The mechanism of anticancer activity of the new synthesized compound - 6,7-Methylenedioxy-4-(2,4-dimethoxyphenyl)quinolin -2(1H)-one(12e) in human ovarian cancer cell lines.

OBJECTIVE: Ovarian cancer is the most lethal of the gynecologic malignancies. Most women have advanced disease at diagnosis and require extensive debulking surgery and aggressive chemotherapy. Induction of apoptosis in cancer cells has been used as an important approach for cancer therapy. We examined the anticancer effect of 6,7-methylenedioxy-4-(2,4-dimethoxyphenyl)quinolin-2(1H)-one (12e) in human ovarian cancer cell line. MATERIALS AND METHODS: The 6,7-methylenedioxy-4- (2,4-dimethoxyphenyl) quinolin-2 (1H) -one (12e) was synthesized and provided by Dr. Li-Jiau Huang of China Medical University. Cell viability analysis showed that 12e inhibited cell growth and induced cell death in time- and dose-dependent manners. In order to study the underlying cell death mechanism, 2774 and SKOV3 cells treated with 12e were studied by morphology, DAPI/TUNEL double staining, DNA gel electrophoresis. To search the mechanisms of anti-proliferative effect of 12e, cell cycle analysis was performed. Changes in proteins related to cell death were analyzed by Western blot. RESULTS: 12e significantly induced apoptosis evidenced by morphological changes, TUNEL-DAPI double-staining and DNA fragmentation. Western blot analysis demonstrated that the protein level of Bcl-2 was decreased after treatment with 12e, while the level of p53 and Bax was increased. 12e treatment resulted in G2/M arrest through down modulation of cyclin B1 and cdk1. CONCLUSION: These results suggested that 12e -induced growth inhibition was associated with cell cycle arrest and apoptosis.

Peroxisome proliferator-activated receptor alpha accelerates neuronal differentiation and this might involve the mitogen-activated protein kinase pathway.

Activation of peroxisome proliferator-activated receptor alpha (PPARα) has been reported to modulate cell proliferation, migration, and differentiation in astrocytes. In this study, we used a retinoic acid (RA)-induced differentiation model of NTERA-2/clone D1 (NT2) cells to explore the functional significance of PPARα in neuronal differentiation. We found that activating PPARα by Wy14643 accelerated neuronal differentiation via regulating the expression of neuronal markers. RT-PCR assays showed a significant increase in NeuroD expression and a decrease in nestin expression in cells treated concomitantly with RA and Wy14643 for 2 days compared to the levels in cells treated with RA alone. Expression of MAP2 protein, a mature neuronal marker, was markedly upregulated at day 10 of Wy14643 treatment, which was maintained after 21 days of neuronal formation. Corresponding to the changes in MAP2 expression, the expression of Cdk5 was upregulated with Wy14643 exposure from day 10 to day 21. Moreover, cells treated with Wy14643 displayed higher expression levels of phospho-ERK and phospho-p38 in the differentiation process than cell treated with RA alone. These results indicated that activation of PPARα accelerated neuronal differentiation through upregulating the expression of NeuroD, MAP2, and Cdk5 and downregulating the expression of nestin. MAPK signals, ERK and p38, might contribute to the accelerated differentiation process. These findings suggest that PPARα plays a role in regulating neuronal differentiation and may be beneficial for functional recovery from neurological disorders.

Human parvovirus B19 antibodies induce altered membrane protein expression and apoptosis of BeWo trophoblasts.

Human parvovirus B19 (B19) is harmful during pregnancy since it can be vertically transmitted to the developing fetus. In addition, the anti­B19 antibodies induced by B19 infection are believed to have a cytopathic role in B19 transmission; however, knowledge regarding the effects of anti­B19 antibodies during pregnancy is limited. To investigate the possible roles of anti­B19 antibodies during pregnancy, the present study examined the effects of anti­B19­VP1 unique region (VP1u), anti­B19­VP2 and anti­B19­nonstructural protein 1 (NS1) immunoglobulin G (IgG) antibodies on BeWo trophoblasts. Briefly, BeWo trophoblasts were incubated with purified IgG against B19­VP1u, B19­VP2 and B19­NS1. Subsequently, the expression of surface proteins and apoptotic molecules were assessed in BeWo trophoblasts using flow cytometry, ELISA and western blotting. The expression levels of human leukocyte antigen (HLA)­G were significantly increased on BeWo trophoblasts treated with rabbit anti­B19­VP1u IgG, and were unchanged in those treated with rabbit anti­B19­NS1 and anti­B19­VP2 IgG, as compared with the control group. Furthermore, the expression levels of globoside (P blood group antigen) and cluster of differentiation (CD)29 (ß1 integrin) were significantly increased in BeWo trophoblasts treated with rabbit anti­B19­NS1 and anti­B19­VP2 IgG, whereas only CD29 was also significantly increased in cells treated with anti­B19­VP1u IgG. In addition, the number of cells at sub­G1 phase; caspase­3 activity; and the expression of intrinsic and extrinsic apoptotic molecules, including Fas­associated death domain protein, activated caspase­8, activated caspase­3, B­cell lymphoma 2­associated X protein, cytochrome c, apoptotic peptidase activating factor 1 and activated caspase­9, were significantly increased in BeWo trophoblasts treated with anti­B19­VP1u and anti­B19­NS1 IgG. In conclusion, the present study demonstrated that antibodies against B19 may have a crucial role in pathological processes during pregnancy. These findings may help to elucidate the mechanisms underlying transmission of the B19 virus during pregnancy.

Ursolic acid plays a protective role in obesity-induced cardiovascular diseases.

The metabolic disturbance of obesity is one of the most common risk factors of atherosclerosis. Resistin, an obesity-induced adipokine, can induce the expression of cell adhesion molecules and the attachment of monocytes to endothelial cells, which play an important role in the development of atherosclerosis. Ursolic acid, a pentacyclic triterpenoid found in fruits and many herbs, exhibits an array of biological effects such as anti-inflammatory and antioxidative properties. The aim of this study was to investigate the potential underlying mechanisms of the effect of ursolic acid on resistin-induced adhesion of U937 cells to human umbilical vein endothelial cells (HUVECs). Our data indicated that ursolic acid suppressed the adhesion of U937 to HUVECs and downregulated the expression of adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1), and E-selectin, in resistin-induced HUVECs by decreasing the production of intracellular reaction oxygen species (ROS) and attenuating the nuclear translocation of NFκB. Ursolic acid appeared to inhibit resistin-induced atherosclerosis, suggesting that ursolic acid may play a protective role in obesity-induced cardiovascular diseases.

Human parvovirus B19 VP1u Protein as inflammatory mediators induces liver injury in naïve mice.

Human parvovirus B19 (B19V) is a human pathogen known to be associated with many non-erythroid diseases, including hepatitis. Although B19V VP1-unique region (B19-VP1u) has crucial roles in the pathogenesis of B19V infection, the influence of B19-VP1u proteins on hepatic injury is still obscure. This study investigated the effect and possible inflammatory signaling of B19-VP1u in livers from BALB/c mice that were subcutaneously inoculated with VP1u-expressing COS-7 cells. The in vivo effects of B19-VP1u were analyzed by using live animal imaging system (IVIS), Haematoxylin-Eosin staining, gel zymography, and immunoblotting after inoculation. Markedly hepatocyte disarray and lymphocyte infiltration, enhanced matrix metalloproteinase (MMP)-9 activity and increased phosphorylation of p38, ERK, IKK-α, IκB and NF-κB (p-p65) proteins were observed in livers from BALB/c mice receiving COS-7 cells expressing B19-VP1u as well as the significantly increased CRP, IL-1ß and IL-6. Notably, IFN-γ and phosphorylated STAT1, but not STAT3, were also significantly increased in the livers of BALB/c mice that were subcutaneously inoculated with VP1u-expressing COS-7 cells. These findings revealed the effects of B19-VP1u on liver injury and suggested that B19-VP1u may have a role as mediators of inflammation in B19V infection.

Synthesis and biological evaluation of novel 3,9-substituted ß-carboline derivatives as anticancer agents.

In our previous studies on 1-benzyl-3-(5-hydroxymethyl-2-furyl)indazole (YC-1) analogs, we synthesised numerous substituted carbazole and α-carboline derivatives, which exhibited anticancer activity. In this study, we designed and synthesised a series of 3,9-substituted ß-carbolines, by replacing the tricyclic rings of carbazole and α-carboline derivatives with isosteric ß-carboline, and evaluated anticancer activity. We observed that 9-(2-methoxybenzyl)-ß-carboline-3-carboxylic acid (11a) inhibited the growth of HL-60 cells by inducing apoptosis, with a half maximal inhibitory concentration of 4.0 µM. Our findings indicate that ß-carboline derivatives can be used as lead compounds for developing novel antitumor agents.

Design and synthesis of 6,7-methylenedioxy-4-substituted phenylquinolin-2(1H)-one derivatives as novel anticancer agents that induce apoptosis with cell cycle arrest at G2/M phase.

Novel 6,7-methylenedioxy-4-substituted phenylquinolin-2(1H)-one derivatives 12a-n were designed and prepared through an intramolecular cyclization reaction and evaluated for in vitro anticancer activity. Among the synthesized compounds, 6,7-methylenedioxy-4-(2,4-dimethoxyphenyl)quinolin-2(1H)-one (12e) displayed potent cytotoxicity against several different tumor cell lines at a sub-micromolar level. Furthermore, results of fluorescence-activated cell sorting (FACS) analysis suggested that 12e induced cell cycle arrest in the G2/M phase accompanied by apoptosis in HL-60 and H460 cells. This action was confirmed by Hoechst staining and caspase-3 activation. Due to their easy synthesis and remarkable biological activities, 4-phenylquinolin-2(1H)-one analogs (4-PQs) are promising new anticancer leads based on the quinoline scaffold. Accordingly, compound 12e was identified as a new lead compound that merits further optimization and development as an anticancer candidate.

Biological evaluation of 9-[(6-chloropyridin-4-yl)methyl]-9H-carbazole-3-carbinol as an anticancer agent.

Most conventional anticancer drugs exert either anti-proliferation or anti-angiogenesis activity. Recently, searching for potential multi-target agents has become an alternative strategy for cancer treatment. Several structurally different carbazole alkaloids from either natural or synthesized sources represent an important and heterogeneous class of anticancer agents. In the present study, we investigated the anticancer activity of a novel synthetic carbazole derivative, 9-[(6-chloropyridin-4-yl)methyl]-9H-carbazole-3-carbinol (HYL-6d), which is structurally different from other previously characterized carbazoles. HYL-6d-treated human breast cancer MCF-7 cells exhibited an increased population arrested at the sub-G1 and S phases, as well as an increase of p53 and decrease of cyclin D1, A and CDK2. Also, HYL-6d treatment induced MCF-7 cell apoptosis and this was accompanied by a decreased expression of Bcl-2, increased levels of p53 and Bcl-XS and the activation of caspase-9. Experimental results from human umbilical vascular endothelial cells (HUVECs) showed that HYL-6d also exerted its anti-angiogenic activity in HUVECs by inhibiting cell proliferation, migration, and tube formation induced by VEGF- or bFGF in vitro. In summary, the data indicate that HYL-6d exhibits both cytotoxic effects against human cancer cells and anti-angiogenic activities, which make it a potential therapeutic drug for cancer treatment.

Co-treating with arecoline and 4-nitroquinoline 1-oxide to establish a mouse model mimicking oral tumorigenesis.

The aim of this study was to establish an effective mouse model of oral cancer and to use this model to identify potential markers of oral tumor progression. C57BL/6JNarl mice were treated with arecoline, 4-nitroquinoline 1-oxide (4-NQO), or both arecoline and 4-NQO in high and low doses for 8 weeks to induce oral tumor. The induced oral lesions were observed for 20 weeks to assess the efficiency of cancer induction and survival rate of the mice. In addition, two target proteins that are frequently overexpressed during tongue cancer tumorigenesis, alphaB-crystallin and Hsp27, were examined by immunohistochemical analysis. In mice exposed to 4-NQO (200 microg/mL) and arecoline (500 microg/mL), the tongue lesions showed evidence of hyperplasia, papilloma, dysplasia, and carcinoma, and the lesions were pathologically similar to those lesions in human oral cancer. The tongue tumor incidence rate was 100% in mice exposed to concomitant 4-NQO (200 microg/mL) and arecoline (500 microg/mL) treatment, 57% in mice exposed to 4-NQO only, and 0% in mice exposed to arecoline only. Immunohistochemical analysis demonstrated that, consistent with human studies, alphaB-crystallin and Hsp27 were upregulated in murine oral tumors. In conclusion, we have established a powerful animal model that enables the study of the promoting effects of arecoline on tongue tumorigenesis. Data subsequently attained from this mouse model support a role for alphaB-crystallin and Hsp27 as clinical markers for tumor progression.

Carnosic acid reduces cytokine-induced adhesion molecules expression and monocyte adhesion to endothelial cells.

BACKGROUND: Expression of cell adhesion molecules on the endothelium and the attachment of monocytes to endothelium may play a major role in the early atherogenic process. AIM OF THE STUDY: We investigated the effects of carnosic acid on the adhesion of U937 cells to IL-1beta-treated human umbilical vein endothelial cells (HUVECs), as well as on the expression of adhesion molecules. RESULTS: Our data showed that pretreatment with 10 and 20 micromol/l carnosic acid significantly reduced the number of U937 cells adhering to IL-1beta-treated HUVECs. In addition, we found that 20 micromol/l carnosic was more effective than 10 micromol/l carnosic acid at inhibiting expression of cell adhesion molecules (ICAM-1, VCAM-1, and E-selectin), the nuclear translocation of NF-kappaB subunits p65 and p50, and the production of ROS in IL-1beta-stimulated HUVECs. CONCLUSIONS: We conclude that carnosic acid inhibits IL-1beta-induced ICAM-1, VCAM-1 and E-selectin expression in HUVECs through a mechanism that involves NFkappaB. We propose that the reduction in binding of human monocytic cell line U937 to IL-1beta-treated HUVECs is due to the anti-inflammatory properties of carnosic acid.

4-Fluoro-N-butylphenylacetamide (H6) inhibits cell growth via cell-cycle arrest and apoptosis in human cervical cancer cells.

Phenylacetate induced tumor cytostasis and differentiation. The chemotherapeutic function of the compound in lung cancer has been previously reported, however, whether or not phenylacetate performs other activities is not known. In this study, the potential usage of synthetic phenylacetate derivatives, 4-fluoro-N-butylphenylacetamides (H6) was investigated in human cervical cancer cells. H6 displayed anti-proliferative and apoptosis effects, with an IC(50) of 1.0-1.5 mM and an ID(50) of about 3days. Moreover, it significantly induced apoptosis as evidenced by morphological changes, DAPI and TUNEL staining and DNA fragmentation. H6 increased the expression of Bax protein, whereas it decreased the expression of Bcl-2 protein. H6 also induced accumulation of cytosolic cytochrome c and activation of caspase cascade (caspase-9 and -3), and then DNA fragmentation and apoptosis occurred. The underlying anti-proliferative mechanism for H6 is likely due to the down-regulation of G2/M-phase association cdks and cyclins and up-regulation of p53 to mediate G2/M-phase arrest. Furthermore, the decrease of Bcl-2 and activation of Bax, caspase-9/caspase-3 may be the effectors of H6-induced apoptosis.

A phenylacetate derivative, SCK6, inhibits cell proliferation via G1 cell cycle arrest and apoptosis.

Phenylacetate is a differentiation agent and has anticancer activity with relatively low toxicity. In the present study, we examined the anticancer effect of six synthetic phenylacetate derivatives in human lung cancer cells in our search for more effective phenylacetate analogous. Results showed that the antiproliferative effects of these synthetic compounds were stronger than those of phenylacetate, and that N-butyl-2-(2-fluorolphenyl)acetamide (SCK6) is the most potent compound. To address the mechanism of the antiproliferative effect of SCK6, cell cycle analysis was performed. Result showed that SCK6 (1 mM) induced G(1) arrest in CH27 cells. Western blot analysis of G(1) phase regulatory proteins demonstrated that the protein levels of cyclin-dependent kinase 2 (Cdk2), Cdk4, Cyclin E and Cyclin D3 were decreased after treatment with SCK6 but not those of Cdk6, Cyclin D1 and D2. In contrast, SCK6 increased the protein levels of p53 and p21(CIP1/WAF1). Data from in situ terminal transferase-mediated dUTP-fluorescensin nick end-labeling (TUNEL) assay and DNA fragmentation analysis demonstrated that SCK6 induced apoptotic cell death in CH27 cells. This SCK6-induced apoptosis was accompanied by a downregulation of Bcl-2 protein and activation of the caspase-9 cascade. Overexpression of Bcl-2 by adeno-Bcl-2 vector infection significantly inhibited SCK6-induced apoptosis. Moreover, treatment with caspase inhibitors also markedly reduced cell death induced by SCK6. Taken together, these results suggest that downregulation of G(1)-associated Cdks and cyclins and upregulation of p53 and p21(CIP1/WAF1) may contribute to SCK6-mediated G(1)-phase arrest. Furthermore, the decrease in Bcl-2 and the activation of caspase-9/caspase-3 may be the effector mechanism through which SCK6 induces apoptosis.

4-Fluoro-N-butylphenylacetamide: a synthetic phenylacetate derivative that upregulates Bcl-X(S), activates caspase cascade and induces apoptosis in human squamous lung cancer CH27 cells.

Phenylacetate (PA) and related aromatic fatty acids induce antiproliferation and differentiation of cancer cell; they have potent anti-tumor properties with relatively low toxicity. To search for more potent analogues of PA, PA derivatives have been synthesized. In this study, we investigated the effects of six synthetic PA derivatives on the growth of human lung cancer cells. Results showed that the anti-proliferative effects of these synthetic compounds were strong than those of PA, 4-fluoro-N-butylphenylacetamide (H6) is the most potent compound. 4,6-Diamidino-2-phenylindole (DAPI) staining, in situ TUNEL assay and DNA gel electrophoresis analysis indicated that a marked reduction in the number of CH27 cells with H6 was related to the induction of apoptosis. The apoptosis triggered by H6 was accompanied by up-regulation of Bcl-X(S), accumulation of cytosolic cytochrome c and activation of caspase cascade (caspase-9 and -3). Furthermore, H6 induces proteolytic cleavage of poly (ADP-ribose) polymerase, which followed the appearance of caspase activity and preceded DNA fragmentation. Pretreatment with caspase inhibitors markedly inhibited H6-induced caspase activity and apoptosis. These results suggest that H6 may induce apoptosis through a Bcl-X(S) and caspase-dependent mechanism.