Transporters MRP2 Research Articles

The Histamine Pathway is a Target to Treat Hepatic Experimental Erythropoietic Protoporphyria.

Erythropoietic protoporphyria (EPP) is caused by mutations in ferrochelatase, which inserts iron into protoporphyrin-IX (PP-IX) to generate heme. EPP is characterized by PP-IX accumulation, skin photosensitivity, cholestasis, and end-stage liver disease. Despite available drugs that address photosensitivity, treatment of EPP-related liver disease remains an unmet need. We administered delta-aminolaevulinic acid (ALA) and deferoxamine (DFO), which results in PP-IX overproduction and accumulation. High-throughput compound screening of ALA+ DFO-treated zebrafish identified chlorcyclizine (first generation H1-antihistamine receptor blocker), as a drug that reduces zebrafish liver PP-IX levels. The effect of chlorcyclizine was validated in porphyrin-loaded primary mouse hepatocytes (PMHs), transgenic Fechm1Pas EPP mice, and mice fed the porphyrinogenic compound 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Plasma and tissue PP-IX were measured by fluorescence; livers were analyzed by histology, immunoblotting, and quantitative polymerase chain reaction. Chlorcyclizine-treated zebrafish larvae and DDC-fed and transgenic EPP mice manifested reduced hepatic PP-IX levels compared with controls. Histamine increased PP-IX accumulation in porphyrin-stressed hepatocytes, whereas H1/H2-receptor blockade decreased PP-IX levels. In both mouse models, chlorcyclizine lowered PP-IX levels in female but not male mice in liver, erythrocytes, and bone marrow; improved liver injury; decreased porphyrin-triggered protein aggregation and oxidation; and increased clearance of stool PP-IX. In PMHs, chlorcyclizine induced nuclear translocation of constitutive androstane and farnesoid X receptors, and transactivated bile acid transporter expression. Knockdown of the transporters BSEP and MRP4 led to increased detection of sequestosome-1 (p62 protein) high-molecular-weight species. Chlorcyclizine also reduced hepatic mast cell number and histamine level in EPP mice. Histamine plays an important role in PP-IX accumulation in zebrafish and 2 experimental EPP models. Chlorcyclizine and/or other antihistamines provide a potential therapeutic strategy to treat EPP-associated liver disease via decreasing PP-IX accumulation.

Enhanced platelet sensitization is accompanied by increased expression of the transporter MRP4 and elevated plasma S1P levels in mild COVID-19 convalescents

Viral infections can lead to platelet activation and hemostatic complications. However, the extent to which platelet reactivity remains altered after convalescence, contributing to long-term health impairments as observed after COVID-19 is not yet fully understood. Therefore, we conducted a cohort study (DRKS00025217) to determine platelet function in individuals convalesced from mild COVID-19. Assays were performed ex vivo with blood from convalescents at 2–15 weeks and 6–10 months after convalescence, focusing on platelet aggregation, activation markers, and thrombin formation. In addition, two other potentially relevant factors for platelet function were examined: the immunomodulatory mediator sphingosine-1-phosphate (S1P) and the platelet expression of the transporter MRP4 (ABCC4). Our findings indicate that robust platelet functions, including platelet aggregation determined by light transmission aggregometry, and thrombin formation, were not altered in convalescents compared to matched control individuals. However, an elevation in subtle platelet activation markers, such as P-selectin surface expression and activation of glycoprotein IIb/IIIa, was observed 2–15 weeks after convalescence. This was accompanied by an increased expression of MRP4 in platelets and significantly elevated levels of S1P in platelet-poor plasma. Our findings suggest increased platelet sensitization and a pro-inflammatory state even after convalescence from mild COVID-19, pointing toward MRP4 and S1P as associated factors.

Dietary protein intake and the tubular handling of indoxyl sulfate.

Chronic kidney disease (CKD) patients are advised to limit their protein intake. A high protein diet is known to induce glomerular hyperfiltration, as well as hypertrophy of the remnant kidney, and glomerulosclerosis. Whether the diet causes changes in kidney tubule transport via gut microbiome metabolites is still unknown. We hypothesized that protein intake affects not only the intestinal generation and absorption, but also the kidney disposal of microbial amino acid metabolites. We combined data from animal models and human studies. 5/6th nephrectomy rats were administered a high (HP) or low-protein (LP) diet for 7 weeks. Plasma and urine concentration of the uremic toxins (UTs) indoxyl sulfate (IS), p-cresyl sulfate (PCS), and p-cresyl glucuronide (PCG) were measured. Their fractional excretion (FE) was calculated. The expression of kidney membrane transporters OAT1, OAT3, BCRP, OCT2 and MRP4 was analyzed. Differences in FE of UTs between individuals with higher and lower protein intake in two CKD cohorts were sought. CKD rats on an HP diet showed increased plasma levels of PCS and PCG but not IS compared to rats on a LP diet. Conversely, urinary excretion and FE of IS were higher in the HP CKD group. BCRP, MRP4 and OCT2 were not influenced by the diet. OAT1 and OAT3 were upregulated in the HP CKD group. In two independent cohorts of CKD patients, individuals with a high dietary protein intake showed a significantly higher FE of IS. A HP diet leads to a higher generation and/or absorption of aminoacid-derived UT precursors in CKD rodent models and humans, most likely via gut microbiome modulation. We demonstrate that dietary protein intake modulates transcription and expression of OAT1 and OAT3, corroborating the existence of the remote sensing and signaling hypothesis. Dietary protein intake influences kidney physiology beyond glomerular filtration.

Effect of allyl isothiocyanate on 4-HNE induced glucocorticoid resistance in COPD and the underlying mechanism

Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory condition, and its clinical management primarily targets bronchodilation and anti-inflammatory therapy. However, these treatments often fail to directly address the progression of COPD, particularly its associated glucocorticoid (GC) resistance. This study elucidates the mechanisms underlying GC resistance in COPD and explores the therapeutic potential of allyl isothiocyanate (AITC) in modulating MRP1 transport. We assessed the levels of the oxidative stress product 4-HNE, HDAC2 protein, inflammatory markers, and pulmonary function indices using animal and cell models of GC-resistant COPD. The cascade effects of these factors were investigated through interventions involving AITC, protein inhibitors, and dexamethasone (DEX). Cigarette smoke-induced oxidative stress in COPD leads to the accumulation of the lipid peroxidation product 4-HNE, which impairs HDAC2 protein activity and diminishes GC-mediated anti-inflammatory sensitivity due to disrupted histone deacetylation. AITC regulates MRP1, facilitating the effective efflux of 4-HNE from cells, thereby reducing HDAC2 protein degradation and restoring dexamethasone sensitivity in COPD. These findings elucidate the mechanism of smoking-induced GC resistance in COPD and highlight MRP1 as a potential therapeutic target, as well as the enormous potential of AITC for combined GC therapy in COPD, promoting their clinical applications.

Tauroursodeoxycholate prevents estradiol 17β-d-glucuronide-induced cholestasis and endocytosis of canalicular transporters by switching off pro-cholestatic signaling pathways

AimsEstradiol 17β-d-glucuronide (E217G) induces cholestasis by triggering endocytosis and further intracellular retention of the canalicular transporters Bsep and Mrp2, in a cPKC- and PI3K-dependent manner, respectively. Pregnancy-induced cholestasis has been associated with E217G cholestatic effect, and is routinely treated with ursodeoxycholic acid (UDCA). Since protective mechanisms of UDCA in E217G-induced cholestasis are still unknown, we ascertained here whether its main metabolite, tauroursodeoxycholate (TUDC), can prevent endocytosis of canalicular transporters by counteracting cPKC and PI3K/Akt activation. Main methodsActivation of cPKC and PI3K/Akt was evaluated in isolated rat hepatocytes by immunoblotting (assessment of membrane-bound and phosphorylated forms, respectively). Bsep/Mrp2 function was quantified in isolated rat hepatocyte couplets (IRHCs) by assessing the apical accumulation of their fluorescent substrates, CLF and GS-MF, respectively. We also studied, in isolated, perfused rat livers (IPRLs), the status of Bsep and Mrp2 transport function, assessed by the biliary excretion of TC and DNP-SG, respectively, and Bsep/Mrp2 localization by immunofluorescence. Key findingsE217G activated both cPKC- and PI3K/Akt-dependent signaling, and pretreatment with TUDC significantly attenuated these activations. In IRHCs, TUDC prevented the E217G-induced decrease in apical accumulation of CLF and GS-MF, and inhibitors of protein phosphatases failed to counteract this protection. In IPRLs, E217G induced an acute decrease in bile flow and in the biliary excretion of TC and DNP-SG, and this was prevented by TUDC. Immunofluorescence studies revealed that TUDC prevented E217G-induced Bsep/Mrp2 endocytosis. SignificanceTUDC restores function and localization of Bsep/Mrp2 impaired by E217G, by preventing both cPKC and PI3K/Akt activation in a protein-phosphatase-independent manner.

Implications of chronic moderate protein-deficiency malnutrition on doxorubicin pharmacokinetics and cardiotoxicity in early post-weaning stage

BackgroundMalnutrition is a common problem in developing countries, and the impact of severe malnutrition on optimal treatment outcomes of chemotherapy in pediatric cancer patients is well documented. However, despite being a more prevalent and distinct entity, moderate malnutrition is until now unexplored for its effects on treatment outcomes. AimsIn this study we aimed to investigate the molecular basis of altered pharmacokinetics and cardiotoxicity of doxorubicin observed in early-life chronic moderate protein deficiency malnutrition. Materials and methodsWe developed an animal model of early-life moderate protein-deficiency malnutrition and validated it using clinical samples. This model was used to study pharmacokinetic and toxicity changes and was further utilized to study the molecular changes in liver and heart to get mechanistic insights. Key findingsHere we show that moderate protein-deficiency malnutrition in weanling rats causes changes in drug disposition in the liver by modification of hepatic ABCC3 and MRP2 transporters through the TNFα signalling axis. Furthermore, malnourished rats in repeat-dose doxorubicin toxicity study showed higher toxicity and mortality. A higher accumulation of doxorubicin in the heart was observed which was associated with alterations in cardiac metabolic pathways and increased cardiotoxicity. SignificanceOur findings indicate that moderate malnutrition causes increased susceptibility towards toxic side effects of chemotherapy. These results may necessitate further investigations and new guidelines on the dosing of chemotherapy in moderately malnourished pediatric cancer patients.

Induction effect of antiretroviral bictegravir on the expression of Abcb1, Abcg2 and Abcc1 genes associated with P-gp, BCRP and MRP1 transporters present in rat peripheral blood mononuclear cells

ABSTRACT Background Antiretrovirals have the potential to cause drug interactions leading to inefficacy or toxicity via induction of efflux transporters through nuclear receptors, altering drug concentrations at their target sites. Research Design and Methods This study used molecular dynamic simulations and qRT-PCR to investigate bictegravir’s interactions with nuclear receptors PXR and CAR, and its effects on efflux transporters (P-gp, BCRP, MRP1) in rat PBMCs. PBMC/plasma drug concentrations were measured using LC-MS/MS to assess the functional impact of transporter expression. Results Bictegravir significantly increased the expression of ABC transporters, with Car identified as a key mediator. This suggests that bictegravir’s influence on nuclear receptors could affect drug transport and efficacy at the cellular level. Conclusions Bictegravir activates nuclear receptors enhancing efflux transporter expression. Understanding these interactions is crucial for preventing drug–drug interactions and reducing toxicity in clinical use. Combining CAR antagonists with bictegravir may prevent drug resistance and toxicity. However, these findings are based on preclinical data and necessitate further clinical trials to confirm their applicability in clinical settings.

Structural basis for the modulation of MRP2 activity by phosphorylation and drugs

Multidrug resistance-associated protein 2 (MRP2/ABCC2) is a polyspecific efflux transporter of organic anions expressed in hepatocyte canalicular membranes. MRP2 dysfunction, in Dubin-Johnson syndrome or by off-target inhibition, for example by the uricosuric drug probenecid, elevates circulating bilirubin glucuronide and is a cause of jaundice. Here, we determine the cryo-EM structure of rat Mrp2 (rMrp2) in an autoinhibited state and in complex with probenecid. The autoinhibited state exhibits an unusual conformation for this class of transporter in which the regulatory domain is folded within the transmembrane domain cavity. In vitro phosphorylation, mass spectrometry and transport assays show that phosphorylation of the regulatory domain relieves this autoinhibition and enhances rMrp2 transport activity. The in vitro data is confirmed in human hepatocyte-like cells, in which inhibition of endogenous kinases also reduces human MRP2 transport activity. The drug-bound state reveals two probenecid binding sites that suggest a dynamic interplay with autoinhibition. Mapping of the Dubin-Johnson mutations onto the rodent structure indicates that many may interfere with the transition between conformational states.

Colchicine disrupts bile acid metabolic homeostasis by affecting the enterohepatic circulation in mice.

Although the medicinal properties of colchicine (COL) have been widely known for centuries, its toxicity has been the subject of controversy. The narrow therapeutic window causes COL to induce gastrointestinal adverse effects even when taken at recommended doses, mainly manifested as nausea, vomiting, and diarrhea. However, the mechanism of COL-induced gastrointestinal toxic reactions remains obscure. In the present study, the mice were dosed with COL (2.5mg/kg b.w./day) for a week to explore the effect of COL on bile acid metabolism and the mechanism of COL-induced diarrhea. The results showed that COL treatment affected liver biochemistry in mice, resulting in a significant down-regulation of the mRNA expression levels of bile acid biosynthesis regulators Cyp7a1, Cyp8b1, Cyp7b1, and Cyp27a1 in liver tissues. The mRNA expression levels of bile acid transporters Ntcp, Oatp1, Mrp2, Ibabp, Mrp3, Osta, and Ostb in liver and ileum tissues were also significantly down-regulated. In addition, COL treatment significantly inhibited the mRNA expression levels of Fxr and its downstream target genes Shp, Lrh1, and Fgf15 in liver and ileum tissues, affecting the feedback regulation of bile acid biosynthesis. More importantly, the inhibition of COL on bile acid transporters in ileal and hepatic tissues affected bile acid recycling in the ileum as well as their reuptake in the liver, leading to a significantly increased accumulation of bile acids in the colon, which may be an important cause of diarrhea. In conclusion, our study revealed that COL treatment affected bile acid biosynthesis and enterohepatic circulation, thereby disrupting bile acid metabolic homeostasis in mice.

The Role of CYPs and Transporters in the Biotransformation and Transport of the Anti-hepatitis C Antiviral Agents Asunaprevir, Daclatasvir, and Beclabuvir: Impact of Liver Disease, Race and Drug-drug Interactions on Safety and Efficacy.

Asunaprevir, daclatasvir, and beclabuvir are direct-acting antiviral agents used in the treatment of patients infected with hepatitis C genotype 1b. This article reviews the biotransformation and disposition of these drugs in relation to the safety and efficacy of therapy. CYP3A4 and 3A5 catalyze the oxidative biotransformation of the drugs, while P-glycoprotein mediates their efflux from tissues. Asunaprevir is also a substrate for the influx transporters OATP1B1 and OATP2B1 and the efflux transporter MRP2, while beclabuvir is also a substrate for the efflux transporter BCRP. Liver disease decreases the expression of CYPs and transporters that mediate drug metabolism and disposition. Serum asunaprevir concentrations, but not those of daclatasvir or beclabuvir, are increased in patients with severe liver disease, which may produce toxicity. Pharmacogenomic variation in CYPs and transporters also has the potential to disrupt therapy with asunaprevir, daclatasvir and beclabuvir; some variants are more prevalent in certain racial groups. Pharmacokinetic drug-drug interactions, especially where asunaprevir, daclatasvir, and beclabuvir are victim drugs, are mediated by coadministered rifampicin, ketoconazole and ritonavir, and are attributable to inhibition and/or induction of CYPs and transporters. Conversely, there is also evidence that asunaprevir, daclatasvir and beclabuvir are perpetrators of drug interactions with coadministered rosuvastatin and dextromethorphan. Together, liver disease, pharmacogenomic variation and drug-drug interactions may disrupt therapy with asunaprevir, daclatasvir and beclabuvir due to the impaired function of important CYPs and transporters.

Gene Expression of Abcc2 and Its Regulation by Chicken Xenobiotic Receptor.

Membrane transporter multidrug resistance-associated protein 2 (MRP2/Abcc2) exhibits high pharmaco-toxicological relevance because it exports multiple cytotoxic compounds from cells. However, no detailed information about the gene expression and regulation of MRP2 in chickens is yet available. Here, we sought to investigate the expression distribution of Abcc2 in different tissues of chicken and then determine whether Abcc2 expression is induced by chicken xenobiotic receptor (CXR). The bioinformatics analyses showed that MRP2 transporters have three transmembrane structural domains (MSDs) and two highly conserved nucleotide structural domains (NBDs), and a close evolutionary relationship with turkeys. Tissue distribution analysis indicated that Abcc2 was highly expressed in the liver, kidney, duodenum, and jejunum. When exposed to metyrapone (an agonist of CXR) and ketoconazole (an antagonist of CXR), Abcc2 expression was upregulated and downregulated correspondingly. We further confirmed that Abcc2 gene regulation is dependent on CXR, by overexpressing and interfering with CXR, respectively. We also demonstrated the induction of Abcc2 expression and the activity of ivermectin, with CXR being a likely mediator. Animal experiments demonstrated that metyrapone and ivermectin induced Abcc2 in the liver, kidney, and duodenum of chickens. Together, our study identified the gene expression of Abcc2 and its regulation by CXR in chickens, which may provide novel targets for the reasonable usage of veterinary drugs.

Distinct bile acid alterations in response to a single administration of PFOA and PFDA in mice

Per- and polyfluoroalkyl substances (PFASs), a group of synthetic chemicals that were once widely used for industrial purposes and in consumer products, are widely found in the environment and in human blood due to their extraordinary resistance to degradation. Once inside the body, PFASs can activate nuclear receptors such as PPARα and CAR. The present study aimed to investigate the impact of perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA) on liver structure and functions, as well as bile acid homeostasis in mice. A single administration of 0.1 mmole/kg of PFDA, not PFOA, elevated serum ALT and bilirubin levels and caused cholestasis in WT mice. PFDA increased total and various bile acid species in serum but decreased them in the liver. Furthermore, in mouse livers, PFDA, not PFOA, down-regulated mRNA expression of uptake transporters (Ntcp, Oatp1a1, 1a4, 1b2, and 2b1) but induced efflux transporters (Bcrp, Mdr2, and Mrp2–4). In addition, PFDA, not PFOA, decreased Cyp7a1, 7b1, 8b1, and 27a1 mRNA expression in mouse livers with concomitant hepatic accumulation of cholesterol. In contrast, in PPARα-null mice, PFDA did not increase serum ALT, bilirubin, or total bile acids, but produced prominent hepatosteatosis; and the observed PFDA-induced expression changes of transporters and Cyps in WT mice were largely attenuated or abolished. In CAR-null mice, the observed PFDA-induced bile acid alterations in WT mice were mostly sustained. These results indicate that, at the dose employed, PFDA has more negative effects than PFOA on liver function. PPARα appears to play a major role in mediating most of PFDA-induced effects, which were absent or attenuated in PPARα-null mice. Lack of PPARα, however, exacerbated hepatic steatosis. Our findings indicate separated roles of PPARα in mediating the adaptive responses to PFDA: protective against hepatosteatosis but exacerbating cholestasis.

Upregulation of hepatic nuclear receptors in extremely preterm ovine fetuses undergoing artificial placenta therapy

Objective Extremely preterm infants have low Nuclear Receptor (NR) expression in their developing hepatobiliary systems, as they rely on the placenta and maternal liver for compensation. NRs play a crucial role in detoxification and the elimination of both endogenous and xenobiotic substances by regulating key genes encoding specific proteins. In this study, we utilized an Artificial Placenta Therapy (APT) platform to examine the liver tissue expression of NRs of extremely preterm ovine fetuses. This fetal model, resembling a “knockout placenta,” lacks placental and maternal support, while maintaining a healthy extrauterine survival. Methods Six ovine fetuses at 95 ± 1 d gestational age (GA; term = ∼150 d)/∼600 g delivery weight were maintained on an APT platform for a period of 120 h (APT Group). Six age-matched, in utero control fetuses were delivered at 99–100 d GA (Control Group). Fetal liver tissue samples and blood samples were collected at delivery from both groups and assessed mRNA expression of NRs and target transporters involved in the hepatobiliary transport system using quantitative PCR. Data were tested for group differences with ANOVA (p < .05 deemed significant). Results mRNA expression of NRs was identified in both the placenta and the extremely preterm ovine fetal liver. The expression of HNF4α, LRH1, LXR, ESR1, PXR, CAR, and PPARα/γ were significantly elevated in the liver of the APT Group compared to the Control Group. Moreover, target transporters NTCP, OATP1B3, BSEP, and MRP4 were upregulated, whereas MRP2 and MRP3 were unchanged. Although there was no evidence of liver necrosis or apoptotic changes histologically, there was an impact in the fetal liver of the ATP group at the tissue level with a significant increase in TNFα mRNA, a cytokine involved in liver inflammation, and blood elevation of transaminases. Conclusion A number of NRs in the fetal liver were significantly upregulated after loss of placental-maternal support. However, the expression of target transporter genes appeared to be insufficient to compensate role of the placenta and maternal liver and avoid fetal liver damage, potentially due to insufficient excretion of organic anions.

Comparison of in vitro thyroxine (T4) metabolism between Wistar rat and human hepatocyte cultures

In vitro assays remain relatively new in exploring human relevance of liver, in particular nuclear receptor-mediated perturbations of the hypothalamus-pituitary-thyroid axis seen in rodents, mainly in the rat. Consistent with in vivo data, we confirm that thyroid hormone thyroxine metabolism was 9 times higher in primary rat hepatocytes (PRH) than in primary human hepatocytes (PHH) cultured in a 2D sandwich (2Dsw) configuration. In addition, thyroxine glucuronide (T4-G) was by far the major metabolite formed in both species (99.1% in PRH and 69.7% in PHH) followed by thyroxine sulfate (T4-S, 0.7% in PRH and 18.1% in PHH) and triiodothyronine/reverse triiodothyronine (T3/rT3, 0.2% in PRH and 12.2% in PHH). After a 7-day daily exposure to orphan receptor-mediated liver inducers, T4 metabolism was strongly increased in PRH, almost exclusively through increased T4-G formation. These results were consistent with the inductions of glucuronosyltransferase Ugt2b1 and canalicular transporter Mrp2. PHH also responded to activation of the three nuclear receptors, with mainly induction of glucuronosyltransferase UGT1A1 and canalicular transporter MRP2. Despite this, T4 disappearance rate and secreted T4 metabolites were only slightly increased in PHH. Overall, our data highlight that cryopreserved hepatocytes in 2Dsw culture allowing long-term exposure and species comparison are of major interest in improving liver-mediated human safety assessment.

Transplacental transport of per- and polyfluoroalkyl substances (PFAS): Mechanism exploration via BeWo cell monolayer model

Per- and polyfluoroalkyl substances (PFAS) have received global concern on adverse effects on pregnancy outcomes. Although human studies have reported fetal exposure to PFAS, the underlying mechanisms driving transplacental transfer of PFAS have not been sufficiently understood. The present study aimed to investigate chemical-specific transplacental transfer of PFAS and potential mechanisms based on a BeWo cell monolayer model. The findings of concentration- and time-dependent transport, asymmetry in bidirectional transport, molecular docking and transporter inhibition experiments indicate that passive diffusion and membrane transporter-involved active transport could collectively determine transplacental transport of PFAS. Membrane transporters could play important roles in chemical-specific transport. The inhibition of OAT transporter resulted in promotion of trans-monolayer transport for most PFAS, while an opposite trend was observed when P-gp, BCRP and MRP transporters were prohibited. By contrast, inhibition of OCT resulted in inhibitory effects on the transport of some PFAS (i.e., PFHxA, PFHpA, PFOA, and PFNA), and promotive effects on the other substances (i.e., PFUdA, PFHpS, PFOS, 6:2 Cl-PFESA and PFOSA). Therefore, simultaneous involvement of diverse membrane transporters in utero could result in complicated influence on transplacental transport. Our work constitutes a solid ground for further exploration of the effects of gestational PFAS exposure on birth outcomes.

In Doxorubicin-Adapted Hodgkin Lymphoma Cells, Acquiring Multidrug Resistance and Improved Immunosuppressive Abilities, Doxorubicin Activity Was Enhanced by Chloroquine and GW4869.

Classical Hodgkin lymphoma (cHL) is a highly curable disease (70-80%), even though long-term toxicities, drug resistance, and predicting clinical responses to therapy are major challenges in cHL treatment. To solve these problems, we characterized two cHL cell lines with acquired resistance to doxorubicin, KM-H2dx and HDLM-2dx (HRSdx), generated from KM-H2 and HDLM-2 cells, respectively. HRSdx cells developed cross-resistance to vinblastine, bendamustin, cisplatin, dacarbazine, gemcitabine, brentuximab vedotin (BV), and γ-radiation. Both HDLM-2 and HDLM-2dx cells had intrinsic resistance to BV but not to the drug MMAE. HDLM-2dx acquired cross-resistance to caelyx. HRSdx cells had in common decreased CD71, CD80, CD54, cyt-ROS, HLA-DR, DDR1, and CD44; increased Bcl-2, CD58, COX2, CD26, CCR5, and invasive capability; increased CCL5, TARC, PGE2, and TGF-β; and the capability of hijacking monocytes. In HRSdx cells less sensitive to DNA damage and oxidative stress, the efflux drug transporters MDR1 and MRP1 were not up-regulated, and doxorubicin accumulated in the cytoplasm rather than in the nucleus. Both the autophagy inhibitor chloroquine and extracellular vesicle (EV) release inhibitor GW4869 enhanced doxorubicin activity and counteracted doxorubicin resistance. In conclusion, this study identifies common modulated antigens in HRSdx cells, the associated cross-resistance patterns, and new potential therapeutic options to enhance doxorubicin activity and overcome resistance.

Cytotoxicity and reversal effect of sertraline, fluoxetine, and citalopram on MRP1- and MRP7-mediated MDR.

Cancer is one of the leading causes of death worldwide, and the development of resistance to chemotherapy drugs is a major challenge in treating malignancies. In recent years, researchers have focused on understanding the mechanisms of multidrug resistance (MDR) in cancer cells and have identified the overexpression of ATP-binding cassette (ABC) transporters, including ABCC1/MRP1 and ABCC10/MRP7, as a key factor in the development of MDR. In this study, we aimed to investigate whether three drugs (sertraline, fluoxetine, and citalopram) from the selective serotonin reuptake inhibitor (SSRI) family, commonly used as antidepressants, could be repurposed as inhibitors of MRP1 and MRP7 transporters and reverse MDR in cancer cells. Using a combination of in silico predictions and in vitro validations, we analyzed the interaction of MRP1 and MRP7 with the drugs and evaluated their ability to hinder cell resistance. We used computational tools to identify and analyze the binding site of these three molecules and determine their binding energy. Subsequently, we conducted experimental assays to assess cell viability when treated with various standard chemotherapies, both with and without the presence of SSRI inhibitors. Our results show that all three SSRI drugs exhibited inhibitory/reversal effects in the presence of chemotherapies on both MRP1-overexpressed cells and MRP7-overexpressed cells, suggesting that these medications have the potential to be repurposed to target MDR in cancer cells. These findings may open the door to using FDA-approved medications in combination therapy protocols to treat highly resistant malignancies and improve the efficacy of chemotherapy treatment. Our research highlights the importance of investigating and repurposing existing drugs to overcome MDR in cancer treatment.

Characterization of a human placental clearance system to regulate serotonin levels in the fetoplacental unit

BackgroundSerotonin (5-HT) is a biogenic monoamine with diverse functions in multiple human organs and tissues. During pregnancy, tightly regulated levels of 5-HT in the fetoplacental unit are critical for proper placental functions, fetal development, and programming. Despite being a non-neuronal organ, the placenta expresses a suite of homeostatic proteins, membrane transporters and metabolizing enzymes, to regulate monoamine levels. We hypothesized that placental 5-HT clearance is important for maintaining 5-HT levels in the fetoplacental unit. We therefore investigated placental 5-HT uptake from the umbilical circulation at physiological and supraphysiological levels as well as placental metabolism of 5-HT to 5-hydroxyindoleacetic acid (5-HIAA) and 5-HIAA efflux from trophoblast cells.MethodsWe employed a systematic approach using advanced organ-, tissue-, and cellular-level models of the human placenta to investigate the transport and metabolism of 5-HT in the fetoplacental unit. Human placentas from uncomplicated term pregnancies were used for perfusion studies, culturing explants, and isolating primary trophoblast cells.ResultsUsing the dually perfused placenta, we observed a high and concentration-dependent placental extraction of 5-HT from the fetal circulation. Subsequently, within the placenta, 5-HT was metabolized to 5-hydroxyindoleacetic acid (5-HIAA), which was then unidirectionally excreted to the maternal circulation. In the explant cultures and primary trophoblast cells, we show concentration- and inhibitor-dependent 5-HT uptake and metabolism and subsequent 5-HIAA release into the media. Droplet digital PCR revealed that the dominant gene in all models was MAO-A, supporting the crucial role of 5-HT metabolism in placental 5-HT clearance.ConclusionsTaken together, we present transcriptional and functional evidence that the human placenta has an efficient 5-HT clearance system involving (1) removal of 5-HT from the fetal circulation by OCT3, (2) metabolism to 5-HIAA by MAO-A, and (3) selective 5-HIAA excretion to the maternal circulation via the MRP2 transporter. This synchronized mechanism is critical for regulating 5-HT in the fetoplacental unit; however, it can be compromised by external insults such as antidepressant drugs.

In vitro metabolism of triclosan and chemoprevention against its cytotoxicity

Triclosan (TCS), a broad-spectrum antibacterial chemical, has been extensively used in personal daily care items, household commodities, and clinical medications; therefore, humans are at risk of being exposed to TCS in their daily lives. This chemical also accumulated in food chains, and potential risks were associated with its metabolism in vivo. The aim of this study was to investigate the difference in metabolic profile of TCS by hepatic P450 enzymes and extrahepatic P450s, and also identify chemical structures of its metabolites. The results showed that RLM mediated the hydroxylation and cleavage of the ether moiety of TCS, resulting in phenolic metabolites that are more polar than the parent compound, including 4-chlorocatechol, 2,4-dichlorophenol and monohydroxylated triclosan. The major metabolite of CYP1A1 and CYP1B1 mediated TCS metabolism is 4-chlorochol. We also performed molecular docking experiments to investigate possible binding modes of TCS in the active sites of human CYP1B1, CYP1A1, and CYP3A4. In addition to in vitro experiments, we further examined the cytotoxic effects of TCS on HepG2 cells expressing hepatic P450 and MCF-7/1B1 cells expressing CYP1B1. It exhibited significant cytotoxicity on HepG2, MCF-10A and MCF-7/1B1 cells, with IC50 values of 70 ± 10 μM, 20 ± 10 μM and 60 ± 20 μM, respectively. The co-incubation of TCS with glutathione (GSH) as a chemopreventive agent could reduce the cytotoxicity of TCS in vitro. The chemopreventive effects of GSH might be ascribed to the promotion of TCS efflux mediated by membrane transporter MRP1 and also its antioxidant property, which partially neutralized the oxidative stress of TCS on mammalian cells. This study contributed to our understanding of the relationship between the P450 metabolism and the toxicity of TCS. It also had implications for the use of specific chemopreventive agents against the toxicity of TCS.

Impact of the cAMP efflux and extracellular cAMP-adenosine pathway on airway smooth muscle relaxation induced by formoterol and phosphodiesterase inhibitors

β2-adrenoceptors agonists and phosphodiesterase (PDE) inhibitors are effective bronchodilators, due to their ability to increase intracellular cyclic AMP (cAMP) levels and induce airway smooth muscle (ASM) relaxation. We have shown that increment of intracellular cAMP induced by β2-adrenoceptors agonist fenoterol is followed by efflux of cAMP, which is converted by ecto-PDE and ecto-5′-nucleotidases (ecto-5′NT) to adenosine, leading to ASM contraction. Here we evaluate whether other classical bronchodilators used to treat asthma and chronic obstructive pulmonary disease (COPD) could induce cAMP efflux and, as consequence, influence the ASM contractility. Our results showed that β2-adrenoceptor agonists formoterol and PDE inhibitors IBMX, aminophylline and roflumilast induced cAMP efflux and a concentration-dependent relaxation of rat trachea precontracted with carbachol. Pretreatment of tracheas with MK-571 (MRP transporter inhibitor), AMP-CP (ecto-5′NT inhibitor) or CGS-15943 (nonselective adenosine receptor antagonist) potentiated the relaxation induced by β2-adrenoceptor agonists but did not change the relaxation induced by PDE inhibitors. These data showed that all bronchodilators tested were able to induce cAMP efflux. However, only β2-adrenoceptor-induced relaxation of tracheal smooth muscle was affected by cAMP efflux and extracellular cAMP-adenosine pathway.