LS180 Cell Research Articles

Copper(II) Complexes with 1-(Isoquinolin-3-yl)heteroalkyl-2-ones: Synthesis, Structure and Evaluation of Anticancer, Antimicrobial and Antioxidant Potential.

Four copper(II) complexes, C1-4, derived from 1-(isoquinolin-3-yl)heteroalkyl-2-one ligands L1-4 were synthesized and characterized using an elemental analysis, IR spectroscopic data as well as single crystal X-ray diffraction data for complex C1. The stability of complexes C1-4 under conditions mimicking the physiological environment was estimated using UV-Vis spectrophotometry. The antiproliferative activity of both ligands L1-4 and copper(II) compounds C1-4 were evaluated using an MTT assay on four human cancer cell lines, A375 (melanoma), HepG2 (hepatoma), LS-180 (colon cancer) and T98G (glioblastoma), and a non-cancerous cell line, CCD-1059Sk (human normal skin fibroblasts). Complexes C1-4 showed greater potency against HepG2, LS180 and T98G cancer cell lines than etoposide (IC50 = 5.04-14.89 μg/mL vs. IC50 = 43.21->100 μg/mL), while free ligands L1-4 remained inactive in all cell lines. The prominent copper(II) compound C2 appeared to be more selective towards cancer cells compared with normal cells than compounds C1, C3 and C4. The treatment of HepG2 and T98G cells with complex C2 resulted in sub-G1 and G2/M cell cycle arrest, respectively, which was accompanied by DNA degradation. Moreover, the non-cytotoxic doses of C2 synergistically enhanced the cytotoxic effects of chemotherapeutic drugs, including etoposide, 5-fluorouracil and temozolomide, in HepG2 and T98G cells. The antimicrobial activities of ligands L2-4 and their copper(II) complexes C2-4 were evaluated using different types of Gram-positive bacteria, Gram-negative bacteria and yeast species. No correlation was found between the results of the antiproliferative and antimicrobial experiments. The antioxidant activities of all compounds were determined using the DPPH and ABTS radical scavenging methods. Antiradical tests revealed that among the investigated compounds, copper(II) complex C4 possessed the strongest antioxidant properties. Finally, the ADME technique was used to determine the physicochemical and drug-likeness properties of the obtained complexes.

Characteristics, Chemical Analysis and Biological Activities of Methanol Extracts of Lichens Pleurosticta Acetabulum and Cladonia Subulata

Abstract The aim of this study is to investigate the chemical composition of methanol extracts of the lichens Pleurosticta acetabulum and Cladonia subulata and their antioxidant, and anticancer activities. The phytochemical analysis of the extracts of lichens was determined by HPLC-UV method. The predominant phenolic compounds in these extracts were norstictic acid and salazinic acids in P. acetabulum, while hypoprotocetraric acid and fumarprotocetraric acid were the major metabolites detected in C. subulata. Total phenolics and flavonoids in the extracts were determined spectrophotometrically, with the varied amount from 21.31 to 73.45 mg GA/g and from 8.48 to 15.42 mg RU/g, respectively. The lichen extracts showed comparable and strong antioxidant activity, exhibited higher DPPH and hydroxyl radical scavengings, inhibitory activity towards lipid peroxidation and reducing power. Cytotoxic effects of lichens were tested against HeLa S3 and LS174 cell lines using MTT method.The cytotoxic effects of P. acetabulum and C. subulata extracts toward two cancer cell lines were in the range from 39.17 to >200 μg/mL IC50 value. The present study showed that the tested extracts of lichens demonstrated important antioxidant and anticancer effects. That suggests that these lichens can be used as new sources of the natural antioxidants and anti-cancer compounds.

Bilirubin, an endogenous antioxidant that affects p53 protein and its downstream apoptosis/autophagy-related genes in LS180 and SW480 cell culture models of colorectal cancer

BackgroundColorectal cancer (CRC) is one of the commonest neoplasms worldwide, which its pathogenesis is strongly correlated with p53 mutations. Antioxidants are believed to decelerate the CRC progression, possibly through interfering with p53 and its downstream target genes and mechanisms. Regarding the potential antioxidant effects of bilirubin, as an incredible endogenous antioxidant, we sought to investigate how bilirubin affected the expression levels of p53 protein and its downstream target genes, including Mdm2, Bcl-2, BECN1 and LC3, in LS180 and SW480 cell culture models of CRC. Methods and resultsUsing the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide) assay, 50 and 100 μM concentrations of bilirubin were determined to be non-toxic for both LS180 and SW480 cell lines. Western blot analysis was employed to evaluate the protein expression levels of p53. The results revealed that p53 protein levels were higher in LS180 cells treated with bilirubin compared to the control group. Notwithstanding, in SW480 cells, no considerable changes were observed in p53 protein levels of treated cells compared to the control ones. The quantitative reverse transcriptase-polymerase chain reaction (q RT-PCR) method was used to measure the mRNA expression levels of the apoptosis/autophagy-related genes, Mdm2, Bcl-2, BECN1, and LC3 , as the p53's downstream target genes. Consequently, the expression of Bcl-2 and Mdm2 genes were affected by p53, while BECN1 and LC3 expression levels were decreased in both cell lines. ConclusionBilirubin is an endogenous antioxidant with significant anti-tumor effects in the studied CRC cell lines, probably through the regulation of p53 protein expression levels and subsequent control of apoptosis and autophagy, as two key processes involved in cell survival and progression of tumor cells.

Anticancer potential of acetone extracts from selected Potentilla species against human colorectal cancer cells.

Cinquefoils have been widely used in local folk medicine in Europe and Asia to manage various gastrointestinal inflammations and/or infections, certain forms of cancer, thyroid gland disorders, and wound healing. In the present paper, acetone extracts from aerial parts of selected Potentilla species, namely P. alba (PAL7), P. argentea (PAR7), P. grandiflora (PGR7), P. norvegica (PN7), P. recta (PRE7), and the closely related Drymocalis rupestris (syn. P. rupestris) (PRU7), were analysed for their cytotoxicity and antiproliferative activities against human colon adenocarcinoma cell line LS180 and human colon epithelial cell line CCD841 CoN. Moreover, quantitative assessments of the total polyphenolic (TPC), total tannin (TTC), total proanthocyanidins (TPrC), total flavonoid (TFC), and total phenolic acid (TPAC) were conducted. The analysis of secondary metabolite composition was carried out by LC-PDA-HRMS. The highest TPC and TTC were found in PAR7 (339.72 and 246.92 mg gallic acid equivalents (GAE)/g extract, respectively) and PN7 (332.11 and 252.3 mg GAE/g extract, respectively). The highest TPrC, TFC, and TPAC levels were found for PAL7 (21.28 mg catechin equivalents (CAT)/g extract, 71.85 mg rutin equivalents (RE)/g extract, and 124.18 mg caffeic acid equivalents (CAE)/g extract, respectively). LC-PDA-HRMS analysis revealed the presence of 83 compounds, including brevifolincarboxylic acid, ellagic acid, pedunculagin, agrimoniin, chlorogenic acid, astragalin, and tiliroside. Moreover, the presence of tri-coumaroyl spermidine was demonstrated for the first time in the genus Potentilla. Results of the MTT assay revealed that all tested extracts decreased the viability of both cell lines; however, a markedly stronger effect was observed in the colon cancer cells. The highest selectivity was demonstrated by PAR7, which effectively inhibited the metabolic activity of LS180 cells (IC50 = 38 μg/ml), while at the same time causing the lowest unwanted effects in CCD841 CoN cells (IC50 = 1,134 μg/ml). BrdU assay revealed a significant decrease in DNA synthesis in both examined cell lines in response to all investigated extracts. It should be emphasized that the tested extracts had a stronger effect on colon cancer cells than normal colon cells, and the most significant antiproliferative properties were observed in the case of PAR7 (IC50 LS180 = 174 μg/ml) and PN7 (IC50 LS180 = 169 μg/ml). The results of LDH assay revealed that all tested extracts were not cytotoxic against normal colon epithelial cells, whereas in the cancer cells, all compounds significantly damaged cell membranes, and the observed effect was dose-dependent. The highest cytotoxicity was observed in LS180 cells in response to PAR7, which, in concentrations ranging from 25 to 250 μg/ml, increased LDH release by 110%–1,062%, respectively. Performed studies have revealed that all Potentilla species may be useful sources for anti-colorectal cancer agents; however, additional research is required to prove this definitively.

DNA-Binding Properties of Bis-N-substituted Tetrandrine Derivatives.

A series of bis-N-substituted tetrandrine derivatives carrying different aromatic substituents attached to both nitrogen atoms of the natural alkaloid were studied with double-stranded model DNAs (dsDNAs) to examine the binding properties and mechanism. Variable-temperature molecular recognition studies using UV–vis and fluorescence techniques revealed the thermodynamic parameters, ΔH, ΔS, and ΔG, showing that the tetrandrine derivatives exhibit high affinity toward dsDNA (K ≈ 105–107 M–1), particularly the bis(methyl)anthraquinone (BAqT) and bis(ethyl)indole compounds (BInT). Viscometry experiments, ethidium displacement assays, and molecular modeling studies enabled elucidation of the possible binding mode, indicating that the compounds exhibit a synergic interaction mode involving intercalation of one of the N-aryl substituents and interaction of the molecular skeleton in the major groove of the dsDNA. Cytotoxicity tests of the derivatives with tumor and nontumor cell lines demonstrated low cytotoxicity of these compounds, with the exception of the bis(methyl)pyrene (BPyrT) derivative, which is significantly more cytotoxic than the remaining derivatives, with IC50 values against the LS-180, A-549, and ARPE-19 cell lines that are similar to natural tetrandrine. Finally, complementary electrochemical characterization studies unveiled good electrochemical stability of the compounds.

Expression and methylation status of BTG2, PPP1CA, and PEG3 genes in colon adenocarcinoma cell lines: promising treatment targets.

This study investigated the association between methylation status and expression levels of BTG2, PPP1CA, and PEG3 genes in colon cancer. Aberrant DNA methylation is one of the most important epigenetic modifications in the development of cancer. Evidence indicates that hypermethylation of various tumor suppressor genes could be a potential mechanism of colon tumorigenesis. The expression levels of BTG2, PPP1CA, and PEG3 genes were evaluated in HT-29/219, HCT116, SW48, SW742, SW480, and LS180 cell lines using quantitative Real-Time PCR. The methylation status of BTG2 and PPP1CA was determined by methylation-specific PCR (MSP) method, and the methylation pattern of PEG3 was evaluated by bisulfite sequencing PCR (BSP). To investigate the effect of methylation on the expression of these genes, all colon cancer cell lines were treated by 5-Azacitidine (5-Aza) and/or Trichostatin A (TSA). The expression levels of BTG2, PPP1CA, and PEG3 were highly heterogeneous and quantitatively correlated to their promoter methylation status in the studied colon cancer cell lines. Treatment by 5-Aza and/or TSA increased the expression of the above-named genes in colon cancer cell lines. Overall, it seems that BTG2, PPP1CA, and PEG3 act as tumor suppressor genes in colon cancer, and methylation is a potential mechanism for their loss of expression. Therefore, these genes may be considered as suitable targets for demethylation approaches and, eventually, colon cancer treatment. Combined treatment by 5-Aza and TSA may be a promising therapeutic strategy for colon cancer treatment. Further studies may contribute to confirm these results.

Effect of Vitamin K-Mediated PXR Activation on Drug-Metabolizing Gene Expression in Human Intestinal Carcinoma LS180 Cell Line.

The pregnane X receptor (PXR) is the key regulator of our defense mechanism against foreign substances such as drugs, dietary nutrients, or environmental pollutants. Because of increased health consciousness, the use of dietary supplements has gradually increased, and most of them can activate PXR. Therefore, an analysis of the interaction between drugs and nutrients is important because altered levels of drug-metabolizing enzymes or transporters can remarkably affect the efficiency of a co-administered drug. In the present study, we analyzed the effect of vitamin K-mediated PXR activation on drug metabolism-related gene expression in intestine-derived LS180 cells via gene expression studies and western blotting analyses. We demonstrated that menaquinone 4 (MK-4), along with other vitamin Ks, including vitamin K1, has the potential to induce MDR1 and CYP3A4 gene expression. We showed that PXR knockdown reversed MK-4-mediated stimulation of these genes, indicating the involvement of PXR in this effect. In addition, we showed that the expression of MDR1 and CYP3A4 genes increased synergistically after 24 h of rifampicin and MK-4 co-treatment. Our study thus elucidates the importance of drug–nutrient interaction mediated via PXR.

Anticancer Potential of Sutherlandia frutescens and Xysmalobium undulatum in LS180 Colorectal Cancer Mini-Tumors

Colorectal cancer remains to be one of the leading causes of death worldwide, with millions of patients diagnosed each year. Although chemotherapeutic drugs are routinely used to treat cancer, these treatments have severe side effects. As a result, the use of herbal medicines has gained increasing popularity as a treatment for cancer. In this study, two South African medicinal plants widely used to treat various diseases, Sutherlandia frutescens and Xysmalobium undulatum, were evaluated for potential activity against colorectal cancer. This potential activity for the treatment of colorectal cancer was assessed relative to the known chemotherapeutic drug, paclitaxel. The cytotoxic activity was considered in an advanced three-dimensional (3D) sodium alginate encapsulated LS180 colorectal cancer functional spheroid model, cultured in clinostat-based rotating bioreactors. The LS180 cell mini-tumors were treated for 96 h with two concentrations of each of the crude aqueous extracts or paclitaxel. S. frutescens extract markedly decreased the soluble protein content, while decreasing ATP and AK per protein content to below detectable limits after only 24 h exposure. X. undulatum extract also decreased the soluble protein content, cell viability, and glucose consumption. The results suggested that the two phytomedicines have potential to become a source of new treatments against colorectal cancer.

Mentha mozaffarianii mediated biogenic zinc nanoparticles target selected cancer cell lines and microbial pathogens

Here we report the biosynthesis of zinc oxide nanoparticles (ZnO-NPs) using aqueous extract of medicinally active Mentha mozaffarianii for biomedical applications. ZnO-NPs were characterized by X-ray diffraction (XRD), Field Emission Scanning Electron Microscope (FE-SEM), Energy-dispersive X-ray (EDX) spectroscopy, and Fourier transform infrared (FT-IR) spectroscopy. The sharp and narrow diffraction peaks in the whole spectrum revealed that the ZnO-NPs are nanosized and well crystalized. FE-SEM analysis exhibited the spherical shape of the biosynthesized ZnO-NPs (mean diameter of about 20–29 nm). EDX analysis confirmed the formation of highly pure ZnO-NPs. ZnO-NPs exerted distinct effects on cancer cell lines while posing no impact on normal fibroblast cells. ZnO-NPs were primarily effective against HeLa (IC50: 50.1 μg/ml) cells, followed by MDA-MD231 (IC50: 54.9 μg/ml), and LS180 (63.4 μg/ml) cell lines. The biogenic ZnO-NPs revealed potent antimicrobial activity against two Gram-negative [Enterobacter aerogenes (maximum zone of inhibition (MZI): 20 mm) and Klebsiella pneumoniae (MZI: 22 mm)] and two Gram-positive [Bacillus subtilis (MZI: 23.2 mm) and Staphylococcus aureus] strains of bacteria, and four strains of fungi [C. glabrata (MZI: 17.5 mm), C. albicans (MZI: 21 mm), P. oryzae (MZI: 15 mm), C. neoformans (MZI: 16 mm)]. These findings provide preliminary information for the development of anticancer and antimicrobial drugs using biosynthesized ZnO-NPs.

Antimigraine Drug Avitriptan Is a Ligand and Agonist of Human Aryl Hydrocarbon Receptor That Induces CYP1A1 in Hepatic and Intestinal Cells.

The efforts for therapeutic targeting of the aryl hydrocarbon receptor (AhR) have emerged in recent years. We investigated the effects of available antimigraine triptan drugs, having an indole core in their structure, on AhR signaling in human hepatic and intestinal cells. Activation of AhR in reporter gene assays was observed for Avitriptan and to a lesser extent for Donitriptan, while other triptans were very weak or no activators of AhR. Using competitive binding assay and by homology docking, we identified Avitriptan as a low-affinity ligand of AhR. Avitriptan triggered nuclear translocation of AhR and increased binding of AhR in CYP1A1 promotor DNA, as revealed by immune-fluorescence microscopy and chromatin immune-precipitation assay, respectively. Strong induction of CYP1A1 mRNA was achieved by Avitriptan in wild type but not in AhR-knockout, immortalized human hepatocytes, implying that induction of CYP1A1 is AhR-dependent. Increased levels of CYP1A1 mRNA by Avitriptan were observed in human colon carcinoma cells LS180 but not in primary cultures of human hepatocytes. Collectively, we show that Avitriptan is a weak ligand and activator of human AhR, which induces the expression of CYP1A1 in a cell-type specific manner. Our data warrant the potential off-label therapeutic application of Avitriptan as an AhR-agonist drug.

Differential activation of human pregnane X receptor PXR by isomeric mono-methylated indoles in intestinal and hepatic in vitro models

Dietary and microbial indoles can act as ligands and activators of pregnane X receptor (PXR), with implications in human intestinal health. In the current study, we examined the effects of simple mono-methylated indoles (MMIs) on the activity and function of PXR, using a series of human hepatic and intestinal cell models. Indoles 1-MMI and 2-MMI strongly induced CYP3A4 and MDR1 mRNAs in human intestinal adenocarcinoma cells LS180, but not in primary human hepatocytes. The levels of CYP3A4 mRNA were increased by 1-MMI and 2-MMI in wild type, but not in PXR-knock-out human hepatic progenitor HepaRG cells, implying the involvement of PXR in CYP3A4 induction by MMIs. Utilizing reporter gene assay, we observed dose-dependent activation of PXR by all MMIs, and their efficacies and potencies were comparable. Tested MMIs also displayed moderate antagonist effects on PXR, revealing about partial agonist effects of these compounds. As demonstrated using the Chromatin immunoprecipitation assay (ChIP),1-MMI increased PXR occupancy of the CYP3A4 promoter. Time-Resolved Fluorescence Resonance Energy Transfer revealed that MMIs are weak ligands of human PXR. Collectively, we show that MMIs are ligands and partial agonists of human PXR, which induce PXR-regulated genes in human intestinal cells.

MLS128 antibody-induced suppression of colon cancer cell growth is mediated by a desmocollin and a 110 kDa glycoprotein.

Protein glycosylation is a diverse form of post-translational modification. Two to three consecutive O-linked N-acetylgalactosamines (Tn-antigens) are recognized by antibodies such as MLS128. MLS128 mAb inhibited cell growth and bound to a 110 kDa glycoprotein (GP) in LS180 and HT29 colon cancer cells. However, purification and identification of the 110 kDa GP was unsuccessful due to its low abundance. The present study used a highly sophisticated and sensitive mass spectrometry method to identify proteins immunoprecipitated with MLS128 and separated by two-dimensional gel electrophoresis. Three desmosome components were identified. Of these, desmocollin and desmoglein shared many similar characteristics, including molecular mass, pI, and potential Tn-antigen sites. Western blotting analyses of LS180 cell lysates revealed a common 110 kDa band recognized by MLS128 and anti-desmocollin, but not by anti-desmoglein. Immunofluorescence microscopy of LS180 cells revealed that desmocollin is membrane-bound, while desmoglein is primarily localized in the cytosol. Confocal microscopy demonstrated colocalization of the desmocollin-specific antibody with the MLS128 antibody on the cell membrane, suggesting that desmocollin may contain Tn-antigens recognized by MLS128. Treatment of LS180 cells with siRNA to knock down desmocollin expression or a desmocollin-specific antibody decreased cell viability, suggesting a critical role for this protein in cell growth and survival. N-glycosidase F digestion of the 110 kDa GP and desmocollin suggested that although both proteins contain N-glycosylation sites, they are not identical. These findings suggest that desmocollin colocalizes with the 110 kDa GP and that growth inhibition induced by the MLS128 antibody may be mediated through a mechanism that involves desmocollin.

Effect of UBE2Q on BECN and CEA Protein in Colorectal Cancer Cells

Background: Expression of UBE2Q1, UBE2Q2, and members of the ubiquitinconjugating enzyme family (E2) are affected in colorectal cancer (CRC). The BECN gene plays a key role in CRC cells. In gastrointestinal carcinoma therapy, tumorassociated antigens such as CEA are typically used.To investigate the association between UBE2Q1 and Beclin1 autophagy marker and CEA protein expression in LS180 CRC cell line. Materials and Methods: In this study, changes in the expression of BECN marker in LS180 cell lines with the vector containing UBE2Q1 were investigated using real-time PCR. The expression of CEA protein was also evaluated by western blotting. Statistical analyses were performed with Graph Pad Prism software. Results: The results indicated reduced expression of BECN autophagy marker (P=0). Therefore, in the presence of UBE2Q1, cancer cells have less ability to induce autophagy. However, CEA protein levels in LS180 transfected cells with a UBE2Q1- ORF-containing plasmid decreased when compared to non-transfected cells. Conclusion: The use of pharmacologic inhibitors related to the autophagy mechanism can be a novel approach in cancer therapy.

Cantharellus cibarius branched mannans inhibits colon cancer cells growth by interfering with signals transduction in NF-ĸB pathway

In order to evaluate the therapeutic potential of polysaccharides obtained from the widely consumed Cantharellus cibarius mushroom on NF-ĸB pathway involved in cancer cells proliferation, survival, and metastasis, their antiproliferative and cytotoxic properties in human colon cancer cells LS180 and human colon epithelial cells CCD841 CoN were studied. BrdU and LDH assays results show that a branched mannan isolated from C. cibarius (CC2a) was selective against colon cancer cells, suppressing their proliferation and destroying membrane integrity but at the same time not adversely affecting colon epithelial cells. Results of Western blotting, immunofluorescence, RealTime PCR revealed that CC2a anticancer abilities were accompanied by disorders in signals transduction in NF-ĸB pathway in particular inhibition of IκBα degradation, attenuation of activated NF-κB phosphorylation and the subsequent decrease of NF-κB nuclear level as well as significant down-regulation of NF-κB target genes BAX, BCL2, CCND1, MMP9, MYC, BIRC5 and the corresponding proteins (except Bax). Furthermore, CC2a treatment of LS180 cells resulted in perturbation in G0/G and S phases of the cell cycle also associated with a marked increase of DNA fragmentation as well as inhibition of cancer cells motility. Obtained results indicated C. cibarius branched mannans as a new option in fighting with colon cancer.

Astaxanthin induces apoptosis and increases activity of antioxidant enzymes in LS-180 cells

Astaxanthin, a Xanthophyll carotenoid, has strong antioxidant properties. Some studies have shown the effectiveness of this compound on the prevention and treatment of cancer. Therefore, the aim of this study was to evaluate the effects of astaxanthin on induction of apoptosis and antioxidant activity in the LS-180 cell line. In this experimental study, after the treatment of LS-180 50, 100 and 150 μm of Astaxanthin for 24 h, the expression levels of Bax, Bcl2 and Caspase3 genes were investigated by Real-time PCR. Also, the level of malondialdehyde, as an indicator of oxidative stress and activity of anti-superoxide dismutase enzymes, catalase and glutathione peroxidase was investigated by colorimetric methods. The results showed that astaxanthin increases the expression of Bax and Caspase3 genes and decreases that of Bcl2, thereby, inducing apoptosis and inhibiting growth and proliferation of the cells. Additionally, reduction in the levels of malondialdehyde was evident with a significant elevation in antioxidant activity mediated by the action of superoxide dismutase, catalase and glutathione peroxidase. These results suggest that astaxanthin has the potency to induce apoptosis in LS-180 cells by increasing the expression of apoptotic genes and activity of antioxidant enzymes. Thus, astaxanthin has potential in the prevention and treatment of cancer.

Development and validation of a rapid reversed-phase liquid chromatography method for CnAMP1 peptide quantification in human intestinal cell lines.

Plant foods are rich sources of biologically active peptides that may have a role in the prevention of diseases. Coconut water is a valuable beverage due to its nutrient composition and the presence of bioactive compounds, such as the peptide CnAMP1. It is unknown if CnAMP1 can be absorbed into intestinal cells. We, therefore, aimed to develop and validate a simple reversed-phase liquid chromatographic method to quantify the peptide in Caco-2 and LS180 cell lysates. CnAMP1 standards (1-200µmol/L) and spiked cell lysates were injected onto a Reprosil-Pur 120 C18-AQ column (4.6 × 250mm) using acetonitrile:water:trifluoroacetic acid (14.0:85.9:0.1, by volume) as mobile phase in isocratic mode at flow rate of 1mL/min. The method achieved rapid separation (total run time of 6min), with linear response, good sensitivity (limit of detection, 8.2ng; lower limit of quantification, 30.6ng) and no interfering peaks. Best recoveries (84-96%), accuracies (7.6-14.8%) and precision (1.5-8%) were found for LS180 cell lysates spiked with medium (50µmol/L) and high (100µmol/L) amounts of the peptide. Uptake assays detected no peptides in the cell lysates; however, after the first 15-min incubation CnAMP1 underwent partial hydrolysis upon incubation with LS180 cells (29%) and extensive hydrolysis with Caco-2 cells (93%).

Potential detoxification effect of active ingredients in liquorice by upregulating efflux transporter

BackgroundAs one of most widely used herbal medicine, liquorice exhibits diverse pharmacological activities, for instance, analgesic, antitussive, antiarrhythmic, anti-inflammatory, and immune regulation. Additionally, detoxification effects were observed in combination of liquorice with other herbal drugs. The mechanism of detoxification of liquorice has been extensively investigated through material basis and interference with CYPs though, investigations of its effect on transporters were very limited, according to the literature. PurposeThe objective of this study was attempt to investigate the effect of active ingredients existing in liquorice on the efflux transporters as to clarify the potential mechanism of detoxification of liquorice. MethodsMultiple analytical approaches have been explored, including flow cytometry, fluorescent detection, RT-PCR, Western blot to measure the function, activity as well as mRNA/protein expression of efflux transporters on LS-180 cell model after treatments with active compounds of liquorice. Additionally, Caco-2 cell model was utilized to further investigate the potential impact of those ingredients on efflux transporter. ResultsThe resulting data indicated that those active ingredients, including flavonoids (liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and licochalcone A) and pentacyclic triterpene saponin (glycyrrhetinic acid) were able to upregulate the expression of efflux transporters, for example P-gp, BCRP and MRP2. The gene expressions were approximately over 2.5 folds by comparison with that of control, and up to 13 folds and 16 folds for BCRP by isoliquiritin and isoliquiritigenin, and further confirmed by Western blot. The functional assay also supported up-regulation of efflux transporter by those ingredients. Flow cytometry study showed that the level of rhodamine123 as probe substrate in LS-180 cells decreased to approximately 50% after treatment with active ingredients of liquorice, compared with that of control. The fluorescent assay confirmed that change of rhodamine 123 was correlated with the concentrations of active ingredients given. The efflux transport of rhodamine 123 was enhanced in Caco-2 cell models as well. ConclusionThe study clarified potential detoxification mechanism of liquorice by up-regulating efflux transporter as to reduce absorption of xenobiotics across small intestinal membrane, which provided a new insight into pharmacological function of liquorice.

Lipovelutibols A-D: Cytotoxic Lipopeptaibols from the Himalayan Cold Habitat Fungus Trichoderma velutinum.

Four novel lipovelutibols A (1), B (2), C (3), and D (4) containing six amino acid residues with leucinol at the C-terminus and a fatty acyl moiety (n-octanoyl) at its N-terminus were isolated from the psychrotrophic fungus Trichoderma velutinum collected from the Himalayan cold habitat. The structures (1-4) were determined by NMR and MS/MS, and the stereochemistry of amino acids by Marfey's method. Lipopeptaibols 2 and 4 were found to contain d-isovaline, a nonproteinogenic amino acid, but lacked α-aminoisobutyric acid, characteristic of peptaibols. Cytotoxic activity of 2 and 4 was observed against HL-60, LS180, MDA-MB-231, and A549 cancer cell lines.

Boeravinone B, A Novel Dual Inhibitor of NorA Bacterial Efflux Pump of Staphylococcus aureus and Human P-Glycoprotein, Reduces the Biofilm Formation and Intracellular Invasion of Bacteria.

This study elucidated the role of boeravinone B, a NorA multidrug efflux pump inhibitor, in biofilm inhibition. The effects of boeravinone B plus ciprofloxacin, a NorA substrate, were evaluated in NorA-overexpressing, wild-type, and knocked-out Staphylococcus aureus (SA-1199B, SA-1199, and SA-K1758, respectively). The mechanism of action was confirmed using the ethidium bromide accumulation and efflux assay. The role of boeravinone B as a human P-glycoprotein (P-gp) inhibitor was examined in the LS-180 (colon cancer) cell line. Moreover, its role in the inhibition of biofilm formation and intracellular invasion of S. aureus in macrophages was studied. Boeravinone B reduced the minimum inhibitory concentration (MIC) of ciprofloxacin against S. aureus and its methicillin-resistant strains; the effect was stronger in SA-1199B. Furthermore, time–kill kinetics revealed that boeravinone B plus ciprofloxacin, at subinhibitory concentration (0.25 × MIC), is as equipotent as that at the MIC level. This combination also had a reduced mutation prevention concentration. Boeravinone B reduced the efflux of ethidium bromide and increased the accumulation, thus strengthening the role as a NorA inhibitor. Biofilm formation was reduced by four–eightfold of the minimal biofilm inhibitory concentration of ciprofloxacin, effectively preventing bacterial entry into macrophages. Boeravinone B effectively inhibited P-gp with half maximal inhibitory concentration (IC50) of 64.85 μM. The study concluded that boeravinone B not only inhibits the NorA-mediated efflux of fluoroquinolones but also considerably inhibits the biofilm formation of S. aureus. Its P-gp inhibition activity demonstrates its potential as a bioavailability and bioefficacy enhancer.

Bortezomib, carfilzomib and ixazomib do not mediate relevant transporter-based drug-drug interactions.

In order to optimize the clinical application of an increasing number of proteasome inhibitors, investigations into the differences between their respective pharmacodynamic and pharmacokinetic profiles, including their ability to act as a perpetrator in drug-drug interactions, are warranted. Therefore, in the present in vitro study, it was investigated whether bortezomib, carfilzomib and ixazomib are able to alter the expression, and/or the activity, of specific drug transporters generally relevant for pharmacokinetic drug-drug interactions. Through induction experiments, the current study demonstrated that the aforementioned three proteasome inhibitors do not induce mRNA expression of the transporter genes ATP binding cassette (ABC)B1, C1, C2 and G2 in the LS180 cell line, which was used as a model for systemic induction. By contrast, in certain myeloma cell lines, ixazomib provoked minor alterations in individual transporter gene expression. None of the proteasome inhibitors tested relevantly inhibited drug transporters within the range of physiological plasma concentrations. Taken together, transporter-based drug-drug interactions are unlikely to be a primary concern in the clinical application of the tested compounds.