Polyalthia longifolia var. pendula is used as an antipyretic agent in indigenous systems of medicine. Microglia-mediated inflammation plays an important role in the pathway leading to neuronal cell death in a number of neurodegenerative diseases. The aim of this study was to investigate the effects of 6-hydroxycleroda-3,13-dien-15,16-olide (PL3) extracted from Polyalthia longifolia var. pendula on lipopolysaccharide(LPS)-induced inflammation in microglia-like HAPI cells and primary microglia cultures. In microglia-neuron co-cultures, LPS decreased the cell viability of neuroblastoma SH-SY5Y cells. LPS-induced cell death was attenuated by the NOS inhibitor, L-NAME, the COX-2 inhibitor, NS-398 or the NADPH oxidase inhibitor, DPI, respectively. In LPS-treated microglia cells, PL3 decreased the expression of iNOS, COX-2, gp91 (phox), and NF- kappaBp65, the degradation of I kappaB alpha, and the production of NO, PGE (2), iROS, and TNF- alpha. PL3 also enhanced the expression of HO-1, a cytoprotective and anti-inflammatory enzyme. Moreover, PL3 reduced LPS-activated microglia-induced cell death. The present results suggest that PL3 inhibits microglia-mediated inflammation and inflammation-related neuronal cell death. Therefore, PL3 has potential use for the treatment of inflammation-related neurodegenerative diseases.
We have studied the role of ATP binding cassette (ABC) transporters in fetal exposure to carcinogens using 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) a known substrate for ABC transporters as a model compound. In perfusion of human term placenta, transfer of (14)C-PhIP (2 microM) through the placenta resulted in fetal-to-maternal concentration ratio (FM ratio) of 0.72+/-0.09 at 6 h. The specific ABCG2 inhibitor KO143 increased the transfer of (14)C-PhIP from maternal to fetal circulation (FM ratio 0.90+/-0.08 at 6 h, p
Mounting evidence underlines the role of inducible nitric oxide synthase (iNOS) in hepatocellular carcinoma (HCC) development, but its functional interactions with pathways involved in HCC progression remain uninvestigated. Here, we analyzed in preneoplastic and neoplastic livers from Fisher 344 and Brown Norway rats, possessing different genetic predisposition to HCC, in transforming growth factor-alpha (TGF-alpha) and c-Myc-TGF-alpha transgenic mice, characterized by different susceptibility to HCC, and in human HCC: (i) iNOS function and interactions with nuclear factor-kB (NF-kB) and Ha-RAS/extracellular signal-regulated kinase (ERK) during hepatocarcinogenesis; (ii) influence of genetic predisposition to liver cancer on these pathways and role of these cascades in determining a susceptible or resistant phenotype and (iii) iNOS prognostic value in human HCC. We found progressive iNos induction in rat and mouse liver lesions, always at higher levels in the most aggressive models represented by HCC of rats genetically susceptible to hepatocarcinogenesis and c-Myc-TGF-alpha transgenic mice. iNOS, inhibitor of kB kinase/NF-kB and RAS/ERK upregulation was significantly higher in HCC with poorer prognosis (as defined by patients' survival length) and positively correlated with tumor proliferation, genomic instability and microvascularization and negatively with apoptosis. Suppression of iNOS signaling by aminoguanidine led to decreased HCC growth and NF-kB and RAS/ERK expression and increased apoptosis both in vivo and in vitro. Conversely, block of NF-kB signaling by sulfasalazine or short interfering RNA (siRNA) or ERK signaling by UO126 caused iNOS downregulation in HCC cell lines. These findings indicate that iNOS cross talk with NF-kB and Ha-RAS/ERK cascades influences HCC growth and prognosis, suggesting that key component of iNOS signaling could represent important therapeutic targets for human HCC.
We analyzed subfraction composition of HDL and cholesterol-acceptor properties of the plasma in Russian men with high and low HDL cholesterol. HDL were subfractionated by two-dimensional electrophoresis in agarose-polyacrylamide gel. The content of pre-beta1 HDL increased in individuals with high concentration of HDL cholesterol and strictly correlated with acception of cellular cholesterol in both groups.
In a previous study, the methanolic extract as well as the chloroform fraction of the aerial parts of Caralluma quadrangula (Forssk.) N.E.Br. indigenous to Saudi Arabia showed significant in vitro cytotoxic activity against breast cancer (MCF7) cell line. In a biologically-guided fractionation approach, four acylated pregnane glycosides were isolated from the chloroform fraction of C. quadrangula. The structures of the isolated compounds were elucidated by the analysis of their MS and NMR data. The compounds were identified as 12,20-di-O-benzoylboucerin 3-O-ß-D-digitoxopyranosyl-(1?4)-ß-D-canaropyranosyl-(1?4)-ß-D-cymaropyranoside (1), 12,20-di-O-benzoylboucerin 3-O-ß-D-cymaropyranosyl-(1?4)-ß-D-canaropyranosyl-(1?4)-ß-D-cymaropyranoside (2), 12,20-di-O-benzoylboucerin 3-O-ß-D-glucopyranosyl-(1?4)-ß-D-digitoxopyranosyl-(1?4)-ß-D-canaropyranosyl-(1?4)-ß-D-cymaropyranoside (3) and 12,20-di-O-benzoyl-3ß,5a,12ß,14ß,20-pentahydroxy-(20R)-pregn-6-ene 3-O-ß-D-glucopyranosyl-(1?4)-ß-D-digitoxopyranosyl-(1?4)-ß-D-canaropyranosyl-(1?4)-ß-D-cymaropyranoside (4). The isolated compounds were tested for their cytotoxic activity against breast cancer (MCF7) cell line.
Although sensitivity to intracellular ATP is considered to be one of the hallmarks of swelling activated Cl- current (I(Cl,swell)) involved in regulatory volume decrease (RVD) following hypotonic stress, the type and manner of such sensitivity seems to vary in different cell types. Here by using whole-cell patch-clamp recording we investigated ATP sensitivity of I(Cl,swell) in LNCaP human prostate cancer cell line. Suppression of endogenous ATP production with metabolic inhibitors (oligomycin, iodoacetate and rotenone) during cell dialysis with ATP- and Mg2+-free pipette solution did not prevent I(Cl,swell) in response to hypotonic exposure. However, supplementing this solution with 5 mM Na-ATP led to the development of I(Cl,swell) with nearly 305 higher density and less pronounced voltage-dependent inactivation (manifested mainly by the increase of non-inactivated current component) at positive potentials. On the contrary, inclusion of 1 mM Mg2+ in the patch pipette resulted in even smaller I(Cl,swell) (30% lower density compared to Mg2+-free conditions), which inactivated completely on prolonged depolarization. The presence of 5 mM Mg-ATP in the pipette did not affect I(Cl,swell) density. Neither intervention significantly altered the rate of I(Cl,swell) development in response to hypotonicity. We conclude that intracellular ATP, a positive modulator of I(Cl,swell)-carrying volume-regulated anion channel (VRAC) in LNCaP cells most likely acts via binding rather than hydrolysis and/or phosphorylation reactions, whereas intracellular Mg2+ is VRAC inhibitor.
The integrin alphavbeta6, a receptor for fibronectin, vitronectin, tenascin and TGF-beta latency-associated peptide (LAP), is not detectable on normal oral epithelium but is neo-expressed in oral squamous cell carcinomas (OSCC) and epithelial dysplasia. Previously it has been shown that alphavbeta6 integrin can up-regulate MMP-3 and -9 expression in OSCC cells. Using beta6-transfected and control OSCC cells we demonstrate that alphavbeta6 integrin down-regulates MMP-13 expression at both mRNA and protein level. Although expressing less MMP-13, beta6-transfected cells were found to have similar collagenolytic activity as control cells and invade at similar levels through type I collagen. Growth of the tumour cells in organotypic culture and confocal microscopy confirmed low levels of MMP-13 in cells with high alphavbeta6 expression. Furthermore, human squamous cell carcinomas of the tongue with high expression of alphavbeta6 showed lower MMP-13 levels than carcinomas with low levels of alphavbeta6. Our results suggest that alphavbeta6 down-regulates MMP-13 expression in OSCC cells and that MMP-13 is not essential for the degradation of type I collagen by OSCC cells.
Increasing population of malignant, apoptosis resistant neuroendocrine (NE) cells due to differentiation of prostate epithelial/basal cells is a hallmark of advanced, androgen-independent prostate cancer, for which there is no successful therapy. Acquisition of apoptosis resistance involves alterations in the mechanisms of cell volume homeostasis, of which volume-regulate anion channels (VRAC) that carry swelling-activated Cl- current (I(Cl,swell)) represent one of the key determinants. Given that VRAC function is generally known to be ATP-dependent, here we investigated how such dependence may evolve during NE differentiation of LNCaP prostate cancer epithelial cells. In the whole-cell patch-clamp recording mode I(Cl,swell) could be activated in response to hypotonicity-induced cell swelling in control and NE-differentiated (by incubation in membrane-permeable cAMP analogs) LNCaP cells even following total depletion of intracellular ATP using a cocktail of metabolic inhibitors. However, this basal I(Cl,swell) had about 30% higher density and was less inactivating in NE-differentiated cells. Inclusion of 5 mM Mg-ATP in the patch pipette caused I(Cl,swell) augmentation in both cell types. The augmentation in the control cells was more prominent and occurred mostly at the expense of a non-inactivating current component. We conclude that I(Cl,swell) in LNCaP cells consists of a non-inactivating, ATP-dependent and inactivating, ATP-independent components. NE differentiation promotes the increase of non-inactivating component and partial loss of its ATP sensitivity making the whole I(Cl,swell) less ATP-sensitive as well. By largely avoiding the ATP metabolic control I(Cl,swell) may contribute to better control of cell volume under metabolic stress and thus enhance the survival rates of apoptosis-resistant NE cells.
The aminothiol WR1065 exerts selective cytoprotective effects in normal cells compared to cancer cells and has clinical applications for the protection of normal cells in cancer patients undergoing radio- or chemotherapy. There is evidence that p53 is activated in response to WR1065. To examine the effects of WR1065 on the signalling pathways controlled by p53, isogeneic human colon carcinoma cell lines (HCT116) differing only in the presence or absence of wild-type p53 were used. Treatment with WR1065 resulted in G1 cell cycle arrest in the p53-positive cell line but not in the p53-negative cell line. Long-term exposure resulted in minimal apoptosis of either cell line. Changes in gene expression in p53-positive or -negative cells treated with WR1065 were examined using commercial human stress and cancer gene arrays (Clontech Atlas arrays). Genes found to be specifically upregulated in a p53-dependent manner included coproporphyrinogen oxidase, ICErel-II cysteine protease, macrophage inhibitory cytokine-1 (also known as placental transforming growth factor beta), S100A4, and Waf1/p21. However, most proapoptotic genes typically upregulated by p53 in response to DNA damage were not activated. These studies show that WR1065 specifically modulates a subset of p53 target genes in a colon carcinoma cell line, consistent with the observation that this agent elicits essentially p53-dependent, cell cycle arrest responses.
Two bacterial isolates from the Barents Sea, both belonging to the genus Algibacter, were found to yield extracts with anti-bacterial bioactivity. Mass spectrometry guided dereplication and purification of the active extracts lead to the isolation of the same active principle in both extracts. The structure of the bioactive compound was identified via mass spectrometry and nuclear resonance spectroscopy and it turned out to be the known lipopeptide Lipid 430. We discovered and determined its previously unknown anti-bacterial activity against Streptococcus agalactiae and revealed a cytotoxic effect against the A2058 human melanoma cell line at significantly lower concentrations compared to its anti-bacterial concentration. Flow cytometry and microscopy investigations of the cytotoxicity against the melanoma cell line indicated that Lipid 430 did not cause immediate cell lysis. The experiments with melanoma cells suggest that the compound functions trough more complex pathways than acting as a simple detergent.