Stereoselective regulation of MDR1 expression in Caco-2 cells by cetirizine enantiomers

MDR1-encoded P-glycoprotein (P-gp) is a drug efflux transporter mainly expressed in liver, kidney, intestine, brain (at the level of the blood-brain barrier), and placenta. It thus plays important roles in drug absorption, distribution, and excretion. Cetirizine is a second-generation nonsedating antihistamine used to treat allergic disease of respiratory system, skin and eyes. To evaluate P-gp expression and function in Caco-2 cells pretreated with cetirizine enantiomers, we assessed the sensitivity of Caco-2 cells to paclitaxel using the MTT assay and the polarized transport of rhodamine-123 and doxorubicin across Caco-2 monolayers. RT-PCR and flow cytometry were used to assay MDR1 mRNA and P-gp protein respectively. The sensitivity of Caco-2 cells to paclitaxel decreased significantly after cells were pretreated with 100 microM R-cetirizine but increased upon treatment with S-cetirizine. The efflux of rhodamine-123 and doxorubicin was enhanced significantly after Caco-2 monolayers were pretreated with 100 microM R-cetirizine but was reduced by S-cetirizine. The MDR1 mRNA and P-gp levels in Caco-2 cells were increased by 100 microM R-cetirizine and decreased by 100 microM S-cetirizine. These results suggest that R-cetirizine up-regulates MDR1 expression while S-cetirizine down-regulates MDR1 expression.     

Stereoselective transport and uptake of propranolol across human intestinal Caco‐2 cell monolayers

The transport and uptake of individual propranolol (PPL) enantiomers were studied in human intestinal Caco‐2 cell monolayers, and a reversed‐phase HPLC‐UV assay was used for quantitative analysis. S‐PPL and R‐PPL across Caco‐2 cell monolayers was determined in the concentrations range of 10–500 μM in both apical (AP) to basolateral (BL) and BL to AP directions. S‐PPL exhibited greater permeability than R‐PPL in the AP to BL direction, whereas in the BL to AP direction S‐enantiomer transported less than R‐enantiomer. Uptake of R‐PPL was significantly higher than that of S‐PPL either from AP side or from BL side. The statistically significant differences in uptake were observed at the concentrations range from 10 to 50 μM. Furthermore, the apparent Michaelis constant (K_m) and maximal velocity (V_max) also showed significant difference between the two enantiomers. Moreover, the AP to BL transport of PPL enantiomer was markedly decreased by lowering the pH of the apical side but it did not affect the stereoselectivity of PPL across Caco‐2 cell monolayers. The transport and uptake of PPL in the BL to AP direction was not influenced by several protein inhibitors. The results suggest that PPL enantiomers showed stereoselective transport and uptake across the Caco‐2 cell monolayers. A special transport mechanism capable of directing the PPL enantiomers might be present in the Caco‐2 monolayers. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Determination of the stereoselectivity of chiral drug transport across Caco-2 cell monolayers

This study aimed to determine the transport characteristics of chiral drug enantiomers across Caco-2 cell monolayers as a model of human intestinal epithelial membrane. Esmolol was chosen as a model drug, and the study focused on the transepithelial transport of esmolol enantiomers in this in vitro model system. Separation and quantitation of (S)- and (R)-esmolol were performed by RP-HPLC with the use of GITC as a precolumn derivatizing agent. Bidirectional transport studies of 5.0-400.0 micromol/l esmolol demonstrated that the two enantiomers were transported mainly by a passive, transcellular mechanism. At concentrations of 5.0-100.0 micromol/l, enantioselective permeability of esmolol was observed. In the absorptive transport, Papp of (S)-esmolol was smaller than (R)-esmolol and vice versa for secretory transport. The enantioselectivity disappeared when the drug concentration was increased to 200.0 micromol/l. In conclusion, the transport characteristics of (S)- and (R)-esmolol were distinctly different. An enantioselective carrier-mediated mechanism in addition to passive diffusion was involved in the transport process of esmolol across Caco-2 cell monolayers.     

Unusual sterolic mixture,and 24‐isopropylcholesterol,from the sponge Ciocalypta sp. reduce cholesterol uptake and basolateral secretion in Caco‐2 cells

An unusual sterolic mixture (82.3% of 24‐isopropylated sterols) and its major component, 24‐isopropylcholesterol, isolated from a marine sponge, Ciocalypta sp. (Halichondriidae), reduce cholesterol uptake, basolateral secretion and ACAT‐2 mRNA expression and increase the expression of ABCA1 mRNA in Caco‐2 cells. The decreases of cholesterol uptake and secretion induced by 24‐isopropylcholesterol alone were more than that of both the sterolic mixture and β‐sitosterol. These data add a new sterol, 24‐isopropylcholesterol, to sterols that may reduce intestinal cholesterol absorption. J. Cell. Biochem. 106: 659–665, 2009. © 2009 Wiley‐Liss, Inc.     

Confirming therapeutic target of protopine using immobilized β2‐adrenoceptor coupled with site‐directed molecular docking and the target‐drug interaction by frontal analysis and injection amount–dependent method

Drug‐protein interaction analysis is pregnant in designing new leads during drug discovery. We prepared the stationary phase containing immobilized β₂‐adrenoceptor (β ₂‐ AR) by linkage of the receptor on macroporous silica gel surface through N ,N ′‐carbonyldiimidazole method. The stationary phase was applied in identifying antiasthmatic target of protopine guided by the prediction of site‐directed molecular docking. Subsequent application of immobilized β ₂‐ AR in exploring the binding of protopine to the receptor was realized by frontal analysis and injection amount–dependent method. The association constants of protopine to β ₂‐ AR by the 2 methods were (1.00 ± 0.06) × 10⁵M⁻¹ and (1.52 ± 0.14) × 10⁴M⁻¹. The numbers of binding sites were (1.23 ± 0.07) × 10⁻⁷M and (9.09 ± 0.06) × 10⁻⁷M, respectively. These results indicated that β ₂‐ AR is the specific target for therapeutic action of protopine in vivo. The target‐drug binding occurred on Ser¹⁶⁹ in crystal structure of the receptor. Compared with frontal analysis, injection amount–dependent method is advantageous to drug saving, improvement of sampling efficiency, and performing speed. It has grave potential in high‐throughput drug‐receptor interaction analysis.     

Cyclo‐oxygenase 2 up‐regulates the effect of multidrug resistance

COX‐2 (cyclo‐oxygenase 2), an inducible form of the enzyme that catalyses the first step in the synthesis of prostanoids, is associated with inflammatory diseases and carcinogenesis, which is suspected to promote angiogenesis and tissue invasion of tumours and resistance to apoptosis. COX‐2 is also involved in drug resistance and poor prognosis of many neoplastic diseases or cancers. The activation of the COX‐2/PGE₂ (prostaglandin E₂)/prostaglandin E receptor signal pathway can up‐regulate the expression of all three ABC (ATP‐binding‐cassette) transporters, MDR1/P‐gp (multidrug resistance/P‐glycoprotein), MRP1 (multidrug‐resistance protein 1) and BCRP (breast‐cancer‐resistance protein), which encode efflux pumps, playing important roles in the development of multidrug resistance. In addition, COX inhibitors inhibit the expression of MDR1/P‐gp, MRP1 and BCRP and enhance the cytotoxicity of anticancer drugs. Therefore we can use the COX inhibitors to potentialize the effects of chemotherapeutic agents and reverse multidrug resistance to facilitate the patient who may benefit from addition of COX inhibitors to standard cytotoxic therapy.     

Comparative Cytotoxicity,Oxidative Stress,and Cytokine Secretion Induced by Two Cyanotoxin Variants,Microcystin LR and RR,in Human Intestinal Caco‐2 Cells

While MC‐LR and MC‐RR share significant structural similarity, MC‐RR is less cytotoxic than MC‐LR. In the current study, we have compared the effects of MC‐LR and MC‐RR in Caco‐2 cells by evaluating cytotoxicity, oxidative stress (reactive oxygen species production), and the cellular proinflammatory response (IL‐6 and IL‐8 production). Following treatment with 100 µM microcystins (MC), cytotoxicity was two‐fold greater with MC‐LR as compared to MC‐RR after 24 h exposure. Whereas the reactive oxygen species production and IL‐6 secretion were similar following a 24‐h treatment with either MC, 100 µM MC‐LR induced a five‐fold greater IL‐8 secretion when compared to MC‐RR. Our study has demonstrated that, although both MC‐LR and MC‐RR induced some cytotoxicity in human intestinal cells, a major difference in IL‐8 production was observed between the two variants. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:253‐258, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21482     

Proinflammatory interleukins' production by adipose tissue‐derived mesenchymal stromal cells: the impact of cell culture conditions and cell‐to‐cell interaction

The impact of culture conditions and interaction with activated peripheral blood mononuclear cells on the interleukin (IL) gene expression profile and proinflammatory IL‐6 and IL‐8 production by adipose‐derived stromal cells (ASCs) was investigated. A microarray analysis revealed a wide range of IL genes either under standard (20%) or hypoxic (5%) O₂ concentrations, some highly up‐regulated at hypoxia. IL‐6 and IL‐8 production was inversely dependent on cell culture density. In early (first–third) passages, IL‐6 and IL‐8 concentration was higher at 20% O₂ and in late (8th‐12th) passages under 5% O₂. Interaction between ASCs and mononuclear cells in indirect setting was accompanied with a significant decrease of IL‐6 and did not result in the elevation of IL‐8 concentration. Thereby, the production of proinflammatory interleukins (IL‐6 and IL‐8) may be affected by the ASC intrinsic features (density in culture, and duration of expansion), as well as by microenvironmental factors, such as hypoxia and the presence of blood‐borne cells. These data are important for elucidating ASC paracrine activity regulation in vitro. They would also be on demand for optimisation of the cell therapy protocols, based on the application of ASC biologically active substances. Copyright © 2015 John Wiley & Sons, Ltd. SIGNIFICANCE PARAGRAPH Ex vivo expansion is widely used for increasing the number of adipose‐derived stromal cells (ASCs) and improving of their quality. The present study was designed to elucidate the particular factors influencing the interleukin production in ASCs. The presented data specified the parameters (i.e. cell density, duration of cultivation, hypoxia, etc.) that should be taken in mind when ASCs are intended to be used in protocols implying their paracrine activity. These data would be of considerable interest for researchers and clinicians working in the biomedical science.     

LysoTracker and MitoTracker Red are transport substrates of P‐glycoprotein: implications for anticancer drug design evading multidrug resistance

LysoTracker and MitoTracker Red are fluorescent probes widely used for viable cell staining of lysosomes and mitochondria, respectively. They are utilized to study organelle localization and their resident proteins, assess organelle functionality and quantification of organelle numbers. The ATP‐driven efflux transporter P‐glycoprotein (P‐gp) is expressed in normal and malignant tissues and extrudes structurally distinct endogenous and exogenous cytotoxic compounds. Thus, once aromatic hydrophobic compounds such as the above‐mentioned fluorescent probes are recognized as transport substrates, efflux pumps including P‐gp may abolish their ability to reach their cellular target organelles. Herein, we show that LysoTracker and MitoTracker Red are expelled from P‐gp‐overexpressing cancer cells, thus hindering their ability to fluorescently mark target organelles. We further demonstrate that tariquidar, a potent P‐gp transport inhibitor, restores LysoTracker and MitoTracker Red cell entry. We conclude that LysoTracker and MitoTracker Red are P‐gp transport substrates, and therefore, P‐gp expression must be taken into consideration prior to cellular applications using these probes. Importantly, as MitoTracker was a superior P‐gp substrate than LysoTracker Red, we discuss the implications for the future design of chemotherapeutics evading cancer multidrug resistance. Furthermore, restoration of MitoTracker Red fluorescence in P‐gp‐overexpressing cells may facilitate the identification of potent P‐gp transport inhibitors (i.e. chemosensitizers).     

MeCP2‐421‐mediated RPE epithelial‐mesenchymal transition and its relevance to the pathogenesis of proliferative vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is a blinding eye disease. Epithelial‐mesenchymal transition (EMT) of RPE cells plays an important role in the pathogenesis of PVR. In the current study, we sought to investigate the role of the methyl‐CpG‐binding protein 2 (MeCP2), especially P‐MeCP2‐421 in the pathogenesis of PVR. The expressions of P‐MeCP2‐421, P‐MeCP2‐80, PPAR‐γ and the double labelling of P‐MeCP2‐421 with α‐SMA, cytokeratin, TGF‐β and PPAR‐γ in human PVR membranes were analysed by immunohistochemistry. The effect of knocking down MeCP2 using siRNA on the expressions of α‐SMA, phospho‐Smad2/3, collagen I, fibronectin and PPAR‐γ; the expression of α‐SMA stimulated by recombinant MeCP2 in ARPE‐19; and the effect of TGF‐β and 5‐AZA treatment on PPAR‐γ expression were analysed by Western blot. Chromatin immunoprecipitation was used to determine the binding of MeCP2 to TGF‐β. Our results showed that P‐MeCP2‐421 was highly expressed in PVR membranes and was double labelled with α‐SMA, cytokeratin and TGF‐β, knocking down MeCP2 inhibited the activation of Smad2/3 and the expression of collagen I and fibronectin induced by TGF‐β. TGF‐β inhibited the expression of PPAR‐γ, silence of MeCP2 by siRNA or using MeCP2 inhibitor (5‐AZA) increased the expression of PPAR‐γ. α‐SMA was up‐regulated by the treatment of recombinant MeCP2. Importantly, we found that MeCP2 bound to TGF‐β as demonstrated by Chip assay. The results suggest that MeCP2 especially P‐MeCP2‐421 may play a significant role in the pathogenesis of PVR and targeting MeCP2 may be a potential therapeutic approach for the treatment of PVR.     

The advanced glycation end product Nϵ‐carboxymethyllysine and its precursor glyoxal increase serotonin release from Caco‐2 cells

Advanced glycation end products (AGEs), comprising a highly diverse class of Maillard reaction compounds formed in vivo and during heating processes of foods, have been described in the progression of several degenerative conditions such as Alzheimer's disease and diabetes mellitus. N^?‐Carboxymethyllysine (CML) represents a well‐characterized AGE, which is frequently encountered in a Western diet and is known to mediate its cellular effects through binding to the receptor for AGEs (RAGE). As very little is known about the impact of exogenous CML and its precursor, glyoxal, on intestinal cells, a genome‐wide screening using a customized microarray was conducted in fully differentiated Caco‐2 cells. After verification of gene regulation by qPCR, functional assays on fatty acid uptake, glucose uptake, and serotonin release were performed. While only treatment with glyoxal showed a slight impact on fatty acid uptake (P < 0.05), both compounds reduced glucose uptake significantly, leading to values of 81.3% ± 22.8% (500 μM CML, control set to 100%) and 68.3% ± 20.9% (0.3 μM glyoxal). Treatment with 500 μM CML or 0.3 μM glyoxal increased serotonin release (P < 0.05) to 236% ± 111% and 264% ± 66%, respectively. Co‐incubation with the RAGE antagonist FPS‐ZM1 reduced CML‐induced serotonin release by 34%, suggesting a RAGE‐mediated mechanism. Similarly, co‐incubation with the SGLT‐1 inhibitor phloridzin attenuated serotonin release after CML treatment by 32%, hinting at a connection between CML‐stimulated serotonin release and glucose uptake. Future studies need to elucidate whether the CML/glyoxal‐induced serotonin release in enterocytes might stimulate serotonin‐mediated intestinal motility.

     

Celecoxib enhanced the sensitivity of cancer cells to anticancer drugs by inhibition of the expression of P‐glycoprotein through a COX‐2‐Independent Manner

The P‐glycoprotein (p170, P‐gp) encoded by human MDR1 gene functions as a pump to extrude anticancer drugs from cancer cells. Over‐expression of p170 is closely related to primary and induced drug resistance phenotype of tumor cells. Recent studies have demonstrated that expression of cyclooxygenase‐2 (COX‐2) is positively correlated with the p170 level, suggesting a potential of COX‐2 specific inhibitors in regulation of cytotoxicity of anticancer agents. Celecoxib is one of the specific inhibitors of COX‐2 and has been widely used in clinic. However, its function in the response of cancer cells to anticancer drugs and the related mechanism are still waiting to be investigated. To explore the correlation of celecoxib and the p170‐mediated drug resistance, the role of celecoxib in drug response of cancer cells was analyzed with flow cytometry, high performance liquid chromatography (HPLC), and colony formation experiments. Celecoxib (50 µM) was found to significantly enhance the sensitivity of MCF‐7 and JAR/VP16 cells to tamoxifen and etoposide, respectively, by inhibition of p170 expression and increase in intracellular accumulation of the drugs. However, celecoxib did not affect pump function of p170. Enzyme activity and methylation analyses demonstrated that the inhibitory effect of celecoxib on p170 was independent on COX‐2 but closely related to hypermethylation of MDR1 gene promoter. Our study suggested that celecoxib was a potential agent for enhancement of the sensitivity of cancer cells to anticancer drugs. It also provided a links between epigenetic change of MDR1 and drug response of cancer cells. J. Cell. Biochem. 108: 181–194, 2009. © 2009 Wiley‐Liss, Inc.     

Dectin‐2‐mediated signaling triggered by the cell wall polysaccharides of Cryptococcus neoformans

Cryptococcus neoformans is rich in polysaccharides of the cell wall and capsule. Dectin‐2 recognizes high‐mannose polysaccharides and plays a central role in the immune response to fungal pathogens. Previously, we demonstrated Dectin‐2 was involved in the activation of dendritic cells upon stimulation with C. neoformans, suggesting the existence of a ligand recognized by Dectin‐2. In the present study, we examined the cell wall structures of C. neoformans contributing to the Dectin‐2‐mediated activation of immune cells. In a NFAT‐GFP reporter assay of the reported cells expressing Dectin‐2, the lysates, but not the whole yeast cells, of an acapsular strain of C. neoformans (Cap67) delivered Dectin‐2‐mediated signaling. This activity was detected in the supernatant of β‐glucanase‐treated Cap67 and more strongly in the semi‐purified polysaccharides of this supernatant using ConA‐affinity chromatography (ConA‐bound fraction), in which a large amount of saccharides, but not protein, were detected. Treatment of this supernatant with periodic acid and the addition of excessive mannose, but not glucose or galactose, strongly inhibited this activity. The ConA‐bound fraction of the β‐glucanase‐treated Cap67 supernatant was bound to Dectin‐2‐Fc fusion protein in a dose‐dependent manner and strongly induced the production of interleukin‐12p40 and tumour necrosis factor‐α by dendritic cells; this was abrogated under the Dectin‐2‐deficient condition. Finally, 98 kDa mannoprotein (MP98) derived from C. neoformans showed activation of the reporter cells expressing Dectin‐2. These results suggested that a ligand with mannose moieties may exist in the cell walls and play a critical role in the activation of dendritic cells during infection with C. neoformans.     

Aromatic‐interaction‐mediated inhibition of β‐amyloid assembly structures and cytotoxicity

Abnormal aggregation of β‐amyloid (Aβ) peptide plays an important role in the onset and progress of Alzheimer's disease (AD); hence, targeting Aβ aggregation is considered as an effective therapeutic strategy. Here, we studied the aromatic‐interaction‐mediated inhibitory effect of oligomeric polypeptides (K8Y8, K4Y8, K8W8) on Aβ42 fibrillization process. The polypeptides containing lysine as well as representative aromatic amino acids of tryptophan or tyrosine were found to greatly suppress the aggregation as evaluated by thioflavin T assay. Circular dichroism spectra showed that the β‐sheet formation of Aβ42 peptides decreased with the polypeptide additives. Molecular docking studies revealed that the oligomeric polypeptides could preferentially bind to Aβ42 through π–π stacking between aromatic amino acids and Phe19, together with hydrogen bonding. The cell viability assay confirmed that the toxicity of Aβ42 to SH‐SY5Y cells was markedly reduced in the presence of polypeptides. This study could be beneficial for developing peptide‐based inhibitory agents for amyloidoses. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Bradykinin‐mediated Ca2+ signalling regulates cell growth and mobility in human cardiac c‐Kit+ progenitor cells

Our recent study showed that bradykinin increases cell cycling progression and migration of human cardiac c‐Kit⁺ progenitor cells by activating pAkt and pERK1/2 signals. This study investigated whether bradykinin‐mediated Ca²⁺ signalling participates in regulating cellular functions in cultured human cardiac c‐Kit⁺ progenitor cells using laser scanning confocal microscopy and biochemical approaches. It was found that bradykinin increased cytosolic free Ca²⁺ () by triggering a transient Ca²⁺ release from ER IP3Rs followed by sustained Ca²⁺ influx through store‐operated Ca²⁺ entry (SOCE) channel. Blockade of B2 receptor with HOE140 or IP3Rs with araguspongin B or silencing IP3R3 with siRNA abolished both Ca²⁺ release and Ca²⁺ influx. It is interesting to note that the bradykinin‐induced cell cycle progression and migration were not observed in cells with siRNA‐silenced IP3R3 or the SOCE component TRPC1, Orai1 or STIM1. Also the bradykinin‐induced increase in pAkt and pERK1/2 as well as cyclin D1 was reduced in these cells. These results demonstrate for the first time that bradykinin‐mediated increase in free via ER‐IP3R3 Ca²⁺ release followed by Ca²⁺ influx through SOCE channel plays a crucial role in regulating cell growth and migration via activating pAkt, pERK1/2 and cyclin D1 in human cardiac c‐Kit⁺ progenitor cells.