Acacia senegal Budmunchiamines as a Potential Adjuvant for Rejuvenating Phenicol Activities towards Escherichia coli-Resistant Strains.

The continuous emergence of bacterial resistance alters the activities of different antibiotic families and requires appropriate strategies to solve therapeutic impasses. Medicinal plants are an attractive source for researching alternative and original therapeutic molecules. In this study, the fractionation of natural extracts from A. senegal and the determination of antibacterial activities are associated with molecular networking and tandem mass spectrometry (MS/MS) data used to characterize active molecule(s). The activities of the combinations, which included various fractions plus an antibiotic, were investigated using the "chessboard" test. Bio-guided fractionation allowed the authors to obtain individually active or synergistic fractions with chloramphenicol activity. An LC-MS/MS analysis of the fraction of interest and molecular array reorganization showed that most identified compounds are Budmunchiamines (macrocyclic alkaloids). This study describes an interesting source of bioactive secondary metabolites structurally related to Budmunchiamines that are able to rejuvenate a significant chloramphenicol activity in strains that produce an AcrB efflux pump. They will pave the way for researching new active molecules for restoring the activity of antibiotics that are substrates of efflux pumps in enterobacterial-resistant strains.

Multidrug resistance-associated protein 2 (MRP2) is an efflux transporter of EGCG and its metabolites in the human small intestine.

Green tea polyphenols have various beneficial effects on human health, such as antiobesity and anti-carcinogenesis. (-)-Epigallocatechin-gallate (EGCG) is one of the major potent green tea catechins; however, detailed mechanisms of EGCG transport and metabolism in the human small intestine remain unknown due to lack of a suitable model. We investigated metabolite profiles of EGCG in the fresh human duodenal biopsy, cryopreserved human duodenal mucosal enterocytes and Caco-2 cells, and found that EGCG was readily metabolized into methylated and sulphate conjugates, which are major metabolites in these models. Next, we examined possible efflux transporters of EGCG and its metabolites using specific inhibitors of MRP2, P-gp and BCRP in Caco-2 cell monolayers. MRP2 was thereby identified as an efflux transporter, and further analysis using MRP2-knockout Caco-2 cells and vesicular transport assays confirmed that MRP2 is a selective efflux transporter of EGCG and its metabolites. Assuming that functional inhibition of MRP2 would result in efficient uptake of EGCG, we screened for MRP2 functional blockade and identified quercetin, which led to increased intracellular accumulation and basal transport of EGCG in Caco-2 cells. This result suggested that co-administration of quercetin and EGCG would enable efficient transport of EGCG in the human intestine. Therefore, we performed co-oral administration of quercetin and EGCG in human subjects to examine whether this occurred in humans. These studies demonstrated that MRP2 is a selective transporter of EGCG and conjugates and Caco-2 is a model to examine transport mechanisms and metabolites of polyphenols in the human small intestine.

MRP5 and MRP9 play a concerted role in male reproduction and mitochondrial function.

Multidrug Resistance Proteins (MRPs) are transporters that play critical roles in cancer even though the physiological substrates of these enigmatic transporters are poorly elucidated. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter because mrp-5 mutants are unviable due to their inability to export heme from the intestine to extraintestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. Correspondingly, cell biological studies show that MRP5 regulates heme levels in the mammalian secretory pathway even though MRP5 knockout (KO) mice do not show reproductive phenotypes. The closest homolog of MRP5 is MRP9/ABCC12, which is absent in C. elegans, raising the possibility that MRP9 may genetically compensate for MRP5. Here, we show that MRP5 and MRP9 double KO (DKO) mice are viable but reveal significant male reproductive deficits. Although MRP9 is highly expressed in sperm, MRP9 KO mice show reproductive phenotypes only when MRP5 is absent. Both ABCC transporters localize to mitochondrial-associated membranes, dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, DKO mice reveal abnormal sperm mitochondria with reduced mitochondrial membrane potential and fertilization rates. Metabolomics show striking differences in metabolite profiles in the DKO testes, and RNA sequencing shows significant alterations in genes related to mitochondrial function and retinoic acid metabolism. Targeted functional metabolomics reveal lower retinoic acid levels in the DKO testes and higher levels of triglycerides in the mitochondria. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating male reproductive functions and mitochondrial sufficiency.

Upregulation of USP22 and ABCC1 during Sorafenib Treatment of Hepatocellular Carcinoma Contribute to Development of Resistance.

Sorafenib is a small molecule that blocks tumor proliferation by targeting the activity of multi-kinases for the treatment of advanced hepatocellular carcinoma (HCC). Increasing sorafenib resistance following long-term treatment is frequently encountered. Mechanisms underlying sorafenib resistance remain not completely clear. To further understand the mechanism of sorafenib resistance in HCC, we established sorafenib-resistant cell lines by slowly increasing sorafenib concentration in cell culture medium. Upregulation of USP22 and ABCC1 were found in Sorafenib-resistant cells. Sorafenib-resistant cells treated with USP22 siRNA showed significant reduction in endogenous mRNA and protein levels of ABCC1. During sorafenib treatment, upregulation of USP22 increases ABCC1 expression and subsequently contributes to sorafenib resistance in HCC cells. Immunohistochemical analysis revealed a positive correlation between USP22 and ABCC1 expression in tissue samples from sorafenib-resistant patients (Pearson's correlation = 0.59, p = 0.03). Our findings indicate that upregulation of USP22 and ABCC1 expression during treatment contribute to sorafenib resistance in HCC cells and that USP22 has strong potential as a therapeutic target for overcoming sorafenib resistance in HCC patients.

Vitamin D and Calcium Supplementation Accelerate Vascular Calcification in a Model of Pseudoxanthoma Elasticum.

Arterial calcification is a common feature of pseudoxanthoma elasticum (PXE), a disease characterized by ABCC6 mutations, inducing a deficiency in pyrophosphate, a key inhibitor of calcium phosphate crystallization in arteries. METHODS: we analyzed whether long-term exposure of Abcc6-/- mice (a murine model of PXE) to a mild vitamin D supplementation, with or without calcium, would impact the development of vascular calcification. Eight groups of mice (including Abcc6-/- and wild-type) received vitamin D supplementation every 2 weeks, a calcium-enriched diet alone (calcium in drinking water), both vitamin D supplementation and calcium-enriched diet, or a standard diet (controls) for 6 months. Aorta and kidney artery calcification was assessed by 3D-micro-computed tomography, Optical PhotoThermal IR (OPTIR) spectroscopy, scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDS) and Yasue staining. RESULTS: at 6 months, although vitamin D and/or calcium did not significantly increase serum calcium levels, vitamin D and calcium supplementation significantly worsened aorta and renal artery calcification in Abcc6-/- mice. CONCLUSIONS: vitamin D and/or calcium supplementation accelerate vascular calcification in a murine model of PXE. These results sound a warning regarding the use of these supplementations in PXE patients and, to a larger extent, patients with low systemic pyrophosphate levels.

Biliary excretion of arsenic by human HepaRG cells is stimulated by selenide and mediated by the multidrug resistance protein 2 (MRP2/ABCC2).

Millions of people worldwide are exposed to unacceptable levels of arsenic, a proven human carcinogen, in drinking water. In animal models, arsenic and selenium are mutually protective through formation and biliary excretion of seleno-bis (S-glutathionyl) arsinium ion [(GS)2AsSe]-. Selenium-deficient humans living in arsenic-endemic regions are at increased risk of arsenic-induced diseases, and may benefit from selenium supplementation. The influence of selenium on human arsenic hepatobiliary transport has not been studied using optimal human models. HepaRG cells, a surrogate for primary human hepatocytes, were used to investigate selenium (selenite, selenide, selenomethionine, and methylselenocysteine) effects on arsenic hepatobiliary transport. Arsenite + selenite and arsenite + selenide at different molar ratios revealed mutual toxicity antagonism, with the latter being higher. Significant levels of arsenic biliary excretion were detected with a biliary excretion index (BEI) of 14 ± 8%, which was stimulated to 32 ± 7% by selenide. Consistent with the formation and biliary efflux of [(GS)2AsSe]-, arsenite increased the BEI of selenide from 0% to 24 ± 5%. Arsenic biliary excretion was lost in the presence of selenite, selenomethionine, and methylselenocysteine. Sinusoidal export of arsenic was stimulated ∼1.6-fold by methylselenocysteine, but unchanged by other selenium forms. Arsenic canalicular and sinusoidal transport (±selenide) was temperature- and GSH-dependent and inhibited by MK571. Knockdown experiments revealed that multidrug resistance protein 2 (MRP2/ABCC2) accounted for all detectable biliary efflux of arsenic (±selenide). Overall, the chemical form of selenium and human MRP2 strongly influenced arsenic hepatobiliary transport, information critical for human selenium supplementation in arsenic-endemic regions.

Antioxidant effect of ascorbic acid against cisplatin-induced nephrotoxicity and P-glycoprotein expression in rats.

Cisplatin (CDDP) is a highly potent anticancer drug that is widely used in the treatment of several cancers. CDDP-induced nephrotoxicity (CIN) is one of the most significant adverse effects, and oxidative stress is thought to be one of the mechanisms underlying CIN. Although there are some studies available on the variability in transporter expression in the kidney after a single CDDP dose, none have reported the change in renal transporter expression after multiple CDDP dose administrations. P-glycoprotein (P-gp), a transporter, is reported to be induced by oxidative stress. Ascorbic acid is a vitamin with antioxidant potential and therefore, may regulate the expression of P-gp transporter and affect CIN. In the present study, our aim was to assess the variability in expression of several renal transporters after multiple CDDP dose administrations and the antioxidant effect of ascorbic acid against transporter expression and CIN. Multiple doses of CDDP affected markers of kidney injury and antioxidants in the kidneys. Also, the expression of P-gp, breast cancer resistance protein, and multidrug resistance-associated protein 4 was upregulated by CDDP. Using a normal kidney cell line, we demonstrated that ascorbic acid attenuated CDDP-induced cytotoxicity due to its high superoxide scavenging ability. CDDP and ascorbic acid were injected into rats once a week for three weeks, and it was observed that co-administration of ascorbic acid attenuated CIN and regulated antioxidant marker. In addition, ascorbic acid reduced P-gp expression, which was upregulated by CDDP. In conclusion, ascorbic acid may attenuate CIN and reverse P-gp-mediated changes in drug pharmacokinetics.

Yinzhihuang Oral Liquid Ameliorates Hyperbilirubinemia Induced by δ-Aminolevulinic Acid and Novobiocin in Neonatal Rats.

Yinzhihuang oral liquid (YZH) is a traditional Chinese medicine that has been widely used in Asia to prevent and treat neonatal hyperbilirubinemia, but the published preclinical studies on its anti-hyperbilirubinemia effect are conducted in adult animals, partly due to the lack of preclinical neonatal hyperbilirubinemia animal models. In the present study, we tested six reagents to induce hyperbilirubinemia in neonatal rats, and established two appropriate neonatal hyperbilirubinemia rat models by subcutaneous injection of δ-Aminolevulinic acid (ALA, 200 mg/kg) or novobiocin (NOVO, 200 mg/kg). Oral treatment of YZH (80, 160 and 320 mg/kg) significantly decreased serum conjugated bilirubin levels in ALA-treated neonatal rats and serum unconjugated bilirubin levels in NOVO-treated neonatal rats, respectively. Additionally, pre-treatment of YZH also prevented the increase of serum bilirubin levels in both ALA- and NOVO-treated rats. Mechanistically, YZH significantly up-regulated the mRNA expression of genes involved in hepatic bilirubin disposition (organic anion-transporting polypeptide 1b2, Oatp1b2; multidrug resistance-associated protein 2, Mrp2) and bilirubin conjugation (UDP-glucuronosyltransferase 1a1, Ugt1a1). Additionally, YZH up-regulated the mRNA expression of cytochrome P450 1A1 (Cyp1a1), the target gene of aryl hydrocarbon receptor (AhR), and increased the nuclear protein levels of AhR in livers of neonatal rats. YZH and its two active ingredients, namely baicalin (BCL) and 4'-hydroxyacetophenone (4-HT), up-regulated the mRNA expression of AhR target genes (CYP1A1 and UGT1A1) and increased nuclear protein levels of AhR in HepG2 cells. In conclusion, the present study provides two neonatal hyperbilirubinemia animal models and evaluates the anti-hyperbilirubinemia effect and mechanisms of YZH in neonatal animals.

ABCC6, Pyrophosphate and Ectopic Calcification: Therapeutic Solutions.

Pathological (ectopic) mineralization of soft tissues occurs during aging, in several common conditions such as diabetes, hypercholesterolemia, and renal failure and in certain genetic disorders. Pseudoxanthoma elasticum (PXE), a multi-organ disease affecting dermal, ocular, and cardiovascular tissues, is a model for ectopic mineralization disorders. ABCC6 dysfunction is the primary cause of PXE, but also some cases of generalized arterial calcification of infancy (GACI). ABCC6 deficiency in mice underlies an inducible dystrophic cardiac calcification phenotype (DCC). These calcification diseases are part of a spectrum of mineralization disorders that also includes Calcification of Joints and Arteries (CALJA). Since the identification of ABCC6 as the "PXE gene" and the development of several animal models (mice, rat, and zebrafish), there has been significant progress in our understanding of the molecular genetics, the clinical phenotypes, and pathogenesis of these diseases, which share similarities with more common conditions with abnormal calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into inorganic pyrophosphate (PPi) and adenosine by the ectonucleotidases NPP1 and CD73 (NT5E). PPi is a potent endogenous inhibitor of calcification, whereas adenosine indirectly contributes to calcification inhibition by suppressing the synthesis of tissue non-specific alkaline phosphatase (TNAP). At present, therapies only exist to alleviate symptoms for both PXE and GACI; however, extensive studies have resulted in several novel approaches to treating PXE and GACI. This review seeks to summarize the role of ABCC6 in ectopic calcification in PXE and other calcification disorders, and discuss therapeutic strategies targeting various proteins in the pathway (ABCC6, NPP1, and TNAP) and direct inhibition of calcification via supplementation by various compounds.

Gut-restricted apical sodium-dependent bile acid transporter inhibitor attenuates alcohol-induced liver steatosis and injury in mice.

BACKGROUND: Recent studies have shown that human and experimental alcohol-related liver disease (ALD) is robustly associated with dysregulation of bile acid homeostasis, which may in turn modulate disease severity. Pharmacological agents targeting bile acid metabolism and signaling may be potential therapeutics for ALD. METHODS: The potential beneficial effects of a gut-restricted apical sodium-dependent bile acid transporter (ASBT) inhibitor were studied in a chronic-plus-binge ALD mouse model. RESULTS: Blocking intestinal bile acid reabsorption by the gut-restricted ASBT inhibitor GSK2330672 attenuated hepatic steatosis and liver injury in a chronic-plus-binge ALD mouse model. Alcohol feeding is associated with intestinal bile acid accumulation but paradoxically impaired ileal farnesoid × receptor (FXR) function, and repressed hepatic cholesterol 7α-hydrolase (CYP7A1) expression despite decreased hepatic small heterodimer partner (SHP) and ileal fibroblast growth factor 15 (FGF15) expression. ASBT inhibitor treatment decreased intestinal bile acid accumulation and increased hepatic CYP7A1 expression, but further decreased ileal FXR activity. Alcohol feeding induces serum bile acid concentration that strongly correlates with a liver injury marker. However, alcohol-induced serum bile acid elevation is not due to intrahepatic bile acid accumulation but is strongly and positively associated with hepatic multidrug resistance-associated protein 3 (MRP4) and MRP4 induction but poorly associated with sodium-taurocholate cotransporting peptide (NTCP) expression. ASBT inhibitor treatment decreases serum bile acid concentration without affecting hepatocyte basolateral bile acid uptake and efflux transporters. CONCLUSION: ASBT inhibitor treatment corrects alcohol-induced bile acid dysregulation and attenuates liver injury in experimental ALD.

Elucidation of underlying molecular mechanism of 5-Fluorouracil chemoresistance and its restoration using fish oil in experimental colon carcinoma.

Latest strategies for cancer treatment primarily focus on the use of chemosensitizers to enhance therapeutic outcome. N-3 PUFAs have emerged as the strongest candidate for the prevention of colorectal cancer (CRC). Our previous studies have demonstrated that fish oil (FO) rich in n-3 PUFAs not only increased therapeutic potential of 5-Fluorouracil(5-FU) in colon cancer but also ameliorated its toxicity. Henceforth, the present study is designed to elucidate mechanistic insights of FO as a chemosensitizer to circumvent drug resistance in experimental colon carcinoma. The colon cancer was induced by 1,2-dimethylhydrazine(DMH)/dextran sulfate sodium(DSS) in male Balb/c mice and these animals were treated with 5-FU(12.5 mg/kg b.w.), FO(0.2 ml), or 5-FU + FO(12.5 mg/kg b.w + 0.2 ml) orally for 14 days. The molecular mechanism of overcoming 5-FU resistance using FO in colon cancer was delineated by estimating expression of cancer stem cell markers using flowcytometric method and drug transporters by immunohistochemistry and immunoblotting. Additionally, distribution profile of 5-FU and its cytotoxic metabolite, 5-FdUMP at target(colon), and non-target sites (serum, kidney, liver, spleen) was assessed using high-performance liquid chromatography(HPLC) method. The observations revealed that expression of CSCs markers was remarkably reduced after using fish oil along with 5-FU in carcinogen-treated animals. Interestingly, the use of FO alongwith 5-FU also significantly declined the expression of drug transporters (ABCB1,ABCC5) and consequently resulted in an increased cellular uptake of 5-FU and its metabolite, 5-FdUMP at target site (colon). It could be possibly associated with change in permeability of cell membrane owing to the alteration in membrane fluidity. The present study revealed the mechanistic insights of FO as a MDR revertant which successfully restored 5-FU-mediated chemoresistance in experimental colon carcinoma.

Dihydromyricetin reverses MRP2-induced multidrug resistance by preventing NF-κB-Nrf2 signaling in colorectal cancer cell.

BACKGROUD: Dihydromyricetin (DMY), a natural flavonoid compound from the leaves of the Chinese medicinal herb Vitis heyneana, has been shown to have the potential to combat chemoresistance by inhibiting Nrf2/MRP2 signaling in colorectal cancer (CRC) cells. However, the precise underlying molecular mechanism and its therapeutic target are not well understood. PURPOSE: Our study aims to investigate the effects of DMY on multidrug resistance (MDR), and elucidate the underlying mechanisms. STUDY DESIGN: In vitro, HCT116/OXA and HCT8/VCR cells were employed as our MDR models. The cells were treated with DMY (50 µM) or MK-571 (50 µM) plus oxaliplatin (OXA) (10 µM) or vincristine (VCR) (10 µM) for 48 h. In vivo, we used BALB/c mice as a CRC xenograft mouse model. BALB/c mice were given DMY (100 mg/kg), OXA (5 mg/kg) and DMY (100 mg/kg) combined with OXA (5 mg/kg) via intraperitoneal route every 2 days per week for 4 weeks. METHODS: We used MTT and colony forming assays to detect DMY's ability to reverse MDR. Flow cytometric analysis was used to detect apoptosis. Immunocytochemistry was used to detect the localization of Nrf2 and NF-κB/p65. Western blot, qRT-PCR and reporter gene assays were employed to measure the protein and gene transcriptional levels (MRP2, Nrf2, NF-κB/p65). Moreover, chromatin immunoprecipitation (ChIP) assay was used to investigate the endogenous promoter occupancy of NF-κB/p65. Finally, immunohistochemistry and TUNEL staining were used to detect protein expression and apoptosis in vivo. RESULTS: DMY restored chemosensitivity (OXA and VCR) by inhibiting both MRP2 expression and its promoter activity in HCT116/OXA and HCT8/VCR cell lines. Furthermore, DMY could inhibit NF-κB/p65 expression, reducing NF-κB/p65 translocation to the nucleus to silence Nrf2 signaling, which is necessary for MRP2 expression. Overexpressing NF-κB/p65 expression reduced the reversal effect of DMY. In addition, NF-κB/p65 regulated Nrf2 expression by directly binding to its specific promoter region and activating its transcription. Finally, we proved that the combination of OXA and DMY has a synergistic tumor suppression effect in vivo. CONCLUSION: Our study provided a novel mechanism of DMY boosted chemosensitivity in human CRC. The downstream signals of DMY, NF-κB or Nrf2 could also be potential targets for the treatment of CRC.

Polysaccharides of vinegar-baked radix bupleuri promote the hepatic targeting effect of oxymatrine by regulating the protein expression of HNF4α, Mrp2, and OCT1.

ETHNOPHARMACOLOGICAL RELEVANCE: Vinegar-baked Radix Bupleuri (VBRB) is a processed form of Bupleurum chinense DC. As a well-known meridian-guiding drug, it is traditionally used as a component of traditional Chinese medicine formulations indicated for the treatment of liver diseases. However, the liver targeting component in VBRB remains unclear. Therefore, this study aims to explore the efficacy and mechanism of PSS (polysaccharides in Vinegar-baked Radix Bupleuri) in enhancing liver targeting. MATERIALS AND METHODS: Drug distribution of OM alone or combined with PSS was investigated in vivo. Relative uptake efficiency (RUE) and relative targeting efficiency (RTE) were calculated to evaluate liver targeting efficiency. The mRNA and protein expression of organic cation transporter 1 (OCT1), multi-drug resistance protein 2 (Mrp2), and hepatocyte nuclear factor 4α (HNF4α) in the liver were determined by q-PCR and Western blot. Then, AZT, the inhibitor of OCT1 and BI6015, the inhibitor of HNF4α were used to investigate regulatory mechanisms involved in the uptake of OM in the cell. At last, the role of PSS in the anti-hepatitis B virus (HBV) was explored on HepG2.2.15. RESULTS: PSS increased the AUC of OM in the liver and increase the RUE and RTE in the liver which indicated a liver targeting enhancing effect. The mRNA and protein expression of OCT1 was increased while Mrp2 and HNF4α decreased. PSS could increase the uptake of OM in HepG2 by increasing the protein expression of HNF4α and OCT1, while inhibited Mrp2. Moreover, PSS combined with OM could enhance the anti-HBV effect of OM. CONCLUSION: PSS enhanced the liver targeting efficiency and the underlying mechanism related to up-regulating the expression of OCT1 and HNF4α, while down-regulating of Mrp2. These results suggest that PSS may become a potential excipient and provide a new direction for new targeted research.

Poncirin downregulates ATP-binding cassette transporters to enhance cisplatin sensitivity in cisplatin-resistant osteosarcoma cells.

Poncirin, a flavanone glycoside with bitter taste extracted from dried immature fruit of Poncirus trifoliate, exhibits multiple biological activities including anti-tumor activity. Our study aimed to determine the effect and potential mechanism of poncirin on cisplatin resistance in osteosarcoma (OS) cells. CCK-8, flow cytometry analysis, and caspase-3/7 activity assays were used to evaluate cisplatin sensitivity. The expression changes of multidrug resistance 1 (MDR1), multidrug resistance-associated protein (MRP1), breast cancer resistance protein (BCRP), and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) pathway-related proteins were detected by RT-qPCR or western blot analyses. Results showed that poncirin exposure enhanced cisplatin sensitivity, promoted apoptosis, and increased caspase-3/7 activity in cisplatin-resistant OS cells. Poncirin decreased the expression levels of MDR1, MRP1, and BCRP, and inhibited the PI3K/Akt signaling in OS cells. Rescue experiments suggested that activation of the PI3K/Akt signaling by 740Y-P abolished poncirin-induced expression reduction of MDR1, MRP1, and BCRP, and attenuated the facilitative effects of poncirin on cisplatin sensitivity and apoptosis in cisplatin-resistant OS cells. In summary, poncirin suppressed cisplatin resistance in cisplatin-resistant OS cells by downregulating the expression of MDR1, MRP1, and BCRP through inhibiting the PI3K/Akt pathway.

Overcoming vincristine resistance in cancer: Computational design and discovery of piperine-inspired P-glycoprotein inhibitors.

P-glycoprotein (P-gp)/MDR-1 plays a major role in the development of multidrug resistance (MDR) by pumping the chemotherapeutic drugs out of the cancer cells and reducing their efficacy. A number of P-gp inhibitors were reported to reverse the MDR when co-administered with chemotherapeutic drugs. Unfortunately, none has approved for clinical use due to toxicity issues. Some of the P-gp inhibitors tested in the clinics are reported to have cross-reactivity with CYP450 drug-metabolizing enzymes, resulting in unpredictable pharmacokinetics and toxicity of co-administered chemotherapeutic drugs. In this study, two piperine analogs (3 and 4) having lower cross-reactivity with CYP3A4 drug-metabolizing enzyme are identified as P-glycoprotein (P-gp) inhibitors through computational design, followed by synthesis and testing in MDR cancer cell lines over-expressing P-gp (KB ChR 8-5, SW480-VCR, and HCT-15). Both the analogs significantly increased the vincristine efficacy in MDR cancer cell lines at low micromole concentrations. Specifically, 3 caused complete reversal of vincristine resistance in KB ChR 8-5 cells and found to act as competitive inhibitor of P-gp as well as potentiated the vincristine-induced NF-KB-mediated apoptosis. Therefore, 3 ((2E,4E)-1-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-5-(4-hydroxy-3-methoxyphenyl)penta-2,4-dien-1-one) can serve as a potential P-gp inhibitor for in vivo investigations, to reverse multidrug resistance in cancer.

Regulation of organic anion transporters: Role in physiology, pathophysiology, and drug elimination.

The members of the organic anion transporter (OAT) family are mainly expressed in kidney, liver, placenta, intestine, and brain. These transporters play important roles in the disposition of clinical drugs, pesticides, signaling molecules, heavy metal conjugates, components of phytomedicines, and toxins, and therefore critical for maintaining systemic homeostasis. Alterations in the expression and function of OATs contribute to the intra- and inter-individual variability of the therapeutic efficacy and the toxicity of many drugs, and to many pathophysiological conditions. Consequently, the activity of these transporters must be highly regulated to carry out their normal functions. This review will present an update on the recent advance in understanding the cellular and molecular mechanisms underlying the regulation of renal OATs, emphasizing on the post-translational modification (PTM), the crosstalk among these PTMs, and the remote sensing and signaling network of OATs. Such knowledge will provide significant insights into the roles of these transporters in health and disease.

A Methanol Extract of Scabiosa atropurpurea Enhances Doxorubicin Cytotoxicity against Resistant Colorectal Cancer Cells In Vitro.

Colorectal cancer is a malignancy with a high incidence. Currently, the drugs used in chemotherapy are often accompanied by strong side effects. Natural secondary metabolites can interfere with chemotherapeutic drugs and intensify their cytotoxic effects. This study aimed to profile the secondary metabolites from the methanol extract of Scabiosa atropurpurea and investigate their in vitro activities, alone or in combination with the chemotherapeutic agent doxorubicin. MTT assay was used to determine the cytotoxic activities. Annexin-V/PI double-staining analysis was employed to evaluate the apoptotic concentration. Multicaspase assay, quantitative reverse transcription real-time PCR (RT-qPCR), and ABC transporter activities were also performed. LC-MS analysis revealed 31 compounds including phenolic acids, flavonoids, and saponins. S. atropurpurea extract intensified doxorubicin anti-proliferative effects against resistant tumor cells and enhanced the cytotoxic effects towards Caco-2 cells after 48 h. The mRNA expression levels of Bax, caspase-3, and p21 were increased significantly whereas Bcl-2 expression level was decreased. Furthermore, the methanol extract reversed P-glycoprotein or multidrug resistance-associated protein in Caco-2 cells. In conclusion, S. atropurpurea improved chemosensitivity and modulated multidrug resistance in Caco-2 cells which makes it a good candidate for further research in order to develop a new potential cancer treatment.

Pharmacokinetic and Pharmacodynamic Factors Contribute to Synergism between Let-7c-5p and 5-Fluorouracil in Inhibiting Hepatocellular Carcinoma Cell Viability.

Pharmacological interventions for hepatocellular carcinoma (HCC) are hindered by complex factors, and rational combination therapy may be developed to improve therapeutic outcomes. Very recently, we have identified a bioengineered microRNA let-7c-5p (or let-7c) agent as an effective inhibitor against HCC in vitro and in vivo. In this study, we sought to identify small-molecule drugs that may synergistically act with let-7c against HCC. Interestingly, we found that let-7c exhibited a strong synergism with 5-fluorouracil (5-FU) in the inhibition of HCC cell viability as manifested by average combination indices of 0.3 and 0.5 in Hep3B and Huh7 cells, respectively. By contrast, coadministration of let-7c with doxorubicin or sorafenib inhibited HCC cell viability with, rather surprisingly, no or minimal synergy. Further studies showed that protein levels of multidrug resistance-associated protein (MRP) ATP-binding cassette subfamily C member 5 (MRP5/ABCC5), a 5-FU efflux transporter, were reduced around 50% by let-7c in HCC cells. This led to a greater degree of intracellular accumulation of 5-FU in Huh7 cells as well as the second messenger cyclic adenosine monophosphate, an endogenous substrate of MRP5. Since 5-FU is an irreversible inhibitor of thymidylate synthetase (TS), we investigated the interactions of let-7c with 5-FU at pharmacodynamic level. Interestingly, our data revealed that let-7c significantly reduced TS protein levels in Huh7 cells, which was associated with the suppression of upstream transcriptional factors as well as other regulatory factors. Collectively, these results indicate that let-7c interacts with 5-FU at both pharmacokinetic and pharmacodynamic levels, and these findings shall offer insight into molecular mechanisms of synergistic drug combinations. SIGNIFICANCE STATEMENT: Combination therapy is a common strategy that generally involves pharmacodynamic interactions. After identifying a strong synergism between let-7c-5p and 5-fluorouracil (5-FU) against hepatocellular carcinoma cell viability, we reveal the involvement of both pharmacokinetic and pharmacodynamic mechanisms. In particular, let-7c enhances 5-FU exposure (via suppressing ABCC5/MRP5 expression) and cotargets thymidylate synthase with 5-FU (let-7c reduces protein expression, whereas 5-FU irreversibly inactivates enzyme). These findings provide insight into developing rational combination therapies based on pharmacological mechanisms.

A Two-Tiered In Vitro Approach to De-Risk Drug Candidates for Potential Bile Salt Export Pump Inhibition Liabilities in Drug Discovery.

Hepatocellular accumulation of bile salts by inhibition of bile salt export pump (BSEP/ABCB11) may result in cholestasis and is one proposed mechanism of drug-induced liver injury (DILI). To understand the relationship between BSEP inhibition and DILI, we evaluated 64 DILI-positive and 57 DILI-negative compounds in BSEP, multidrug resistance protein (MRP) 2, MRP3, and MRP4 vesicular inhibition assays. An empirical cutoff (5 µM) for BSEP inhibition was established based on a relationship between BSEP IC50 values and the calculated maximal unbound concentration at the inlet of the human liver (fu*Iin,max, assay specificity = 98%). Including inhibition of MRP2-4 did not increase DILI predictivity. To further understand the potential to inhibit bile salt transport, a selected subset of 30 compounds were tested for inhibition of taurocholate (TCA) transport in a long-term human hepatocyte micropatterned co-culture (MPCC) system. The resulting IC50 for TCA in vitro biliary clearance and biliary excretion index (BEI) in MPCCs were compared with the compound's fu*Iin,max to assess potential risk for bile salt transport perturbation. The data show high specificity (89%). Nine out of 15 compounds showed an IC50 value in the BSEP vesicular assay of <5µM, but the BEI IC50 was more than 10-fold the fu*Iin,max, suggesting that inhibition of BSEP in vivo is unlikely. The data indicate that although BSEP inhibition measured in membrane vesicles correlates with DILI risk, that measurement of this assay activity is insufficient. A two-tiered strategy incorporating MPCCs is presented to reduce BSEP inhibition potential and improve DILI risk. SIGNIFICANCE STATEMENT: This work describes a two-tiered in vitro approach to de-risk compounds for potential bile salt export pump inhibition liabilities in drug discovery utilizing membrane vesicles and a long-term human hepatocyte micropatterned co-culture system. Cutoffs to maximize specificity were established based on in vitro data from a set of 121 DILI-positive and -negative compounds and associated calculated maximal unbound concentration at the inlet of the human liver based on the highest clinical dose.

Effects of Chrysin and Its Major Conjugated Metabolites Chrysin-7-Sulfate and Chrysin-7-Glucuronide on Cytochrome P450 Enzymes and on OATP, P-gp, BCRP, and MRP2 Transporters.

Chrysin is an abundant flavonoid in nature, and it is also contained by several dietary supplements. Chrysin is highly biotransformed in the body, during which conjugated metabolites chrysin-7-sulfate and chrysin-7-glucuronide are formed. These conjugates appear at considerably higher concentrations in the circulation than the parent compound. Based on previous studies, chrysin can interact with biotransformation enzymes and transporters; however, the interactions of its metabolites have been barely examined. In this in vitro study, the effects of chrysin, chrysin-7-sulfate, and chrysin-7-glucuronide on cytochrome P450 enzymes (2C9, 2C19, 3A4, and 2D6) as well as on organic anion-transporting polypeptides (OATPs; 1A2, 1B1, 1B3, and 2B1) and ATP binding cassette [P-glycoprotein, multidrug resistance-associated protein 2, and breast cancer resistance protein (BCRP)] transporters were investigated. Our observations revealed that chrysin conjugates are strong inhibitors of certain biotransformation enzymes (e.g., CYP2C9) and transporters (e.g., OATP1B1, OATP1B3, OATP2B1, and BCRP) examined. Therefore, the simultaneous administration of chrysin-containing dietary supplements with medications needs to be carefully considered due to the possible development of pharmacokinetic interactions. SIGNIFICANCE STATEMENT: Chrysin-7-sulfate and chrysin-7-glucuronide are the major metabolites of flavonoid chrysin. In this study, we examined the effects of chrysin and its conjugates on cytochrome P450 enzymes and on organic anion-transporting polypeptides and ATP binding cassette transporters (P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2). Our results demonstrate that chrysin and/or its conjugates can significantly inhibit some of these proteins. Since chrysin is also contained by dietary supplements, high intake of chrysin may interrupt the transport and/or the biotransformation of drugs.