Mechanism Of Cross-resistance Research Articles

Targeting the Androgen Receptor Confers In Vivo Cross-resistance Between Enzalutamide and Docetaxel, But Not Cabazitaxel, in Castration-resistant Prostate Cancer

Treatment options for metastatic castration-resistant prostate cancer (CRPC) have evolved with the established benefit of the novel androgen receptor (AR)-targeted agents abiraterone and enzalutamide in the prechemotherapy setting. However, concerns regarding cross-resistance between the taxanes docetaxel and cabazitaxel and these AR-targeted agents have arisen, and the optimal drug treatment sequence is unknown. We investigated the in vivo efficacy of docetaxel and cabazitaxel in enzalutamide-resistant CRPC, and mechanisms of cross-resistance between these agents. Castrated mice harboring enzalutamide-resistant tumors and enzalutamide-naïve tumors were treated with docetaxel and cabazitaxel. Tumor growth kinetics, AR nuclear localization, AR-regulated gene expression, Ki67 expression, and serum levels of prostate-specific antigen, docetaxel, and cabazitaxel were analyzed. Docetaxel inhibited tumor growth, AR nuclear localization, and AR-regulated gene expression in enzalutamide-naïve tumors, but did not in enzalutamide-resistant tumors, demonstrating in vivo cross-resistance. By contrast, cabazitaxel remained highly effective in enzalutamide-resistant tumors and demonstrated superior antitumor activity compared to docetaxel, independent of the AR pathway. These findings demonstrate that the AR pathway is able to confer in vivo cross-resistance between enzalutamide and docetaxel, but not cabazitaxel, in CRPC. Patient summaryWe found reduced efficacy of docetaxel, but not cabazitaxel, in enzalutamide-resistant prostate cancer.

Altered radiation responses of breast cancer cells resistant to hormonal therapy.

Endocrine therapy agents (the selective estrogen receptor (ER) modulators such as tamoxifen or the selective ER down-regulators such as ICI 182,780) are key treatment regimens for hormone receptor-positive breast cancers. While these drugs are very effective in controlling ER-positive breast cancer, many tumors that initially respond well to treatment often acquire drug resistance, which is a major clinical problem. In clinical practice, hormonal therapy agents are commonly used in combination or sequence with radiation therapy. Tamoxifen treatment and radiotherapy improve both local tumor control and patient survival. However, tamoxifen treatment may render cancer cells less responsive to radiation therapy. Only a handful of data exist on the effects of radiation on cells resistant to hormonal therapy agents. These scarce data show that cells that were resistant to tamoxifen were also resistant to radiation. Yet, the existence and mechanisms of cross-resistance to endocrine therapy and radiation therapy need to be established. Here, we for the first time examined and compared radiation responses of MCF-7 breast adenocarcinoma cells (MCF-7/S0.5) and two antiestrogen resistant cell lines derived from MCF-7/S0.5: the tamoxifen resistant MCF-7/TAMR-1 and ICI 182,780 resistant MCF-7/182R-6 cell lines. Specifically, we analyzed the radiation-induced changes in the expression of genes involved in DNA damage, apoptosis, and cell cycle regulation. We found that the tamoxifen-resistant cell line in contrast to the parental and ICI 182,780-resistant cell lines displayed a significantly less radiation-induced decrease in the expression of genes involved in DNA repair. Furthermore, we show that MCF-7/TAMR-1 and MCF-7/182R-6 cells were less susceptible to radiation-induced apoptosis as compared to the parental line. These data indicate that tamoxifen-resistant breast cancer cells have a reduced sensitivity to radiation treatment. The current study may therefore serve as a roadmap to the future analysis of the mechanisms of cross-resistance between hormonal therapy and radiation.

Selection for resistance: Cross-resistance, inheritance, synergists and biochemical mechanisms of resistance to acequinocyl in Phytoseiulus persimilis A.H. (Acari: Phytoseiidae)

The predatory mite Phytoseiulus persimilis is effective in controlling tetranychids on many cultivated crops. The LC50 and LC60 values of acequinocyl were determined for P. persimilis using a leaf-disc method and a spraying tower. A laboratory population ACE6 had a 32.75-fold increase in resistance to acequinocyl after six selection cycles. This resistant population also developed low resistance to spinosad, chlorantraniliprole, and deltamethrin and moderate cross-resistance to acetamiprid, indoxacarb, milbemectin and spiromesifen. The synergists piperonyl butoxide (PBO) S-benzyl-O,O-diisopropyl phosphorothioate (IBP) and diethyl maleate (DEM) increased the resistance 2.03-, 2.33- and ˂1.00-fold, respectively. Crossing experiments revealed that acequinocyl resistance in the ACE6 population was intermediately dominant and polygenic. Additionally, in the ACE6 population, cytochrome P450 monooxygenase (P450) and esterase detoxifying enzyme activities increased 2.40-fold and 1.85-fold, respectively. These results indicated that P450 and esterase were important enzymes in the development of acequinocyl resistance in the ACE6 population.

Cross resistances in spinosad-resistant Culex quinquefasciatus (Diptera: Culicidae).

A Culex quinquefasciatus Say colony was selected for 45 generations at LC70-90 levels using Natular XRG, a granular formulation of 2.5% spinosad for induction of spinosad resistance. Resistance to spinosad was noticed in early generations (F1-F9). Resistance levels increased gradually from generations F11-F35, and elevated significantly from generation F37 through F47, when resistance ratios reached 2,845-2,907-fold at LC50 and 11,948-22,928-fold at LC90 The spinosad-resistant Cx. quinquefasciatus colony was found not to be cross-resistant to Bacillus thuringiensis israelensis (Bti), a combination of Bti and Bacillus sphaericus, methoprene, pyriproxyfen, diflubenzuron, novaluron, temephos, or imidacloprid. However, it showed various levels of cross-resistance to B. sphaericus, spinetoram, abamectin, and fipronil. Conversely, a laboratory colony of Cx. quinquefasciatus that is highly resistant to B. sphaericus did not show cross-resistance to spinosad and spinetoram. Field-collected and laboratory-selected Cx. quinquefasciatus that showed low to moderate resistance to methoprene did not show cross-resistance to spinosad and spinetoram. Mechanisms of cross-resistance among several biorational pesticides were discussed according to their modes of actions.

Biochemical and molecular characterisation and cross‐resistance in field and laboratory chlorpyrifos‐resistant strains of Laodelphax striatellus (Hemiptera: Delphacidae) from eastern China

Laboratory selection is often employed in resistance mechanism studies because field-derived populations commonly do not have high enough resistance for such studies. In the present study, a field-collected Laodelphax striatellus population from eastern China was laboratory selected for chlorpyrifos resistance and susceptibility, and the developed strains, along with a field population, were studied for cross-resistance and resistance mechanisms at biochemical and molecular levels. A 158.58-fold chlorpyrifos-resistant strain (JH-chl) and a chlorpyrifos-susceptible strain (JHS) were established after laboratory selection of 25 generations. Cross-resistance to deltamethrin, diazinon, methomyl, carbosulfan, acephate and imidacloprid were detected in JH-chl and a field-collected strain (JHF). Synergism and enzyme activity data suggested potential involvement of P450s and esterases in JH-chl as well as AChE alteration. Furthermore, CYP6AY3v2, CYP306A2v2, CYP353D1v2 and LSCE36 genes were significantly overexpressed in JH-chl (6.87-12.14-fold). Feeding of dsRNAs reduced the expression of the four target genes (35.6-56.8%) and caused significant adult mortality (75.21-88.45%), implying resistance reduction. However, mechanism(s) conferring chlorpyrifos resistance in JHF were unclear. In contrast to previous reports, multiple overexpressed detoxification genes were potentially associated with chlorpyrifos resistance, as confirmed by RNAi feeding tests. Chlorpyrifos resistance exhibits cross-resistance with insecticides in the same and different classes.

Cross-resistance between taxanes and new hormonal agents abiraterone and enzalutamide may affect drug sequence choices in metastatic castration-resistant prostate cancer

IntroductionTreatment options for patients with metastatic castration-resistant prostate cancer (mCRPC) have expanded in recent years with the introduction of cabazitaxel, abiraterone and enzalutamide. With new systemic therapies available, the optimal treatment sequence of these drugs in mCRPC becomes increasingly important. As shown recently, patients who had previously been treated with abiraterone showed impaired responses to docetaxel, suggesting clinical cross-resistance [1]. In the present study, we aimed to identify cross-resistance between taxanes (docetaxel and cabazitaxel) and the new hormonal agents abiraterone and enzalutamide. As a potential mechanism for cross-resistance, we investigated the effects on androgen receptor (AR) nuclear translocation of these compounds. MethodsTo identify cross-resistance, we determined the effects of docetaxel, cabazitaxel, abiraterone and enzalutamide on cell viability in prostate cancer cell lines with acquired resistance to abiraterone and enzalutamide. Time-lapse confocal microscopy was used to study the dynamics of AR nuclear translocation. ResultsWe observed impaired efficacy of docetaxel, cabazitaxel and enzalutamide in the abiraterone-resistant cell line, compared to the non-resistant cell line, providing evidence for in vitro cross-resistance. Impaired efficacy of docetaxel, cabazitaxel and abiraterone was observed in the enzalutamide-resistant cell line. Furthermore, docetaxel and cabazitaxel inhibited AR nuclear translocation, which was also observed for abiraterone and enzalutamide. ConclusionsIn conclusion we found substantial preclinical evidence for cross-resistance between the taxanes docetaxel and cabazitaxel, and AR targeting agents abiraterone and enzalutamide. Since these compounds all interfere with AR-signalling, this strongly suggests a common mechanism of action, and thus a potential mechanism for cross-resistance in mCRPC.

Species differences in chlorantraniliprole and flubendiamide insecticide binding sites in the ryanodine receptor

Anthranilic and phthalic diamides exemplified by chlorantraniliprole (Chlo) or cyantraniliprole (Cyan) and flubendiamide (Flu), respectively, are the newest major chemotype of insecticides with outstanding potency, little or no cross resistance with other classes and low mammalian toxicity. They are activators of the ryanodine (Ry) receptor (RyR)-Ca2+ channel, based on Ca2+ flux and electrophysiology investigations. The goal of this study is to define species differences in the degree and mechanisms of diamide selective action by radioligand specific binding studies at the [3H]Ry, [3H]Chlo and [3H]Flu sites. The [3H]Ry site is observed in muscle of lobster, rabbit and four insect species (Musca domestica, Apis mellifera, Heliothis virescens and Agrotis ipsilon) whereas the [3H]Chlo site is evident in the four insects and the [3H]Flu site in only the two lepidoptera (Agrotis and Heliothis). [3H]Ry binding is significantly stimulated by Chlo, Cyan and Flu with the insects (except Flu with Musca) but not the lobster and rabbit. [3H]Chlo binding is stimulated by Ry and Flu in Musca and Apis but not in the lepidoptera, while Flu and Cyan are inhibitory. [3H]Flu binding is strongly inhibited by Chlo and Cyan in Agrotis and Heliothis. [3H]Chlo and [3H]Flu binding are not dependent on added Ca2+ or ATP in Heliothis and Agrotis whereas the other radioligand-receptor combinations are usually enhanced by Ca2+ and ATP. More generally, there are species differences in the Ry, Chlo and Flu binding sites of the RyR that may confer selective toxicity and determine target site cross resistance mechanisms.

Insecticide resistance and, efficacy of space spraying and larviciding in the control of dengue vectors Aedes aegypti and Aedes albopictus in Sri Lanka

Unprecedented incidence of dengue has been recorded in Sri Lanka in recent times. Source reduction and use of insecticides in space spraying/fogging and larviciding, are the primary means of controlling the vector mosquitoes Aedes aegypti and Ae. albopictus in the island nation. A study was carried out to understand insecticide cross-resistance spectra and mechanisms of insecticide resistance of both these vectors from six administrative districts, i.e. Kandy, Kurunegala, Puttalam, Gampaha, Ratnapura and Jaffna, of Sri Lanka. Efficacy of the recommended dosages of frequently used insecticides in space spraying and larviciding in dengue vector control programmes was also tested.Insecticide bioassay results revealed that, in general, both mosquito species were highly resistant to DDT but susceptible to propoxur and malathion except Jaffna Ae. aegypti population. Moderate resistance to malathion shown by Jaffna Ae. aegypti population correlated with esterase and malathion carboxylesterase activities of the population. High levels of acetylcholinesterase (AChE) insensitivity in the absence of malathion and propoxur resistance may be due to non-synaptic forms of AChE proteins. Moderate pyrethroid resistance in the absence of high monooxygenase levels indicated the possible involvement of ‘kdr’ type resistance mechanism in Sri Lankan dengue vectors.Results of the space spraying experiments revealed that 100% mortality at a 10m distance and >50% mortality at a 50m distance can be achieved with malathion, pesguard and deltacide even in a ground with dense vegetation. Pesguard and deltacide spraying gave 100% mortality up to 50m distance in open area and areas with little vegetation. Both species gave >50% mortalities for deltacide at a distance of 75m in a dense vegetation area. Larval bioassays conducted in the laboratory showed that a 1ppm temephos solution can maintain a larval mortality rate of 100% for ten months, and the mortality rate declined to 0% in the eleventh month. In the field, where 1ppm concentration is gradually decreased with water usage, 100% mortality was observed only for the first four months, <50% mortality for the next two months, and 0% mortality was observed eight months after the application of temephos. Deltacide can be effectively used for space spraying programmes in Sri Lanka. Larval control can be successfully achieved through temephos with public participation.

Effects on androgen receptor nuclear import by docetaxel, cabazitaxel, abiraterone, and enzalutamide: Potential mechanism for cross-resistance in castration-resistant prostate cancer (CRPC).

5064 Background: Recent reports have suggested that paclitaxel and docetaxel, which exert their therapeutic activity primarily through inhibition of microtubule function and mitosis, also impair androgen receptor (AR) signaling. AR signaling is the key therapeutic target for the newly available agents abiraterone and enzalutamide. A recent study suggested impaired efficacy of docetaxel when given after progression on abiraterone in patients with CRPC. To identify potential mechanisms of cross-resistance, we investigated whether and to what extent AR nuclear translocation is affected by docetaxel, cabazitaxel, abiraterone and enzalutamide. Methods: Hep3B cells stably expressing GFP labeled AR were used for AR translocation studies. Cells were seeded on a cover slip in steroid-stripped medium (DCC) and treated with docetaxel (1 µM), cabazitaxel (1 µM), abiraterone (6 µM) and enzalutamide (1 µM). The anthracenedione mitoxantrone (100 nM) that does not target the microtubule cytoskeleton was used as a control cytotoxic agent. Following incubation for 48 hours, the synthetic androgen R1881 was added to study dynamics of AR nuclear import by time-lapse confocal microscopy. Results: Docetaxel and cabazitaxel inhibited R1881 induced AR nuclear translocation with 21% and 34% respectively compared to control, while mitoxantrone did not affect AR transport. Abiraterone inhibited AR translocation with 58% and enzalutamide completely blocked AR translocation. Conclusions: These data point towards a role for microtubules in AR nuclear transport and an additional mechanism of taxane activity in CRPC. Docetaxel, cabazitaxel, abiraterone and enzalutamide all interfere with AR nuclear transport, which is a crucial step in AR signaling. Our findings identify and provide insight in a potential mechanism of clinical cross-resistance between the two taxanes currently registered for treatment in CRPC and the novel agents abiraterone and enzalutamide. Further investigations are warranted in order to define if this potential cross-resistance impacts the most suitable treatment sequence for these drugs in CRPC.

Exploring the Molecular Mechanism of Cross-Resistance to HIV-1 Integrase Strand Transfer Inhibitors by Molecular Dynamics Simulation and Residue Interaction Network Analysis

The rapid emergence of cross-resistance to the integrase strand transfer inhibitors (INSTIs) has become a serious problem in the therapy of human immunodeficiency virus type 1 (HIV-1) infection. Understanding the detailed molecular mechanism of INSTIs cross-resistance is therefore critical for the development of new effective therapy against cross-resistance. On the basis of the homology modeling constructed structure of tetrameric HIV-1 intasome, the detailed molecular mechanism of the cross-resistance mutation E138K/Q148K to three important INSTIs (Raltegravir (RAL, FDA approved in 2007), Elvitegravir (EVG, FDA approved in 2012), and Dolutegravir (DTG, phase III clinical trials)) was investigated by using molecular dynamics (MD) simulation and residue interaction network (RIN) analysis. The results from conformation analysis and binding free energy calculation can provide some useful information about the detailed binding mode and cross-resistance mechanism for the three INSTIs to HIV-1 intasome. Binding free energy decomposition analysis revealed that Pro145 residue in the 140s 1oop (Gly140 to Gly149) of the HIV-1 intasome had strong hydrophobic interactions with INSTIs and played an important role in the binding of INSTIs to HIV-1 intasome active site. A systematic comparison and analysis of the RIN proves that the communications between the residues in the resistance mutant is increased when compared with that of the wild-type HIV-1 intasome. Further analysis indicates that residue Pro145 may play an important role and is relevant to the structure rearrangement in HIV-1 intasome active site. In addition, the chelating ability of the oxygen atoms in INSTIs (e.g., RAL and EVG) to Mg(2+) in the active site of the mutated intasome was reduced due to this conformational change and is also responsible for the cross-resistance mechanism. Notably, the cross-resistance mechanism we proposed could give some important information for the future rational design of novel INSTIs overcoming cross-resistance. Furthermore, the combination use of molecular dynamics simulation and residue interaction network analysis can be generally applicable to investigate drug resistance mechanism for other biomolecular systems.

Pyrethroid resistance in Anopheles gambiae from the rubber cultivated area of Niete, South Region of Cameroon

Anti-vector intervention remains the most effective way of controlling malaria. Although in Cameroon and elsewhere focus is on the use of long-lasting insecticidal nets and indoor residual spraying, the efficacy of both methods greatly depends on the continuing susceptibility of the vectors to the insecticides used. The emergence and spread of insecticide resistance in the major malaria vectors constitute a huge challenge to control programmes. Consequently, routine monitoring and evaluation of vector resistance status to insecticides are mandatory for early detection of resistance should it arise, and effectively planning future anti-vector interventions especially in areas reputed for routine application in agriculture. The WHO bioassay kit was used to determine the susceptibility status of Anopheles gambiae s.l. populations to seven insecticides belonging to four classes (organochlorine, organophosphate, carbamate and pyrethroids) in Niete, an area of intense rubber cultivation in southern forested Cameroon. Species and molecular forms of An. gambiae s.l. as well as the presence of knock down resistance (kdr) mutations were determined using polymerase chain reaction (PCR) techniques. All Anopheles tested was identified as An. gambiae s.s. and of the M molecular form. Based on WHO classification, while the mosquitoes were fully (100%) susceptible to malathion and bendiocarb, resistance was confirmed to DDT and the pyrethroids, permethrin and lambda-cyhalothrin. The other pyrethroids (deltamethrin and cyfluthrin) showed signs of developing resistance. Resistance to DDT and pyrethroids is indicative of existing cross resistance mechanisms between these insecticides. The increase in knockdown times was greater than twofold that of the reference susceptible strain, suggesting the possible involvement of kdr mutations, also confirmed in this study. The findings highlight the need for constant evaluation, re-evaluation and monitoring of the insecticides for malaria vector control in Cameroon. However, bendiocarb and malathion can be used and may require alternation or combination with insecticides of other classes to better manage the occurrence and spread of resistance in Niete.

The PARP inhibitor olaparib (AZD2281) as potent radiosensitizer of head and neck cancer cells.

e16018 Background: Current combined radiotherapy regimens for squamous cell carcinoma of the head and neck (SCCHN) are frequently not curative, necessitating novel therapeutic strategies. Inhibitors of poly(ADP-ribose) polymerases (PARPi), either alone or in combination with chemo- and radiotherapy have been shown to be highly active in tumor cells with intrinsic defects in DNA repair. Despite the frequent occurrence of genomic alterations in SCCHN cells also affecting their capacity of DNA repair the radiosensitizing potential of PARPi has not been addressed in detail so far. In this study, the efficacy of PARPi as radiosensitizer in SCCHN and possible mechanisms of cross-resistance between PARPi and cisplatin were evaluated. Methods: Usingthe clonogenic survival assay and a panel of 10 SCCHN cell lines the sensitivity of SCCHN cells to olaparib alone (0 to 500 nM) or in combination with irradiation (0 to 4 Gy) was determined. Survival fractions for given treatments were calculated on the basis of survival of untreated cells. In addition, the activity of cisplatin to inhibit clonogenic growth and its radiosensitizing potential was determined. From the dose-effect curves the IC50 values and the combinatory indices were calculated using the CalcuSyn Software. Results: In 9 of 10 cell lines, olaparib monotherapy showed significant inhibitory activity on clonogenic survival. Synergistic activity of olaparib in combination with irradiation was observed in all cell lines. No correlation between sensitivity of cells to olaparib and cisplatin (IC50 cisplatin vs IC50 olaparib: r2=0.042, p=.57; IC50 cisplatin vs CI [IR+olaparib]: r2=0.005, p=.95) was observed. Furthermore, the activity of olaparib was not dependent on the p53 genotype. Conclusions: The combination of PARPi with radiotherapy represents an active therapeutic regimen in SCCHN. The observed high activity of this combination even in cells with reduced sensitivity to cisplatin and p53 dysfunction suggests its clinical usefulness also in the group of patients with more aggressive disease and the second-line setting. Currently, detailed molecular characterization for identification of potential biomarker for tumor susceptibility to PARP inhibition is ongoing.

Interaction and cross-resistance of cisplatin and pemetrexed in malignant pleural mesothelioma cell lines

Although cisplatin and pemetrexed are key drugs in the treatment of malignant pleural mesothelioma, their drug-drug interactions, cross-resistance and resistance mechanisms in malignant pleural mesothelioma are not well understood. In the present study, the interaction of these 2 agents was determined by clonogenic assays followed by isobologram analysis of 4 human malignant pleural mesothelioma cell lines. The cell lines were exposed to the agents using a stepwise dose-escalation method to establish drug-resistant sublines. Thymidylate synthase mRNA expression was evaluated in the drug-resistant sublines. As a consequence, cisplatin and pemetrexed had synergistic effects in 3 cell lines and an additive effect in the fourth cell line. The former 3 cell lines showed similar pemetrexed sensitivity in the parental cells and their cisplatin-resistant sublines, whereas the fourth cell line exhibited cross-resistance. In contrast, cisplatin had diverse effects on pemetrexed-resistant sublines. High thymidylate synthase expression did not correlate with natural pemetrexed resistance. Elevated thymidylate synthase expression correlated with acquired pemetrexed resistance in 2 sublines. In conclusion, cisplatin and pemetrexed showed synergistic activity and no cross-resistance in 3 of the 4 malignant pleural mesothelioma cell lines, suggesting the clinical relevance of their combination in chemotherapy. Thymidylate synthase expression did not necessarily correlate with pemetrexed resistance. The information together with the experimental model presented here would be useful for further investigating therapeutic targets of malignant mesothelioma.

Abstract 3821: Interaction and cross-resistance of cisplatin and pemetrexed in malignant pleural mesothelioma cell lines

Abstract Background: Malignant pleural mesothelioma (MPM) is one of the most lethal cancers with a rapidly increasing incidence worldwide. Although cisplatin and pemetrexed are recognized as key drugs, and their combination chemotherapy is a standard therapy for MPM, their drug-to-drug interactions, cross-resistance, and resistance mechanisms are not well understood. Methods: The interaction between cisplatin and pemetrexed was evaluated by colonogenic assays followed by isobologram analysis in 4 human MPM cell lines. The cell lines were exposed to the agents using a stepwise dose-escalation method to establish drug-resistant sublines. Thymidylate synthase (TS) mRNA expression was evaluated by RT-PCR, and gene expression profiles were assessed by cDNA microarray analysis and gene ontology analysis using Ingenuity Pathway Analisis (IPA) in the drug-resistant sublines. Results: Cisplatin and pemetrexed had synergistic effects in 3 cell lines and an additive effect in the fourth cell line. The former 3 cell lines showed similar sensitivity to pemetrexed in the parental cells and their cisplatin-resistant sublines, whereas the fourth cell line exhibited cross-resistance. In contrast, cisplatin had diverse effects on pemetrexed-resistant sublines. High TS expression did not correlate with natural pemetrexed resistance and its elevatedexpression correlated with acquired pemetrexed resistance in 2 sublines. Microarray analysis disclosed that the genes which related to “cellular development”,” small molecule biochemistry,” “cellular movement,” “cell death, and “cellular growth and proliferation” affected drug-resistant mechanisms deeply, and that this tendency was common to cisplatin-resistant sublines and pemetrexed-resistant sublines. Conclusion: Cisplatin and pemetrexed showed synergistic activity and no cross-resistance in 3 of the 4 MPM cell lines, suggesting the clinical relevance of their combination chemotherapy. TS expression did not necessarily correlate with pemetrexed resistance. Multiple mechanisms of molecular and cellular functions may underlie acquired resistance to cisplatin and pemetrexed in MPM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3821. doi:1538-7445.AM2012-3821

Modulating activity of vancomycin and daptomycin on the expression of autolysis cell-wall turnover and membrane charge genes in hVISA and VISA strains.

Glycopeptides are still the gold standard to treat MRSA (Methicillin Resistant Staphylococcus aureus) infections, but their widespread use has led to vancomycin-reduced susceptibility [heterogeneous Vancomycin-Intermediate-Staphylococcus aureus (hVISA) and Vancomycin-Intermediate-Staphylococcus aureus (VISA)], in which different genetic loci (regulatory, autolytic, cell-wall turnover and cell-envelope positive charge genes) are involved. In addition, reduced susceptibility to vancomycin can influence the development of resistance to daptomycin. Although the phenotypic and molecular changes of hVISA/VISA have been the focus of different papers, the molecular mechanisms responsible for these different phenotypes and for the vancomycin and daptomycin cross-resistance are not clearly understood. The aim of our study was to investigate, by real time RT-PCR, the relative quantitative expression of genes involved in autolysis (atl-lytM), cell-wall turnover (sceD), membrane charges (mprF-dltA) and regulatory mechanisms (agr-locus-graRS-walKR), in hVISA and VISA cultured with or without vancomycin and daptomycin, in order to better understand the molecular basis of vancomycin-reduced susceptibility and the modulating activity of vancomycin and daptomycin on the expression of genes implicated in their reduced susceptibility mechanisms. Our results show that hVISA and VISA present common features that distinguish them from Vancomycin-Susceptible Staphylococcus aureus (VSSA), responsible for the intermediate glycopeptide resistance i.e. an increased cell-wall turnover, an increased positive cell-wall charge responsible for a repulsion mechanism towards vancomycin and daptomycin, and reduced agr-functionality. Indeed, VISA emerges from hVISA when VISA acquires a reduced autolysis caused by a down-regulation of autolysin genes, atl/lytM, and a reduction of the net negative cell-envelope charge via dltA over-expression. Vancomycin and daptomycin, acting in a similar manner in hVISA and VISA, can influence their cross-resistance mechanisms promoting VISA behavior in hVISA and enhancing the cell-wall pathways responsible for the intermediate vancomycin resistance in VISA. Daptomycin can also induce a charge repulsion mechanism both in hVISA and VISA increasing the activity of the mprF.

Differential Resistance and Cross-Resistance to Three Phenylpyrazole Insecticides in the Planthopper Nilaparvata lugens (Hemiptera: Delphacidae)

Cross-resistance to two fipronil analogs, butene-fipronil and ethiprole, was detected in fipronil-resistant field populations and a resistant laboratory strain of the planthopper Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), although the two analogs have not been used widely in rice-growing areas in China. The results showed that six field populations with 23.8-43.3-fold resistance to fipronil had reached a higher level of cross-resistance to ethiprole (resistance ratio [RR] = 47.1-100.9-fold) and had a minor level of cross-resistance (RR = 3.4- 8.1-fold) to butene-fipronil. After 10 generations of selection, the RR to fipronil increased from 7.3-fold to 41.3-fold. At the same time, the insect increased cross-RR to ethiprole from 16.3-fold to 65.6-fold, whereas it had only minor increase in cross-resistance to butene-fipronil from 2.8-fold to 4.0-fold. These results confirmed that fipronil-resistant N. lugens could develop a higher level of cross-resistance to ethiprole, although it still maintained a lower level cross-resistance to butene-fipronil. Our data suggest that ethiprole is not a suitable alternative for controlling N. lugens, once the insect has developed a high level resistance to fipronil. Further investigation is necessary to understand the cross-resistance mechanisms in N. lugens.

Cross-resistance and biochemical mechanisms of abamectin resistance in the western flower thrips, Frankliniella occidentalis

Abamectin resistance was selected in the western flower thrips [ Frankliniella occidentalis (Pergande)] under the laboratory conditions, and cross-resistance patterns and possible resistance mechanisms in the abamectin-resistant strain (ABA-R) were investigated. Compared with the susceptible strain (ABA-S), the ABA-R strain displayed 45.5-fold resistance to abamectin after 15 selection cycles during 18 generations. Rapid reversion of abamectin resistance was observed in the ABA-R strain in the absence of the insecticide selection pressure. Moderate and low levels of cross-resistance to chlorpyrifos (RR 11.4) and lambda-cyhalothrin (3.98) were observed in the ABA-R strain, but no significant cross-resistance was found to spinosad (2.00), acetamiprid (1.47) and chlorfenapyr (0.26). Our studies also showed that the esterase inhibitor S, S, S-tributyl phosphorotrithioate (DEF) and glutathione S-transferase inhibitor diethyl maleate (DEM) were not able to synergize the toxicity of abamectin, whereas the oxidase inhibitor piperonyl butoxide (PBO) conferred a significant synergism on abamectin in the ABA-R strain (SR 3.00). Biochemical analysis showed that cytochrome P450 monooxygenase activity of the ABA-R strain was 6.66-fold higher than that of the ABA-S strain. It appears that enhanced oxidative metabolism mediated by cytochrome P450 monooxygenases was a major mechanism for abamectin resistance in the western flower thrips.

Molecular mechanisms of cross-resistance to azole antifungal agents in a clinical isolate of Aspergillus fumigatus: a preliminary study

Objective To investigate the molecular mechanisms of cross-resistance to azoles in a clinical isolate of Aspergillus fumigatus. Methods A. fumigatus was isolated from a patient with invasive aspergillosis.Clinical Laboratory Standard Institute M38-A2 broth microdilution method and E-test method were used to determine the minimum inhibitory concentrations (MICs) or minimum effective concentration (MEC) of itraconazole, voriconazole, amphotericin B, posaconazole and caspofungin for the A. fumigatus isolate. DNA was extracted from the isolate and subjected to the amplification of cyp51A gene encoding the target enzyme of azole antifungal agents followed by sequence analysis. Results The broth microdilution test showed that the MEC of caspofungin was 0.5 mg/L, and MICs of itraconazole, voriconazole and amphotericin B were ≥ 16 mg/L,8 mg/L and 1 mg/L, respectively, for this isolate; while E-test assay revealed that the MICs of caspofungin,itraconazole, voriconazole, amphotericin B and posaconazole were 0.047 mg/L, ≥32 mg/L,≥32 mg/L, 12 mg/L and ≥32 mg/L, respectively. Sequence analysis showed an insertion of a 34-bp tandem sequence in the promoter region of the cyp51A gene as well as a T364A point mutation causing the substitution of leucine 98 (L98H). In addition, there were some other mutations in the cyp51A gene of this isolate, such as A137T,G585A, C814A, G836C, T991C and A1350G, which could result in corresponding amino acid substitutions.Conclusions An A. fumigatus strain with cross-resistance to azole antifungal agents is isolated. There is an insertion of a 34-bp tandem sequence into the promoter region as well as a T364A point mutation in the cyp51A gene, which contribute to the cross resistance to azole antifungal agents including itraconazole, voriconazole,and posaconazole. In addition, other mutations causing amino acid substitutions have also been detected in the cyp51 A gene of this isolate. Key words: Aspergillus fumigatus; Azoles; Drug resistance, multiple, fungal; cyp51A

Mechanism of action and limited cross-resistance of new lipopeptide MX-2401.

MX-2401 is a semisynthetic calcium-dependent lipopeptide antibiotic (analogue of amphomycin) in preclinical development for the treatment of serious Gram-positive infections. In vitro and in vivo, MX-2401 demonstrates broad-spectrum bactericidal activity against Gram-positive organisms, including antibiotic-resistant strains. The objective of this study was to investigate the mechanism of action of MX-2401 and compare it with that of the lipopeptide daptomycin. The results indicated that although both daptomycin and MX-2401 are in the structural class of Ca²⁺-dependent lipopeptide antibiotics, the latter has a different mechanism of action. Specifically, MX-2401 inhibits peptidoglycan synthesis by binding to the substrate undecaprenylphosphate (C₅₅-P), the universal carbohydrate carrier involved in several biosynthetic pathways. This interaction resulted in inhibition, in a dose-dependent manner, of the biosynthesis of the cell wall precursors lipids I and II and the wall teichoic acid precursor lipid III, while daptomycin had no significant effect on these processes. MX-2401 induced very slow membrane depolarization that was observed only at high concentrations. Unlike daptomycin, membrane depolarization by MX-2401 did not correlate with its bactericidal activity and did not affect general membrane permeability. In contrast to daptomycin, MX-2401 had no effect on lipid flip-flop, calcein release, or membrane fusion with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG) liposomes. MX-2401 adopts a more defined structure than daptomycin, presumably to facilitate interaction with C₅₅-P. Mutants resistant to MX-2401 demonstrated low cross-resistance to other antibiotics. Overall, these results provided strong evidence that the mode of action of MX-2401 is unique and different from that of any of the approved antibiotics, including daptomycin.

Comparison of clinical isolates and in vitro selected mutants reveals that tlyA is not a sensitive genetic marker for capreomycin resistance in Mycobacterium tuberculosis

The aim of this study was to clarify the conflicting data regarding cross-resistance and drug-resistance mechanisms for the cyclic peptide capreomycin and the aminoglycosides amikacin and kanamycin by comparing genotypes and phenotypes of clinical isolates and in vitro selected mutants of Mycobacterium tuberculosis. The genes rrs and tlyA and the promoter region of eis of 152 M. tuberculosis clinical isolates (including 55 capreomycin resistant) and 44 in vitro selected capreomycin-, amikacin- and kanamycin-resistant mutants were sequenced. In addition, MICs of capreomycin, amikacin and kanamycin on Middlebrook 7H10 were determined. The results clearly show major differences in genotypes and cross-resistance patterns to amikacin and kanamycin between the capreomycin-resistant clinical isolates and in vitro selected mutants. tlyA mutations were found almost exclusively among the in vitro selected capreomycin-resistant mutants, while only four were found among the clinical isolates, of which two were capreomycin susceptible. In contrast, 53 of the 55 capreomycin-resistant clinical isolates had a mutation at position 1401 in rrs and were resistant to capreomycin, amikacin and kanamycin. Low-level resistance to kanamycin was correlated to mutations in the promoter region of eis. Our findings are consistent with the belief that a mutation at position 1401 in rrs leads to resistance to capreomycin, amikacin and kanamycin. The data also show that tlyA is not a sensitive genetic marker for capreomycin resistance in clinical isolates of M. tuberculosis, as mutations in this gene are infrequent and not all mutations in tlyA lead to capreomycin resistance.