Tetsuo Yamagishi

P-Glycoprotein Mediates Drug Resistance via a Novel Mechanism Involving Lysosomal Sequestration

Background: Localization of the drug transporter P-glycoprotein (Pgp) to the plasma membrane is thought to be the only contributor of Pgp-mediated multidrug resistance (MDR). Results: Lysosomal Pgp sequesters ionizable chemotherapeutics into lysosomes to prevent interaction with molecular targets, resulting in drug resistance. Conclusion: Lysosomal Pgp mediates drug resistance. Significance: Pgp-mediated sequestration of chemotherapeutics into lysosomes can be exploited pharmacologically. Localization of the drug transporter P-glycoprotein (Pgp) to the plasma membrane is thought to be the only contributor of Pgp-mediated multidrug resistance (MDR). However, very little work has focused on the contribution of Pgp expressed in intracellular organelles to drug resistance. This investigation describes an additional mechanism for understanding how lysosomal Pgp contributes to MDR. These studies were performed using Pgp-expressing MDR cells and their non-resistant counterparts. Using confocal microscopy and lysosomal fractionation, we demonstrated that intracellular Pgp was localized to LAMP2-stained lysosomes. In Pgp-expressing cells, the Pgp substrate doxorubicin (DOX) became sequestered in LAMP2-stained lysosomes, but this was not observed in non-Pgp-expressing cells. Moreover, lysosomal Pgp was demonstrated to be functional because DOX accumulation in this organelle was prevented upon incubation with the established Pgp inhibitors valspodar or elacridar or by silencing Pgp expression with siRNA. Importantly, to elicit drug resistance via lysosomes, the cytotoxic chemotherapeutics (e.g. DOX, daunorubicin, or vinblastine) were required to be Pgp substrates and also ionized at lysosomal pH (pH 5), resulting in them being sequestered and trapped in lysosomes. This property was demonstrated using lysosomotropic weak bases (NH4Cl, chloroquine, or methylamine) that increased lysosomal pH and sensitized only Pgp-expressing cells to such cytotoxic drugs. Consequently, a lysosomal Pgp-mediated mechanism of MDR was not found for non-ionizable Pgp substrates (e.g. colchicine or paclitaxel) or ionizable non-Pgp substrates (e.g. cisplatin or carboplatin). Together, these studies reveal a new mechanism where Pgp-mediated lysosomal sequestration of chemotherapeutics leads to MDR that is amenable to therapeutic exploitation.

Antitumor activity and mechanism of action of the iron chelator, Dp44mT, against leukemic cells

Iron chelators have been reported to induce apoptosis and cell cycle arrest in cancer cells. Recent studies suggest broad and selective antitumor activity of the new iron chelator, di‐2‐pyridylketone‐4,4‐dimethyl‐3‐thiosemicarbazone (Dp44mT; Whitnall et al., Proc Natl Acad Sci USA 2006;103:14901–14906). However, little is known concerning its effects on hematological malignancies. Using acute leukemia cells, the effect of Dp44mT on apoptosis, cell cycle, caspase‐3 activation, and mitochondrial trans‐membrane potential has been examined by flow cytometry. Dp44mT acted to induce a G1/S arrest in NB4 promyelocytic leukemia cells at low concentrations (0.5–2.5 μM), being far more effective than the clinically used chelator, desferrioxamine (DFO). Moreover, Dp44mT induced apoptosis of NB4 cells in a dose‐ and time‐dependent manner with markedly less effect on nonproliferating cells. The apoptosis‐inducing activity of Dp44mT was significantly more effective than DFO. Furthermore, this study also showed that Dp44mT had broad activity, inducing apoptosis in several types of acute leukemia and also multiple myeloma cell lines. Additional studies examining the cytotoxic mechanisms of Dp44mT showed that a reduction in the mitochondrial trans‐membrane potential and caspase‐3 activation could be involved in the mechanism of apoptosis. Our results suggest that Dp44mT possesses potential as an effective cytotoxic agent for the chemotherapeutic treatment of acute leukemia. Am. J. Hematol. 2009.

Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes multidrug resistance by a novel mechanism involving the hijacking of lysosomal P-Glycoprotein (Pgp)

Background: There is a critical need for chemotherapeutics that overcome multidrug resistance (MDR). Results: Dp44mT is transported into the lysosome by Pgp, causing lysosomal targeting of Dp44mT and resulting in enhanced cytotoxicity in vitro and in vivo. Conclusion: Dp44mT overcomes MDR via utilization of lysosomal Pgp transport activity. Significance: DpT thiosemicarbazones offer a new therapeutic strategy to overcome MDR via utilization of lysosomal Pgp transport activity. Multidrug resistance (MDR) is a major obstacle in cancer treatment. More than half of human cancers express multidrug-resistant P-glycoprotein (Pgp), which correlates with a poor prognosis. Intriguingly, through an unknown mechanism, some drugs have greater activity in drug-resistant tumor cells than their drug-sensitive counterparts. Herein, we investigate how the novel anti-tumor agent di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes MDR. Four different cell types were utilized to evaluate the effect of Pgp-potentiated lysosomal targeting of drugs to overcome MDR. To assess the mechanism of how Dp44mT overcomes drug resistance, cellular studies utilized Pgp inhibitors, Pgp silencing, lysosomotropic agents, proliferation assays, immunoblotting, a Pgp-ATPase activity assay, radiolabeled drug uptake/efflux, a rhodamine 123 retention assay, lysosomal membrane permeability assessment, and DCF (2′,7′-dichlorofluorescin) redox studies. Anti-tumor activity and selectivity of Dp44mT in Pgp-expressing, MDR cells versus drug-sensitive cells were studied using a BALB/c nu/nu xenograft mouse model. We demonstrate that Dp44mT is transported by the lysosomal Pgp drug pump, causing lysosomal targeting of Dp44mT and resulting in enhanced cytotoxicity in MDR cells. Lysosomal Pgp and pH were shown to be crucial for increasing Dp44mT-mediated lysosomal damage and subsequent cytotoxicity in drug-resistant cells, with Dp44mT being demonstrated to be a Pgp substrate. Indeed, Pgp-dependent lysosomal damage and cytotoxicity of Dp44mT were abrogated by Pgp inhibitors, Pgp silencing, or increasing lysosomal pH using lysosomotropic bases. In vivo, Dp44mT potently targeted chemotherapy-resistant human Pgp-expressing xenografted tumors relative to non-Pgp-expressing tumors in mice. This study highlights a novel Pgp hijacking strategy of the unique dipyridylthiosemicarbazone series of thiosemicarbazones that overcome MDR via utilization of lysosomal Pgp transport activity.

Clonal expansions of cytotoxic T cells exist in the blood of patients with Waldenström macroglobulinemia but exhibit anergic properties and are eliminated by nucleoside analogue therapy

T cells contribute to host-tumor interactions in patients with monoclonal gammopathies. Expansions of CD8(+)CD57(+) T-cell receptor Vbeta-positive (TCRVbeta(+))-restricted cytotoxic T-cell (CTL) clones are found in 48% of patients with multiple myeloma and confer a favorable prognosis. We now report that CTL clones with varying TCRVbeta repertoire are present in 70% of patients with Waldenström macroglobulinemia (WM; n = 20). Previous nucleoside analog (NA) therapy, associated with increased incidence of transformation to aggressive lymphoma, significantly influenced the presence of TCRVbeta expansions (chi(2) = 11.6; P < .001), as 83% of patients without (n = 6) and only 7% with (n = 14) TCRVbeta expansions had received NA. Clonality of CD3(+)CD8(+)CD57(+)TCRVbeta(+)-restricted CTLs was confirmed by TCRVbeta CDR3 size analysis and direct sequencing. The differential expression of CD3(+)CD8(+)CD57(+)TCRVbeta(+) cells was profiled using DNA microarrays and validated at mRNA and protein level. By gene set enrichment analysis, CTL clones expressed not only genes from cytotoxic pathways (GZMB, PRF1, FGFBP2) but also genes that suppress apoptosis, inhibit proliferation, arrest cell-cycle G1/S transition, and activate T cells (RAS, CSK, and TOB pathways). Proliferation tracking after stimulation confirmed their anergic state. Our studies demonstrate the incidence, NA sensitivity, and nature of clonal CTLs in WM and highlight mechanisms that cause anergy in these cells.

Presence of Hoechst low side populations in multiple myeloma

Multiple myeloma (MM) is an incurable B cell malignancy characterised by the accumulation of monoclonal plasma cells in the bone marrow and the presence of high level of monoclonal immunoglobulin in the blood [1]. Relapse of the disease after chemotherapy is inevitable and may be due to the persistence of drug-resistant myeloma cancer stem cells (MCSC) that remain even after intensive therapy. Therefore, it may be important to target not only the morphologically visible malignant plasma cells but also the MCSC with appropriate therapy to achieve lasting cure. The presence of cancer stem cells has been demonstrated in a number of solid and non-solid tumors including colon cancers [2] and acute myeloid leukemia (AML) [3]. Matsui et al. [4] demonstrated the existence of a subpopulation of CD138 myeloma cells which has the capability to proliferate, differentiate, and initiate tumors in NOD-SCID mice effectively. However, their study provides no information of specific surface markers that may allow positive identification of the MCSC. Side population (SP) cells were originally identified in murine bone marrow and SP have been defined by Goodell et al. [5] as a population of cells that have the capability to efflux the fluorescent dye Hoechst 33342 in a unique pattern. The differential blue (450 nm) versus red (670 nm) emission fluorescence upon UV excitation allows clear identification of a cell population that locates sideways from the diagonal and thus named ‘side’ population. SP cells have been isolated from various cancer cell lines as well as from samples of primary tumors [6,7]. In the primary samples, SP cells have been identified in the neuroblastoma tumor cell samples from patients with relapsed disease [8], and from the bone marrow and/or peripheral blood of human AML patients. In an in vivo study, Wulf et al. [9] transplanted an average of 340 CD34 SP cells (range, 3.8610 to 1610) per NOD/SCID mouse and successfully resulted in engraftment of AML-like disease in three of the recipient mice, each resulting in growth of high number of AML cells (1610 to 1610). Other evidence for repopulating ability by SP to regenerate a population resembling the original population includes human lung cancer [10] and human nasopharyngeal carcinoma [11]. This study aims to investigate if SP cells are present in myeloma cell lines as well as fresh myeloma samples. We have examined the expression of two phenotypic markers CD38 and CD138, two important surface markers and examined the relationship between SP cells and known clinical indicators. RPMI 8226, U266 and OPM2, human myeloma cell lines were obtained from ATCC (Manassas, VA). KMS-11 was obtained from JCRB (HSRRB, Osaka, Japan). Bone marrow samples of myeloma

Abstract C40: Understanding the mechanism of action of novel iron chelators on drug-resistant neoplastic cells.

Introduction: Deprivation of cellular iron (Fe) may represent a novel and effective anti-cancer strategy1. Our laboratory has developed iron chelators with marked and selective anti-tumor activity against a variety of tumor types in vitro and in vivo. One of our leading compounds, Dp44mT, demonstrated potent anti-tumor activity against drug-resistant cell lines1,2 over-expressing P-gp and MRP1. Moreover, these compounds possess high activity at releasing cellular 59Fe3. Aims: To further understand the mechanism of chelator-mediated cellular 59Fe release we investigated: The difference in iron efflux between drug-resistant MCF7-VP (MRP1 hyper-expressing) cells and their wild-type counterparts (MCF7); Whether chelator-mediated Fe release was MRP1 and/or GSH-dependent and; If chelator-mediated cellular Fe release was temperature-dependent. Results: These studies demonstrated that MRP1 over-expressing MCF7-VP cells showed reduced chelator-mediated59Fe release compared to MCF-7 cells (P Interestingly, MCF7-VP cells showed higher 59Fe uptake from 59Fe-transferrin than wild-type cells. This was due to marked transferrin receptor-1 expression in MCF7-VP compared to MCF7 cells. Additionally, the only known Fe efflux pump, ferroportin1, was down-regulated in MCF7-VP cells. This latter observation was intriguing as chelator-mediated Fe efflux was depressed in this cell type. These studies indicate that chelator-mediated 59Fe release is temperature-dependent and MRP-independent. Further studies will examine the role of MRP1 in Fe uptake and metabolism. References: 1. Whitnall, M., Howard, J., Ponka, P., and Richardson, D. R., Proc Natl Acad Sci U S A103 (40), 14901 (2006). 2. Richardson, D. R. and Milnes, K., Blood89 (8), 3025 (1997). 3. Richardson, D. R., Biochim Biophys Acta1320 (1), 45 (1997). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C40.