Mediated Multidrug Resistance Research Articles

Abstract 3102: A high-throughput screening method for discovering potent P-glycoprotein modulators from the chemical library in Tohoku University

Abstract Background: Drug development is important for cancer therapy to improve patient outcomes. In most cases, drug development starts in discovering candidate compounds from a chemical library in high-throughput screening (HTS). Tohoku University Graduate School of Pharmaceutical Science owns the original chemical library consisting of thousands of original chemical compounds. P-glycoprotein (P-gp), a major drug efflux transporter mediates multi-drug resistance (MDR) in cancer cells. Therefore, P-gp modulators could revere MDR and increase chemosensitivity. The aim of this study is to reveal an adequate method of HTS for discovering potent P-gp modulators from the chemical library in Tohoku University. Methods: The chemical library in Tohoku University had 5861 compounds in the present study, and the collection contains over 7 thousand compounds in 2023. P-gp overexpressing KB-V1, human epidermal carcinoma cell lines were obtained, and calcein AM, P-gp substrate was employed for fluorescent substrate efflux assays. A plate reader-based calcein AM efflux assay for HTS was established to select candidate compounds as P-gp modulators. After the primary HTS, structures of the candidate compounds were analyzed, then several analogous compounds were extracted from the chemical library. For the validation of the hit compounds in HTS and the analogs, a flow cytometry-based calcein AM efflux assay was conducted. In addition, reversal effects of P-gp modulators on the cytotoxicity of paclitaxel were measured in a cell proliferation assay. Results: Of 5861 compounds in the chemical library, 53 compounds showed 2-fold or higher fluorescence intensity, and then 13 compounds showed 3-fold or higher fluorescence intensity in a plate reader-based assay for the HTS. Especially 2 candidate compounds showed over 5-fold fluorescence intensity. In the analysis for structures of the candidate compounds, 20 analogous compounds were extracted from the chemical library. To verify the result of the plate reader-based assay, a flow cytometry-based assay was conducted for 53 hits and 20 analogs. As a result, the 2 candidate compounds that selected in the plate reader-based assay showed 60-fold, the highest fluorescence intensity in a flow cytometry-based assay. The other compounds showed less fluorescence intensity than these 2 candidates. In addition, both candidates enhanced the paclitaxel-induced cytotoxicity in a cell proliferation assay. Conclusions: A plate reader-based calcein AM efflux assay in HTS was an adequate method for discovering potent P-gp modulators from the chemical library in Tohoku University. Citation Format: Norihiko Sugisawa, Shinobu Ohnuma, Takayuki Doi, Michiaki Unno. A high-throughput screening method for discovering potent P-glycoprotein modulators from the chemical library in Tohoku University [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3102.

Design and Synthesis of Cyclolipopeptide Mimics of Dysoxylactam A and Evaluation of the Reversing Potencies against P-Glycoprotein-Mediated Multidrug Resistance.

Inspired by the structure of dysoxylactam A (DLA) that has been demonstrated to reverse P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) effectively, 61 structurally simplified cyclolipopeptides were thus designed and synthesized via an effective method, and their reversing P-gp-mediated MDR potentials were evaluated, which provided a series of more potent analogues and allowed us to explore their structure-activity relationship (SAR). Among them, a well-simplified compound, 56, with only two chiral centers that all derived from amino acids dramatically reversed drug resistance in KBV200 cells at 10 μM in combination with vinorelbine (VNR), paclitaxel (PTX), and adriamycin (ADR), respectively, which is more promising than DLA. The mechanism study showed that 56 reversed the MDR of tumor cells by inhibiting the transport function of P-gp rather than reducing its expression. Notably, compound 56 effectively restored the sensitivity of MDR tumors to VNR in vivo at a dosage without obvious toxicity.

Reversal of Chemoresistance via Staged Liberation of Chemodrug and siRNA in Hierarchical Response to ROS Gradient.

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) often leads to the failure of antitumor chemotherapy, and codelivery of chemodrug with P-gp siRNA (siP-gp) represents a promising approach for treating chemoresistant tumors. To maximize the antitumor efficacy, it is desired that the chemodrug be latently released upon completion of siP-gp-mediated gene silencing, which however, largely remains an unmet demand. Herein, core-shell nanocomplexes (NCs) are developed to overcome MDR via staged liberation of siP-gp and chemodrug (doxorubicin, Dox) in hierarchical response to reactive oxygen species (ROS) concentration gradients. The NCs are constructed from mesoporous silica nanoparticles (MSNs) surface-decorated with cRGD-modified, PEGylated, ditellurium-crosslinked polyethylenimine (RPPT), wherein thioketal-linked dimeric doxorubicin (TK-Dox2 ) and photosensitizer are coencapsulated inside MSNs while siP-gp is embedded in the RPPT polymeric layer. RPPT with ultrahigh ROS-sensitivity can be efficiently degraded by the low-concentration ROS inside cancer cells to trigger siP-gp release. Upon siP-gp-mediated gene silencing and MDR reversal, light irradiation is performed to generate high-concentration, lethal amount of ROS, which cleaves thioketal with low ROS-sensitivity to liberate the monomeric Dox. Such a latent release profile greatly enhances Dox accumulation in Dox-resistant cancercells (MCF-7/ADR) in vitro and in vivo, which cooperates with the generated ROS to efficiently eradicate MCF-7/ADR xenograft tumors.

Outer Membrane Proteins and Efflux Pumps Mediated Multi-Drug Resistance in Salmonella: Rising Threat to Antimicrobial Therapy.

Despite colossal achievements in antibiotic therapy in recent decades, drug-resistant pathogens have remained a leading cause of death and economic loss globally. One such WHO-critical group pathogen is Salmonella. The extensive and inappropriate treatments for Salmonella infections have led from multi-drug resistance (MDR) to extensive drug resistance (XDR). The synergy between efflux-mediated systems and outer membrane proteins (OMPs) may favor MDR in Salmonella. Differential expression of the efflux system and OMPs (influx) and positional mutations are the factors that can be correlated to the development of drug resistance. Insights into the mechanism of influx and efflux of antibiotics can aid in developing a structurally stable molecule that can be proficient at escaping from the resistance loops in Salmonella. Understanding the strategic responsibilities and developing policies to address the surge of drug resistance at the national, regional, and global levels are the needs of the hour. In this Review, we attempt to aggregate all the available research findings and delineate the resistance mechanisms by dissecting the involvement of OMPs and efflux systems. Integrating major OMPs and the efflux system's differential expression and positional mutation in Salmonella may provide insight into developing strategic therapies for one health application.

Repositioning of HMG-CoA Reductase Inhibitors as Adjuvants in the Modulation of Efflux Pump-Mediated Bacterial and Tumor Resistance.

Efflux pump (EP)-mediated multidrug resistance (MDR) seems ubiquitous in bacterial infections and neoplastic diseases. The diversity and lack of specificity of these efflux mechanisms raise a great obstacle in developing drugs that modulate efflux pumps. Since developing novel chemotherapeutic drugs requires large investments, drug repurposing offers a new approach that can provide alternatives as adjuvants in treating resistant microbial infections and progressive cancerous diseases. Hydroxy-methyl-glutaryl coenzyme-A (HMG-CoA) reductase inhibitors, also known as statins, are promising agents in this respect. Originally, statins were used in the therapy of dyslipidemia and for the prevention of cardiovascular diseases; however, extensive research has recently been performed to elucidate the functions of statins in bacterial infections and cancers. The mevalonate pathway is essential in the posttranslational modification of proteins related to vital eukaryotic cell functions. In this article, a comparative review is given about the possible role of HMG-CoA reductase inhibitors in managing diseases of bacterial and neoplastic origin. Molecular research and clinical studies have proven the justification of statins in this field. Further well-designed clinical trials are urged to clarify the significance of the contribution of statins to the lower risk of disease progression in bacterial infections and cancerous diseases.

Pregnane steroid glycosides with multidrug resistance reversal activity from the stems of Marsdenia tenacissima

Eighteen previously unreported pregnane glycosides, namely marsdenosides S1–S18, along with 15 known analogues, have been isolated from the stems of Marsdenia tenacissima. The structures of the undescribed compounds were elucidated by spectroscopic means, and their absolute configurations were established on the basis of time-dependent density functional theory (TD-DFT) based electronic circular dichroism (ECD) calculation, X-ray crystallography and acid hydrolysis. All the isolates were evaluated for their chemo-reversal ability against P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in MCF-7/ADR cell line, and nine ones displayed moderate MDR reversal activity with reversal folds in the range of 2.45–9.01. The most active 12-O-acetyl-20-O-benzoyl-(14,17,18-orthoacetate)-dihydrosarcostin-3-O-β-d-thevetopyranosyl-(1 → 4)-O-β-d-oleandropyranosyl-(1 → 4)-O-β-d-cymaropyranoside increased the sensitivity of MCF-7/ADR cell to adriamycin comparably to the reference drug verapamil (RF = 8.93).

Pregnane steroids and steroid glycosides with multidrug resistance reversal activity from Marsdenia tenacissima

Twenty compounds comprising four pregnane steroids (2-4 & 20) and 16 pregnane glycosides (1 & 5-19) have been obtained from the ethanol extract of the roots of a Dai ethnological herb, Marsdenia tenacissima. Their structures were characterized on the basis of comprehensive spectroscopic analyses with 17 ones (1-17) being reported for the first time, including the rare cases (2 & 3) of free C21 steroids with 17α-acetyl substitution, compounds 4-7 bearing an unusual 14α-OH, and the first examples with simultaneous 14α-OH/17α-acetyl substitution (7) and glycosylation at C-12 position (10 & 11). An empirical rule for the identification of C-17 configuration, in C21 steroids incorporating the marsdenin constitution structure, was also proposed. All the isolates, along with an array of previously reported analogues in our compound library, were screened for their chemo-reversal ability against P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in MCF-7/ADR cell line, and six compounds exhibited moderate MDR reversal activity with reversal folds ranging from 1.92 to 4.44.

A multi-functional nano-system combining PI3K-110α/β inhibitor overcomes P-glycoprotein mediated MDR and improves anti-cancer efficiency

P-glycoprotein (P-gp/ABCB1)-mediated multidrug resistance (MDR) in cancers severely limit chemotherapeutic efficacy. We recently reported that phosphatidylinositol-3-kinase (PI3K) 110α and 110β subunits can be novel targets for reversal of P-gp mediated MDR in cancers, and BAY-1082439 as an inhibitor specific for PI3K 110α and 110β subunits could reverse P-gp-mediated MDR by downregulating P-gp expression in cancer cells. However, BAY-1082439 has very low solubility, short half-life and high in-vivo clearance rate. Till now, nano-system with the functions to target PI3K P110α and P110β and reverse P-gp mediated MDR in cancers has not been reported. In our study, a tumor targeting drug delivery nano-system PBDF was established, which comprised doxorubicin (DOX) and BAY-1082439 respectively encapsulated by biodegradable PLGA-SH nanoparticles (NPs) that were grafted to gold nanorods (Au NRs) modified with FA-PEG-SH, to enhance the efficacy to reverse P-gp mediated MDR and to target tumor cells, further, to enhance the efficiency to inhibit MDR tumors overexpressing P-gp. In-vitro experiments indicated that PBDF NPs greatly enhanced uptake of DOX, improved the activity to reverse MDR, inhibited the cell proliferation, and induced S-phase arrest and apoptosis in KB-C2 cells, as compared with free DOX combining free BAY-1082439. In-vivo experiments further demonstrated that PBDF NPs improved the anti-tumor ability of DOX and inhibited development of KB-C2 tumors. Notably, the metastasis of KB-C2 cells in livers and lungs of nude mice were inhibited by treatment with PBDF NPs, which showed no obvious in-vitro or in-vivo toxicity.

Abstract 2843: RN486, a Bruton’s tyrosine kinase inhibitor, reverses multidrug resistance in ABCG2-overexpressing cancer cells

Abstract 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 leading to MDR is the overexpression of ABCG2. Identification of novel chemotherapeutic reagents as inhibitors of ABCG2 has been considered an effective strategy. RN486, a Bruton’s Tyrosine Kinase (BTK) inhibitor, was discovered to potentially reverse ABCB1-mediated MDR. In this study, we reported that RN486 effectively antagonizes ABCG2-mediated MDR in cancer cells. 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 increased the accumulation and decreased the efflux of ABCG2 substrate drug in ABCG2-overexpressing cancer cells. In addition, the inhibitory effect of RN486 on ABCG2-associated ATPase activity was also verified. Docking analysis indicated a strong binding between RN486 and ABCG2 transporter. Meanwhile, the AGCG2 subcellular localization and expression level were not altered by the treatment of RN486. Taken together, our studies suggest that RN486 can antagonize ABCG2-mediated MDR in cancer cells via interacting with ABCG2 and inhibiting the transporting function. RN486 could be potentially used in combination with chemotherapy against ABCG2-mediated MDR in cancers. Citation Format: Xing-Duo Dong, Qisi Lu, Yi-Dong Li, Qiu-Xu Teng, Zi-Ning Lei, Zhe-Sheng Chen. RN486, a Bruton’s tyrosine kinase inhibitor, reverses multidrug resistance in ABCG2-overexpressing cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2843.

Exploration of the inhibition action of TPGS on tumor cells and its combined use with chemotherapy drugs

D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) is a commonly used nonionic surfactant used as a pharmaceutical carrier in different drug delivery systems. TPGS can reverse P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) and also has anticancer activities. It suggests that when designing antitumor drug preparation, it’s necessary to take into account the antitumor activity of TPGS. Our in vivo studies showed that TPGS exerted the strongest cytotoxicity in MCF-7-ADR cells when compared with seven other tumor cell lines. The further study revealed TPGS caused apoptosis and blocked MCF-7 cell growth in G2/M phase. Mechanistic insights suggested that TPGS increased intracellular calcium ion concentrations, leading to apoptosis via the mitochondrial pathway. Furthermore, two in vivo experiments were performed. One was TPGS, and DOX solution was administered by tail vein injection on MCF-7-ADR tumor bearing nude mice. The other was temperature sensitive TPGS gel (TPGS-TG) was administered by intratumoral injection on MCF-7-ADR tumor bearing nude mice combined with paclitaxel albumin nanoparticles (Abraxane®) administered by tail vein injection. The findings confirmed that TPGS could play its role in anti-tumor to reduce the toxicity of chemotherapeutic drugs and improve the efficiency of drug-resistant tumors, thereby enhancing the development of safe oncology therapeutics.

Enhanced delivery of quercetin and doxorubicin using β-cyclodextrin polymer to overcome P-glycoprotein mediated multidrug resistance

In this study, we prepared a β-cyclodextrin polymer (β-CDP) co-loaded quercetin (QCT) and doxorubicin (DOX) nanocarrier (β-CDP/QD NCs) by freeze-dried method to combat P-glycoprotein (P-gp) mediated multidrug resistance (MDR) in KB-ChR 8–5 cancer cells. Various microscopic and spectroscopic techniques were employed to characterize the prepared nanocarrier. The molecular docking studies confirm the effective binding interactions of QCT and DOX with the synthesized β-CD polymer. The in vitro drug release study illustrates the sustainable release of DOX and QCT from the β-CDP nanocarrier. Further, we noticed that the QCT released from the β-CDP nanocarrier improved the intracellular availability of DOX via modulating P-gp drug efflux function in KB-ChR 8–5 cells and MCF-7/DOX cancer cells. Cell uptake results confirmed the successful internalization of DOX in KB-ChR 8–5 cells compared with free DOX. Cell-based assays such as nuclear condensation, alteration in the mitochondrial membrane potential (MMP), and apoptosis morphological changes confirmed the enhanced anticancer effect of β-CDP/QD NCs in the resistant cancer cells. Hence, QCT and DOX co-loaded β-CDP may be considered effective in achieving maximum cell death in the P-gp overexpressing MDR cancer cells.

Modular Biomimetic Strategy Enabled Discovery of Simplified Pseudo-Natural Macrocyclic P-Glycoprotein Inhibitors Capable of Overcoming Multidrug Resistance.

Natural macrocycles have shown impressive activity to overcome P-glycoprotein (P-gp)-mediated multidrug resistance (MDR). However, the total synthesis and structural modification of natural macrocycles are challenging, which would hamper the deeper investigations of their structure-activity relationship (SAR) and drug likeness. Herein, we describe a modular biomimetic strategy to expeditiously achieve a new class of macrocycles featuring polysubstituted 1,3-diene, which efficiently inhibited P-gp and reversed MDR in cancer cells. The SAR analysis revealed that the size and linker of the macrocycles are important structural characteristics to restore activity. Particularly, 32 containing a naphthyl group and (d)-Phe moiety has higher potency with an excellent reversal fold than verapamil at a concentration of 5 μM, which induces conformational change of P-gp and inhibits its function instead of altering P-gp expression. Furthermore, 23 and 32 were identified to be attractive leads, which possess a good pharmacokinetic profile and antitumor activity in a KBV200 xenograft mouse model.

Concise Total Synthesis of Dysoxylactam A and a Simplified Analog

Comprehensive SummaryA total synthesis of 17‐membered macrocyclolipopeptide dysoxylactam A, a potent agent reversing P‐glycoprotein (P‐gp)‐mediated multidrug resistance (MDR) in cancer cells, was developed from the starting material (S)‐2‐methylbutanal in a linear sequence of 12 steps with 23.2% overall yield. The key steps include proline‐catalyzed asymmetric aldol reaction, Evans aldol reaction and Krische allylation to construct the multiple stereocenters containing fragment, and ring‐closing metathesis to furnish the macrocycle. This total synthesis facilitates the preparation of large amount samples of dysoxylactam A and the side chain simplified derivatives for further biological tests and preliminary structure‐activity relationship exploration.

A New Twist in ABC Transporter Mediated Multidrug Resistance – Pdr5 is a Drug/proton Co-transporter

The two major efflux pump systems that are involved in multidrug resistance (MDR) are (i) ATP binding cassette (ABC) transporters and (ii) secondary transporters. While the former use binding and hydrolysis of ATP to facilitate export of cytotoxic compounds, the latter utilize electrochemical gradients to expel their substrates. Pdr5 from Saccharomyces cerevisiae is a prominent member of eukaryotic ATP binding cassette (ABC) transporters that are involved in multidrug resistance (MDR) and used as a frequently studied model system. Although investigated for decades, the underlying molecular mechanisms of drug transport and substrate specificity remain elusive. Here, we provide electrophysiological data on the reconstituted Pdr5 demonstrating that this MDR efflux pump does not only actively translocate its substrates across the lipid bilayer, but at the same time generates a proton motif force in the presence of Mg2+-ATP and substrates by acting as a proton/drug co-transporter. Importantly, a strictly substrate dependent co-transport of protons was also observed in in vitro transport studies using Pdr5-enriched plasma membranes. We conclude from these results that the mechanism of MDR conferred by Pdr5 and likely other transporters is more complex than the sole extrusion of cytotoxic compounds and involves secondary coupled processes suitable to increase the effectiveness.

Interaction of crown ethers with the ABCG2 transporter and their implication for multidrug resistance reversal.

Overexpression of ABC transporters, such as ABCB1 and ABCG2, plays an important role in mediating multidrug resistance (MDR) in cancer. This feature is also attributed to a subpopulation of cancer stem cells (CSCs), having enhanced tumourigenic potential. ABCG2 is specifically associated with the CSC phenotype, making it a valuable target for eliminating aggressive and resistant cells. Several natural and synthetic ionophores have been discovered as CSC-selective drugs that may also have MDR-reversing ability, whereas their interaction with ABCG2 has not yet been explored. We previously reported the biological activities, including ABCB1 inhibition, of a group of adamantane-substituted diaza-18-crown-6 (DAC) compounds that possess ionophore capabilities. In this study, we investigated the mechanism of ABCG2-inhibitory activity of DAC compounds and the natural ionophores salinomycin, monensin and nigericin. We used a series of functional assays, including real-time microscopic analysis of ABCG2-mediated fluorescent substrate transport in cells, and docking studies to provide comparative aspects for the transporter-compound interactions and their role in restoring chemosensitivity. We found that natural ionophores did not inhibit ABCG2, suggesting that their CSC selectivity is likely mediated by other mechanisms. In contrast, DACs with amide linkage in the side arms demonstrated noteworthy ABCG2-inhibitory activity, with DAC-3Amide proving to be the most potent. This compound induced conformational changes of the transporter and likely binds to both Cavity 1 and the NBD-TMD interface. DAC-3Amide reversed ABCG2-mediated MDR in model cells, without affecting ABCG2 expression or localization. These results pave the way for the development of new crown ether compounds with improved ABCG2-inhibitory properties.

Cytotoxicity of cinchona alkaloid organocatalysts against MES-SA and MES-SA/Dx5 multidrug-resistant uterine sarcoma cell lines

Since the first application of natural quinine as an anti-malarial drug, cinchona alkaloids and their derivatives have been exhaustively studied for their biological activity. In our work, we tested 13 cinchona alkaloid organocatalysts, synthesised from quinine. These derivatives were screened against MES-SA and Dx5 uterine sarcoma cell lines for in vitro anticancer activity and to investigate their potential to overcome P-glycoprotein (P-gp) mediated multidrug resistance (MDR). Decorating quinine with hydrogen-bond donor units, such as thiourea and (thio)squaramide, resulted in decreased half-maximal growth inhibition values on both cell lines (1.3–21 µM) compared to quinine and other cinchona alcohols (47–111 µM). Further cytotoxicity studies conducted in the presence of the P-gp inhibitor tariquidar indicated that several analogues, especially cinchona amines and squaramides, but not thiosquaramide, were expelled from MDR cells by P-gp. Similarly to the established P-gp inhibitor quinine, 6 cinchona analogues were shown to inhibit calcein-AM efflux. Interestingly, quinine and didehydroquinine exhibited a marginally increased toxicity against the multidrug resistant Dx5 cells. Collateral sensitivity of the MDR cell line was more pronounced when the cinchona thiosquaramide was complexed with Cu(II) acetate. Based on the results, cinchona derivatives are good anticancer candidates for further drug development.

CDK6-PI3K signaling axis is an efficient target for attenuating ABCB1/P-gp mediated multi-drug resistance (MDR) in cancer cells

BackgroundMultidrug resistance (MDR) mediated by ATP binding cassette subfamily B member 1 (ABCB1/P-gp) is a major cause of cancer chemotherapy failure, but the regulation mechanisms are largely unknown.MethodsBased on single gene knockout, we studied the regulation of CDK6-PI3K axis on ABCB1-mediated MDR in human cancer cells. CRISPR/Cas9 technique was performed in KB-C2 cells to knockout cdk6 or cdk4 gene. Western blot, RT-PCR and transcriptome analysis were performed to investigate target gene deletion and expression of critical signaling factors. The effect of cdk4 or cdk6 deficiency on cell apoptosis and the cell cycle was analyzed using flow cytometry. In vivo studies were performed to study the sensitivity of KB-C2 tumors to doxorubicin, tumor growth and metastasis.ResultsDeficiency of cdk6 led to remarkable downregulation of ABCB1 expression and reversal of ABCB1-mediated MDR. Transcriptomic analysis revealed that CDK6 knockout regulated a series of signaling factors, among them, PI3K 110α and 110β, KRAS and MAPK10 were downregulated, and FOS-promoting cell autophagy and CXCL1-regulating multiple factors were upregulated. Notably, PI3K 110α/110β deficiency in-return downregulated CDK6 and the CDK6-PI3K axis synergizes in regulating ABCB1 expression, which strengthened the regulation of ABCB1 over single regulation by either CDK6 or PI3K 110α/110β. High frequency of alternative splicing (AS) of premature ABCB1 mRNA induced by CDK6, CDK4 or PI3K 110α/110β level change was confirmed to alter the ABCB1 level, among them 10 common skipped exon (SE) events were found. In vivo experiments demonstrated that loss of cdk6 remarkably increased the sensitivity of KB-C2 tumors to doxorubicin by increasing drug accumulation of the tumors, resulting in remarkable inhibition of tumor growth and metastasis, as well as KB-C2 survival in the nude mice.ConclusionsCDK6-PI3K as a new target signaling axis to reverse ABCB1-mediated MDR is reported for the first time in cancers. Pathways leading to inhibition of cancer cell proliferation were revealed to be accompanied by CDK6 deficiency.

Double-Coated Poly(butyl Cyanoacrylate) Nanoparticles as a Potential Carrier for Overcoming P-Gp- and BCRP-Mediated Multidrug Resistance in Cancer Cells

The present study evaluates poly (butyl cyanoacrylate) nanoparticles (PBCA-NPs), double-coated with Tween 80 and polyethylene glycol (PEG) 20,000 as a potential carrier system for overcoming P-glycoprotein (P-gp) and breast cancer resistant protein (BCRP)-mediated multidrug resistance (MDR) in cancer cell lines. Doxorubicin-loaded PBCA-NPs were prepared by the anionic polymerization method and were successively double-coated with Tween 80 and PEG 20000 at varied concentrations. MDR reversing potential was investigated by cellular uptake in P-gp overexpressing cell line. And, the outcomes were verified by modified MTT assay in P-gp or BCRP overexpressing cell lines. The findings from the cell uptake study indicate that double-coated PBCA-NPs significantly enhanced doxorubicin accumulation within the cells. MTT assays revealed that double-coated PBCA-NPs significantly potentiated the sensitivity of doxorubicin in P-gp overexpressing cells, in comparison to free doxorubicin, single-, and un-coated PBCA-NPs, respectively. Moreover, further increase in concentration with Tween 80, double-coated PBCA-NPs significantly enhanced the sensitivity of doxorubicin in BCRP overexpressing cell line, in comparison to single- and double-coated formulations (with lower concentration of Tween 80). Hence, it could be concluded that double-coated PBCA-NPs can be used as a potential carrier for enhancing doxorubicin accumulation in MDR cancer cells.

Overexpression of ABCG2 Confers Resistance to MLN7243, a Ubiquitin-Activating Enzyme (UAE) Inhibitor.

Overexpression of ATP-binding cassette transporter superfamily G member 2 (ABCG2), is known as a major mechanism mediating multidrug resistance (MDR) in cancer cells. MLN7243 is a small-molecule ubiquitin activating enzyme inhibitor currently under clinical investigation. The aim of the current study is to determine if MLN7243 is a substrate of MDR-related ABCG2 transporter. Our results showed that cancer cells overexpressing ABCG2 transporter were resistant to MLN7243 compared to the parental cells, while knockout of ABCG2 gene or pharmacological inhibition of ABCG2 efflux function completely reversed the drug resistance. Unexpectedly, the endogenous low expression of ABCG2 is sufficient to confer cancer cells resistance to MLN7243. The ABCG2 ATPase assay and HPLC assay suggested that MLN7243 can significantly stimulate ABCG2 ATPase activity and be pumped out from ABCG2-overexpressing cells by ABCG2. The docking analysis also implied that MLN7243 binds to ABCG2 drug-binding pocket with optimal binding affinity. However, MLN7243 did not competitively inhibit the efflux of other ABCG2 substrate drugs, indicating it may not serve as an MDR reversal agent. In conclusion, our study provides direct in vitro evidence to show that MLN7243 is a potent ABCG2 substrate. If our results can be translated to humans, it suggests that combining MLN7243 with ABCG2 inhibitors may enhance the anticancer efficacy for patients with high tumor ABCG2 level.

Rational design of phenyl thiophene (pyridine) derivatives that overcome P-glycoprotein mediated MDR in MCF-7/ADR cell

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) has become an important hindrance in the clinical treatment of malignant tumors. In this paper, based on our lead compound 5m and strategies of bioisosteric replacement and alkoxy effect, phenylthiophene and phenylpyridine derivatives were designed as chemosensitizers for front-line antineoplastic agents and overcomes P-gp mediated MDR in MCF-7/ADR cell. Generally, 4-phenylthiophene-2-carboxamide derivative 60 has been screened and obtained with optimal activity against P-gp mediated MDR in MCF-7/ADR (IC50 (doxorubicin) = 1.02 μM, RF = 49.9 with 5 μM 60 treated). The results of western blot and Rh123 accumulation assays showed that 60 effectively inhibited P-gp efflux function but not its expression. It is noted that compound 60 is a potentially broad-spectrum chemosensitizer in combination with commonly used anti-tumor drugs, such as doxorubicin, paclitaxel, daunorubicin and vincristine with RF value of 20–80.