Chemical molecular‐based approach to overcome multidrug resistance in cancer by targeting P‐glycoprotein (P‐gp)

Multidrug resistance (MDR) remains one of the major impediments for efficacious cancer chemotherapy. Increased efflux of multiple chemotherapeutic drugs by transmembrane ATP‐binding cassette (ABC) transporter superfamily is considered one of the primary causes for cancer MDR, in which the role of P‐glycoprotein (P‐gp/ABCB1) has been most well‐established. The clinical co‐administration of P‐gp drug efflux inhibitors, in combination with anticancer drugs which are P‐gp transport substrates, was considered to be a treatment modality to surmount MDR in anticancer therapy by blocking P‐gp‐mediated multidrug efflux. Extensive attempts have been carried out to screen for sets of nontoxic, selective, and efficacious P‐gp efflux inhibitors. In this review, we highlight the recent achievements in drug design, characterization, structure–activity relationship (SAR) studies, and mechanisms of action of the newly synthetic, potent small molecules P‐gp inhibitors in the past 5 years. The development of P‐gp inhibitors will increase our knowledge of the mechanisms and functions of P‐gp‐mediated drug efflux which will benefit drug discovery and clinical cancer therapeutics where P‐gp transporter overexpression has been implicated in MDR.     

Transgene‐mediated suppression of the RNA interference pathway in Aedes aegypti interferes with gene silencing and enhances Sindbis virus and dengue virus type 2 replication

RNA interference (RNAi) is the major innate antiviral pathway in Aedes aegypti that responds to replicating arboviruses such as dengue virus (DENV) and Sindbis virus (SINV). On the one hand, the mosquito's RNAi machinery is capable of completely eliminating DENV2 from Ae. aegypti. On the other, transient silencing of key genes of the RNAi pathway increases replication of SINV and DENV2, allowing the viruses to temporally overcome dose‐dependent midgut infection and midgut escape barriers (MEB) more efficiently. Here we expressed Flock house virus B2 (FHV‐B2) from the poly‐ubiquitin (PUb) promoter in Ae. aegypti using the ΦC31 site‐directed recombination system to investigate the impact of transgene‐mediated RNAi pathway suppression on infections with SINV‐TR339eGFP and DENV2‐QR94, the latter of which has been shown to be confronted with a strong MEB in Ae. aegypti. FHV‐B2 was constitutively expressed in midguts of sugar‐ and blood‐fed mosquitoes of transgenic line PUbB2 P61. B2 over‐expression suppressed RNA silencing of carboxypeptidase A‐1 (AeCPA‐1) in midgut tissue of PUbB2 P61 mosquitoes. Following oral challenge with SINV‐TR339eGFP or DENV2‐QR94, mean titres in midguts of PUbB2 P61 females were significantly higher at 7 days post‐bloodmeal (pbm) than in those of nontransgenic control mosquitoes. At 14 days pbm, infection rates of carcasses were significantly increased in PubB2 P61 mosquitoes infected with SINV‐TR339eGFP. Following infection with DENV2‐QR94, midgut infection rates were significantly increased in the B2‐expressing mosquitoes at 14 days pbm. However, B2 expression in PUbB2 P61 did not increase the DENV2‐QR94 dissemination rate, indicating that the infection phenotype was not primarily controlled by RNAi.     

Validation of novel promoter sequences derived from two endogenous ubiquitin genes in transgenic Aedes aegypti

To date, only a limited number of promoter sequences have been described to drive transgene expression in the disease vector Aedes aegypti. We sought to increase this repertoire by characterizing the ability of upstream sequences derived from the Ae. aegypti Ub_L40 and polyubiquitin genes to drive the expression of marker proteins. Both genomic fragments were able to drive robust expression of luciferase in cultured mosquito cells. Following Mos1‐transformation, the Ub_L40 promoter drove strong expression of a fluorescent marker in early larvae and in ovaries, while the polyubiquitin promoter drove robust EGFP expression in all stages of development, including constitutive expression throughout the midgut. These promoter fragments provide two new expression profiles for future Ae. aegypti genetic experiments.     

浙江省宁波地区登革热疫情前后白纹伊蚊抗药性调查

  目的   调查浙江省宁波地区登革热疫情前后白纹伊蚊对常用杀虫剂的抗药性变化,为抗药性治理和登革热防控提供科学依据。  方法   登革热疫情发生前（2018年）在宁波市辖10个县（市、区）采集白纹伊蚊幼蚊,疫情发生后（2019年）在登革热旧疫点采集白纹伊蚊幼蚊。采集的幼蚊在实验室繁殖1～2代,分别采用浸渍法和接触法测定幼蚊和成蚊抗药性。  结果   2018年,白纹伊蚊幼蚊对溴氰菊酯、高效氯氰菊酯、残杀威、敌敌畏和双硫磷的抗性倍数分别为45.43、18.85、4.81、1.55和4.13,2019年抗性倍数分别为82.00、29.43、7.14、3.27和6.25。 将白纹伊蚊成蚊暴露于0.4%高效氯氰菊酯、0.1%溴氰菊酯、3%氯菊酯、0.05%残杀威和0.5%马拉硫磷各1 h,2018年试虫24 h死亡率分别为98.33%、83.33%、88.52%、95.24%和76.67%,2019年试虫24 h死亡率分别为98.89%、92.22%、98.89%、100.00%和98.89%。  结论   宁波地区白纹伊蚊幼蚊对测试的杀虫剂均产生了抗药性,且较登革热疫情前有了较大提高,而成蚊对测试的杀虫剂则相对敏感,抗药性未出现明显提高。 幼蚊和成蚊对杀虫剂的抗性水平并不一致,蚊虫防控应根据不同虫态的抗药性采取相应杀虫剂使用策略。     

Low density lipopolyprotein inhibits flavivirus acquisition in Aedes aegypti

Aedes aegypti is the primary vector of a number of human pathogens including dengue virus (DENV) and Zika virus (ZIKV). Ae. aegypti acquires these viruses during the processing of bloodmeals obtained from an infected vertebrate host. Vertebrate blood contains a number of factors that have the potential to modify virus acquisition in the mosquito. Interestingly, low density lipopolyprotein (LDL) levels are decreased during severe DENV infection. Accordingly, we hypothesized that LDL is a modifiable factor that can influence flavivirus acquisition in the mosquito. We found that LDL is endocytosed by Ae. aegypti cells in a dynamin‐dependent manner. LDL is also endocytosed by midgut epithelial cells and accumulates at the luminal midgut epithelium during bloodmeal digestion. Importantly, pretreatment with LDL, but not high density lipopolyprotein (HDL), significantly inhibited both DENV and ZIKV infection in vitro, and LDL inhibited ZIKV infection in vivo. This study identifies human LDL or ‘bad cholesterol’ as a modifiable factor that can inhibit flavivirus acquisition in Ae. aegypti. Identification of modifiable blood factors and critical cellular interactions that mediate pathogen acquisition may lead to novel strategies to disrupt the transmission cycle of vector‐borne diseases.     

Intestinal permeability and P‐glycoprotein‐mediated efflux transport of ticagrelor in Caco‐2 monolayer cells

Ticagrelor is the unique reversible oral antiplatelet drug commercialized today. During this study, the intestinal permeability of ticagrelor and its potential P‐glycoprotein (P‐gp)‐mediated active transport were assessed. To this end, bidirectional transport of ticagrelor was performed across Caco‐2 (human epithelial colorectal adenocarcinoma) monolayer model in the presence and absence of potent P‐gp inhibitor valspodar. Ticagrelor presented an apical–basolateral apparent permeability coefficient (P_app) of 6.0 × 10^?6 cm/s. On the other hand, mean efflux ratio (ER) of 2.71 was observed for ticagrelor describing a higher efflux permeability compared to the influx component. Valspodar showed a significant inhibitory effect on the efflux of ticagrelor suggesting involvement of P‐gp in its oral disposition. Co‐incubation of the P‐gp inhibitor decreased the efflux P_app of ticagrelor from 1.60 × 10^?5 to 1.13 × 10^?5 cm/s and decreased its ER by 70%. Results suggest a modest active transport of ticagrelor by P‐gp across the Caco‐2 cell monolayer. The co‐administration of ticagrelor with a P‐gp inhibitor seems altogether unlikely to have an extended impact on pharmacokinetics of ticagrelor and cause bleeding events in patients.     

Knockout of a P‐glycoprotein gene increases susceptibility to abamectin and emamectin benzoate in Spodoptera exigua

P‐glycoprotein [P‐gp or the ATP‐binding cassette transporter B1 (ABCB1)] is an important participant in multidrug resistance of cancer cells, yet the precise function of this arthropod transporter is unknown. The aim of this study was to determine the importance of P‐gp for susceptibility to insecticides in the beet armyworm (Spodoptera exigua) using clustered regularly interspaced short palindromic repeats/CRISPR‐associated 9 (CRISPR/Cas9) gene‐editing technology. We cloned an open reading frame (ORF) encoding the S. exigua P‐gp protein (SeP‐gp) predicted to display structural characteristics common to P‐gp and other insect ABCB1 transporters. A knockout line with a frame shift deletion of four nucleotides in the SeP‐gp ORF was established using the CRISPR/Cas9 gene‐editing system to test its potential role in determining susceptibility to chemical insecticides or insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). Results from comparative bioassays demonstrate that knockout of SeP‐gp significantly increases susceptibility of S. exigua by around threefold to abamectin and emamectin benzoate (EB), but not to spinosad, chlorfenapyr, beta‐cypermethrin, carbosulfan indoxacarb, chlorpyrifos, phoxim, diafenthiuron, chlorfluazuron, chlorantraniliprole or two Bt toxins (Cry1Ca and Cry1Fa). Our data support an important role for SeP‐gp in susceptibility of S. exigua to abamectin and EB and imply that overexpression of SeP‐gp may contribute to abamectin and EB resistance in S. exigua.     

Exome‐wide association of deltamethrin resistance in Aedes aegypti from Mexico

Aedes aegypti is the major vector of a number of arboviruses that cause disease in humans. Without vaccines or pharmaceuticals, pyrethroid insecticides remain the major tool for public health protection. Pyrethroid resistance is now widespread. Replacement substitutions in the voltage‐gated sodium channel (vgsc) that reduce the stability of pyrethroid binding account for most of the resistance, but metabolic mechanisms also inactivate pyrethroids. High‐throughput sequencing and the A. aegypti L5 annotated physical map has allowed interrogation of the exome for genes and single‐nucleotide polymorphisms associated with pyrethroid resistance. We exposed females of A. aegypti from Mexico to a deltamethrin discriminating dose to designate them as resistant (active after 1 h) or susceptible (knocked down with no recovery after 4 h). The vgsc on chromosome 3 had the highest association, followed by genes proximal to vgsc. We identified potential detoxification genes located singly (eg HPX8C) or within clusters in chromosome 2 [three esterase clusters, two of cytochrome P450 monooxygenases (CYP)] and chromosome 3 (one cluster of 16 CYP325 and seven CYP9 genes). Deltamethrin resistance in A. aegypti is associated with mutations in the vgsc gene and a large assortment of genes.     

Horizontal transmission of an R4 clade non‐long terminal repeat retrotransposon between the divergent Aedes and Anopheles mosquito genera

AaegR4_1 and AgamR4_1 are the sole R4 clade non‐long terminal repeat (non‐LTR) retrotransposons in Aedes aegypti and Anopheles gambiae, two species that diverged approximately 145–200 million years ago. Twelve full‐length copies were found in Ae. aegypti and have less than 1% nucleotide (nt) divergence, suggesting recent activity on an evolutionary time scale. Five of these copies have intact open reading frames and the 3.6 kb open reading frame of AaegR4_1.1 has 78% nt identity to AgamR4_1.1. No intact copies were found in An. gambiae. Searches of 25 genomic databases for 22 mosquito species from three genera revealed R4 clade representatives in Aedes and Anopheles genera but not in Culex. Interestingly, these elements are present in all six species of the An. gambiae species complex that were searched but not in 13 other anopheline species. These results combined with divergence vs. age analysis suggest that horizontal transfer is the most likely explanation for the low divergence between R4 clade retrotransposon sequences of the divergent mosquito species from the Aedes and Anopheles genera. This is the first report of the horizontal transfer of an R4 clade non‐LTR retrotransposon and the first report of the horizontal transfer of a non‐LTR retrotransposon in mosquitoes.     

FISH landmarks for Aedes aegypti chromosomes

Aedes aegypti metaphase chromosome landmarks have been developed so that each chromosome of the haploid genome can be unambiguously identified and oriented by fluorescence In situ hybridization (FISH) and digital imaging microscopy. The FISH tags were derived from three cosmids that contain repetitive Ae. aegypti sequences and their unique FISH tagging characteristics are demonstrated. The sequence of the three chromosomal tags revealed that the chromosome 1 tag is an 18S fragment from the ribosomal cistron, and the other two chromosomal tags are repeats found in Ae, aegypti with no apparent similarity to known sequences. A single plasmid that contains the three chromosome tag sequences has been constructed to simplify future FISH physical mapping.     

First screening of Aedes albopictus immunogenic salivary proteins

Study of the human antibody (Ab) response to Aedes salivary proteins can provide new biomarkers to evaluate human exposure to vector bites. The identification of genus‐ and/or species‐specific proteins is necessary to improve the accuracy of biomarkers. We analysed Aedes albopictus immunogenic salivary proteins by 2D immunoproteomic technology and compared the profiles according to human individual exposure to Ae. albopictus or Ae. aegypti bites. Strong antigenicity to Ae. albopictus salivary proteins was detected in all individuals whatever the nature of Aedes exposure. Amongst these antigenic proteins, 68% are involved in blood feeding, including D7 protein family, adenosine deaminase, serpin and apyrase. This study provides an insight into the repertoire of Ae. albopictus immunogenic salivary proteins for the first time.     

Characterization and Mosquitocidal Potential of the Soil Bacteria <Emphasis Type="Italic">Aneurinibacillus aneurinilyticus</Emphasis> Isolated from Burdwan,West Bengal,India

Aedes aegypti, Anopheles subpictus and Culex quinquefasciatus are the principal vector mosquitoes globally, especially in tropical and subtropical countries. It is very essential to control the mosquito population to prevent mosquito-borne disease transmission. The major goal of the present study was to isolate an alternative for the microbial mosquito control agents like Bacillus thuringiensis and Bacillus sphaericus. The spore forming soil bacteria isolated from Burdwan, West Bengal, India were assessed for mosquitocidal potential. One potent mosquitocidal isolate was characterized by phenotypic, biochemical and 16S rDNA sequence (GU244410) analysis. The isolate was identified as Aneurinibacillus aneurinilyticus. Five ml bacterial suspension of A. aneurinilyticus (22 × 10⁹ colony forming unit/milliliter)/100 ml water resulted in 34, 59.6 and 100 % mortality of Ae. aegypti, Cx. quinquefasciatus and An. subpictus larvae respectively within 72 h, whereas, 10 ml suspension having same concentration showed 95 and 97 % death of Ae. aegypti and Cx. quinquefasciatus larvae respectively within the same time period.     

Improved transient silencing of gene expression in the mosquito female Aedes aegypti

Gene silencing using RNA interference (RNAi) has become a widely used genetic technique to study gene function in many organisms. In insects, this technique is often applied through the delivery of dsRNA. In the adult female Aedes aegypti, a main vector of human-infecting arboviruses, efficiency of gene silencing following dsRNA injection varies greatly according to targeted genes. Difficult knockdowns using dsRNA can thus hamper gene function analysis. Here, by analysing silencing of three different genes in female Ae. aegypti (p400, ago2 and E75), we show that gene silencing can indeed be dsRNA sequence dependent but different efficiencies do not correlate with dsRNA length. Our findings suggest that silencing is likely also gene dependent, probably due to gene-specific tissue expression and/or feedback mechanisms. We demonstrate that use of high doses of dsRNA can improve knockdown efficiency, and injection of a transfection reagent along with dsRNA reduces the variability in efficiency between replicates. Finally, we show that gene silencing cannot be achieved using siRNA injection in Ae. aegypti adult females. Overall, this work should help future gene function analyses using RNAi in adult females Ae. aegypti, leading toward a better understanding of physiological and infectious processes.     

Recent attempts at RNAi-mediated P-glycoprotein downregulation for reversal of multidrug resistance in cancer

Multidrug resistance (MDR) is among the major mechanisms leading to failure in chemotherapy of cancer patients. The ATP‐binding cassette proteins are major contributors to MDR, involved in the active efflux of xenobiotics out of cancer cells. Among them, P‐glycoprotein (P‐gp) is the most dominant protein involved in the efflux of drugs. For more than 30 years, scientists have searched for the ideal P‐gp inhibitor to modulate drug resistance activity of P‐gp. This inhibitor should be tissue and cell specific with side effects on other tissues, must not provoke immune responses from the host, should provide sustained inhibition, and must be synthesized readily with low cost. Chemical P‐gp inhibitors tested to date, have shown nonspecific toxic effects limiting their clinical applications. Sequence‐specific P‐gp gene silencing by RNA interference (RNAi) may provide a more effective approach for downregulation of specific protein targets due to high specificity, limited toxicity and immunogenicity, and relative ease in synthesis. RNAi can be implemented by delivery of synthetic small interfering RNAs (siRNAs) or by gene expression of short hairpin RNAs using gene expressing vectors. Specific delivery systems and expression vectors have been designed for this purpose and many researchers have explored their effectiveness for P‐gp downregulation. In this report, we review the efficiency of various methods for siRNA delivery and transfection for P‐gp downregulation in cancer cells for MDR reversal. Novel ideas and observations by different research groups were discussed for future improvement in this essential field. © 2011 Wiley Periodicals, Inc. Med Res Rev