Identification and functional analysis of cyclooxygenase-1 as a molecular target of boswellic acids

Boswellic acids (BAs) are assumed as the anti-inflammatory principles of Boswellia species. Initially, it was found that BAs inhibit leukotriene biosynthesis and 5-lipoxygenase (EC number 1.13.11.34), whereas suppression of prostaglandin formation and inhibition of cyclooxygenases (COX, EC number 1.14.99.1) has been excluded. Recently, we demonstrated that BAs also interfere with platelet-type 12-lipoxygenase. Here, we show that BAs, preferably 3-O-acetyl-11-keto-beta-BA (AKBA), concentration-dependently inhibit COX-1 product formation in intact human platelets (IC(50)=6 microM) as well as the activity of isolated COX-1 enzyme in cell-free assays (IC(50)=32 microM). The inhibitory effect of AKBA is reversible, and increased levels of arachidonic acid (AA) as substrate for COX-1 impair the efficacy. COX-1 in platelet lysates or isolated COX-1 selectively bound to an affinity matrix composed of immobilized BAs linked via glutaric acid to sepharose and this binding was reversed by ibuprofen or AA. Automated molecular docking of BAs into X-ray structures of COX-1 yielded positive Chemscore values for BAs, indicating favorable binding to the active site of the enzyme. In contrast, COX-2 was less efficiently inhibited by BAs as compared to COX-1, and pull-down experiments as well as docking studies exclude strong affinities of BAs towards COX-2. In conclusion, BAs, in particular AKBA, directly interfere with COX-1 and may mediate their anti-inflammatory actions not only by suppression of lipoxygenases, but also by inhibiting cyclooxygenases, preferentially COX-1.     

Identification of drugs and drug metabolites as substrates of multidrug resistance protein 2 (MRP2) using triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells

BACKGROUND AND PURPOSE

The coordinate activity of hepatic uptake transporters [e.g. organic anion transporting polypeptide 1B1 (OATP1B1)], drug-metabolizing enzymes [e.g. UDP-glucuronosyltransferase 1A1 (UGT1A1)] and efflux pumps (e.g. MRP2) is a crucial determinant of drug disposition. However, limited data are available on transport of drugs (e.g. ezetimibe, etoposide) and their glucuronidated metabolites by human MRP2 in intact cell systems.

EXPERIMENTAL APPROACH

Using monolayers of newly established triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells as well as MDCK control cells, single- (OATP1B1) and double-transfected (OATP1B1-UGT1A1, OATP1B1-MRP2) MDCK cells, we therefore studied intracellular concentrations and transcellular transport after administration of ezetimibe or etoposide to the basal compartment.

KEY RESULTS

Intracellular accumulation of ezetimibe was significantly lower in MDCK-OATP1B1-UGT1A1-MRP2 triple-transfected cells compared with all other cell lines. Considerably higher amounts of ezetimibe glucuronide were found in the apical compartment of MDCK-OATP1B1-UGT1A1-MRP2 monolayers compared with all other cell lines. Using HEK cells, etoposide was identified as a substrate of OATP1B1. Intracellular concentrations of etoposide equivalents (i.e. parent compound plus metabolites) were affected only to a minor extent by the absence or presence of OATP1B1/UGT1A1/MRP2. In contrast, apical accumulation of etoposide equivalents was significantly higher in monolayers of both cell lines expressing MRP2 (MDCK-OATP1B1-MRP2, MDCK-OATP1B1-UGT1A1-MRP2) compared with the single-transfected (OATP1B1) and the control cell line.

CONCLUSIONS AND IMPLICATIONS

Ezetimibe glucuronide is a substrate of human MRP2. Moreover, etoposide and possibly also its glucuronide are substrates of MRP2. These data demonstrate the functional interplay between transporter-mediated uptake, phase II metabolism and export by hepatic proteins involved in drug disposition.     

Synthesis,SAR and docking studies of substituted aryl phenylthiazolyl phenylcarboxamide as potential protein tyrosine phosphatase 1B (PTP1B) inhibitors

In our continued effort to discover novel PTP1B inhibitor with improved in vivo activity, we attempted to optimize our previously discovered lead compound by replacing the sulfonyl group with benzoyl group to yield compound II . Additional structural modifications were performed on compound II to yield a series of 24 aryl phenylthiazolyl phenylcarboxamides as potential PTP1B inhibitors. Of the 24 tested, 6 compounds showed good PTP1B inhibitory activity while compound 38 as the most promising one. The plausible PTP1B‐binding site interaction of compound 38 showed favourable binding similar to known PTP1B binders and suggests its selectivity towards PTP1B. Compound 38 also showed promising antihyperglycaemic, antidyslipidaemic and insulin resistant reversal activities in vivo in STZ model and db/db mice model. Altogether, the compound 38 presents an excellent candidate for future PTP1B targeted drug discovery.     

A clonal cell line (BME-UV1) as a possible model to study bovine mammary epithelial metabolism: metabolism and cytotoxicity of aflatoxin B1

Despite the toxicological risks to which humans and animals are exposed due to the transfer of toxic xenobiotic metabolites into milk of domestic animals, studies on the metabolizing mechanisms occurring in ruminant mammary gland are totally lacking. To investigate the possible biotransformation capabilities of a bovine mammary epithelial cell line (BME-UV1), monolayers were exposed to aflatoxin B1 (AFB1 - 1.0-8.0 μM). Starting from 4 h of exposure, the hydroxylate metabolite aflatoxin M1 (AFM1) was detected in media by high performance liquid chromatography. AFM1 concentration increased linearly with time for 36-48 h and the percent biotransformation of AFB1 (2-4 μM) at 48 h was about 12-14%. Parallel cytotoxicity assays (neutral red uptake-NRU and MTT assays) were performed to investigate the possible interference of AFB1 cytotoxicity with cellular metabolism. MTT assay (from 24 h of cell exposure) and NRU assay (from 16 h of cell exposure) showed time-dependent and time/concentration-dependent decrease of cell viability, respectively, and the former assay being more successful at revealing cytotoxic effects (NRU: CC₅₀ at 48 h = 12.00 ± 2.66 μM; MTT: CC₅₀ at 72 h = 20.42 ± 7.30 μM). The results suggest that BME-UV1 cells express metabolizing enzymes having catalytic activity, thus representing a potential in vitro model for studying biotransformation in bovine mammary gland.     

Potential role of EPB41L3 (Protein 4.1B/Dal-1) as a target for treatment of advanced prostate cancer

Background: Loss of erythrocyte membrane protein band 4.1-like 3 (EPB41L3; aliases: protein 4.1B, differentially expressed in adenocarcinoma of the lung-1 (Dal-1)) expression has been implicated in tumor progression. Objective: To evaluate literature describing the role of EPB41L3 in tumorigenesis and metastasis, and to consider whether targeting this gene would be useful in the treatment of prostate cancer. Methods: A literature review of studies describing EPB41L3 and its aliases was conducted. Online databases (NCBI, SwissProt) were also interrogated to collect further data. Results/conclusion: A growing body of evidence supports a role for loss of EPB41L3 in tumor progression, including in prostate cancer. Therapeutic strategies that could be harnessed to upregulate EPB41L3 gene expression in prostate cancer cells are currently being developed.     

The adipocyte cGMP-inhibited cyclic nucleotide phosphodiesterase (PDE3B) as a target for lipolytic and thermogenic agents for the treatment of obesity

Agents that stimulate breakdown of adipocyte triglyceride to glycerol and free fatty acids (lipolysis) and increase basal metabolic rate (thermogenesis) have potential benefit as therapeutics for obesity. In the adipocyte, cAMP levels regulate both lipolysis and thermogenesis. PDE3B is one of the major cAMP-hydrolysing cyclic nucleotide phosphodiesterases in the adipocyte and PDE3 inhibitors induce lipolysis in vitro and in vivo. PDE3 inhibitors also elevate metabolic rate in human subjects, though the mechanism of this effect has not yet been determined. Hence, PDE3 inhibitors have a combination of metabolic properties that suggests their utility for the treatment of obesity. PDE3 inhibitors may also have effects on glucose homeostasis, and the relevance of this finding to the possible use of PDE3 inhibitors in obese diabetics is discussed.     

Effects of rosmarinic acid against aflatoxin B1 and ochratoxin-A-induced cell damage in a human hepatoma cell line (Hep G2)

Recent findings have suggested that oxidative damage might contribute to the cytotoxicity and carcinogenicity of aflatoxin B(1) (AFB(1)). The induction of oxidative stress also plays an important role in the toxicity of another mycotoxin: ochratoxin A (OTA). In this study, the protective effect of rosmarinic acid (Ros A) against AFB(1) and OTA-induced cytotoxicity was investigated in a human hepatoma-derived cell line (Hep G2). Rosmarinic acid, a natural phenolic compound contained in many Lamiaceae herbs such as Perilla frutescens, sage, basil and mint, inhibits complement-dependent inflammatory processes and may have therapeutic potential.The ability of Ros A to reduce radical oxygen species (ROS) production, protein and DNA synthesis inhibition and apoptosis caused by the two mycotoxins was also investigated. Our experiments proved the significant cytoprotective effect of Ros A in vitro from OTA- and AFB(1)-induced cell damage. In particular, 24-h pretreatment with 50 micro M Ros A inhibited the cytotoxicity of 10 micro M AFB(1) (by 45%) and 10 micro M OTA (by 35%) in Hep G2 cells (P < 0.001). Moreover, Ros A dose dependently attenuated ROS production and DNA and protein synthesis inhibition induced by both of the toxins. Similarly, apoptosis cell death was prevented, as demonstrated by reduction of DNA fragmentation and inhibition of caspase-3 activation (P < 0.001).     

Protective effect of Ocimum sanctum on 3-methylcholanthrene, 7,12-dimethylbenz(a)anthracene and aflatoxin B1 induced skin tumorigenesis in mice

A study on the protective effect of alcoholic extract of the leaves of Ocimum sanctum on 3-methylcholanthrene (MCA), 7,12-dimethylbenzanthracene (DMBA) and aflatoxin B1 (AFB1) induced skin tumorigenesis in a mouse model has been investigated. The study involved pretreatment of mice with the leaf extract prior to either MCA application or tetradecanoyl phorbol acetate (TPA) treatment in a two-stage tumor protocol viz a viz, DMBA/TPA and AFB1/TPA. The results of the present study indicate that the pretreatment with alcoholic extract of the leaves of O. sanctum decreased the number of tumors in MCA, DMBA/TPA and AFB1/TPA treated mice. The skin tumor induced animals pretreated with alcoholic extract led to a decrease in the expression of cutaneous gamma-glutamyl transpeptidase (GGT) and glutathione-S-transferase-P (GST-P) protein. The histopathological examination of skin tumors treated with leaf extract showed increased infiltration of polymorphonuclear, mononuclear and lymphocytic cells, decreased ornithine decarboxylase activity with concomitant enhancement of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in the serum, implying the in vivo antiproliferative and immunomodulatory activity of leaf extract. The decrease in cutaneous phase I enzymes and elevation of phase II enzymes in response to topical application of leaf extract prior to MCA, AFB1, DMBA/TPA and AFB1/TPA treatment indicate the possibility of impairment in reactive metabolite(s) formation and thereby reducing skin carcinogenicity. Furthermore, pretreatment of leaf extract in the carcinogen induced animals resulted in elevation of glutathione levels and decrease in lipid peroxidation along with heat shock protein expression, indicating a scavenging or antioxidant potential of the extract during chemical carcinogenesis. Thus it can be concluded that leaf extract of O. sanctum provides protection against chemical carcinogenesis in one or more of the following mechanisms: (i) by acting as an antioxidant; (ii) by modulating phase I and II enzymes; (iii) by exhibiting antiproliferative activity.     

Thymoquinone (2-Isopropyl-5-methyl-1, 4-benzoquinone) as a chemopreventive/anticancer agent: Chemistry and biological effects

Cancer remains the topmost disorder of the mankind and number of cases is unceasingly growing at unprecedented rates. Although the synthetic anti-cancer compounds still hold the largest market in the modern treatment of cancer, natural agents have always been tried and tested for potential anti-cancer properties. Thymoquinone (TQ), a monoterpene and main ingredient in the essential oil of Nigella sativa L. has got very eminent rankings in the traditional systems of medicine for its anti-cancer pharmacological properties. In this review we summarized the diverse aspects of TQ including its chemistry, biosynthesis, sources and pharmacological properties with a major concern being attributed to its anti-cancer efficacies. The role of TQ in different aspects involved in the pathogenesis of cancer like inflammation, angiogenesis, apoptosis, cell cycle regulation, proliferation, invasion and migration have been described. The mechanism of action of TQ in different cancer types has been briefly accounted. Other safety and toxicological aspects and some combination therapies involving TQ have also been touched. A detailed literature search was carried out using various online search engines like google scholar and pubmed regarding the available research and review accounts on thymoquinone upto may 2019. All the articles reporting significant addition to the activities of thymoquinone were selected. Additional information was acquired from ethno botanical literature focusing on thymoquinone. The compound has been the centre of attention for a long time period and researched regularly in quite considerable numbers for its various physicochemical, medicinal, biological and pharmacological perspectives. Thymoquinone is studied for various chemical and pharmacological activities and demonstrated promising anti-cancer potential. The reviewed reports confirmed the strong anti-cancer efficacy of thymoquinone. Further in-vitro and in-vivo research is strongly warranted regarding the complete exploration of thymoquinone in ethnopharmacological context.     

Inhibition of interleukin-8 (CXCL8/IL-8) responses by repertaxin, a new inhibitor of the chemokine receptors CXCR1 and CXCR2

Repertaxin is a new non-competitive allosteric blocker of interleukin-8 (CXCL8/IL-8) receptors (CXCR1/R2), which by locking CXCR1/R2 in an inactive conformation prevents receptor signaling and human polymorphonuclear leukocyte (PMN) chemotaxis. Given the unique mode of action of repertaxin it was important to examine the ability of repertaxin to inhibit a wide range of biological activities induced by CXCL8 in human leukocytes. Our results show that repertaxin potently and selectively blocked PMN adhesion to fibrinogen and CD11b up-regulation induced by CXCL8. Reduction of CXCL8-mediated PMN adhesion by repertaxin was paralleled by inhibition of PMN activation including secondary and tertiary granule release and pro-inflammatory cytokine production, whereas PMN phagocytosis of Escherichia coli bacteria was unaffected. Repertaxin also selectively blocked CXCL8-induced T lymphocyte and natural killer (NK) cell migration. These data suggest that repertaxin is a potent and specific inhibitor of a wide range of CXCL8-mediated activities related to leukocyte recruitment and functional activation in inflammatory sites.