ABCG2-mediated Drug Resistance Research Articles

Tumor-acquired somatic mutation affects conformation to abolish ABCG2-mediated drug resistance

ABCG2 is an important ATP-binding cassette transporter impacting the absorption and distribution of over 200 chemical toxins and drugs. ABCG2 also reduces the cellular accumulation of diverse chemotherapeutic agents. Acquired somatic mutations in the phylogenetically conserved amino acids of ABCG2 might provide unique insights into its molecular mechanisms of transport. Here, we identify a tumor-derived somatic mutation (Q393K) that occurs in a highly conserved amino acid across mammalian species. This ABCG2 mutant seems incapable of providing ABCG2-mediated drug resistance. This was perplexing because it is localized properly and retained interaction with substrates and nucleotides. Using a conformationally sensitive antibody, we show that this mutant appears “locked” in a non-functional conformation. Structural modeling and molecular dynamics simulations based on ABCG2 cryo-EM structures suggested that the Q393K interacts with the E446 to create a strong salt bridge. The salt bridge is proposed to stabilize the inward-facing conformation, resulting in an impaired transporter that lacks the flexibility to readily change conformation, thereby disrupting the necessary communication between substrate binding and transport.

Carboranes as Potent Phenyl Mimetics: A Comparative Study on the Reversal of ABCG2-Mediated Drug Resistance by Carboranylquinazolines and Their Organic Isosteres.

Multidrug resistance is a major challenge in clinical cancer therapy. In particular, overexpression of certain ATP-binding cassette (ABC) transporter proteins, like the efflux transporter ABCG2, also known as breast cancer resistance protein (BCRP), has been associated with the development of resistance to applied chemotherapeutic agents in cancer therapies, and therefore targeted inhibition of BCRP-mediated transport might lead to reversal of this (multidrug) resistance (MDR). In a previous study, we have described the introduction of a boron-carbon cluster, namely closo-dicarbadodecaborane or carborane, as an inorganic pharmacophore into a polymethoxylated 2-phenylquinazolin-4-amine backbone. In this work, the scope was extended to the corresponding amide derivatives. As most of the amide derivatives suffered from poor solubility, only the amide derivative QCe and the two amine derivatives DMQCc and DMQCd were further investigated. Carboranes are often considered as sterically demanding phenyl mimetics or isosteres. Therefore, the organic phenyl and sterically demanding adamantyl analogues of the most promising carborane derivatives were also investigated. The studies showed that the previously described DMQCd, a penta-methoxylated N-carboranyl-2-phenylquinazolin-4-amine, was by far superior to its organic analogues in terms of cytotoxicity, inhibition of the human ABCG2 transporter, as well as the ability to reverse BCRP-mediated mitoxantrone resistance in MDCKII-hABCG2 and HT29 colon cancer cells. Our results indicate that DMQCd is a promising candidate for further in vitro as well as in vivo studies in combination therapy for ABCG2-overexpressing cancers.

Enhanced reversal of ABCG2-mediated drug resistance by replacing a phenyl ring in baicalein with a meta-carborane.

Success of chemotherapy is often hampered by multidrug resistance. One mechanism for drug resistance is the elimination of anticancer drugs through drug transporters, such as breast cancer resistance protein (BCRP; also known as ABCG2), and causes a poor 5-year survival rate of human patients. Co-treatment of chemotherapeutics and natural compounds, such as baicalein, is used to prevent chemotherapeutic resistance but is limited by rapid metabolism. Boron-based clusters as meta-carborane are very promising phenyl mimetics to increase target affinity; we therefore investigated the replacement of a phenyl ring in baicalein by a meta-carborane to improve its affinity towards the human ABCG2 efflux transporter. Baicalein strongly inhibited the ABCG2-mediated efflux and caused a fivefold increase in mitoxantrone cytotoxicity. Whereas the baicalein derivative 5,6,7-trimethoxyflavone inhibited ABCG2 efflux activity in a concentration of 5 μm without reversing mitoxantrone resistance, its carborane analogue 5,6,7-trimethoxyborcalein significantly enhanced the inhibitory effects in nanomolar ranges (0.1 μm) and caused a stronger increase in mitoxantrone toxicity reaching similar values as Ko143, a potent ABCG2 inhibitor. Overall, in silico docking and in vitro studies demonstrated that the modification of baicalein with meta-carborane and three methoxy substituents leads to an enhanced reversal of ABCG2-mediated drug resistance. Thus, this seems to be a promising basis for the development of efficient ABCG2 inhibitors.

Novel chlorin e6-based conjugates of tyrosine kinase inhibitors: Synthesis and photobiological evaluation as potent photosensitizers for photodynamic therapy

Since tyrosine kinase inhibitor (TKI) could reverse ABCG2-mediated drug-resistance, novel chlorin e6-based conjugates of Dasatinib and Imatinib as photosensitizer (PS) were designed and synthesized. The results demonstrated that conjugate 10b showed strongest phototoxicity against HepG2 and B16-F10 cells, which was more phototoxic than chlorin e6 and Talaporfin. It could reduce efflux of intracellular PS by inhibiting ABCG2 in HepG2 cells, and localize in mitochondria, lysosomes, golgi and ER, resulting in higher cell apoptosis rate and ROS production than Talaporfin. Moreover, it could induce cell autophagy and block cell cycle in S phase, and significantly inhibit tumor growth and prolong survival time on BALB/c nude mice bearing HepG2 xenograft tumor to a greater extent than chlorin e6. Consequently, compound 10b could be applied as a promising candidate PS due to its good water-solubility and stability, low drug-resistance, high quantum yield of 1O2 and excellent antitumor efficacy in vitro and in vivo.

The AKT inhibitor, MK-2206, attenuates ABCG2-mediated drug resistance in lung and colon cancer cells.

Introduction: The overexpression of ATP-binding cassette (ABC) transporters, ABCB1 and ABCG2, are two of the major mediators of multidrug resistance (MDR) in cancers. Although multiple ABCB1 and ABCG2 inhibitors have been developed and some have undergone evaluation in clinical trials, none have been clinically approved. The compound, MK-2206, an inhibitor of the protein kinases AKT1/2/3, is undergoing evaluation in multiple clinical trials for the treatment of certain types of cancers, including those resistant to erlotinib. In this in vitro study, we conducted in vitro experiments to determine if MK-2206 attenuates multidrug resistance in cancer cells overexpressing the ABCB1 or ABCG2 transporter. Methodology: The efficacy of MK-2206 (0.03-1μM), in combination with the ABCB1 transporter sub-strates doxorubicin and paclitaxel, and ABCG2 transporter substrates mitoxantrone, SN-38 and topotecan, were determined in the cancer cell lines, KB-C2 and SW620/Ad300, which overexpress the ABCB1 transporter or H460/MX20 and S1-M1-80, which overexpress the ABCG2 transporter, respectively. The expression level and the localization of ABCG2 transporter on the cancer cells membranes were determined using western blot and immunofluorescence assays, respectively, following the incubation of cells with MK-2206. Finally, the interaction between MK-2206 and human ABCG2 transporter was predicted using computer-aided molecular modeling. Results: MK-2206 significantly increased the efficacy of anticancer compounds that were substrates for the ABCG2 but not the ABCB1 transporter. MK-2206 alone (0.03-1μM) did not significantly alter the viability of H460/MX20 and S1-M1-80 cancer cells, which overexpress the ABCG2 transporter, compared to cells incubated with vehicle. However, MK-2206 (0.3 and 1μM) significantly increased the anticancer efficacy of mitoxantrone, SN-38 and topotecan, in H460/MX20 and S1-M1-80 cancer cells, as indicated by a significant decrease in their IC50 values, compared to cells incubated with vehicle. MK-2206 significantly increased the basal activity of the ABCG2 ATPase (EC50 = 0.46μM) but did not significantly alter its expression level and sub-localization in the membrane. The molecular modeling results suggested that MK-2206 binds to the active pocket of the ABCG2 transporter, by a hydrogen bond, hydrophobic interactions and π-π stacking. Conclusion: These in vitro data indicated that MK-2206 surmounts resistance to mitoxantrone, SN-38 and topotecan in cancer cells overexpressing the ABCG2 transporter. If these results can be translated to humans, it is possible that MK-2206 could be used to surmount MDR in cancer cells overexpressing the ABCG2 transporter.

Monoacylglycerol Lipase Inhibitor JJKK048 Ameliorates ABCG2 Transporter-Mediated Regorafenib Resistance Induced by Hypoxia in Triple Negative Breast Cancer Cells

Triple negative breast cancer (TNBC) is among the most aggressive and deadly cancer subtypes. Intra-tumoral hypoxia is associated with aggressiveness and drug resistance in TNBC. One of the underlying mechanisms of hypoxia-induced drug resistance is the elevated expression of efflux transporters such as breast cancer resistant protein (ABCG2). In the present study, we investigated the possibility of ameliorating ABCG2-mediated drug resistance in hypoxic TNBC cells by monoacylglycerol lipase (MAGL) inhibition and the consequent downregulation of ABCG2 expression.The effect of MAGL inhibition on ABCG2 expression, function, and efficacy of regorafenib, an ABCG2 substrate was investigated in cobalt dichloride (CoCl2) induced pseudohypoxic TNBC (MDA-MB-231) cells, using quantitative targeted absolute proteomics, qRT-PCR, anti-cancer drug accumulation in the cells, cell invasiveness and resazurin-based cell viability assays.Our results showed that hypoxia-induced ABCG2 expression led to low regorafenib intracellular concentrations, reduced the anti-invasiveness efficacy, and elevated half maximal inhibitory concentration (IC50) of regorafenib in vitro MDA-MB-231 cells. MAGL inhibitor, JJKK048, reduced ABCG2 expression, increased regorafenib cell accumulation, which led to higher regorafenib efficacy.In conclusion, hypoxia-induced regorafenib resistance due to ABCG2 over-expression in TNBC cells can be ameliorated by MAGL inhibition.

Abstract 407: OTS964, a TOPK inhibitor, is susceptible to ABCG2-mediated drug resistance

Abstract The overexpression of ATP-binding cassette (ABC) transporters has known to be one of the most important mechanisms responsible for the development of multidrug resistance (MDR). OTS964 is a potent T-LAK cell-originated protein kinase (TOPK) inhibitor. Herein, we investigated the interaction of OTS964 and MDR-associated ATP-binding cassette sub-family G member 2 (ABCG2, breast cancer resistance protein/BCRP). The cell viability assay indicated that the effectiveness of OTS964 is limited in drug-resistant and gene-transfected cells overexpressing ABCG2. We found that the known ABCG2 inhibitor is able to sensitize ABCG2-overexpressing cells to OTS964. In mechanism-based studies, OTS964 shows inhibitory effect on the efflux function mediated by ABCG2, and in turn, affects the pharmacokinetic profile of other ABCG2 substrate-drugs. Furthermore, OTS964 upregulates ABCG2 protein and mRNA expression levels, resulting in enhanced resistance to ABCG2 substrate-drugs. The ATPase assay demonstrated that OTS964 stimulates ATPase activity of ABCG2 in a concentration-dependent manner, and that this stimulation can be antagonized by a verified ABCG2 ATPase inhibitor. Additionally, the computational molecular docking analysis combined with results from ATPase assay suggested that OTS964 interacts with drug-binding pocket of ABCG2 protein and has substrate-like behaviors. Thus, OTS964 is an MDR-susceptible agent due to its interactions with ABCG2, and overexpression of ABCG2 transporter may attenuate its therapeutic effect in cancer cells. Citation Format: Yuqi Yang, Zhuo-Xun Wu, Jing-Quan Wang, Qiu-Xu Teng, Zi-Ning Lei, Sabrina Lusvarghi, Suresh V. Ambudkar, Ning Ji, Zhe-Sheng Chen. OTS964, a TOPK inhibitor, is susceptible to ABCG2-mediated drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 407.

Development of Simple and Accurate in Silico Ligand-Based Models for Predicting ABCG2 Inhibition.

The ATP binding cassette transporter ABCG2 is a physiologically important drug transporter that has a central role in determining the ADMET (absorption, distribution, metabolism, elimination, and toxicity) profile of therapeutics, and contributes to multidrug resistance. Thus, development of predictive in silico models for the identification of ABCG2 inhibitors is of great interest in the early stage of drug discovery. In this work, by exploiting a large public dataset, a number of ligand-based classification models were developed using partial least squares-discriminant analysis (PLS-DA) with molecular interaction field- and fingerprint-based structural description methods, regarding physicochemical and fragmental properties related to ABCG2 inhibition. An in-house dataset compiled from recently experimental studies was used to rigorously validated the model performance. The key molecular properties and fragments favored to inhibitor binding were discussed in detail, which was further explored by docking simulations. A highly informative chemical property was identified as the principal determinant of ABCG2 inhibition, which was utilized to derive a simple rule that had a strong capability for differentiating inhibitors from non-inhibitors. Furthermore, the incorporation of the rule into the best PLS-DA model significantly improved the classification performance, particularly achieving a high prediction accuracy on the independent in-house set. The integrative model is simple and accurate, which could be applied to the evaluation of drug-transporter interactions in drug development. Also, the dominant molecular features derived from the models may help medicinal chemists in the molecular design of novel inhibitors to circumvent ABCG2-mediated drug resistance.

OTS964, a TOPK Inhibitor, Is Susceptible to ABCG2-Mediated Drug Resistance.

OTS964 is a potent T-LAK cell-originated protein kinase (TOPK) inhibitor. Herein, we investigated the interaction of OTS964 and multidrug resistance (MDR)-associated ATP-binding cassette sub-family G member 2 (ABCG2). The cell viability assay indicated that the effect of OTS964 is limited in cancer drug-resistant and transfected cells overexpressing ABCG2. We found that the known ABCG2 transporter inhibitor has the ability to sensitize ABCG2-overexpressing cells to OTS964. In mechanism-based studies, OTS964 shows inhibitory effect on the efflux function mediated by ABCG2, and in turn, affects the pharmacokinetic profile of other ABCG2 substrate-drugs. Furthermore, OTS964 upregulates ABCG2 protein expression, resulting in enhanced resistance to ABCG2 substrate-drugs. The ATPase assay demonstrated that OTS964 stimulates ATPase activity of ABCG2 in a concentration-dependent manner. The computational molecular docking analysis combined with results from ATPase assay suggested that OTS964 interacts with drug-binding pocket of ABCG2 and has substrate-like behaviors. Thus, OTS964 is an MDR-susceptible agent due to its interactions with ABCG2, and overexpression of ABCG2 transporter may attenuate its therapeutic effect in cancer cells.

BMS-599626, a Highly Selective Pan-HER Kinase Inhibitor, Antagonizes ABCG2-Mediated Drug Resistance

Simple SummaryABC transporters comprise a large group of ATP binding plasma membrane proteins, classified into subfamilies A-G, that transport substrates out of cells to maintain homeostasis. Prolonged exposure to chemotherapeutic drugs leads to increased expression of ABC transporters in cancer cells, resulting in increased efflux and decreased efficacy of anti-neoplastic agents. We found that BMS-599626, at 300 nM, inhibited the function of ABCG2, thereby increasing the efficacy of substrate chemotherapeutic drugs in wild-type as well as mutant ABCG2 overexpressing cells. In addition, BMS-599626 did not alter the expression or intracellular localization of ABCG2 but produced its reversal effect by decreasing efflux and increasing the intracellular accumulation of substrate chemotherapeutic drugs. Finally, BMS-5999626 also inhibited ABCG2 mediated ATP hydrolysis. Overall, our results show that administration of BMS-599626 along with chemotherapeutic drugs can improve the efficacy of chemotherapy in ABC transporter overexpressing cancer cells.Multidrug resistance (MDR) associated with the overexpression of ABC transporters is one of the key causes of chemotherapy failure. Various compounds blocking the function and/or downregulating the expression of these transporters have been developed over the last few decades. However, their potency and toxicity have always been a concern. In this report, we found that BMS-599626 is a highly potent inhibitor of the ABCG2 transporter, inhibiting its efflux function at 300 nM. Our study repositioned BMS-599626, a highly selective pan-HER kinase inhibitor, as a chemosensitizer in ABCG2-overexpressing cell lines. As shown by the cytotoxicity assay results, BMS-599626, at noncytotoxic concentrations, sensitizes ABCG2-overexpressing cells to topotecan and mitoxantrone, two well-known substrates of ABCG2. The results of our radioactive drug accumulation experiment show that the ABCG2-overexpressing cells, treated with BMS-599626, had an increase in the accumulation of substrate chemotherapeutic drugs, as compared to their parental subline cells. Moreover, BMS-599626 did not change the protein expression or cell surface localization of ABCG2 and inhibited its ATPase activity. Our in-silico docking study also supports the interaction of BMS-599626 with the substrate-binding site of ABCG2. Taken together, these results suggest that administration of chemotherapeutic drugs, along with nanomolar concentrations (300 nM) of BMS-599626, may be effective against ABCG2-mediated MDR in clinical settings.

Reversal Effect of ALK Inhibitor NVP-TAE684 on ABCG2-Overexpressing Cancer Cells.

Failure of cancer chemotherapy is mostly due to multidrug resistance (MDR). Overcoming MDR mediated by overexpression of ATP binding cassette (ABC) transporters in cancer cells remains a big challenge. In this study, we explore whether NVP-TAE684, a novel ALK inhibitor which has the potential to inhibit the function of ABC transport, could reverse ABC transporter-mediated MDR. MTT assay was carried out to determine cell viability and reversal effect of NVP-TAE684 in parental and drug resistant cells. Drug accumulation and efflux assay was performed to examine the effect of NVP-TAE684 on the cellular accumulation and efflux of chemotherapeutic drugs. The ATPase activity of ABCG2 transporter in the presence or absence of NVP-TAE684 was conducted to determine the impact of NVP-TAE684 on ATP hydrolysis. Western blot analysis and immunofluorescence assay were used to investigate protein molecules related to MDR. In addition, the interaction between NVP-TAE684 and ABCG2 transporter was investigated via in silico analysis. MTT assay showed that NVP-TAE684 significantly decreased MDR caused byABCG2-, but not ABCC1-transporter. Drug accumulation and efflux tests indicated that the effect of NVP-TAE684 in decreasing MDR was due to the inhibition of efflux function of ABCG2 transporter. However, NVP-TAE684 did not alter the expression or change the subcellular localization of ABCG2 protein. Furthermore, ATPase activity analysis indicated that NVP-TAE684 could stimulate ABCG2 ATPase activity. Molecular in silico analysis showed that NVP-TAE684 interacts with the substrate binding sites of the ABCG2 transporter. Taken together, our study indicates that NVP-TAE684 could reduce the resistance of MDR cells to chemotherapeutic agents, which provides a promising strategy to overcome MDR.

Overexpressing microRNA-34a overcomes ABCG2-mediated drug resistance to 5-FU in side population cells from colon cancer via suppressing DLL1.

Colon cancer side population (SP) cells are a small subset of cancer cells that have cancer stemness capacity and enhanced drug resistance. ABCG2 is a multidrug resistance-related protein in SP cells and has been demonstrated to be regulated by Notch signalling pathway. Recently, microRNAs are reported to play a critical role in SP cell fate. However, their role in ABCG2-mediated drug resistance in colon cancer SP cells remains unclear. In the current study, the different expressions of miR-552, miR-611, miR-34a and miR-5000-3p were compared within SP and non-SP cells, which were separated from human colon cancer cell lines (SW480 and LoVo). We found that miR-34a was significantly down-regulated in SP cells and that overexpressing miR-34a overcame drug resistance to 5-fluorouracil (5-FU). The luciferase reporter assay indicated that miR-34a negatively regulated DLL1, a ligand of Notch signalling pathway, via binding with 3'-untranslated region of its messenger RNA. In addition, overexpressing miR-34a overcame ABCG2-mediated resistance to 5-FU via DLL1/Notch pathway in vitro, and suppressed tumour growth under 5-FU treatment in vivo. In conclusion, our findings suggest that miR-34a acts as a tumour suppressor via enhancing chemosensitivity to 5-FU in SP cells, which provides a novel therapeutic target in chemotherapy-resistant colon cancer.

Tivantinib, A c-Met Inhibitor in Clinical Trials, Is Susceptible to ABCG2-Mediated Drug Resistance.

Tivantinib, also known as ARQ-197, is a potent non-ATP competitive selective c-Met inhibitor currently under phase 3 clinical trial evaluation for liver and lung cancers. In this study, we explored factors that may lead to tivantinib resistance, especially in regards to its interaction with ATP-binding cassette super-family G member 2 (ABCG2). ABCG2 is one of the most important members of the ATP-binding cassette (ABC) transporter family, a group of membrane proteins that play a critical role in mediating multidrug resistance (MDR) in a variety of cancers, including those of the liver and lung. Tivantinib received a high score in docking analysis, indicating a strong interaction between tivantinib and ABCG2, and an ATPase assay indicated that tivantinib stimulated ABCG2 ATPase activity in a concentration-dependent manner. An MTT assay showed that ABCG2 overexpression significantly desensitized both the cancer cells and ABCG2 transfected-HEK293 cells to tivantinib and that this drug resistance can be reversed by ABCG2 inhibitors. Furthermore, tivantinib upregulated the protein expression of ABCG2 without altering the cell surface localization of ABCG2, leading to increased resistance to substrate drugs, such as mitoxantrone. Altogether, these data demonstrate that tivantinib is a substrate of ABCG2, and, therefore, ABCG2 overexpression may decrease its therapeutic effect. Our study provides evidence that the overexpression of ABCG2 should be monitored in clinical settings as an important risk factor for tivantinib drug resistance.

Abstract 3796: Selonsertib, an ASK1 inhibitor, antagonizes ABCB1- and ABCG2-mediated chemotherapeutic drug resistance

Abstract The overexpression of ATP-binding cassette (ABC) transporters has known to be one of the most important mechanisms responsible for the development of multidrug resistance (MDR). Selonsertib, an apoptosis signal-regulating kinase 1 (ASK1) inhibitor, is in phase III clinical trial for the treatment of non-alcoholic steatohepatitis (NASH). In this study, we investigated whether selonsertib could antagonize MDR mediated by ABC transporters, involving in ABCB1, ABCG2, ABCC1 and ABCC10. The results showed that selonsertib significantly reversed ABCB1- and ABCG2-mediated MDR, but not MDR-mediated by ABCC1 or ABCC10. Mechanistically, our studies indicated that the reversal effect of selonsertib was related to the attenuation of the efflux function of ABCB1 and ABCG2 transporters, consequently enhancing intracellular accumulation of substrate drugs. Meanwhile, selonsertib, at reversal concentration, affected neither the expression level of ABCB1 and ABCG2 nor the localization of corresponding proteins in subcellular level. Selonsertib stimulated the ATPase activity of both ABCB1 and ABCG2 in a concentration-dependent manner. Our in silico docking study showed that selonsertib could interact with the substrate-binding sites of both ABCB1 and ABCG2. This study provides a clue into a novel treatment strategy, which includes a combination of selonsertib with antineoplastic drugs to circumvent ABCB1- or ABCG2- mediated MDR. Citation Format: Ning Ji, Yuqi Yang, Chao-Yun Cai, Zi-Ning Lei, Jing-Quan Wang, Pranav Gupta, Suneet Shukla, Suresh V. Ambudkar, Dexin Kong, Zhe-Sheng Chen. Selonsertib, an ASK1 inhibitor, antagonizes ABCB1- and ABCG2-mediated chemotherapeutic drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3796.

Structure activity relationships, multidrug resistance reversal and selectivity of heteroarylphenyl ABCG2 inhibitors

An overexpression of the transmembrane ATP-binding cassette transporter G2 (ABCG2, BCRP) in cancer tissues is supposed to play a role in the multidrug resistance (MDR) of tumors resulting in an inefficient chemotherapy. Therefore, co-administration of selective and non-toxic ABCG2 inhibitors is a promising strategy for improving the efficacy of chemotherapy by blocking ABCG2-mediated export of the cytostatic drugs. In the present study, we designed a small library of 38 novel compounds containing a heteroaryl-phenyl scaffold possessing several (bioisosteric) moieties, and twelve new precursors. We investigated the library for ABCG2 inhibition, for the selectivity against MDR-involved efflux pump ABCB1 (P-gp) and for toxicity. Structure activity relationship (SAR) studies revealed that, at least a phenylheteroaryl-phenylamide scaffold is necessary for observing an ABCG2 inhibition. 4-Methoxy-N-(2-(2-(6-methoxypyridin-3-yl)-2H-tetrazol-5-yl)phenyl)benzamide (43) exhibited a high potency (IC50 = 61 nM)), selectivity, low intrinsic toxicity and reversed the ABCG2-mediated drug resistance in presence of only 0.1 μM.

Hedyotis diffusa Willd overcomes 5-fluorouracil resistance in human colorectal cancer HCT-8/5-FU cells by downregulating the expression of P-glycoprotein and ATP-binding casette subfamily G member 2.

Previous studies have demonstrated that Hedyotis diffusa Willd (HDW), a traditional Chinese herbal medicine, exhibits potent anticancer activity in models of colorectal cancer (CRC). Aggressive forms of CRC exhibit resistance to widely used chemotherapeutic drugs, including the antimetabolite, 5-fluorouracil (5-FU); however, less is known with regard to the activity of HDW against 5-FU-resistant cancer. In the present study, the mechanism of action and the potency of ethanol extracts of HDW (EEHDW) were investigated on a multidrug-resistant CRC HCT-8/5-FU cell line. Using an MTT cell proliferation assay, EEHDW treatment was shown to significantly reduce the cell viability of HCT-8/5-FU cells in a dose- and time-dependent manner. Furthermore, EEHDW significantly increased the retention of the ATP-binding cassette (ABC) transporter substrate, rhodamine-123, as compared with the untreated controls. To further investigate the molecular mechanisms targeted by EEHDW in the resistant cells, the expression levels of the ABC drug transporter protein, P-glycoprotein (P-gp), and ABC subfamily G member 2 (ABCG2), were analyzed using reverse-transcription polymerase chain reaction and western blot analysis. The mRNA and protein expression levels of P-gp and ABCG2 were reduced in the HCT-8/5-FU cells following EEHDW treatment, indicating that EEHDW inhibits ABCG2-mediated drug resistance by downregulating the expression of ABCG2 and P-gp. Therefore, the potential application of EEHDW as a chemotherapeutic adjuvant represents a promising alternative approach to the treatment of drug-resistant CRC.

Overcoming ABCG2-mediated drug resistance with imidazo-[1,2-b]-pyridazine-based Pim1 kinase inhibitors.

Multidrug efflux pumps such as ABCG2 confer drug resistance to a number of cancer types, leading to poor prognosis and outcome. To date, the strategy of directly inhibiting multidrug efflux pumps in order to overcome drug resistance in cancer has been unsuccessful. An alternative strategy is to target proteins involved in the regulation of multidrug efflux pump activity or expression. Pim1 kinase has been demonstrated to phosphorylate ABCG2, promote its oligomerisation and contribute to its ability to confer drug resistance. In the present manuscript, imidazo-pyridazine-based inhibitors of Pim1 were examined for their ability to overcome ABCG2-mediated drug resistance. Drug efficacy was measured as a cytotoxic response or an effect on transport by ABCG2. Protein expression patterns were assessed using western immuno-blotting. The two Pim1 inhibitors increased the potency of flavopiridol, mitoxantrone, topotecan and doxorubicin, specifically in ABCG2-expressing cells. This effect was associated with an increase in the cellular accumulation of [(3)H]-mitoxantrone, suggesting direct impairment of the transporter. However, prolonged pre-incubation with the studied inhibitors greatly enhanced the effect on mitoxantrone accumulation. The inhibitors caused a significant time-dependent reduction in the expression of ABCG2 in the resistant cells, an effect that would improve drug efficacy. Consequently, it appears that the Pim1 inhibitors display a dual-mode effect on ABCG2-expressing cancer cells. This may provide a powerful new strategy in overcoming drug resistance by targeting proteins that regulate expression of efflux pumps.

FL118, a novel camptothecin derivative, is insensitive to ABCG2 expression and shows improved efficacy in comparison with irinotecan in colon and lung cancer models with ABCG2-induced resistance.

BackgroundIrinotecan is a camptothecin analogue currently used in clinical practice to treat advanced colorectal cancer. However, acquired resistance mediated by the drug efflux pump ABCG2 is a recognized problem. We reported on a novel camptothecin analogue, FL118, which shows anticancer activity superior to irinotecan. In this study, we sought to investigate the potency of FL118 versus irinotecan or its active metabolite, SN-38, in both in vitro and in vivo models of human cancer with high ABCG2 activity. We also sought to assess the potency and ABCG2 affinity of several FL118 analogues with B-ring substitutions.MethodsColon and lung cancer cells with and without ABCG2 overexpression were treated with FL118 in the presence and absence of Ko143, an ABCG2-selective inhibitor, or alternatively by genetically modulating ABCG2 expression. Using two distinct in vivo human tumor animal models, we further assessed whether FL118 could extend time to progression in comparison with irinotecan. Lastly, we investigated a series of FL118 analogues with B-ring substitutions for ABCG2 sensitivity.ResultsBoth pharmacological inhibition and genetic modulation of ABCG2 demonstrated that, in contrast to SN-38, FL118 was able to bypass ABCG2-mediated drug resistance. FL118 also extended time to progression in both in vivo models by more than 50% compared with irinotecan. Lastly, we observed that FL118 analogues with polar substitutions had higher affinity for ABCG2, suggesting that the nonpolar nature of FL118 plays a role in bypassing ABCG2-mediated resistance.ConclusionsOur results suggest that in contrast to SN-38 and topotecan, FL118 is a poor substrate for ABCG2 and can effectively overcome ABCG2-mediated drug resistance. Our findings expand the uniqueness of FL118 and support continued development of FL118 as an attractive therapeutic option for patients with drug-refractory cancers resulting from high expression of ABCG2.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0362-9) contains supplementary material, which is available to authorized users.

WHI-P154 enhances the chemotherapeutic effect of anticancer agents in ABCG2-overexpressing cells.

ATP-binding cassette (ABC) transmembrane proteins evidently decrease the intracellular accumulation of substrate chemotherapeutic drugs by extruding them against a concentration gradient, thereby inducing drug resistance. Here we reported the effect of WHI-P154, an irreversible inhibitor of Janus kinase 3 and epidermal growth factor receptor tyrosine kinases, on reversing ABC transporters-mediated drug resistance. We found that WHI-P154 significantly enhanced the sensitivity of ABCG2-overexpressing cells to its substrates. WHI-P154 moderately sensitized ABCB1-overexpressing KB-C2 cells to its substrates whereas showed no sensitizing effect on ABCC1-, ABCC2 or ABCC10-mediated drug resistance. Moreover, WHI-P154 produced a significant increase in the intracellular accumulation of [³H]-mitoxantrone in ABCG2-overexpressing cells. The expression levels nor the localization of the ABCG2 protein was altered after treatment of ABCG2-overexpressing cells with WHI-P154. Further studies indicated that WHI-P154 enhanced the ATPase activity of ABCG2 at low concentrations (<10 μM). Additionally, a docking model predicted the binding conformation of WHI-P154 within the transmembrane region of homology-modeled human ABCG2 transporter. Collectively, these findings highlighted WHI-P154 could significantly reverse ABCG2-mediated multidrug drug resistance by directly blocking the efflux function.

The Overexpression of ABCG2 Reduces the Efficacy of Volasertib (BI 6727) and GSK641364 in Human S1-M1-80 Colon Carcinoma Cells

The polo-like kinase 1 (Plk1) is one of the key regulators in cell cycle progression. Plk1 is overexpressed in many types of cancer and promotes the proliferation of cancer cells. Inhibition of Plk1 activity induces G2/M cell cycle arrest and reduces cancer cell viability. Volasertib and GSK461364 are selective inhibitors of Plk1, active against a wide range of tumor cells at nanomolar concentrations. In this study, while examining the effectiveness of Plk1 inhibitors against multiple human colon cancer cell lines, we discovered that the overexpression of ATP-binding cassette (ABC) drug transporter ABCG2 in human S1-M1-80 colon cancer cells confers resistance to volasertib and GSK461364. Moreover, we found that ABCG2-transfected HEK293 cells were also resistant to both Plk1 inhibitors. We revealed that volasertib and GSK461364 inhibited the function of ABCG2 in a concentration dependent manner, and had no significant effect on the protein expression of ABCG2. More importantly, we showed that the G2/M cell cycle arrest induced by volasertib or GSK461364 was significantly reduced in S1-M1-80 cells, and that ABCG2-mediated drug resistance to Plk1 inhibitors can be restored by inhibition of ABCG2 function. Therefore, the development of ABCG2-mediated drug resistance to volasertib and GSK461364 in cancer clearly present a significant therapeutic challenge, and a better treatment strategy should be further investigated.