Increased P-glycoprotein Expression Research Articles

Generation of Two Paclitaxel-Resistant High-Grade Serous Carcinoma Cell Lines With Increased Expression of P-Glycoprotein.

Debulking surgery followed by chemotherapy are the standard of care for high-grade serous carcinoma. After an initial good response to treatment, the majority of patients relapse with a chemoresistant profile, leading to a poor overall survival. Chemotherapy regimens used in high-grade serous carcinomas are based in a combination of classical chemotherapeutic drugs, namely, Carboplatin and Paclitaxel. The mechanisms underlying drug resistance and new drug discovery are crucial to improve patients’ survival. To uncover the molecular mechanisms of chemoresistance and test drugs capable of overcoming this resistant profile, it is fundamental to use good cellular models capable of mimicking the chemoresistant disease. Herein, we established two high-grade serous carcinoma cell lines with intrinsic resistance to Carboplatin and induced Paclitaxel resistance (OVCAR8 PTX R C and OVCAR8 PTX R P) derived from the OVCAR8 cell line. These two chemoresistant cell line variants acquired an enhanced resistance to Paclitaxel-induced cell death by increasing the drug efflux capacity, and this resistance was stable in long-term culture and following freeze/thaw cycles. The mechanism underlying Paclitaxel resistance resides in a significant increase in P-glycoprotein expression and, when this drug efflux pump was blocked with Verapamil, cells re-acquired Paclitaxel sensitivity. We generated two high-grade serous carcinoma cell lines, with a double-chemoresistant (Carboplatin and Paclitaxel) phenotype that mimics the majority of tumor recurrences in ovarian cancer context. This robust tool is suitable for preliminary drug testing towards the development of therapeutic strategies to overcome chemoresistance.

Cystine supplementation rebalances the redox homeostasis of microenvironment in non-small cell lung cancer cells and reverses their resistance to docetaxel.

Continuous docetaxel (DTX) treatment of non-small cell lung cancer induces development of drug resistance, but the mechanism is poorly understood. In this study we performed metabolomics analysis to characterize the metabolic patterns of sensitive and resistant A549 non-small cell lung cancer cells (A549/DTX cells). We showed that the sensitive and resistant A549 cells exhibited distinct metabolic phenotypes: the resistant cells were characterized by an altered microenvironment of redox homeostasis with reduced glutathione and elevated reactive oxygen species (ROS). DTX induction reprogrammed the metabolic phenotype of the sensitive cells, which acquired a phenotype similar to that of the resistant cells: it reduced cystine influx, inhibited glutathione biosynthesis, increased ROS and decreased glutathione/glutathione disulfide (GSH/GSSG); the genes involved in glutathione biosynthesis were dramatically depressed. Addition of the ROS-inducing agent Rosup (25, 50 μg/mL) significantly increased P-glycoprotein expression and reduced intracellular DTX in the sensitive A549 cells, which ultimately acquired a phenotype similar to that of the resistant cells. Supplementation of cystine (1.0 mM) significantly increased GSH synthesis, rebalanced the redox homeostasis of A549/DTX cells, and reversed DTX-induced upregulation of P-glycoprotein, and it markedly improved the effects of DTX and inhibited the growth of A549/DTX in vitro and in vivo. These results suggest that microenvironmental redox homeostasis plays a key role in the acquired resistance of A549 cancer cells to DTX. The enhancement of GSH synthesis by supplementary cystine is a promising strategy to reverse the resistance of tumor cells and has potential for translation in the clinic.

Sulfocoumarins, specific carbonic anhydrase IX and XII inhibitors, interact with cancer multidrug resistant phenotype through pH regulation and reverse P-glycoprotein mediated resistance

New 6-triazolyl-substituted sulfocoumarins were described as potent inhibitors of the transmembrane human carbonic anhydrase isoforms, CAIX and CAXII. These membrane associated enzymes that maintain pH and CO2 homeostasis are involved in cancer progression, invasion, and resistance to therapy. Recently, it was shown that CAXII expression associates with the expression of P-glycoprotein in multidrug resistant cancer cells. CAXII regulates P-glycoprotein activity by maintaining high intracellular pHi. In this study, the activity of three new sulfocoumarins was evaluated in three sensitive and corresponding multidrug resistant cancer cell lines with increased P-glycoprotein expression (non-small cell lung carcinoma, colorectal carcinoma and glioblastoma). Compound 3 showed the highest potential for cancer cell growth inhibition in all tested cell lines. Flow cytometric analyses showed that compound 3 induced intracellular acidification, cell cycle arrest in G2/M phase and necrosis in non-small cell lung carcinoma cells. Compound 3 demonstrated irreversible, concentration- and time-dependent inhibition of P-glycoprotein activity in multidrug resistant non-small cell lung carcinoma cells. The suppression of P-glycoprotein activity was accompanied with increased P-glycoprotein expression suggesting a compensatory mechanism of multidrug resistant cancer cells. In addition, compound 3 was able to sensitize multidrug resistant non-small cell lung carcinoma cells to doxorubicin. Overall, results imply that compound 3 has multidrug resistance modulating effect through intracellular acidification and subsequent inhibition of P-glycoprotein activity.

Bronchiolitis obliterans syndrome is associated with increased p-glycoprotein expression and loss of glucocorticoid receptor from steroid-resistant proinflammatory CD8+ T cells.

Immunosuppressive therapy fails to suppress the production of proinflammatory cytokines, particularly by CD8+ T cells, in stable lung transplant recipients and those undergoing chronic rejection, suggesting that some patients may become relatively resistant to immunosuppressants such as glucocorticoids (GC). We have shown loss of GC receptor (GCR) from the CD8+ cells, and we hypothesized that the drug membrane efflux pump, p-glycoprotein-1 (Pgp), may also be involved in lymphocyte steroid resistance following lung transplant. Pgp/GCR expression and interferon (IFN)-γ/tumour necrosis factor (TNF)-α proinflammatory cytokine production was measured in blood lymphocytes from 15 stable lung transplant patients, 10 patients with bronchiolitis obliterans syndrome (BOS) and 10 healthy aged-matched controls (±prednisolone±Pgp inhibitor, cyclosporin A±GCR activator, Compound A) using flow cytometry. Both Pgp+ and Pgp- lymphocyte subsets from all subjects produced IFN-γ/TNF-α proinflammatory cytokines. Pgp expression was increased in CD8+ Pgp+ T cells and correlated with IFN-γ/TNF-α expression and BOS grade. Reduced GCR was observed in CD8+ Pgp- T, natural killer (NK)T-like and NK cells from stable patients compared with controls, and reduced further in CD8+ Pgp- T cells in BOS. The addition of 2·5ng/ml cyclosporin A and 1µM prednisolone inhibit IFN-γ/TNF-α production significantly by CD8+ Pgp+ T cells from BOS patients. The addition of 10µM Compound A and 1 µM prednisolone inhibit IFN-γ/TNF-α production significantly by CD8+ Pgp- T cells from BOS patients. BOS is associated with increased Pgp expression and loss of GCR from steroid-resistant proinflammatory CD8+ T cells. Treatments that inhibit Pgp and up-regulate GCR in CD8+ T cells may improve graft survival.

BRONCHIOLITIS OBLITERANS SYNDROME IS ASSOCIATED WITH INCREASED P‐GLYCOPROTEIN EXPRESSION AND LOSS OF GLUCOCORTICOID RECEPTOR FROM STEROID RESISTANT PRO‐INFLAMMATORY CD8+T CELLS

RespirologyVolume 22, Issue S3 p. 16-16 Cell and Molecular Biology BRONCHIOLITIS OBLITERANS SYNDROME IS ASSOCIATED WITH INCREASED P-GLYCOPROTEIN EXPRESSION AND LOSS OF GLUCOCORTICOID RECEPTOR FROM STEROID RESISTANT PRO-INFLAMMATORY CD8+T CELLS First published: 23 November 2017 https://doi.org/10.1111/resp.13206_30Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume22, IssueS3Supplement: 22nd Congress of the Asian Pacific Society of Respirology, International Convention Centre, Sydney, Australia, 23–26 November 2017November 2017Pages 16-16 RelatedInformation

DTA0100, dual topoisomerase II and microtubule inhibitor, evades paclitaxel resistance in P-glycoprotein overexpressing cancer cells

The efficacy of microtubule targeting agents in cancer treatment has been compromised by the development of drug resistance that may involve both, P-glycoprotein overexpression and the changes in β-tubulin isoforms' expression. The anti-Topoisomerase II activity of methyl 4-((E)-2-(methoxycarbonyl)vinyloxy)oct-2-ynoate (DTA0100) was recently reported. Herein, we further evaluated this propargylic enol ether derivative and found that it exerts inhibitory effect on tubulin polymerization by binding to colchicine binding site. DTA0100 mitotic arrest properties were investigated in two multi-drug resistant cancer cell lines with P-glycoprotein overexpression (colorectal carcinoma and glioblastoma). The sensitivity of multi-drug resistant cancer cell lines to DTA0100 was not significantly changed in contrast to microtubule targeting agents such as paclitaxel, vinblastine and colchicine. DTA0100 clearly induced microtubule depolymerization, leading to disturbance of cell cycle kinetics and subsequent apoptosis. The fine-tuning in β-tubulin isoforms expression observed in multi-drug resistant cancer cells may influence the efficacy of DTA0100. Importantly, DTA0100 blocked the P-glycoprotein function in both multi-drug resistant cancer cell lines without inducing the increase in P-glycoprotein expression. Therefore, DTA0100 acting as dual inhibitor of Topoisomerase II and microtubule formation could be considered as multi-potent anticancer agent. Besides, it is able to overcome the problem of drug resistance that emerges in the therapeutic approaches with either Topoisomerase II or microtubule targeting agents.

PPAR-α, a lipid-sensing transcription factor, regulates blood–brain barrier efflux transporter expression

Lipid sensor peroxisome proliferator-activated receptor alpha (PPAR-α) is the master regulator of lipid metabolism. Dietary release of endogenous free fatty acids, fibrates, and certain persistent environmental pollutants, e.g. perfluoroalkyl fire-fighting foam components, are peroxisome proliferator-activated receptor alpha ligands. Here, we define a role for peroxisome proliferator-activated receptor alpha in regulating the expression of three ATP-driven drug efflux transporters at the rat and mouse blood–brain barriers: P-glycoprotein (Abcb1), breast cancer resistance protein (Bcrp/Abcg2), and multidrug resistance-associated protein 2 (Mrp2/Abcc2). Exposing isolated rat brain capillaries to linoleic acid, clofibrate, or PKAs increased the transport activity and protein expression of the three ABC transporters. These effects were blocked by the PPAR-α antagonist, GW6471. Dosing rats with 20 mg/kg or 200 mg/kg of clofibrate decreased the brain accumulation of the P-glycoprotein substrate, verapamil, by 50% (in situ brain perfusion; effects blocked by GW6471) and increased P-glycoprotein expression and activity in capillaries ex vivo. Fasting C57Bl/6 wild-type mice for 24 h increased both serum lipids and brain capillary P-glycoprotein transport activity. Fasting did not alter P-glycoprotein activity in PPAR-α knockout mice. These results indicate that hyperlipidemia, lipid-lowering fibrates and exposure to certain fire-fighting foam components activate blood–brain barrier peroxisome proliferator-activated receptor alpha, increase drug efflux transporter expression and reduce drug delivery to the brain.

Multi-drug resistance in a canine lymphoid cell line due to increased P-glycoprotein expression, a potential model for drug-resistant canine lymphoma

Canine lymphoma is routinely treated with a doxorubicin-based multidrug chemotherapy protocol, and although treatment is initially successful, tumor recurrence is common and associated with therapy resistance. Active efflux of chemotherapeutic agents by transporter proteins of the ATP-Binding Cassette superfamily forms an effective cellular defense mechanism and a high expression of these transporters is frequently observed in chemotherapy-resistant tumors in both humans and dogs.In this study we describe the ABC-transporter expression in a canine lymphoid cell line and a sub-cell line with acquired drug resistance following prolonged exposure to doxorubicin. This sub-cell line was more resistant to doxorubicin and vincristine, but not to prednisolone, and had a highly increased P-glycoprotein (P-gp/abcb1) expression and transport capacity for the P-gp model-substrate rhodamine123. Both resistance to doxorubicin and vincristine, and rhodamine123 transport capacity were fully reversed by the P-gp inhibitor PSC833. No changes were observed in the expression and function of the ABC-transporters MRP-1 and BCRP.It is concluded that GL-40 cells represent a useful model for studying P-gp dependent drug resistance in canine lymphoid neoplasia, and that this model can be used for screening substances as potential P-gp substrates and their capacity to modulate P-gp mediated drug resistance.

Zoledronic Acid Restores Doxorubicin Chemosensitivity and Immunogenic Cell Death in Multidrug-Resistant Human Cancer Cells

Durable tumor cell eradication by chemotherapy is challenged by the development of multidrug-resistance (MDR) and the failure to induce immunogenic cell death. The aim of this work was to investigate whether MDR and immunogenic cell death share a common biochemical pathway eventually amenable to therapeutic intervention. We found that mevalonate pathway activity, Ras and RhoA protein isoprenylation, Ras- and RhoA-downstream signalling pathway activities, Hypoxia Inducible Factor-1alpha activation were significantly higher in MDR+ compared with MDR− human cancer cells, leading to increased P-glycoprotein expression, and protection from doxorubicin-induced cytotoxicity and immunogenic cell death. Zoledronic acid, a potent aminobisphosphonate targeting the mevalonate pathway, interrupted Ras- and RhoA-dependent downstream signalling pathways, abrogated the Hypoxia Inducible Factor-1alpha-driven P-glycoprotein expression, and restored doxorubicin-induced cytotoxicity and immunogenic cell death in MDR+ cells. Immunogenic cell death recovery was documented by the ability of dendritic cells to phagocytise MDR+ cells treated with zoledronic acid plus doxorubicin, and to recruit anti-tumor cytotoxic CD8+ T lymphocytes. These data indicate that MDR+ cells have an hyper-active mevalonate pathway which is targetable with zoledronic acid to antagonize their ability to withstand chemotherapy-induced cytotoxicity and escape immunogenic cell death.

Role of ABCB1 Variants on Postoperative Seizure Recurrence in Patients with AED-Resistant Temporal Lobe Epilepsy (P05.079)

Objective: To investigate the role of single nucleotide polymorphisms (SNPs) in ATP-binding cassette family gene, ABCB1 in predicting the postoperative seizure recurrence in antiepileptic drug (AED)-resistant temporal lobe epilepsy (TLE) patients from Southern India. Background Even though several electro-clinical and imaging characteristics could predict postoperative seizure outcome in patients with drug-resistant TLE patients, none of them have consistently been shown to be associated with the outcome. The genetic factors can play a critical role in attaining postoperative seizure freedom, and if identified, they could serve as biological markers of immense clinical importance. Increased P-glycoprotein expression in the temporal lobe has been shown to be associated with AED resistance and seizure recurrence after surgery. We hypothesized that genetic variation in ABCB1 could influence postoperative seizure outcome in TLE patients. Design/Methods: We assembled 200 patients, who had undergone anterior temporal lobectomy (ATL) for AED-resistant TLE and had been followed up for 5 years after surgery. We genotyped seven SNPs in the ABCB1 gene using PCR-RFLP and compared the distribution of SNPs between patients who were seizure-free and those who had seizure recurrence. Results: Genotypic frequencies were in Hardy-Weinberg equilibrium with exception to rs1045642 in seizure-free group. There was no significant difference in allele and genotype frequencies of the SNPs in ABCB1 gene between patients who were seizure-free and those who had seizure recurrence. Conclusions: Even though studies have suggested that P-glycoprotein expression in temporal lobe may be associated with postoperative seizure recurrence, we could not find any association in genotypic level with the studied SNPs, so as to predict seizure recurrence. Supported by: BRNS and KSCSTE, Govt. of India. Disclosure: Dr. Balan has nothing to disclose. Dr. Sumitha has nothing to disclose. Dr. Bharadwaj has nothing to disclose. Dr. Lekshmi has nothing to disclose. Dr. Sathyan has nothing to disclose. Dr. Radhakrishnan has nothing to disclose. Dr. Banerjee has nothing to disclose.

ABC Transporter Expression, Function and Regulation at the Blood‐Spinal Cord Barrier

The blood‐spinal cord barrier (BSCB), which resides within spinal cord capillaries, is a major impediment to the delivery of drugs used to treat, e.g., traumatic spinal cord injury and amyotrophic lateral sclerosis. These capillaries appear structurally similar to brain capillaries, the site of the blood‐brain barrier (BBB), but we have little information about the molecular basis of BSCB function. Here we demonstrate protein expression (western blots) and transport function (confocal‐based assay) for three ATP‐driven, drug efflux pumps: P‐glycoprotein, multidrug resistance protein 2 (Mrp2) and breast cancer related protein (Bcrp) in capillaries isolated from rat and mouse spinal cords. We show upregulation of transporter function and expression in capillaries exposed to pregnenolone‐16α‐carbonitrile (activates pregnane X receptor), phenobarbital (activates constitutive androstane receptor) and dioxin (activates aryl hydrocarbon receptor). We also show increased P‐glycoprotein expression and transport function in brain capillaries from rats dosed with 5μg/kg TCDD. These results indicate that the BBB and BSCB are remarkably similar with regard to efflux transporter function and regulation.

Progress in the multidrug resistance of refractory epilepsy

Multidrug resistance is a major obstacle in the chemotherapy of refractory epilepsy. P-glyco- protein, an efflux transporter, which may play an important role in refractory epilepsy. Some studies point out that increased P-glycoprotein expression and activity limit pharrnacotherapy with antiepilepfic drugs. Other studies indicate that glutamate released during seizures signals through the NMDA receptor and COX-2 to increase P-glycoprotein. These evidences may lead to a new therapy of drug-resistantepilepsy. Key words: Multidrug resistance; Cyclooxygenase-2 ; P-glycoprotein; Refractory epilepsy

Increased P-glycoprotein expression in mitochondria is related to acquired multidrug resistance in human hepatoma cells depleted of mitochondrial DNA

Mitochondrial DNA-depleted ρ0 cells are resistant to apoptosis, but the mechanism remains unclear. A human hepatoma cell line (SK-Hep1) depleted of mtDNA (ρ0SK-Hep1) was induced by ethidium bromide treatment. The ρ0SK-Hep1 cells were resistant to both doxorubicin- and cisplatin-induced apoptosis, while cybrids (SK-Hep1Cyb) prepared by fusing ρ0SK-Hep1 cells with platelets showed restored susceptibility to both drugs. We observed P-glycoprotein and MRP1 were both overexpressed in ρ0 cells, and more P-glycoproteins were localized in the mitochondria and were functionally active. ρ0 cells showed resistance to chemotherapeutic drug-induced apoptosis. The increased expression and localization of P-glycoproteins in the mitochondria of ρ0 cells may facilitate the exclusion of chemotherapeutic drugs from the mitochondria to the cytosol.

The B-cell lymphoma 2 (BCL2)-inhibitors, ABT-737 and ABT-263, are substrates for P-glycoprotein

Inhibition of BCL2 proteins is one of the most promising new approaches to targeted cancer therapy resulting in the induction of apoptosis. Amongst the most specific BCL2-inhibitors identified are ABT-737 and ABT-263. However, targeted therapy is often only effective for a limited amount of time because of the occurrence of drug resistance. In this study, the interaction of BCL2-inhibitors with the drug efflux transporter P-glycoprotein was investigated. Using 3H labelled ABT-263, we found that cells with high P-glycoprotein activity accumulated less drug. In addition, cells with increased P-glycoprotein expression were more resistant to apoptosis induced by either ABT-737 or ABT-263. Addition of tariquidar or verapamil sensitized the cells to BCL2-inhibitor treatment, resulting in higher apoptosis. Our data suggest that the BCL2-inhibitors ABT-737 and ABT-263 are substrates for P-glycoprotein. Over-expression of P-glycoprotein may be, at least partly, responsible for resistance to these BCL2-inhibitors.

Indomethacin and SC236 enhance the cytotoxicity of doxorubicin in human hepatocellular carcinoma cells via inhibiting P-glycoprotein and MRP1 expression

Doxorubicin is a chemotherapeutic drug widely used for the treatment of hepatocellular carcinoma but its efficacy is restricted by multidrug resistance. Non-steroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase (COX)-2-selective inhibitors exhibit anti-cancer properties as well as abilities to overcome drug resistance. In the present study, indomethacin (a NSAID) and SC236 (a COX-2-selective inhibitor) enhanced the cytotoxicity of doxorubicin in the hepatocellular carcinoma cell line HepG2 and its drug-resistant sub-line R-HepG2. Both drugs increased the intracellular accumulation and retention of doxorubicin in vitro. The effects were not reversed by prostaglandin E 2, implicating a COX-independent mechanism. Indomethacin and SC236 partially reversed the increase in expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) induced by doxorubicin in R-HepG2 cells. In conclusion, indomethacin and SC236 increased the intracellular accumulation and retention of doxorubicin and thus its cytotoxicity in HepG2 and drug-resistant HepG2 cells. These effects, mediated through decrease in P-gp and MRP1 expression and/or direct inhibition of P-gp activity, may improve multidrug resistant-cancer chemotherapy.

The C3435T polymorphism in ABCB1 influences atorvastatin efficacy and muscle symptoms in a high-risk vascular cohort

The CC genotype of the C3435T polymorphism in ABCB1 is associated with increased P-glycoprotein expression, reduced low-density lipoprotein cholesterol (LDL-C) response to atorvastatin, and a reduced area-under-the-curve in pharmacokinetic studies. We sought to assess the relationship between 1) genotype and Atorvastatin efficacy, independently of variation in cholesterol metabolism and 2) genotype and myalgia. High-risk vascular patients were genotyped and treated with atorvastatin 80 mg for 6 weeks. The lipid panel and percent LDL-C reduction with atorvastatin were related to C3435T genotype. Genotypes and allele frequency were assessed in patients with and without myalgia. A total of 117 patients were recruited and genotyped. Of these, 98 completed the study with adequate atorvastatin adherence, and 10 reported myalgia. T and C allele frequencies were 0.63 and 0.37, respectively. A 6-week course of atorvastatin (80 mg/day) reduced LDL-C by 58% ± 11% (mean ± SD). Patients with the CC genotype showed less LDL-C reduction with atorvastatin compared with the TT/TC genotype (53% vs 59%, respectively, P = .034), and this finding was independent of variation in cholesterol metabolism (P = .045 after correction for desmosterol and cholestanol/cholesterol ratio). The T allele was more frequent in patients with myalgia than those without (0.80 vs 0.62) and the C allele less frequent (0.20 vs 0.38, P = .043). In patients treated with atorvastatin, the CC genotype at the C3435T polymorphism in ABCB1 is associated with reduced atorvastatin efficacy independently of cholesterol metabolism. The T allele is more frequent and the C allele less frequent in patients with myalgia.

Aryl hydrocarbon receptor‐mediated up‐regulation of ATP‐driven xenobiotic efflux transporters at the blood‐brain barrier

Many widespread and persistent organic pollutants, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), activate the aryl hydrocarbon receptor (AhR), causing it to translocate to the cell nucleus, where it transactivates target genes. AhR's ability to target the blood-brain barrier is essentially unexplored. We show here that exposing isolated rat brain capillaries to 0.05-0.5 nM TCDD roughly doubled transport activity and protein expression of P-glycoprotein, an ATP-driven drug efflux pump and a critical determinant of drug entry into the CNS. These effects were abolished by actinomycin D or cycloheximide or by the AhR antagonists resveratrol and α-naphthoflavone. Brain capillaries from TCDD-dosed rats (1-5 μg/kg, i.p.) exhibited increased transport activity and protein expression of 3 xenobiotic efflux pumps, P-glycoprotein, multidrug resistance-associated protein 2, and breast cancer resistance polypeptide, as well as expression of Cyp1a1 and Cyp1b1, both AhR target genes. Consistent with increased P-glycoprotein expression in capillaries from TCDD-dosed rats, in situ brain perfusion indicated significantly reduced brain accumulation of verapamil, a P-glycoprotein substrate. These findings suggest a new paradigm for the field of environmental toxicology: toxicants acting through AhR to target xenobiotic efflux transporters at the blood-brain barrier and thus reduce brain accumulation of CNS-acting therapeutic drugs.

Anthelmintic resistance: markers for resistance, or susceptibility?

The Consortium for Anthelmintic Resistance and Susceptibility (CARS) brings together researchers worldwide, with a focus of advancing knowledge of resistance and providing information on detection methods and treatment strategies. Advances in this field suggest mechanisms and features of resistance that are shared among different classes of anthelmintic. Benzimidazole resistance is characterized by specific amino acid substitutions in beta-tubulin. If present, these substitutions increase in frequency upon drug treatment and lead to treatment failure. In the laboratory, sequence substitutions in ion-channels can contribute to macrocyclic lactone resistance, but there is little evidence that they are significant in the field. Changes in gene expression are associated with resistance to several different classes of anthelmintic. Increased P-glycoprotein expression may prevent drug access to its site of action. Decreased expression of ion-channel subunits and the loss of specific receptors may remove the drug target. Tools for the identification and genetic analysis of parasitic nematodes and a new online database will help to coordinate research efforts in this area. Resistance may result from a loss of sensitivity as well as the appearance of resistance. A focus on the presence of anthelmintic susceptibility may be as important as the detection of resistance.

6-Thioguanine Selectively Kills BRCA2-Defective Tumors and Overcomes PARP Inhibitor Resistance

Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor-resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair-independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair-independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy.

COX-2 inhibition controls P-glycoprotein expression and promotes brain delivery of phenytoin in chronic epileptic rats

Epileptic seizures drive expression of the blood-brain barrier efflux transporter P-glycoprotein via a glutamate/cyclooxygenase-2 mediated signalling pathway. Targeting this pathway may represent an innovative approach to control P-glycoprotein expression in the epileptic brain and to enhance brain delivery of antiepileptic drugs. Therefore, we tested the effect of specific cyclooxygenase-2 inhibition on P-glycoprotein expression in two different status epilepticus models. Moreover, the impact of a cyclooxygenase-2 inhibitor on expression of the efflux transporter and on brain delivery of an antiepileptic drug was evaluated in rats with recurrent spontaneous seizures. The highly selective cyclooxygenase-2 inhibitors SC-58236 and NS-398 both counteracted the status epilepticus-associated increase in P-glycoprotein expression in the parahippocampal cortex and the ventral hippocampus. In line with our working hypothesis, a sub-chronic 2-week treatment with SC-58236 in the chronic epileptic state kept P-glycoprotein expression at control levels. As described previously, enhanced P-glycoprotein expression in chronic epileptic rats was associated with a significant reduction in the brain penetration of the antiepileptic drug phenytoin. Importantly, the brain delivery of phenytoin was significantly enhanced by sub-chronic cyclooxygenase-2 inhibition in rats with recurrent seizures. In conclusion, the data substantiate targeting of cyclooxygenase-2 in the chronic epileptic brain as a promising strategy to control the expression levels of P-glycoprotein despite recurrent seizure activity. Cyclooxygenase-2 inhibition may therefore help to increase concentrations of antiepileptic drugs at the target sites in the epileptic brain. It needs to be further evaluated whether the approach also enhances efficacy.