COH-203, a novel microtubule inhibitor, exhibits potent anti-tumor activity via p53-dependent senescence in hepatocellular carcinoma.

5-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-3H-1,2-dithiol-3-one (COH-203) is a novel synthesized analogue of combretastatin A-4 that can be classified as a microtubule inhibitor. In this study, we evaluated the anti-hepatoma effect of COH-203 in vitro and in vivo and explored the underlying molecular mechanisms. COH-203 was shown to be more effective in inhibiting the proliferation of liver cancer cells compared with normal liver cells. COH-203 also displayed potent anti-tumor activity in a hepatocellular carcinoma xenograft model without significant toxicity. Mechanistic studies demonstrated that treatment with COH-203 induced mitotic arrest by inhibiting tubulin polymerization in BEL-7402 liver cancer cells. Long-term COH-203 treatment in BEL-7402 cells led to mitotic slippage followed by senescence via the p14(Arf)-p53-p21 and p16(INK4α)-Rb pathways. Furthermore, suppression of p53 via pifithrin-α (p53 inhibitor) and p53-siRNA attenuated COH-203-induced senescence in BEL-7402 cells, suggesting that COH-203 induced senescence p53-dependently. In conclusion, we report for the first time that COH-203, one compound in the combretastatin family, promotes anti-proliferative activity through the induction of p-53 dependent senescence. Our findings will provide a molecular rationale for the development of COH-203 as a promising anti-tumor agent.

Amyloid ß 3-10 DNA vaccination suggests a potential new treatment for Alzheimer's disease in BALB/c mice.

BACKGROUND: Amyloid ß(1-42) (Aß(42)) peptide vaccination has been proved to be effective in reducing amyloid burden in brain and improving cognitive function in Alzheimer's disease (AD) mouse models. But the phase II trial of Aß(42) peptide vaccine was halted because of T cell-mediated meningoencephalitis. In this study, a DNA vaccine, p(Aß(3-10))(10)-CpG, was constructed to test whether it would induce predominant T(H)2 immune response upon immunization of BALB/c mice. METHODS: BALB/c mice were vaccinated intramuscularly with p(Aß(3-10))(10)-CpG plasmids. Aß(42) peptide, pcDNA3.1(+) empty vector and PBS were injected to the control groups. Expression of interesting gene in injected muscle was identified by immunohistochemistry. Anti-Aß antibody titers, isotype profiles as well as cytokines in ex vivo splenocytes culture supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA). RESULTS: P(Aß(3-10))(10)-CpG plasmid was expressed in muscle after injection detected by immunohistochemistry. The p(Aß(3-10))(10)-CpG vaccine induced high titers of anti-Aß antibodies in BALB/c mice. And isotype of the antibodies was mainly IgG1, the IgG1/IgG2a ratio for the p(Aß(3-10))(10)-CpG group was approximately 5 times greater than that for the Aß(42) peptide group. Ex vivo cultured splenocytes isolated from mice immunized with p(Aß(3-10))(10)-CpG exhibited high interleukin-4 response and low interleukin-γ (IFN-γ) response. CONCLUSIONS: Immunization with p(Aß(3-10))(10)-CpG vaccine primarily induces a T(H)2 type of response, thus reduces the probability of inflammation. This p(Aß(3-10))(10)-CpG vaccine possesses the basic factors required for a safe and effective AD vaccine.

Improving memory and decreasing cognitive impairment in Tg-APPswe/PSEN1dE9 mice with Aß3-10 repeat fragment plasmid by reducing Aß deposition and inflammatory response.

The present study aimed to investigate the effects of Aß3-10 repeat fragment plasmid for the treatment of Tg-APPswe/PSEN1dE9 (Tg) mice. The plasmid pcDNA3.1-(Aß3-10)10-CpG was constructed and intramuscularly injected into 12-month-old Tg mice. Through the use of behavioral tests, anti-Aß antibody and Aß assays, cytokine assay, Aß deposition, and astrocytes analysis results demonstrated that Aß3-10 repeat fragment plasmid exhibited immunogenicity and reduced memory impairment in Tg mice via clearance of cerebral Aß deposition, without significant side effects. Aß3-10 repeat fragment plasmid immunization reduced Th1 cell-mediated immunity, secretion of interferon-γ, and stimulation to astrocytes. These data showed that the Aß3-10 repeat fragment plasmid improved memory and decreased cognitive impairment in Tg mice by reducing Aß deposition and inflammatory responses.

Design and synthesis of biphenyl derivatives as mushroom tyrosinase inhibitors.

Two new series of biphenyls, analogs of aglycone of natural product fortuneanoside E, were prepared using Suzuki-Miyaura cross-coupling and selective magnesium iodide demethylation/debenzylation, and their mushroom tyrosinase inhibitory activity was evaluated. Most of the 4-hydroxy-3,5-dimethoxyphenyl biphenyl compounds (series II, 20-36) were in general more active than 3,4,5-trimethoxyphenyl biphenyl compounds (series I, 1-19). Structure-activity relationships study showed that monosaccharide substituents, such as glucose, were not necessary and the presence of 4-hydroxy-3,5-dimethoxyphenyl moiety was crucial for inhibitory activity. Among the compounds synthesised, compound 21 (IC50=0.02 mM) was found to be the most active one, which exhibited an activity that was 7 times higher than that of fortuneanoside E (IC50=0.14 mM) and 10 times higher than that of arbutin (IC50=0.21 mM), known as potent tyrosinase inhibitors. The inhibition kinetics analyzed by Lineweaver-Burk plots revealed that compound 21 was a competitive inhibitor (Ki=0.015 mM).

In vitro effects of erythromycin on RANKL and nuclear factor-kappa B by human TNF-alpha stimulated Jurkat cells.

The receptor activator of NF-kappaB ligand (RANKL) and its signal downstream nuclear factor-kappaB (NF-kappaB) are critical regulators for immune responses as well as bone remodeling. The present study aimed to examine the effects of erythromycin (EM) on the activation of RANKL, correlation with NF-kappaB expression, proliferation and apoptosis of human Jurkat T cells. Jurkat T cells were pretreated with 100 pmol/l tumor necrosis factor-alpha (TNF-alpha) for 1 h followed by various concentrations of EM for 24 h. The mRNA expressions of RANKL and NF-kappaB were examined by RT-PCR. The protein expression of NF-kappaB was analyzed by Western blot. The protein level of RANKL was examined by flow cytometry, immunofluorescence microscopy and Western blot analyses. We also examined proliferation of Jurkat T cells by MTT assay, apoptosis by flow cytometry analysis after staining with PI and morphological observation after AO/EB staining. The results showed that EM inhibited TNF-alpha-induced expressions of RANKL and NF-kappaB at both mRNA and protein levels in a concentration-dependent manner. The expression of RANKL was correlated with the expression of NF-kappaB. Moreover, EM influenced the proliferation and apoptosis of human Jurkat T cells. These data suggest that EM acts as an anti-inflammatory agent not only to interact with the expression of NF-kappaB and the proliferation of human Jurkat T cells, but also to reduce the level of RANKL.

Synthesis of (+/-) homoisoflavanone and corresponding homoisoflavane.

The total synthesis of racemic 3-(4'-methoxybenzyl)-7,8-methylenedioxy-chroman-4-one, a homoisoflavanone with antimycobacterial activity isolated recently from Chlorophytum inornatum, was described. During this research, the first approach for the conversion of homoisoflavonoids into homoisoflavanes was also developed.

Synthesis and antiproliferative in-vitro activity of natural flavans and related compounds.

The total synthesis of natural flavan racemates (+/-) 1, (+/-) 2 and natural flavones 3, 4 had thus been achieved. A straightforward synthetic procedure of flavans via the Pd-C catalyzed hydrogenation/hydrogenolysis of corresponding flavones was developed. Furthermore, the antiproliferative activities of racemic flavans (+/-) 1, (+/-) 2, flavones 3, 4, and five synthetic intermediates toward human SGC-7901, BEL-7402, HeLa, and HL-60 cell lines in vitro were evaluated by MTT assay, and the racemic flavans (+/-) 1 were found to have significant antiproliferative activity against all four cell lines.

Synthesis and anti-proliferative in-vitro activity of two natural dihydrostilbenes and their analogues.

A total synthetic route for two natural dihydrostilbenes with significant cytotoxicity toward human cancer cell lines, (3-(2-(7-methoxybenzo[d][1,3]dioxol-5-yl)ethyl)phenol 1a and 6-(3-hydroxyphenethyl)benzo[d][1,3]dioxol-4-ol 1b), which were isolated from Bulbophyllum odoratissimum Lindl, was developed via Wittig-Horner reaction. The natural products 1a and 1b were obtained in 28% and 20% overall yield, respectively. Additionally, nine analogues, 1c-1k, of the two natural dihydrostilbenes were synthesized and evaluated for their anti-proliferative activity against human SGC-7901, KB and HT-1080 cell lines by MTT assay. The activities of 1c and 1d were in the same range as those of the natural products 1a and 1b.

Involvement of mitochondria and caspase pathways in N-demethyl-clarithromycin-induced apoptosis in human cervical cancer HeLa cell.

AIM: To study the mechanisms by which N-demethyl-clarithromycin (NDC) induces human cervical cancer HeLa cell apoptosis in vitro. METHODS: The viability of N-demethyl-clarithromycin-induced HeLa cells was measured by MTT assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. Measurement of mitochondrial transmembrane potential was analyzed by a FACScan flowcytometer. Caspase-3, poly-(ADP-ribose) polymerase (PARP), caspase-activated DNase (ICAD), Bcl-2, Bax, p53, and SIRT1 protein expression and the release of cytochrome c were detected by Western blot analysis. RESULTS: N-demethyl-clarithromycin, an anti-inflammatory substance, inhibited HeLa cell growth in a dose- and time-dependent manner. N-demethyl-clarithro-mycin induced HeLa cell death through the apoptotic pathways. The pan-caspase inhibitor (z-VAD-fmk), caspase-3 inhibitor (z-DEVD-fmk) and the caspase-9 inhibitor (z-LEHD-fmk) partially enhanced cell viability induced by N-demethyl-clarithromycin, but the caspase-8 inhibitor (z-IETD-fmk) had almost no effect. Caspase-3 was activated then followed by the degradation of caspase-3 substrates, the inhibitor of ICAD and PARP. Simultaneously, mitochondrial transmembrane potential was markedly reduced and the release of cytochrome c in the cytosol was increased. N-demethyl-clarithromycin upregulated the expression ratio of mitochondrial Bax/Bcl-2, and significantly increased the expression of the p53 protein. It also downregulated anti-apoptotic protein SIRT1 expression. CONCLUSION: N-demethyl-clarithromycin induced apoptosis in HeLa cells via the mitochondrial pathway.