Multidrug Resistance Protein Research Articles

Bim downregulation by activation of NF-κB p65, Akt, and ERK1/2 is associated with adriamycin and dexamethasone resistance in multiple myeloma cells.

Multiple myeloma (MM) frequently acquires multidrug resistance (MDR), which is due to poor prognosis. Our previous study indicated that high expression of Survivin and multidrug resistance protein 1 (MDR1) and decreased expression of Bim are associated with MDR in adriamycin- and dexamethasone-resistant cells. However, the fundamental mechanism of MDR in adriamycin- and dexamethasone-resistant MM cells is still unidentified. In this study, we examined the MDR mechanism in adriamycin- and dexamethasone-resistant cells. RPMI8226/ADM, ARH-77/ADM, RPMI8226/DEX, and ARH-77/DEX cells exhibited enhanced nuclear factor κB (NF-κB) p65, Akt, and extracellular signal-regulated kinase 1/2 (ERK1/2) activation. Combination treatment with NF-κB p65, phosphoinositide 3-kinase (PI3K), and mitogen-activated protein kinase 1/2 (MEK1/2) inhibitors resensitized to adriamycin and dexamethasone via increased Bim expression. Although treatment with MDR1 or Survivin siRNA did not overcome adriamycin and dexamethasone resistance in RPMI8226/ADM and RPMI8226/DEX cells, administration of Bim siRNA induced adriamycin and dexamethasone resistance in RPMI8226 cells. Moreover, low expression of Bim was related to poor prognosis in MM patients. These results indicate that activation of NF-κB p65, Akt, and ERK1/2 is associated with adriamycin and dexamethasone resistance via decreasing Bim expression, and these signal inhibitor combinations overcome drug resistance in MM. These findings suggest that combination treatment with these inhibitors and adriamycin or dexamethasone may be a promising therapy for adriamycin- and dexamethasone-resistant MM.

Inhibition of multidrug resistance proteins by MK571 restored the erectile function in obese mice through cGMP accumulation.

Intracellular levels of cyclic nucleotides can also be controlled by the action of multidrug resistance protein types 4 (MRP4) and 5 (MRP5). To date, no studies evaluated the role of their inhibition in an animal model of erectile dysfunction (ED). To evaluate the effect of a 2-week treatment with MK571, an inhibitor of the efflux of cyclic nucleotides in the ED of obese mice. Mice were divided in three groups: (i) lean, (ii) obese, and (iii) obese+MK571. The corpus cavernosum (CC) were isolated, and concentration-response curves to acetylcholine (ACh), sodium nitroprusside (SNP), and tadalafil in addition to electrical field stimulation (EFS) were carried out in phenylephrine pre-contracted tissues. Expression of ABCC4 and ABCC5, intracellular levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), the protein levels for pVASPSer157 and pVASPSer239 , and the intracavernous pressure (ICP) were also determined. The intracellular and extracellular (supernatant) ratios in CC from obese and lean stimulated with a cGMP-increasing substance (BAY 58-2667) in the absence and presence of MK571 (20μM, 30min) were also assessed. The treatment with MK571 completely reversed the lower relaxing responses induced by EFS, ACh, SNP, and tadalafil observed in obese mice CC in comparison with untreated obese mice. Cyclic GMP and p-VASPSer239 expression were significantly reduced in CC from obese groups. MK571 promoted a sixfold increase in cGMP without interfering in the protein expression of p-VASPSer239 . Neither the cAMP levels nor p-VASPSer157 were altered in MK571-treated animals. The ICP was ∼50% lower in obese than in the lean mice; however, the treatment with MK571 fully reversed this response. Expressions of ABCC4 and ABCC5 were not different between groups. The intra/extracellular ratio of cGMP was similar in CC from obese and lean mice stimulated with BAY 58-2667. The MRPs inhibition by MK571 favored the accumulation of cGMP in the smooth muscle cells, thus improving the smooth muscle relaxation and the erectile function in obese mice.

Inhibition of MRP1 Induces Fetal Hemoglobin through NRF2 Activation to Protect Human Erythroid Cells from Sickling

Introduction: Sickle cell disease (SCD) is a genetic disorder caused by a single nucleotide substitution (Glu6→Val) in the HBB gene coding for the β-globin of the hemoglobin A (HbA; α2β2) in erythroid cells. This point mutation leads to abnormal hemoglobin (HbS), which polymerizes in low oxygen condition and, ultimately, causes sickling of circulating erythrocytes leading to hemolysis, vascular inflammation, and painful vaso-occlusive crises. Hereditary persistence of fetal hemoglobin (HbF; α2γ2) in sickle cell patients significantly reduces clinical symptoms by preventing polymerization of HbS and subsequent cell sickling. Therefore, raising HbF expression in SCD patients is a promising therapeutic strategy. However, hydroxyurea, the standard of care for SCD and only FDA-approved HbF inducer, carries concerns for carcinogenicity-associated effects and the non-responders rate for this drug is relatively high (~30%). Consequently, there is a large unmet need for new therapies in SCD. Methods: To discover potential new HbF inducers, we conducted a genome-wide association study of HbF levels in 11,004 healthy donors from the INTERVAL study. We identified ABCC1, which encodes the multidrug resistant protein 1 (MRP1) and efflux pump for glutathione, as a putative causal gene modulating HbF expression. We detected a specific missense variant in ABCC1 which increases HbF expression in erythrocytes and strongly associates with plasma glutathione levels (β=0.8SD, P=3x10-38). To elucidate the role of MRP1 in HbF regulation, we performed a CRISPR-Cas9-mediated ABCC1 knockdown in erythroid HUDEP-2 cells and CD34+ hematopoietic stem cells and quantified the expression of HbF by flow cytometry, Western blot and high-performance liquid chromatography (HPLC) after erythroid differentiation (2-phase culture). We also generated HUDEP-2 clones homozygous for the p.R433S variant using CRISPR-Cas9 gene editing and measured HbF levels using the methods previously described. In CD34+ cells, MRP1 effect on HbF expression was also evaluated by using the selective MRP1-inhibitor, MK571. Transcriptomic, proteomics and phospho-proteomic analysis were performed to identify the mechanism of action involved in HbF induction after MRP1 inhibition. Expression of erythroid differentiation markers GATA1, ALAS2, LRF and Band 3 was assessed by Western blot to evaluate the impact of MRP1 inhibition on erythroid differentiation. Finally, we conducted sickling assays on MK571-treated CD34+ cells from SCD patients. Results:ABCC1 knockdown (90% gene silencing compared to control) in HUDEP-2 cells increased intracellular glutathione level (7−fold, P<0.01) and HbF (2.5−fold, P<0.05), as shown by flow cytometry, HPLC and Western blot analysis, after 7-day differentiation. Similarly, HUDEP-2 clones homozygous for the ABCC1-R433S allele presented an increase in intracellular glutathione (6-fold, P<0.05) and HbF expression levels (6-fold, P<0.01) compared to non-treated cells. In CD34+ cells knockdown for ABCC1 (95% gene silencing compared to control), HbF was increased (1.5−fold, P<0.05) after 14-day differentiation. Furthermore, pharmacological inhibition of MRP1, by MK571, in CD34+ cells led to an induction in glutathione (5-fold, P<0.01) and HbF (2-fold, P<0.05), and prevented sickling of CD34+ cells from SCD patients (2-fold decrease, P<0.05) in hypoxic condition (2% O2), without impacting erythroid differentiation. Finally, transcriptomic, proteomic, and phospho-proteomic integrated analyses in MK571-treated CD34+ cells revealed an activation of the NRF2-mediated oxidative stress pathway. Addition of ML385 (10µM), an NRF2 inhibitor, in MK571-treated CD34+ cells completely abolished HbF induction, confirming a NRF2-dependent HbF induction. Conclusion: In summary, we identified MRP1 as a HbF modulator in human erythroid cells and demonstrated that MRP1 inhibition induces HbF and prevents sickling of CD34+ cells through NRF2-mediated oxidative stress pathway activation without affecting erythroid differentiation, establishing MRP1 as a potential new therapeutic target for SCD. Future directions include replication of the ABCC1 genetic association in an independent cohort, and in vivo studies aiming to confirm the efficacy of MRP1 inhibition on improving SCD disease biomarkers.

Tailored modulation of stemness and drug resistance marker characteristics in K-Ras mutant lung cancer cells via PD-L1 gene suppression

AimsWe aimed to analyze the association of tumor cell-specific Programmed Cell Death Ligand 1 (PD-L1) expression with stemness markers and multi-drug resistance genes in non-small cell lung cancer. Main methodsATP Binding Cassette Subfamily G Member 2 (ABCG2), Aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), CD44, Epithelial cell adhesion molecule (EPCAM), Kruppel-like factor 4 (KLF4), MYC Proto-Oncogene (MYC), Nanog Homeobox (NANOG), SRY-Box Transcription Factor 2 (SOX2) as well as ATP Binding Cassette Subfamily C Member (ABCC) 1–6, ABCC10–12 with ATP Binding Cassette Subfamily B Member (ABCB) 1 and ABCB4–9 belongs to ABC transporters family were analyzed and their correlation with PD-L1 was evaluated using Cancer Cell Line Encyclopedia and The Cancer Genome Atlas data sets. We validated potential lung cancer stemness markers harboring ABCG2, CD44, ALDH1A1 and SOX-2, which are affected as a result of siRNA-mediated suppression of PD-L1 via flow cytometry. K-Ras downstream signaling proteins as well as multidrug resistance proteins were also investigated. Key findingsPD-L1 was found to be positively correlated with CD44, whereas negatively correlated with ALDH1A1 (Pearson r = 0.44, r = −0.48; respectively) in 45 K-Ras mutated NSCLC cells based on CCLE database. While ABCC5 was dominantly decreased in K-Ras mutant lung cancer cells affected by PD-L1 gene suppression, expression changes were observed in the activation of distinct signaling pathways in a cell line-dependent manner. SignificanceThe evaluation of markers in lung adenocarcinoma with different types of K-Ras mutations in terms of both stemness and drug resistance markers will contribute to the development of personalized immunotherapy regimens.

The potential involvement of MRP5 pump in urethral dysfunction in streptozotocin-induced diabetic rats.

To examine the effects of i.v. administration of MK-571, a MRP4/5 pump inhibitor, on urethral function in the urethane-anesthetized rat, and the changes of urethral multidrug resistance protein 5 (MRP5) pump in streptozotocin (STZ)-induced diabetes mellitus (DM) rats. Isovolumetric cystometry and urethral perfusion pressure (UPP) measurements were carried out in normal control (NC) group and 8week DM groups under urethane anesthesia. When stable rhythmic bladder contractions were showed, UPP parameters were recorded after successive administration of various dose of MK-571. Additionally, urethral cyclic guanosine monophosphate (cGMP) protein level was evaluated by ELISA, and changes of MRP5 pump and neurogenic nitric oxide synthase (nNOs) in the urethra were examined with immunohistochemical staining and Western blot analysis. In NC group, UPPnadir was significantly decreased but UPP change increased after administration of MK-571, while no significant differences in UPP parameters were observed in 8-week DM group. Furthermore, urethral MRP5 protein level was up-regulated, whereas urethral cGMP and nNOS protein levels were down-regulated in 8-week DM group. MK-571 could not restore NO-mediated urethral relaxation dysfunction in DM rats, which may be attributed to the up-regulation of urethral MRP5 pump, and thus decrease of intracellular cGMP concentration in the urethra. These novel results would be useful for a better understanding of DM-related lower urinary tract dysfunction LUT (LUTD). Also, they could be helpful to study the importance of MRP pumps in the control of urethral relaxation mechanisms under physiological and pathological states.

Sex-specific effects of excipients on oral drug bioavailability

The mechanism of action of excipients eliciting sex differences in drug bioavailability is poorly understood. In this study, the excipients Cremophor RH 40 (PEG 40 hydrogenated castor oil), Poloxamer 188 (2-methyloxirane) and Tween 80 (polyoxyethylene (80) sorbitan monooleate) were screened at 0.07 – 5% concentrations for their effect on ranitidine bioavailability in male and female Wistar rats. We show that all excipient concentrations significantly increased ranitidine bioavailability in male, but not female, rats. The effect of these excipients on the intestinal efflux transporters P-glycoprotein (P-gp), breast cancer resistant protein (BCRP) and multi-drug resistant protein 2 (MRP2) were also monitored. Measured by ELISA assay, in male rats, peak reductions in intestinal P-gp protein expression occurred in the presence of 1% Cremophor RH 40 and Poloxamer 188 and 0.5% Tween 80. In contrast, no distinct changes were observed in female intestinal P-gp expression. Unlike P-gp, all excipients had a positive effect on MRP2 protein expression – albeit only in males – in a concentration-dependent manner. The excipients did not modulate intestinal BCRP protein expression in either sex. Endogenous hormones and a nuclear receptor (testosterone, oestradiol and pregnane X receptor; PXR) that are purported to regulate intestinal efflux membrane transporter expression were also quantified. In the presence of all excipients, testosterone levels significantly elevated in males, although PXR levels reduced at similar rates in both sexes. No significant effects were identified in oestradiol levels in male and female rats. It is clear that excipients are not inert and their pathway for modulating drug response is multi-dimensional and specific between sexes. This study showed that excipients increased drug bioavailability of a P-gp drug substrate due to its reductive effect on intestinal P-gp expression; we propose that this link may be due to the excipients modulating fundamental testosterone levels. Understanding the implication of excipients on intestinal physiology and hormone levels can therefore improve pharmaceutical design, clinical efficacy and instigate next generation personalised, sex-specific formulations.

ODP560 Thyroid Hormones membrane receptor, Integrin αvβ3, as a potential prognostic marker of chemotherapy response in breast cancer

Abstract Despite the development of novel therapies, breast cancer (BC) tumors are treated with conventional therapy eventually. Due to tumor heterogeneity, it is necessary to define new biomarkers to identify groups with different molecular characteristic and on this basis improve the treatment decision. The impact of thyroid status in BC growth has previously been studied. In particular, alterations of thyroid function during chemotherapy for BC treatment have been found and it has been demonstrated that T3 induces chemosensitization of BC cells. However, the relationship between thyroid hormones (THs) membrane receptor, integrin αvβ3, and BC response to chemotherapy remains unclear. The aim of this study is to evaluate the relationship between THs and integrin αvβ3 expression as prognostic value in BC patients. The mRNA expression of ITGB3 in the TCGA-PanCancer Atlas BC dataset (1084 patients) was associated with worse overall survival. Moreover, ITGB3 mRNA expression was significantly correlated with genes encoding multidrug resistance transporter proteins MDR1 and BCRP, (Pearson, R score &amp;gt;0.3 and p&amp;lt;0,05). The diagnostic role of ITGB3 in BC was assessed by receptor operating characteristic (ROC) curve analysis. ITGB3 mRNA levels were higher in non-responder patients treated with anthracycline (AUC=0,673, p=1.8e-10) and taxanes (AUC=0.675, p=1.9e-07). To confirm the association between the integrinαvβ3-mediated effects and multidrug resistance protein transporters, we conducted in vitro assays ofMDA-MB-231 cells treated with physiological concentrations of THs and evaluated their action on MDR1 protein levels and Rhodamine efflux as an indicator of the transporter activity. We found that THs induce MDR1 protein expression and transporter activation and this effect is blocked by the presence of the integrin selective inhibitor cilengitide. Furthermore, in MDA-MB-231 doxorubicin-resistant cells THs induce MDR1 and BCRP greater protein expression than in MDA-MB-231 parental cells and this effect is abrogated by cilengitide. Therefore, we found that integrin β3 is correlated with worse overall survival and response to anthracyclines and taxanes pointing out its role as acancer biomarker with potential clinical utility. In patients with high integrin β3 mRNA expression, we found a significant correlation with proteins involved in drug transport such as MDR1 and BCRP. We also demonstrated that THs through integrin αvβ3 modulate the expression and activity of drug transporters. These results pointout the role of THs, and their membrane receptor, in BC response to treatments and introduce a new biomarker with potential clinical significance. Presentation: No date and time listed

Copper induces a downregulation of the Multidrug resistance protein 4 (MRP4) and Copper-transporting ATPase-RAN1 (RAN1) genes in red pine (Pinus resinosa) at low concentrations

ABSTRACT Copper is required in plant tissues at small concentrations for several physiological processes; however, excess metal can cause toxicity. The main objectives of this study were to (1) evaluate Pinus resinosa resistance to different concentrations of copper, and (2) assess changes induced by copper ions on gene expression. The targeted genes include the Multidrug resistance protein 4 (MRP4) and Copper-transporting ATPase-RAN1 (RAN1). P. resinosa seedlings were treated with different concentrations of copper sulphate and corresponding salt controls in growth chamber screening tests. These concentrations include 13, 130 mg and 1300 mg of copper per 1 kg of dry soil. Potassium sulfate and water were used as controls. The expression of MRP4 and RANI genes was assessed using RT-qPCR. All the genotypes tested were resistant to copper. There was a significant suppression of the MRP4 and RANI genes at the lowest concentration of copper (13 mg/kg) in comparison to the water control. Further increases in the copper concentrations (130 mg /kg and 1300 mg/kg) did not induce any significant change in gene expression. Overall, this study showed that P. resinosa is in general resistant to copper contaminants. However, copper ions affect gene expression, even at low concentrations.

Apicoplast ribosomal protein S10-V127M enhances artemisinin resistance of a Kelch13 transgenic Plasmodium falciparum

BackgroundThe resistance of Plasmodium falciparum to artemisinin-based (ART) drugs, the front-line drug family used in artemisinin-based combination therapy (ACT) for treatment of malaria, is of great concern. Mutations in the kelch13 (k13) gene (for example, those resulting in the Cys580Tyr [C580Y] variant) were identified as genetic markers for ART-resistant parasites, which suggests they are associated with resistance mechanisms. However, not all resistant parasites contain a k13 mutation, and clearly greater understanding of resistance mechanisms is required. A genome-wide association study (GWAS) found single nucleotide polymorphisms associated with ART-resistance in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2), and crt (chloroquine resistance transporter), in addition to k13 gene mutations, suggesting that these alleles contribute to the resistance phenotype. The importance of the FD and ARPS10 variants in ART resistance was then studied since both proteins likely function in the apicoplast, which is a location distinct from that of K13.MethodsThe reported mutations were introduced, together with a mutation to produce the k13-C580Y variant into the ART-sensitive 3D7 parasite line and the effect on ART-susceptibility using the 0−3 h ring survival assay (RSA0−3 h) was investigated.Results and conclusionIntroducing both fd-D193Y and arps10-V127M into a k13-C580Y-containing parasite, but not a wild-type k13 parasite, increased survival of the parasite in the RSA0−3 h. The results suggest epistasis of arps10 and k13, with arps10-V127M a modifier of ART susceptibility in different k13 allele backgrounds.

Scintigraphy in Thyroid Nodular Pathology

Thyroid scintigraphy is a method that formed more than 60 years ago and retains its importance for the diagnosis and assessment of the risk of nodular malignancy. The review examines the use of the scintigraphic method in functional and metabolic assessment of nodular goiter. Prerequisites for functional thyroid nodes screening with short-lived 99mTc-pertechnetate are outlined. Information is provided on the principles of its preparation and pharmacokinetics, as well as variants of the thyroid nodes functional activity, their possible malignant potential and indications for metabolic screening. The characteristic of metabolic activity allows to assess the risk of functionally “cold” thyroid nodes malignancy. The main stages of the use of nonspecific tumorotropic radiopharmaceuticals, the mechanism of their accumulation and metabolism in the thyroid tumors, the possibilities of the differential diagnosis of various types of nodular formations are presented. Modern views on the diagnostic capabilities of two-phase scintigraphy with 99mTс-methylisobutylisonitrile are reflected taking into account the relationship of radiopharmaceuticals accumulation with factors of proliferative activity and ultrastructural cell types, a variant of oncocytic pathology and multidrug-resistant protein expression.

Metformin modified chitosan as a multi-functional adjuvant to enhance cisplatin-based tumor chemotherapy efficacy

Recently, it was newly revealed that the DNA damage induced by cis‑platinum (Cis-Pt) mediated chemotherapy was significantly impaired by the highly expressed programmed death ligand-1 (PD-L1) in tumor cells. Besides, the efficacy of Cis-Pt was also limited due to its severe side effects, especially enhanced drug efflux induced by multidrug resistance protein 1 (MDR-1) and increased tumor metastasis. Up to now, few drugs or carbohydrates could simultaneously solve these defects of Cis-Pt mediated chemotherapy. Here, we newly found that metformin-modified chitosan (Ch-Met) possessed ideal selective mitochondria accumulation capacity, leading to the further disrupted mitochondrial function, which then effectively inhibited the upregulated PD-L1 expression to inhibit DNA damage repair in tumor cells, as well as impaired drug efflux and lowered tumor metastasis. Therefore, it was demonstrated that Ch-Met could sensitize the chemotherapy efficacy of Cis-Pt.

Lactate drives cellular DNA repair capacity: Role of lactate and related short-chain fatty acids in cervical cancer chemoresistance and viral infection

The characteristic feature of a cancer microenvironment is the presence of a highly elevated concentration of L-lactate in the tumor niche. The lactate-rich environment is also maintained by commensal mucosal microbiota, which has immense potential for affecting cancer cells through its receptoric and epigenetic modes of action. Some of these lactate activities might be associated with the failure of anticancer therapy as a consequence of the drug resistance acquired by cancer cells. Upregulation of cellular DNA repair capacity and enhanced drug efflux are the most important cellular mechanisms that account for ineffective radiotherapy and drug-based therapies. Here, we present the recent scientific knowledge on the role of the HCA1 receptor for lactate and lactate intrinsic activity as an HDAC inhibitor in the development of an anticancer therapy-resistant tumor phenotype, with special focus on cervical cancer cells. In addition, a recent study highlighted the viable role of interactions between mammalian cells and microorganisms in the female reproductive tract and demonstrated an interesting mechanism regulating the efficacy of retroviral transduction through lactate-driven modulation of DNA-PKcs cellular localization. To date, very few studies have focused on the mechanisms of lactate-driven enhancement of DNA repair and upregulation of particular multidrug-resistance proteins in cancer cells with respect to their intracellular regulatory mechanisms triggered by lactate. This review presents the main achievements in the field of lactate impact on cell biology that may promote undesirable alterations in cancer physiology and mitigate retroviral infections.

Ginsenosides Rg1 and CK Control Temozolomide Resistance in Glioblastoma Cells by Modulating Cholesterol Efflux and Lipid Raft Distribution.

Background Cholesterol efflux and lipid raft redistribution contribute to attenuating temozolomide resistance of glioblastoma. Ginsenosides are demonstrated to modify cholesterol metabolism and lipid raft distribution, and the brain distribution and central nervous effects of whose isoforms Rb1, Rg1, Rg3, and CK have been identified. This study aimed to reveal the role of Rb1, Rg1, Rg3, and CK in the drug resistance of glioblastoma. Methods The effects of ginsenosides on cholesterol metabolism in temozolomide-resistant U251 glioblastoma cells were evaluated by cholesterol content and efflux assay, confocal laser, qRT-PCR, and Western blot. The roles of cholesterol and ginsenosides in temozolomide resistance were studied by CCK-8, flow cytometry, and Western blot, and the mechanism of ginsenosides attenuating resistance was confirmed by inhibitors. Results Cholesterol protected the survival of resistant U251 cells from temozolomide stress and upregulated multidrug resistance protein (MDR)1, which localizes in lipid rafts. Resistant cells tended to store cholesterol intracellularly, with limited cholesterol efflux and LXRα expression to maintain the distribution of lipid rafts. Ginsenosides Rb1, Rg1, Rg3, and CK reduced intracellular cholesterol and promoted cholesterol efflux in resistant cells, causing lipid rafts to accumulate in specific regions of the membrane. Rg1 and CK also upregulated LXRα expression and increased the cytotoxicity of temozolomide in the presence of cholesterol. We further found that cholesterol efflux induction, lipid raft redistribution, and temozolomide sensitization by Rg1 and CK were induced by stimulating LXRα. Conclusions Ginsenosides Rg1 and CK controlled temozolomide resistance in glioblastoma cells by regulating cholesterol metabolism, which are potential synergists for temozolomide therapy.

Circ-LIMK1 regulates cisplatin resistance in lung adenocarcinoma by targeting miR-512-5p/HMGA1 axis.

This study aimed to unveil the detailed role and new mechanism of circ-LIMK1 in lung adenocarcinoma. Real-time quantitative polymerase chain reaction was performed to analyze the expression of circ-LIMK1, miR-512-5p, and HMGA1. 3-(4,5)-Dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide assay was employed to test the half maximal inhibitory concentration of cisplatin (DDP). Western blot was used to measure the expression of HMGA1, multidrug resistance protein 1, mitochondrial 37S ribosomal protein, and vascular endothelial growth factor A. Colony formation assay, flow cytometry, transwell assay, and tube formation assay were performed to analyze cell functions. Animal models were established to assay the role of circ-LIMK1 in vivo. The expression of circ-LIMK1 was up-regulated in DDP-resistant tumor tissues and cells. Knockdown of circ-LIMK1 reduced DDP resistance, impaired cancer cell growth, migration, invasion, and angiogenesis. circ-LIMK1 targeted miR-512-5p, and HMGA1 was targeted by miR-512-5p. MiR-512-5p absence could restore the repressive effects of circ-LIMK1 knockdown on lung adenocarcinoma cell phenotypes. Overexpression of HMGA1 could restore the inhibitory effects of miR-512-5p enrichment on lung adenocarcinoma cell malignant phenotypes. Knockdown of circ-LIMK1 could reduce growth of DDP-resistant tumors in vivo. Collectively, circ-LIMK1 regulated DDP resistance in lung adenocarcinoma by targeting miR-512-5p/HMGA1 axis.

Fluorophenylalkyl-substituted cyanoguanidine derivatives as bacteria-selective MATE transporter inhibitors for the treatment of antibiotic-resistant infections

Drug efflux pump inhibitors for the multidrug resistance protein HmrM, a member of the multidrug and toxin extrusion (MATE) family of transporters, were investigated to increase the drug susceptibility of multidrug-resistant bacteria and restore the antimicrobial effect of fluoroquinolones, such as norfloxacin. The lead inhibitor, prepared from the known hMATE1 inhibitor cimetidine, reduced the norfloxacin resistance of HmrM-expressing strains by 92% at non-cytotoxic concentrations in human cells, and multidrug resistance protein MdtK-expressing strains by 86%. These results indicated that the inhibitor is a lead candidate for the development of drugs with a novel mechanism of action against infections caused by multidrug-resistant bacteria that act synergistically with antimicrobial drugs.

Inhibitory effect of DL-butionine sulfoximine on P-glycoprotein activity in vitro

Background. P-glycoprotein (Pgp) is a multidrug resistance protein 1 with broad substrate specificity. Its functioning can change under the influence of various substances, so the search for endogenous and exogenous compounds that modulate the activity of the transporter protein is an important area of research.&#x0D; Aim. To evaluate the effect of DL-butionine sulfoximine on the activity and amount of the Pgp transporter protein in Caco-2 cells.&#x0D; Material and methods. The study was performed on a human colon adenocarcinoma cell line (Caco-2). Cells were incubated with DL-butionine sulfoximine and quinidine (a classical Pgp inhibitor) at concentrations of 1, 5, 10, 50, 100, and 500 M for 3 hours. Pgp activity was evaluated by the transport of its substrate, fexofenadine, which concentration was determined by high performance liquid chromatography with ultraviolet detection (Stayer, Russia). The results were analyzed using StatSoft Statistica 13.0 (ANOVA), IC50 was calculated using GraphPad Prism 8 software. Differences were considered statistically significant at p 0.05.&#x0D; Results. Incubation with DL-butionine sulfoximine and quinidine at concentrations of 1500 M for 3 hours did not affect the amount of Pgp in Caco-2 cells. Pgp activity decreased when using DL-butionine sulfoximine at concentrations of 50500 M by a maximum of 47.7% (p=0.040). Quinidine at concentrations of 5500 M reduced Pgp activity by a maximum of 79.1% (p=0.0002) at a concentration of 500 M. Quinidine inhibited Pgp activity at lower concentrations compared to DL-butionine sulfoximine: the IC50 of fexofenadine with quinidine was 5.160.59 mol/l, for DL-butionine sulfoximine it was 17.212.46 mol/l (p= 0.001).&#x0D; Conclusion. DL-butionine sulfoximine has a direct inhibitory effect on the activity of the Pgp transporter protein on the Caco-2 cell line.

Sulforaphane has an additive anticancer effect to FOLFOX in highly metastatic human colon carcinoma cells.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Patients with CRC may need chemotherapy (CTx) in a neoadjuvant, adjuvant or palliative setting through the course of the disease. Unfortunately, its effect is limited by chemoresistance and chemotoxicity. Novel more effective and non‑toxic CTx regimens are needed to further improve CRC treatment outcomes. Thus, the present study was designed to test the hypothesis that non‑toxic sulforaphane (SF) is effective against CRC and has additive effects in combination with conventional 5‑fluorouracil, oxaliplatin and folinic acid (FOLFOX) CTx invitro. Highly metastatic human colon cancer cells, CX‑1, and fibroblasts were treated with FOLFOX ± SF. Cell viability was assessed using an MTT assay. The level of apoptosis and the expression of apoptotic proteins were measured by TUNEL assay and quantitative PCR analysis. Aldehyde dehydrogenase isoform 1 (ALDH1) and multidrug resistance protein 2 (MRP2) levels were evaluated. The ability of cells to form spheroids was measured in three‑dimensional cell culture. SF alone and in combination with FOLFOX effectively decreased the viability of the CX‑1 cells, promoted apoptosis within the CX‑1 cells, prevented cellular spheroid formation and decreased ALDH1 activity. However, SF promoted MRP2 expression and protein levels. In conclusion, SF together with conventional FOLFOX has additive anticancer effects against highly metastatic human CRC invitro.

Vasor: Accurate prediction of variant effects for amino acid substitutions in multidrug resistance protein 3.

The phosphatidylcholine floppase multidrug resistance protein 3 (MDR3) is an essential hepatobiliary transport protein. MDR3 dysfunction is associated with various liver diseases, ranging from severe progressive familial intrahepatic cholestasis to transient forms of intrahepatic cholestasis of pregnancy and familial gallstone disease. Single amino acid substitutions are often found as causative of dysfunction, but identifying the substitution effect in in vitro studies is time and cost intensive. We developed variant assessor of MDR3 (Vasor), a machine learning‐based model to classify novel MDR3 missense variants into the categories benign or pathogenic. Vasor was trained on the largest data set to date that is specific for benign and pathogenic variants of MDR3 and uses general predictors, namely Evolutionary Models of Variant Effects (EVE), EVmutation, PolyPhen‐2, I‐Mutant2.0, MUpro, MAESTRO, and PON‐P2 along with other variant properties, such as half‐sphere exposure and posttranslational modification site, as input. Vasor consistently outperformed the integrated general predictors and the external prediction tool MutPred2, leading to the current best prediction performance for MDR3 single‐site missense variants (on an external test set: F1‐score, 0.90; Matthew's correlation coefficient, 0.80). Furthermore, Vasor predictions cover the entire sequence space of MDR3. Vasor is accessible as a webserver at https://cpclab.uni‐duesseldorf.de/mdr3_predictor/ for users to rapidly obtain prediction results and a visualization of the substitution site within the MDR3 structure. The MDR3‐specific prediction tool Vasor can provide reliable predictions of single‐site amino acid substitutions, giving users a fast way to initially assess whether a variant is benign or pathogenic.

AMP-activated protein kinase α2 contributes to acute and chronic hyperuricemic nephropathy via renal urate deposition in a mouse model

Hyperuricemia can induce acute and chronic kidney damage, but the pathological mechanism remains unclear. The potential role of AMP-activated protein kinase (AMPK) α2 in hyperuricemia-induced renal injury was investigated in this study. Acute and chronic hyperuricemic nephropathy was induced by administering intraperitoneal injections of uric acid and oxonic acid to AMPK α2 knockout and wild-type mice. Changes in renal function, histopathology, inflammatory cell infiltration, renal interstitial fibrosis, and urate deposition were analyzed. In both acute and chronic hyperuricemic nephropathy mouse models, knockout of AMPK α2 significantly reduced serum creatinine levels and renal pathological changes. The tubular expression of kidney injury molecule-1 was also reduced in hyperuricemic nephropathy mice deficient in AMPK α2. In addition, knockout of AMPK α2 significantly suppressed the infiltration of renal macrophages and progression of renal interstitial fibrosis in mice with chronic hyperuricemic nephropathy. Knockout of AMPK α2 reduced renal urate crystal deposition, probably through increasing the expression of the uric acid transporter, multidrug resistance protein 4. In summary, AMPK α2 is involved in acute and chronic hyperuricemia-induced kidney injury and may be associated with increased urate crystal deposition in the kidney.

Co-administration of MDR1 and BCRP or EGFR/PI3K inhibitors overcomes lenvatinib resistance in hepatocellular carcinoma.

Lenvatinib is the first-line treatment for hepatocellular carcinoma (HCC), the most common type of primary liver cancer; however, some patients become refractory to lenvatinib. The underlying mechanism of lenvatinib resistance (LR) in patients with advanced HCC remains unclear. We focused on exploring the potential mechanism of LR and novel treatments of lenvatinib-resistant HCC. In particular, we established a Huh7 LR cell line and performed in vitro, bioinformatic, and biochemical assays. Additionally, we used a Huh7-LR cell-derived xenograft mouse model to confirm the results in vivo. Following LR induction, multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP) transporters were markedly upregulated, and the epidermal growth factor receptor (EGFR), MEK/ERK, and PI3K/AKT pathways were activated. In vitro, the co-administration of elacridar, a dual MDR1 and BCRP inhibitor, with lenvatinib inhibited proliferation and induced apoptosis of LR cells. These effects might be due to inhibiting cancer stem-like cells (CSCs) properties, by decreasing colony formation and downregulating CD133, EpCAM, SOX-9, and c-Myc expression. Moreover, the co-administration of gefitinib, an EGFR inhibitor, with lenvatinib retarded proliferation and induced apoptosis of LR cells. These similar effects might be caused by the inhibition of EGFR-mediated MEK/ERK and PI3K/AKT pathway activation. In vivo, co-administration of lenvatinib with elacridar or gefitinib suppressed tumour growth and angiogenesis. Therefore, inhibiting MDR1 and BCRP transporters or targeting the EGFR/PI3K pathway might overcome LR in HCC. Notably, lenvatinib should be used to treat HCC after LR induction owing to its role in inhibiting tumour proliferation and angiogenesis. Our findings could help develop novel and effective treatment strategies for HCC.