5-Hydroxytryptamine promotes hepatocellular carcinoma proliferation by influencing ß-catenin.

5-Hydroxytryptamine (5-HT), a neurotransmitter and vasoactive factor, has been reported to promote proliferation of serum-deprived hepatocellular carcinoma (HCC) cells but the detailed intracellular mechanism is unknown. As Wnt/ß-catenin signalling is highly dysregulated in a majority of HCC, this study explored the regulation of Wnt/ß-catenin signalling by 5-HT. The expression of various 5-HT receptors was studied by quantitative real-time polymerase chain reaction (qPCR) in HCC cell lines as well as in 33 pairs of HCC tumours and corresponding adjacent non-tumour tissues. Receptors 5-HT1D (21/33, 63.6%), 5-HT2B (12/33, 36.4%) and 5-HT7 (15/33, 45.4%) were overexpressed whereas receptors 5-HT2A (17/33, 51.5%) and 5-HT5 (30/33, 90.1%) were reduced in HCC tumour tissues. In vitro data suggests 5-HT increased total ß-catenin, active ß-catenin and decreased phosphorylated ß-catenin protein levels in serum deprived HuH-7 and HepG2 cells compared to control cells under serum free medium without 5-HT. Activation of Wnt/ß-catenin signalling was evidenced by increased expression of ß-catenin downstream target genes, Axin2, cyclin D1, dickoppf-1 (DKK1) and glutamine synthetase (GS) by qPCR in serum-deprived HCC cell lines treated with 5-HT. Additionally, biochemical analysis revealed 5-HT disrupted Axin1/ß-catenin interaction, a critical step in ß-catenin phosphorylation. Increased Wnt/ß-catenin activity was attenuated by antagonist of receptor 5-HT7 (SB-258719) in HCC cell lines and patient-derived primary tumour tissues in the presence of 5-HT. SB-258719 also reduced tumour growth in vivo. This study provides evidence of Wnt/ß-catenin signalling activation by 5-HT and may represent a potential therapeutic target for hepatocarcinogenesis.

Anti-proliferative activities of sinigrin on carcinogen-induced hepatotoxicity in rats.

Liver cancer is one of the leading causes of cancer death worldwide. A very high incidence of new liver cancer cases is diagnosed every year, and metastasis has been found to correlate to poor prognoses in humans. Better treatments for liver cancer are thus clearly needed. Sinigrin is one of the major ingredients present in Brassica nigra, which has been used in combination with other herbs for treatment of various diseases. The anti-proliferative activities of sinigrin were studied in a model of carcinogen-induced hepatotoxicity in rats. Rats were orally administered with sinigrin on a daily basis for three months before sacrifice. Sinigrin was found to significantly inhibit the proliferation of liver tumor cells; the number of surface tumors in the rat liver was dramatically reduced. Sinigrin induced apoptosis of liver cancer cells through up-regulation of p53 and down-regulation of Bcl-2 family members and caspases. Our findings indicated that the liver functions were gradually restored after treatment with sinigrin and that the agent did not cause liver toxicity. Cell cycle analysis indicated that sinigrin caused cell cycle arrest in G0/G1 phase. The results suggest that sinigrin exerts important anti-proliferative activities in carcinogen-induced hepatocarcinogenesis in rats, and highlight the potential of sinigrin as an anti-cancer agent for liver cancer.

Combination of phytochemicals as adjuvants for cancer therapy.

Newer treatments of advanced human cancer are based on combination of cancer drugs that have different mechanism of actions yet the combination strategy may potentiate the anti-cancer effects and cytotoxicity. Recent studies suggest that cancer growth can be inhibited more effectively by combination of phytochemicals that affect different pathways. The apoptotic activity can be modulated by intrinsic and extrinsic molecules. The combination of anti-tumor phytochemicals can be more effective in modulating different signaling pathways associated with tumor cell growth which is the common target for anti-tumor action. Combinations of cytotoxic anti-tumor agents and inhibitors from phytochemicals are believed to act together producing inhibitory mechanisms on cancer growth. This combination strategy shows promise on cancer therapy. However, the combination of phytochemicals in cancer therapy needs to be further investigated to develop a better treatment strategy. Recent patents on anti-tumor phytochemicals are reviewed in this article.

Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis.

Alkaline exonuclease and single-strand DNA (ssDNA) annealing proteins (SSAPs) are key components of DNA recombination and repair systems within many prokaryotes, bacteriophages and virus-like genetic elements. The recently sequenced ß-proteobacterium Laribacter hongkongensis (strain HLHK9) encodes putative homologs of alkaline exonuclease (LHK-Exo) and SSAP (LHK-Bet) proteins on its 3.17 Mb genome. Here, we report the biophysical, biochemical and structural characterization of recombinant LHK-Exo protein. LHK-Exo digests linear double-stranded DNA molecules from their 5'-termini in a highly processive manner. Exonuclease activities are optimum at pH 8.2 and essentially require Mg(2+) or Mn(2+) ions. 5'-phosphorylated DNA substrates are preferred over dephosphorylated ones. The crystal structure of LHK-Exo was resolved to 1.9 Å, revealing a 'doughnut-shaped' toroidal trimeric arrangement with a central tapered channel, analogous to that of λ-exonuclease (Exo) from bacteriophage-λ. Active sites containing two bound Mg(2+) ions on each of the three monomers were located in clefts exposed to this central channel. Crystal structures of LHK-Exo in complex with dAMP and ssDNA were determined to elucidate the structural basis for substrate recognition and binding. Through structure-guided mutational analysis, we discuss the roles played by various active site residues. A conserved two metal ion catalytic mechanism is proposed for this class of alkaline exonucleases.

Effects of oxymatrine from Ku Shen on cancer cells.

Oxymatrine is one of active constituents isolated from Ku Shen, which is the dried root of Sophora flavescens Ait. The herb used in different herbal formulations is commonly known with specific pharmacological properties for treatment of liver disorders and other diseases such as arrhythmia, eczema and skin disorders, leukopenia and bronchitis. Sophora flavescens Ait is known to enhance liver functions and reduce hepatotoxicity due to oxidative stress and liver injury. The protection of cells from chemical toxicity is important in reducing liver damage. Reduction of oxidative stress by active components of herbal medicines is shown to be beneficial and important in regulating the normal functions of the liver. In this study, effects of oxymatrine on cancer cells after treatment of the cell line with DMSO were reported. This review described for cells without oxymatrine pre-treatment, cell injury was implicated as indicated by the decrease in cell viability. Ku Shen showed protective effects on cells from the DMSO-induced toxicity. The results show that oxymatrine can inhibit the G(2) and M phase of H4IIE. The findings suggest that anti-inflammatory constituents such as oxymatrine could mediate cell division of cancer cells and reduce cell cytotoxicity due probably to its capacity to inhibit the metabolic activation of hepato-toxin, a critical factor in the pathogenesis of chemical-induced liver injury.

Catheter-directed thrombolysis with the Endowave system in the treatment of acute massive pulmonary embolism: a retrospective multicenter case series.

PURPOSE: To evaluate the efficacy of thrombolysis with the EndoWave peripheral infusion system in the treatment of patients with massive pulmonary embolism (PE) as compared to patients treated with catheter-directed thrombolysis. MATERIALS AND METHODS: Ten patients (five men and five women; age range, 31-85 years; mean age, 54.20 years) with massive acute PE (17 lesions) were treated with ultrasonography (US)-assisted catheter-directed thrombolysis with the Endowave system. All patients had hypoxia and dyspnea. No patient had contraindication for thrombolysis. Angiographic findings, duration of lysis, dose of thrombolytics used, and procedural complications were recorded. Thrombolytics used were urokinase, tissue-type plasminogen activator (tPA), and Reteplase. RESULTS: Complete thrombus removal was achieved in 13 of the 17 lesions (76%), near complete thrombolysis was achieved in three lesions (18%), and partial thrombolysis was achieved in one lesion (6%). The mean time of thrombolysis was 24.76 hours +/- 8.44 (median, 24 hours). The mean dose of tPA used for the Endowave group was 0.88 mg/h +/- 0.19 (13 lesions). CONCLUSIONS: US-assisted catheter-directed thrombolysis is an effective method for treating massive thrombolysis. It has the potential to shorten the time of lysis and lower the dose of thrombolytics.

Pharmacological activity of cardiovascular agents from herbal medicine.

Some of the active phytochemicals in herbal medicine are finding therapeutic use. For example, patients with heart disease are reported to benefit from treatment with herbal medicine with fewer side effects. Previous studies showed the inhibitory effects of tetramethylpyrazine, an active component of medicinal herb, on phosphodiesterase that is associated with heart disease and the cardio-protective effects of other herbal medicine that was used to protect ischemia-reperfusion injury of rat hearts. Individual herbal medicines show antipyretic, analgesic and anti-inflammatory and anti-cancer effects. In addition to sharing many therapeutic activities, the active components of herbal medicine are also used in nutrient supplement for cardiovascular disease. Numerous in vitro studies of herbal medicine on different cell lines and in vivo study of herbal medicine have been reported. However, the mechanism of actions remains unclear. The present review aims to give an overview of the recent development of herbal medicine in treatment of cardiovascular disease, and covers the possible mechanism of action of some of active principles. The study will provide insights into drug action and demonstrate the therapeutic benefits of herbal medicine for the treatment of cardiovascular disease.

Identification of histidine-60 interaction with copper in activation of Chironomidae ferrochelatase.

Site-directed mutagenesis study of the conserved residue in ferrochelatase of chironomidae showed the binding interaction of copper with histidine-60. The activities of the variants increase by > 4-fold with H60N and 2 fold with H60D. The study identifies for the first time that the highly conserved H60 is a key molecular determinant in directing a catalytically competent mode of metal binding in the active site.

Deciphering signaling control by phosphoproteome using mass spectrometry.

Analysis of changes in genes and protein profiles provides invaluable information to understand the activities and functions of proteins. However, their activities are further regulated by post-translational modifications, such as glycosylation and phosphorylation. Cell growth and apoptotic pathways are good examples of demonstrating the roles of phosphorylation in activation of protein cascades upon stimulation.

Potential and cytotoxicity of cis-platinum complex with anti-tumor activity in combination therapy.

The use of anticancer agents forms an important part for treatment of cancer of various types. Complexes with cis-platinum compounds have been used for the prevention and treatment of cancers. Quite a number of these metal-containing complexes have been isolated, chemically prepared and characterized for the treatment of cancer. Many of these compounds display potent cytotoxic effects, although there is a considerable progress made in the design of novel anticancer agents. Some of these compounds showed strong inhibitory effects on cancer growth with a potential to become anti-cancer drugs. However, the unwanted deleterious effects hamper the common use of these agents as anticancer drugs. Nevertheless, the use of protective agents during, before or after treatment with anti-cancer agents in combination therapy has proven effective in the treatment. The results prompt the study of the biologic activities and the design of better modality for treatment and prevention of cancer. Here, we review the potential and reduction of cytotoxic properties of the prominent member of this class of metal compounds for the treatment of cancer.

Activation of p53 by specific agents in potential cancer therapy.

Tumor suppressor genes can promote p53-mediated apoptosis. Apoptosis is an important protective mechanism for normal cell growth. Aberrant regulation of p53 expression is linked to cancer development. The loss of function of p53 often results in tumorigenicity. It is reported that the high incidence of tumors in p53-deficient animals is highly attributed to p53-induced apoptosis. Malignancies that retain the wild-type p53 gene are associated with the biologic activity of p53 function. Most cancer cells show defects in p53 or inhibition in the associated pathways. A lot of effort has been focused on reactivating mutant p53, or recombinant technique to incorporate p53 in cells. Regulation of p53 has been described at both transcription and translation level. Activation of the p53 pathway appears to be an effective approach in inhibiting tumor development. In the present study, we have reviewed the recent developments of specific compounds that can regulate p53 expression and its function in cell growth and development. Integral to this is the function of other proteins that affect p53 activity.

Cloning and characterization of chironomidae ferrochelatase: copper activation of the purified ferrochelatase.

All organisms utilize ferrochelatase (EC 4.99.1.1) to catalyze the insertion of ferrous iron into protoposphyrin IX in the terminal step of the heme biosynthetic pathway. Different metal-binding affinity for the enzyme leads to changes in enzyme activity. In this work, we have cloned and over-expressed the enzyme from chironomidae in E. coli. The enzyme was purified and characterized. The recombinant enzyme showed higher enzymatic activity (four-fold increase) in the presence of copper ions and unaffected by calcium ions. Other divalent metal ions including magnesium, manganese, lead, reduced the enzyme activity by >60%. Over 90% of the enzyme activity was inhibited by Zn2+. The sequence alignment of amino acid residues reveals 83% homology with other ferrochelatases. The results of electron proton resonance (EPR) analysis showed that Fe2+ ion was present in the cluster of the recombinant enzyme complex. The recombinant enzyme also contained the [2Fe-2S] center with two-fold higher enzymatic activity than human ferrochelatase.

Transcriptional regulation of fosl-1 by licorice in rat Clone 9 cells.

Licorice is a commonly used herbal medicine for treatment of liver disorders. Its biological activities have been widely studied. However, little information on its transcriptional regulation has been reported. In the present study, the effect of an aqueous extract of licorice on the gene expression in rat liver cells (Clone 9) was investigated. The results show the expression of GST-pi, DT-diaphorase, PAI-1, fosl-1 and uPAR were over two-fold increased. Northern blot analysis revealed that the over-expression of these genes was concentration-dependent (0.25-3 mg/ml) but the temporal expression profile (8-48 h) of each individual gene varied. The over-expression of fosl-1 could be related to the event in the induction process leading to the expression of GST-pi, DT-diaphorase, uPAR and PAI-1 through AP-1. Induction of the over-expression of GST-pi and DT-diaphorase genes may contribute to the hepatoprotective properties of licorice whereas activation of uPAR and PAI-1 together with down-regulation of TIMP-3 suggest a role of LE in the regulation of cell mobility.

Simultaneous detection of trichloroethylene alcohol and acetate in rat urine by gas chromatography-mass spectrometry.

In order to better understand the cytotoxic effects of trichloroethylene (TCE) and its metabolites in TCE-induced carcinogenicity, it is necessary to determine the molecular species in biological samples. We have developed an efficient gas chromatography-mass spectrometry assay for the quantitative analysis of trichloroethylene alcohol and acetate. This method utilizes a simple esterification procedure, and a single liquid-liquid extraction with hexane-dichloromethane (1:1) that allows >90% recovery of the metabolites, followed by gas chromatography-mass spectrometry. This protocol allows for the accurate, sensitive, and reproducible analysis of the toxic TCE metabolites. The utility of the assay is demonstrated through the analysis of TCE metabolites in urine from rats administered with TCE. The limit of quantitation (precision and accuracy<20%) was 1.7 ng/ml for TCE alcohol and 2.3 ng/ml for TCE acetate.

Inhibition of glycyrrhizic acid on aflatoxin B1-induced cytotoxicity in hepatoma cells.

Aflatoxin B(1) (AFB(1)) causes oxidative stress and ROS formation via metabolic activation of AFB(1). Glycyrrhizic acid (GA) has been reported to have antioxidative properties. The present study was to investigate the effect of GA, a major component of licorice on AFB(1)-induced cytotoxicity in human hepatoma cell line (HepG2). GA displayed protective effects against AFB(1) treatment. Both CYP1A1, and glutathione S-transferase (GST) activities were increased in cells after treatment with the GA, while CYP1A2 did not seem to be affected by GA. For cells without GA pre-treatment, cell injury was implicated as indicated by the decrease in cell viability. The time-course study of GA showed pretreatment of cells with GA for 24 h was effective. The treatment of cells with GA and AFB(1) enhanced the detoxifying enzyme activity. The pre-treatment of cells with GA provides protective effects in terms of the enzyme activity and increase in cell viability. The results suggest that GA protects against aflatoxin-induced oxidative stress. This may contribute to its anticarcinogenic capability. The protective effect is likely due to its capacity to inhibit the metabolic activation of hepato-toxin, a critical factor in the pathogenesis of chemical-induced carcinogenicity.

Induction of rcl, a novel growth-related gene by coptidis rhizoma in rat H4IIE cells.

Exposure of rat hepatoma cells to a low concentration of Coptidis Rhizoma reduces cell viability and probably induces apoptotic cell death. However, Coptidis Rhizoma treatment increased the expression of a putative c-myc-responsive gene rcl and could increase the activity of a transcription factor in inhibiting the growth of cancer cells. This increase was accompanied by an increment in the expression of mRNA for c-myc-responsive gene. The expression was analysed by PCR and confirmed by Northern blot analysis. The rcl expression level increases with the Coptidis Rhizoma concentration, and in the time-course study. The results suggest the expression of rcl is important to the fate of cell growth, since overexpression of the c-myc proto-oncogene cell proliferation, differentiation, and apoptosis can be regulated by the treatment of Coptidis rhizoma. Additionally, difference between overexpression of c-myc-responsive gene in the control suggested that this protein was responsible for the inhibitory effect of a transcriptional factor on cell growth. The results support the notion of rcl as an important antiapoptotic protein mediating sensitivity to Coptidis Rhizoma induction in cancer cells. rcl may play an important role during cellular proliferation and c-myc-mediated transformation.

Induction of UDP-glucuronosyltransferase 1A8 mRNA by 3-methylcholanthene in rat hepatoma cells.

UDP-glucuronosyltransferases (UGTs) catalyze the glucuronidation of a broad spectrum of endobiotic and xenobiotic compounds, which leads to the excretion of hydrophilic glucuronides via bile or urine. By a mechanism of exon sharing, isoforms of the UGT1 family are made from the complex gene locus by an alternative combination of one of the unique first exons with the other commonly used exons. This study demonstrates that the expression of the UGT1 gene UGT1A6, 1A7 and 1A8 is regulated at the transcriptional level by 3-methylcholanthene (3-MC) in rat hepatoma H-4-II-E cells. Following 3-MC treatment, there is a gradual increase in the amount of UGT1A6 and UGT1A7 mRNA to the maximum levels after 16hr of treatment. The induction effect of 3-MC led to the expression of UGT1A8 which has not been reported before. This induction is suppressed by the RNA synthesis inhibitor actinomycin D, indicating that the inducer does not act at the level of mRNA stabilization. Northern blot analysis showed a 4-fold increase in UGT1A8 transcription after treatment with 3-MC. The prolonged treatment with the protein synthesis inhibitor did not affect the induction process. The results provide experimental evidence for a transcriptional control of UGT1A8 synthesis. Transcriptional activation of the UGT1A8 by 3-MC does not appear to require de novo protein synthesis. 3-MC dependent activation is probably the result of a direct action of the compound on the aryl hydrocarbon receptor complex (AhR).