Design, synthesis, and biological evaluation of phenylurea indole derivatives as ABCG2 inhibitors.

Three series of phenylurea indole derivatives were synthesized with potent inhibitory activities on ABCG2 with simple and efficient synthetic routes. Among these compounds, four phenylurea indole derivatives 3c-3f with extended π system were discovered as the most potent ABCG2 inhibitors, while these compounds showed no inhibition on ABCB1. Compounds 3c and 3f were selected for further investigation to explore the mechanisms of action on reversing ABCG2-mediated multidrug resistance (MDR). The results revealed that compounds 3c and 3f increased the accumulation of mitoxantrone (MX) in ABCG2-overexpressing cells, but they did not alter the expression level or localization of ABCG2 in cells. In addition, both 3c and 3f significantly stimulated the ATP hydrolysis of ABCG2 transporter indicating that they can be competitive substrates of ABCG2 transporter, and thereby increase the accumulation of mitoxantrone in ABCG2-overexpressing H460/MX20 cells. Both 3c and 3f was docked into the drug-binding site of the human ABCG2 transporter protein (PDB 6FFC) with high affinities. This study showed that extending the π system of phenylurea indole derivatives enhanced their inhibitory activities on ABCG2, which may provide a clue for the further research to discover more potent ABCG2 inhibitors.

Hsa-miR-3178/RhoB/PI3K/Akt, a novel signaling pathway regulates ABC transporters to reverse gemcitabine resistance in pancreatic cancer.

BACKGROUND: Although gemcitabine has been considered as the first-line drug for advanced pancreatic cancer (PC), development of resistance to gemcitabine severely limits the effectiveness of this chemotherapy, and the underlying mechanism of gemcitabine resistance remains unclear. Various factors, such as ATP binding cassette (ABC) transporters, microRNAs and their downstream signaling pathways are included in chemoresistance to gemcitabine. This study investigated the potential mechanisms of microRNAs and ABC transporters related signaling pathways for PC resistance to gemcitabine both in vivo and in vitro. METHODS: Immunohistochemistry and Western blotting were applied to detect the expression of ABC transporters. Molecular docking analysis was performed to explore whether gemcitabine interacted with ABC transporters. Gain-of-function and loss-of-function analyses were performed to investigate the functions of hsa-miR-3178 in vitro and in vivo. Bioinformatics analysis, Western blotting and dual-luciferase reporter assay were used to confirm the downstream regulatory mechanisms of hsa-miR-3178. RESULTS: We found that P-gp, BCRP and MRP1 were highly expressed in gemcitabine-resistant PC tissues and cells. Molecular docking analysis revealed that gemcitabine can bind to the ABC transporters. Hsa-miR-3178 was upregulated in gemcitabine resistance PANC-1 cells as compared to its parental PANC-1 cells. Moreover, we found that hsa-miR-3178 promoted gemcitabine resistance in PC cells. These results were also verified by animal experiments. RhoB was down-regulated in gemcitabine-resistant PC cells and it was a downstream target of hsa-miR-3178. Kaplan-Meier survival curve showed that lower RhoB expression was significantly associated with poor overall survival in PC patients. Rescue assays demonstrated that RhoB could reverse hsa-miR-3178-mediated gemcitabine resistance. Interestingly, hsa-miR-3178 promoted gemcitabine resistance in PC by activating the PI3K/Akt pathway-mediated upregulation of ABC transporters. CONCLUSIONS: Our results indicate that hsa-miR-3178 promotes gemcitabine resistance via RhoB/PI3K/Akt signaling pathway-mediated upregulation of ABC transporters. These findings suggest that hsa-miR-3178 could be a novel therapeutic target for overcoming gemcitabine resistance in PC.

Multidrug resistance proteins (MRPs): Structure, function and the overcoming of cancer multidrug resistance.

ATP-binding cassette (ABC) transporters mediate the ATP-driven translocation of structurally and mechanistically distinct substrates against steep concentration gradients. Among the seven human ABC subfamilies namely ABCA-ABCG, ABCC is the largest subfamily with 13 members. In this respect, 9 of the ABCC members are termed "multidrug resistance proteins" (MRPs1-9) due to their ability to mediate cancer multidrug resistance (MDR) by extruding various chemotherapeutic agents or their metabolites from tumor cells. Furthermore, MRPs are also responsible for the ATP-driven efflux of physiologically important organic anions such as leukotriene C4, folic acid, bile acids and cAMP. Thus, MRPs are involved in important regulatory pathways. Blocking the anticancer drug efflux function of MRPs has shown promising results in overcoming cancer MDR. As a result, many novel MRP modulators have been developed in the past decade. In the current review, we summarize the structure, tissue distribution, biological and pharmacological functions as well as clinical insights of MRPs. Furthermore, recent updates in MRP modulators and their therapeutic applications in clinical trials are also discussed.

Targeting the ubiquitin-proteasome pathway to overcome anti-cancer drug resistance.

Drug resistance is a major obstacle in the field of pre-clinical and clinical therapeutics. The development of novel technologies and targeted therapies have yielded new modalities to overcome drug resistance, but multidrug resistance (MDR) remains one of the major challenges in the treatment of cancer. The ubiquitin-proteasome system (UPS) has a central role in regulating the levels and activities of a multitude of proteins as well as regulation of cell cycle, gene expression, response to oxidative stress, cell survival, cell proliferation and apoptosis. Therefore, inhibition of the UPS could represent a novel strategy for the treatment and overcoming of drug resistance in chemoresistant malignancies. In 2003, bortezomib was approved by the FDA for the treatment of multiple myeloma (MM). However, due to its limitations, second generation proteasome inhibitors (PIs) like carfilzomib, ixazomib, oprozomib, delanzomib and marizomib were introduced which displayed clinical activity in bortezomib-resistant tumors. Past studies have demonstrated that proteasome inhibition potentiates the anti-cancer efficacy of other chemotherapeutic drugs by: i) decreasing the expression of anti-apoptotic proteins such as TNF-α and NF-kB, ii) increasing the levels of Noxa, a pro-apoptotic protein, iii) activating caspases and inducing apoptosis, iv) degrading the pro-survival protein, induced myeloid leukemia cell differentiation protein (MCL1), and v) inhibiting drug efflux transporters. In addition, the mechanism of action of the immunoproteasome inhibitors, ONX-0914 and LU-102, suggested their therapeutic role in the combination treatment with PIs. In the current review, we discuss various PIs and their underlying mechanisms in surmounting anti-tumor drug resistance when used in combination with conventional chemotherapeutic agents.

Overexpression of ABCB1 Transporter Confers Resistance to mTOR Inhibitor WYE-354 in Cancer Cells.

The overexpressing ABCB1 transporter is one of the key factors leading to multidrug resistance (MDR). Thus, many ABCB1 inhibitors have been found to be able to overcome ABCB1-mediated MDR. However, some inhibitors also work as a substrate of ABCB1, which indicates that in order to achieve an effective reversal dosage, a higher concentration is needed to overcome the pumped function of ABCB1, which may concurrently increase the toxicity. WYE-354 is an effective and specific mTOR (mammalian target of rapamycin) inhibitor, which recently has been reported to reverse ABCB1-mediated MDR. In the current study, 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out to determine the cell viability and reversal effect of WYE-354 in parental and drug-resistant cells. Drug accumulation was performed to examine the effect of WYE-354 on the cellular accumulation of chemotherapeutic drugs. The ATPase (adenosine triphosphatase) activity of the ABCB1 transporter in the presence or absence of WYE-354 was conducted in order to determine the impact of WYE-354 on ATP hydrolysis. Western blot analysis and immunofluorescence assay were used to investigate the protein molecules related to MDR. In addition, the interaction between the WYE-354 and ABCB1 transporter was investigated via in silico analysis. We demonstrated that WYE-354 is a substrate of ABCB1, that the overexpression of the ABCB1 transporter decreases the efficacy of WYE-354, and that the resistant WYE-354 can be reversed by an ABCB1 inhibitor at a pharmacological achievable concentration. Furthermore, WYE-354 increased the intracellular accumulation of paclitaxel in the ABCB1-mediated MDR cell line, without affecting the corresponding parental cell line, which indicated that WYE-354 could compete with other chemotherapeutic drugs for the ABCB1 transporter substrate binding site. In addition, WYE-354 received a high score in the docking analysis, indicating a strong interaction between WYE-354 and the ABCB1 transporter. The results of the ATPase analysis showed that WYE-354 could stimulate ABCB1 ATPase activity. Treatment with WYE-354 did not affect the protein expression or subcellular localization of the ABCB1. This study provides evidence that WYE-354 is a substrate of the ABCB1 transporter, implicating that WYE-354 should be avoided for use in ABCB1-mediated MDR cancer.

Chk1 Inhibitor MK-8776 Restores the Sensitivity of Chemotherapeutics in P-glycoprotein Overexpressing Cancer Cells.

P-glycoprotein (P-gp), which is encoded by the ATP-binding cassette (ABC) transporter subfamily B member 1 (ABCB1) gene, is one of the most pivotal ABC transporters that transport its substrates across the cell membrane. Its overexpression is one of the confirmed causes of multidrug resistance (MDR), which results in the failure of cancer treatment. Here, we report that checkpoint kinase (Chk) 1 inhibitor MK-8776, a drug candidate in clinical trial, can restore the sensitivity of chemotherapeutics that are substrates of P-gp in KB-C2, SW620/Ad300 cells and human embryonic kidney (HEK)293/ABCB1 cells that overexpress P-gp. MK-8776 remarkably enhanced the cellular [3H]-paclitaxel accumulation and suppressed the efflux function of P-gp without reducing its expression and affecting its cellular localization in cancer cells. Furthermore, MK-8776 (0-40 µM) stimulated the activity of ATPase in P-gp, which was 4.1-fold greater than the control. In addition, MK-8776 formed a cation-π bond and π-π interaction with key residues of the substrate-binding site in P-gp, as indicated by computer-aided molecular docking study. Our study indicated that MK-8776 may significantly enhance the sensitivity of chemotherapeutics that are substrates of P-gp, providing important information for its application in the reversal of MDR.

Tetrandrine Interaction with ABCB1 Reverses Multidrug Resistance in Cancer Cells Through Competition with Anti-Cancer Drugs Followed by Downregulation of ABCB1 Expression.

The overexpression of ABC transporters induced by anticancer drugs has been found to be the main cause of multidrug resistance. It is actually also a strategy by which cancer cells escape being killed. Tetrandrine is a natural product extracted from the stem of Tinospora crispa. In this study, tetrandrine showed synergistic cytotoxic activity in combinational use with chemotherapeutic drugs, such as Doxorubicin, Vincristine, and Paclitaxel, in both drug-induced and MDR1 gene-transfected cancer cells that over-expressed ABCB1/P-glycoprotein. Tetrandrine stimulated P-glycoprotein ATPase activity, decreased the efflux of [3H]-Paclitaxel and increased the intracellular accumulation of [3H]-Paclitaxel in KB-C2 cells. Furthermore, SW620/Ad300 and KB-C2 cells pretreated with 1 µM tetrandrine for 72 h decreased P-glycoprotein expression without changing its cellular localization. This was demonstrated through Western blotting and immunofluorescence analysis. Interestingly, down-regulation of P-glycoprotein expression was not correlated with gene transcription, as the MDR1 mRNA level exhibited a slight fluctuation in SW620/Ad300 and KB-C2 cells at 0, 24, 48, and 72 h treatment time points. In addition, molecular docking analysis predicted that tetrandrine had inhibitory potential with the ABCB1 transporter. Our results suggested that tetrandrine can antagonize MDR in both drug-selected and MDR1 gene-transfected cancer cells by down regulating the expression of the ABCB1 transporter, followed by increasing the intracellular concentration of chemotherapeutic agents. The combinational therapy using tetrandrine and other anticancer drugs could promote the treatment efficiency of drugs that are substrates of ABCB1.

Synthesis of 3-acyloxyxanthone derivatives as α-glucosidase inhibitors: A further insight into the 3-substituents' effect.

Considerable interest has been attracted in xanthone and its derivatives because of their important biological activities. In this paper, a series of novel 3-arylacyloxyxanthone derivatives 2a-p were synthesized and evaluated for their biological activities toward α-glucosidase. In comparison to the parent 1,3-dihydroxylxanthone 1a, 3-arylacyloxy derivatives 2a-p with additional aromatic ester groups at 3-position show up to 13.7-fold higher inhibitory activities. In particular, the IC50 values of compounds 2i, 2m, 2p reach 13.3, 10.6, 11.6 µM, respectively. These results suggest that addition of aromatic moieties by esterification at the 3-OH of the parent 1,3-dihydroxylxanthone is an efficient way to increase the inhibition against α-glucosidase. Different from previous multi-hydroxylxanthones, these 3-arylacyloxyxanthone derivatives show efficient inhibitory activities may due to the π-stacking or hydrophobic effects of the additional aromatic moieties rather than the H-bonding donor interaction of 3-OH. Structure-activity relationship analysis shows that the substituents on the additional aromatic ring also influence the inhibition. All the oxygen or nitrogen-containing groups, like hydroxyl, methoxy, methaminyl, and alkylsilyloxy, can enhance the inhibitory activities. In addition, the kinetics of enzyme inhibition measured by using Lineweaver-Burk plots shows that selected compounds 2i, 2m and 2p are non-competitive inhibitors. Docking simulations further support our structure-activity relationship analysis that additional aromatic moieties enhance inhibitory activities via hydrophobic effects. The new developed 3-arylacyloxyxanthone derivatives probably bind with α-glucosidase in an allosteric site different from traditional multi-hydroxylxanthones.

Mobocertinib antagonizes multidrug resistance in ABCB1- and ABCG2-overexpressing cancer cells: in vitro and in vivo studies.

Overexpression of ATP-binding cassette (ABC) transporters, particularly ABCB1 and ABCG2, strongly correlates with multidrug resistance (MDR), rendering cancer chemotherapy ineffective. Exploration and identification of novel inhibitors targeting ABCB1 and ABCG2 are necessary to overcome the related MDR. Mobocertinib is an approved EGFR/HER2 inhibitor for non-small cell lung cancer (NSCLC) with EGFR exon 20 insertion mutations. This study demonstrates that mobocertinib can potentially reverse ABCB1- and ABCG2-mediated MDR. Our findings indicate a strong interaction between mobocertinib and these two proteins, supported by its high binding affinity with ABCB1 and ABCG2 models. Through inhibiting the drug efflux function of ABCB1 and ABCG2, mobocertinib facilitates substrate drugs accumulation, thereby re-sensitizing substrate drugs in drug-resistant cancer cells. Additionally, mobocertinib inhibited the ATPase activity of ABCB1 and ABCG2 without changing the expression levels or subcellular localization. In the tumor-bearing mouse model, mobocertinib boosted the antitumor effect of paclitaxel and topotecan, resulting in tumor regression. In summary, our study uncovers a novel potential for repurposing mobocertinib as a dual inhibitor of ABCB1 and ABCG2, and suggests the combination of mobocertinib with substrate drugs as a strategy to counteract MDR.

RN486, a Bruton's Tyrosine Kinase inhibitor, antagonizes multidrug resistance in ABCG2-overexpressing cancer cells.

Background and Objectives: Overcoming ATP-binding cassette subfamily G member 2 (ABCG2)-mediated multidrug resistance (MDR) has attracted the attention of scientists because one of the critical factors resulting in MDR in cancer is the overexpression of ABCG2. RN486, a Bruton's Tyrosine Kinase (BTK) inhibitor, was discovered to potentially reverse ABCB1-mediated MDR. However, there is still uncertainty about whether RN486 has a reversal off-target impact on ABCG2-mediated MDR. Methods: MTT assay was used to detect the reversal effect of RN486 on ABCG2-overexpressing cancer cells. The ABCG2 expression level and subcellular localization were examined by Western blotting and immunofluorescence. Drug accumulation and eflux assay and ATPase assay were performed to analyze the ABCG2 transporter function and ATPase activity. Molecular modeling predicted the binding between RN486 and ABCG2 protein. Results: Non-toxic concentrations of RN486 remarkably increased the sensitivity of ABCG2-overexpressing cancer cells to conventional anticancer drugs mitoxantrone and topotecan. The reversal mechanistic studies showed that RN486 elevated the drug accumulation because of reducing the eflux of ABCG2 substrate drug in ABCG2-overexpressing cancer cells. In addition, the inhibitory efect of RN486 on ABCG2-associated ATPase activity was also verified. Molecular docking study implied a strong binding afinity between RN486 and ABCG2 transporter. Meanwhile, the ABCG2 subcellular localization was not altered by the treatment of RN486, but the expression level of ABCG2 was down-regulated. Conclusions: Our studies propose that RN486 can antagonize ABCG2-mediated MDR in cancer cells via down-regulating the expression level of ABCG2 protein, reducing ATPase activity of ABCG2 transporter, and inhibiting the transporting function. RN486 could be potentially used in conjunction with chemotherapy to alleviate MDR mediated by ABCG2 in cancer.

Design, Synthesis and Biological Evaluation of Quinazolinamine Derivatives as Breast Cancer Resistance Protein and P-Glycoprotein Inhibitors with Improved Metabolic Stability.

A series of twenty-two quinazolinamine derivatives showing potent inhibitory activities on breast cancer resistance protein (BCRP) and p-glycoprotein (P-gp) were synthesized. A cyclopropyl-containing quinazolinamine 22 was identified as a dual BCRP and P-gp inhibitor, while azide-containing quinazolinamine 33 showed BCRP inhibitory activity. These lead compounds were further investigated in a battery of mechanistic experiments. Compound 22 changed the localization of BCRP and P-gp in cells, thus inhibiting the efflux of anticancer drugs by the two ATP-binding cassette (ABC) transporters. In addition, both 22 and 33 significantly stimulated the ATP hydrolysis of the BCRP transporter, indicating that they can be competitive substrates of the BCRP transporter, and thereby increase the accumulation of mitoxantrone in BCRP-overexpressing H460/MX20 cells. Azide derivative 33, exhibited a greater inhibitory effect on BCRP after UV activation and can serve as a valuable probe for investigating the interactions of quinazolinamine derivatives with BCRP. Notably, the dual BCRP and P-gp inhibitors 4-5, 22-24, 27, and BCRP inhibitor 33 showed improved metabolic stability compared to Ko143.

Developing 3-(2-Isocyano-6-methylbenzyl)-1H-indole Derivatives to Enhance the Susceptibility of Serratia marcescens by Occluding Quorum Sensing.

Quorum sensing (QS) inhibition is recognized as a novel antimicrobial target for infections caused by drug-resistant pathogens and is an attractive strategy for antipathogenic agent development. We designed and synthesized three parts of 3-(2-isocyanobenzyl)-1H-indole derivatives and tested their activity as novel quorum sensing inhibitors (QSIs). 3-(2-Isocyanobenzyl)-1H-indole derivatives demonstrated promising QS, biofilms, and prodigiosin inhibitory activities against Serratia marcescens at subminimum inhibitory concentrations (sub-MICs). In particular, 3-(2-isocyano-6-methylbenzyl)-1H-indole (IMBI, 32) was identified as the best candidate based on several screening assays, including biofilm and prodigiosin inhibition. Further studies demonstrated that exposure to IMBI at 1.56 µg/mL to S. marcescens NJ01 significantly inhibited the formation of biofilms by 42%. The IMBI treatment on S. marcescens NJ01 notably enhanced the susceptibility of the formed biofilms, destroying the architecture of the biofilms by up to 40%, as evidenced by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). For interference of virulence factors in S. marcescens NJ01, IMBI at 3.12 µg/mL inhibited the activity of protease and extracellular polysaccharides (EPS) by 17% and 51%, respectively, which were higher than that of the positive control vanillic acid (VAN). Furthermore, IMBI downregulated the expression of QS- and biofilm-related genes fimA, bsmA, pigP, flhC, rssB, fimC, and rsmA by 1.02- to 2.74-fold. To confirm these findings, molecular docking was performed, which indicated that the binding of IMBI to SmaR, RhlI, RhlR, LasR, and CviR could antagonize the expression of QS-linked traits. In addition, molecular dynamic simulations (MD) and energy calculations indicated that the binding of receptors with IMBI was extremely stable. The biofilms of S. marcescens NJ01 were markedly reduced by 50% when IMBI (0.39 µg/mL) was combined with kanamycin (0.15 µg/mL). In conclusion, this study highlights the potency of IMBI in inhibiting the virulence factors of S. marcescens. IMBI has all the potential to be developed as an effective and efficient QS inhibitor and antibiofilm agent in order to restore or improve antimicrobial drug sensitivity.

Cycloruthenated Self-Assembly with Metabolic Inhibition to Efficiently Overcome Multidrug Resistance in Cancers.

The synthesis and the evaluation of the efficacy of a cycloruthenated complex, RuZ, is reported, to overcome multi-drug resistance (MDR) in cancer cells. RuZ can self-assemble into nanoaggregates in the cell culture medium, resulting in a high intracellular concentration of RuZ in MDR cancer cells. The self-assembly significantly decreases oxygen consumption and inhibits glycolysis, which decreases cellular adenosine triphosphate (ATP) levels. The decrease in ATP levels and its low affinity for the ABCB1 and ABCG2 transporters (which mediate MDR) significantly increase the retention of RuZ by MDR cancer cells. Furthermore, RuZ increases cellular oxidative stress, inducing DNA damage, and, in combination with the aforementioned effects of RuZ, increases the apoptosis of cancer cells. Proteomic profiling analysis suggests that the RuZ primarily decreases the expression of proteins that mediate glycolysis and aerobic mitochondrial respiration and increases the expression of proteins involved in apoptosis. RuZ inhibits the proliferation of 35 cancer cell lines, of which 7 cell lines are resistant to clinical drugs. It is also active in doxorubicin-resistant MDA-MB-231/Adr mouse tumor xenografts. To the best of our knowledge, the results are the first to show that self-assembled cycloruthenated complexes are efficacious in inhibiting the growth of MDR cancer cells.

Overexpression of ABCB1 Associated With the Resistance to the KRAS-G12C Specific Inhibitor ARS-1620 in Cancer Cells.

The KRAS-G12C inhibitor ARS-1620, is a novel specific covalent inhibitor of KRAS-G12C, possessing a strong targeting inhibitory effect on KRAS-G12C mutant tumors. Overexpression of ATP-binding cassette super-family B member 1 (ABCB1/P-gp) is one of the pivotal factors contributing to multidrug resistance (MDR), and its association with KRAS mutations has been extensively studied. However, the investigations about the connection between the inhibitors of mutant KRAS and the level of ABC transporters are still missing. In this study, we investigated the potential drug resistance mechanism of ARS-1620 associated with ABCB1. The desensitization effect of ARS-1620 was remarkably intensified in both drug-induced ABCB1-overexpressing cancer cells and ABCB1-transfected cells as confirmed by cell viability assay results. This desensitization of ARS-1620 could be completely reversed when co-treated with an ABCB1 reversal agent. In mechanism-based studies, [3H] -paclitaxel accumulation assay revealed that ARS-1620 could be competitively pumped out by ABCB1. Additionally, it was found that ARS-1620 remarkably stimulated ATPase activity of ABCB1, and the HPLC drug accumulation assay displayed that ARS-1620 was actively transported out of ABCB1-overexpressing cancer cells. ARS-1620 acquired a high docking score in computer molecular docking analysis, implying ARS-1620 could intensely interact with ABCB1 transporters. Taken all together, these data indicated that ARS-1620 is a substrate for ABCB1, and the potential influence of ARS-1620-related cancer therapy on ABCB1-overexpressing cancer cells should be considered in future clinical applications.

VKNG-1 Antagonizes ABCG2-Mediated Multidrug Resistance via p-AKT and Bcl-2 Pathway in Colon Cancer: In Vitro and In Vivo Study.

The emergence of multidrug resistance (MDR) to chemotherapeutic drugs is a major problem in the therapy of cancer. Knowledge of the mechanisms of drug resistance in cancer is necessary for developing efficacious therapies. ATP-binding cassette (ABC) transporters are transmembrane proteins that efflux chemotherapeutic drugs from cancer cells, thereby producing MDR. Our research efforts have led to the discovery of VKNG-1, a compound that selectively inhibits the ABCG2 transporter and reverses resistanctabe to standard anticancer drugs both in vitro and in vivo. VKNG-1, at 6 µM, selectively inhibited ABCG2 transporter and sensitized ABCG2-overexpressing drug-resistant cancer cells to the ABCG2 substrate anticancer drugs mitoxantrone, SN-38, and doxorubicin in ABCG2-overexpressing colon cancers. VKNG- 1 reverses ABCG2-mediated MDR by blocking ABCG2 efflux activity and downregulating ABCG2 expression at the mRNA and protein levels. Moreover, VKNG-1 inhibits the level of phosphorylated protein kinase B (PKB/p-AKT), and B-cell lymphoma-2 (Bcl-2) protein which may overcome resistance to anticancer drugs. However, the in vitro translocation of ABCG2 protein did not occur in the presence of 6 µM of VKNG-1. In addition, VKNG-1 enhanced the anticancer efficacy of irinotecan in ABCG2- overexpressing mouse tumor xenografts. Overall, our results suggest that VKNG-1 may, in combination with certain anticancer drugs, represent a treatment to overcome ABCG2-mediated MDR colon cancers.

Establishment and Characterization of a Topotecan Resistant Non-small Cell Lung Cancer NCI-H460/TPT10 Cell Line.

While topotecan (TPT) is a first- and second-line chemotherapeutic drug in treating lung cancer, the development of drug resistance in tumors still reserves as a major obstacle to chemotherapeutic success. Therefore, a better understanding of the mechanisms of topotecan resistance is critical. In this study, the first topotecan-resistant human non-small cell lung cancer (NSCLC) cell line, termed NCI-H460/TPT10, was established from the parental NCI-H460 cell line. NCI-H460/TPT10 cells exhibited a 394.7-fold resistance to TPT, and cross-resistance to SN-38, mitoxantrone, and doxorubicin, compared to parental NCI-H460 cells. Overexpression of ABCG2 localized on the cell membrane, but not ABCB1 or ABCC1, was found in NCI-H460/TPT10 cells, indicating that ABCG2 was likely to be involved in topotecan-resistance. This was confirmed by the abolishment of drug resistance in NCI-H460/TPT10 cells after ABCG2 knockout. Moreover, the involvement of functional ABCG2 as a drug efflux pump conferring multidrug resistance (MDR) was indicated by low intracellular accumulation of TPT in NCI-H460/TPT10 cells, and the reversal effects by ABCG2 inhibitor Ko143. The NCI-H460/TPT10 cell line and its parental cell line can be useful for drug screening and developing targeted strategies to overcome ABCG2-mediated MDR in NSCLC.

Sitravatinib, a Tyrosine Kinase Inhibitor, Inhibits the Transport Function of ABCG2 and Restores Sensitivity to Chemotherapy-Resistant Cancer Cells in vitro.

Sitravatinib, also called MGCD516 or MG-516, is a broad-spectrum tyrosine kinase inhibitor (TKI) under phase III clinical evaluation. Herein, we explored the activity of sitravatinib toward multidrug resistance (MDR) by emphasizing its inhibitory effect on ATP-binding cassette super-family G member 2 (ABCG2). ABCG2 is a member of ATP-binding cassette (ABC) transporter family and plays a critical role in mediating MDR. Sitravatinb received an outstanding docking score for binding to the human ABCG2 model (PDB code: 6ETI) among thirty screened TKIs. Also, an MTT assay indicated that sitravatinib at 3 µM had the ability to restore the antineoplastic effect of various ABCG2 substrates in both drug-selected and gene-transfected ABCG2-overexpressing cell lines. In further tritium-labeled mitoxantrone transportation study, sitravatinib at 3 µM blocked the efflux function mediated by ABCG2 and as a result, increased the intracellular concentration of anticancer drugs. Interestingly, sitravatinib at 3 µM altered neither protein expression nor subcellular localization of ABCG2. An ATPase assay demonstrated that ATPase activity of ABCG2 was inhibited in a concentration-dependent manner with sitravatinib; thus, the energy source to pump out compounds was interfered. Collectively, the results of this study open new avenues for sitravatinib working as an ABCG2 inhibitor which restores the antineoplastic activity of anticancer drugs known to be ABCG2 substrates.

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.

Modulating the function of ABCB1: in vitro and in vivo characterization of sitravatinib, a tyrosine kinase inhibitor.

BACKGROUND: Overexpression of ATP-binding cassette (ABC) transporter is a major contributor to multidrug resistance (MDR), in which cancer cells acquire resistance to a wide spectrum of chemotherapeutic drugs. In this work, we evaluated the sensitizing effect of sitravatinib, a broad-spectrum tyrosine kinase inhibitor (TKI), on ATP-binding cassette subfamily B member 1 (ABCB1)- and ATP-binding cassette subfamily C member 10 (ABCC10)-mediated MDR. METHODS: MTT assay was conducted to examine cytotoxicity and evaluate the sensitizing effect of sitravatinib at non-toxic concentrations. Tritium-labeled paclitaxel transportation, Western blotting, immunofluorescence analysis, and ATPase assay were carried out to elucidate the mechanism of sitravatinib-induced chemosensitization. The in vitro findings were translated into preclinical evaluation with the establishment of xenograft models. RESULTS: Sitravatinib considerably reversed MDR mediated by ABCB1 and partially antagonized ABCC10-mediated MDR. Our in silico docking simulation analysis indicated that sitravatinib strongly and stably bound to the transmembrane domain of ABCB1 human-mouse chimeric model. Furthermore, sitravatinib inhibited hydrolysis of ATP and synchronously decreased the efflux function of ABCB1. Thus, sitravatinib could considerably enhance the intracellular concentration of anticancer drugs. Interestingly, no significant alterations of both expression level and localization of ABCB1 were observed. More importantly, sitravatinib could remarkably restore the antitumor activity of vincristine in ABCB1-mediated xenograft model without observable toxic effect. CONCLUSIONS: The findings in this study suggest that the combination of sitrvatinib and substrate antineoplastic drugs of ABCB1 could attenuate the MDR mediated by the overexpression of ABCB1.

Biological evaluation of non-basic chalcone CYB-2 as a dual ABCG2/ABCB1 inhibitor.

The enhancement of drug efflux caused by ATP-binding cassette (ABC) transporters (including ABCG2 and ABCB1) overexpression is an important factor for multidrug resistance (MDR) in cancers. After testing the reversal activities of 19 chalcone and bis-chalcone derivatives on MDR cancer cell lines, we found that non-basic chalcone CYB-2 exhibited the most potent reversal activities against both ABCG2- and ABCB1-mediated MDR. The mechanistic studies show that this compound can increase the accumulation of anticancer drugs in both ABCG2- and ABCB1-overexpressing cancer cell lines, resulting from the blocked efflux function of the MDR cancer cell lines. This inhibition is due to the barred ABCG2 and ABCB1 ATPase activities rather than altering the expression or localization of ABCG2 or ABCB1 transporters. The previous studies showed that non-basic chalcones were ABCG2-specific inhibitors; however, we found that non-basic chalcone CYB-2 can be developed as an ABCG2/ABCB1 dual inhibitor to overcome MDR in cancers that co-express both ABCG2 and ABCB1. Moreover, non-basic chalcone CYB-2 has synthetic tractability compared to other chalcone-based derivatives.