Overcoming multidrug resistance via photodestruction of ABCG2-rich extracellular vesicles sequestering photosensitive chemotherapeutics

Multidrug resistance (MDR) remains a dominant impediment to curative cancer chemotherapy. Efflux transporters of the ATP-binding cassette (ABC) superfamily including ABCG2, ABCB1 and ABCC1 mediate MDR to multiple structurally and functionally distinct antitumor agents. Recently we identified a novel mechanism of MDR in which ABCG2-rich extracellular vesicles (EVs) form in between attached neighbor breast cancer cells and highly concentrate various chemotherapeutics in an ABCG2-dependent manner, thereby sequestering them away from their intracellular targets. Hence, development of novel strategies to overcome MDR modalities is a major goal of cancer research. Towards this end, we here developed a novel approach to selectively target and kill MDR cancer cells. We show that illumination of EVs that accumulated photosensitive cytotoxic drugs including imidazoacridinones (IAs) and topotecan resulted in intravesicular formation of reactive oxygen species (ROS) and severe damage to the EVs membrane that is shared by EVs-forming cells, thereby leading to tumor cell lysis and the overcoming of MDR. Furthermore, consistent with the weak base nature of IAs, MDR cells that are devoid of EVs but contained an increased number of lysosomes, highly accumulated IAs in lysosomes and upon photosensitization were efficiently killed via ROS-dependent lysosomal rupture. Combining targeted lysis of IAs-loaded EVs and lysosomes elicited a synergistic cytotoxic effect resulting in MDR reversal. In contrast, topotecan, a bona fide transport substrate of ABCG2, accumulated exclusively in EVs of MDR cells but was neither detected in lysosomes of normal breast epithelial cells nor in non-MDR breast cancer cells. This exclusive accumulation in EVs enhanced the selectivity of the cytotoxic effect exerted by photodynamic therapy to MDR cells without harming normal cells. Moreover, lysosomal alkalinization with bafilomycin A1 abrogated lysosomal accumulation of IAs, consequently preventing lysosomal photodestruction of normal breast epithelial cells. Thus, MDR modalities including ABCG2-dependent drug sequestration within EVs can be rationally converted to a pharmacologically lethal Trojan horse to selectively eradicate MDR cancer cells.     

A Novel Two Mode-Acting Inhibitor of ABCG2-Mediated Multidrug Transport and Resistance in Cancer Chemotherapy

Background

Multidrug resistance (MDR) is a major problem in successful treatment of cancers. Human ABCG2, a member of the ATP-binding cassette transporter superfamily, plays a key role in MDR and an important role in protecting cancer stem cells. Knockout of ABCG2 had no apparent adverse effect on the mice. Thus, ABCG2 is an ideal target for development of chemo-sensitizing agents for better treatment of drug resistant cancers and helping eradicate cancer stem cells.

Methods/Preliminary Findings

Using rational screening of representatives from a chemical compound library, we found a novel inhibitor of ABCG2, PZ-39 (N-(4-chlorophenyl)-2-[(6-{[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]amino}-1,3-benzothiazol-2-yl)sulfanyl]acetamide), that has two modes of actions by inhibiting ABCG2 activity and by accelerating its lysosome-dependent degradation. PZ-39 has no effect on ABCB1 and ABCC1-mediated drug efflux, resistance, and their expression, indicating that it may be specific to ABCG2. Analyses of its analogue compounds showed that the pharmacophore of PZ-39 is benzothiazole linked to a triazine ring backbone.

Conclusion/Significance

Unlike any previously known ABCG2 transporter inhibitors, PZ-39 has a novel two-mode action by inhibiting ABCG2 activity, an acute effect, and by accelerating lysosome-dependent degradation, a chronic effect. PZ-39 is potentially a valuable probe for structure-function studies of ABCG2 and a lead compound for developing therapeutics targeting ABCG2-mediated MDR in combinational cancer chemotherapy.     

上皮细胞黏附分子增强细胞间紧密连接促进乳腺癌细胞耐药

乳腺癌是致死率很高的恶性肿瘤,由ABCG2 (ATP-binding cassette G2)介导的多药耐药(multidrug resistance,MDR)是导致其化疗失败的重要原因,探讨ABCG2介导的耐药机制并探寻其关键分子是当前亟待解决的难题。上皮细胞黏附分子(epithelial cell adhesion molecule,EpCAM)参与多种肿瘤耐药,且与乳腺癌MDR密切相关,但它在ABCG2介导的乳腺癌耐药中的作用尚未阐明。本研究目的在于探究EpCAM对于ABCG2介导的乳腺癌细胞的多药耐药的调节作用及其机制。CCK8细胞毒性结果证实,相对于人乳腺癌药物敏感株MCF-7,耐药株MCF-7/MX对米托蒽醌(mitoxantrone,MX)的耐药性显著增强;Western 印迹结果显示,与MCF-7相比,MCF-7/MX细胞中ABCG2高表达,EpCAM表达上调。siRNA法敲低MCF-7/MX细胞中EpCAM可下调其ABCG2表达,并恢复对MX的敏感性。倒置显微镜观察细胞形态,发现敲低EpCAM可减少MCF-7/MX细胞间连接。免疫荧光双染法观察到EpCAM与密封蛋白1(claudin 1)在MCF-7/MX细胞共定位;进一步Western 印迹结果表明,敲低EpCAM减少MCF-7/MX细胞中密封蛋白1表达。综上所述,EpCAM可能通过与密封蛋白1相互作用,增强细胞间紧密连接,促进ABCG2介导的乳腺癌多药耐药。     

Structure and function of ABCG2-rich extracellular vesicles mediating multidrug resistance

Multidrug resistance (MDR) is a major impediment to curative cancer chemotherapy. The ATP-Binding Cassette transporters ABCG2, ABCB1 and ABCC2 form a unique defense network against multiple structurally and functionally distinct chemotherapeutics, thereby resulting in MDR. Thus, deciphering novel mechanisms of MDR and their overcoming is a major goal of cancer research. Recently we have shown that overexpression of ABCG2 in the membrane of novel extracellular vesicles (EVs) in breast cancer cells results in mitoxantrone resistance due to its dramatic sequestration in EVs. However, nothing is known about EVs structure, biogenesis and their ability to concentrate multiple antitumor agents. To this end, we here found that EVs are structural and functional homologues of bile canaliculi, are apically localized, sealed structures reinforced by an actin-based cytoskeleton and secluded from the extracellular milieu by the tight junction proteins occludin and ZO-1. Apart from ABCG2, ABCB1 and ABCC2 were also selectively targeted to the membrane of EVs. Moreover, Ezrin-Radixin-Moesin protein complex selectively localized to the border of the EVs membrane, suggesting a key role for the tethering of MDR pumps to the actin cytoskeleton. The ability of EVs to concentrate and sequester different antitumor drugs was also explored. Taking advantage of the endogenous fluorescence of anticancer drugs, we found that EVs-forming breast cancer cells display high level resistance to topotecan, imidazoacridinones and methotrexate via efficient intravesicular drug concentration hence sequestering them away from their cellular targets. Thus, we identified a new modality of anticancer drug compartmentalization and resistance in which multiple chemotherapeutics are actively pumped from the cytoplasm and highly concentrated within the lumen of EVs via a network of MDR transporters differentially targeted to the EVs membrane. We propose a composite model for the structure and function of MDR pump-rich EVs in cancer cells and their ability to confer multiple anticancer drug resistance.     

The Spleen Tyrosine Kinase Inhibitor,Entospletinib (GS-9973) Restores Chemosensitivity in Lung Cancer Cells by Modulating ABCG2-mediated Multidrug Resistance

Tyrosine kinase inhibitors (TKIs) are important in managing lymphoid malignancies by targeting B-cell receptor signaling pathways. Entospletinib (GS-9973) is an oral, selective inhibitor of spleen tyrosine kinase (Syk), currently in the phase II clinical trials for the treatment of chronic lymphocytic leukemia. Syk is abundantly present in the cells of hematopoietic lineage that mediates cell proliferation, differentiation, and adhesion. In this current study, we evaluated the efficacy of GS-9973 to overcome multidrug resistance (MDR) due to the overexpression of the ABCG2 transporter in the non-small cell lung cancer (NSCLC) cell line, NCI-H460/MX20. In vitro, 3 μM of GS-9973 reversed the drug resistance of NCI-H460/MX20 cell line to mitoxantrone or doxorubicin. GS-9973, at 3 μM reverses ABCG2-mediated MDR by blocking ABCG2 efflux activity and downregulating ABCG2 expression at the protein level but did not alter the ABCG2 mRNA expression and subcellular localization of the ABCG2 protein compared to drug-resistant cells incubated with the vehicle. GS-9973 produced a moderate concentration-dependent increase in the ATPase activity of ABCG2 (EC₅₀ = 0.42 µM) and molecular docking data indicated that GS-9973 had a high affinity (-10.226 kcal/mol) for the substrate-binding site of ABCG2. Finally, HPLC analysis proved that the intracellular concentration of GS-9973 is not significantly different in both parental and resistant cell lines. In conclusion, our study suggests that in vitro, GS-9973 in combination with certain anticancer drugs, represent a strategy to overcome ABCG2-mediated MDR cancers.     

Reversal of ABCG2-mediated multidrug resistance by human cathelicidin and its analogs in cancer cells

Multidrug resistance (MDR) of cancer cells to a wide spectrum of anticancer drugs is a major obstacle to successful chemotherapy. It is usually mediated by the overexpression of one of the three major ABC transporters actively pumping cytotoxic drugs out of the cells. There has been great interest in the search for inhibitors toward these transporters with an aim to circumvent resistance. This is usually achieved by screening from natural product library and the subsequent structural modifications. This study reported the reversal of ABCG2-mediated MDR in drug-selected resistant cancer cell lines by a class of host defense antimicrobial peptides, the human cathelicidin LL37 and its fragments. The effective human cathelicidin peptides (LL17-32 and LL13-37) were found to increase the accumulation of mitoxantrone in cancer cell lines with ABCG2 overexpression, thereby circumventing resistance to mitoxantrone. At the effective concentrations of the cathelicidin peptides, cell proliferation of the parental cells without elevated ABCG2 expression was not affected. Result from drug efflux and ATPase assays suggested that both LL17-32 and LL13-37 interact with ABCG2 and inhibit its transport activity in an uncompetitive manner. The peptides were also found to downregulate ABCG2 protein expression in the resistant cells, probably through a lysosomal degradation pathway. Our data suggest that the human cathelicidin may be further developed for sensitizing resistant cancer cells to chemotherapy.     

Autophagy inhibition suppresses the tumorigenic potential of cancer stem cell enriched side population in bladder cancer

The mechanisms that underlie tumor formation and progression have not been elucidated in detail in cancer biology. Recently, the identification of a tumor cell subset defined as cancer stem cells (CSCs), which is enriched for tumor initiating capacity, has engendered new perspectives towards selective targeting of tumors. In this study, we isolated the side population (SP) cells which share characteristics of CSCs from bladder cancer cell lines, T24 and UM-UC-3 by fluorescence activated cell sorting. The cells were cultured in serum free medium and expression profile of stem cell like markers (SOX-2, NANOG, KLF-4 and OCT-4), drug resistant genes (ABCG2 and MDR1) and spheroid forming capability were examined in SP, non-side population (NSP) and bulk T24 and UM-UC-3 cells. We observed that SP cells possessed a higher mRNA expression of SOX-2, NANOG, KLF-4, OCT-4, ABCG2, and MDR1 as well as a higher spheroid forming ability as compared to other bulk cells or NSP cells. The SP cells had low ROS levels and high GSH/GSSG ratio which may contribute to radio-resistance. The SP cells also showed substantial resistance to gemcitabine, mitomycin and cisplatin compared with the NSP counterpart. A high autophagic flux was observed in the SP cells. Both pharmacological and siRNA mediated inhibition of autophagy potentiated the chemotherapeutic effects of gemcitabine, mitomycin and cisplatin in these cells. We concluded that the ABCG2 expressing SP cells show autophagy associated cell survival and may be a potent target for developing more effective treatment in bladder carcinoma to enhance patient survival.     

Linsitinib (OSI-906) antagonizes ATP-binding cassette subfamily G member 2 and subfamily C member 10-mediated drug resistance

In this study we investigated the effect of linsitinib on the reversal of multidrug resistance (MDR) mediated by the overexpression of the ATP-binding cassette (ABC) subfamily members ABCB1, ABCG2, ABCC1 and ABCC10. Our results indicate for the first time that linsitinib significantly potentiate the effect of anti-neoplastic drugs mitoxantrone (MX) and SN-38 in ABCG2-overexpressing cells; paclitaxel, docetaxel and vinblastine in ABCC10-overexpressing cells. Linsitinib moderately enhanced the cytotoxicity of vincristine in cell lines overexpressing ABCB1, whereas it did not alter the cytotoxicity of substrates of ABCC1. Furthermore, linsitinib significantly increased the intracellular accumulation and decreased the efflux of [³H]-MX in ABCG2-overexpressing cells and [³H]-paclitaxel in ABCC10-overexpressing cells. However, linsitinib, at a concentration that reversed MDR, did not significantly alter the expression levels of either the ABCG2 or ABCC10 transporter proteins. Furthermore, linsitinib did not significantly alter the intracellular localization of ABCG2 or ABCC10. Moreover, linsitinib stimulated the ATPase activity of ABCG2 in a concentration-dependent manner. Overall, our study suggests that linsitinib attenuates ABCG2- and ABCC10-mediated MDR by directly inhibiting their function as opposed to altering ABCG2 or ABCC10 protein expression.     

Riboflavin concentration within ABCG2-rich extracellular vesicles is a novel marker for multidrug resistance in malignant cells

We have previously shown that overexpression of the multidrug resistance (MDR) efflux transporter ABCG2 in the membrane of novel extracellular vesicles that are confined to breast cancer cell-cell attachment zones confers mitoxantrone resistance and mediates a marked intravesicular concentration of an unknown endogenous green fluorescent compound (I. Ifergan, G.L. Scheffer, Y.G. Assaraf, Novel extracellular vesicles mediate an ABCG2-dependent anticancer drug sequestration and resistance, Cancer Res. 65 (2005) 10952-10958). Here we identified the latter as riboflavin (vitamin B2) and further demonstrated that the marked intravesicular concentration of riboflavin in ABCG2-overexpressing breast and lung cancer cells tightly correlates with the extent of ABCG2 overexpression and its differential localization to the vesicular membrane and not to the plasma membrane surrounded by growth medium. We hence propose that the ABCG2-dependent concentration of riboflavin in these intercellular compartments may serve as a novel, sensitive, and non-cytotoxic (i.e. based on vitamin accumulation) functional marker for the quantification of the levels of MDR mediated by ABCG2-rich extracellular vesicles in multiple malignant cells.     

ABCG2-mediated DyeCycle Violet efflux defined side population in benign and malignant prostate

The efflux of Hoechst 33342 by ATP-binding cassette protein G2 (ABCG2) membrane pump allows reproducible identification of a subpopulation of cells by flow cytometric analysis termed the “side population” (SP). The SP identified by constitutive Hoechst efflux contains the stem/progenitor cell population from bone marrow and many solid organs, including prostate. DyeCycle Violet (DCV) is a cell membrane permeable, fluorescent vital dye that intercalates into DNA and is a substrate for ABCG2-mediated efflux. Therefore, DCV was evaluated in this study as a tool for identification of the SP from prostate cancer cell lines and from freshly harvested human prostate tissue. SPs that demonstrated ABCG2-mediated efflux of DCV were identified in the human prostate cancer cell lines CWR-R1, DU-145, and RWPE-1, but not in the BPH-1, LAPC-4, or PC-3 cell lines. Additionally, a SP was identified in enzymatically disaggregated prostate tumors from Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) human benign prostate tissue, and human prostate cancer tissue. The causal role of ABCG2-mediated efflux of DCV in the identification of the SP was confirmed by loss of the SP by incubation with the specific inhibitor of ABCG2, Fumitremorgin C. Expression of ABCG2 in the SP cells was confirmed by qRT-PCR and immunofluorescence analysis. Consequently, DCV represents an important new tool for isolation of viable candidate stem cells/cancer stem cells as a SP from cultured prostate cell lines, and prostate tissue specimens, without the requirement for instrumentation with ultra-violet excitation capability and minimizing the risk of damage to DNA in the sorted population.     

BIRB796, the Inhibitor of p38 Mitogen-Activated Protein Kinase,Enhances the Efficacy of Chemotherapeutic Agents in ABCB1 Overexpression Cells

ATP-binding-cassette family membrane proteins play an important role in multidrug resistance. In this study, we investigated BIRB796, an orally active inhibitor of p38 mitogen-activated protein kinase, reversed MDR induced by ABCB1, ABCG2 and ABCC1. Our results showed that BIRB796 could reverse ABCB1-mediated MDR in both the drug selected and transfected ABCB1-overexpressing cell models, but did not enhance the efficacy of substrate-chemotherapeutical agents in ABCC1 or ABCG2 overexpression cells and their parental sensitive cells. Furthermore, BIRB796 increased the intracellular accumulation of the ABCB1 substrates, such as rhodamine 123 and doxorubicin. Moreover, BIRB796 bidirectionally mediated the ATPase activity of ABCB1, stimulating at low concentration, inhibiting at high concentration. However, BIRB796 did not alter the expression of ABCB1 both at protein and mRNA level. The down-regulation of p38 by siRNA neither affected the expression of ABCB1 nor the cytotoxic effect of paclitaxel on KBV200. The binding model of BIRB796 within the large cavity of the transmembrane region of ABCB1 may form the basis for future lead optimization studies. Importantly, BIRB796 also enhanced the effect of paclitaxel on the inhibition of growth of the ABCB1-overexpressing KBV200 cell xenografts in nude mice. Overall, we conclude that BIRB796 reverses ABCB1-mediated MDR by directly inhibiting its transport function. These findings may be useful for cancer combinational therapy with BIRB796 in the clinic.     

Low Molecular Weight Heparin Ablates Lung Cancer Cisplatin-Resistance by Inducing Proteasome-Mediated ABCG2 Protein Degradation

Cancer side population (SP) cells, which are often referred to as cancer stem cells, are thought to be responsible for lung cancer chemotherapy resistance, and currently no drug can specifically target these cells. We hypothesize low-molecular-weight heparin (LMWH) may affect the biological properties of SP cells and could be used to clinically target these cells. To test this, SP cells were isolated from cisplatin (DDP)-resistant lung adenocarcinoma A549/DDP cells by flow cytometric sorting. Compared to non-SP cells, SP cells formed increased numbers of colonies in vitro, and had a 1000-fold increase in tumorigenicity in vivo. Proliferation and apoptosis assays demonstrated LMWH had no significant effect on lung SP cell proliferation or apoptosis. However, LMWH reduced lung SP cell colony formation ability and protein expression of the multidrug transporter, ABCG2, by FACS and western blot analyses without affecting its mRNA levels by RT-PCR. Consistently, immunohistochemistry stainings of ABCG2 in LMWH-treated tumor tissues were significantly reduced compared with those in controls. Further, we found proteasomal inhibitor MG132, but not lysosomal inhibitors leupeptin and pepstatin A, could restore ABCG2 protein levels in LMWH-treated SP cells. These suggest LMWH ablates lung SP cell chemoresistance by proteasome-mediated reduction of ABCG2 protein levels without affecting its mRNA levels. We also determined LMWH combined with cisplatin could overcome cisplatin-resistance and induced lung SP cells apoptosis both in vitro and in vivo. This study provides an experimental basis for using a combination of LMWH, which targets lung SP cells, with chemotherapy to improve lung cancer survival.     

Lapatinib Antagonizes Multidrug Resistance–Associated Protein 1–Mediated Multidrug Resistance by Inhibiting Its Transport Function

Lapatinib, a tyrosine kinase inhibitor, is used in the treatment of advanced or metastatic breast cancer overexpressing human epidermal receptor 2 (HER2). Lapatinib can modulate the function of ATP-binding cassette (ABC) transporters (ABCB1 and ABCG2), which are the major mechanism responsible for multidrug resistance (MDR) in cancer. In this study, we investigated the effect of lapatinib on multidrug resistance–associated protein 1 (MRP1 [ABCC1]), MRP2 (ABCC2), MRP4 (ABCC4) and lung relative resistance protein (LRP) drug efflux pumps. We demonstrated that lapatinib could enhance the efficacy of conventional chemotherapeutic agents in MRP1-overexpressing cells in vitro and in vivo, but no effect in MRP2-, MPR4- and LRP-overexpressing cells. Furthermore, lapatinib significantly increased the accumulation of rhodamine 123 (Rho123) and doxorubicin (DOX) in MRP1-overexpressing cells. However, lapatinib did not alter the protein or mRNA expression levels of MRP1. Further studies showed that the level of phosphorylation of AKT and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) were not altered at the indicated concentrations of lapatinib. In conclusion, lapatinib enhanced the efficacy of conventional chemotherapeutic agents in MRP1-overexpressing cells by inhibiting MRP1 transport function without altering the level of AKT or ERK1/2 phosphorylation. These findings will encourage the clinical research of lapatinib combined with conventional chemotherapeutic drugs in MRP1-overexpressing cancer patients.     

半分子转运蛋白ABCG2的肿瘤多药耐药性研究及其应用

肿瘤常对临床上传统使用的多种化学治疗显示其内源性或获得性的药物耐受性即多药耐药性(multidrug resistance,MDR)．这种多药耐药性主要是由一类称为ABC(ATP-binding cassette)转运体蛋白超家族的跨膜蛋白引起的,它们结合并利用水解ATP提供的能量来转运药物,导致肿瘤细胞呈现抗药性．半分子转运蛋白ABCG2是近年来才发现的可归于ABC转运体大家族中的一个新成员,在肠、肝、胎盘和血脑屏障等部位大量表达,与全分子转运蛋白如P-gp (P-glycoprotein)和多药耐药蛋白(multi-drug resistance protein,MRP)相似,可以主动地把具有不同化学结构和作用于细胞内不同靶位点的化疗药物泵出胞外,从而引起肿瘤对多种抗癌药物(包括最新开发的药物)产生抗性．最近的一些十分有趣的研究还表明,ABCG2可能与干细胞分化状态和保护干细胞发育过程中免受周围环境的影响有关,而且还发现,它在侧群骨髓和神经干细胞内大量存在,因此,ABCG2可能在基因治疗中作为选择性的蛋白质标记正受到研究者们的极大关注．同时,ABCG2的单核苷酸多态性影响其结合并转运不同的底物/药物．鉴于ABCG2在肿瘤抗药性研究中的重要性以及它的一些新功能的初步研究表明,对ABCG2的生物学功能和作用机理以及在医学实践中的应用研究必将受到更大的关注．主要阐述了半分子ABC转运蛋白ABCG2的发现、重要的生化特性和生理功能及其相关的新研究进展和问题．     

Complete reversal of ABCG2-depending atypical multidrug resistance by RNA interference in human carcinoma cells

In the chemotherapeutic treatment of patients with disseminated neoplasms, multidrug resistance (MDR) is a major obstacle. ABCG2 (BCRP/MXR), a member of the superfamily of adenosine triphosphate-binding cassette (ABC) transporters, was demonstrated to be associated with "atypical" forms of multidrug-resistant phenotypes of cancer cells. To overcome the ABCG2-depending MDR, two specific anti-ABCG2 small interfering RNAs (siRNAs) were designed for transient triggering of the gene-silencing RNA interference (RNAi) pathway in the human gastric carcinoma cell line EPG85-257RNOV, exhibiting an atypical MDR phenotype. Because both siRNAs showed biological activity, for stable inhibition of ABCG2 corresponding short hairpin RNA (shRNA) expression vectors were constructed. By treatment of EPG85-257RNOV cells with these constructs, expression of the targeted ABCG2-encoding mRNA and transport protein was inhibited completely. Furthermore, anti-ABCG2 shRNA-treated cells increased cellular drug accumulation to the same level measured in drug-sensitive parental cells. These effects were accompanied by complete reversal of the drug-resistant phenotype. Thus, the data indicate that siRNA- and shRNA-mediated RNAi-based gene therapy may be applicable in preventing and reversing ABCG2-depending atypical MDR.     

Tumor necrosis factor alpha induces stronger cytotoxicity in ABCG2-overexpressing resistant breast cancer cells compared with their drug-sensitive parental line

Tumor necrosis factor alpha (TNF-α) has been reported to modulate the multidrug resistance (MDR) phenotype in vitro and in vivo. Multidrug-resistant cells overexpressing the ABCB1 transporter are more susceptible to inhibition of proliferation and induction of apoptosis by TNF-α than their drug-sensitive counterparts. This study was aimed to investigate TNF-α modulatory and antiproliferative effects on drug-resistant cells overexpressing ABCG2. The effects of TNF-α on viability and proliferation rate of MCF-7 breast cancer cells and their ABCG2-overexpressing sublines MCF-7/mitoxantrone (MX) cells were studied using dye exclusion assay, dimethylthiazolyl-2,5-diphenyl tetrazolium bromide technique, and flow cytometric analysis of cell cycle. TNF-α influence on MX accumulation was investigated by flow cytometry. ABCG2-overexpressing cells were more susceptible to antiproliferative and cytotoxic effects of TNF-α than their parental cells. TNF-α increased accumulation of MX in both parental and resistant cells. Higher sensitivity of MDR cells to TNF-α cytotoxicity would help in characterization of its complex modulatory effects on cancer cells and benefit us in designing new approaches to overcome MDR.     

Characterization of 3-methoxy flavones for their interaction with ABCG2 as suggested by ATPase activity

Breast Cancer Resistance Protein (BCRP/ABCG2) belongs to the superfamily of ATP binding cassette (ABC) transporters. Characteristic of some of these transporter proteins is the transport of a variety of structurally unrelated substances against a concentration gradient by using the energy of ATP hydrolysis. ABCG2 has been found to confer multidrug resistance (MDR) in cancer cells. Several anticancer drugs have been identified as ABCG2 substrates including mitoxantrone, etoposide and topotecan. As inhibition of the transporter is one of the strategies to overcome MDR, we have synthesized and tested several 3-methoxy flavones and investigated them for their ABCG2 inhibition. Among these, pentamethyl quercetin (compound 4) and pentamethyl morin (compound 5) were found to be fluorescent and hence screened for their possible transport by ABCG2 using confocal microscopy. This study showed that pentamethyl quercetin was far less accumulated in ABCG2 overexpressing MDCK BCRP cells as compared to MDCK sensitive cells, suggesting possible efflux of this compound by ABCG2. Pentamethyl morin showed no visible difference in both cell lines. Based on this observation, we studied several other fluorescent 3-methoxy flavones for their accumulation in ABCG2 overexpressing cells. To confirm the substrate or inhibitor nature of the tested compounds, these compounds were further investigated by ATPase assay. If stimulation of the transporter ATPase activity is detected, one can conclude that the compound is probably a transported substrate. All compounds except pentamethyl morin (compound 5) and tetramethyl quercetin (compound 6) were found to stimulate ATPase activity pointing to possible substrates despite being potent inhibitors of ABCG2.