Pharmacokinetic Estimation Models-based Approach to Predict Clinical Implications for CYP Induction by Calcitriol in Human Cryopreserved Hepatocytes and HepaRG Cells

Calcitriol, a vitamin D3 metabolite, is approved for various indications because it is the bioactive form of vitamin D in the body. The purpose of this study was to predict the clinical significance of cytochrome P450 (CYP) induction by calcitriol using in vitro human cryopreserved hepatocytes, HepaRG experimental systems, and various pharmacokinetic estimation models. CYP2B6, 3A4, 2C8, and 2C9 mRNA levels increased in a concentration-dependent manner in the presence of calcitriol in human cryopreserved hepatocytes and HepaRG cells. Using the half maximal effective concentration (EC50) and maximum induction effect (Emax) obtained from the in vitro study, a basic kinetic model was applied, suggesting clinical relevance. In addition, a static mechanistic model showed the improbability of a clinically significant effect; however, the calculated area under the plasma concentration–time curve ratio (AUCR) was marginal for CYP3A4 in HepaRG cells. To clarify the effect of CYP3A4 in vivo, physiologically based pharmacokinetic (PBPK) modeling was applied as a dynamic mechanistic model, revealing a low clinically significant effect of CYP3A4 induction by calcitriol. Therefore, we conclude that CYP induction by calcitriol treatment would not be clinically significant under typical clinical conditions.

Download Full-text

Nanocurcumin: A Double-Edged Sword for Microcancers

Current Pharmaceutical Design ◽

10.2174/1381612826666201118100045 ◽

2020 ◽

Vol 26 (45) ◽

pp. 5783-5792

Author(s):

Kholood Abid Janjua ◽

Adeeb Shehzad ◽

Raheem Shahzad ◽

Salman Ul Islam ◽

Mazhar Ul Islam

Keyword(s):

Intracellular Signaling ◽

Dosage Forms ◽

The Body ◽

In Vivo Studies ◽

Mrna Levels ◽

Anticancer Activities ◽

Road Map ◽

Anticancer Effects

There is compelling evidence that drug molecules isolated from natural sources are hindered by low systemic bioavailability, poor absorption, and rapid elimination from the human body. Novel approaches are urgently needed that could enhance the retention time as well as the efficacy of natural products in the body. Among the various adopted approaches to meet this ever-increasing demand, nanoformulations show the most fascinating way of improving the bioavailability of dietary phytochemicals through modifying their pharmacokinetics and pharmacodynamics. Curcumin, a yellowish pigment isolated from dried ground rhizomes of turmeric, exhibits tremendous pharmacological effects, including anticancer activities. Several in vitro and in vivo studies have shown that curcumin mediates anticancer effects through the modulation (upregulation and/or downregulations) of several intracellular signaling pathways both at protein and mRNA levels. Scientists have introduced multiple modern techniques and novel dosage forms for enhancing the delivery, bioavailability, and efficacy of curcumin in the treatment of various malignancies. These novel dosage forms include nanoparticles, liposomes, micelles, phospholipids, and curcumin-encapsulated polymer nanoparticles. Nanocurcumin has shown improved anticancer effects compared to conventional curcumin formulations. This review discusses the underlying molecular mechanism of various nanoformulations of curcumin for the treatment of different cancers. We hope that this study will make a road map for preclinical and clinical investigations of cancer and recommend nano curcumin as a drug of choice for cancer therapy.

Download Full-text

The impact of indole-3-lactic acid on immature intestinal innate immunity and development: a transcriptomic analysis

Scientific Reports ◽

10.1038/s41598-021-87353-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Wuyang Huang ◽

Ky Young Cho ◽

Di Meng ◽

W. Allan Walker

Keyword(s):

Lactic Acid ◽

Mechanistic Model ◽

Transcriptomic Analysis ◽

Dependent Manner ◽

Protective Effects ◽

Very Preterm Infants ◽

Anti Inflammatory ◽

The Impact

AbstractAn excessive intestinal inflammatory response may have a role in the pathogenesis of necrotizing enterocolitis (NEC) in very preterm infants. Indole-3-lactic acid (ILA) of breastmilk tryptophan was identified as the anti-inflammatory metabolite involved in probiotic conditioned media from Bifidobacteria longum subsp infantis. This study aimed to explore the molecular endocytic pathways involved in the protective ILA effect against inflammation. H4 cells, Caco-2 cells, C57BL/6 pup and adult mice were used to compare the anti-inflammatory mechanisms between immature and mature enterocytes in vitro and in vivo. The results show that ILA has pleiotropic protective effects on immature enterocytes including anti-inflammatory, anti-viral, and developmental regulatory potentials in a region-dependent and an age-dependent manner. Quantitative transcriptomic analysis revealed a new mechanistic model in which STAT1 pathways play an important role in IL-1β-induced inflammation and ILA has a regulatory effect on STAT1 pathways. These studies were validated by real-time RT-qPCR and STAT1 inhibitor experiments. Different protective reactions of ILA between immature and mature enterocytes indicated that ILA’s effects are developmentally regulated. These findings may be helpful in preventing NEC for premature infants.

Download Full-text

Estrogen Up-Regulates Hepatic Expression of Suppressors of Cytokine Signaling-2 and -3 in Vivo and in Vitro

Endocrinology ◽

10.1210/en.2004-0061 ◽

2004 ◽

Vol 145 (12) ◽

pp. 5525-5531 ◽

Cited By ~ 50

Author(s):

Gary M. Leong ◽

Sofia Moverare ◽

Jesena Brce ◽

Nathan Doyle ◽

Klara Sjögren ◽

...

Keyword(s):

Promoter Activity ◽

Hepatoma Cells ◽

Cytokine Signaling ◽

Mrna Levels ◽

Dependent Manner ◽

Wild Type ◽

Hepatic Expression ◽

Suppressors Of Cytokine Signaling

Abstract Suppressors of cytokine signaling (SOCS) are important negative regulators of cytokine action. We recently reported that estrogen stimulates SOCS-2 expression and inhibits GH signaling in kidney cells. The effects of estrogen on SOCS expression in other tissues are unclear. The aim of this study was to investigate in vivo and in vitro whether estrogen affected SOCS expression in the liver, a major target organ of GH. The in vivo hepatic effects of estrogen on ovariectomized mice lacking estrogen receptor (ER)-α, ERβ, or both and their wild-type littermates were examined by DNA microarray analysis. In vitro, the effects of estrogen on SOCS expression in human hepatoma cells were examined by reverse transcription quantitative PCR. Long-term (3 wk) estrogen treatment induced a 2- to 3-fold increase in hepatic expression of SOCS-2 and -3 in wild-type and ERβ knockout mice but not in those lacking ERα or both ER subtypes. Short-term treatment (at 24 h) increased the mRNA level of SOCS-3 but not SOCS-2. In cultured hepatoma cells, estrogen increased SOCS-2 and -3 mRNA levels by 2-fold in a time- and dose-dependent manner (P < 0.05). Estrogen induced murine SOCS-3 promoter activity by 2-fold (P < 0.05) in constructs containing a region between nucleotides −1862 and −855. Moreover, estrogen and GH had additive effects on the SOCS-3 promoter activity. In summary, estrogen, via ERα, up-regulated hepatic expression of SOCS-2 and -3, probably through transcriptional activation. This indicates a novel mechanism of estrogen regulation of cytokine action.

Download Full-text

Tumor-Stroma Crosstalk Enhances REG3A Expressions that Drive the Progression of Hepatocellular Carcinoma

International Journal of Molecular Sciences ◽

10.3390/ijms21020472 ◽

2020 ◽

Vol 21 (2) ◽

pp. 472 ◽

Cited By ~ 2

Author(s):

Yuri Cho ◽

Min Ji Park ◽

Koeun Kim ◽

Jae-Young Park ◽

Jihye Kim ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Microarray Analysis ◽

Cdna Microarray ◽

Tumor Stroma ◽

Mrna Levels ◽

Dependent Manner ◽

Cdna Microarray Analysis ◽

Hcc Cells

Abstract: Background: Crosstalk between tumors and their microenvironment plays a crucial role in the progression of hepatocellular carcinoma (HCC). However, there is little existing information about the key signaling molecule that modulates tumor-stroma crosstalk. Methods: Complementary DNA (cDNA) microarray analysis was performed to identify the key molecule in tumor-stroma crosstalk. Subcutaneous xenograft in vivo murine model, immunoblotting, immunofluorescence, and real-time polymerase chain reaction using HCC cells and tissues were performed. Results: The key molecule, regenerating gene protein-3A (REG3A), was most significantly enhanced when coculturing HCC cells and activated human hepatic stellate cells (HSCs) (+8.2 log) compared with monoculturing HCC cells using cDNA microarray analysis. Downregulation of REG3A using small interfering RNA significantly decreased the proliferation of HSC-cocultured HCC cells in vitro and in vivo, and enhanced deoxycholic acid-induced HCC cell apoptosis. Crosstalk-induced REG3A upregulation was modulated by platelet-derived growth factor ββ (PDGF-ββ) in p42/44-dependent manner. REG3A mRNA levels in human HCC tissues were upregulated 1.8-fold compared with non-tumor tissues and positively correlated with PDGF-ββ levels. Conclusions: REG3A/p42/44 pathway/PDGF-ββ signaling plays a significant role in hepatocarcinogenesis via tumor-stroma crosstalk. Targeting REG3A is a potential novel therapeutic target for the management of HCCs by inhibiting crosstalk between HCC cells and HSCs.

Download Full-text

Human Proximal Tubule Epithelial Cells (HK-2) as a Sensitive In Vitro System for Ochratoxin A Induced Oxidative Stress

Toxins ◽

10.3390/toxins13110787 ◽

2021 ◽

Vol 13 (11) ◽

pp. 787

Author(s):

Enrique García-Pérez ◽

Dojin Ryu ◽

Hwa-Young Kim ◽

Hae Dun Kim ◽

Hyun Jung Lee

Keyword(s):

Oxidative Stress ◽

Ochratoxin A ◽

Cell Lines ◽

Time Dependent ◽

Mrna Levels ◽

Dependent Manner ◽

In Vitro System ◽

Glutathione Peroxidase 1 ◽

Glucose 6 Phosphate Dehydrogenase

Ochratoxin A (OTA) is a mycotoxin that is potentially carcinogenic to humans. Although its mechanism remains unclear, oxidative stress has been recognized as a plausible cause for the potent renal carcinogenicity observed in experimental animals. The effect of OTA on oxidative stress parameters in two cell lines of LLC-PK1 and HK-2 derived from the kidneys of pig and human, respectively, were investigated and compared. We found that the cytotoxicity of OTA on LLC-PK1 and HK-2 cells was dose- and time-dependent in both cell lines. Furthermore, increased intracellular reactive oxygen species (ROS) induced by OTA in both cell lines were observed in a time-dependent manner. Glutathione (GSH) was depleted by OTA at >48 h in HK-2 but not in LLC-PK1 cells. While the mRNA levels of glucose-6-phosphate dehydrogenase (G6PD) and glutathione peroxidase 1 (GPX1) in LLC-PK1 were down-regulated by 0.67- and 0.66-fold, respectively, those of catalase (CAT), glutathione reductase (GSR), and superoxide dismutase 1 (SOD) in HK-2 were up-regulated by 2.20-, 2.24-, and 2.75-fold, respectively, after 72 h exposure to OTA. Based on these results, we conclude that HK-2 cells are more sensitive to OTA-mediated toxicity than LLC-PK1, and OTA can cause a significant oxidative stress in HK-2 as indicated by changes in the parameter evaluated.

Download Full-text

Transendothelial movement of adiponectin is restricted by glucocorticoids

Journal of Endocrinology ◽

10.1530/joe-16-0363 ◽

2017 ◽

Vol 234 (2) ◽

pp. 101-114 ◽

Cited By ~ 2

Author(s):

Thanh Q Dang ◽

Nanyoung Yoon ◽

Helen Chasiotis ◽

Emily C Dunford ◽

Qilong Feng ◽

...

Keyword(s):

Critical Role ◽

Endothelial Permeability ◽

The Body ◽

Diabetic Rat ◽

Mrna Levels ◽

Endothelial Monolayers ◽

Basolateral Side ◽

Induced Changes

Altered permeability of the endothelial barrier in a variety of tissues has implications both in disease pathogenesis and treatment. Glucocorticoids are potent mediators of endothelial permeability, and this forms the basis for their heavily prescribed use as medications to treat ocular disease. However, the effect of glucocorticoids on endothelial barriers elsewhere in the body is less well studied. Here, we investigated glucocorticoid-mediated changes in endothelial flux of Adiponectin (Ad), a hormone with a critical role in diabetes. First, we used monolayers of endothelial cells in vitro and found that the glucocorticoid dexamethasone increased transendothelial electrical resistance and reduced permeability of polyethylene glycol (PEG, molecular weight 4000 Da). Dexamethasone reduced flux of Ad from the apical to basolateral side, measured both by ELISA and Western blotting. We then examined a diabetic rat model induced by treatment with exogenous corticosterone, which was characterized by glucose intolerance and hyperinsulinemia. There was no change in circulating Ad but less Ad protein in skeletal muscle homogenates, despite slightly higher mRNA levels, in diabetic vs control muscles. Dexamethasone-induced changes in Ad flux across endothelial monolayers were associated with alterations in the abundance of select claudin tight junction (TJ) proteins. shRNA-mediated knockdown of one such gene, claudin-7, in HUVEC resulted in decreased TEER and increased adiponectin flux, confirming the functional significance of Dex-induced changes in its expression. In conclusion, our study identifies glucocorticoid-mediated reductions in flux of Ad across endothelial monolayers in vivo and in vitro. This suggests that impaired Ad action in target tissues, as a consequence of reduced transendothelial flux, may contribute to the glucocorticoid-induced diabetic phenotype.

Download Full-text

A preprogalanin cDNA from the turtle pituitary and regulation of its gene expression

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00452.2006 ◽

2007 ◽

Vol 292 (4) ◽

pp. R1649-R1656 ◽

Cited By ~ 2

Author(s):

John Yuh-Lin Yu ◽

Chin-Hon Pon ◽

Hui-Chen Ku ◽

Chih-Ting Wang ◽

Yung-Hsi Kao

Keyword(s):

Mrna Expression ◽

Untranslated Region ◽

Signal Sequence ◽

Biological Effects ◽

In Vitro Study ◽

The Body ◽

Mrna Levels ◽

Coding Region ◽

Reading Frame

Galanin is a hormone 29 or 30 amino acids (aa) long that is widely distributed within the body and exerts numerous biological effects in vertebrates. To fully understand its physiological roles in reptiles, we analyzed preprogalanin cDNA structure and expression in the turtle pituitary. Using the Chinese soft-shell turtle ( Pelodiscus sinensis order Testudines), we obtained a 672-base pair (bp) cDNA containing a 99-bp 5′-untranslated region, a 324-bp preprogalanin coding region, and a 249-bp 3′-untranslated region. The open-reading frame encoded a 108-aa preprogalanin protein with a putative 23-aa signal sequence at the NH2 terminus. Based on the location of putative Lys-Arg dibasic cleavage sites and an amidation signal of Gly-Lys-Arg, we propose that turtle preprogalanin is processed to yield a 29-aa galanin peptide with Gly1 and Thr29 substitutions and a COOH-terminal amidation. Sequence comparison revealed that turtle preprogalanin and galanin-29 had 48–81% and 76–96% aa identities with those of other vertebrates, respectively, suggesting their conservative nature. Expression of the turtle galanin gene was detected in the pituitary, brain, hypothalamus, stomach, liver, pancreas, testes, ovaries, and intestines, but not in the adipose or muscle tissues, suggesting tissue-dependent differences. An in vitro study that used pituitary tissue culture indicated that treatment with 17β-estradiol, testosterone, or gonadotropin-releasing hormone resulted in increased galanin mRNA expression with dose- or time-dependent differences, whereas leptin and neuropeptide Y reduced galanin mRNA levels. These results suggest a hormone-dependent effect on hypophyseal galanin mRNA expression.

Download Full-text

ZAP-70 Expression Is Associated with Increased Migration to SDF-1 in Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v108.11.587.587 ◽

2006 ◽

Vol 108 (11) ◽

pp. 587-587

Author(s):

Yuji Miura ◽

Elinor Lee ◽

Federica Gibellini ◽

Therese White ◽

Gerald Marti ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cxcr4 Expression ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Short Exposure ◽

Mrna Levels ◽

Dependent Manner ◽

Western Blots ◽

Stroma Cell

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature B lymphocytes in the peripheral blood (PB), lymph nodes (LN) and bone marrow (BM). Increasing evidence suggests that CLL cells depend on survival and proliferation signals provided by stroma cells in LN and BM. The chemokine receptor CXCR4 (CD184) and its ligand stromal cell-derived factor-1 (SDF-1) play an important role in trafficking of lymphocytes and may guide CLL cells to stroma cell niches. ZAP70 expression has prognostic value in CLL but the functional consequences of ZAP70 expression remain incompletely defined. Given that ZAP70 has been implicated in CXCR4 signaling its expression could enhance migration to SDF-1 and thereby promote interactions with stroma cells. As measured by flow cytometry, CXCR4 expression on leukemic cells obtained from different anatomic sites differed; cells from the PB (n=24, median 71% above isotype control) expressed CXCR4 more strongly than cells from BM (n=21, median 39%) and from LN (n=9, median 24%). Expression of CD69, an activation marker, followed a reverse pattern with cells from LN and BM typically showing higher expression than cells from PB, albeit with not detectable difference in expression in several patients. In vitro CLL cells from PB migrated in a dose dependent manner to SDF-1, and cells that had migrated down-modulated CXCR4 expression (89% before migration - 54% after migration). After exposure to SDF-1 CXCR4 expression decreased rapidly and remained virtually absent for at least 24 hours. Several mechanisms apparently decrease CXCR4 expression after contact with SDF-1, including internalization (given rapid re-expression of CXCR4 when SDF-1 is washed off after short exposure), protein degradation or inhibition of translation (evidenced by a decrease in total CXCR4 protein on Western blots), and mRNA degradation or transcriptional inhibition (decrease in mRNA levels more than 6 hours from SDF-1 exposure). In vitro migration of ZAP70(+) CLL cells toward SDF-1 through a 5μm membrane (Migration Index [MI] of 12.0, n=5) was significantly increased compared to ZAP70(−) CLL cells (MI of 2.9, n=4, p<0.05). To exclude effects of contaminating cells we repeated these assays with purified CLL cells (negative selection) with similar results. To model the complex interactions of CLL cells with stroma, we cultured PB derived leukemic cells with or without murine marrow stroma cells (S17). CXCR4 expression on CD19+ cells decreased from 90% without S17 to 50% when cultured on S17 cells, consistent with the known SDF-1 secretion by the murine stroma cell line. Conversely, CD69 expression increased from 58% without S17 to 71% with S17 cells. In addition, culturing of CLL cells on an S17 stroma cell layer extended their survival by several weeks when compared to cultures without S17 cells. Our data is consistent with a model in which CLL cells migrate along an SDF-1 gradient to stroma cell niches in BM and LN where they are activated. ZAP70 expression is associated with more effective migration in an SDF-1 gradient and thereby may facilitate access to growth and survival signals which then could contribute to the more progressive nature of ZAP70(+) CLL. The interaction between leukemic cells and stroma may represent a novel target for therapy of patients with CLL.

Download Full-text

Comparison of the Effects on Reversal of Multi-Drug Resistance of 5-Bromotetrandrine and Tetrandrine in K562/A02 Cell Line in Vivo and in Vitro

Blood ◽

10.1182/blood.v112.11.5059.5059 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5059-5059

Author(s):

Bao-An Chen ◽

Jue-qiong Wang ◽

Jian Cheng ◽

Feng Gao ◽

Wen-lin Xu ◽

...

Keyword(s):

Cell Line ◽

Nude Mice ◽

Leukemia Cell Line ◽

Multi Drug Resistance ◽

Human Leukemia ◽

Mrna Levels ◽

Dependent Manner ◽

Intracellular Accumulation

Abstract Objective This study was to compare the reversal effect of 5-bromotetrandrine (BrTet) with Tetrandrine (Tet) when combined with ADM on multidrug resistance cell line K562/A02 and to investigate the reversal mechanism of this new derivative. Methods The protein levels of P-glycoprotein (P-gp) were detected by fluorospectrophotometry and Western blot. The mRNA levels of P-gp were determined by RT-PCR. The in vivo effect of Tet was investigated using nude mice grafted with sensitive human leukemia cell line K562 and MDR cell line K562/A02. Results Flow cytometry assay showed that 1.0 μMol/L BrTet significantly increased the apoptosis percentage. BrTet also enhanced the intracellular accumulation of ADM in K562/A02 cells and its potency was greater than that of Tet at the same concentrations. BrTet inhibited the overexpression of P-gp and down regulated MDR1 mRNA expression in K562/A02 cells in a dose-dependent manner. In nude mice bearing K562 xenografts on the left flank and K562/A02 xenografts on the right flank, i.p. injection of 10 mg/kg BrTet significantly enhanced the antitumor activity of ADM against K562/A02 xenografts with inhibitory rates of 26.1%, while ADM alone inhibited the growth of KBv200 xenografts by only 5.8%. Conclusion BrTet showed significant MDR reversal activity in vitro and in vivo. Its activity may be related to the inhibition of P-gp overexpression and the increase in intracellular accumulation of anticancer drugs, which lead to more K562/A02 cells apoptosis.

Download Full-text

A Dairy Bacterium Displays InVitro Probiotic Properties for the Pharyngeal Mucosa by Antagonizing Group A Streptococci and Modulating the Immune Response

Infection and Immunity ◽

10.1128/iai.00559-10 ◽

2010 ◽

Vol 78 (11) ◽

pp. 4734-4743 ◽

Cited By ~ 24

Author(s):

Simone Guglielmetti ◽

Valentina Taverniti ◽

Mario Minuzzo ◽

Stefania Arioli ◽

Ivan Zanoni ◽

...

Keyword(s):

Immune Response ◽

The Body ◽

Lactobacillus Helveticus ◽

Hypopharyngeal Carcinoma ◽

Probiotic Properties ◽

Dependent Manner ◽

Necrosis Factor Alpha ◽

Gm Csf ◽

Tnf Α

ABSTRACT The probiotic approach represents an alternative strategy in the prevention and treatment of infectious diseases, not only at the intestinal level but also at other sites of the body where the microbiota plays a role in the maintenance of physiological homeostasis. In this context, we evaluated in vitro the potential abilities of probiotic and dairy bacteria in controlling Streptococcus pyogenes infections at the pharyngeal level. Initially, we analyzed bacterial adhesion to FaDu hypopharyngeal carcinoma cells and the ability to antagonize S. pyogenes on FaDu cell layers and HaCat keratinocytes. Due to its promising adhesive and antagonistic features, we studied the dairy strain Lactobacillus helveticus MIMLh5, also through in vitro immunological experiments. First, we performed quantification of several cytokines and measurement of NF-κB activation in FaDu cells. MIMLh5 efficiently reduced the induction of interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha (TNF-α), in a dose-dependent manner. After stimulation of cells with IL-1β, active NF-κB was still markedly lowered. Nevertheless, we observed an increased secretion of IL-6, gamma interferon (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) under these conditions. These effects were associated with the ability of MIMLh5 to enhance the expression of the heat shock protein coding gene hsp70. In addition, MIMLh5 increased the GM-CSF/G-CSF ratio. This is compatible with a switch of the immune response toward a TH1 pathway, as supported by our observation that MIMLh5, once in contact with bone marrow-derived dendritic cells, triggered the secretion of TNF-α and IL-2. In conclusion, we propose MIMLh5 as a potential probiotic bacterium for the human pharynx, with promising antagonistic and immunomodulatory properties.

Download Full-text