Drug Efflux Pump Activity Research Articles

Discovery of Potent Isoquinolinequinone N-Oxides to Overcome Cancer Multidrug Resistance.

Multidrug resistance (MDR) of human tumors has resulted in an immediate need to develop appropriate new drugs. This work outlines the development of 20 potent IQQ N-oxide derivatives in two isomeric families, both exhibiting nanomolar GI50 against human tumor cell lines. Preliminary NCI-60 tumor screening sees the C(6) isomers achieve a mean GI50 > 2 times lower than the corresponding C(7) isomers. MDR evaluation of nine selected compounds reveals that each presents lower GI50 concentrations in two MDR tumor cell lines. Four of the series display nanomolar GI50 values against MDR cells, having selectivity ratios up to 2.7 versus the sensitive (parental) cells. The most potent compound 25 inhibits the activity of drug efflux pumps in MDR cells, causes significant ROS accumulation, and potently inhibits cell proliferation, causing alterations in the cell cycle profile. Our findings are confirmed by 3D spheroid models, providing new candidates for studies against MDR cancers.

Tryptophan transport gene inactivation promotes the development of antibiotic resistance in Escherichia coli.

Indole serves as a signaling molecule that could regulate different bacterial physiological processes, including antibiotic resistance through biofilm formation and drug efflux pump activity. In Escherichia coli, indole is produced through the tryptophan pathway, which involves three permeases (Mtr, AroP, and TnaB) that can transport the amino acid tryptophan. Although these permeases play distinct roles in the secretion of indole biosynthesis, their impact on multidrug resistance mediated by indole remaines unclear. This study was designed to investigate the connection between the tryptophan transport system and antibiotic resistance by constructing seven gene deletion mutants from E. coli MG1655 (wild type). Our result showed that deletion of the aroP or tnaB gene led to increased antibiotic resistance as evaluated by MICs for different antibiotics. Efflux activity test results revealed that the increased antibiotic resistance was related with the AcrAB-Tolc drug efflux pump in the mutants. The transcriptome analysis further demonstrated that decreased susceptibility to kanamycin and ampicillin in E. coli was accompanied by reduced accumulation of reactive oxygen species and decreased motility. These findings highlight the substantial influence of the tryptophan transport system on antibiotic resistance in E. coli, which is crucial for developing strategies against antibiotic resistance in bacterial infections.

Reduction in Rumen Tetracycline-Insensitive Bacteria during a Grain Challenge Using the Isoflavone Biochanin A.

The isoflavone biochanin A was previously shown to promote weight gain in growing steers by selectively inhibiting rumen bacteria-like growth-promoting feed antibiotics. The hypothesis that biochanin A inhibited the action of drug efflux pumps was tested by enumerating tetracycline-insensitive bacteria from steers in a subacute rumen acidosis (SARA) challenge. Steers (n = 3/group) treatment groups were forage only, SARA control, SARA with monensin (0.2 g d-1), and SARA with biochanin A (6.0 g d-1). As the steers were stepped up from the forage-only basal diet to 70% cracked corn, the number of rumen bacteria enumerated on two tetracycline-containing media types (nutrient glucose agar and tetracycline, and bile esculin azide and tetracycline) increased (p < 0.05) from as little as 1.7(105) to as great as 6.7(106) cfu mL-1 on the nutrient glucose agar in the SARA and monensin control groups. The biochanin A group maintained the same number of tetracycline-insensitive bacteria as the forage-only controls (p > 0.05). The effects were similar to the more selective media type, but the differences were smaller. These results support the hypothesis that biochanin A inhibits the activity of drug efflux pumps in vivo.

Antimicrobial Peptides: From Design to Clinical Application.

Infection of multidrug-resistant (MDR) bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Enterobacteriaceae (CRE), and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli, brings public health issues and causes economic burden. Pathogenic bacteria develop several methods to resist antibiotic killing or inhibition, such as mutation of antibiotic function sites, activation of drug efflux pumps, and enzyme-mediated drug degradation. Antibiotic resistance components can be transferred between bacteria by mobile genetic elements including plasmids, transposons, and integrons, as well as bacteriophages. The development of antibiotic resistance limits the treatment options for bacterial infection, especially for MDR bacteria. Therefore, novel or alternative antibacterial agents are urgently needed. Antimicrobial peptides (AMPs) display multiple killing mechanisms against bacterial infections, including directly bactericidal activity and immunomodulatory function, as potential alternatives to antibiotics. In this review, the development of antibiotic resistance, the killing mechanisms of AMPs, and especially, the design, optimization, and delivery of AMPs are reviewed. Strategies such as structural change, amino acid substitution, conjugation with cell-penetration peptide, terminal acetylation and amidation, and encapsulation with nanoparticles will improve the antimicrobial efficacy, reduce toxicity, and accomplish local delivery of AMPs. In addition, clinical trials in AMP studies or applications of AMPs within the last five years were summarized. Overall, AMPs display diverse mechanisms of action against infection of pathogenic bacteria, and future research studies and clinical investigations will accelerate AMP application.

Nanomedicines for combating multidrug resistance of cancer.

Chemotherapy typically involves the use of specific chemodrugs to inhibit the proliferation of cancer cells, but the frequent emergence of a variety of multidrug-resistant cancer cells poses a tremendous threat to our combat against cancer. The fundamental causes of multidrug resistance (MDR) have been studied for decades, and can be generally classified into two types: one is associated with the activation of diverse drug efflux pumps, which are responsible for translocating intracellular drug molecules out of the cells; the other is linked with some non-efflux pump-related mechanisms, such as antiapoptotic defense, enhanced DNA repair ability, and powerful antioxidant systems. To overcome MDR, intense efforts have been made to develop synergistic therapeutic strategies by introducing MDR inhibitors or combining chemotherapy with other therapeutic modalities, such as phototherapy, gene therapy, and gas therapy, in the hope that the drug-resistant cells can be sensitized toward chemotherapeutics. In particular, nanotechnology-based drug delivery platforms have shown the potential to integrate multiple therapeutic agents into one system. In this review, the focus was on the recent development of nanostrategies aiming to enhance the efficiency of chemotherapy and overcome the MDR of cancer in a synergistic manner. Different combinatorial strategies are introduced in detail and the advantages as well as underlying mechanisms of why these strategies can counteract MDR are discussed. This review is expected to shed new light on the design of advanced nanomedicines from the angle of materials and to deepen our understanding of MDR for the development of more effective anticancer strategies. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Presence of a mutation in PSEN1 or PSEN2 gene is associated with an impaired brain endothelial cell phenotype in vitro

BackgroundAlzheimer’s disease (AD) is the most common form of neurodegenerative disease. It is an irreversible condition marked by irreversible cognitive loss, commonly attributed to the loss of hippocampal neurons due to the formation of senile plaques and neurofibrillary tangles. Although the sporadic form is the most prevalent, the presence of familial form (involving several genes such as APP, PSEN1, and PSEN2) of the disease is commonly used as a model for understanding the pathophysiology of the disease. The aim of this study is to investigate the effect of a mutation on PSEN1 and PSEN2 genes on the BBB function using induced pluripotent stem cells (iPSCs).Methods iPSC lines from patients suffering from a familial form of Alzheimer’s disease and harboring mutations in PSEN1 or PSEN2 were used in this study and compared to a control iPSC line. Cells were differentiated into brain microvascular endothelial cells (BMECs) following established differentiation protocols. Barrier function was assessed by measuring TEER and fluorescein permeability, drug transporter activity was assessed by uptake assay, glucose uptake and metabolism assessed by cell flux analyzer, mitochondrial potential by JC-1, and lysosomal acidification by acridine orange.ResultsiPSC-derived BMECs from the FAD patient presenting a mutation in the PSEN1 gene showed impaired barrier function compared to the FAD patient harboring a mutation in PSEN2 and to the control group. Such impaired barrier function correlated with poor tight junction complexes and reduced drug efflux pump activity. In addition, both PSEN1 and PSEN2-BMECs displayed reduced bioenergetics, lysosomal acidification, autophagy, while showing an increase in radical oxygen species (ROS) production. Finally, PSEN1- and PSEN2-BMECs showed an elevated secretion of Aβ1–40 peptides compared to control-BMECs.ConclusionsOur study reports that iPSC-derived BMECs obtained from FAD patients showed impaired barrier properties and BMEC metabolism. In particular, mutation in the PSEN1 gene was associated with a more detrimental phenotype than mutation in PSEN2, as noted by a reduced barrier function, reduced drug efflux pump activity, and diminished glucose metabolism. Therefore, assessing the contribution of genetic mutations associated with Alzheimer’s disease will allow us to better understand the contribution of the BBB in dementia, but also other neurodegenerative diseases.

Quercetin potentializes the respective cytotoxic activity of gemcitabine or doxorubicin on 3D culture of AsPC-1 or HepG2 cells, through the inhibition of HIF-1α and MDR1.

The impact of cancer on lifespan is significantly increasing worldwide. Enhanced activity of drug efflux pumps and the incidences of the tumor microenvironment such as the apparition of a hypoxic gradient inside of the bulk tumor, are the major causes of chemotherapy failure. For instance, expression of Hypoxia-inducible factor (HIF-1α) has been associated with metastasis, resistance to chemotherapy and reduced survival rate. One of the current challenges to fight against cancer is therefore to find new molecules with therapeutic potential that could overcome this chemoresistance. In the present study, we focused on the bioactive plant flavonoid quercetin, which has strong antioxidant and anti-proliferative properties. We examined the efficacy of combined treatments of quercetin and the anti-cancer drugs gemcitabine and doxorubicin, known to specifically act on human pancreatic and hepatic cancer cells, respectively. Moreover, our study aimed to investigate more in-depth the implication of the multidrug transporter MDR1 and HIF-1α n chemoresistance and if quercetin could act on the activity of the drug efflux pumps and the hypoxia-associated effects. We observed that the anti-cancer drugs, were more effective when administered in combination with quercetin, as shown by an increased percentage of dead cells up to 60% in both 2D and 3D cultures. In addition, our results indicated that the combination of anti-cancer drugs and quercetin down-regulated the expression of HIF-1α and increased the expression levels of the regulator of apoptosis p53. Moreover, we observed that quercetin could inhibit the efflux activity of MDR1. Finally, our in vitro study suggests that the efficiency of the chemotherapeutic activity of known anti-cancer drugs might be significantly increased upon combination with quercetin. This flavonoid may therefore be a promising pharmacological agent for novel combination therapy since it potentializes the cytotoxic activity of gemcitabine and doxorubicin on by targeting the chemoresistance developed by the pancreatic and liver cancer cells respectively.

Mitochondria-Mediated Azole Drug Resistance and Fungal Pathogenicity: Opportunities for Therapeutic Development.

In recent years, the role of mitochondria in pathogenic fungi in terms of azole resistance and fungal pathogenicity has been a rapidly developing field. In this review, we describe the molecular mechanisms by which mitochondria are involved in regulating azole resistance and fungal pathogenicity. Mitochondrial function is involved in the regulation of drug efflux pumps at the transcriptional and posttranslational levels. On the one hand, defects in mitochondrial function can serve as the signal leading to activation of calcium signaling and the pleiotropic drug resistance pathway and, therefore, can globally upregulate the expression of drug efflux pump genes, leading to azole drug resistance. On the other hand, mitochondria also contribute to azole resistance through modulation of drug efflux pump localization and activity. Mitochondria further contribute to azole resistance through participating in iron homeostasis and lipid biosynthesis. Additionally, mitochondrial dynamics play an important role in azole resistance. Meanwhile, mitochondrial morphology is important for fungal virulence, playing roles in growth in stressful conditions in a host. Furthermore, there is a close link between mitochondrial respiration and fungal virulence, and mitochondrial respiration plays an important role in morphogenetic transition, hypoxia adaptation, and cell wall biosynthesis. Finally, we discuss the possibility for targeting mitochondrial factors for the development of antifungal therapies.

Abstract 2622: Paclitaxel directed screening to identify novel anti-cancer drugs

Abstract Among the main challenges for the treatment of breast cancers is disease spread which is often coupled with resistance to chemotherapy. Furthermore, patients with late stage breast cancer go through first- and second-line therapies before they are candidates for new and/or experimental therapies. At such late stages, tumors that might have responded to new therapies have evolved to express multi-drug resistance - conceivably as a consequence of exposure to the initial therapies. Many mechanisms have been proposed to contribute to drug resistance, ranging from (but not limited to) reduced drug penetration and the activity of drug efflux pumps through to cell-adhesion mediated resistance to cell death. To address these challenges we sought to determine whether; a) tumor metastasis to different secondary sites generates location-specific tumor phenotypes that alter sensitivity to chemotherapy and, b) whether pre-treatment of tumors to conventional paclitaxel (PTX) chemotherapy alters their sensitivity to new drugs. First, we implanted 2x10(5) human luciferase-positive LM2-4 human breast cancer cells intracardiacally into SCID mice (n=15), which resulted 2 weeks later in the development of widespread metastases to the lungs, liver, spleen, and bone. For some of the mice bearing such metastases, PTX therapy (20mg/kg, i.p.) was administered 24 hours before the mice were euthanized. Next, we used RNA sequencing of isolated LM2-4 metastatic nodules to determine the impact of the location of metastases on transcriptome profiles (assessed by 40 million reads per each total RNA sample, followed by Principal Component Analysis). This study revealed that lung metastases have similar signatures to metastases to spleen and ovary, and to metastases in those organs following PTX therapy, while such transcriptome profiles are all quite distinct from those of metastases of the same tumor to the liver. These changes presumably are due to different cell-cell interactions of tumor cells in the different organs, as well as a consequence of the different microenvironments. Second, we set up a drug screen of LM2-4 breast cancer cells recovering from 1nM paclitaxel 24hr exposure in vitro to test for drugs that are toxic to the tumor cells following PTX pre-exposure. A screen of 30,000 small molecule drugs thus far identified 7 hit compounds. Our results serve a proof of concept that the location of metastases can alter the tumor cell phenotype, as can pre-treatment with PTX, and suggest that in some cases therapy ought to be tailored to the location of metastasis. Based on those results we identified drugs that may be useful in hampering the tumor cell recovery from PTX chemotherapy. Our work serves as a test ground for stratifying tumors based on their tissue of tumor spread, and provide a rationale for identifying drugs that hamper recovery of tumor cells to chemotherapy agents often incorporated into first and second line therapies, such as paclitaxel. Citation Format: Joseph Wingate, Diana Gonzalez Garcia, Karla Parra, Serina Batson, Joel Martinez, Ivan Sosa Ontiveros, Renato Aguilera, Armando Varela, Paloma Valenzuela, Marcos Armendariz, Chloe Clifton, Arlene Levario, Angelica Chacon, Andres Villagrana, Emmanuel Sanchez, Guido Bocci, Giulio Francia. Paclitaxel directed screening to identify novel anti-cancer drugs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2622.

Abstract 2998: Linear confined space migration induces treatment resistance and gene expression changes in glioblastoma multiforme

Abstract Background: Glioblastoma Multiforme (GBM), an aggressive brain cancer, is known for its treatment resistant recurrence and lethality. The diffuse migration and invasion of GBM cells commonly occurs along brain white matter tracts in linear confined spaces between axons and has significant clinical implications as these cells survive initial therapies forming the main source of recurrent tumors resistant to therapies. In this context, this project aims to analyze the treatment resistance mechanism associated with linear confined space migrating cells (LCSM) to devise a better treatment strategy for GBM. Methods: We used a combination of microfluidic technology (5 µM wide channels in PDMS block) emulating linear white matter tracts in brain and standard molecular analysis (confocal microscopy, high content imaging, western blot, and flow cytometry) to investigate LCSM cells compared to Standard Monolayer Culture cells (SMCs) for the mechanism of drug resistance. Both established and patient derived GBM cell lines were used in the study. Results: LCSM cells were more resistant to chemo-drugs (Temozolomide and Doxorubicin) than SMCs as observed in cell viability assays. Enhanced drug efflux was observed in LCSM cells compared to SMC cells indicating the activation of drug efflux pumps. Protein expression analysis indicated that ABC drug efflux transporters (ABCB1, ABCC1, ABCG2) were activated or enhanced at varying levels in different LCSM cells compared to SMCs. Pharmacological inhibition of the prominently expressed ABCG2 protein using KO143 resulted in enhanced cell death by chemo drugs, suggesting that chemo resistance in LCSM, at least in part, is conferred by ABCG2 or other efflux transporters. In addition, CD133 (a marker of treatment resistant stem cell population in GBM) and AQP4 (Aquaporin 4; pro-migratory marker in GBM) were also enhanced in LCSM compared to SMC suggesting their possible roles in treatment resistance. Conclusion: Preliminary studies reveal that the enhanced expression ABC transporters, prominently ABCG2, might be responsible for enhanced drug efflux and low therapeutic sensitivity in GBM- LCSM cells. The over expression of AQP4 and CD133, and differential expression of other genes suggest that more complex mechanisms might be involved, which are currently under investigation. Citation Format: Anish Babu, Patrick McKernan, Xue Cai, James Battiste. Linear confined space migration induces treatment resistance and gene expression changes in glioblastoma multiforme [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2998.

Octyl gallate reduces ABC multidrug transporter CaCdr1p expression and leads to its mislocalisation in azole-resistant clinical isolates of Candida albicans

ObjectivesFungal pathogens pose a serious threat to public health. Widespread and prolonged use of antifungal drugs has led to the development of multidrug resistance in the human fungal pathogen Candida albicans. Among several mechanisms leading to drug resistance in C. albicans, overexpression of drug efflux transporters remains by far the leading cause of multidrug resistance, facilitated by overexpression of ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporters. Hence, targeting efflux pumps still represents a promising approach to combat multidrug resistance. In this study, the effect of octyl gallate (OG), a natural food additive, on drug efflux pump activity of C. albicans was analysed. MethodsDrug efflux pump activity was determined by rhodamine 6G (R6G) efflux and Nile red accumulation assay in a Candida drug resistance protein 1 (CaCdr1p)-overexpressing strain. Gene expression and protein expression and localisation were studied by RT-PCR, Western blot and confocal microscopy. Ergosterol content was measured by the alcoholic KOH method. ResultsOG specifically inhibits the activity of CaCdr1p, belonging to the ABC superfamily. The underlying mechanism was confirmed as competitive mode of inhibition by OG as revealed by Lineweaver–Burk plot. Furthermore, OG leads to reduced expression of CDR1 and CaCdr1p and mislocalisation of CaCdr1p. Additionally, OG sensitises azole-susceptible and -resistant clinical matched-pair isolates Gu4 & Gu5 and leads to impeded R6G efflux and depleted ergosterol content. ConclusionThe ability of OG as a potent inhibitor of CaCdr1p that chemosensitises drug-resistant C. albicans warrants further studies to be exploited as an effective antifungal agent.

Talazoparib nanoparticles for overcoming multidrug resistance in triple-negative breast cancer.

Herein, we investigated efflux pumps-mediated talazoparib-resistance in the treatment of triple-negative breast cancer (TNBC). Furthermore, we produced a novel talazoparib-solid lipid nanoparticles (SLNs) and then explored in vitro therapeutic efficacy of talazoparib-SLNs to overcome talazoparib-resistance in TNBC cells. Talazoparib-SLNs formulation was produced and then characterized. Calcein and Rho-123 were used to analyze the functional activity of drug efflux pumps in these cells. Additionally, RT-PCR, western blot and immunofluorescence analysis were used to detect the messenger RNA, and protein expression level, and cellular localization of the multidrug resistance (MDR1), breast cancer resistance protein (BCRP), and MRP1. We found that talazoparib efflux was mediated by BCRP and MRP1 pumps in TNBC cells. Talazoparib-SLNs could significantly enhance therapeutic efficacy of talazoparib. Furthermore, talazoparib-SLNs were more effective in the suppression of MDR1, BCRP, and MRP1 gene and protein expression levels than talazoparib. Consequently, this study suggests that talazoparib-SLNs formulation represents a promising therapeutic carrier to reverse MDR-mediated resistance in TNBC.

Vanillin confers antifungal drug synergism in Candida albicans by impeding CaCdr2p driven efflux

AimAmong the most common mechanisms of multidrug resistance (MDR) in prevalent human fungal pathogen, Candida albicans, overexpression of drug efflux pumps remains the predominant mechanism. Hence to inhibit efflux pumps and chemosensitize C. albicans against traditional antifungal drugs still represents an attractive approach. The present study aimed to analyze the effect of Vanillin (Van), a natural food flavoring agent, on drug efflux pump activity of Candida albicans. Methods and resultsWe observed that Van specifically inhibits Candida drug resistance protein 2 (CaCdr2p) activity belonging to ATP Binding Cassette (ABC) superfamily as revealed by abrogated rhodamine 6G efflux and nile red accumulation assay with CaCdr2p over expressing strain. Insight studies into the mechanisms suggested that abrogated efflux by CaCdr2p is due to competitive mode of inhibition by Van as depicted by Lineweaver-Burk plot. RT-PCR, western blot and confocal microscopy further unraveled that Van leads to reduced expression of CDR2 and CaCdr2p mislocalization respectively. Furthermore, Van sensitizes the azole sensitive and resistant clinical matched pair of isolates Gu4/Gu5 and led to abrogated rhodamine 6G efflux and depleted ergosterol. Furthermore, Van synergizes with membrane targeting drugs fluconazole and amphotericin B as their fractional inhibitory coefficient index was less than 0.5. ConclusionVan being a potent inhibitor of CaCdr2p and chemosensitizing of drug resistant C. albicans warrants further studies to be exploited as effective antifungal agent.

Abstract 4412: Piperine enhances the efficacy of 5 FU against liver cancer

Abstract Liver cancer is one of the most devastating malignancies. Hepatocellular carcinoma (HCC) accounts for &amp;gt;90% of primary liver malignancies and is the third-leading cause of cancer-related deaths worldwide. Systemic chemotherapy is one of the most important treatment modalities for advanced HCC. Unfortunately, long-term treatment acquired resistance in HCC due to the activation of drug efflux pumps. A natural compound in combination with chemotherapeutic drugs is a potential approach to combat the side effects. In this study, we have demonstrated whether piperine (natural compound) could increase the effect of 5-FU in HCC cells. The cell viability (MTT) assay was performed to determine the optimal IC50 values of Piperine, 5-FU alone and in combination. Immunofluorescence study confirmed the effectiveness of combined drug; dead nuclei were found more within the cells treated by the combination compared to piperine or 5-FU alone. The combined drug treatment was most effective in inhibiting proliferation and inducing apoptosis; accordingly, it showed noticeable increases in pro-apoptotic (BAK, BID), PARP and decreases in anti-apoptotic genes (BCL-XL) gene in a western blot and RT-PCR analysis. Further, in SK-HEP-1 cells, the combination upregulated the expression of cell cycle inhibitors (p21WAF1/CIP1, p27KIP), which, in turn, inhibited the expression of CDK2/ Cyclin D1 complex. Therefore, it blocks the transition of cells into G0/G1 to S phase. Taken together, we observed that the combination of 5-FU and Piperine resulted in a significant cytotoxicity and apoptosis as compared to alone. Our results suggest that piperine can be a potential agent in enhancing the efficacy of 5-FU in liver cancer treatment. These finding highlights the promising role of natural bioactive compounds and provides the rationale for further transitional researches. Citation Format: David Anwanwan, Santosh Kumar Singh, James W. Lillard, Manoj K. Mishra, Rajesh Singh. Piperine enhances the efficacy of 5 FU against liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4412.

Calcium signaling pathway is involved in non-CYP51 azole resistance in Aspergillus fumigatus.

The opportunistic fungal pathogen Aspergillus fumigatus, which is one of the primary airborne ascomycete pathogens and allergens worldwide, causes invasive fungal infections, which have high morbidity and mortality rates among immunosuppressed patients. The abuse of azole antifungals results in serious drug resistance in clinical therapy. Thus, a thorough understanding of the azole drug resistance mechanism and screening of antifungal agents with a novel mode of action and new drug targets are required to fight against drug resistance. Current studies suggest that there are three major azole resistance mechanisms in fungal pathogens, including changes of the drug target Cyp51, activation of drug efflux pumps and induction of cellular stress responses. Fungi must adapt to a variety of external environmental stressors to survive. These obstacles include stress to the plasma membrane after azole antifungal treatments, high temperature, pH variation, and oxidative stress. As a filamentous fungus, A. fumigatus has evolved numerous signal-transduction systems to sense and respond to azole stresses to survive and proliferate in harsh environmental conditions. Among these signal-transduction systems, the Ca2+ signaling pathway is one of the most important response systems, which has been verified to be involved in stress adaptation. In this review, we have summarized how the components of the calcium-signaling pathway and their interaction network are involved in azole stress response in A. fumigatus.

Altered drug efflux under iron deprivation unveils abrogated MmpL3 driven mycolic acid transport and fluidity in mycobacteria.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is a global threat to human health hence better understanding of the MTB pathogenesis for improved therapeutics requires immediate attention. Emergence of drug-resistant strains has stimulated an urgent need for adopting new strategies that could be implemented to control TB. One of the contributing mechanisms by which MTB evades drug doses is overexpression of drug efflux pumps. Thus blocking or modulating the functionality of efflux pumps represents an attractive approach to combat drug resistance. Iron is a critical micronutrient required for MTB survival and not freely available inside the host. In this study, we demonstrated that iron deprivation impairs drug efflux pump activity and confers synergism for anti-TB drugs in presence of efflux pump inhibitors against MTB. Mechanistic insights revealed that iron deprivation inhibit resistance nodulation division superfamily transporter activity. This was evident from enhanced Nile red accumulation and reduced expression of MmpL3, a transmembrane promising target involved in mycolic acid transport across membrane. Furthermore, iron deprivation led to abrogated MA transport particularly of class methoxy-MA which was confirmed by TLC and mass spectrometry based lipidome analysis. Additionally, iron deprivation leads to enhanced membrane fluidity in MTB. Together, MmpL3 being a promiscuous anti-TB target, metal chelation strategy could be adopted to boost the effectiveness of current anti-TB drug regimes to combat drug resistance TB.

Inhibition of mutagenic translesion synthesis: A possible strategy for improving chemotherapy?

While DNA damaging chemotherapy can be very effective and even curative in the treatment of certain cancers, intrinsic and acquired drug resistance underlies tumor progression and morbidity in many cancer patients. Intrinsic resistance defines a cell state that is inherently tolerant of drug action. This can include the activation of drug efflux pumps or detoxifying processes that effectively reduce intracellular drug concentration [1]. This can also include a change in the recognition or persistence of DNA damage, mediated by an enhanced DNA repair capability, a blunted DNA damage response, or the ability to proliferate in the presence of DNA damage. Conversely, acquired drug resistance represents a mutational or epigenetic process by which a chemosensitive cell develops 1 or more of the characteristics of an intrinsically resistant cancer cell. Thus, the mechanisms underlying intrinsic and acquired drug resistance are quite distinct. One describes a cell state, and the other describes the capability of reaching that cell state. Yet, these processes are very much coupled in the context of mutagenic translesion synthesis (TLS). As discussed throughout this review, mutagenic TLS polymerases underlie 2 important phenotypes in response to genotoxic chemotherapy. First, they allow for the bypass of modified DNA bases during DNA synthesis, allowing proliferation to continue in the presence of chemotherapy. Second, the low fidelity replication performed by TLS polymerases results in the introduction of inappropriate, nonpairing bases across from modified nucleotides. The bypass function of TLS polymerases is particularly relevant to intrinsic drug resistance. Many tumors, including most pancreatic adenocarcinomas, nonsmall cell lung cancers, and aggressive brain tumors, as well as most metastatic malignancies, fail to significantly regress following chemotherapy [2]. In these tumors, TLS activity contributes to a drug resistant state by promoting the tolerance of DNA damage [3–6]. Conversely, the mutational role of TLS polymerases is central to process of acquired drug resistance. Tumor regression and relapse following chemotherapy is almost always accompanied by the development of drug resistant disease. This may not occur at initial relapse, but upon serial cycles of treatment patients generally succumb to tumors that have acquired intrinsically resistant disease. In fact, for certain cancers the overall prognosis is not dictated by the initial response of the tumor to chemotherapy. Rather, the response of the relapsed tumor to therapy is a significantly better determinant of overall survival. For instance, a high error-prone TLS activity translates into greater tumor adaptation to chemotherapy, while a low error-prone TLS activity leaves tumor in a treatment-naive state. This latter state is amenable to continued long-term treatment of tumors that remain response to treatment with the initial therapy. The dual functions of mutagenic TLS polymerases in intrinsic and acquired chemoresistance make these proteins very attractive potential targets for adjuvant therapy. When combined with front-line genotoxic therapy, these TLS inhibitors would be expected to sensitize tumors to chemotherapy while blocking drug-induced mutation. Consequently, while the generation of such inhibitors is complex, their route to the clinic is more apparent. TLS inhibitors could be applied in combination with the standard of care for many malignancies. By effectively increasing the effects of chemotherapy in target cells, these agents may also allow for a reduction in chemotherapy dose regimens. An added benefit of these agents may be a reduction in the rate of secondary chemotherapy-driven malignancies that occur in patients following successful treatment of the primary disease.

Interspecies cross-talk between co-cultured Pseudomonas putida and Escherichia coli.

Pseudomonas putida and Escherichia coli are ubiquitous microorganisms that can be isolated from soil rhizosphere, the surface of vegetables, fresh waters and wastewaters - environments in which they likely co-exist. Despite this, the potential interactions between these microbes have not been studied in detail. To analyse these interactions, we carried out RNA-seq transcriptomic analysis of these microbes as monocultures and as co-cultures. Our results show that co-culture of these microbes significantly alters transcriptional profiles. The most dramatic transcriptional changes in both microorganisms were involved in central carbon metabolism, as well as adhesion to surfaces and the activation of drug efflux pumps. We also found that acetate production was one of the mechanisms used by E. coli K-12 MG1655 in response to the presence of P. putida DOT-T1E.

ATP-dependent activity and mitochondrial localization of drug efflux pumps in doxorubicin-resistant breast cancer cells

BackgroundWe hypothesized that, among the mechanisms of drug-resistance acquired by doxorubicin (DOX)-resistant breast cancer cells to maintain cell survival, ATP-dependent drug efflux pumps could be expressed in their mitochondrial membranes and this might limit the accumulation of DOX in this subcellular compartment in relation to mitochondrial ATP production. Methods/resultsMitochondrial DOX accumulation: the presence and the activity of mitochondrial efflux pumps and their relationship with mitochondrial ATP synthesis were analyzed in DOX-resistant (MCF-7doxR) and -sensitive (MCF-7S) breast cancer cells. Mitochondrial accumulation of DOX (autofluorescence) was decreased when ATP was produced, but only in MCF-7doxR. In these DOX-resistant cells, breast cancer resistance protein (BCRP) and multidrug resistance-associated protein (MRP1) were expressed and localized in mitochondria (confocal microscopy and confocal spectral imaging studies). In addition, mitochondrial accumulation of DOX was increased by BCRP and MRP1 inhibitors and, to a lower extent, by the mitochondrial ATP synthase inhibitor, oligomycin, in MCF-7doxR. ConclusionsBoth BCRP and MRP1 were localized in mitochondria and participated to the reduction of mitochondrial accumulation of DOX in MCF-7doxR. This process was partly dependent of mitochondrial ATP synthesis. General Significance. The present study provides novel insights in the involvement of mitochondria in the underlying mechanisms of DOX-resistance in breast cancer cells.

Quinone derivatives isolated from the endolichenic fungus Phialocephala fortinii are Mdr1 modulators that combat azole resistance in Candida albicans.

One of the main azole-resistance mechanisms in Candida pathogens is the upregulation of drug efflux pumps, which compromises the efficacy of azoles and results in treatment failure. The combination of azole-antifungal agents with efflux pump inhibitors represents a promising strategy to combat fungal infection. High-throughput screening of 150 extracts obtained from endolichenic fungal cultures led to the discovery that the extract of Phialocephala fortinii exhibits potent activity for the reversal of azole resistance. From P. fortinii cultures, a total of 15 quinone derivatives, comprising 11 new derivatives and 4 known compounds, were obtained. Among these compounds, palmarumycin P3 (3) and phialocephalarin B (8) specifically modulate the expression of MDR1 to inhibit the activity of drug efflux pumps and therefore reverse azole resistance. The present study revealed Mdr1 targeting as an alternative mechanism for the discovery of new agents to fight antifungal drug resistance.