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We explored whether the anti-prostate cancer (PC) activity of the androgen receptor-axis-targeted agents (ARATs) abiraterone and enzalutamide is enhanced by metformin. Using complementary biological and molecular approaches, we determined the associated underlying mechanisms in pre-clinical androgen-sensitive PC models. ARATs increased androgren receptors (ARs) in LNCaP and AR/ARv7 (AR variant) in VCaP cells, inhibited cell proliferation in both, and induced poly(ADP-ribose) polymerase-1 (PARP-1) cleavage and death in VCaP but not LNCaP cells. Metformin decreased AR and ARv7 expression and induced cleaved PARP-1-associated death in both cell lines. Metformin with abiraterone or enzalutamide decreased AR and ARv7 expression showed greater inhibition of cell proliferation and greater induction of cell death than single agent treatments. Combination treatments led to increased cleaved PARP-1 and enhanced PARP-1 activity manifested by increases in poly(ADP-ribose) (PAR) and nuclear accumulation of apoptosis inducing factor (AIF). Enhanced annexin V staining occurred in LNCaP cells only with metformin/ARAT combinations, but no caspase 3 recruitment occurred in either cell line. Finally, metformin and metformin/ARAT combinations increased lysosomal permeability resulting in cathepsin G-mediated PARP-1 cleavage and cell death. In conclusion, metformin enhances the efficacy of abiraterone and enzalutamide via two PARP-1-dependent, caspase 3-independent pathways, providing a rationale to evaluate these combinations in castration-sensitive PC.

Expression of DAPK1, a critical regulator of autophagy and apoptosis, is lost in a wide variety of tumors, although the mechanisms are unclear. A transcription factor complex consisting of ATF6 (an endoplasmic reticulum-resident factor) and C/EBP-β is required for the IFN-γ-induced expression of DAPK1 IFN-γ-induced proteolytic processing of ATF6 and phosphorylation of C/EBP-β are obligatory for the formation of this transcriptional complex. We report that defects in this pathway fail to control growth of chronic lymphocytic leukemia (CLL). Consistent with these observations, IFN-γ and chemotherapeutics failed to activate autophagy in CLL patient samples lacking ATF6 and/or C/EBP-β. Together, these results identify a molecular basis for the loss of DAPK1 expression in CLL., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique - usually untranslated - first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

Phosphorylated cyclic-AMP (cAMP) response element binding protein (p-CREB) is a downstream effector of a variety of important signaling pathways. We investigated whether the human BCRP promoter contains a functional cAMP response element (CRE). 8Br-cAMP, a cAMP analogue, increased the activity of a BCRP promoter reporter construct and BCRP mRNA in human carcinoma cells. Epidermal growth factor receptor (EGFR) pathway activation also led to an increase in p-CREB and in BCRP promoter reporter activity via two major downstream EGFR signaling pathways: the phosphotidylinositol-3-kinase (PI3K)/AKT pathway and the mitogen-activated protein kinase (MAPK) pathway. EGF treatment increased the phosphorylation of EGFR, AKT, ERK and CREB, while simultaneously enhancing BCRP mRNA and functional protein expression. EGF-stimulated CREB phosphorylation and BCRP induction were diminished by inhibition of EGFR, PI3K/AKT or RAS/MAPK signaling. CREB silencing using RNA interference reduced basal levels of BCRP mRNA and diminished the induction of BCRP by EGF. Chromatin immunoprecipitation assays confirmed that a putative CRE site on the BCRP promoter bound p-CREB by a point mutation of the CRE site abolished EGF-induced stimulation of BCRP promoter reporter activity. Furthermore, the CREB co-activator, cAMP-regulated transcriptional co-activator (CRTC2), is involved in CREB-mediated BCRP transcription: androgen depletion of LNCaP human prostate cancer cells increased both CREB phosphorylation and CRTC2 nuclear translocation, and enhanced BCRP expression. Silencing CREB or CRTC2 reduced basal BCRP expression and BCRP induction under androgen-depletion conditions. This novel CRE site plays a central role in mediating BCRP gene expression in several human cancer cell lines following activation of multiple cancer-relevant signaling pathways., (Published by Elsevier B.V.)

Alternative promoter usage is typically associated with mRNAs with differing first exons that contain or consist entirely of a 5' untranslated region. The murine Bcrp1 (Abcg2) transporter has three alternative promoters associated with mRNAs containing alternative untranslated first exons designated as E1A, E1B, and E1C. The E1B promoter regulates Bcrp1 transcription in mouse intestine. Here, we report the identification and characterization of a novel Bcrp1 promoter and first exon, E1U, located upstream from the other Bcrp1 promoters/first exons, which is the predominant alternative promoter utilized in murine testis. Using in silico analysis we identified a putative steroidogenic factor-1 (SF-1) response element that was unique to the Bcrp1 E1U alternative promoter. Overexpression of SF-1 in murine TM4 Sertoli cells enhanced Bcrp1 E1U mRNA expression and increased Bcrp1 E1U alternative promoter activity in a reporter assay, whereas mutation of the SF-1 binding site totally eliminated Bcrp1 E1U alternative promoter activity. Moreover, expression of Bcrp1 E1U and total mRNA and Bcrp1 protein was markedly diminished in the testes from adult Sertoli cell-specific SF-1 knockout mice, in comparison to the testes from wild-type mice. Binding of SF-1 to the SF-1 response element in the E1U promoter was demonstrated by chromatin immunoprecipitation assays. In conclusion, nuclear transcription factor SF-1 is involved with the regulation of a novel promoter of Bcrp1 that governs transcription of the E1U mRNA isoform in mice. The present study furthers understanding of the complex regulation of Bcrp1 expression in specific tissues of a mammalian model., (© 2013.)

Dasatinib is effective in most chronic phase chronic myeloid leukemia patients both in first-line therapy and following imatinib failure. While imatinib uptake into CD34(+) cells is low compared to mononuclear cells, few data evaluate how well dasatinib targets primitive CML cells. This study compares intracellular concentration of dasatinib and Bcr-Abl kinase inhibition in CML-CD34(+) progenitors and mononuclear cells induced by dasatinib. The intracellular concentrations of dasatinib were similar between CML-CD34(+) and mononuclear cells (P=0.8). Similarly, there was no significant difference in the degree of dasatinib-mediated Bcr-Abl kinase inhibition. ABCB1 (MDR1) and ABCG2 inhibitors neither increased dasatinib intracellular concentration nor enhanced dasatinib-mediated Bcr-Abl kinase inhibition. In contrast to nilotinib, we show that dasatinib is not an ABCB1 inhibitor. Thus, dasatinib targets CML-CD34(+) progenitors as effectively as it targets mononuclear cells. ABCB1 and ABCG2 efflux pumps do not appear to influence the intracellular dasatinib concentration in CML-CD34(+) progenitors.

Purpose: To determine the maximum-tolerated dose (MTD) of the histone deacetylase inhibitor vorinostat combined with fixed doses of cytarabine (ara-C or cytosine arabinoside) and etoposide in patients with poor-risk or advanced acute leukemia, to obtain preliminary efficacy data, describe pharmacokinetics, and in vivo pharmacodynamic effects of vorinostat in leukemia blasts., Experimental Design: In this open-label phase I study, vorinostat was given orally on days one to seven at three escalating dose levels: 200 mg twice a day, 200 mg three times a day, and 300 mg twice a day. On days 11 to 14, etoposide (100 mg/m(2)) and cytarabine (1 or 2 g/m(2) twice a day if ≥65 or <65 years old, respectively) were given. The study used a standard 3+3 dose escalation design., Results: Eighteen of 21 patients with acute myelogenous leukemia (AML) treated on study completed planned therapy. Dose-limiting toxicities [hyperbilirubinemia/septic death (1) and anorexia/fatigue (1)] were encountered at the 200 mg three times a day level; thus, the MTD was established to be vorinostat 200 mg twice a day. Of 21 patients enrolled, seven attained a complete remission (CR) or CR with incomplete platelet recovery, including six of 13 patients treated at the MTD. The median remission duration was seven months. No differences in percentage S-phase cells or multidrug resistance transporter (MDR1 or BCRP) expression or function were observed in vivo in leukemia blasts upon vorinostat treatment., Conclusions: Vorinostat 200 mg twice a day can be given safely for seven days before treatment with cytarabine and etoposide. The relatively high CR rate seen at the MTD in this poor-risk group of patients with AML warrants further studies to confirm these findings., (©2013 AACR.)

The multidrug transporter, breast cancer resistance protein, ABCG2, is up-regulated in certain chemoresistant cancer cells and in the mammary gland during lactation. We investigated the role of the lactogenic hormone prolactin (PRL) in the regulation of ABCG2. PRL dose-dependently induced ABCG2 expression in T-47D human breast cancer cells. This induction was significantly reduced by short-interfering RNA-mediated knockdown of Janus kinase 2 (JAK2). Knockdown or pharmacologic inhibition of the down-stream signal transducer and activator of transcription-5 (STAT5) also blunted the induction of ABCG2 by PRL, suggesting a role for the JAK2/STAT5 pathway in PRL-induced ABCG2 expression. Corroborating these findings, we observed PRL-stimulated STAT5 recruitment to a region containing a putative γ-interferon activation sequence (GAS) element at -434 base pairs upstream of the ABCG2 transcription start site. Introduction of a single mutation to the -434 GAS element significantly attenuated PRL-stimulated activity of a luciferase reporter driven by the ABCG2 gene promoter and 5'-flanking region containing the -434 GAS motif. In addition, this GAS element showed strong copy number dependency in its response to PRL treatment. Interestingly, inhibitors against the mitogen-activated protein kinase and phosphoinositide-3-kinase signaling pathways significantly decreased the induction of ABCG2 by PRL without altering STAT5 recruitment to the GAS element. We conclude that the JAK2/STAT5 pathway is required but not sufficient for the induction of ABCG2 by PRL.

Since cloning of the ATP-binding cassette (ABC) family member breast cancer resistance protein (BCRP/ABCG2) and its characterization as a multidrug resistance efflux transporter in 1998, BCRP has been the subject of more than two thousand scholarly articles. In normal tissues, BCRP functions as a defense mechanism against toxins and xenobiotics, with expression in the gut, bile canaliculi, placenta, blood-testis and blood-brain barriers facilitating excretion and limiting absorption of potentially toxic substrate molecules, including many cancer chemotherapeutic drugs. BCRP also plays a key role in heme and folate homeostasis, which may help normal cells survive under conditions of hypoxia. BCRP expression appears to be a characteristic of certain normal tissue stem cells termed "side population cells," which are identified on flow cytometric analysis by their ability to exclude Hoechst 33342, a BCRP substrate fluorescent dye. Hence, BCRP expression may contribute to the natural resistance and longevity of these normal stem cells. Malignant tissues can exploit the properties of BCRP to survive hypoxia and to evade exposure to chemotherapeutic drugs. Evidence is mounting that many cancers display subpopulations of stem cells that are responsible for tumor self-renewal. Such stem cells frequently manifest the "side population" phenotype characterized by expression of BCRP and other ABC transporters. Along with other factors, these transporters may contribute to the inherent resistance of these neoplasms and their failure to be cured., (Published by Elsevier Inc.)

Breast cancer resistance protein (BCRP)/ATP-binding cassette subfamily G member 2 (ABCG2) is an ATP-binding cassette (ABC) transporter identified as a molecular cause of multidrug resistance (MDR) in diverse cancer cells. BCRP physiologically functions as a part of a self-defense mechanism for the organism; it enhances elimination of toxic xenobiotic substances and harmful agents in the gut and biliary tract, as well as through the blood-brain, placental, and possibly blood-testis barriers. BCRP recognizes and transports numerous anticancer drugs including conventional chemotherapeutic and targeted small therapeutic molecules relatively new in clinical use. Thus, BCRP expression in cancer cells directly causes MDR by active efflux of anticancer drugs. Because BCRP is also known to be a stem cell marker, its expression in cancer cells could be a manifestation of metabolic and signaling pathways that confer multiple mechanisms of drug resistance, self-renewal (stemness), and invasiveness (aggressiveness), and thereby impart a poor prognosis. Therefore, blocking BCRP-mediated active efflux may provide a therapeutic benefit for cancers. Delineating the precise molecular mechanisms for BCRP gene expression may lead to identification of a novel molecular target to modulate BCRP-mediated MDR. Current evidence suggests that BCRP gene transcription is regulated by a number of trans-acting elements including hypoxia inducible factor 1α, estrogen receptor, and peroxisome proliferator-activated receptor. Furthermore, alternative promoter usage, demethylation of the BCRP promoter, and histone modification are likely associated with drug-induced BCRP overexpression in cancer cells. Finally, PI3K/AKT signaling may play a critical role in modulating BCRP function under a variety of conditions. These biological events seem involved in a complicated manner. Untangling the events would be an essential first step to developing a method to modulate BCRP function to aid patients with cancer. This review will present a synopsis of the impact of BCRP-mediated MDR in cancer cells, and the molecular mechanisms of acquired MDR currently postulated in a variety of human cancers.

To graduate internal medicine residents with basic competency in palliative care, we employ a two-pronged strategy targeted at both residents and attending physicians as learners. The first prong provides a knowledge foundation using web-based learning programs designed specifically for residents and clinical faculty members. The second prong is assessment of resident competency in key palliative care domains by faculty members using direct observation during clinical rotations. The faculty training program contains Competency Assessment Tools addressing 19 topics distributed amongst four broad palliative care domains designed to assist faculty members in making the clinical competency assessments. Residents are required to complete their web-based training by the end of their internship year; they must demonstrate competency in one skill from each of the four broad palliative care domains prior to graduation. Resident and faculty evaluation of the training programs is favorable. Outcome-based measures are planned to evaluate long-term program effectiveness.

Mouse models are often used to predict drug absorption in humans. Mouse Bcrp1 protein exhibits sequence and functional homology with human BCRP protein. Additionally, BCRP/Bcrp1 expression is regulated by alternative promoter usage in humans and mice; however, the precise intestine-specific alternative promoter utilized in either species is yet to be determined. Therefore we sought to identify and characterize the mouse intestinal Bcrp1 promoter. Using real-time quantitative RT-PCR and 5' RACE PCR we first established the predominance of a single Bcrp1 first exon (E1b) in the Bcrp1 mRNA isolated throughout the mouse intestine. Simultaneously using 5' RACE PCR we identified E1C as the predominant BCRP 5' UTR expressed in the human intestine. Next we established functional activity for the murine promoter upstream of E1b using reporter assays. Subsequently using deletion-construct analysis we found the core promoter region to span -231 to -42bps from the transcriptional start site of E1b. We then predicted a cAMP response element (CRE) as a transcription factor binding site unique only to the E1b promoter region, using in silico methods. We finally established functional interaction of phospho-CREB (p-CREB) protein with the CRE on the E1b promoter using both functional assays and chromatin immunoprecipitation assays. In conclusion, mouse intestinal Bcrp1 expression is regulated by a single alternative promoter upstream of E1b, the predominant Bcrp1 mRNA isoform expressed in the mouse intestine. Furthermore, Bcrp1 E1b mRNA expression is regulated by binding of p-CREB to its cis site on the mouse E1b promoter region., (Published by Elsevier B.V.)

Preclinical investigations have identified insulin-like growth factor (IGF) signaling as a key mechanism for cancer growth and resistance to clinically useful therapies in multiple tumor types including breast cancer. Thus, agents targeting and blocking IGF signaling have promise in the treatment of solid tumors. To identify possible mechanisms of resistance to blocking the IGF pathway, we generated a cell line that was resistant to the IGF-1R/InsR benzimidazole inhibitors, BMS-554417 and BMS-536924, and compared expression profiles of the parental and resistant cells lines using Affymetrix GeneChip Human Genome U133 arrays. Compared with MCF-7 cells, breast cancer resistance protein (BCRP) expression was increased 9-fold in MCF-7R4, which was confirmed by immunoblotting and was highly statistically significant (P = 7.13E-09). BCRP was also upregulated in an independently derived resistant cell line, MCF-7 924R. MCF-7R4 cells had significantly lower intracellular accumulation of BMS-536924 compared with MCF-7 cells. Expression of BCRP in MCF-7 cells was sufficient to reduce sensitivity to BMS-536924. Furthermore, knockdown of BCRP in MCF-7R4 cells resensitized cells to BMS-536924. Four cell lines selected for resistance to the pyrrolotriazine IGF-1R/InsR inhibitor, BMS-754807, did not have upregulation of BCRP. These data suggest that benzimidazole IGF-1R/InsR inhibitors may select for upregulation and be effluxed by the ATP-binding cassette transporter, BCRP, contributing to resistance. However, pyrrolotriazine IGF-1R/InsR inhibitors do not appear to be affected by this resistance mechanism., (©2010 AACR.)

Induction of the breast cancer resistance protein (BCRP/ABCG2) expression has been found in various tissues and cell-types after exposure to chemicals including 17β-estradiol, rosiglitazone, imatinib, as well as aryl hydrocarbon receptor (AhR) activators such as 2,3,7,8-tetrachlorodibenzodioxin, 3-methylcholanthrene (3MC), and omeprazole. However, the mechanism(s) underlying AhR-related induction of ABCG2 is largely unknown. Here, we demonstrate the AhR-dependent induction of ABCG2 expression in human colon adenocarcinoma LS174T cells. Importantly, a novel distal AhR-responsive element (AhRE5) located -2357/-2333bp upstream of the ABCG2 transcriptional start site has been identified and characterized as a functional unit pivotal to 3MC-mediated induction of ABCG2. Cell-based reporter assays revealed that deletion of AhRE5 and 4 dramatically attenuated 3MC-induced activation of ABCG2 reporter activity, while further deletion of the proximal AhRE3 and 2 only moderately changed the luciferase activities. Notably, site-directed mutation of the AhRE5 in the BCRP-3.8kb reporter construct alone resulted in approximately 80% decrease in 3MC activation of the ABCG2 promoter; additional mutation of the AhRE4 site had negligible effect on the ABCG2 promoter activity. Moreover, chromatin immunoprecipitation assays demonstrated that treatment with 3MC significantly enhanced the recruitment of AhR to the AhRE5 occupied region, and mutation of the AhRE5 site clearly dissociated AhR protein from this promoter region. Together, these data show that the novel distal AhRE5 is critical for AhR-mediated transcriptional activation of ABCG2 gene expression in LS174T cells, and it may offer new strategies for early identification of ABCG2 inducers, which would be of benefit for preventing transporter-associated drug-drug interactions., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Amino acids activate nutrient signaling via the mammalian target of rapamycin (mTOR), we therefore evaluated the relationship between amino acid transporter gene expression and proliferation in human ovarian cancer cell lines. Expression of three cancer-associated amino acid transporter genes, LAT1, ASCT2 and SN2, was measured by qRT-PCR and Western blot. The effects of silencing the LAT1 gene and its inhibitor BCH on cell growth were evaluated by means of cell proliferation and colony formation assays. The system L amino acid transporter LAT1 was up-regulated in human ovarian cancer SKOV3, IGROV1, A2780, and OVCAR3 cells, compared to normal ovarian epithelial IOSE397 cells, whereas ASCT2 and SN2 were not. BCH reduced phosphorylation of p70S6K, a down-stream effector of mTOR, in SKOV3 and IGROV1 cells, and decreased their proliferation by 30% and 28%, respectively. Although proliferation of SKOV3 (S1) or IGROV1 (I10) cells was unaffected by LAT1-knockdown, plating efficiency in colony formation assays was significantly reduced in SKOV3(S1) and IGROV1(I10) cells to 21% and 52% of the respective plasmid transfected control cells, SKOV3(SC) and IGROV(IC), suggesting that LAT1 affects anchorage-independent cell proliferation. Finally, BCH caused 10.5- and 4.3-fold decrease in the IC(50) value of bestatin, an anti-proliferative aminopeptidase inhibitor, in IGROV1 and A2780 cells, respectively, suggesting that the combined therapy is synergistic. Our findings indicate that LAT1 expression is increased in human ovarian cancer cell lines; LAT1 may be a target for combination therapy with anti-proliferative aminopeptidase inhibitors to combat ovarian cancer., (Copyright 2010 Elsevier Inc. All rights reserved.)

Breast cancer resistance protein (BCRP/ABCG2) is a membrane-bound efflux transporter important in cellular detoxification and multidrug resistance. Some aryl hydrocarbon receptor (AHR) agonists were reported to induce BCRP expression in human colon carcinoma cells. However, a direct involvement of AHR transcriptional regulation remains unexplored. In this study, we show that BCRP induction by AHR ligands occurs in human intestinal, liver, and mammary carcinoma cells and in primary colonocytes and hepatocytes. Increased BCRP transporter activity consistent with gene induction was also evident in the Caco2 subclone C2bbe1 cells. Using RNA interference and ectopic expression techniques to manipulate cellular AHR status, we confirmed AHR dependence of ABCG2 gene regulation. By gene promoter analysis, chromatin immunoprecipitation, and electrophoretic mobility shift assays, an active, proximal dioxin-response element at -194/-190 base pairs upstream of the transcription start site of the human ABCG2 gene was identified. Despite a common observation in human-derived cells, our in vitro and in vivo studies supported by phylogenetic footprinting analysis did not find that mouse Abcg2 is subject to AHR regulation. We conclude that AHR is a direct transcriptional regulator of human BCRP and provide an unprecedented role of AHR in cellular adaptive response and cytoprotection by up-regulating an important ATP-binding cassette efflux transporter.

Plasma membrane transporter proteins play an important role in taking up nutrients into and effluxing xenobiotics out of cells to sustain cell survival. In the last decade, a number of studies have shown that these physiologically important transporters affect absorption, distribution, and excretion of major anticancer agents in clinical use. More importantly, many transporters have been reported to be differentially upregulated in cancer cells compared to normal tissues, suggesting that the differential expression of transporters in cancer cells may become good targets for enhancing drug delivery as well as diagnostic markers for cancer therapy. Hence, utilizing the knowledge of transporter functions likely provides us with the possibility of delivering a drug to the target tissues, avoiding distribution to other tissues, and improving oral bioavailability. This chapter focuses on methodology to analyze the activity of transporters that are involved in drug transport.

Breast cancer resistance protein (BCRP/ABCG2) was discovered in multidrug resistant breast cancer cells having an ATP-dependent transport-based resistance phenotype. This ABC transporter functions (at least in part) as a xenobiotic protective mechanism for the organism: in the gut and biliary tract, it prevents absorption and enhances elimination of potentially toxic substances. As a placental barrier, it protects the fetus; similarly, it serves as a component of blood-brain and blood-testis barrier; BCRP is expressed in stem cells and may protect them from potentially harmful agents. Therefore, BCRP could influence cancer outcomes by (a) endogenous BCRP affecting the absorption, distribution, metabolism, and elimination of anticancer drugs; (b) BCRP expression in cancer cells may directly cause resistance by active efflux of anticancer drugs; (c) BCRP expression in cancer cells could be a manifestation of the activity of metabolic and signaling pathways that impart multiple mechanisms of drug resistance, self-renewal (stemness), and invasiveness (aggressiveness)--i.e. impart a poor prognosis--to cancers. This chapter presents a synopsis of translational clinical studies relating BCRP expression in leukemias, lymphomas, and a variety of solid tumors with clinical outcome. Data are emerging that expression of BCRP, like P-glycoprotein/ABCB1, is associated with adverse outcomes in a variety of human cancers. Whether this adverse prognostic effect results from resistance imparted to the cancer cells as the direct result of BCRP efflux of anticancer drugs, or whether BCRP expression (and also Pgp expression - coexpression of these transporters is common among poor risk cancers) serves as indicators of the activity of signaling pathways that enhance cancer cellular proliferation, metastases, genomic instability, enhance drug resistance, and oppose programmed cell death mechanisms is yet unknown.

The purpose of this study was to examine whether histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA; Zolinza/vorinostat) could sensitize tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant breast carcinoma in vivo. BALB/c nude mice were orthotopically implanted with TRAIL-resistant MDA-MB-468 cells and treated i.v. with SAHA, TRAIL, or SAHA followed by TRAIL for four times during first 3 weeks. The effects of drugs on tumor growth and markers of apoptosis, metastasis, and angiogenesis were examined. SAHA sensitized TRAIL-resistant xenografts to undergo apoptosis through multiple mechanisms. Whereas TRAIL alone was ineffective, SAHA inhibited growth of MDA-MB-468 xenografts in nude mice by inhibiting markers of tumor cell proliferation, angiogenesis, and metastasis and inducing cell cycle arrest and apoptosis. The sequential treatment of nude mice with SAHA followed by TRAIL was more effective in inhibiting tumor growth, angiogenesis, and metastasis and inducing apoptosis than SAHA alone, without overt toxicity. Treatment of nude mice with SAHA resulted in down-regulation of nuclear factor-kappaB and its gene products (cyclin D1, Bcl-2, Bcl-X(L), vascular endothelial growth factor, hypoxia-inducible factor-1alpha, interleukin-6, interleukin-8, matrix metalloproteinase-2, and matrix metalloproteinase-9) and up-regulation of DR4, DR5, Bak, Bax, Bim, Noxa, PUMA, p21(CIP1), tissue inhibitor of metalloproteinase-1, and tissue inhibitor of metalloproteinase-2 in tumor cells. Furthermore, control mice showing increased rate of tumor growth had increased numbers of CD31(+) or von Willebrand factor-positive blood vessels and increased circulating vascular endothelial growth factor receptor 2-positive endothelial cells compared with SAHA-treated or SAHA plus TRAIL-treated mice. In conclusion, sequential treatment with SAHA followed by TRAIL may target multiple pathways in tumor progression, angiogenesis, and metastasis and represents a novel therapeutic approach to treat breast cancer.

Purpose: Vorinostat [suberoylanilide hydroxamic acid (SAHA)] is a potent histone deacetylase inhibitor with promising clinical efficacy as an anticancer agent. In this preclinical study, we evaluated combining cytosine arabinoside [1-beta-D-arabinofuranosylcytosine (ara-C)] and/or etoposide with vorinostat for use in the treatment of acute leukemias., Experimental Design: Cell survival was examined in vitro in HL-60 human myeloid leukemia cells and K562 myeloid blast crisis chronic myelogenous leukemia cells, using the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt and/or fluorescein diacetate/propidium iodide assays. Drug interactions were analyzed by the combination index method (CalcuSyn) and by a novel statistical method that we developed (SynStat). Cell cycle phase distribution was measured by flow cytometry., Results: Cytotoxic antagonism resulted when vorinostat was combined concomitantly with ara-C; however, when vorinostat was given first followed by a drug-free interval before ara-C treatment, this sequential combination was mostly synergistic. Etoposide combined with vorinostat was additive to synergistic, and the synergism became more pronounced when etoposide was given after vorinostat. Cell cycle analyses revealed that the sequence-dependent interaction of vorinostat and ara-C or etoposide reflected the arrest of cells in G1 or G2 phase during vorinostat treatment and recovery into S phase after removal of vorinostat., Conclusions: These findings using two independent methods to assess drug combination effects provide a preclinical rationale for phase I trials of the sequential combination of vorinostat followed by ara-C and etoposide in patients with advanced or refractory leukemias. CalcuSyn findings were concordant with those of SynStat, validating the use of the latter in analyzing drug interactions.

Drug combination is a major treatment approach in cancer and antiviral therapies. A key issue is to find which combinations are additive, synergistic or antagonistic. In this paper, we develop statistical methods for experimental design and data analysis of combination studies of drugs that have log-linear dose-response curves. This class of dose response includes the Hill, sigmoid and simple exponential models. The experimental design (dose-finding and sample size determination) is derived by means of uniform measures that maximize the minimum power of the F-test to detect any departures from additive action and at the same time minimizes the maximum bias due to lack of fit among all potential departures of a given meaningful magnitude. Furthermore, we propose a model-free interaction index surface to capture the interaction of two drugs. The nonparametric function of the interaction index is estimated using the technique developed in thin plate splines. These methods are applicable to both in vivo and in vitro experiments. A study of two anticancer drugs, suberoylanilide hydroxamic acid (Vorinostat) and Etoposide applied sequentially against the cell line HL-60, is given to illustrate the proposed methods of experimental design and interaction analysis.

Breast cancer resistance protein (BCRP) plays a significant role in drug disposition and in conferring multidrug resistance in cancer cells. Previous studies have shown that steroid hormones such as 17beta-estradiol and progesterone can affect BCRP expression in cancer cells. In this study, we investigated the molecular mechanism by which BCRP expression in human placental choriocarcinoma BeWo cells is regulated by progesterone. Transfection of the progesterone receptor (PR) isoforms PRA and PRB resulted in a similarly increased expression of PRA and PRB, respectively. However, progesterone significantly increased BCRP expression and activity only in PRB-transfected cells. This stimulatory effect of progesterone was abrogated by the PR antagonist mifepristone (RU-486). Consistently, transcriptional activity of the BCRP promoter was induced 2- to 6-fold by 10(-8) to 10(-5) M progesterone in PRB-transfected cells. Progesterone had little effect on BCRP expression and activity and transcriptional activity of the BCRP promoter in PRA-transfected cells; however, cotransfection of PRA and PRB significantly decreased the progesterone-response compared with that in cells transfected with only PRB. Mutations in a novel progesterone response element (PRE) identified between -243 to -115 bp of the BCRP promoter region significantly attenuated the progesterone-response in PRB-transfected cells, and deletion of the PRE nearly completely abrogated the progesterone effect. Specific binding of both PRA and PRB to the BCRP promoter through the identified PRE was confirmed using the electrophoretic mobility shift assay. Collectively, progesterone induces BCRP expression in BeWo cells via PRB but not PRA. PRA represses the PRB activity. Thus, PRA and PRB differentially regulate BCRP expression in BeWo cells.

We previously showed that the 44-kDa serine/threonine kinase Pim-1 (Pim-1L) can protect prostate cancer cells from apoptosis induced by chemotherapeutic drugs (Xie, Y., Xu, K., Dai, B., Guo, Z., Jiang, T., Chen, H., and Qiu, Y. (2006) Oncogene 25, 70-78). To further explore the mechanisms of Pim-1L-mediated resistance to chemotherapeutic drugs in prostate cancer cells, we employed a yeast two-hybrid screening to identify cellular proteins that were associated with Pim-1L, and we found the ABC transporter BCRP/ABCG2 as one of the potential interacting partners of Pim-1L. We also showed that the expression level of Pim-1L and BCRP was up-regulated in mitoxantrone and docetaxel-resistant prostate cancer cell lines. Pim-1L was co-localized with BCRP on the plasma membrane and induced phosphorylation of BCRP at threonine 362. Knocking-down Pim-1L expression in the drug-resistant prostate cancer cells abolished multimer formation of endogenous BCRP and resensitized the resistant cells to chemotherapeutic drugs suggesting that BCRP phosphorylation induced by Pim-1L was essential for its functionality. This is further corroborated by our finding that the plasma membrane localization and drug-resistant activity of BCRP were compromised by T362A mutation. Our data suggest that Pim-1L may protect prostate cancer cells from apoptosis, at least in part, through regulation of transmembrane drug efflux pump. These findings may provide a potential therapeutic approach by disrupting Pim-1 signaling to reverse BCRP-mediated multidrug resistance.

Cells with increasing resistance to the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin (TG), ranging from 60-fold (PC3/TG(10) cells) to 1350-fold (PC3/TG(2000) cells), were derived from PC3 cells. SERCA2 is overexpressed in all PC3/TG cells but retains sensitivity to TG. siRNA-mediated downregulation of SERCA completely or partially reverses TG resistance in PC3/TG(10) or PC3/TG(2000) cells, respectively; thus SERCA overexpression mediates resistance in PC3/TG(10) cells but is not the only resistance mechanism in PC3/TG(2000) cells. By contrast, SERCA is not overexpressed in TG-resistant DU145/TG cells derived from DU145 cells. DU145/TG cells retain resistance while in PC3/TG cells resistance decreases upon removal of TG selection. The transport proteins PGP/BCRP/MRP1 and anti-apoptotic proteins Bcl2/Bcl(XL) are not involved in mediating resistance in either cell line. PARP and caspase 3 cleavage in response to other drugs demonstrate that the apoptotic pathways tested remain intact in these cells. Further, no cross-resistance occurs to other drugs. Thus, novel TG-specific resistance mechanisms are recruited by these cancer cells.

Purpose: Flavopiridol is known to modulate the transcription of genes. We investigated the effect of flavopiridol pretreatment on TRAIL cytotoxicity and on the expression of FLIP(L) in different TRAIL-resistant cell lines, because FLIP expression is known to confer TRAIL-resistance., Methods: Apoptosis was assessed by PI staining and protein expression by Western blotting. RT-PCR was used for mRNA quantitation. siRNA gene silencing was used to knock down FLIP(L)., Results: Flavopiridol pretreatment synergized TRAIL-induced apoptosis in human myeloma and breast cancer cells. Flavopiridol treatment repressed the transcription of FLIP(L) and downregulated its expression in both myeloma and breast cancer cells. Silencing of FLIP(L) gene by siRNA sensitized myeloma cells to TRAIL. Flavopiridol treatment downregulated the expression of the proapoptotic members of the Bcl-2 family proteins (Bak, Bax and PUMA-alpha). The expression of the antiapoptotic Bcl-2 members (Bcl-2 and Bcl-X(L)) was not altered by flavopiridol treatment in myeloma cells., Conclusion: Our data indicate that flavopiridol synergizes TRAIL cytotoxicity by downregulation of FLIP(L) and this synergistic effect is Bcl-2 family independent.

Purpose: Several studies have demonstrated significant interactions between immunosuppressants (e.g., cyclosporin A) and chemotherapeutic drugs that are BCRP substrates (e.g., irinotecan), resulting in increased bioavailability and reduced clearance of these agents. One possible mechanism underlying this observation is that the immunosuppressants modulate the pharmacokinetics of these drugs by inhibiting BCRP. Therefore, the aim of this study was to determine whether the immunosuppressants cyclosporin A, tacrolimus and sirolimus are inhibitors and/or substrates of BCRP., Methods: First, the effect of the immunosuppressants on BCRP efflux activity in BCRP-expressing HEK cells was measured by flow cytometry., Results: Cyclosporin A, tacrolimus and sirolimus significantly inhibited BCRP-mediated efflux of pheophorbide A, mitoxantrone and BODIPY-prazosin. The EC(50) values of cyclosporin A, tacrolimus and sirolimus for inhibition of BCRP-mediated pheophorbide A efflux were 4.3 +/- 1.9 microM, 3.6 +/- 1.8 microM and 1.9 +/- 0.4 microM, respectively. Cyclosporin A, tacrolimus and sirolimus also effectively reversed resistance of HEK cells to topotecan and mitoxantrone conferred by BCRP. When direct efflux of cyclosporin A, tacrolimus and sirolimus was measured, these compounds were found not to be transported by BCRP. Consistent with this finding, BCRP did not confer resistance to the immunosuppressants in HEK cells., Conclusion: These results indicate that cyclosporin A, tacrolimus and sirolimus are effective inhibitors but not substrates of BCRP. These findings could explain the altered pharmacokinetics of BCRP substrate drugs when co-administered with the immunosuppressants and suggest that pharmacokinetic modulation by the immunosuppressants may improve the therapeutic outcome of these drugs.

Imatinib, a potent tyrosine kinase inhibitor, is effluxed from cells by the breast cancer resistance protein (BCRP/ABCG2), yet published studies to date fail to demonstrate resistance to imatinib cytotoxicity in BCRP-overexpressing cells in vitro. We investigated cellular resistance to imatinib in BCR-ABL-expressing cells transduced and selected to overexpress BCRP (K562/BCRP-MX10). These cells exhibited a 2- to 3-fold increase in resistance to imatinib (P < .05) and a 7- to 12-fold increase in resistance to mitoxantrone, a known BCRP substrate. Resistance to imatinib was completely abolished by the specific BCRP inhibitor fumitremorgin C. Studies of the mechanism of the diminished resistance to imatinib compared with mitoxantrone revealed that imatinib decreased the expression of BCRP in K562/BCRP-MX10 cells without affecting mRNA levels. BCRP levels in cells that do not express BCR-ABL were not affected by imatinib. Loss of BCRP expression was accompanied by imatinib-induced reduction of phosphorylated Akt in the BCRP-expressing K562 cells. The phosphoinositol-3 kinase (PI3K) inhibitor LY294002 also decreased BCRP levels in K562/BCRP-MX10 cells. These studies show that BCRP causes measurable imatinib resistance, but this effect is attenuated by imatinib-mediated inhibition of BCR-ABL, which in turn downregulates overall BCRP levels posttranscriptionally via the PI3K-Akt pathway.

To investigate transcriptional activation of the breast cancer resistance protein gene (BCRP/ABCG2), we examined the 5' untranslated region of BCRP mRNA in cell lines with high BCRP transcriptional activity and in normal tissues. Human choriocarcinoma cells with high endogenous BCRP expression (JAR and BeWo) and human cancer cells (MCF-7 and Igrov1) and their BCRP-overexpressing, drug-selected, multidrug-resistant derivatives (MCF-7/AdrVp, Igrov1/MX3, and Igrov1/T8) were studied. Rapid amplification of 5'-cDNA ends-PCR (5'RACE-PCR) revealed at least three novel forms of the untranslated exon 1 (E1a, E1b, and E1c) that are spliced to a common exon 2, with differential expression of these splice variants in the drug-selected cell lines. Additionally, sequence analysis of the 5'RACE-PCR products revealed multiple transcriptional start sites for each variant, particularly in the drug-selected cells. The E1c isoform predominated in drug-selected MCF-7 cell lines and was translated more efficiently in MCF-7 cells than the E1a isoform. Varying patterns of expression of the exon 1 isoforms were observed in a variety of human tissues, suggesting that tissue-specific alternative promoters of BCRP exist. In summary, we find that BCRP overexpression in the drug-selected cells is accompanied by multiple transcriptional start sites and predominance of the more efficiently translated E1c isoform. The exon 1 variation we observe suggests that alternative promoters of the BCRP gene exist.

Purpose: The serine/threonine kinase inhibitor flavopiridol targets multiple cyclin-dependent kinases, induces checkpoint arrest, and interrupts transcriptional elongation. We designed a phase I clinical trial using a timed sequential therapy approach where flavopiridol was given for the dual purpose of initial cytoreduction and enhancing cell cycle progression of the remaining leukemia cell cohort followed by cycle-dependent drugs 1-beta-D-arabinofuranosylcytosine (ara-C) and mitoxantrone., Experimental Design: Flavopiridol was given by 1-hour infusion daily for 3 days beginning day 1 followed by 2 g/m2/72 h ara-C beginning day 6 and 40 mg/m2 mitoxantrone beginning day 9. In vivo correlates included pharmacokinetics, modulation of blast cycle regulators, and serum and marrow supernatant vascular endothelial growth factor levels., Results: Of 34 adults receiving induction therapy, 16 (47%) evinced direct leukemia cytotoxicity with > or =50% drop in peripheral blast counts and tumor lysis in 9 (26%). Four (12%) died during therapy (two fungal infections and two sudden death). Dose-limiting toxicity occurred at 60 mg/m2/d with profound neutropenia >40 days duration, and maximal tolerated dose was 50 mg/m2/d. Overall response rate was 31% in 26 acute myelogenous leukemia and 12.5% in acute lymphoblastic leukemia. Pharmacokinetics showed that a linear two-compartment model with first-order elimination provided the best fit of the observed concentration versus time data. Flavopiridol down-regulated one or more target proteins in marrow blasts in vivo. Vascular endothelial growth factor was detected in sera and marrow supernatant pretreatment, and sera obtained on day 3 inhibited bovine aortic endothelial cell proliferation by a mean of 32% (range, 10-80%)., Conclusions: Our data suggest that flavopiridol is cytotoxic to leukemic cells and, when followed by ara-C and mitoxantrone, exerts biological and clinical effects in patients with relapsed and refractory acute leukemias. These findings warrant continuing development of flavopiridol at 50 mg/m2/d x 3 days in combination with cytotoxic and biological agents for acute leukemias.

In the present study, we aimed to elucidate the mechanism responsible for the interactive effects of histone deacetylase (HDAC) inhibitors [suberoylanilide hydroxamic acid (SAHA), MS-275, m-carboxycinnamic acid bishydroxamide (CBHA), and trichostatin-A (TSA)] and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on apoptosis in leukemia cells. HDAC inhibitors enhance the apoptosis-inducing potential of TRAIL in leukemia cells (HL60, Jurkat, K562, and U937) through multiple mechanisms; up-regulation of DR4, DR5, Bak, Bax, Bim, Noxa and PUMA, down-regulation of IAPs, Mcl-1, Bcl-2, Bcl-XL and cFLIP, release of mitochondrial proteins (cytochrome c, Smac/DIABLO and Omi/Htr2) to the cytosol, induction of p21WAF1/CIP1 and p27KIP1, activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP). The sequential treatment of cells with HDAC inhibitors followed by TRAIL was more effective in inducing apoptosis than the concurrent treatment or single agent alone. The up-regulation of death receptors and inhibition of cFLIP by HDAC inhibitors will increase the ability of TRAIL to induce apoptosis, due to enhance activation of caspase-8, cleavage of Bid, and release of mitochondrial proteins to the cytosol, and subsequent activation of caspase-9 and caspase-3. Thus, the combination of HDAC inhibitors and TRAIL can be used as a new therapeutic approach for the treatment of leukemia.

Background: In response to the nationwide need for improved care of patients at end of life, our medical school implemented approximately 20 hours of mandatory coursework on the care of dying patients for all students, with satisfactory completion required for graduation., Objective: We now report a long-term evaluation of this coursework. DESIGN/SUBJECTS/MEASUREMENTS: A 74-item questionnaire concerning attainment of palliative care and other medical school course objectives, postgraduate practice encounters with patients at end of life, and postgraduate behaviors in palliative care was mailed to all students who graduated from our medical school in 2000, 2001, and 2002. Responses were graded by a Likert-type scale. Additionally, the Association of American Medical Colleges Medical School Graduation Questionnaire (AAMC/GQ) reports for all schools and for our medical school were queried for data regarding palliative care., Results: The return rate of the questionnaire was 54%. The respondents perceived the training to be valuable and effective. They displayed good postgraduate palliative care practices such as choosing to use opiates other than meperidine for pain in the end-of-life setting. Fifty-three percent of the respondents reported that dying patients were often or frequently under their care since graduation. Despite evidence for the efficacy of the training, the respondents perceived that preparedness in certain palliative care domains was somewhat below preparedness in benchmark medical school competencies such as assessment/management of hypertension and diabetes. The AAMC/GQ surveys indicated that for the years 2000-2002, at least 20% more of our graduates perceived that their training in pain management and palliative care was adequate than did all other medical students graduating nationwide., Conclusions: The evaluation provides support for the conclusion that mandatory training in palliative and end-of-life care is effective, perceived to be valuable, and contributes to good palliative and end-of-life care practices in our graduates. Furthermore, the training meets a significant demand in our graduates' clinical practice: end-of-life care. However, expanded medical school emphasis and curriculum hours are still needed for palliative care topics, because preparedness in palliative care skills was perceived to be inferior to benchmark medical skills. To our knowledge, this is the first report of a rigorous summative evaluation of the efficacy of required coursework in palliative and end-of-life care in a U.S. medical school curriculum.

Down-regulation of the androgen receptor (AR) is being evaluated as an effective therapy for the advanced stages of prostate cancer. We report that Ebp1, a protein identified by its interactions with the ErbB3 receptor, down-regulates expression of AR and AR-regulated genes in the LNCaP prostate cancer cell line. Using microarray analysis, we identified six endogenous AR target genes, including the AR itself, that are down-regulated by ebp1 overexpression. Chromatin immunoprecipitation assays revealed that Ebp1 was recruited to the prostate-specific antigen gene promoter in response to the androgen antagonist bicalutamide, suggesting that Ebp1 directly affected the expression of AR-regulated genes in response to androgen antagonists. Ebp1 expression was reduced in cells that had become androgen-independent. Androgens failed to stimulate either the growth of ebp1 transfectants or transcription of AR-regulated reporter genes in these cells. The agonist activity of the antiandrogen cyproterone acetate was abolished in ebp1 transfectants. In severe combined immunodeficient mice, Ebp1 overexpression resulted in a reduced incidence of LNCaP tumors and slower tumor growth. These findings suggest that Ebp1 is a previously unrecognized therapeutic target for treatment of hormone refractory prostate cancer.

In this study, we have evaluated the cytotoxic effect of combining two HDAC inhibitors, SAHA and TSA, with TRAIL in human multiple myeloma cell lines. Low doses of SAHA or TSA enhanced the cytotoxic and apoptotic effects of TRAIL and upregulated the surface expression of TRAIL death receptors (DR4 and/or DR5). SAHA and TSA induced G1 phase cell cycle growth arrest by upregulating p21(WAF1) and p27(Kip1) expression and by inhibiting E2F transcriptional activity. The enhanced TRAIL effect after pretreatment with HDAC inhibitors was consistent with the upregulation of the proapoptotic Bcl-2 family members (Bim, Bak, Bax, Noxa, and PUMA), the downregulation of the anti-apoptotic members of the Bcl-2 family (Bcl-2 and Bcl-X(L)), and IAPs. SAHA and TSA dissipated the mitochondrial membrane potential and enhanced the release of Omi/HtrA2 and AIF from the mitochondria to the cytosol. The cytotoxic effect of both SAHA and TSA was caspase- and calpain-independent. Inhibition of NF(kappa)B activation by the proteasome inhibitor, MG132, enhanced the apoptotic effect of TSA. Our study demonstrated the enhancing effects of HDAC inhibitors on apoptosis when combined with TRAIL and, for the first time, emphasized the role of AIF in mediating the cytotoxic effects of HDAC inhibitors.

This study was conducted to define the maximum tolerated dose (MTD), dose limiting toxicity (DLT), and pharmacokinetics of idarubicin when administered with and without the P-glycoprotein inhibitor PSC-833 in combination with cytarabine, and etoposide. Fifteen patients with relapsed and refractory acute leukemia were enrolled and received cytarabine as a 7-day continuous infusion, with etoposide and idarubicin administered for any three consecutive days during the cytarabine infusion. Two hours prior to the second dose of idarubicin, PSC-833 administration was initiated. The pharmacokinetics of idarubicin alone and with PSC-833 was assessed at three idarubicin dose levels (6, 8 and 10 mg/m(2)). The MTD of idarubicin in this combination was 8 mg/(m(2) day) with a DLT of oral mucositis. The complete remission rate (on an intent-to-treat basis) for this regimen was 33%, with a median duration of 6 months. The clearance of idarubicin was 140 +/- 200 and 181 +/- 94.3 l/h for idarubicin alone and with PSC-833, respectively. The volume of distribution of the central compartment was 423 +/- 443 and 337 +/- 394 l for idarubicin alone and in combination with PSC-833, respectively. This combination including PSC-833 was well tolerated. Although a pharmacokinetic interaction might have been expected, PSC-833 did not significantly alter the disposition of idarubicin.

Overexpression of the breast cancer resistance protein (BCRP/ABCG2) confers multidrug resistance (MDR) to tumor cells and often limits the efficacy of chemotherapy. To circumvent BCRP-mediated MDR, a common approach is the use of potent and specific inhibitors of BCRP transport such as fumitremorgin C, novobiocin, and GF120918. Here, we evaluated a new approach using RNA interference for the specific knockdown of BCRP. We designed and synthesized small interfering RNA (siRNA) using T7 RNA polymerase and showed that siRNAs markedly down-regulated both exogenous and endogenous expression of BCRP. As a functional consequence, knockdown of BCRP by siRNAs increased the sensitivity of human choriocarcinoma BeWo cells to mitoxantrone and topotecan by 10.5- and 8.2-fold, respectively. Using flow cytometry, we found that introduction of siRNAs also enhanced the intracellular accumulation of topotecan. We have previously identified an estrogen response element in the BCRP promoter and have shown that 17beta-estradiol increased BCRP mRNA expression. Furthermore, in the present study, we found that expression of BCRP protein was inducible by 17beta-estradiol and that this effect was ameliorated by the introduction of siRNAs. These studies indicate that siRNAs could modulate MDR in vitro and may present a new approach to overcome BCRP-mediated drug resistance.

Resistance to chemotherapy is an obstacle to the successful treatment of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The failure of therapeutic treatment may be due to the development of multidrug resistance (MDR), mechanisms of which include upregulation of membrane-resident transporters which efflux chemotherapeutic drugs from tumor cells, and failure of the cancer cell to undergo apoptosis in response to chemotherapy. Membrane transporter-based drug efflux transporters have been extensively studied, and agents that block drug efflux have been found and investigated. Presence of P-glycoprotein (Pgp, MDR1, ABCB1), a member of the ATP-binding cassette (ABC) transporter family, has been reported to correlate with poor prognosis in AML and MDS. In MDS, Pgp expression increases as the disease progresses. Overexpression of other transporters, such as the multidrug resistance protein (MRP1, ABCC1), and the vault-associated transporter lung resistance protein have been shown as well in both MDS and AML, but their prognostic relevance is not clear. Recently, a novel ABC half-transporter, the breast cancer resistance protein (ABCG2) has been found in approximately 30% of AML cases, and may play a role in resistance to chemotherapy. In clinical trials in MDS, first-generation Pgp blockers, such as cyclosporin-A and verapamil, were minimally effective, non-specific, and toxic. However, another first-generation blocker, quinine, was used in MDS and may specifically benefit MDS patients overexpressing Pgp. A second-generation drug, the non-immunosuppressive cyclosporine analog valspodar (PSC833), was studied in AML and MDS, and was highly toxic, resulting in the need to reduce the dosage of the chemotherapeutic drugs as a result of valspodar reducing the clearance of the chemotherapeutic agents. Third-generation drugs, which are highly specific for Pgp and which seem to have only modest effects on drug clearance, include tariquidar, zosuquidar, laniquidar, and ONT-093. These are all in phase I/II trials and show promise for future treatment.

Drugs used in treatment of adult acute lymphoblastic leukaemia (ALL) are substrates for breast cancer resistance protein (BCRP, MXR, ABCG2), which may thus play a role in resistance in this disease. Pretreatment blasts from 30 adult ALL patients were studied for BCRP mRNA by quantitative reverse transcription polymerase chain reaction analysis, BCRP protein by immunophenotyping with three antibodies and BCRP function by fumitremorgin C modulation of intracellular mitoxantrone retention, measured by flow cytometry. BCRP mRNA in all cases encoded wild type protein (BCRP(R482)), which mediates mitoxantrone and methotrexate resistance, but only low-level anthracycline resistance. The BXP-21, BXP-34 and anti-ABCG2 antibodies stained blasts in 13, 11 and 14 cases (43%, 37% and 47%); BXP-21 correlated well with BXP-34 and anti-ABCG2, but BXP-34 and anti-ABCG2 did not correlate, and antibody staining did not correlate with mRNA levels. BCRP function was seen in 21 cases (70%), but correlated poorly with antibody staining. An exploratory statistical analysis indicated that BXP-21 staining was predictive of shorter disease-free survival (DFS) (P = 0.0374) in this small patient population. Poor correlations between mRNA, protein and function indicate the complex biology of BCRP in adult ALL, and the possible correlation of BCRP expression with DFS should be studied in larger series.

Fumitremorgin C (FTC) was recently discovered to be a potent and selective inhibitor of the breast cancer resistance protein (BCRP/ABCG2). FTC was shown to reverse multidrug resistance mediated by BCRP and to increase the cytotoxicity of several anticancer agents in vitro. To support in vivo studies a reverse phase HPLC method with ultraviolet detection was developed to quantitate FTC in mouse plasma and tissues. Further, assay method validation was performed for the determination of FTC in mouse plasma. Plasma standard curves ranged from 0.03 to 30 microg/ml, while the various tissue assay ranges differed to some extent. The sample preparation consisted of acetonitrile precipitation with separation accomplished with a C18 Novapak column and a C18 pre-column utilizing an isocratic mobile phase of ammonium acetate and acetonitrile. UV detection was set at 225 nm for FTC and at 312 nm for roquefortine, the internal standard. The retention times were approximately 9.5 min for FTC and 13.0 min for roquefortine. The recoveries for FTC and roquefortine from plasma were 90.8+/-5.8% and 111.6+/-13.6, respectively. The reported assay can be used for future study of BCRP resistance in vivo in different biological matrices. Further, we found that a more potent analogue of FTC, Ko143, was able to be extracted and detected, with a maximal UV absorbance at 320 nm under the conditions reported.

The association of transporter proteins and cancer drug resistance has been known for approximately 25 years, with recent discoveries pointing to an ever-increasing number of ATP binding cassette (ABC) transporter proteins involved with the response of cancer cells to pharmacotherapy. As reported in this issue of Cancer Cell, Szakács et al. couple quantitative, real-time PCR assays for all 48 human ABC transporters with chemosensitivity information mined from the NCI-60 cancer cell line database. Predictions of transporter involvement in drug effect were validated in selected cases, and furthermore produced novel leads relating ABC transporter expression and chemoresistance or chemosensitivity.

Our studies demonstrate that the ABC transporter and marker of stem and progenitor cells known as the breast cancer resistance protein (BCRP or ABCG2) confers a strong survival advantage under hypoxic conditions. We show that, under hypoxia, progenitor cells from Bcrp(-)/(-)mice have a reduced ability to form colonies as compared with progenitor cells from Bcrp(+/+) mice. Blocking BCRP function in Bcrp(+/+) progenitor cells markedly reduces survival under hypoxic conditions. However, blocking heme biosynthesis reverses the hypoxic susceptibility of Bcrp(-/-) progenitor cells, a finding that indicates that heme molecules (i.e. porphyrins) are detrimental to Bcrp(-/-) cells under hypoxia. BCRP specifically binds heme, and cells lacking BCRP accumulate porphyrins. Finally, Bcrp expression is up-regulated by hypoxia, and we demonstrate that this up-regulation involves the hypoxia-inducible transcription factor complex HIF-1. Collectively, our findings suggest that cells can, upon hypoxic demand, use BCRP to reduce heme or porphyrin accumulation, which can be detrimental to cells. Our findings have implications for the survival of stem cells and tumor cells in hypoxic environments.

Background: The need for better care for terminally ill patients led us to create an educational program to provide internal medicine residents and medical oncology fellows basic competency in palliative and end-of-life care., Methods: An interdisciplinary team identified educational strategies, course objectives, content, and evaluation instruments., Results and Conclusions: Our strategy is to use a required Web-based course to establish a knowledge base upon which specific training during clinical rotations build skills. Field testing of the Web course showed it was an effective tool for delivering clinically applicable content. Skill building experiences are now being integrated into selected clinical rotations.

The breast cancer resistance protein (BCRP) is an ATP-binding cassette half transporter that confers resistance to anticancer drugs such as mitoxantrone, anthracyclines, topotecan, and SN-38. Initial characterization of the BCRP promoter revealed that it is TATA-less with 5 putative Sp1 sites downstream from a putative CpG island and several AP1 sites (K. J. Bailey-Dell et al., Biochim. Biophys. Acta, 1520: 234-241, 2001). Here, we examined the sequence of the 5'-flanking region of the BCRP gene and found a putative estrogen response element (ERE). We showed that estrogen enhanced the expression of BCRP mRNA in the estrogen receptor (ER)-positive T47D:A18 cells and PA-1 cells stably expressing ERalpha. In BCRP promoter-luciferase assays, sequential deletions of the BCRP promoter showed that the region between -243 and -115 is essential for the ER effect. Mutation of the ERE found within this region attenuated the estrogen response, whereas deletion of the site completely abrogated the estrogen effect. Furthermore, electrophoretic mobility shift assays revealed specific binding of ERalpha to the BCRP promoter through the identified ERE. Taken together, we provide evidence herein for a novel ERE in the BCRP promoter.

Breast cancer resistance protein (BCRP/ABCG2) of an ATP-binding cassette half-transporter confers resistance against mitoxantrone and camptothecin derivatives of topotecan and irinotecan. Novobiocin, a coumermycin antibiotic, is known to enhance anticancer drug sensitivity of cancer cells in vitro and in vivo, the mechanism of which remains undetermined. Here we focused on drug efflux pump and examined whether novobiocin reversed drug resistance in multidrug-resistant cells highly expressing BCRP. To explore the reversal mechanisms, intracellular drug accumulation was measured by flow cytometry, and a topotecan transport study using plasma membrane vesicles was performed. We used PC-6/SN2-5H2 small cell lung cancer and MCF-7/MX breast cancer cells selected with SN-38 of the active irinotecan metabolite and mitoxantrone, respectively, and the BCRP cDNA transfectant MCF-7/clone 8 cells. These cells expressed high levels of BCRP mRNA but not other known transporters. Compared to the parental PC-6 cells, PC-6/SN2-5H2 cells were 141-, 173- and 57.2-fold resistant to topotecan, SN-38 and mitoxantrone, respectively. Novobiocin at 60 microM decreased the degree of the above resistance by approximately 26-fold in PC-6/SN2-5H2 cells, and similarly reversed resistance in MCF-7/MX, MCF-7/clone 8 and un-selected NCI-H460 cells highly expressing BCRP. Furthermore, novobiocin increased the intracellular topotecan accumulation in these cells and inhibited the topotecan transport into the membrane vesicles of PC-6/SN2-5H2 cells. No effects of novobiocin in these assay were observed in the parental PC-6 and MCF-7 cells. The kinetic parameters in the transport study indicated that novobiocin was a inhibitor for BCRP, resulting in competitive inhibition of BCRP-mediated topotecan transport. These findings suggest that novobiocin effectively overcomes BCRP-mediated drug resistance at acceptable concentrations., (Copyright 2004 Wiley-Liss, Inc.)

To evaluate the function and substrate specificity of human breast cancer resistance protein (BCRP, ABCG2) in the absence of cofactors or heterologous partner proteins, Xenopus laevis oocytes were injected with cRNA of wild-type or mutant (R482T) BCRP. High expression of BCRP was observed on the oocyte surface. Accumulation and efflux assays revealed that oocytes expressing R482T transported daunorubicin (DNR), mitoxantrone (MX), rhodamine 123, and flavopiridol (FLV), whereas wild-type BCRP transported only MX and FLV, in agreement with observations in mammalian and other systems. Transport activity was completely inhibited by fumitremorgin C, a known inhibitor of BCRP. Injection of oocytes with cRNA containing mutations of serine 187 in the ATP-binding cassette signature motif (S187T or S187A) resulted in strong expression of the mutant forms; however, these oocytes were devoid of transporter activity. When oocytes were coinjected with R482T and R482T/S187T, DNR transport was inhibited in a manner dependent on the amount of R482T/S187T cRNA added, consistent with the idea that the active form of BCRP is a homodimer or homomultimer. Substrate interaction studies found that no two substrates reciprocally inhibited the efflux of the other. Although FLV proved to be an effective inhibitor of both MX and DNR transport, and MX inhibited DNR transport, the other substrates tested had only weak or no inhibitory activity, indicating a complex nature of substrate interaction with the BCRP homodimer. We conclude that the X. laevis oocyte heterologous expression system is a valid and effective means of studying BCRP function and substrate specificity.

Observations of functional adenosine triphosphate (ATP)-dependent drug efflux in certain multidrug-resistant cancer cell lines without overexpression of P-glycoprotein or multidrug resistance protein (MRP) family members suggested the existence of another ATP-binding cassette (ABC) transporter capable of causing cancer drug resistance. In one such cell line (MCF-7/AdrVp), the overexpression of a novel member of the G subfamily of ABC transporters was found. The new transporter was termed the breast cancer resistance protein (BCRP), because of its identification in MCF-7 human breast carcinoma cells. BCRP is a 655 amino-acid polypeptide, formally designated as ABCG2. Like all members of the ABC G (white) subfamily, BCRP is a half transporter. Transfection and enforced overexpression of BCRP in drug-sensitive MCF-7 or MDA-MB-231 cells recapitulates the drug-resistance phenotype of MCF-7/AdrVp cells, consistent with current evidence suggesting that functional BCRP is a homodimer. BCRP maps to chromosome 4q22, downstream from a TATA-less promoter. The spectrum of anticancer drugs effluxed by BCRP includes mitoxantrone, camptothecin-derived and indolocarbazole topoisomerase I inhibitors, methotrexate, flavopiridol, and quinazoline ErbB1 inhibitors. Transport of anthracyclines is variable and appears to depend on the presence of a BCRP mutation at codon 482. Potent and specific inhibitors of BCRP are now being developed, opening the door to clinical applications of BCRP inhibition. Owing to tissue localization in the placenta, bile canaliculi, colon, small bowel, and brain microvessel endothelium, BCRP may play a role in protecting the organism from potentially harmful xenobiotics. BCRP expression has also been demonstrated in pluripotential "side population" stem cells, responsible for the characteristic ability of these cells to exclude Hoechst 33342 dye, and possibly for the maintenance of the stem cell phenotype. Studies are emerging on the role of BCRP expression in drug resistance in clinical cancers. More prospective studies are needed, preferably combining BCRP protein or mRNA quantification with functional assays, in order to determine the contribution of BCRP to drug resistance in human cancers.

The methylthioadenosine phosphorylase (MTAP) gene gained considerable interest as therapeutic target for tumors with the 9p21 deletion. This gene maps to 9p21 and loss of this chromosomal region in tumors offers an unique opportunity for chemoselective treatment, since MTAP is an important salvage enzyme for the formation of adenine that is needed for DNA synthesis. L-Alanosine, an antibiotic from Streptomyces alanosinicus, blocks the common de novo purine biosynthesis pathway and, thereby, inhibits tumor cells with MTAP deficiency. Normal cells escape the detrimental effects of L-alanosine due to their proficiency in the MTAP salvage pathway. The present analysis was undertaken to gain insights into the molecular architecture of tumor cells that determines the response to L-alanosine apart from the MTAP gene. Analysis of cell doubling times and IC(50) values for L-alanosine showed that slowly growing cell lines were more resistant to L-alanosine than rapidly growing ones. Mining the database of the National Cancer Institute (N.C.I.), for the mRNA expression of 9706 genes in 60 cell lines by means of Kendall's tau-test, false discovery rate calculation, and hierarchical cluster analysis pointed to 11 genes or expressed sequence tags whose mRNA expression correlated with the IC(50) values for L-alanosine. Furthermore, we tested L-alanosine for cross-resistance in multidrug-resistant cell lines which overexpress selectively either the P-glycoprotein/MDR1 (CEM/ADR5000), MRP1 (HL-60/AR), or BCRP (MDA-MB-231-BCRP) genes. None of the multidrug-resistant cell lines was cross-resistant to L-alanosine indicating that L-alanosine may be suitable to treat multidrug-resistant, refractory tumors in the clinic. Finally, the IC(50) values for L-alanosine of the 60 cell lines were correlated to the p53 mutational status and expression of p53 downstream genes. We found that p53 mutated cell lines were more resistant to L-alanosine than p53 wild type cell lines.

Purpose: Flavopiridol is a cyclin-dependent kinase inhibitor currently undergoing human clinical trials. As clinical development is pursued, it becomes important to evaluate resistance mechanisms to flavopiridol. To elucidate the contribution of breast cancer resistance protein (BCRP) to cellular resistance to flavopiridol in acute myeloid leukemia, we studied the relationship between cellular resistance to flavopiridol and mRNA expression of BCRP or P-glycoprotein (P-gp, product of MDR1gene) in blast cells from adult patients with acute leukemia., Experimental Design: Twenty-one blast cell samples from 20 patients were studied. The expression of BCRP, P-gp, or beta-actin mRNA was determined by real-time reverse transcription-PCR, using fluorescent hybridization probes to evaluate codon 482, a known site of mutations in BCRP mRNA. In vitro cell viability and apoptosis were examined after 24 h exposure to flavopiridol., Results: BCRP mRNA expression varied over a 200-fold range. In the blast cell samples with BCRP mRNA expression > 10000 copies/pg beta-actin (n = 9), BCRP mRNA correlated proportionally with cell viability in the presence of 250 nM flavopiridol (r = 0.86, P = 0.003) and with apoptosis induced by flavopiridol (r = 0.71, P = 0.031). In contrast, MDR1mRNA expression did not correlate with either flavopiridol cytotoxicity or induction of apoptosis. Melting point analysis of the hybridization probes determined that all 21 patient samples had arginine at codon 482 of BCRP mRNA, the wild-type form., Conclusions: These results suggest that unlike P-gp, BCRP may play a role in leukemia cellular resistance to flavopiridol. No mutations at codon 482 were observed in BCRP mRNA in this group of patients.

A profound cytotoxic action of the antimalarial, artesunate (ART), was identified against 55 cancer cell lines of the U.S. National Cancer Institute (NCI). The 50% inhibition concentrations (IC50 values) for ART correlated significantly to the cell doubling times (P = 0.00132) and the portion of cells in the G0/G1 (P = 0.02244) or S cell cycle phases (P = 0.03567). We selected mRNA expression data of 465 genes obtained by microarray hybridization from the NCI data base. These genes belong to different biological categories (drug resistance genes, DNA damage response and repair genes, oncogenes and tumor suppressor genes, apoptosis-regulating genes, proliferation-associated genes, and cytokines and cytokine-associated genes). The constitutive expression of 54 of 465 (=12%) genes correlated significantly to the IC50 values for ART. Hierarchical cluster analysis of these 12 genes allowed the differentiation of clusters with ART-sensitive or ART-resistant cell lines (P = 0.00017). For exemplary validation, cell lines transduced with 3 of the 12 genes were used to prove a causative relationship. The cDNAs for a deletion-mutated epidermal growth factor receptor (EGFR) and for gamma-glutamylcysteine synthetase increased resistance to ART. The conditional expression of the CDC25A gene using a tetracycline repressor expression vector increased sensitivity toward ART. Multidrug-resistant cells differentially expressing the MDR1, MRP1, or BCRP genes were not cross-resistant to ART. ART acts via p53-dependent and- independent pathways in isogenic p53+/+ p21WAF1/CIP1+/+, p53-/- p21WAF1/CIP1+/+, and p53+/+ p21WAF1/CIP1-/- colon carcinoma cells.