Expression Of Drug Efflux Transporters Research Articles

Diethyldithiocarbamate-ferrous oxide nanoparticles inhibit human and mouse glioblastoma stemness: aldehyde dehydrogenase 1A1 suppression and ferroptosis induction.

The development of effective therapy for eradicating glioblastoma stem cells remains a major challenge due to their aggressive growth, chemoresistance and radioresistance which are mainly conferred by aldehyde dehydrogenase (ALDH)1A1. The latter is the main stemness mediator via enhancing signaling pathways of Wnt/β-catenin, phosphatidylinositol 3-kinase/AKT, and hypoxia. Furthermore, ALDH1A1 mediates therapeutic resistance by inactivating drugs, stimulating the expression of drug efflux transporters, and detoxifying reactive radical species, thereby apoptosis arresting. Recent reports disclosed the potent and broad-spectrum anticancer activities of the unique nanocomplexes of diethyldithiocarbamate (DE, ALDH1A1 inhibitor) with ferrous oxide nanoparticles (FeO NPs) mainly conferred by inducing lipid peroxidation-dependent non-apoptotic pathways (iron accumulation-triggered ferroptosis), was reported. Accordingly, the anti-stemness activity of nanocomplexes (DE-FeO NPs) was investigated against human and mouse glioma stem cells (GSCs) and radioresistant GSCs (GSCs-RR). DE-FeO NPs exhibited the strongest growth inhibition effect on the treated human GSCs (MGG18 and JX39P), mouse GSCs (GS and PDGF-GSC) and their radioresistant cells (IC50 ≤ 70 and 161μg/mL, respectively). DE-FeO NPs also revealed a higher inhibitory impact than standard chemotherapy (temozolomide, TMZ) on self-renewal, cancer repopulation, chemoresistance, and radioresistance potentials. Besides, DE-FeO NPs surpassed TMZ regarding the effect on relative expression of all studied stemness genes, as well as relative p-AKT/AKT ratio in the treated MGG18, GS and their radioresistant (MGG18-RR and GS-RR). This potent anti-stemness influence is primarily attributed to ALDH1A1 inhibition and ferroptosis induction, as confirmed by significant elevation of cellular reactive oxygen species and lipid peroxidation with significant depletion of glutathione and glutathione peroxidase 4. DE-FeO NPs recorded the optimal LogP value for crossing the blood brain barrier. This in vitro novel study declared the potency of DE-FeO NPs for collapsing GSCs and GSCs-RR with improving their sensitivity to chemotherapy and radiotherapy, indicating that DE-FeO NPs may be a promising remedy for GBM. Glioma animal models will be needed for in-depth studies on its safe effectiveness.

Abstract 1990: TIP60-mediated acetylation of ΔNp63α is associated with cisplatin resistance

Abstract The chemotherapeutic drug Cisplatin is often used to treat squamous cell carcinoma (SCC) patients, but low response rates and disease recurrence are common. About 5.4 million basal and squamous cell skin cancers are diagnosed each year in the US. ΔNp63α, a member of the p53 family of transcription factors, is overexpressed and considered oncogenic in non-melanoma skin cancer where it regulates cell survival, promotes proliferation, and inhibits cell apoptosis. ΔNp63α has also been shown to promote resistance to cisplatin by orchestrating the transcriptional regulation of several DNA damage response (DDR) genes. Our previous study showed that the histone acetyltransferase (HAT) TIP60 promotes SCC proliferation by positively regulating ΔNp63α protein levels in a manner reliant on the catalytic activity of TIP60. This observation suggested that TIP60 may contribute to the failure of platinum-based drugs in SCC and led us to hypothesize that TIP60-mediated acetylation of ΔNp63α regulates its stability and transcriptional activity to promote chemoresistance. We found that TIP60 levels positively correlated with ΔNp63α stability, protein levels, and cisplatin resistance. Further, silencing endogenous TIP60 led to a decrease in ΔNp63α transcript and protein levels in multiple cisplatin resistant SCC cell lines. Further, stable expression of TIP60 or ΔNp63α individually promoted resistance to cisplatin and reduced cell death, whereas loss of ΔNp63α or TIP60 induced G2/M arrest, increased cell death, and sensitized cells to cisplatin. Additionally, pharmacological inhibition of TIP60 reduced acetylation of ΔNp63α, decreased colony formation, and sensitized resistant cells to cisplatin. Finally, we demonstrated that ΔNp63α and TIP60 levels positively correlated with DNA repair capacity and negatively correlated with cisplatin-DNA adduct formation. Silencing of either TIP60 or ΔNp63α intensified cisplatin-DNA adduct formation and significantly reduced the expression of drug efflux transporters. Taken together, our data indicate that TIP60-mediated stabilization of ΔNp63α increases cisplatin resistance and provides critical insights into the mechanisms by which ΔNp63α confers cisplatin resistance in SCC. Our findings suggest that the inhibition of TIP60 may be therapeutically advantageous in overcoming cisplatin resistance in SCC and other epithelial cancers. Citation Format: Akshay Hira, Jin Zhang, Mike Kemp, Madhavi P. Kadakia. TIP60-mediated acetylation of ΔNp63α is associated with cisplatin resistance [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 1990.

Evaluation of ABT-751, a novel anti-mitotic agent able to overcome multi-drug resistance, in melanoma cells

PurposeDrug efflux transporter associated multi-drug resistance (MDR) is a potential limitation in the use of taxane chemotherapies for the treatment of metastatic melanoma. ABT-751 is an orally bioavailable microtubule-binding agent capable of overcoming MDR and proposed as an alternative to taxane-based therapies.MethodsThis study compares ABT-751 to taxanes in vitro, utilizing seven melanoma cell line models, publicly available gene expression and drug sensitivity databases, a lung cancer cell line model of MDR drug efflux transporter overexpression (DLKP-A), and drug efflux transporter ATPase assays.ResultsMelanoma cell lines exhibit a low but variable protein and RNA expression of drug efflux transporters P-gp, BCRP, and MDR3. Expression of P-gp and MDR3 correlates with sensitivity to taxanes, but not to ABT-751. The anti-proliferative IC50 profile of ABT-751 was higher than the taxanes docetaxel and paclitaxel in the melanoma cell line panel, but fell within clinically achievable parameters. ABT-751 IC50 was not impacted by P-gp-overexpression in DKLP-A cells, which display strong resistance to the P-gp substrate taxanes compared to DLKP parental controls. The addition of ABT-751 to paclitaxel treatment significantly decreased cell proliferation, suggesting some reversal of MDR. ATPase activity assays suggest that ABT-751 is a potential BCRP substrate, with the ability to inhibit P-gp ATPase activity.ConclusionOur study confirms that ABT-751 is active against melanoma cell lines and models of MDR at physiologically relevant concentrations, it inhibits P-gp ATPase activity, and it may be a BCRP and/or MDR3 substrate. ABT-751 warrants further investigation alone or in tandem with other drug efflux transporter inhibitors for hard-to-treat MDR melanoma.

ATP-binding cassette (ABC) drug transporters in the developing blood-brain barrier: role in fetal brain protection.

The blood-brain barrier (BBB) provides essential neuroprotection from environmental toxins and xenobiotics, through high expression of drug efflux transporters in endothelial cells of the cerebral capillaries. However, xenobiotic exposure, stress, and inflammatory stimuli have the potential to disrupt BBB permeability in fetal and post-natal life. Understanding the role and ability of the BBB in protecting the developing brain, particularly with respect to drug/toxin transport, is key to promoting long-term brain health. Drug transporters, particularly P-gp and BCRP are expressed in early gestation at the developing BBB and have a crucial role in developmental homeostasis and fetal brain protection. We have highlighted several factors that modulate drug transporters at the developing BBB, including synthetic glucocorticoid (sGC), cytokines, maternal infection, and growth factors. Some factors have the potential to increase expression and function of drug transporters and increase brain protection (e.g., sGC, transforming growth factor [TGF]-β). However, others inhibit drug transporters expression and function at the BBB, increasing brain exposure to xenobiotics (e.g., tumor necrosis factor [TNF], interleukin [IL]-6), negatively impacting brain development. This has implications for pregnant women and neonates, who represent a vulnerable population and may be exposed to drugs and environmental toxins, many of which are P-gp and BCRP substrates. Thus, alterations in regulated transport across the developing BBB may induce long-term changes in brain health and compromise pregnancy outcome. Furthermore, a large portion of neonatal adverse drug reactions are attributed to agents that target or access the nervous system, such as stimulants (e.g., caffeine), anesthetics (e.g., midazolam), analgesics (e.g., morphine) and antiretrovirals (e.g., Zidovudine); thus, understanding brain protection is key for the development of strategies to protect the fetal and neonatal brain.

442 Repurposing FDA-approved PI3K/Akt Inhibitors to Improve Anti-Cancer Drug Brain Uptake in Glioblastoma Resection Models

OBJECTIVES/GOALS: We have shown that glioblastoma upregulates blood-brain barrier drug efflux transporters via a mechanism that likely involves TNFα and PI3K/Akt. Our goal is to repurpose FDA-approved PI3K/Akt inhibitors to increase anticancer drug brain concentrations, which holds the potential for translation into the neuro-oncology clinic. METHODS/STUDY POPULATION: GL261 Red-FLuc and MBR525-1 Red-FLuc cells (2μl; 2.5K cells/μl; 1μl/min) were injected into the right hemisphere of 8-week old female J:NU mice (coordinates relative to bregma: AP -2 mm, ML -2 mm, DV -3 mm). Tumor burden was assessed weekly with IVIS® Spectrum in vivo imaging; tumor volume and invasiveness were measured by MRI and histopathology, respectively. On day 14 post-injection, mice received 5-ALA (200 mg/kg ip), and tumors were resected with a 2 mm punch biopsy tool and surgical fluorescence microscope (ex/em: 405/635nm). Drug efflux transporter expression and activity in isolated brain capillaries were determined by Western blot and substrate fluorescence assays, respectively. Cytotoxicity was assessed after 48-hour drug incubation using CyQuant MTT Cell Proliferation Assay kits. RESULTS/ANTICIPATED RESULTS: IC50 values of temozolomide, lapatinib, alpelisib, and miltefosine were N/A, 32, 20, and 190 μM for GL261 Red-FLuc cells and N/A, 49, 36, and 148 μM for MBR525-1 Red-FLuc cells, respectively. Median survival of GL261 Red-FLuc mice was 26.5d and significantly increased to 34d with resection (p=0.116). In GBM mice, drug efflux transporter expression and activity levels in brain capillaries isolated from the contralateral hemisphere were significantly upregulated compared to sham controls. Furthermore, treatment with FDA-approved PI3K/Akt inhibitors, alpelisib and miltefosine, significantly reduced drug efflux transporter expression and activity to control levels. In PK and survival studies, we expect that PI3K/Akt inhibition will increase brain uptake of anticancer drugs and prolong GBM mouse survival. DISCUSSION/SIGNIFICANCE: We have previously shown that PI3K/Akt inhibition reduces P-gp/BCRP levels in brain capillaries. Here, we vertically extend this strategy by repurposing the FDA-approved PI3K/Akt inhibitors alpelisib/miltefosine to improve brain uptake of anticancer drugs in GBM resection models.

Altered hippocampal expression and function of cytosolic phospholipase A2 (cPLA2) in temporal lobe epilepsy (TLE)

ABSTRACT Objectives Temporal lobe epilepsy (TLE) is the most common form of drug-resistant epilepsy. Blood–brain barrier (BBB) leakage occurs during epileptogenesis and several pieces of evidence suggest that this might contribute to the progression of epilepsy. Seizures trigger a pathway involving glutamate signalling through cytosolic phospholipase A2 (cPLA2). This pathway leads to BBB leakage and induces the expression of drug efflux transporters, leading to drug resistance. Therefore, this study aims to determine the mRNA and protein levels of cPLA2, along with its functional activity, in the hippocampus of pilocarpine model of TLE as well as in the surgically resected hippocampal samples of patients with TLE. Methods mRNA levels and protein levels of cPLA2 were evaluated by real-time PCR and western blot analysis respectively in animal model of TLE as well as surgically resected hippocampal tissue specimens of TLE. cPLA2 functional activity was measured spectrophotometrically. Results Significant up-regulation of cPLA2 mRNA was observed in the hippocampal samples obtained from TLE rats (p < 0.05) and-TLE patients (p < 0.01). Increased protein expression of cPLA2 was also demonstrated in the hippocampal samples of TLE rats (p < 0.01) as well as TLE patients (p < 0.01). Similarly, functional activity of cPLA2 was found to be up-regulated in the hippocampus of pilocarpine model of TLE rats (p < 0.01) as well as in the TLE patients (p < 0.01). Discussion These findings suggest that alterations in cPLA2 expression and activity level in the hippocampus could potentially be a part of dynamic changes associated with TLE.

ROLE OF ANTIDEPRESSANTS AGAINST LEISHMANIASIS

The disease, Leishmaniasis is a forlorn, tropical, vector-borne disease caused by the kinetoplast protozoan, an obligate intracellular parasite of the class Leishmania, which is transmitted by the nibble of female sandfly (Phlebotomus). Leishmaniasis is an incurable rare disease, since the organism developed resistance towards the currently used drugs, including pentavalent antimonials, amphotericin B and miltefosine. The mechanisms involved in drug resistance include reduced expression and mutations in AQP1, mutations in miltefosine transporters like, LMT and/or LRos3 and increased expression of drug efflux transporters like, ABC transporters in the parasitic cells. Hence alternate therapeutics against leishmaniasis is the need of the hour. Here we discuss the therapeutic potential of antidepressants as potential antileishmanial agents.

Alisertib Synergistically Strengthens the Anti-Leukemia Activity of Venetoclax in TCF3-Hlf B-ALL

Background: Despite maximally-intensive chemotherapy and stem cell transplantation, survival of patients with the very rare t(17;19)/TCF3-HLF B-acute lymphoblastic leukemia (B-ALL) subtype remains effectively 0%. Prior studies have demonstrated association of the oncogenic TCF3-HLF fusion protein with multi-drug resistance via increased expression of ABC and P-glycoprotein drug efflux transporters, as well as via upregulation of pro-survival Ras and BCL-2 pathways. Preclinical studies and small clinical case series of targeted inhibitor addition to chemotherapy or antibody-based and cellular immunotherapies have aimed to improve outcomes for children with TCF3-HLF ALL. Unfortunately, targeting of these activated pathways with the BCL-2 inhibitor venetoclax or other small molecule inhibitors (SMIs) has been minimally or only transiently effective, suggesting more complex mechanism(s) of chemoresistance. In recent years, many patients with relapsed TCF3-HLF ALL have enrolled on clinical trials of CD19- or CD22-targeted immunotherapies. However, TCF3-HLF ALL frequently harbours deactivating mutations in PAX5, a major B-cell regulator and indispensable CD19 transcription factor, placing immunotherapy-treated patients at higher risk of CD19 antigen-loss relapse. New therapies remain needed to prevent relapse and attempt cure.Methods: We designed an unbiased kinome-wide CRISPR/Cas9 library to identify essential drivers in TCF3-HLF leukemogenesis. We screened the human TCF3-HLF ALL cell line HAL-01 and our TCF3-HLF ALL patient-derived xenograft (PDX) model ALL1807 (Hurtz JCI 2020, Schultz Genome Biol 2021), then validated identified targets using 49 SMIs targeting receptor tyrosine kinases (RTK), MEK signaling, and cell cycle pathways. We selected promising candidate inhibitor pairings with non-overlapping mechanisms of action and assessed for in vitro drug synergy via SynergyFinder analyses. Finally, we assessed the in vivo activity of targeted inhibitors in ALL1807 and two newly established TCF3-HLF ALL PDX models (CPCT-0002, CPCT-0003) created from primary pediatric specimens obtained via the LEAP Consortium (Pikman Cancer Disc 2021).Results: RNA-sequencing of HAL-01 and ALL1807 cells followed by functional protein association (STRING) analysis confirmed a network of significantly upregulated (>3-fold) plasma membrane and cytoplasm components of RTK pathways as well as BCL-2. The intersection of the results of the SMI drug library screen with the top 1% targets identified in CRIPSR/Cas9 screen determined p120-RasGAP and Aurora kinase A (AURKA) as therapeutic targets in TCF3-HLF ALL. In vitro treatment of HAL-01 or ALL1807 cells with the RasGAP inhibitor, pluripotin, or the AURKA inhibitor, alisertib, across a range of concentrations demonstrated robust anti-ALL activity. AURKA and RasGAP co-immunoprecipitated and this protein complex was disrupted with alisertib or pluripotin treatment. The AURKB inhibitor barisertib had minimal activity against TCF3-HLF ALL cells, confirming preferential dependency of these cells upon AURKA. Treatment of TCF3-HLF ALL cells with the BCL-2i venetoclax did not disrupt the AURKA/RasGAP complex, suggesting its different mechanism of action and potential for combinatorial drug therapy. Next, we found that alisertib and venetoclax synergistically killed TCF3-HLF ALL cells. Finally, we observed superior inhibition of in vivo leukemia with dual AURKA and BCL-2 inhibitor treatment of three TCF3-HLF ALL PDX models compared to single-agent alisertib or venetoclax (Figure 1).Conclusions: We identified AURKA as a critical new driver in TCF3-HLF ALL via orthogonal genetic and functional assays and confirmed prior observations of BCL-2 dependency in our models. We validated these key targets via in vitro and in vivo pharmacologic inhibition studies with drug synergy detected with combined alisertib and venetoclax in human TCF3-HLF ALL cell lines and PDX models. We posit that dual AURKA and BCL-2 inhibition is a clinically-pragmatic and potentially effective therapeutic strategy for patients with this rare, but highly fatal, leukemia subtype that merits formal clinical investigation. [Display omitted] DisclosuresCarroll: Incyte Pharmaceuticals: Research Funding; Janssen Pharmaceutical: Consultancy. Stegmaier: Auron Therapeutics, Kronos Bio, AstraZeneca, Novartis Institute of Biomedical Research: Consultancy, Research Funding. Tasian: Incyte Corporation: Research Funding; Gilead Sciences: Research Funding; Kura Oncology: Consultancy; Aleta Biotherapeutics: Consultancy.

Regulation of ABC Drug Efflux Transporters in Human T-Cells Exposed to an HIV Pseudotype.

ATP-binding cassette (ABC) drug efflux transporters could contribute to low intracellular concentrations of antiretroviral drugs in HIV-1 cell reservoirs and sanctuary sites. Furthermore, the functional expression of these transporters could be induced in activated T-cells. Therefore, we investigated the expression of ABC drug efflux transporters in human T-cells exposed to an HIV pseudotype virus (pHIVNL4-3), and further examined the potential involvement of the mammalian target of rapamycin (mTOR) signaling pathway in regulating their expression following exposure to pHIVNL4-3. Additionally, we investigated the contribution of the drug efflux transporters to the inflammatory response following pHIVNL4-3-induced T-cell activation. Human peripheral blood mononuclear cells (PBMCs) were exposed to HIV-1 envelope glycoprotein gp120IIIB, pHIVNL4-3 and/or mTOR inhibitors. The expression of ABC transporters, T-cell activation marker CD69, mTOR and pHIVNL4-3 was assessed in CD4+ T-cells by Flow cytometry. mRNA and protein levels of proinflammatory cytokines (IL6, TNFα and INFγ) were examined in PBMCs by qPCR and ELISA analyses, respectively, following exposure to pHIVNL4-3 with or without inhibitors of mTOR or ABC transporters. The expression of ABC transporters (P-glycoprotein, breast cancer resistance protein and multi-drug resistance associated protein-1) was significantly increased in CD4+ T-cells exposed to pHIVNL4-3. Treatment with mTOR inhibitors attenuated pHIVNL4-3-induced transporter expression, as well as mRNA and protein levels of IL6, TNFα and INFγ. Additionally, inhibition of P-gp or MRP1 activity resulted in lower concentrations of proinflammatory cytokines in supernatants of PBMC exposed to pHIVNL4-3. Herein we present novel data demonstrating that upregulation of ABC drug efflux transporters could involve the mTOR signaling pathway in CD4+ T-cells exposed to an HIV pseudotype. These transporters could limit antiretroviral drug penetration in HIV target T-cells. Furthermore, ABC transporters could potentially contribute to HIV-associated proinflammatory cytokine secretion.

Messing Up the Cancer Stem Cell Chemoresistance Mechanisms Supported by Tumor Microenvironment.

Despite the recent advances in cancer patient management and in the development of targeted therapies, systemic chemotherapy is currently used as a first-line treatment for many cancer types. After an initial partial response, patients become refractory to standard therapy fostering rapid tumor progression. Compelling evidence highlights that the resistance to chemotherapeutic regimens is a peculiarity of a subpopulation of cancer cells within tumor mass, known as cancer stem cells (CSCs). This cellular compartment is endowed with tumor-initiating and metastasis formation capabilities. CSC chemoresistance is sustained by a plethora of grow factors and cytokines released by neighboring tumor microenvironment (TME), which is mainly composed by adipocytes, cancer-associated fibroblasts (CAFs), immune and endothelial cells. TME strengthens CSC refractoriness to standard and targeted therapies by enhancing survival signaling pathways, DNA repair machinery, expression of drug efflux transporters and anti-apoptotic proteins. In the last years many efforts have been made to understand CSC-TME crosstalk and develop therapeutic strategy halting this interplay. Here, we report the combinatorial approaches, which perturb the interaction network between CSCs and the different component of TME.

Pharmacogenomics of Impaired Tyrosine Kinase Inhibitor Response: Lessons Learned From Chronic Myelogenous Leukemia.

The use of small molecules became one key cornerstone of targeted anti-cancer therapy. Among them, tyrosine kinase inhibitors (TKIs) are especially important, as they were the first molecules to proof the concept of targeted anti-cancer treatment. Since 2001, TKIs can be successfully used to treat chronic myelogenous leukemia (CML). CML is a hematologic neoplasm, predominantly caused by reciprocal translocation t(9;22)(q34;q11) leading to formation of the so-called BCR-ABL1 fusion gene. By binding to the BCR-ABL1 kinase and inhibition of downstream target phosphorylation, TKIs, such as imatinib or nilotinib, can be used as single agents to treat CML patients resulting in 80 % 10-year survival rates. However, treatment failure can be observed in 20-25 % of CML patients occurring either dependent or independent from the BCR-ABL1 kinase. Here, we review approved TKIs that are indicated for the treatment of CML, their side effects and limitations. We point out mechanisms of TKI resistance focusing either on BCR-ABL1-dependent mechanisms by summarizing the clinically observed BCR-ABL1-mutations and their implications on TKI binding, as well as on BCR-ABL1-independent mechanisms of resistances. For the latter, we discuss potential mechanisms, among them cytochrome P450 implications, drug efflux transporter variants and expression, microRNA deregulation, as well as the role of alternative signaling pathways. Further, we give insights on how TKI resistance could be analyzed and what could be learned from studying TKI resistance in CML in vitro.

Electronic cigarette aerosols alter the expression of cisplatin transporters and increase drug resistance in oral cancer cells

Tobacco smoking is the leading preventable cause of cancer. Moreover, continued smoking during cancer therapy reduces overall survival. Aware of the negative consequences of tobacco smoking and the challenges of smoking cessation, cancer patients are inquiring whether they should switch to electronic cigarettes (e-cigarettes). To obtain evidence-based data to inform this decision, we examined the effects of e-cigarette aerosol exposure on cisplatin resistance in head and neck cancer cells. Our results show that cancer cells exposed to e-cigarette aerosol extracts and treated with cisplatin have a significant decrease in cell death, increase in viability, and increase in clonogenic survival when compared to non-exposed cells. Moreover, exposure to e-cigarette aerosol extracts increased the concentration of cisplatin needed to induce a 50% reduction in cell growth (IC50) in a nicotine-independent manner. Tobacco smoke extracts induced similar increases in cisplatin resistance. Changes in the expression of drug influx and efflux transporters, rather than activation of cell growth-promoting pathways or DNA damage repair, contribute to e-cigarette induced cisplatin resistance. These results suggest that like combustible tobacco, e-cigarette use might increase chemotherapy resistance, and emphasize the urgent need for rigorous evaluation of e-cigarettes health effects to ensure evidence-based public health policies.

Hypoxia Dictates Metabolic Rewiring of Tumors: Implications for Chemoresistance.

Hypoxia is a condition commonly observed in the core of solid tumors. The hypoxia-inducible factors (HIF) act as hypoxia sensors that orchestrate a coordinated response increasing the pro-survival and pro-invasive phenotype of cancer cells, and determine a broad metabolic rewiring. These events favor tumor progression and chemoresistance. The increase in glucose and amino acid uptake, glycolytic flux, and lactate production; the alterations in glutamine metabolism, tricarboxylic acid cycle, and oxidative phosphorylation; the high levels of mitochondrial reactive oxygen species; the modulation of both fatty acid synthesis and oxidation are hallmarks of the metabolic rewiring induced by hypoxia. This review discusses how metabolic-dependent factors (e.g., increased acidification of tumor microenvironment coupled with intracellular alkalinization, and reduced mitochondrial metabolism), and metabolic-independent factors (e.g., increased expression of drug efflux transporters, stemness maintenance, and epithelial-mesenchymal transition) cooperate in determining chemoresistance in hypoxia. Specific metabolic modifiers, however, can reverse the metabolic phenotype of hypoxic tumor areas that are more chemoresistant into the phenotype typical of chemosensitive cells. We propose these metabolic modifiers, able to reverse the hypoxia-induced metabolic rewiring, as potential chemosensitizer agents against hypoxic and refractory tumor cells.

Suppression of HIV-1 Viral Replication by Inhibiting Drug Efflux Transporters in Activated Macrophages.

Ethanol has been shown to increase oxidative stress, drug efflux transporter expression, and promote HIV progression. Macrophages, which express drug efflux transporters, serve as an essential sanctuary site for HIV. The antiretroviral drug lopinavir, a protease inhibitor, is a substrate of the drug efflux transporters P-glycoprotein and multidrug resistance-associated protein 1. The NF-κB signaling pathway is associated with inflammation and drug efflux transporter expression. To examine the effects of ethanol on drug efflux transporters and HIV replication of macrophages and develop strategies to increase the efficacy of the protease inhibitor. The expression of PGP and MRP1 was examined with western blot. The NF- κB inhibition was assessed with nuclear western blot. LC-MS/MS and p24 ELISA were used to assess intracellular LPV and viral replication. Ethanol at 40mM slightly increased drug efflux transporter PGP and MRP1 expression in activated macrophages. IKK-16, an NF- κB inhibitor, counteracted the increased transporter expression caused by ethanol exposure. MK571, an MRP1 inhibitor, and IKK-16 significantly increased intracellular LPV concentration with or without ethanol treatment. MK571 significantly increased LPV efficacy in suppressing viral replication with or without ethanol treatment. A decreasing trend and a significant decrease were observed with IKK-16+LPV treatment compared with LPV alone in the no ethanol treatment and ethanol treatment groups, respectively. In activated macrophages, inhibiting drug efflux transporter MRP1 activity and reducing its expression may represent a promising approach to suppress viral replication by increasing intracellular antiretroviral concentrations. However, different strategies may be required for ethanolrelated vs. untreated groups.

Apigenin by targeting hnRNPA2 sensitizes triple-negative breast cancer spheroids to doxorubicin-induced apoptosis and regulates expression of ABCC4 and ABCG2 drug efflux transporters

Acquired resistance to doxorubicin is a major hurdle in triple-negative breast cancer (TNBC) therapy, emphasizing the need to identify improved strategies. Apigenin and other structurally related dietary flavones are emerging as potential chemo-sensitizers, but their effect on three-dimensional TNBC spheroid models has not been investigated. We previously showed that apigenin associates with heterogeneous ribonuclear protein A2/B1 (hnRNPA2), an RNA-binding protein involved in mRNA and co-transcriptional regulation. However, the role of hnRNPA2 in apigenin chemo-sensitizing activity has not been investigated. Here, we show that apigenin induced apoptosis in TNBC spheroids more effectively than apigenin-glycoside, owing to higher cellular uptake. Moreover, apigenin inhibited the growth of TNBC patient-derived organoids at an in vivo achievable concentration. Apigenin sensitized spheroids to doxorubicin-induced DNA damage, triggering caspase-9-mediated intrinsic apoptotic pathway and caspase-3 activity. Silencing of hnRNPA2 decreased apigenin-induced sensitization to doxorubicin in spheroids by diminishing apoptosis and partly abrogated apigenin-mediated reduction of ABCC4 and ABCG2 efflux transporters. Together these findings provide novel insights into the critical role of hnRNPA2 in mediating apigenin-induced sensitization of TNBC spheroids to doxorubicin by increasing the expression of efflux transporters and apoptosis, underscoring the relevance of using dietary compounds as a chemotherapeutic adjuvant.

TGF-β induced chemoresistance in liver cancer is modulated by xenobiotic nuclear receptor PXR

ABSTRACT Hepatocellular carcinoma appears as an extremely angiogenic solid tumor marked by apoptosis evasion, dysregulated cell cycle and low sensitivity to chemotherapy. TGF-β, a multifunctional cytokine, plays a pleiotropic role in the tumor microenvironment and has implications in cancer drug resistance. The current study provides novel evidence that TGF-β signaling contributes to drug resistance in liver cancer cells by inducing the expression of xenobiotic nuclear receptor PXR. We observed that PXR increases the expression of drug efflux transporters; therefore, accounting for exacerbated drug resistance. Additionally, anti-apoptotic nature of PXR contributes to TGF-β mediated chemoresistance as seen by procaspase-3 and Mcl-1 cellular levels. TGF-β binding to the TGF-β receptor triggers a complex downstream signaling cascade through a non-canonical SMAD-independent ERK pathway that leads to increased PXR expression. Activated ERK activates ETS1 transcription factor which is a critical regulator of endogenous PXR expression in hepatic cells. Loss of function of ETS1 abrogates the TGF-β induced PXR expression. Together these findings indicate that PXR modulates TGF-β induced resistance to chemotherapy in liver cancer cells. This underscores the need for combinatorial approaches with focus on PXR antagonism to improve drug effectiveness in hepatocellular carcinoma. Abbreviations HCC: Hepatocellular Carcinoma; FDA: Food and Drug Administration; TGF-β: Transforming growth factor-β; PXR: Pregnane X receptor; CAR: Constitutive androstane receptor; P-gp/ABCB1: P-glycoproteins/ATP-binding cassette transporter subfamily B member 1; MRP1/ABCC1 and MRP2/ABCC2: Multidrug-resistance associated proteins; BCRP/ABCG2: Breast cancer resistant protein; DMEs: Drug-metabolizing enzymes; CFDA: 5,6-carboxyfluorescein diacetate; ETS1: Transcription factor E26 transformation specific sequence 1.

Ex vivo resistance in childhood acute lymphoblastic leukemia: Correlations between BCRP, MRP1, MRP4 and MRP5 ABC transporter expression and intracellular methotrexate polyglutamate accumulation

Chemoresistance is an important factor in the treatment failure of childhood acute lymphoblastic leukemia (ALL). One underlying mechanism of chemoresistance involves (over)expression of ATP-dependent drug efflux transporters such as multidrug resistance protein 1–5 (MRP1–5) and breast cancer resistance protein (BCRP), which can extrude the important antileukemia drug methotrexate (MTX). Survival of childhood ALL critically depends on the leukemic blasts' capacity for intracellular retention of MTX and MTX-polyglutamates. This pilot study assessed whether expression of MRP1, MRP4, MRP5 and BCRP (real-time PCR) in primary childhood ALL blasts (n = 23) correlated with ex vivo resistance to MTX (assayed by in situ thymidylate synthase inhibition assay (TSIA)), ex vivo accumulation of (radioactive) MTX polyglutamates, and patient survival. Results show that high MRP4 expression is correlated with ex vivo MTX resistance assayed by TSIA (P = 0.01). Moreover, elevated MRP4 and BCRP expression correlated with lower accumulation of MTX-PGs (P = 0.004 and P = 0.03, respectively). Combined high expression of BCRP and MRP4 even further impacted reduced MTX-PG accumulation (P = 0.02). Overall survival was lower (P logrank = 0.04) in children with ALL cells which featured a relatively high expression of both BCRP and MRP4 transporters. These results underscore the impact of high drug efflux transporter expression, notably MRP4 and BCRP, in diminished MTX response in childhood ALL.

Cx32 mediates cisplatin resistance in human ovarian cancer cells by affecting drug efflux transporter expression and activating the EGFR‑Akt pathway

Our previous study demonstrated that connexin 32 (Cx32) was upregulated and redistributed to the cytoplasm in A2780 human ovarian cancer cells with acquired resistance to cisplatin; this increased Cx32 feedback promoted cisplatin resistance. To further investigate the mechanism underlying Cx32‑mediated cisplatin resistance, alterations in drug transporters, the DNA repair system and the anti‑apoptotic signalling pathway were investigated by overexpressing or knocking down Cx32 in parental cells (A2780); cisplatin‑resistant human ovarian cancer cells (A2780/CDDP) were also acquired. Upregulation of efflux transporters [multi‑drug resistance protein 2 (MRP‑2), ATPase copper transporting α (ATP7A) and ATPase copper transporting β] and downregulation of the influx transporter copper uptake protein 1 mediated cisplatin resistance in A2780/CDDP cells. A2780/CDDP cells also exhibited increased expression of the DNA repair enzyme excision repair cross‑complementation group1 (ERCC1) and activation of the epidermal growth factor receptor (EGFR) signalling pathway. Small interfering RNA‑mediated knockdown of Cx32 in A2780/CDDP cells decreased the expression of efflux transporters (MRP‑2 and ATP7A). Knockdown of Cx32 in A2780/CDDP cells also decreased the expression of ERCC1, inhibited the activation of the EGFR signalling pathway and enhanced the cytotoxicity of cisplatin. When Cx32 was overexpressed in A2780 cells, an opposite effect on the expression of efflux transporters (MRP‑2 and ATP7A) and the activation of the EGFR signalling pathway was observed, which resulted in insensitivity to cisplatin‑induced apoptosis. Thus, Cx32 expression may induce cisplatin resistance by modulating drug efflux transporter expression and activating the EGFR‑protein kinase B signalling pathway in ovarian cancer cells.

Tobacco and Antiretrovirals Modulate Transporter, Metabolic Enzyme, and Antioxidant Enzyme Expression and Function in Polarized Macrophages.

Background: Cigarette smoking increases systemic oxidative stress, inflammation, and viral replication in individuals with HIV. Macrophages are infected during HIV infection and serve as an important reservoir throughout the process. Macrophages exist in two phenotypes, the classically acti-vated M1 macrophage and alternatively activated M2 macrophage. The expression of drug efflux transporters and metabolic enzymes, which have direct effects on intracellular drug concentrations, differ between the pro-inflammatory M1 macrophage and the anti-inflammatory M2 macrophage.Objective: To further explain the role of tobacco use in worsened outcomes in the HIV + population receiving antiretroviral therapy.Methods: Western blotting was used to examine macrophage polarization and expression of drug efflux transporters, CYP enzymes, and antioxidant enzymes. The arginase assay was used to measure arginase activity. Cytokine production was measured using the human multiplex inflammatory cytokine assay kit. The 8-OHdG DNA Damage Quantification Direct Kit was used to quantify DNA damage. Viral replication under the influence of tobacco and antiretroviral drug use was measured by p24 Elisa.Results: We observed phenotypic shifts from M1 to M2 with both individual and combination treatments with cigarette smoke condensate and the protease inhibitor antiretroviral drug lopinavir. These shifts lead to changes in cytokine production, the expression of CYP enzymes, anti-oxidant enzymes, and drug efflux transporters, as well as changes in viral replication.Conclusion: This data suggest a mechanism by which tobacco use impairs HIV antiretroviral therapy to increase intracellular drug concentrations in this important cellular reservoir.

A human xenobiotic nuclear receptor contributes to nonresponsiveness of Mycobacterium tuberculosis to the antituberculosis drug rifampicin

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB). It acquires phenotypic drug resistance inside macrophages, and this resistance mainly arises from host-induced stress. However, whether cellular drug-efflux mechanisms in macrophages contribute to nonresponsiveness of M. tuberculosis to anti-TB drugs is unclear. Here, we report that xenobiotic nuclear receptors mediate TB drug nonresponsiveness by modulating drug-efflux transporters in macrophages. This was evident from expression analysis of drug-efflux transporters in macrophages isolated from TB patients. Among patients harboring rifampicin-susceptible M. tuberculosis, we observed increased intracellular survival of M. tuberculosis upon rifampicin treatment of macrophages isolated from patients not responding to anti-TB drugs compared with macrophages from patients who did respond. Of note, M. tuberculosis infection and rifampicin exposure synergistically modulated macrophage drug-efflux transporters in vitro We also found that the xenobiotic nuclear receptor pregnane X receptor (PXR) modulates macrophage drug-efflux transporter expression and activity, which compromised the anti-TB efficacy of rifampicin. We further validated this finding in a TB mouse model in which use of the PXR antagonist ketoconazole rescued rifampicin anti-TB activity. We conclude that PXR activation in macrophages compromises the efficacy of the anti-TB drug rifampicin. Alternative therapeutic strategies, such as use of the rifampicin derivatives rifapentine and rifabutin, which do not activate PXR, or of a PXR antagonist, may be effective for tackling drug nonresponsiveness of M. tuberculosis that arises from drug-efflux systems of the host.