Mechanistic insights into P-glycoprotein ligand transport and inhibition revealed by enhanced molecular dynamics simulations.

P-glycoprotein (P-gp) plays a crucial role in cellular detoxification and drug efflux processes, transitioning between inward-facing (IF) open, occluded, and outward-facing (OF) states to facilitate substrate transport. Its role is critical in cancer therapy, where P-gp contributes to the multidrug resistance phenotype. In our study, classical and enhanced molecular dynamics (MD) simulations were conducted to dissect the structural and functional features of the P-gp conformational states. Our advanced MD simulations, including kinetically excited targeted MD (ketMD) and adiabatic biasing MD (ABMD), provided deeper insights into state transition and translocation mechanisms. Our findings suggest that the unkinking of TM4 and TM10 helices is a prerequisite for correctly achieving the outward conformation. Simulations of the IF-occluded conformations, characterized by kinked TM4 and TM10 helices, consistently demonstrated altered communication between the transmembrane domains (TMDs) and nucleotide binding domain 2 (NBD2), suggesting the implication of this interface in inhibiting P-gp's efflux function. A particular emphasis was placed on the unstructured linker segment connecting the NBD1 to TMD2 and its role in the transporter's dynamics. With the linker present, we specifically noticed a potential entrance of cholesterol (CHOL) through the TM4-TM6 portal, shedding light on crucial residues involved in accommodating CHOL. We therefore suggest that this entry mechanism could be employed for some P-gp substrates or inhibitors. Our results provide critical data for understanding P-gp functioning and developing new P-gp inhibitors for establishing more effective strategies against multidrug resistance.

BI-2865, a pan-KRAS inhibitor, reverses the P-glycoprotein induced multidrug resistance in vitro and in vivo.

BACKGROUND: Multidrug resistance (MDR) limits successful cancer chemotherapy. P-glycoprotein (P-gp), BCRP and MRP1 are the key triggers of MDR. Unfortunately, no MDR modulator was approved by FDA to date. Here, we will investigate the effect of BI-2865, a pan-KRAS inhibitor, on reversing MDR induced by P-gp, BCRP and MRP1 in vitro and in vivo, and its reversal mechanisms will be explored. METHODS: The cytotoxicity of BI-2865 and its MDR removal effect in vitro were tested by MTT assays, and the corresponding reversal function in vivo was assessed through the P-gp mediated KBv200 xenografts in mice. BI-2865 induced alterations of drug discharge and reservation in cells were estimated by experiments of Flow cytometry with fluorescent doxorubicin, and the chemo-drug accumulation in xenografts' tumor were analyzed through LC-MS. Mechanisms of BI-2865 inhibiting P-gp substrate's efflux were analyzed through the vanadate-sensitive ATPase assay, [125I]-IAAP-photolabeling assay and computer molecular docking. The effects of BI-2865 on P-gp expression and KRAS-downstream signaling were detected via Western blotting, Flow cytometry and/or qRT-PCR. Subcellular localization of P-gp was visualized by Immunofluorescence. RESULTS: We found BI-2865 notably fortified response of P-gp-driven MDR cancer cells to the administration of chemo-drugs including paclitaxel, vincristine and doxorubicin, while such an effect was not observed in their parental sensitive cells and BCRP or MRP1-driven MDR cells. Importantly, the mice vivo combination study has verified that BI-2865 effectively improved the anti-tumor action of paclitaxel without toxic injury. In mechanism, BI-2865 prompted doxorubicin accumulating in carcinoma cells by directly blocking the efflux function of P-gp, which more specifically, was achieved by BI-2865 competitively binding to the drug-binding sites of P-gp. What's more, at the effective MDR reversal concentrations, BI-2865 neither varied the expression and location of P-gp nor reduced its downstream AKT or ERK1/2 signaling activity. CONCLUSIONS: This study uncovered a new application of BI-2865 as a MDR modulator, which might be used to effectively, safely and specifically improve chemotherapeutic efficacy in the clinical P-gp mediated MDR refractory cancers.

Interdomain communication in a homodimeric ABC transporter.

ABC transporters are found in all organisms and almost every cellular compartment. They mediate the transport of various solutes across membranes, energized by ATP binding and hydrolysis. Dysfunctions can result in severe diseases, such as cystic fibrosis or antibiotic resistance. In type IV ABC transporters, each of the two nucleotide-binding domains is connected to a transmembrane domain by two coupling helices, which are part of cytosolic loops. Although there are many structural snapshots of different conformations, the interdomain communication is still enigmatic. Therefore, we analyzed the function of three conserved charged residues in the intracytosolic loop 1 of the human homodimeric, lysosomal peptide transporter TAPL (transporter associated with antigen processing-like). Substitution of D278 in coupling helix 1 by alanine interrupted peptide transport by impeding ATP hydrolysis. Alanine substitution of R288 and D292, both localized next to the coupling helix 1 extending to transmembrane helix 3, reduced peptide transport but increased basal ATPase activity. Surprisingly, the ATPase activity of the R288A variant dropped in a peptide-dependent manner, whereas ATPase activity of wildtype and D292A was unaffected. Interestingly, R288A and D292A mutants did not differentiate between ATP and GTP in respect of hydrolysis. However, in contrast to wildtye TAPL, only ATP energized peptide transport. In sum, D278 seems to be involved in bidirectional interdomain communication mediated by network of polar interactions, whereas the two residues in the cytosolic extension of transmembrane helix 3 are involved in regulation of ATP hydrolysis, most likely by stabilization of the outward-facing conformation.

Minimal Involvement of P-gp and BCRP in Oral Absorption of Ensitrelvir, An Oral SARS-CoV-2 3C-like Protease Inhibitor, in a Non-Clinical Investigation.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are important transporters causing drug-drug interaction (DDI). Here, we investigated the involvement of P-gp and BCRP in the oral absorption of ensitrelvir in non-clinical studies and estimated the DDI risk mediated by P-gp and BCRP inhibition in humans. Although ensitrelvir is an in vitro P-gp and BCRP substrate, it demonstrated high bioavailability in rats and monkeys after oral administration. Plasma exposures of ensitrelvir following oral administration were comparable in wild type (WT) and Bcrp (-/-) mice. On the other hand, the area under the plasma concentration-time curve (AUC) ratio of ensitrelvir in the Mdr1a/1b (-/-) mice to the WT mice was 1.92, indicating that P-gp, but not BCRP, was involved in the oral absorption of ensitrelvir. Based on our previous retrospective analyses, such a low AUC ratio (<3) in the Mdr1a/1b (-/-) mice indicates a minimal impact of P-gp on the oral absorption in humans. In conclusion, our studies demonstrate that the involvement of both P-gp and BCRP in the oral absorption of ensitrelvir is minimal, and suggest that ensitrelvir has a low risk for DDIs mediated by P-gp and BCRP inhibition in humans.

The impact of ATP-binding cassette transporters in the diseased brain: Context matters.

ATP-binding cassette (ABC) transporters facilitate the movement of diverse molecules across cellular membranes, including those within the CNS. While most extensively studied in microvascular endothelial cells forming the blood-brain barrier (BBB), other CNS cell types also express these transporters. Importantly, disruptions in the CNS microenvironment during disease can alter transporter expression and function. Through this comprehensive review, we explore the modulation of ABC transporters in various brain pathologies and the context-dependent consequences of these changes. For instance, downregulation of ABCB1 may exacerbate amyloid beta plaque deposition in Alzheimer's disease and facilitate neurotoxic compound entry in Parkinson's disease. Upregulation may worsen neuroinflammation by aiding chemokine-mediated CD8 T cell influx into multiple sclerosis lesions. Overall, ABC transporters at the BBB hinder drug entry, presenting challenges for effective pharmacotherapy. Understanding the context-dependent changes in ABC transporter expression and function is crucial for elucidating the etiology and developing treatments for brain diseases.

New uracil analog as inhibitor/modulator of ABC transporters or/and NF-κB in taxol-resistant MCF-7/Tx cell line.

PURPOSE: The global increase in breast cancer cases necessitates ongoing exploration of advanced therapies. Taxol (Tx), an initial breast cancer treatment, induces mitotic arrest but faces limitations due to side effects and the development of resistance. Addressing Tx resistance involves understanding the complex molecular mechanisms, including alterations in tubulin dynamics, NF-κB signaling, and overexpression of ABC transporters (ABCB1 and ABCG2), leading to multidrug resistance (MDR). METHODS: Real-time PCR and ELISA kits were used to analyze ABCB1, ABCG2 and NF-κB gene and protein expression levels, respectively. An MDR test assessed the resistance cell phenotype. RESULTS: MCF-7/Tx cells exhibited a 24-fold higher resistance to Tx. Real-time PCR and ELISA analysis revealed the upregulation of ABCB1, ABCG2, and NF-κB. U-359 significantly downregulated both ABCB1 and ABCG2 gene and protein levels. Co-incubation with Tx and U-359 further decreased the mRNA and protein expression of these transporters. The MDR test indicated that U-359 increased MDR dye retention, suggesting its potential as an MDR inhibitor. U-359 and Tx, either individually or combined, modulated NF-κBp65 protein levels. CONCLUSION: The development of a Taxol-resistant MCF-7 cell line provided valuable insights. U-359 demonstrated effectiveness in reducing the expression of ABC transporters and NF-κB, suggesting a potential solution for overcoming multidrug resistance in breast cancer cells. The study recommends a strategy to enhance the sensitivity of cancer cells to chemotherapy by integrating U-359 with traditional drugs.

Impact of ABCB1 and ABCG2 Transporter in Outcome of Gallbladder Cancer.

Background: Gallbladder cancer (GBC) is a biologically aggressive malignancy requiring appropriate biomarkers to improve its outcome. Role of ABC transporters (ABCB1 and ABCG2) has been linked to cancer aggressiveness, tumorigenesis and multidrug resistance. Herein, we studied the prognostic implication of ABCB1 and ABCG2 in GBC. Methods: Fresh tissue (tumour & normal) samples collected from 54 patients who underwent R0 resection, were analysed for mRNA and protein expression of ABCB1 and ABCG2 by quantitative Real-Time PCR and western blotting, respectively, in this prospective study. The molecular findings were correlated with clinical-pathological parameters using χ2 and fisher exact test. The molecular changes in ABCB1 and ABCG2 were analysed for predicting overall survival (OS), disease-free survival (DFS) and response to chemotherapy using Kaplan-Meier log-rank test and Cox regression multivariate analysis. Results: The mean age of the cohort was 50 ± 13.2 with 26 (48.1%) in patients having early stage gallbladder cancer (GBC). Over-expression of ABCB1 and ABCG2 was noted in 32/54 (59%) and 40/54 (74%) cases, respectively. The protein expression of ABCB1(P-glycoprotein) and ABCG2 (BCRP) was higher in 27/54 (50%) and 37/54 (59%) cases, respectively. The mean OS and DFS was 20.7 ± 11.5 and 19.3 ± 12.2 months at median follow-up of 24 months. The TNM stage, lymph node metastasis, and presence of gallstone were significant factors for predicting OS and DFS on multivariate analysis. Both ABCB1 and ABCG2 did not show any significant correlation with OS and DFS with similar incidences of late death and recurrence among over-expression and down-expression. Sub-group comparison suggests that change in expression pattern of ABCB1 and ABCG2 may not affect response to chemotherapy in GBC. Conclusion: Altered expression of ABCB1 and ABCG2 may not be a useful prognostic marker for survival or response to chemotherapy in GBC. Presently, histo-pathological characteristics and associated gallstones are the important predictors for survival and recurrence in GBC.

Influence of Human Bone Marrow Mesenchymal Stem Cells Secretome from Acute Myeloid Leukemia Patients on the Proliferation and Death of K562 and K562-Lucena Leukemia Cell Lineages.

Leukemias are among the most prevalent types of cancer worldwide. Bone marrow mesenchymal stem cells (MSCs) participate in the development of a suitable niche for hematopoietic stem cells, and are involved in the development of diseases such as leukemias, to a yet unknown extent. Here we described the effect of secretome of bone marrow MSCs obtained from healthy donors and from patients with acute myeloid leukemia (AML) on leukemic cell lineages, sensitive (K562) or resistant (K562-Lucena) to chemotherapy drugs. Cell proliferation, viability and death were evaluated, together with cell cycle, cytokine production and gene expression of ABC transporters and cyclins. The secretome of healthy MSCs decreased proliferation and viability of both K562 and K562-Lucena cells; moreover, an increase in apoptosis and necrosis rates was observed, together with the activation of caspase 3/7, cell cycle arrest in G0/G1 phase and changes in expression of several ABC proteins and cyclins D1 and D2. These effects were not observed using the secretome of MSCs derived from AML patients. In conclusion, the secretome of healthy MSCs have the capacity to inhibit the development of leukemia cells, at least in the studied conditions. However, MSCs from AML patients seem to have lost this capacity, and could therefore contribute to the development of leukemia.

Extracellular Vesicles as Surrogates for the Regulation of the Drug Transporters ABCC2 (MRP2) and ABCG2 (BCRP).

Drug efflux transporters of the ATP-binding-cassette superfamily play a major role in the availability and concentration of drugs at their site of action. ABCC2 (MRP2) and ABCG2 (BCRP) are among the most important drug transporters that determine the pharmacokinetics of many drugs and whose overexpression is associated with cancer chemoresistance. ABCC2 and ABCG2 expression is frequently altered during treatment, thus influencing efficacy and toxicity. Currently, there are no routine approaches available to closely monitor transporter expression. Here, we developed and validated a UPLC-MS/MS method to quantify ABCC2 and ABCG2 in extracellular vesicles (EVs) from cell culture and plasma. In this way, an association between ABCC2 protein levels and transporter activity in HepG2 cells treated with rifampicin and hypericin and their derived EVs was observed. Although ABCG2 was detected in MCF7 cell-derived EVs, the transporter levels in the vesicles did not reflect the expression in the cells. An analysis of plasma EVs from healthy volunteers confirmed, for the first time at the protein level, the presence of both transporters in more than half of the samples. Our findings support the potential of analyzing ABC transporters, and especially ABCC2, in EVs to estimate the transporter expression in HepG2 cells.

Magnolol derivatives as specific and noncytotoxic inhibitors of breast cancer resistance protein (BCRP/ABCG2).

The breast cancer resistance protein (BCRP/ABCG2) transporter mediates the efflux of numerous antineoplastic drugs, playing a central role in multidrug resistance related to cancer. The absence of successful clinical trials using specific ABCG2 inhibitors reveals the urge to identify new compounds to attend this critical demand. In this work, a series of 13 magnolol derivatives was tested as ABCG2 inhibitors. Only two compounds, derivatives 10 and 11, showed partial and complete ABCG2 inhibitory effect, respectively. This inhibition was selective toward ABCG2, since none of the 13 compounds inhibited neither P-glycoprotein nor MRP1. Both inhibitors (10 and 11) were not transported by ABCG2 and demonstrated a low cytotoxic profile even at high concentrations (up to 100 µM). 11 emerged as the most promising compound of the series, considering the ratio between cytotoxicity (IG50) and ABCG2 inhibition potency (IC50), showing a therapeutic ratio (TR) higher than observed for 10 (10.5 versus 1.6, respectively). This derivative showed a substrate-independent and a mixed type of inhibition. The effect of compound 11 on the ABCG2 ATPase activity and thermostability revealed allosteric protein changes. This compound did not affect the expression levels of ABCG2 and increased the binding of the conformational-sensitive antibody 5D3. A docking study showed that 11 did not share the same binding site with ABCG2 substrate mitoxantrone. Finally, 11 could revert the chemoresistance to SN-38 mediated by ABCG2.

Increased expression of ATP-binding cassette transporters in enfortumab vedotin-resistant urothelial cancer.

Introduction: In addition to reduced nectin-4 expression, the upregulation of ATP-binding cassette transporters has been suggested as a potential mechanism of resistance to enfortumab vedotin. Case presentation: A 76-year-old man previously treated with platinum-containing chemotherapy and pembrolizumab for metastatic bladder cancer was administered enfortumab vedotin because of disease progression. Subsequently, metastasectomy was performed for oligometastatic lesions (in the lung and adrenal gland) that exhibited growth during enfortumab vedotin therapy. Immunostaining analysis revealed decreased nectin-4 expression and elevated MDR1, MRP1, and BCRP expression in the metastatic lesions. Conclusion: Decreased nectin-4 expression and increased ATP-binding cassette transporter expression are potential factors in the development of enfortumab vedotin resistance in urothelial carcinoma. Immunohistochemical evaluation of these proteins may aid in predicting treatment efficacy.

Cadmium transport by mammalian ATP-binding cassette transporters.

Cellular responses to toxic metals depend on metal accessibility to intracellular targets, reaching interaction sites, and the intracellular metal concentration, which is mainly determined by uptake pathways, binding/sequestration and efflux pathways. ATP-binding cassette (ABC) transporters are ubiquitous in the human body-usually in epithelia-and are responsible for the transfer of indispensable physiological substrates (e.g. lipids and heme), protection against potentially toxic substances, maintenance of fluid composition, and excretion of metabolic waste products. Derailed regulation and gene variants of ABC transporters culminate in a wide array of pathophysiological disease states, such as oncogenic multidrug resistance or cystic fibrosis. Cadmium (Cd) has no known physiological role in mammalians and poses a health risk due to its release into the environment as a result of industrial activities, and eventually passes into the food chain. Epithelial cells, especially within the liver, lungs, gastrointestinal tract and kidneys, are particularly susceptible to the multifaceted effects of Cd because of the plethora of uptake pathways available. Pertinent to their broad substrate spectra, ABC transporters represent a major cellular efflux pathway for Cd and Cd complexes. In this review, we summarize current knowledge concerning transport of Cd and its complexes (mainly Cd bound to glutathione) by the ABC transporters ABCB1 (P-glycoprotein, MDR1), ABCB6, ABCC1 (multidrug resistance related protein 1, MRP1), ABCC7 (cystic fibrosis transmembrane regulator, CFTR), and ABCG2 (breast cancer related protein, BCRP). Potential detoxification strategies underlying ABC transporter-mediated efflux of Cd and Cd complexes are discussed.

NAT10-mediated mRNA N4-acetylcytidine modification of MDR1 and BCRP promotes breast cancer progression.

BACKGROUND: N-acetyltransferase 10 (NAT10) serves as a critical enzyme in mediating the N4-acetylcytidine (ac4C) that ensures RNA stability and effective translation processes. The role of NAT10 in driving the advancement of breast cancer remains uninvestigated. METHODS: We observed an increase in NAT10 expression, both at mRNA level through the analysis of the Cancer Genome Atlas (TCGA) database and at the protein level of tumor tissues from breast cancer patients. We determined that a heightened expression of NAT10 served as a predictor of an unfavorable clinical outcome. By screening the Cancer Cell Line Encyclopedia (CCLE) cell bank, this expression pattern of NAT10 was consistency found across almost all the classic breast cancer cell lines. RESULTS: Functionally, interference of NAT10 expression exerts an inhibitory effect on proliferation and invasion of breast cancer cells. By using ac4C RNA immunoprecipitation (ac4c-RIP) and acRIP-qPCR assays, we identified a reduction of ac4C enrichment within the ATP binding cassette (ABC) transporters, multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP), consequent to NAT10 suppression. Expressions of MDR1 and BCRP exhibited a positive correlation with NAT10 expression in tumor tissues, and the inhibition of NAT10 in breast cancer cells resulted in a decrease of MDR1 and BCRP expression. Therefore, the overexpressing of MDR1 and BCRP could partially rescue the adverse consequences of NAT10 depletion. In addition, we found that, remodelin, a NAT10 inhibitor, reinstated the susceptibility of capecitabine-resistant breast cancer cells to the chemotherapy, both in vitro and in vivo. CONCLUSION: The results of our study demonstrated the essential role of NAT10-mediated ac4c-modification in breast cancer progression and provide a novel strategy for overcoming chemoresistance challenges.

Natural products for combating multidrug resistance in cancer.

Cancer cells frequently develop resistance to chemotherapeutic therapies and targeted drugs, which has been a significant challenge in cancer management. With the growing advances in technologies in isolation and identification of natural products, the potential of natural products in combating cancer multidrug resistance has received substantial attention. Importantly, natural products can impact multiple targets, which can be valuable in overcoming drug resistance from different perspectives. In the current review, we will describe the well-established mechanisms underlying multidrug resistance, and introduce natural products that could target these multidrug resistant mechanisms. Specifically, we will discuss natural compounds such as curcumin, resveratrol, baicalein, chrysin and more, and their potential roles in combating multidrug resistance. This review article aims to provide a systematic summary of recent advances of natural products in combating cancer drug resistance, and will provide rationales for novel drug discovery.

Structural insights into AtABCG25, an angiosperm-specific abscisic acid exporter.

Cellular hormone homeostasis is essential for precise spatial and temporal signaling responses and plant fitness. Abscisic acid (ABA) plays pivotal roles in orchestrating various developmental and stress responses and confers fitness benefits over ecological and evolutionary timescales in terrestrial plants. Cellular ABA level is regulated by complex processes, including biosynthesis, catabolism, and transport. AtABCG25 is the first ABA exporter identified through genetic screening and affects diverse ABA responses. Resolving the structural basis of ABA export by ABCG25 is critical for further manipulations of ABA homeostasis and plant fitness. We used cryo-electron microscopy to elucidate the structural dynamics of AtABCG25 and successfully characterized different states, including apo AtABCG25, ABA-bound AtABCG25, and ATP-bound AtABCG25 (E232Q). Notably, AtABCG25 forms a homodimer that features a deep, slit-like cavity in the transmembrane domain, and we precisely characterized the critical residues in the cavity where ABA binds. ATP binding triggers closure of the nucleotide-binding domains and conformational transitions in the transmembrane domains. We show that AtABCG25 belongs to a conserved ABCG subfamily that originated during the evolution of angiosperms. This subfamily neofunctionalized to regulate seed germination via the endosperm, in concert with the evolution of this angiosperm-specific, embryo-nourishing tissue. Collectively, these findings provide valuable insights into the intricate substrate recognition and transport mechanisms of the ABA exporter AtABCG25, paving the way for genetic manipulation of ABA homeostasis and plant fitness.

Zinc finger proteins and ATP-binding cassette transporter-dependent multidrug resistance.

BACKGROUND: Multidrug resistance (MDR) remains a significant challenge in cancer treatment, leading to poor clinical outcomes. Dysregulation of ATP-binding cassette (ABC) transporters has been identified as a key contributor to MDR. Zinc finger proteins (ZNPs) are key regulators of transcription and have emerged as potential contributors to cancer drug resistance. Bridging the knowledge gap between ZNPs and MDR is essential to understand a source of heterogeneity in cancer treatment. This review sought to elucidate how different ZNPs modulate the transcriptional regulation of ABC genes, contributing to resistance to cancer therapies. METHODS: The search was conducted using PubMed, Google Scholar, EMBASE and Web of Science. RESULTS: In addition to ABC-blockers, the transcriptional features regulated by ZNP are expected to play a role in reversing ABC-mediated MDR and predicting the efficacy of anticancer treatments. Among the ZNP-induced epithelial to mesenchymal transition, SNAIL, SLUG and Zebs have been identified as important factors in promoting MDR through activation of ATM, NFκB and PI3K/Akt pathways, exposing the metabolism to potential ZNP-MDR interactions. Additionally, nuclear receptors, such as VDR, ER and PXR have been found to modulate certain ABC regulations. Other C2H2-type zinc fingers, including Kruppel-like factors, Gli and Sp also have the potential to contribute to MDR. CONCLUSION: Besides reviewing evidence on the effects of ZNP dysregulation on ABC-related chemoresistance in malignancies, significant markers of ZNP functions are discussed to highlight the clinical implications of gene-to-gene and microenvironment-to-gene interactions on MDR prospects. Future research on ZNP-derived biomarkers is crucial for addressing heterogeneity in cancer therapy.

Linking ABC transporters to the hallmarks of cancer.

Human ATP-binding cassette (ABC) transporters are ubiquitously expressed and transport a broad range of endogenous and xenobiotic substrates across extra- and intracellular membranes. Mutations in ABC genes cause 21 monogenic diseases, and polymorphisms in these genes are associated with susceptibility to complex diseases. ABC transporters also play a major role in drug bioavailability, and they mediate multidrug resistance in cancer. At least 13 ABC transporters were shown to be involved in drug resistance in vitro. In the past decade, efforts have been made to elucidate their roles in tumor biology. Herein, we explore their involvement in tumorigenesis, focusing on the hallmarks of cells as they make their way from normalcy to neoplastic growth states.

Multidrug resistance protein 5 affects cell proliferation, migration and gemcitabine sensitivity in pancreatic cancer MIA Paca­2 and PANC­1 cells.

Gemcitabine­based chemotherapy has been widely adopted as the standard and preferred chemotherapy regimen for treating advanced pancreatic cancer. However, the contribution of multidrug resistance protein 5 (MRP5) to gemcitabine resistance and pancreatic cancer progression remains controversial. In the present study, the effect of silencing MRP5 on gemcitabine resistance and cell proliferation and migration of human pancreatic cancer MIA Paca­2 and PANC­1 cells was investigated by using short­hairpin RNA delivered by lentiviral vector transduction. The knockdown of MRP5 was confirmed on both mRNA and protein levels using qPCR and surface staining assays, respectively. MRP5­regulated gemcitabine sensitivity was assessed by MTT, PrestoBlue and apoptosis assays. The effect of MRP5 on pancreatic cancer cell proliferation and migration was determined using colony­formation, wound­healing and Transwell migration assays. The interaction of gemcitabine and cyclic guanosine monophosphate (cGMP) with MRP5 protein was explored using molecular docking. The results indicated that the MRP5 mRNA and protein levels were significantly reduced in all the MIA Paca­2 and PANC­1 clones. MRP5 affected gemcitabine cytotoxicity and the rate of gemcitabine­induced apoptosis. Silencing MRP5 decreased cell proliferation and migration in both MIA Paca­2 and PANC­1 cells. Docking studies showed high binding affinity of cGMP towards MRP5, indicating the potential of MRP5­mediated cGMP accumulation in the microenvironment. In conclusion, MRP5 has an important role in cancer proliferation and migration in addition to its drug efflux functions in two widely available pancreatic tumour cell lines (MIA Paca­2 and PANC­1).

Cardiac PET Imaging of ATP Binding Cassette (ABC) Transporters: Opportunities and Challenges.

Adenosine triphosphate binding cassette (ABC) transporters are a broad family of membrane protein complexes that use energy to transport molecules across cells and/or intracellular organelle lipid membranes. Many drugs used to treat cardiac diseases have an affinity for these transporters. Among others, P-glycoprotein (P-gp) plays an essential role in regulating drug concentrations that reach cardiac tissue and therefore contribute to cardiotoxicity. As a molecular imaging modality, positron emission tomography (PET) has emerged as a viable technique to investigate the function of P-gp in organs and tissues. Using PET imaging to evaluate cardiac P-gp function provides new insights for drug development and improves the precise use of medications. Nevertheless, information in this field is limited. In this review, we aim to examine the current applications of ABC transporter PET imaging and its tracers in the heart, with a specific emphasis on P-gp. Furthermore, the opportunities and challenges in this novel field will be discussed.