expressions. Knockdown of eIF4E significantly reduced the inhibitory effect of CPT in K562/ADM, confirming the participation of eIF4E during CPT function process. More importantly, the relative inhibitory efficiency of CPT positively correlated with the reductions on eIF4E in primary CML specimens. These results demonstrated that CPT played antitumor roles in K562/ADM cells by inhibiting the eIF4E regulatory system. Our results provide a novel anticancer mechanism of CPT in human CML cells.

Abstract 767: CXL017 re-sensitizes multidrug resistant leukemia cells to chemotherapy via modulating Bcl-2 family proteins and SERCA proteins

Abstract Multidrug resistance (MDR) is a major hurdle in the treatment of cancer and there is a pressing need for new therapies. We have recently developed CXL017, derived from a Bcl-2 and SERCA dual inhibitor, with selective cytotoxicity towards MDR cancer cells. In this study, we present new evidence for its therapeutic potential in treatment of MDR cancers and offer mechanistic insights. CXL017 significantly and selectively suppresses the growth of tumors derived from the MDR cancer cell line, HL60/MX2, in vivo. In addition, HL60/MX2 failed to develop stable resistance to CXL017 upon prolonged exposure. In comparison, HL60/MX2 acquired greater than 2000-fold resistance to cytarabine (Ara-C). Remarkably, instead of acquiring cross-resistance, HL60/MX2 cells exposed to CXL017 were re-sensitized to standard therapies (10 to 100-fold). Western blotting analyses revealed that HL60/MX2 has significantly elevated levels of Mcl-1, Bax and Bak relative to HL60. HL60/MX2 also over-expresses SERCA2 and SERCA3 with elevated endoplasmic reticulum (ER) calcium content. CXL017 exposure significantly down-regulates Mcl-1 and Bax but causes up-regulation of Bcl-XL, SERCA2, and SERCA3 proteins, along with a reduction in ER calcium content. Given the well-established functions of Bcl-2 family proteins and ER calcium in drug resistance, our results suggest that the reduction in Mcl-1 and the decrease in ER calcium content are responsible for CXL017 induced chemosensitization of MDR cancer cells. These results support the strategy to develop dual inhibitors of anti-apoptotic Bcl-2 proteins and SERCA to treat MDR cancers. 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 767. doi:1538-7445.AM2012-767

MiR-181a sensitizes a multidrug-resistant leukemia cell line K562/A02 to daunorubicin by targeting BCL-2

The aim of this study was to investigate whether miR-181a could modulate the sensitivity of the leukemia drug-resistant cell line K562/A02 to the chemotherapeutic agent daunorubicin (DNR), and explore the mechanism of miR-181a on the DNR sensitivity of K562/A02 cells. MicroRNA microarray and stem-loop reverse transcription-polymerase chain reaction were used to detect the expression of miR-181a. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay was performed to quantify the effect of miR-181a on K562 cells growth and viability. Apoptotic cells were quantitatively detected using Annexin V/FITC and PI apoptosis detection kit. BCL-2 protein expression was measured by western blot. Luciferase reporter vector with the putative BCL-2 3' untranslated region was constructed to explore whether BCL-2 was a direct target gene of miR-181a. BCL-2 siRNA was transfected into the cell to explore the relationship between BCL-2 and DNR resistance. The miR-181a expression level was lower in the K562/A02 cells than in the K562 cells (P< 0.05). K562 cells that were transfected with miR-181a inhibitor had a significantly higher survival than K562 cells, and K562/A02 cells that were transfected with the miR-181a mimic had a significantly lower survival than K562/A02 cells (P< 0.05). miR-181a could enhance DNR-induced apoptosis in K562/A02 cells. BCL-2 siRNA transfected K562/A02 cells had decreased survival compared with the K562/A02 control group. In conclusion, miR-181a could play a role in the development of DNR resistance in K562/A02 cells and the over-expression of miR-181a could sensitize K562/A02 cells to DNR by targeting BCL-2.

Acquisition of MDR phenotype by leukemic cells is associated with increased caspase‐3 activity and a collateral sensitivity to cold stress

The acquisition of a multidrug-resistant (MDR) phenotype by tumor cells that renders them unsusceptible to anti-neoplasic agents is one of the main causes of chemotherapy failure in human malignancies. The increased expression of P-glycoprotein (MDR1, P-gp, ABCB1) in tumor cells contributes to drug resistance by extruding chemotherapeutic agents or by regulating programmed cell death. In a study of MDR cell survival under cold stress conditions, it was found that resistant leukemic cells with P-gp over-expression, but not their sensitive counterparts, are hypersensitive to cold-induced cell death when exposed to temperatures below 4 °C. The transfection of parental cells with a P-gp-expressing plasmid makes these cells sensitive to cold stress, demonstrating an association between P-gp expression and cell death at low temperatures. Furthermore, we observed increased basal expression and activity of effector caspase-3 at physiological temperature (37 °C) in MDR cells compared with their parental cell line. Treatment with a caspase-3 inhibitor partially rescues MDR leukemic cells from cold-induced apoptosis, which suggests that the cell death mechanism may require caspase-3 activity. Taken together, these findings demonstrate that P-gp expression plays a role in MDR cell survival, and is accompanied by a collateral sensitivity to death induced by cold stress. These findings may assist in the design of specific therapeutic strategies to complement current chemotherapy treatment against cancer.

Abstract B11: Expression of microRNAs-135b and -196b in response to cellular stress in leukemia cells

Abstract MicroRNAs are a class of non-coding small RNA molecules that have roles in important biological processes. Multidrug resistance is a key obstacle to successful cancer therapy in human cancers. To determine whether microRNAs have a role in the development or maintenance of multidrug resistance, we used the TaqMan™ Human MicroRNA Array to investigate the expression of microRNAs in multidrug resistant (MDR) CCRF-CEM human T-cell leukemia cells (CEM/VM-1-5) and drug-sensitive parental line, CCRF-CEM (CEM). The MDR line was selected for resistance to teniposide and is cross-resistant to etoposide and other drugs. Our profiling study of 365 microRNAs revealed that certain microRNAs are differentially expressed between CEM/VM-1-5 and CEM cells, and we focused our subsequent work on two of these: miR-135b and -196b. Since MDR cells were selected and developed by stepwise increases in concentrations of teniposide, we therefore asked whether these altered microRNAs in MDR CEM/VM-1-5 cells are associated with cellular responses to genotoxic agents. We observed substantial increases in miR-135b and -196b expression in drug-sensitive CEM cells following their short exposure (3 to 48 hours) with etoposide, doxorubicin and topotecan, but not in cells treated with vinblastine or paclitaxel, suggesting that the upregulation of these two microRNAs might be the consequence of DNA damage. We also found similar increases in miR-135b and -196b in other leukemia cell lines (RPMI 8226 [myeloma], HL-60 [acute promyelocytic leukemia], Jurkat and MOLT-4 [both acute T cell leukemia]), but not in solid tumor-derived cell lines (MCF7 breast cancer and A2780 ovarian cancer). This suggests that induction of expression of these microRNAs is histiotype-specific. To further confirm that upregulation of these two microRNAs is a consequence of DNA damage, we examined expression of miR-135b and -196b following ionizing radiation. When we treated CEM pairs with ionizing radiation, MDR CEM/VM-1-5 cells were more radioresistant compared to CEM cells. As expected, higher doses of radiation induced miR-135b and -196b expression in MDR CEM/VM-1-5 cells compared to CEM cells, in accordance with what we observed in cells treated with the DNA damaging agent etoposide. Moreover, time-course and dose-response studies showed that the elevation of miR-135b and -196b correlated positively with the exposure time and radiation dosage. Together, our results suggest that microRNAs might play a significant role in cellular response to genotoxic agents and upregulation of miR-135b and -196b appears to be a marker of DNA damage in leukemia cells. (Supported in part by RO1CA40570 from NCI to WTB, and in part by UIC.) Citation Format: Tsui-Ting Ho, Xiaolong He, Ahmet Dirim Arslan, William T. Beck. Expression of microRNAs-135b and -196b in response to cellular stress in leukemia cells [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B11.

Antileukemic Activity and Cellular Metabolism of the Aryl Phosphate Derivative of Bromo-Methoxy Zidovudine (Compound WHI-07)

The novel aryl phosphate derivative of bromo-methoxy zidovudine (ZDV/AZT) (compound WHI-07, CAS 213982-96-8) was found to be a potent antileukemic agent against human leukemia, lymphoma, and multiple myeloma cell lines in MTT and clonogenic assays with low micromolar IC50 values. In addition, WHI-07 was antimitotic, leading to cell fusion and developmental arrest in the Zebrafish model of rapid cell proliferation. WHI-07 was cytotoxic to drug-sensitive (NALM-6, MOLT-3, HL-60, P388) and multi-drug resistant (MDR) leukemia cell lines (HL-60/VCR, HL-60/ADR, P388/ ADR). Treatment of leukemia cells with WHI-07 showed rapid and dramatic depletion of all cellular nucleoside diphosphate and triphosphate (NDP/NTP) pools, which would contribute to the overall reduction of nucleic acid synthesis and cell death. WHI-07 was rapidly metabolized to alaninyl ZDV monophosphate (Ala-ZDV-MP), the levels of which inversely correlated with cytotoxic IC50 values of WHI-07. Glutathione was found to mediate the in vitro and in vivo detoxification pathway of WHI-07 to 3'-azidothymidine-5'-p-bromophenylmethoxyalaninyl phosphate and Ala-ZDV-MP, respectively. The proposed intracellular metabolic pathway for WHI-07 involves a thiol-mediated dehalogenation step followed by the paraoxon-sensitive carboxylesterase-mediated reaction leading to the formation of Ala-ZDV-MP as the major intracellular metabolite.

Enhanced invasiveness in multidrug resistant leukemic cells is associated with overexpression of P-glycoprotein and cellular inhibitor of apoptosis protein

Multidrug resistance (MDR) and multi-organ infiltration are the major obstacles to the successful treatment of leukemia. It is known that the drug efflux protein, P-glycoprotein (P-gp), and inhibitors of apoptosis proteins (IAPs) are involved in the MDR of leukemic cells, but their roles in leukemia infiltration have not been clearly elucidated. In this study, leukemic cell lines K562 and HL60 and their MDR variants K562R and HL60R have been used to analyze their infiltrative ability. MDR variants display enhanced invasion compared with parental cells. Results from xenografts in SCID (severe combined immunodeficiancy) mice are consistent with these in vitro observations. Furthermore, P-gp and cIAP are overexpressed and co-localize with protein kinase C-ε (PKC-ε) in MDR variants. Our study shows that overexpression of P-gp and cIAP may enhance the infiltration of leukemic cells.

Abstract 4302: Targeting the sphingolipid pathway in NK large granular lymphocyte leukemia

Abstract Large granular lymphocytes (LGL) are a lymphoid subset that consists of 10-15% of peripheral blood mononuclear cells. LGL can be divided into (2) major lineages of CD3- NK cells and CD3+ T Cells. Symptoms may include rheumatoid arthritis and neutropenia which leads to recurrent bacterial infections. The sphingolipid metabolism pathway has been found to be deregulated in NK-LGL leukemia. Glucosylceramide synthase (GCS), a key component of this pathway is an enzyme that catalyzes the conversion of proapoptotic ceramide to inactive glucosylceramide by transferring a glucose residue from UDP-glucose to ceramide. GCS has been found to be upregulated in cancer cells and its overexpression has been associated with multidrug resistance in leukemia cells. Both rat and human leukemic LGL cells have been treated with a combination treatment targeting two key components of the sphingolipid pathway. One of the agents in use is the C6-ceramide (C6) nanoliposome which has led to caspase-dependent apoptosis in a time- and dose-dependent manner. Systemic i.v. delivery of nanoliposomal ceramide led to complete remission in 5/14 syngeneic Fischer F344 rat model of NK-LGL leukemia. However a greater response will be achieved by combination treatment of C6 and through targeting a second key component of ceramide metabolism with DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP). PPMP is a GCS inhibitor that prevents the endogenous ceramide already present in the cell from being glycosylated into its inactive form where it can no longer induce cell death. Overall, this combination treatment shows a synergistic effect with increase of ceramide levels in order to induce apoptosis and therefore death of these persistent LGL cells. Cell flow cytometry examining 7AAD/Annexin V staining has shown ∼80% apoptosis when 6.25 uM C6 ceramide and 15 uM PPMP are used in combination on aggressive rat LGL cells. Whereas, only ∼19% is observed with C6 and ∼17% of PPMP are observed individually in treatment. Overall, apoptosis appeared increased in a dose response manner after treatment with PPMP and C6. This is a significant finding of P&amp;lt;0.005, when compared to DMSO and ghost nanoliposome controls which only induce ∼15% apoptosis. In addition, this combination treatment has been utilized on normal human peripheral blood mononuclear cells where only 11.2% apoptosis was induced and 9% in the normal spleen mononuclear cells in rats. As a result, this combination treatment appears promising in specifically targeting LGL cells while sparing normal cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4302. doi:10.1158/1538-7445.AM2011-4302

WAVE1 regulates P-glycoprotein expression via Ezrin in leukemia cells

For children with acute myeloblastic leukemia (AML), multidrug resistance (MDR) reduces treatment effectiveness, and often leads to poor patient survival. While a number of factors have been described that affect MDR, the mechanisms underlying this effect remain unclear. In this study, the role of WAVE1 in MDR was investigated. Among 62 children with AML, high levels of WAVE1 were associated with poor patient outcomes. Proteomic techniques were used to identify novel WAVE1-interacting proteins from leukemia cells, one of which was the cytoskeleton regulator Ezrin. In leukemia cells, WAVE1 co-localized with both Ezrin and P-glycoprotein (P-gp), a critical regulator of the MDR phenotype. Overexpression of WAVE1 in K562 leukemia cells up-regulated P-gp and Ezrin, and reduced K562 cells' sensitivity to the chemotherapy drug adriamycin. The opposite effect was seen when WAVE1 expression was reduced via RNA interference. Critically, overexpression of WAVE1 in the absence of Ezrin did not affect P-gp levels or MDR. These data suggest that WAVE1 affects P-gp and MDR of leukemia cells through Ezrin.

Reverse-phase protein microarrays (RPPA) as a diagnostic and therapeutic guide in multidrug resistant leukemia

Reverse-phase microarray assays using phospho-specific antibodies (RPPA) can directly measure levels of phosphorylated protein isoforms. In the current study, lysates from parental and multidrug resistant (MDR) CEM leukemia cells were spotted onto reverse-phase protein microarrays and probed with a panel of phospho-antibodies to ERK, PCK and Akt pathways. In particular, the Akt pathway is considered to play significant roles in leukemia and Akt inhibitor therapy has been proposed as a potential tool in the treatment of this disease. The RPPA data prompted us to investigate deeper this pathway. Here, we found that whereas total Akt1 protein level is higher in parental CEM cells, the activated isoform content, p-Akt1, increases in doxorubicin-selected CEM cells (MDR-CEM). This was backed up by Western blot analysis, confirming that Akt1 activity/phosphorylation may be up-regulated in MDR-CEM cells. Further exploration of inhibitory therapy in this system was evaluated. The TNF-related apoptosis-inducing ligand, TRAIL, has been shown to selectively kill tumor cells. Herein, we describe that in MDR-CEM cells TRAIL responsiveness correlates with a reduced expression of endogenous Akt1, suggesting that the MDR phenotype associated to P-gp sensitizes cells to TRAIL therapy.