MRP1 Gene Research Articles

NUDT15 c.415C>T Polymorphism Predicts 6-MP Induced Early Myelotoxicity in Patients with Acute Lymphoblastic Leukemia Undergoing Maintenance Therapy.

PurposeSevere myelosuppression in patients with acute lymphoblastic leukemia (ALL) undergoing 6-MP-based maintenance therapy is attributed to TPMT gene polymorphisms, which is rare in Asian populations. This study aims to evaluate the role of selected polymorphisms in NUDT15, ITPA, and MRP4 genes in addition to TPMT in predicting 6-MP intolerance during ALL maintenance therapy.Patients and MethodsWe screened for the presence of NUDT15*3 (c.415 C>T, rs116855232); MRP4 c.2269 C>T (rs3765534), ITPA c.94 C>A (rs1127354) polymorphisms in addition to TPMT *2 (rs1800462), *3A (*3B and *3C; rs1800460 and rs1142345) in ALL patients with documented severe neutropenia (cohort-1; n=42). These polymorphisms were then screened in a prospective cohort of ALL patients (cohort-2; n=133) and compared with 6-MP dose reduction, early/late myelotoxicity.ResultsNineteen (45%) patients in cohort-1 and 18 (14%) in cohort-2 had NUDT15 c.415 C>T variant while 4 (3%) patients in cohort-2 had TPMT*3C variant. Five (12%) in cohort-1 and 30 (24%) in cohort-2 had ITPA c.94 C>A variant while 9 (22%) and 15 (12%) had MRP4 c.2269 C>T variant in cohorts-1 and 2, respectively. All in cohort-1 and 36 (27%) in cohort-2 had severe myelotoxicity. Twenty-eight patients (66.6%) in cohort-1 and 40 (30%) patients in cohort-2 had significant 6-MP dose reduction. NUDT15 c.415 C>T variant explained severe myelotoxicity in 63% and 33% in cohort 1 and 2. TPMT*3C and ITPA c.94 C>A variants also explained myelotoxicity in cohort-2 (Median ANC: 376 vs 1014 mm3; p=0.04 and 776 vs 1023 mm3; p=0.04 respectively). NUDT15 c.415 C>T polymorphism explained significant myelotoxicity (507 vs 1298 mm3; p<0.0001) in the multivariate analysis as well (β=−0.314, p<0.0001).ConclusionNUDT15 c.415 C>T (15*3), TPMT*3C, as well as ITPA c.94 C>A and MRP4 c.2269 C>T polymorphisms explain hematotoxicities. Preemptive genotype-based (NUDT15*3, TPMT, ITPA c.94 C>A) 6-MP dosing could improve the outcome after maintenance therapy.

Virulence associated gene profiling and antimicrobial resistance pattern of Streptococcus suis isolated from clinically healthy pigs from North East India.

This study revealed the prevalence of Streptococcus suis in 20·39% clinically healthy pigs from North East India. All these isolates were screened for the presence of virulence- associated genes such as suilysin (sly), muramidase released protein (mrp), extracellular protein factor (epf) and arginine deiminase (arcA). Of these 62 isolates, 29 isolates carried mrp gene, 17 isolates carried sly gene, 57 isolates carried arcA gene, whereas all isolates were negative for epf gene. The most prevalent genotype was mrp- sly- epf- arcA+ (45·16%) followed by genotypes mrp+ sly- epf- arcA+ (27·41%), mrp+ sly+ epf- arcA+ (19·35%) and mrp- sly+ epf- arcA- (8·06%). High frequency of resistance was observed for antimicrobials such as tetracycline (93·54%), clindamycin (91·93%), co-trimoxazole (88·70%) and erythromycin (85·48%). Antimicrobial resistance patterns of the S. suis isolates revealed 16 resistance groups (R1 to R16), where 93·54% isolates showed multi-drug resistance (≥3 antimicrobial agents). It has also been observed that 57 (91·93%) isolates were resistant to at least four antimicrobials. The most predominant resistance pattern observed was CD-COT-E-TE, which accounted for 38·70% of the isolates. The occurrence of relatively high levels of resistance of S. suis to some antimicrobials (e.g., macrolides, tetracyclines, and sulphonamides) as observed in this study may represent a human health concern. In addition, a relatively higher percentage of S. suis isolated from clinically healthy pigs indicates a carrier status with risk of dissemination to other pigs in the herd as well as to humans.

CYP3A5 and UGT1A9 Polymorphisms Influence Immunosuppressive Therapy in Pediatric Kidney Transplant Recipients

Background: Tacrolimus (TAC) and mycophenolic acid (MPA) are the main immunosuppressive drugs used in pediatric kidney transplantation. Single nucleotide polymorphisms (SNPs) in metabolizing enzymes and transporters might influence plasma levels of these drugs. Herein, we sought to determine the influence of SNPs on CYP3A5, MRP2 and UGT1A9 genes in Chilean pediatric kidney recipients using TAC and MPA. Patients and Methods: A prospective study was performed on 104 pediatric kidney recipients that used TAC and MPA for immunosuppression. The median age at the time of transplantation was 8.1 years [Q1–Q3 4.5–11.6 years] and the main clinical diagnosis was a structural anomaly. In a subgroup of patients, a complete steroid withdrawal was made at day 7. The CYP3A5 polymorphism (ancestral allele *1; variant allele *3) was determined in the entire cohort, while MRP2 -24G > A, UGT1A9 -275T > A, and UGT1A9 -2152C > T polymorphisms were determined in 53 patients. Genotypes were associated with trough drug concentrations (C0), dose requirements normalized by weight (TAC-D mg/kg) or body surface (MPA-D mg/m2), trough levels normalized by dose requirements (C0/D), and area under the curve in 12 h normalized by dose requirements (AUC0–12h/D). Results: The frequencies of the variant alleles CYP3A5*3, MRP2-24A, UGT1A9-275A, and UGT1A9-2152T were 76.9, 22.1, 6.6, and 2.9%, respectively. AUC0–12h/TAC-D were 1.6-fold higher in CYP3A5*3/*3 patients than in CYP3A5*1 carriers (CYP3A5*1/*3 and CYP3A5*1/*1). When analyzing patients with steroid withdrawal, CYP3A5*3/*3 patients had 1.7-fold higher AUC0–12h/TAC-D than the other genotypes. Patients carrying the CYP3A5*3/*3 genotype had higher TAC-C0, lower TAC-D and higher TAC-C0/D, consistently in a 6-months follow-up. Creatinine clearance was stable during the follow-up, regardless of the genotype. No significant differences between MRP2 and UGT1A9 genotypes were observed in MPA-C0, MPA-D or MPA-C0/D. However, patients carrying the UGT1A9-275A allele had lower AUC0–12h/MPA-D than those carrying the UGT1A9-275T ancestral allele. Conclusions: These results support that CYP3A5 and UGT1A9 genotyping in pediatric recipients might be useful and advisable to guide TAC and MPA dosing and monitoring in children that undergo kidney transplantation.

Persistent pesticides: effects of endosulfan at the molecular level on the aquatic invertebrate Chironomus riparius.

Although banned in multiple areas, due to its persistence in the environment, endosulfan constitutes a significant environmental concern. In this work, fourth instar Chironomus riparius larvae were exposed at environmentally relevant endosulfan concentrations of 0.1, 1, and 10 μg/L for 24 h to analyze the possible effects of this acaricide on gene expression and enzymatic activity. Transcriptional changes were studied through the implementation of a real-time polymerase chain reaction array with 42 genes related to several metabolic pathways (endocrine system, detoxification response, stress response, DNA reparation, and immune system). Moreover, glutathione-S-transferase (GST), phenoloxidase (PO), and acetylcholinesterase (AChE) activities were assessed. The five pathways were differentially altered by endosulfan exposure with significant changes in the E93, Dis, MAPR, Met, InR, GSTd3, GSTt3, MRP1, hsp70, hsp40, hsp24, ATM, PARP, Proph, and Def genes. Besides, all of the measured enzymatic activities were modified, with increased activity of GST, followed by PO and AChE. In summary, the results reflected the effects provoked in C. riparius at molecularlevel despite the absence of lethality. These data raise concerns about the strong alteration on different metabolic routes despite the low concentrations used. Therefore, new risk assessment strategies should consider include the effects at the sub-organismal level as endpoints in addition to the classical ecologically relevant parameters (such as survival). This endeavor will facilitate a comprehensive evaluation of toxicants in the environment.

The Effectiveness of Dietary Byproduct Antioxidants on Induced CYP Genes Expression and Histological Alteration in Piglets Liver and Kidney Fed with Aflatoxin B1 and Ochratoxin A.

The purpose of this study was to investigate the potential of a byproduct mixture derived from grapeseed and sea buckthorn oil industry to mitigate the harmful damage produced by ochratoxin A and aflatoxin B1 at hepatic and renal level in piglets after weaning. Forty cross-bred TOPIGS-40 hybrid piglets after weaning were assigned to three experimental groups (E1, E2, E3) and one control group (C), and fed with experimental diets for 30 days. The basal diet was served as a control and contained normal compound feed for starter piglets without mycotoxins. The experimental groups were fed as follows: E1—basal diet plus a mixture (1:1) of two byproducts (grapeseed and sea buckthorn meal); E2—the basal diet experimentally contaminated with mycotoxins (479 ppb OTA and 62ppb AFB1); and E3—basal diet containing 5% of the mixture (1:1) of grapeseed and sea buckthorn meal and contaminated with the mix of OTA and AFB1. After 4 weeks, the animals were slaughtered, and tissue samples were taken from liver and kidney in order to perform gene expression and histological analysis. The gene expression analysis showed that when weaned piglets were fed with contaminated diet, the expression of most analyzed genes was downregulated. Among the CYP450 family, CYP1A2 was the gene with the highest downregulation. According to these results, in liver, we found that mycotoxins induced histomorphological alterations in liver and kidney and had an effect on the expression level of CYP1A2, CYP2A19, CYP2E1, and CYP3A29, but we did not detect important changes in the expression level of CY4A24, MRP2 and GSTA1 genes.

If Artificial In Vitro Microenvironment Can Influence Tumor Drug Resistance Network via Modulation of lncRNA Expression?\u2014Comparative Analysis of Glioblastoma-Derived Cell Culture Models and Initial Tumors In Vivo

The tumor resistance of glioblastoma cells in vivo is thought to be enhanced by their heterogeneity and plasticity, which are extremely difficult to curb in vitro. The external microenvironment shapes the molecular profile of tumor culture models, thus influencing potential therapy response. Our study examines the expression profile of selected lncRNAs involved in tumor resistance network in three different glioblastoma-derived models commonly utilized for testing drug response in vitro. Differential expression analysis revealed significant divergence in lncRNA profile between parental tumors and tumor-derived cell cultures in vitro, including the following particles: MALAT1, CASC2, H19, TUSC7, XIST, RP11-838N2.4, DLX6-AS1, GLIDR, MIR210HG, SOX2-OT. The examined lncRNAs influence the phenomenon of tumor resistance via their downstream target genes through a variety of processes: multi-drug resistance, epithelial–mesenchymal transition, autophagy, cell proliferation and viability, and DNA repair. A comparison of in vivo and in vitro expression identified differences in the levels of potential lncRNA targets, with the highest discrepancies detected for the MDR1, LRP1, BCRP and MRP1 genes. Co-expression analyses confirmed the following interrelations: MALAT1–TYMS, MALAT1–MRP5, H19–ZEB1, CASC2–VIM, CASC2–N-CAD; they additionally suggest the possibility of MALAT1–BCRP, MALAT1–mTOR and TUSC7–PTEN interconnections in glioblastoma. Although our results clearly demonstrate that the artificial ex vivo microenvironment changes the profile of lncRNAs related to tumor resistance, it is difficult to anticipate the final phenotypic effect, since this phenomenon is a complex one that involves a network of molecular interactions underlying a variety of cellular processes.

Expression analysis of mammalian mitochondrial ribosomal protein genes

Mitochondrial ribosomal proteins (MRPs) are essential components for the structural and functional integrity of the mitoribosome complex. Throughout evolution, the mammalian mitoribosome has acquired new Mrp genes to compensate for loss of ribosomal RNA. More than 80 MRPs have been identified in mammals. Here we document expression pattern of 79 Mrp genes during mouse development and adult tissues and find that these genes are consistently expressed throughout early embryogenesis with little stage or tissue specificity. Further investigation of the amino acid sequence reveals that this group of proteins has little to no protein similarity. Recent work has shown that the majority of Mrp genes are essential resulting in early embryonic lethality, suggesting no functional redundancy among the group. Taken together, these results indicate that the Mrp genes are not a gene family descended from a single ancestral gene, and that each MRP has unique and essential role in the mitoribosome complex. The lack of functional redundancy is surprising given the importance of the mitoribosome for cellular and organismal viability. Further, these data suggest that genomic variants in Mrp genes may be causative for early pregnancy loss and should be evaluated as clinically.

In vitro function and in situ localization of Multidrug Resistance-associated Protein (MRP)1 (ABCC1) suggest a protective role against methyl mercury-induced oxidative stress in the human placenta

Methyl mercury (MeHg) is an organic highly toxic compound that is transported efficiently via the human placenta. Our previous data suggest that MeHg is taken up into placental cells by amino acid transporters while mercury export from placental cells mainly involves ATP binding cassette (ABC) transporters. We hypothesized that the ABC transporter multidrug resistance-associated protein (MRP)1 (ABCC1) plays an essential role in mercury export from the human placenta. Transwell transport studies with MRP1-overexpressing Madin-Darby Canine Kidney (MDCK)II cells confirmed the function of MRP1 in polarized mercury efflux. Consistent with this, siRNA-mediated MRP1 gene knockdown in the human placental cell line HTR-8/SVneo resulted in intracellular mercury accumulation, which was associated with reduced cell viability, accompanied by increased cytotoxicity, apoptosis, and oxidative stress as determined via the glutathione (GSH) status. In addition, the many sources claiming different localization of MRP1 in the placenta required a re-evaluation of its localization in placental tissue sections by immunofluorescence microscopy using an MRP1-specific antibody that was validated in-house. Taken together, our results show that (1) MRP1 preferentially mediates apical-to-basolateral mercury transport in epithelial cells, (2) MRP1 regulates the GSH status of placental cells, (3) MRP1 function has a decisive influence on the viability of placental cells exposed to low MeHg concentrations, and (4) the in situ localization of MRP1 corresponds to mercury transport from maternal circulation to the placenta and fetus. We conclude that MRP1 protects placental cells from MeHg-induced oxidative stress by exporting the toxic metal and by maintaining the placental cells' GSH status in equilibrium.

Changes of Transporters and Drug-metabolizing Enzymes in Nephrotic Syndrome.

Drug-metabolizing enzymes and transporters play key roles in drug disposition and drug interactions. The alterations of their expression will influence drug pharmacokinetics and pharmacodynamics. However, the changes in the expression of enzymes and transporters in the disease state are still unclear. Our study was to investigate the changes in the expression of main enzymes and drug transporters distributed in Adriamycin nephropathy rat liver, kidney, and intestine. An intravenous injection with a single dose of Adriamycin (6mg/kg) was made to establish Adriamycin nephropathy (AN) model and normal groups were injected with normal saline. Serum was collected for lipid metabolism, renal, and hepatic function measurement. The real-time PCR and western blot were applied to determine the mRNA and protein expression of drug enzymes and transporters. In the kidney, a greater expression of Mdr1, Mrp2, Mrp4 Oat2 and Oct2 mRNA was found in AN rats as compared with control rats. In the liver, the expression of Bcrp mRNA was more doubled or tripled than control groups and downregulation of Mdr1, Mrp2, Mrp4 and Bsep gene expression was found in AN rats. Besides, we observed a downward trend of Cyp1a2, Cyp3a4 and Cyp2c9 mRNA levels in AN groups. In the duodenum, the expression of Mdr1 and Mrp3 mRNA level was decreased, while Bcrp and Mrp2 mRNA were increased. The changes in drug-metabolizing enzymes and transporters expression in AN rats were clarified, which may be beneficial for understanding the altered pharmacokinetics and pharmacodynamics of clinical drugs and reduce unexpected clinical findings for nephropathy patients.

The potential impact of trigonelline loaded micelles on Nrf2 suppression to overcome oxaliplatin resistance in colon cancer cells.

Nuclear factor erythroid 2-related factor 2 (Nrf2) has a pivotal role in promoting chemoresistance by regulation of antioxidants and detoxification enzymes. Trigonelline is one of the majoralkaloids inrawcoffee which has been recently introduced as potent inhibitor of Nrf2. This study investigated the role of trigonelline and trigonelline loaded micelles in Nrf2 inhibition to break down oxaliplatin resistance in colon cancer cells. The PCL-PEG-PCL and PLA-PCL-PEG-PCL-PLA copolymers and trigonelline loaded micelles were prepared and characterized for fourier transforms infrared (FTIR), hydrogen nuclear magnetic resonance (1H-NMR), carbon nuclear magnetic resonance (13C-NMR) spectroscopy, particle size, zeta potential, scanning electron microscopy (SEM) and entrapment efficiency. Cell viability and apoptosis were evaluated by using MTT and flow cytometry assays, respectively. Nrf2, MRP1, NQO1, HO-1, Bax, and Bcl2 gene expressions were examined by qRT-PCR. Our results revealed that micelles had spherical shapes with narrow sizes and zeta potential indexes of - 9.06 ± 6.94mV for trigonelline loaded 3Block and - 7.47 ± 6.08mV for trigonelline loaded 5Block micelles. After Nrf2 inhibition by trigonelline, antioxidant response element (ARE) related gene expressions were decreased (p < 0.05) with a significantly higher impact by trigonelline loaded micelles (p < 0.05). Trigonelline loaded micelles also strongly decreased IC50 value of oxaliplatin in resistant colon cancer cells (p < 0.05). Furthermore, trigonelline loaded 5Block micelle increased oxaliplatin-induced apoptosis in a Nrf2/ARE dependent manner. Altogether, the current study suggests that delivery of trigonelline loaded micelles as potent Nrf2 inhibitors can be considered as a promising strategy to overcome oxaliplatin resistance in colon cancer patients.

Arsenic induced alteration in Mrp-1 like activity leads to zebrafish hepatocyte apoptosis: The cellular GSH connection

Multidrug-resistance protein-1 facilitates the efflux of arsenic conjugated with reduced glutathione nonetheless; the relation between Mrp-1 ATPase activity and cellular GSH levels is contentious. To study this, Mrp-1-ATPase activity was measured in 5 μM arsenic trioxide exposed zebrafish hepatocytes (ZFH) and correlated with intracellular GSH levels. Alongside, mrp-1 gene expression as well as Mrp-1 protein level was also monitored. Diverse mode of Mrp-1 inhibition was reflected from differential level of Km and Vmax of Mrp-1 at different time points. 3 h post-arsenic treatment demonstrated non-competitive inhibition. At 6 h, there was significant increase in Km and ZFH death, suggesting reduced binding affinity of Mrp-1 for ATP. Increased caspase-9-cytochromeC-ATP levels (putative apoptosome), reinforced ZFH apoptosis. The increase in Vmax coupled with reduced substrate affinity of Mrp-1 suggests malfunctioning in arsenic- tolerance mechanisms. We posit the triggering glutathione level regulate arsenic tolerance in ZFH. Irreversible impairment of ATP binding to Mrp-1 culminates in arsenic-induced ZFH apoptosis.

Ablation of Myeloid Cell MRP8 Ameliorates Nephrotoxic Serum-induced Glomerulonephritis by Affecting Macrophage Characterization through Intraglomerular Crosstalk

Toll-like receptor 4 (TLR4) and one of its endogenous ligands myeloid-related protein 8 (MRP8 or S100A8), especially expressed in macrophages, play an important role in diabetic nephropathy and autoimmune disorders. However, detailed mechanisms and consequence of MRP8 expression remain unknown, partly due to embryonic lethality of MRP8 knockout mice. In this study, Myeloid lineage cell-specific MRP8 knockout mice were generated, and nephrotoxic serum-induced glomerulonephritis was developed. Mice with conditional ablation of MRP8 gene in myeloid cells exhibited less severe histological damage, proteinuria and inflammatory changes compared to control mice. Mechanism of MRP8 upregulation was investigated using cultured cells. Co-culture of macrophages with mesangial cells or mesangial cell-conditioned media, but not with proximal tubules, markedly upregulated MRP8 gene expression and inflammatory M1 phenotype in macrophages, which was attenuated in MRP8-deleted bone marrow-derived macrophages. Effects of MRP8 deletion was further studied in the context of macrophage-inducible C-type lectin (Mincle), which is critically involved in maintenance of M1 phenotype of macrophages. MRP8 ablation in myeloid cells suppressed the induction of Mincle expression on macrophages in glomerulonephritis. Thus, we propose that intraglomerular crosstalk between mesangial cells and macrophages plays a role in inflammatory changes in glomerulonephritis, and MRP8-dependent Mincle expression in macrophage may be involved in the process.

Norfloxacin Sub-Inhibitory Concentration Affects Streptococcus suis Biofilm Formation and Virulence Gene Expression

Streptococcus suis (S. suis) is a major pathogen causing economic losses to the swine industry. Norfloxacins are usually used at sub-MIC (Minimum Inhibitory Concentration) doses to prevent S. suis infection. This study demonstrates the effect of norfloxacin sub-MIC on biofilm formation and virulence gene expression in S. suis.It was found that 1/4 MIC of norfloxacin increased biofilm formation in S. suis, the biofilms formed contained a higher number of viable bacteria. Additionally, bacterial growth rates were inhibited at 1/2 MIC of norfloxacin. Furthermore, the mRNA level of S. suis virulence gene cps, ef, sly, fpbs, gdh and gapdh increased by real-time PCR, while the virulence gene mrp decreased at 1/4 MIC. In conclusion, Norfloxacin sub-MICs affects biofilm formation and virulence gene expression in S. suis. These findings suggest that investigating the effect of the administration of antibiotics sub-MICs on bacterial biofilms and infection may lead to the development of future antibiotic treatments modalities.

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.

Bioinformatics analysis of structure and function in the MRP gene family and its expression in response to various drugs in Bursaphelenchus xylophilus

Genes homologous to members of the MRP gene family in Caenorhabditis elegans are important in drug resistance. To further explore the molecular mechanism of drug resistance in pine wood nematode (Bursaphelenchus xylophilus), we used bioinformatics approaches to analyze genomic data for B. xylophilus and identified Bx-MRP genes. We predicted the structure and function of the genes and encoded proteins. Using bioinformatics programs to predict and analyze various properties of the predicted proteins, including hydrophobicity, transmembrane regions, phosphorylation sites, and topologically isomeric structures, of these Bx-MRP genes, we determined that they function in transmembrane transport. From the results of RT-qPCR, the Bx-MRP family members confer significant differential resistance to different drug treatments. After treatment with different concentrations of emamectin benzoate, avermectin and matrine, the expression of each gene increased with increasing drug concentrations, indicating that the family members play a positive role in the regulation of multidrug resistance.

Combined effects of benzophenone-3 and temperature on gene expression and enzymatic activity in the aquatic larvae Chironomus riparius

Climate change and pollution are two of the main environmental problems living organisms currently face. Temperature can modify a toxicant's effects and the organism's response to it. Global warming is expected to increase the temperature of freshwater ecosystems. In this work, we analyzed the effect of a mild temperature increase on the acute response of the aquatic larvae Chironomus riparius to the ultraviolet filter benzophenone-3 (BP3). This substance is commonly used in sunscreens and other commercial products and can reach the environment in different ways. We exposed larvae to BP3 at 18.5 or 23 °C for 8 or 24 h and analyzed the acute response at the molecular level. By quantitative real-time polymerase chain reaction (q-PCR), we studied altered messenger RNA (mRNA) levels of genes related to the endocrine system (EcR, InR and Met), detoxification mechanisms (Cyp4d2, Cyp6b7, GST d6, GST o1 and MRP-1) and stress response (Hsp22, Hsp27, Hsp70, HYOU and Gp93). Moreover, enzyme activity was evaluated, with a focus on glutathione-S-transferase (GST), phenoloxidase (PO) and acetylcholinesterase (AChE). Results showed that temperature affected the acute response of this organism by modifying the expression of EcR, Cyp6b7, GST d6, GST o1, MRP-1, Hsp22, Hsp27 and Hsp70 genes. These results suggest that even mild temperature change can affect the response of this organism to BP3 influencing short-term progress of the population. Although longer exposures are required to determine the ability of C. riparius to manage the pollutants in this novel environmental conditions, in order to know the possible mechanisms of detoxification or adaptation that may develop. This research represents a first step in the analysis of multi-stress response in this animal, and opens new possibilities in the toxicity evaluation of this organism in line with the real scenario that organisms face today.

The Response of Cupriavidus metallidurans CH34 to Cadmium Involves Inhibition of the Initiation of Biofilm Formation, Decrease in Intracellular c-di-GMP Levels, and a Novel Metal Regulated Phosphodiesterase.

Cadmium is a highly toxic heavy metal for biological systems. Cupriavidus metallidurans CH34 is a model strain to study heavy metal resistance and bioremediation as it is able to deal with high heavy metal concentrations. Biofilm formation by bacteria is mediated by the second messenger bis-(3′–5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). The aim of this study was to characterize the response of C. metallidurans CH34 planktonic and biofilm cells to cadmium including their c-di-GMP regulatory pathway. Inhibition of the initiation of biofilm formation and EPS production by C. metallidurans CH34 correlates with increased concentration of cadmium. Planktonic and biofilm cells showed similar tolerance to cadmium. During exposure to cadmium an acute decrease of c-di-GMP levels in planktonic and biofilm cells was observed. Transcription analysis by RT-qPCR showed that cadmium exposure to planktonic and biofilm cells induced the expression of the urf2 gene and the mercuric reductase encoding merA gene, which belong to the Tn501/Tn21 mer operon. After exposure to cadmium, the cadA gene involved in cadmium resistance was equally upregulated in both lifestyles. Bioinformatic analysis and complementation assays indicated that the protein encoded by the urf2 gene is a functional phosphodiesterase (PDE) involved in the c-di-GMP metabolism. We propose to rename the urf2 gene as mrp gene for metal regulated PDE. An increase of the second messenger c-di-GMP content by the heterologous expression of the constitutively active diguanylate cyclase PleD correlated with an increase in biofilm formation and cadmium susceptibility. These results indicate that the response to cadmium in C. metallidurans CH34 inhibits the initiation of biofilm lifestyle and involves a decrease in c-di-GMP levels and a novel metal regulated PDE.

Abstract 4743: Using genome-wide CRISPR screen to understand resistance mechanisms to PCA062, a P-cadherin targeting antibody-drug conjugate

Abstract P-cadherin (PCAD) is a member of the cadherin family that mediates calcium dependent cell-cell contacts in adherens-type junctions of epithelium. Expression of P-cadherin is high in malignant tumors of epithelial origin, such as breast, esophagus, head and neck cancers, but low in normal tissues. This expression pattern makes P-cadherin a potential good target for antibody-drug conjugates (ADCs). PCA062 is a first-in-class antibody drug conjugate targeting P-cadherin. PCA062 consists of a fully human anti-P-cadherin antibody of the IgG1/κ subtype, a non-cleavable bi-functional linker (SMCC) and a maytansine-derived cytotoxic payload (DM1, with a target average drug to antibody ratio (DAR) of 3.8). PCA062 activity was examined in a collection of cell lines expressing high level of PCAD. A subset of these PCAD high cell lines are resistant to PCA062 treatment while they remain sensitive to DM1, suggesting defects in the process of PCA062 uptake or the processing and release of DM1 into the cytoplasmic compartment. PCA062 internalization rate was measured by the uptake of a fluorescent dye labeled anti-PCAD antibody (CQY684, the Ab portion of PCA062) in both PCA062 sensitive and resistant lines. PCA062 resistant lines show slower CQY684 internalization as compared to PCA062 sensitive lines, indicating a defect in ADC internalization may contribute to PCA062 resistance. To explore additional resistance mechanisms to PCA062 as well as to find critical components for PCA062 internalization, a genome wide CRISPR screen was performed in PCA062 sensitive HCC1954 and in PCA062 resistant KYSE510 cell line in the presence and absence of PCA062 and DM1 to look for genes that when knocked out may specifically modulate PCA062 sensitivity. The multi-drug resistant gene MRP1 is a strong hit for PCA062 sensitization in both PCA062 resistant KYSE510 and PCA062 sensitive HCC1954 cells. Lysosomal transporter SLC46A3 and Saga transcription complex components are strong rescue hits for PCA062 in HCC1954. These data suggest that in addition to target expression level and cell intrinsic sensitivity to payload, genes involved in ADC internalization and payload cytoplasmic accumulation will also impact tumor cell sensitivity to ADCs. Citation Format: Angela Tam, Mark Zambrowski, Katherine Seiss, Si-Qi Liu, Tinya Abrams, Giordano Caponigro, William Tschantz, Jennifer Campbell, Tony DAlessio, Qing Sheng. Using genome-wide CRISPR screen to understand resistance mechanisms to PCA062, a P-cadherin targeting antibody-drug conjugate [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 4743.

The long non-coding RNA HOTAIRM1 suppresses cell progression via sponging endogenous miR-17-5p/ B-cell translocation gene 3 (BTG3) axis in 5-fluorouracil resistant colorectal cancer cells

5-Fluorouracil (5-FU)-based chemotherapy has always been the first-line treatment of colorectal cancer (CRC). However, the occurrence of clinical 5-FU resistance is a major reason for CRC therapy failure. This study intended to explore the possible role of long non-coding RNA HOTAIRM1 (HOTAIRM1) in the pathogenesis of 5-FU resistant CRC and its underlying mechanism. Our data showed that HOTAIRM1 was downregulated in CRC tissues and cell lines (HCT116 and SW480), and even lower in 5-FU resistant CRC tissues and cell lines (HCT116/5-FU and SW480/5-FU). In vitro, effects of HOTAIRM1 dysregulation in 5-FU resistant CRC cells were investigated and its overexpression could reduce cell viability, invasion, migration, and multi-drug resistance as evidenced by MTT assay, Transwell assay, epithelial-mesenchymal transition (EMT), and western blot analyzing expression of drug-resistant genes MRP1 and MDR1, respectively. Mechanically, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) identified that HOTAIRM1 and B-cell translocation gene 3 (BTG3) were target genes of miR-17-5p. Moreover, miR-17-5p was upregulated and BTG3 was downregulated in HCT116/5-FU and SW480/5-FU cells. Silencing of miR-17-5p showed suppressive role on cell viability, invasion, migration, and multi-drug resistance in HCT116/5-FU and SW480/5-FU cells, which could be abolished by HOTAIRM1 knockdown. Similarly, ectopic expression of miR-17-5p reversed BTG3-mediated inhibition on cell viability, invasion, migration, and multi-drug resistance. In vivo, the tumorigenesis of HCT116/5-FU cells when highly expressed HOTAIRM1 by lentivirus infection was inhibited through downregulating miR-17-5p and upregulating BTG3. In conclusion, HOTAIRM1 might act as a tumor-suppressor in 5-FU resistant CRC cells in vitro and in vivo through downregulating miR-17-5p/BTG3 pathway and inhibiting multi-drug resistance.

Saikosaponin b2 enhances the hepatotargeting effect of anticancer drugs through inhibition of multidrug resistance-associated drug transporters

Aims: Vinegar-baked Radix Bupleuri (VBRB) potentiates the activity of anticancer drugs in the liver by increasing their hepatic distribution. However, this phenomenon may be associated with drug transporters. We investigated the effect of saikosaponin b2 (SSb2; the main component of VBRB) on the activity and expression of different drug transporters in both normal cells and those that overexpress the transporter.Main methods: The activities of transporters were analyzed by concentration of their cellular substrates. Concentrations of colchicine (substrate of Pgp and MRP1) and cisplatin (substrate of OCT2 and MRP2) were determined by high-performance liquid chromatography (HPLC). The concentration of rhodamine B was determined by flow cytometry. The expression of transporter gene and protein were determined by qRT-PCR and Western blotting analysis.Key findings: SSb2 increased colchicine efflux in HEK293 cells by primarily increasing Mrp1 activity, independent of gene and protein expression. SSb2 enhanced Mrp2 function and increased cisplatin efflux in BRL3A cells by upregulating Mrp2 gene expression, with a marginal effect on Pgp in normal cells. SSb2 increased OCT2 activity in OCT2-HEK293 cells by increasing the expression of OCT2 protein and mRNA; however, SSb2 inhibited MRP2 activity in MRP2-HEK293 cells by decreasing MRP2 protein expression, and decreased Pgp and MRP1 activity in Pgp- and MRP1-HEK293 cells.Significance: SSb2 might potentially be the key active component of VBRB that enhances the hepatotargeting of anticancer drugs through the inhibition of multidrug resistance-associated drug transporters (Pgp, MRP1, and MRP2) in an environment-dependent manner.