Dysregulation and oncogenic activities of ubiquitin specific peptidase 2a in the pathogenesis of hepatocellular carcinoma.

Ubiquitin specific peptidase 2a (USP2a) plays critical roles in protein degradation and other cellular activities. Currently, our understanding on USP2a dysregulation in subjects with hepatocellular carcinoma (HCC) and its roles in HCC pathogenesis is limited. In this study, we found that USP2a mRNA and protein levels were significantly upregulated in HCC tumors from both human and mice. USP2a overexpression in HepG2 and Huh 7 cells significantly increased cell proliferation while inhibition of USP2a activity by chemical inhibitor or stable knockout of USP2 by CRISPR markedly reduced cell proliferation. In addition, USP2a overexpression significantly augmented the resistance while knockout of USP2a markedly increased the susceptibility of HepG2 cells to bile acid-induced apoptosis and necrosis. Consistent with the oncogenic activities detected in vitro, overexpression of USP2a promoted de novo HCC development in mice with significantly increased tumor occurrence rates, tumor sizes and liver/body ratios. Further investigations with unbiased co-immunoprecipitation (Co-IP)-coupled proteomic analysis and Western blot identified novel USP2a target proteins involved in cell proliferation, apoptosis, and tumorigenesis. Analysis of those USP2a target proteins revealed that USP2a's oncogenic activities are mediated through multiple pathways, including modulating protein folding and assembling through regulating protein chaperones/co-chaperones HSPA1A, DNAJA1 and TCP1, promoting DNA replication and transcription through regulating RUVBL1, PCNA and TARDBP, and altering mitochondrial apoptotic pathway through regulating VDAC2. Indeed, those newly identified USP2a target proteins were markedly dysregulated in HCC tumors. In summary, USP2a was upregulated in HCC subjects and acted as an oncogene in the pathogenesis of HCC through multiple downstream pathways. The findings provided molecular and pathogenesis bases for developing interventions to treat HCC by targeting USP2a or its downstream pathways.

Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma.

Ubiquitin specific peptidase-2 (USP2) plays important roles in a myriad of cellular activities through deubiquitinating target proteins and its implications in various diseases, especially cancers, are starting to emerge. Our current understanding on USP2 expression in subjects with hepatocellular carcinoma (HCC) and its roles in the pathogenesis of HCC is limited. In this study, we found that USP2 protein and mRNA levels were significantly dysregulated in HCC tumor (HCC-T) when compared to adjacent non-tumor (HCC-NT) or normal liver tissues from both human and mouse HCC model. Among the USP2 isoforms, USP2b was the predominant isoform in the normal liver and markedly down-regulated in HCC-T tissues in both human and mice. Data from overexpression, chemical inhibition and knockout studies consistently demonstrated that USP2b promoted cell proliferation, colony formation and wound healing in HepG2 and Huh 7 cells. On the other hand, USP2b exhibited proapoptotic and pronecrtotic activities through enhancing bile acid-induced apoptosis and necrosis in both HepG2 and Huh 7 cells. Unbiased proteomic analysis of USP2-knockout (KO) and parental HepG2 cells resulted in identification of USP2-regulated downstream target proteins involved in cell proliferation, apoptosis, and tumorigenesis, including serine/threonine kinase 4 (STK4), epidermal growth factor receptor (EGFR), dipeptidyl peptidase 4 (DPP4) and fatty acid binding protein 1 (FABP1). In conclusion, USP2b expression was dysregulated in subjects with HCC and contributed to the pathogenesis of HCC by promoting cell proliferation and exerting proapoptotic and pronecrotic activities. The findings provide the molecular basis for developing therapies for HCC through modulating USP2b expression or activities.

Dysregulation of bile acids increases the risk for preterm birth in pregnant women.

Preterm birth (PTB) is the leading cause of perinatal mortality and newborn complications. Bile acids are recognized as signaling molecules regulating a myriad of cellular and metabolic activities but have not been etiologically linked to PTB. In this study, a hospital-based cohort study with 36,755 pregnant women is conducted. We find that serum total bile acid levels directly correlate with the PTB rates regardless of the characteristics of the subjects and etiologies of liver disorders. Consistent with the findings from pregnant women, PTB is successfully reproduced in mice with liver injuries and dysregulated bile acids. More importantly, bile acids dose-dependently induce PTB with minimal hepatotoxicity. Furthermore, restoring bile acid homeostasis by farnesoid X receptor activation markedly reduces PTB and dramatically improves newborn survival rates. The findings thus establish an etiologic link between bile acids and PTB, and open an avenue for developing etiology-based therapies to prevent or delay PTB.

Dysregulation of Δ4-3-oxosteroid 5ß-reductase in diabetic patients: Implications and mechanisms.

Aldo-keto reductase family 1 member D1 (AKR1D1) is a Δ4-3-oxosteroid 5ß-reductase required for bile acid synthesis and steroid hormone metabolism. Both bile acids and steroid hormones, especially glucocorticoids, play important roles in regulating body metabolism and energy expenditure. Currently, our understanding on AKR1D1 regulation and its roles in metabolic diseases is limited. We found that AKR1D1 expression was markedly repressed in diabetic patients. Consistent with repressed AKR1D1 expression, hepatic bile acids were significantly reduced in diabetic patients. Mechanistic studies showed that activation of peroxisome proliferator-activated receptor-α (PPARα) transcriptionally down-regulated AKR1D1 expression in vitro in HepG2 cells and in vivo in mice. Consistently, PPARα signaling was enhanced in diabetic patients. In summary, dysregulation of AKR1D1 disrupted bile acid and steroid hormone homeostasis, which may contribute to the pathogenesis of diabetes. Restoring bile acid and steroid hormone homeostasis by modulating AKR1D1 expression may represent a new approach to develop therapies for diabetes.

Bitter taste receptors as targets for tocolytics in preterm labor therapy.

Preterm birth (PTB) is the leading cause of neonatal mortality and morbidity, with few prevention and treatment options. Uterine contraction is a central feature of PTB, so gaining new insights into the mechanisms of this contraction and consequently identifying novel targets for tocolytics are essential for more successful management of PTB. Here we report that myometrial cells from human and mouse express bitter taste receptors (TAS2Rs) and their canonical signaling components (i.e., G-protein gustducin and phospholipase C ß2). Bitter tastants can completely relax myometrium precontracted by different uterotonics. In isolated single mouse myometrial cells, a phenotypical bitter tastant (chloroquine, ChQ) reverses the rise in intracellular Ca2+ concentration ([Ca2+]i) and cell shortening induced by uterotonics, and this reversal effect is inhibited by pertussis toxin and by genetic deletion of α-gustducin. In human myometrial cells, knockdown of TAS2R14 but not TAS2R10 inhibits ChQ's reversal effect on an oxytocin-induced rise in [Ca2+]i Finally, ChQ prevents mouse PTBs induced by bacterial endotoxin LPS or progesterone receptor antagonist mifepristone more often than current commonly used tocolytics, and this prevention is largely lost in α-gustducin-knockout mice. Collectively, our results reveal that activation of the canonical TAS2R signaling system in myometrial cells produces profound relaxation of myometrium precontracted by a broad spectrum of contractile agonists, and that targeting TAS2Rs is an attractive approach to developing effective tocolytics for PTB management.-Zheng, K., Lu, P., Delpapa, E., Bellve, K., Deng, R., Condon, J. C., Fogarty, K., Lifshitz, L. M., Simas, T. A. M., Shi, F., ZhuGe, R. Bitter taste receptors as targets for tocolytics in preterm labor therapy.

Synthesis and biological evaluations of chalcones, flavones and chromenes as farnesoid x receptor (FXR) antagonists.

Farnesoid X receptor (FXR), a nuclear receptor mainly distributed in liver and intestine, has been regarded as a potential target for the treatment of various metabolic diseases, cancer and infectious diseases related to liver. Starting from two previously identified chalcone-based FXR antagonists, we tried to increase the activity through the design and synthesis of a library containing chalcones, flavones and chromenes, based on substitution manipulation and conformation (ring closure) restriction strategy. Many chalcones and four chromenes were identified as microM potent FXR antagonists, among which chromene 11c significantly decreased the plasma and hepatic triglyceride level in KKay mice.

Differential Feedback Regulation of Δ4-3-Oxosteroid 5ß-Reductase Expression by Bile Acids.

Δ4-3-oxosteroid 5ß-reductase is member D1 of the aldo-keto reductase family 1 (AKR1D1), which catalyzes 5ß-reduction of molecules with a 3-oxo-4-ene structure. Bile acid intermediates and most of the steroid hormones carry the 3-oxo-4-ene structure. Therefore, AKR1D1 plays critical roles in both bile acid synthesis and steroid hormone metabolism. Currently our understanding on transcriptional regulation of AKR1D1 under physiological and pathological conditions is very limited. In this study, we investigated the regulatory effects of primary bile acids, chenodeoxycholic acid (CDCA) and cholic acid (CA), on AKR1D1 expression. The expression levels of AKR1D1 mRNA and protein in vitro and in vivo following bile acid treatments were determined by real-time PCR and Western blotting. We found that CDCA markedly repressed AKR1D1 expression in vitro in human hepatoma HepG2 cells and in vivo in mice. On the contrary, CA significantly upregulated AKR1D1 expression in HepG2 cells and in mice. Further mechanistic investigations revealed that the farnesoid x receptor (FXR) signaling pathway was not involved in regulating AKR1D1 by bile acids. Instead, CDCA and CA regulated AKR1D1 through the mitogen-activated protein kinases/c-Jun N-terminal kinases (MAPK/JNK) signaling pathway. Inhibition of the MAPK/JNK pathway effectively abolished CDCA and CA-mediated regulation of AKR1D1. It was thus determined that AKR1D1 expression was regulated by CDCA and CA through modulating the MAPK/JNK signaling pathway. In conclusion, AKR1D1 expression was differentially regulated by primary bile acids through negative and positive feedback mechanisms. The findings indicated that both bile acid concentrations and compositions play important roles in regulating AKR1D1 expression, and consequently bile acid synthesis and steroid hormone metabolism.

Estrogen and Estrogen Receptor-α-Mediated Transrepression of Bile Salt Export Pump.

Among diseases unique to pregnancy, intrahepatic cholestasis of pregnancy is the most prevalent disorder with elevated serum bile acid levels. We have previously shown that estrogen 17ß-estradiol (E2) transrepresses bile salt export pump (BSEP) through an interaction between estrogen receptor (ER)-α and farnesoid X receptor (FXR) and transrepression of BSEP by E2/ERα is an etiological contributing factor to intrahepatic cholestasis of pregnancy. Currently the mechanistic insights into such transrepression are not fully understood. In this study, the dynamics of coregulator recruitment to BSEP promoter after FXR activation and E2 treatment were established with quantitative chromatin immunoprecipitation assays. Coactivator peroxisome proliferator-activated receptor-γ coactivator-1 was predominantly recruited to the BSEP promoter upon FXR activation, and its recruitment was decreased by E2 treatment. Meanwhile, recruitment of nuclear receptor corepressor was markedly increased upon E2 treatment. Functional evaluation of ERα and ERß chimeras revealed that domains AC of ERα are the determinants for ERα-specific transrepression on BSEP. Further studies with various truncated ERα proteins identified the domains in ERα responsible for ligand-dependent and ligand-independent transrepression. Truncated ERα-AD exhibited potent ligand-independent transrepressive activity, whereas ERα-CF was fully capable of transrepressing BSEP ligand dependently in vitro in Huh 7 cells and in vivo in mice. Both ERα-AD and ERα-CF proteins were associated with FXR in the coimmunoprecipitation assays. In conclusion, E2 repressed BSEP expression through diminishing peroxisome proliferator-activated receptor-γ coactivator-1 recruitment with a concurrent increase in nuclear receptor corepressor recruitment to the BSEP promoter. Domains AD and CF in ERα mediated ligand-independent and ligand-dependent transrepression on BSEP, respectively, through interacting with FXR.

Transcriptional dynamics of bile salt export pump during pregnancy: mechanisms and implications in intrahepatic cholestasis of pregnancy.

UNLABELLED: Bile salt export pump (BSEP) is responsible for biliary secretion of bile acids, a rate-limiting step in the enterohepatic circulation of bile acids and transactivated by nuclear receptor farnesoid X receptor (FXR). Intrahepatic cholestasis of pregnancy (ICP) is the most prevalent disorder among diseases unique to pregnancy and primarily occurs in the third trimester of pregnancy, with a hallmark of elevated serum bile acids. Currently, the transcriptional regulation of BSEP during pregnancy and its underlying mechanisms and involvement in ICP are not fully understood. In this study the dynamics of BSEP transcription in vivo in the same group of pregnant mice before, during, and after gestation were established with an in vivo imaging system (IVIS). BSEP transcription was markedly repressed in the later stages of pregnancy and immediately recovered after parturition, resembling the clinical course of ICP in human. The transcriptional dynamics of BSEP was inversely correlated with serum 17ß-estradiol (E2) levels before, during, and after gestation. Further studies showed that E2 repressed BSEP expression in human primary hepatocytes, Huh 7 cells, and in vivo in mice. Such transrepression of BSEP by E2 in vitro and in vivo required estrogen receptor α (ERα). Mechanistic studies with chromatin immunoprecipitation (ChIP), protein coimmunoprecipitation (Co-IP), and bimolecular fluorescence complementation (BiFC) assays demonstrated that ERα directly interacted with FXR in living cells and in vivo in mice. CONCLUSION: BSEP expression was repressed by E2 in the late stages of pregnancy through a nonclassical E2/ERα transrepressive pathway, directly interacting with FXR. E2-mediated repression of BSEP expression represents an etiological contributing factor to ICP and therapies targeting the ERα/FXR interaction may be developed for prevention and treatment of ICP.

Low-dose chemotherapy of hepatocellular carcinoma through triggered-release from bilayer-decorated magnetoliposomes.

Low-dose (LD) chemotherapy is a promising treatment strategy that may be improved by controlled delivery. Polyethylene glycol-stabilized bilayer-decorated magnetoliposomes (dMLs) have been designed as a stimuli-responsive LD chemotherapy drug delivery system and tested in vitro using Huh-7 hepatocellular carcinoma cell line. The dMLs contained hydrophobic superparamagnetic iron oxide nanoparticles within the lipid bilayer and doxorubicin hydrochloride (DOX, 2 µM) within the aqueous core. Structural analysis by cryogenic transmission electron microscopy and dynamic light scattering showed that the assemblies were approximately 120 nm in diameter. Furthermore, the samples consisted of a mixture of dMLs and bare liposomes (no nanoparticles), which provided dual burst and spontaneous DOX release profiles, respectively. Cell viability results show that the cytotoxicity of DOX-loaded dMLs was similar to that of bare dMLs (∼10%), which indicates that spontaneous DOX leakage had little cytotoxic effect. However, when subjected to a physiologically acceptable radiofrequency (RF) electromagnetic field, cell viability was reduced up to 40% after 8h and significant cell death (>90%) was observed after 24h. The therapeutic mechanism was intracellular RF-triggered DOX release from the dMLs and not intracellular hyperthermia due to nanoparticle heating via magnetic losses.

Mechanistic insights into isoform-dependent and species-specific regulation of bile salt export pump by farnesoid X receptor.

Expression of bile salt export pump (BSEP) is regulated by the bile acid/farnesoid X receptor (FXR) signaling pathway. Two FXR isoforms, FXRα1 and FXRα2, are predominantly expressed in human liver. We previously showed that human BSEP was isoform-dependently regulated by FXR and diminished with altered expression of FXRα1 and FXRα2 in patients with hepatocellular carcinoma. In this study, we demonstrate that FXRα1 and FXRα2 regulate human BSEP through two distinct FXR responsive elements (FXRE): IR1a and IR1b. As the predominant regulator, FXRα2 potently transactivated human BSEP through IR1a, while FXRα1 weakly transactivated human BSEP through a newly identified IR1b. Relative expression of FXRα1 and FXRα2 affected human BSEP expression in vitro and in vivo. Electrophoretic mobility shift and chromatin immunoprecipitation assays confirmed the binding and recruitment of FXRα1 and FXRα2 to IR1b and IR1a. Sequence analysis concluded that IR1b was completely conserved among species, whereas IR1a exhibited apparent differences across species. Sequence variations in IR1a were responsible for the observed species difference in BSEP transactivation by FXRα1 and FXRα2. In conclusion, FXR regulates BSEP in an isoform-dependent and species-specific manner through two distinct FXREs, and alteration of relative FXR isoform expression may be a potential mechanism for FXR to precisely regulate human BSEP in response to various physiological and pathological conditions.

Chiral cationic polyamines for chiral microcapsules and siRNA delivery.

Reported herein is the use of chiral cationic polyamines for two intriguing applications: fabrication of chiral covalently-linked microcapsules, and enantiospecific delivery of siRNA to Huh 7 cells. The microcapsules are easily fabricated from homochiral polymers, and the resulting architectures can be used for supramolecular chiral catalysis and many other potential applications. Enantiospecific delivery of siRNA to Huh 7 cells is seen by one 'enantiomer' of the polymers delivering siRNA with significantly improved transfection efficiency and reduced toxicity compared to the 'enantiomeric' polymer and commercially available transfection reagents. Taken together, the use of these easily accessible polyamine structures for diverse applications is highlighted in this Letter herein and can lead to numerous future research efforts.

Bile salt export pump is dysregulated with altered farnesoid X receptor isoform expression in patients with hepatocellular carcinoma.

UNLABELLED: As a canalicular bile acid effluxer, the bile salt export pump (BSEP) plays a vital role in maintaining bile acid homeostasis. BSEP deficiency leads to severe cholestasis and hepatocellular carcinoma (HCC) in young children. Regardless of the etiology, chronic inflammation is the common pathological process for HCC development. Clinical studies have shown that bile acid homeostasis is disrupted in HCC patients with elevated serum bile acid level as a proposed marker for HCC. However, the underlying mechanisms remain largely unknown. In this study, we found that BSEP expression was severely diminished in HCC tissues and markedly reduced in adjacent nontumor tissues. In contrast to mice, human BSEP was regulated by farnesoid X receptor (FXR) in an isoform-dependent manner. FXR-α2 exhibited a much more potent activity than FXR-α1 in transactivating human BSEP in vitro and in vivo. The decreased BSEP expression in HCC was associated with altered relative expression of FXR-α1 and FXR-α2. FXR-α1/FXR-α2 ratios were significantly increased, with undetectable FXR-α2 expression in one third of the HCC tumor samples. A similar correlation between BSEP and FXR isoform expression was confirmed in hepatoma Huh7 and HepG2 cells. Further studies showed that intrahepatic proinflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α), were significantly elevated in HCC tissues. Treatment of Huh7 cells with IL-6 and TNF-α resulted in a marked increase in FXR-α1/FXR-α2 ratio, concurrent with a significant decrease in BSEP expression. CONCLUSION: BSEP expression is severely diminished in HCC patients associated with alteration of FXR isoform expression induced by inflammation. Restoration of BSEP expression through suppressing inflammation in the liver may reestablish bile acid homeostasis.

A review of the hypoglycemic effects of five commonly used herbal food supplements.

Hyperglycemia is a pathological condition associated with prediabetes and diabetes. The incidence of prediabetes and diabetes is increasing and imposes great burden on healthcare worldwide. Patients with prediabetes and diabetes have significantly increased risk for cardiovascular diseases and other complications. Currently, management of hyperglycemia includes pharmacological interventions, physical exercise, and change of life style and diet. Food supplements have increasingly become attractive alternatives to prevent or treat hyperglycemia, especially for subjects with mild hyperglycemia. This review summarized current patents and patent applications with relevant literature on five commonly used food supplements with claims of hypoglycemic effects, including emblica officinalis (gooseberry), fenugreek, green tea, momordica charantia (bitter melon) and cinnamon. The data from human clinical studies did not support a recommendation for all five supplements to manage hyperglycemia. Fenugreek and composite supplements containing emblica officinalis showed the most consistency in lowering fasting blood sugar (FBS) or glycated hemoglobin (HbA1c) levels in diabetic patients. The hypoglycemic effects of cinnamon and momordica charantia were demonstrated in most of the trials with some exceptions. However, green tea exhibited limited benefits in reducing FBS or HbA1c levels and should not be recommended for managing hyperglycemia. Certain limitations are noticed in a considerable number of clinical studies including small sample size, poor experimental design and considerable variations in participant population, preparation format, daily dose, and treatment duration. Future studies with more defined participants, standardized preparation and dose, and improved trial design and size are warranted.

Hypolipidemic agent Z-guggulsterone: metabolism interplays with induction of carboxylesterase and bile salt export pump.

Z-Guggulsterone is a major ingredient in the Indian traditional hypolipidemic remedy guggul. A study in mice has established that its hypolipidemic effect involves the farnesoid X receptor (FXR), presumably by acting as an antagonist of this receptor. It is generally assumed that the antagonism leads to induction of cytochrome P450 7A1 (CYP7A1), the rate-limiting enzyme converting free cholesterol to bile acids. In this study, we tested whether Z-guggulsterone indeed induces human CYP7A1. In addition, the expression of cholesteryl ester hydrolase CES1 and bile salt export pump (BSEP) was monitored. Contrary to the general assumption, Z-guggulsterone did not induce CYP7A1. Instead, this phytosterol significantly induced CES1 and BSEP through transactivation. Z-Guggulsterone underwent metabolism by CYP3A4, and the metabolites greatly increased the induction potency on BSEP but not on CES1. BSEP induction favors cholesterol elimination, whereas CES1 involves both elimination and retention (probably when excessively induced). Interestingly, clinical trials reported the hypolipidemic response rates from 18% to 80% and showed that higher dosages actually increased VLDL cholesterol. Our findings predict that better hypolipidemic outcomes likely occur in individuals who have a relatively higher capacity of metabolizing Z-guggulsterone with moderate CES1 induction, a scenario possibly achieved by lowering the dosing regimens.

Scoparone potentiates transactivation of the bile salt export pump gene and this effect is enhanced by cytochrome P450 metabolism but abolished by a PKC inhibitor.

BACKGROUND AND PURPOSE: Hyperbilirubinaemia and cholestasis are two major forms of liver abnormality. The Chinese herb Yin Chin has been used for thousands of years to treat liver dysfunctions. In mice, this herb and its principal ingredient scoparone were found to accelerate the clearance of bilirubin accompanied by the induction of uridine diphosphate-5'-glucuronosyltransferase-1A1 (UGT1A1), a bilirubin processing enzyme. The aim of this study was to determine whether scoparone induces the expression of human UGT1A1. In addition, the expression of the bile salt export pump (BSEP), a transporter of bile acids, was determined. EXPERIMENTAL APPROACH: Primary human hepatocytes and hepatoma line Huh7 were treated with scoparone, chenodeoxycholic acid (CDCA) or both. The expression of UGT1A1 and BSEP mRNA was determined. The activation of the human BSEP promoter reporter by scoparone was determined in Huh7 cells by transient transfection and in mice by bioluminescent imaging. The metabolism of scoparone was investigated by recombinant CYP enzymes and pooled human liver microsomes. KEY RESULTS: Scoparone did not enhance the expression of either human BSEP or, surprisingly, UGT1A1. However, scoparone significantly potentiated the expression of BSEP induced by CDCA. Consistent with this, scoparone potentiated the stimulant effect of CDCA on the human BSEP promoter. This potentiation was enhanced by co-transfection of cytochrome P4501A2 but abolished by the PKC inhibitor GF109203X. CONCLUSIONS AND IMPLICATIONS: Scoparone and Yin Chin normalize liver function primarily by enhancing the secretion of bile acids, and this effect probably varies depending on the metabolic rate of scoparone.

Hypolipidemic, antioxidant, and antiinflammatory activities of microalgae Spirulina.

Spirulina is free-floating filamentous microalgae growing in alkaline water bodies. With its high nutritional value, Spirulina has been consumed as food for centuries in Central Africa. It is now widely used as nutraceutical food supplement worldwide. Recently, great attention and extensive studies have been devoted to evaluate its therapeutic benefits on an array of diseased conditions including hypercholesterolemia, hyperglycerolemia, cardiovascular diseases, inflammatory diseases, cancer, and viral infections. The cardiovascular benefits of Spirulina are primarily resulted from its hypolipidemic, antioxidant, and antiinflammatory activities. Data from preclinical studies with various animal models consistently demonstrate the hypolipidemic activity of Spirulina. Although differences in study design, sample size, and patient conditions resulting in minor inconsistency in response to Spirulina supplementation, the findings from human clinical trials are largely consistent with the hypolipidemic effects of Spirulina observed in the preclinical studies. However, most of the human clinical trials are suffered with limited sample size and some with poor experimental design. The antioxidant and/or antiinflammatory activities of Spirulina were demonstrated in a large number of preclinical studies. However, a limited number of clinical trials have been carried out so far to confirm such activities in human. Currently, our understanding on the underlying mechanisms for Spirulina's activities, especially the hypolipidemic effect, is limited. Spirulina is generally considered safe for human consumption supported by its long history of use as food source and its favorable safety profile in animal studies. However, rare cases of side-effects in human have been reported. Quality control in the growth and process of Spirulina to avoid contamination is mandatory to guarantee the safety of Spirulina products.

Differential modulation of farnesoid X receptor signaling pathway by the thiazolidinediones.

Thiazolidinediones (TZD), including troglitazone, rosiglitazone, and pioglitazone, are agonists of peroxisome proliferator-activated receptor (PPAR)-gamma and belong to a class of insulin-sensitizing drugs for type 2 diabetes mellitus. However, member-specific, PPARgamma-independent activities and toxicity have been reported, especially for troglitazone. Currently, the underlying mechanisms are not fully understood. In this study, we demonstrated that troglitazone but not rosiglitazone or pioglitazone modulated expression of farnesoid X receptor (FXR) target genes bile salt export pump (BSEP) and small heterodimer partner (SHP) in Huh-7 cells. More specifically, troglitazone acted as a partial agonist of FXR to weakly increase BSEP and SHP expression but functioned as a potent antagonist to significantly suppress bile acid-induced expression. Consistent with the finding, troglitazone partially induced but markedly antagonized bile acid-mediated BSEP promoter transactivation. However, such modulating effects were not detected with rosiglitazone or pioglitazone. Using the crystal structure of ligand-bound FXR ligand binding domain (LBD), molecular docking predicted that troglitazone, but not rosiglitazone or pioglitazone, could form a stable complex with FXR LBD. The specific alpha-tocopherol side chain of troglitazone significantly contributed to the formation of such a stable complex through extensive interactions with FXR LBD. The docking model was further validated by functional analyses of a series of docking-guided FXR mutants. In summary, the data demonstrated that troglitazone, but not rosiglitazone or pioglitazone, was an FXR modulator and potently antagonized bile acid-induced expression of FXR target genes. Such differential modulation of FXR signaling pathway by TZDs may represent one of the mechanisms for member-specific, PPARgamma-independent activities and toxicity.

Pyrethroid insecticides: isoform-dependent hydrolysis, induction of cytochrome P450 3A4 and evidence on the involvement of the pregnane X receptor.

Pyrethroids account for more than one-third of the insecticides currently marketed in the world. In mammals, these insecticides undergo extensive metabolism by carboxylesterases and cytochrome P450s (CYPs). In addition, some pyrethroids are found to induce the expression of CYPs. The aim of this study was to determine whether pyrethroids induce carboxylesterases and CYP3A4, and whether the induction is correlated inversely with their hydrolysis. Human liver microsomes were pooled and tested for the hydrolysis of 11 pyrethroids. All pyrethroids were hydrolyzed by the pooled microsomes, but the hydrolytic rates varied by as many as 14 fold. Some pyrethroids such as bioresmethrin were preferably hydrolyzed by carboxylesterase HCE1, whereas others such as bifenthrin preferably by HCE2. In primary human hepatocytes, all pyrethroids except tetramethrin significantly induced CYP3A4. In contrast, insignificant changes were detected on the expression of carboxylesterases. The induction of CYP3A4 was confirmed in multiple cell lines including HepG2, Hop92 and LS180. Overall, the magnitude of the induction was correlated inversely with the rates of hydrolysis, but positively with the activation of the pregnane X receptor (PXR). Transfection of a carboxylesterase markedly decreased the activation of PXR, and the decrease was in agreement with carboxylesterase-based preference for hydrolysis. In addition, human PXR variants as well as rat PXR differed from human PXR (wild-type) in responding to certain pyrethroids (e.g., lambda-cyhalothrin), suggesting that induction of PXR target genes by these pyrethroids varies depending on polymorphic variants and the PXR species identity.