Non-homologous end joining pathway is the major route of protection against 4ß-hydroxywithanolide E-induced DNA damage in MCF-7 cells.

4ß-Hydroxywithanolide E is a bioactive withanolide extracted from Physalis peruviana. 4ß-Hydroxywithanolide E caused reactive oxygen species production and cell apoptosis in human breast cancer MCF-7 cells. We further found that 4ß-hydroxywithanolide E induced DNA damage and regulated the DNA damage signaling in MCF-7 cells. The DNA damage sensors and repair proteins act promptly to remove DNA lesions by 4ß-hydroxywithanolide E. The ataxia-telangiectasia mutated protein (ATM)-dependent DNA damage signaling pathway is involved in 4ß-hydroxywithanolide E-induced apoptosis of MCF-7 cells. Non-homologous end joining pathway, but not homologous recombination, is the major route of protection of MCF-7 cells against 4ß-hydroxywithanolide E-induced DNA damage. 4ß-Hydroxywithanolide E had no significant impact on the base excision repair pathway. In this study, we examined the 4ß-hydroxywithanolide E-induced DNA damage as a research tool in project investigating the DNA repair signaling in breast cancer cells. We also suggest that 4ß-hydroxywithanolide E assert its anti-tumor activity in carcinogenic progression and develop into a dietary chemopreventive agent.

Molecular modelling, synthesis, cytotoxicity and anti-tumour mechanisms of 2-aryl-6-substituted quinazolinones as dual-targeted anti-cancer agents.

BACKGROUND AND PURPOSE: Our previous study demonstrated that 6-(pyrrolidin-1-yl)-2-(3-methoxyphenyl)quinazolin-4-one (HMJ38) was a potent anti-tubulin agent. Here, HMJ38 was used as a lead compound to develop more potent anti-cancer agents and to examine the anti-cancer mechanisms. EXPERIMENTAL APPROACH: Using computer-aided drug design, 2-aryl-6-substituted quinazolinones (MJ compounds) were designed and synthesized by introducing substituents at C-2 and C-6 positions of HMJ38. The cytotoxicity of MJ compounds towards human cancer cells was examined by Trypan blue exclusion assay. Microtubule distribution was visualized using TubulinTracker(TM) Green reagent. Protein expression of cell cycle regulators and JNK was assessed by Western blot analysis. KEY RESULTS: Compounds MJ65-70 exhibited strong anti-proliferative effects towards melanoma M21, lung squamous carcinoma CH27, lung non-small carcinoma H460, hepatoma Hep3B and oral cancer HSC-3 cells, with one compund MJ66 (6-(pyrrolidin-1-yl)-2-(naphthalen-1-yl)quinazolin-4-one) highly active against M21 cells (IC50 about 0.033 µM). Treatment of CH27 or HSC-3 cells with MJ65-70 resulted in significant mitotic arrest accompanied by increasing multiple asters of microtubules. JNK protein expression was involved in the MJ65-70-induced CH27 and M21 cell death. Consistent with the cell cycle arrest at G2/M phase, marked increases in cyclin B1 and Bcl-2 phosphorylation were also observed, after treatment with MJ65-70. CONCLUSIONS AND IMPLICATION: MJ65-70 are dual-targeted, tubulin- and JNK-binding, anti-cancer agents and induce cancer cell death through up-regulation of JNK and interfering in the dynamics of tubulin. Our work provides a new strategy and mechanism for developing dual-targeted anti-cancer drugs, contributing to clinical anti-cancer drug discovery and application.

Genetic polymorphisms in androgen receptor-binding sites predict survival in prostate cancer patients receiving androgen-deprivation therapy.

BACKGROUND: Activated androgen receptor binds to androgen-responsive elements (AREs) in genome to regulate target gene transcription and, consequently, mediates physiological or tumorigenic processes of the prostate. Our aim was to determine whether genetic variants in AREs are associated with clinical outcomes after androgen-deprivation therapy (ADT) in prostate cancer patients. PATIENTS AND METHODS: We systematically investigated 55 common single-nucleotide polymorphisms (SNPs) in the genome-wide insilico-predicted AREs in a cohort of 601 men with advanced prostate cancer treated with ADT. The prognostic significance of these SNPs on disease progression, prostate cancer-specific mortality (PCSM) and all-cause mortality (ACM) after ADT was assessed by Kaplan-Meier analysis and Cox regression model. RESULTS: In univariate analysis, two, five, and four SNPs were associated with disease progression, PCSM, and ACM, respectively. After adjusting for known prognostic factors, ARRDC3 rs2939244, FLT1 rs9508016, and SKAP1 rs6504145 remained as significant predictors for PCSM and FBXO32 rs7830622 and FLT1 rs9508016 remained as significant predictors for ACM in multivariate analysis. Moreover, strong combined genotype effects on PCSM and ACM were also observed (P(trend) < 0.001). CONCLUSION: Our results suggest that SNPs in AREs influence prostate cancer survival and may further advance our understanding of the disease progression.

Genetic polymorphisms in oestrogen receptor-binding sites affect clinical outcomes in patients with prostate cancer receiving androgen-deprivation therapy.

BACKGROUND: Accumulating evidence indicates that oestrogens have significant direct effects on normal prostate development and carcinogenesis. The majority of the biological activities of oestrogens are mediated through the oestrogen receptor (ER), which functions as a hormone-inducible transcription factor to regulate target gene expression by binding to oestrogen response elements (EREs) in the regulatory regions of target genes. Sequence variants in EREs might affect the ER-ERE interaction and subsequent physiological activities. Therefore, we tested whether common single-nucleotide polymorphisms (SNPs) inside EREs are related to the clinical outcomes of androgen-deprivation therapy (ADT) in men with prostate cancer. METHODS: We systematically evaluated 49 ERE SNPs predicted using a genome-wide database in a cohort of 601 men with advanced prostate cancer treated with ADT. The prognostic significance of these SNPs on disease progression, prostate cancer-specific mortality (PCSM) and all-cause mortality (ACM) after ADT was assessed using Kaplan-Meier analysis and a Cox regression model. RESULTS: Based on multiple hypothesis testing, BNC2 rs16934641 was found to be associated with disease progression; in addition, TACC2 rs3763763 was associated with PCSM, and ALPK1 rs2051778 and TACC2 rs3763763 were associated with ACM. These SNPs remained significant in multivariate analyses that included known clinicopathological predictors. Moreover, a combined genotype effect on ACM was observed when ALPK1 rs2051778 and TACC2 rs3763763 were analysed in combination. Patients with a greater number of unfavourable genotypes had a shorter time to ACM during ADT (P for trend <0.001). CONCLUSION: The incorporation of ERE SNPs into models with known predictors might improve outcome prediction in patients with prostate cancer receiving ADT.

Production and diagnostic application of a purified, E. coli-expressed, serological-specific chicken anaemia virus antigen VP3.

The aim of this study was to evaluate the production of chicken anaemia virus VP3 protein in different Escherichia coli strains and to address the diagnostic application of purified E. coli-expressed VP3 protein for the detection of chicken anaemia virus (CAV) infection and the development of an ELISA kit. Three E. coli strains, BL21, BL21 codonplus RP and BL21 pLysS, each harbouring a VP3 protein expressing plasmid, were investigated after induction to produce recombinant VP3 protein. After isopropyl-ß-D-thiogalactoside (IPTG) induction, VP3 protein was successfully expressed in all three E. coli strains. The BL21 pLysS strain gave the best performance in terms of protein productivity and growth profile. In addition, the optimal culture temperature and IPTG concentration were found to be 0.25 mM and 20 °C, respectively. Using Ni-NTA-purified VP3 protein as an ELISA coating antigen, the purified VP3 was shown to be highly antigenic and able to discriminate sera from chickens infected with CAV from those that were uninfected during an evaluation of CAV infection serodiagnosis. A VP3-based ELISA demonstrated 100% (6/6 x 100%) specificity and sensitivities of 91.3% (21/23 x 100%) and 82.6% (19/23 x 100%) using cut-off values of the mean plus 2 SD and the mean plus 3 SD, respectively.

Oxidative stress involvement in Physalis angulata-induced apoptosis in human oral cancer cells.

In this report, we investigated the role of oxidative stress in Physalis angulata-induced apoptosis of human oral cancer cells. P. angulata-induced apoptosis was characterized by nuclear morphological changes, membrane blebbing and activation of caspase-9. Exposure of HSC-3 cells to P. angulata caused production of reactive oxygen species and up-regulation of oxidative stress markers heme oxygenase-1 (HO-1), superoxide dismutase (SOD), heat shock protein 70 (HSP70) and caspase-4. Down-regulation of HO-1, SOD and HSP70 proteins expression by attenuation of oxidative stress, pretreatment with glutathione or N-acetylcysteine, significantly decreased P. angulata-triggered cell death. The present study also demonstrated that the mitochondria and the endoplasmic reticulum are the targets of P. angulata in HSC-3 cells. Our results revealed that: (1) reactive oxygen species may play a dominant role in this process, (2) P. angulata induces oxidative stress in HSC-3 cells, (3) P. angulata-initiated apoptosis is caused through oxidative stress-dependent induction of heme oxygenase-1, Cu/Zn SOD and HSP70 proteins expression and (4) antioxidants inhibited P. angulata-induced cell death through inhibition of the proteins expression of HO-1, Cu/Zn SOD and HSP70.

P38-associated pathway involvement in apoptosis induced by photodynamic therapy with Lonicera japonica in human lung squamous carcinoma CH27 cells.

Photodynamic therapy (PDT) is an effective therapy for local malignant tumors. Lonicera japonica was found to have the anti-tumor effect. The aim of this study is to explore the mechanisms of apoptosis induced by PDT in lung CH27 carcinoma cells with alcohol extract from Lonicera japonica as photosensitizer. Our study indicated that Lonicera japonica extracts exhibited significant photocytotoxicity in CH27 cells at a concentration range of 50-150 microg/ml, with 0.4-1.2J/cm2 light dose. PDT with Lonicera japonica extracts-induced cell death is a typical apoptosis that was accompanied by DNA condensation, externalization of phosphatidylserine and formation of apoptotic bodies. PDT with Lonicera japonica extracts was shown to be caspase-3-independent apoptosis via activation of AIF in this study. P38-associated pathway may be involved in apoptosis induced by PDT with Lonicera japonica extracts in CH27 cells. We also have demonstrated that PDT with Lonicera japonica extracts-induced CH27 cells apoptosis was probably related to its ability to change the protein expression and distribution of heat shock protein 27.

Kaempferol induces apoptosis in human lung non-small carcinoma cells accompanied by an induction of antioxidant enzymes.

Kaempferol (3, 4',5,7-tetrahydroxyflavone) is one of the most commonly found dietary flavonols. The biological and pharmacological effects of kaempferol may depend upon its behavior as either an antioxidant or a prooxidant. However, the clear biological effects of prooxidant or antioxidant character of kaempferol has not been clarified yet. The overall objective of the present study is to explore the role of prooxidant or antioxidant in kaempferol-induced cell toxicity. In this paper, we have proved that antioxidant pathway may be involved in kaempferol induces H460 cell apoptosis. Kaempferol-induced H460 cell apoptosis is a typical apoptosis that was accompanied by a significant DNA condensation and increasing intracellular ATP levels. Kaempferol-induced apoptosis is related to its ability to change the expression of apoptotic markers, such as caspase-3 (caspase-dependent) and AIF (caspase-independent). The overexpression of antioxidant enzyme Mn SOD protein levels, which was promoted to a new type tumor suppressor gene in several human cancer cells recently, may be an important role in kaempferol-induced H460 cell apoptosis.

Inhibition of 12-lipoxygenase during baicalein-induced human lung nonsmall carcinoma H460 cell apoptosis.

Baicalein is known as a 12-lipoxygenase (12-LOX) inhibitor. The 12-LOX is found to be involved in the progression of human cancers and the inhibitor of 12-LOX offers a target for the prevention cancer. We demonstrated the inhibitory effect of baicalein on the gene and protein expression of 12-LOX in H460 human lung nonsmall carcinoma cell line. Treatment of baicalein inhibited the growth of H460 cells in a dose-dependent manner. Following 24h exposure to 50muM baicalein, cell cycle analysis revealed an increase in the cell population in S-phase. During the S-phase arrest, baicalein decreased the protein levels of cdk1 and cyclin B1, which are the regulating proteins of S-phase transition to G2/M-phase, in this study. Furthermore, baicalein induced the most of H460 cell apoptosis after treatment for 48h. H460 cells formed vesicles and apoptotic body, and then floated after treatment with baicalein. Baicalein-induced H460 cell apoptosis was confirmed by DNA condensation and fragmentation. Baicalein-induced apoptosis were also accompanied by decreasing in Bcl-2 and proform of caspase-3 and increasing p53 and Bax protein levels. Pretreatment with a specific caspase-3 inhibitor, Ac-DEVD-CHO, partially reduced baicalein-induced cell death, indicating baicalein induces apoptosis is partially dependent on caspase-3 pathway in H460 cells. These data suggest that baicalein, a 12-LOX inhibitor, inhibits the proliferation of H460 cells via S-phase arrest and induces apoptosis in association with the regulation of molecules in the cell cycle and apoptosis-related proteins.

Effects and mechanisms of aloe-emodin on cell death in human lung squamous cell carcinoma.

Aloe-emodin (1,8-dihydroxy-3-(hydroxymethyl)-anthraquinone) is an active component from the root and rhizome of Rheum palmatum. The study investigated the effects and mechanisms of aloe-emodin-induced cell death in human lung squamous cell carcinoma cell line CH27. Aloe-emodin (40 microM)-induced CH27 cell apoptosis was confirmed by DNA fragmentation (DNA ladders and sub-G(1) formation). Aloe-emodin-induced apoptosis of CH27 cells involved modulation of the expression of Bcl-2 family proteins, such as BclX(L), Bag-1, and Bak, and was associated with the translocation of Bak and Bax from cytosolic to particulate fractions. Aloe-emodin-treated CH27 cells had an increased relative abundance of cytochrome c in the cytosolic fraction. Results demonstrated that the activation of caspase-3, caspase-8, and caspase-9 is an important determinant of apoptotic death induced by aloe-emodin. These results suggest that aloe-emodin induces CH27 cell death by the Bax and Fas death pathway.

Protein kinase C involvement in aloe-emodin- and emodin-induced apoptosis in lung carcinoma cell.

1. This study demonstrated aloe-emodin- and emodin-induced apoptosis in lung carcinoma cell lines CH27 (human lung squamous carcinoma cell) and H460 (human lung non-small cell carcinoma cell). Aloe-emodin- and emodin-induced apoptosis was characterized by nuclear morphological changes and DNA fragmentation. 2. During apoptosis, an increase in cytochrome c of cytosolic fraction and activation of caspase-3, identified by the cleavage of its proform, were observed. 3. To elucidate whether the expression of protein kinase C (PKC) isozymes are involved in aloe-emodin- and emodin-induced apoptosis, this study examined the changes of PKC isozymes by Western blotting techniques during aloe-emodin- and emodin-induced apoptosis. 4. The expression of PKC isozymes involved in aloe-emodin- and emodin-induced apoptosis of CH27 and H460 cells. In this study, aloe-emodin and emodin induced the changes of each of PKC isozymes in CH27 and H460 cells. 5. The decrease in the expression of PKC delta and epsilon may play a critical role in aloe-emodin- and emodin-induced apoptosis in CH27 and H460 cells. 6. The present study also demonstrated that PKC stimulation occurs at a site downstream of caspase-3 in the emodin-mediated apoptotic pathway.

Effects and mechanisms of emodin on cell death in human lung squamous cell carcinoma.

1. Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an active component from the root and rhizome of Rheum palmatum that has been reported to exhibit antitumour effects, but the mechanism is not known. The study investigated the effects and mechanisms of emodin-induced cell death in human lung squamous carcinoma cell line CH27. 2. Emodin (50 microM)-induced CH27 cell apoptosis was confirmed by cell morphological change, sub-G1 formation in flow cytometry analysis, viability assay and degradation of focal adhesion kinase in this study. 3. Emodin-induced apoptosis of CH27 cells does not involve modulation of endogenous Bcl-X(L) protein expression, but appears to be associated with the increased expression of cellular Bak and Bax proteins. This study also demonstrated the translocation of Bak and Bax from cytosolic to particulate fractions. 4. This study has shown that emodin-treated CH27 cells revealed the increases in the relative abundance of cytochrome c for the indicated time intervals in cytosolic fraction. 5. This study demonstrates that the activation of caspase-3, caspase-9 and caspase-8 is an important determinant of apoptotic death induced by emodin. 6. These results suggested that emodin induces CH27 cell death by Bax death pathway and Fas pathway.

Serotonin-induced protein kinase C activation in cultured rat heart endothelial cells.

This study examined whether serotonin can activate protein kinase C in rat heart endothelial cells. Protein kinase C isozyme translocation was examined by Western blot analysis with isozyme-specific anti-protein kinase C antibody. In this study, only alpha protein kinase C isozyme was found to be translocated from the cytosolic to the particulate fractions after serotonin stimulation. The effect of serotonin on the incorporation of 32P from [gamma-32P]ATP into peptide substrate was studied as another indicator of protein kinase C activation. The experiments in this study demonstrated that the Ca(2+)-phospholipid-dependent protein kinase, protein kinase C, was activated by serotonin. By investigating [3H]phorbol 12,13-dibutyrate binding to protein kinase C and trypsin-treated protein kinase C activity, we demonstrated that the site of action of serotonin is probably the regulatory domain of protein kinase C. Finally, we also demonstrated that serotonin had no effect on the intracellular concentration of cyclic nucleotides (cAMP, cGMP). These findings support the hypothesis that protein kinase C may be an important participant in serotonin-induced endothelial cell contraction and barrier dysfunction.

Serotonin-stimulated increase in cytosolic Ca(2+) in cultured rat heart endothelial cells.

This study was designed to investigate the effects of serotonin on changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) in cultured rat heart endothelial cells. Serotonin stimulated a biphasic change in cytosolic Ca(2+) of rat heart endothelial cells: an initial transient increase, which primarily reflects the release of Ca(2+) from internal stores, followed by a slow rise in [Ca(2+)](i) during the incubation with serotonin. Our study also demonstrated that the pattern of the serotonin-induced increase in [Ca(2+)](i) was different from that induced by thrombin in rat heart endothelial cells. In this study, the role of [Ca(2+)](i) on endothelial paracellular barrier function was also investigated. Serotonin induced an increase in endothelial permeability which paralleled the rise in [Ca(2+)](i) and was blocked by the 5-HT(2) receptor antagonist cyproheptadine. Therefore, the serotonin-stimulated increase in cytosolic Ca(2+) and macromolecular permeability was receptor-mediated in rat heart endothelial cells. Further experiments demonstrated that the serotonin-induced increase in [Ca(2+)](i) was inhibited by the phospholipase C inhibitors, neomycin and [6-[[17beta-3-methoxyestra-1,3, 5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122). Experiments involving the rapid depletion of intracellular Ca(2+) stores and Ca(2+)-free medium demonstrated that the biphasic response of endothelial Ca(2+) to serotonin was related to the release of Ca(2+) from intracellular stores and to the influx of extracellular Ca(2+). We also suggest that serotonin-induced changes in [Ca(2+)](i) are related to Ca(2+) channels sensitive to voltage-operated and inorganic Ca(2+) channel blockers.

2-Phenyl-4-quinolone prevents serotonin-induced increases in endothelial permeability to albumin.

To investigate the role of 2-phenyl-4-quinolone in enhancing endothelial monolayer paracellular barrier function and preventing the disturbance of paracellular barrier function by vasoactive agents, the study examined the effect of 2-phenyl-4-quinolone on serotonin-mediated macromolecule transfer and microfilament changes in cultured rat heart endothelial cells. Serotonin-treated endothelial cells induced concentration-dependent increases in the passage of Evans blue dye-bound bovine serum albumin. Incubation of the endothelial monolayers with 2-phenyl-4-quinolone antagonized serotonin- and cytochalasin B-induced macromolecular permeability. 2-Phenyl-4-quinolone also opposed the effect of serotonin or cytochalasin B on the distribution and quantity of actin filaments in the endothelial cytoskeleton. Furthermore, 2-phenyl-4-quinolone alone led to an apparent quantitative increase in F actin fluorescence in endothelial cells. The addition of 10(-7) M 2-phenyl-4-quinolone had an effect on serotonin-induced changes in the myosin and distribution of myosin were comparable to that on serotonin monolayers. In conclusion, 2-phenyl-4-quinolone attenuated the serotonin-induced permeability of rat heart endothelial cells and this was associated with stabilization of F actin microfilaments and changes in the myosin organization. This result suggests that influences on cytoskeletal assembly may be involved in this process.

Decreased protein kinase C activation mediates inhibitory effect of norathyriol on serotonin-mediated endothelial permeability.

We examined the mechanisms of norathyriol on the serotonin-induced increased permeability of rat heart endothelial cell monolayers. The present study showed that the activation of rat heart endothelial cell protein kinase C by phorbol myristate acetate led to the dose-dependent increase in endothelial permeability to albumin, an effect that was inhibited by staurosporine (a protein kinase inhibitor). Staurosporine also attenuated the serotonin-induced increase in permeability. Norathyriol abolished both serotonin- and phorbol myristate acetate-induced permeability. We investigated whether norathyriol, by inhibiting protein kinase C activation, attenuated the serotonin-induced permeability. Immunofluorescence studies demonstrated that norathyriol prevented the redistribution of protein kinase C isozymes following stimulation with serotonin. Western blot analysis showed that norathyriol significantly inhibited the serotonin-induced translocation of the alpha protein kinase C isozyme from the cytosolic to the particulate fraction. In conclusion, norathyriol attenuates the serotonin-induced permeability of rat heart endothelial cells to macromolecules in association with inhibition of protein kinase C activation. This decrease in endothelial cell permeability may be one of the mechanisms for the protective effects of norathyriol against edema formation in response to inflammatory agonists in vivo.

Inhibitory effect of 2-phenyl-4-quinolone on serotonin-mediated changes in the morphology and permeability of endothelial monolayers.

The integrity of endothelial cell monolayers, a critical requirement for barrier maintenance, is needed for the prevention of edema formation. To investigate the mechanisms by which 2-phenyl-4-quinolone (YT-1) provided protection against serotonin-induced exudation, rat heart endothelial cell cultures were used. In this study, serotonin and phorbol myristate acetate (PMA) caused endothelial cells to became permeable to macromolecules by causing cell contraction and intercellular gap formation. These responses were attenuated by staurosporine, a protein kinase C inhibitor. Further experiments showed that YT-1 (1) did not alter serotonin-mediated early signal events such as protein kinase C activation, (2) protected against serotonin-induced endothelial barrier dysfunction by increasing intracellular cAMP levels, (3) played a role in regulating adenylate cyclase activity, (4) reversed serotonin-induced permeability to macromolecules, an effect which did not correlate with intracellular cGMP concentrations. This study demonstrates a possible mechanism by which YT-1 protects endothelial function and preserves the microvasculature from pharmacologic injury by vasoactive agents.

Synthesis and cytotoxicity of 1,6,7,8-substituted 2-(4'-substituted phenyl)-4-quinolones and related compounds: identification as antimitotic agents interacting with tubulin.

A series of 1,6,7,8-substituted 2-(4'-substituted phenyl)-4-quinolones and related compounds have been synthesized and evaluated as cytotoxic compounds and as antimitotic agents interacting with tubulin. The 2-phenyl-4-quinolones (22-30) with substituents (e.g. F, Cl, and OCH3) at C-6, C-7, and C-8 show, in general, potent cytotoxicity against human lung carcinoma (A-549), ileocecal carcinoma (HCT-8), melanoma (RPMI-7951), and epidermoid carcinoma of the nasopharynx (KB) and two murine leukemia lines (P-388 and L1210). Introduction of alkyl groups at N-1 or C-4 oxygen led to inactive compounds (35-43 and 50). In addition, compounds 24, 26, and 27 were evaluated in the National Cancer Institute's 60 human tumor cell line in vitro screen. These compounds demonstrated the most marked effects in the screen on two colon carcinoma cell lines (COLO-205 and KM-20L2) and on a central nervous system tumor cell line (SF-539) with compound 26 the most potent of the three agents. Compounds 24, 26, and 27 were potent inhibitors of tubulin polymerization, with activity nearly comparable to that of the potent antimitotic natural products colchicine, podophyllotoxin, and combretastatin A-4. The three agents also inhibited the binding of radiolabeled colchicine to tubulin, but this inhibition was less potent than that obtained with the natural products.