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Flavonoids, a class of natural polyphenolic compounds, inhibit cell cycle progression and induce apoptosis. This study was performed to investigate the antiproliferative effect of luteolin, the flavonoid isolated from Ixeris sonchifolia Hance, and to elucidate the detailed apoptotic mechanism in HCC cells. According to the result of MTT assay luteolin possessed antiproliferative effect, and HepG2 cells were the most sensitive to luteolin. Propidium iodide staining, fluorescence activated cell sorting analysis, western blot analysis and RT-PCR were applied to compare the difference of apoptotic event between the two HCC cell lines, with wild-type p53 (HepG2) or not (Hep3B) based on time and concentration. The treatment of luteolin upregulated the expression levels of transforming growth factor β1 (TGF‑β1), p21WAF1/CIP1, p27KIP1, Smad4, and Fas in HCC cells. Thus, the expression of p21WAF1/CIP1 was controlled by another factor, such as TGF‑β1 in addition to p53, and notably the key factor might be p21WAF1/CIP1 in the remarkable switch to G1 cell cycle arrest in HepG2 cells rather than p27KIP1. Luteolin induced apoptotic cell death in Hep3B cells while caused G1 arrest in HepG2 cells. Taken together, we conclude that luteolin induces apoptosis from G1 arrest via three signaling pathways of TGF‑β1, p53, and Fas/Fas-ligand in HCC cells.

Since elevated ethanol is a major stress during ethanol fermentation, yeast strains tolerant to ethanol are highly desirable for the industrial scale ethanol production. A technology called global transcriptional machinery engineering (gTME), which exploits a mutant library of SPT15 encoding the TATA-binding protein of Saccharomyces cerevisiae (Alper et al., 2006; Science 314: 1565–1568), seems to a powerful tool for creating ethanol-tolerant strains. However, the ability of created strains to tolerate high ethanol on rich media remains unproven. In this study, a similar strategy was used to obtain five strains with enhanced ethanol tolerance (ETS1–5) of S. cerevisiae. Comparing global transcriptional profiles of two selected strains ETS2 and ETS3 with that of the control identified 42 genes that were commonly regulated with twofold change. Out of 34 deletion mutants available from a gene knockout library, 18 were ethanol sensitive, suggesting that these genes were closely associated with ethanol tolerance. Eight of them were novel with most being functionally unknown. To establish a basis for future industrial applications, strains iETS2 and iETS3 were created by integrating the SPT15 mutant alleles of ETS2 and ETS3 into the chromosomes, which also exhibited enhanced ethanol tolerance and survival upon ethanol shock on a rich medium. Fermentation with 20% glucose for 24 h in a bioreactor revealed that iETS2 and iETS3 grew better and produced approximately 25% more ethanol than a control strain. The ethanol yield and productivity were also substantially enhanced: 0.31 g/g and 2.6 g/L/h, respectively, for control and 0.39 g/g and 3.2 g/L/h, respectively, for iETS2 and iETS3. Thus, our study demonstrates the utility of gTME in generating strains with enhanced ethanol tolerance that resulted in increase of ethanol production. Strains with enhanced tolerance to other stresses such as heat, fermentation inhibitors, osmotic pressure, and so on, may be further created by using gTME. Biotechnol. Bioeng. 2011; 108:1776–1787. © 2011 Wiley Periodicals, Inc.

In a preliminary study, we found that benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD- fmk), unlike Boc-aspartyl(OMe)-fluoromethylketone (BocD-fmk), at usual dosage could not prevent genistein-induced apoptosis of p815 mastocytoma cells. This study was undertaken to reveal the mechanism underlying the incapability of zVAD-fmk in preventing this type of apoptosis. We observed that 14-3-3 protein level was reduced in genistein-treated cells and that BocD-fmk but not zVAD-fmk prevented the reduction of 14-3-3 protein level and the release of Bad from 14-3-3. We also demonstrated that truncated Bad to Bcl-xL interaction in genistein- treated cells was prevented by BocD-fmk but not by zVAD-fmk treatment. Our data indicate that BocD- fmk, compared to zVAD-fmk, has a certain preference for inhibiting 14-3-3/Bad signalling pathway. We also elucidated that this differential efficacy of BocD-fmk and zVAD-fmk resulted from the different effect in inhibiting caspase-6 and that co-treatment of zVAD-fmk and caspase-6 specific inhibitor substantially prevented genistein-induced apoptosis. Our data shows that caspase-6 plays a role on Bad/14-3-3 pathway in genistein-induced apoptosis of p815 cells, and that the usual dose of zVAD-fmk, in contrast to BocD-fmk, did not prevent caspase-6 acting on 14-3-3/Bad-mediated event.

We investigated the possible mechanisms by which petrotetrayndiol A, a polyacetylene from the sponge Petrosia sp., exerts its anti-proliferative activity in cultured SK-MEL-2 human melanoma cells. Petrotetrayndiol A-treated SK-MEL-2 cells showed growth inhibition and induction of apoptosis in a dose-dependent manner as measured by MTT assay, fluorescent microscopy and flow cytometric analysis. Flow cytometric analysis revealed that petrotetrayndiol A resulted in G2/M arrest in the cell cycle progression which was associated with a marked decrease in the protein expression of cyclin B1 and its activating partner Cdc2 with concomitant inductions of p21WAF1/CIP1. The increase in apoptosis was associated with a dose-dependent up-regulation of cytosolic factor, such as Bax and release of cytochrome c, and down-regulation of Bcl-2. We also observed activation of caspase-9 and caspase-3, DNA ladder formation, proteolytic degradation of poly(ADP-ribose)-polymerase (PARP), and selective down-regulation of cIAP-1. The apoptotic manifestations, such as PARP cleavage and DNA fragmentation, were abolished in the presence of the tripeptide caspase inhibitor z-VAD-fmk and a caspase-3-specific inhibitor Ac-DEVD-cho. Our data thus demonstrate that petrotetrayndiol A-induced apoptosis and growth inhibition of SK-MEL-2 cells is dependent on caspase activation.

We investigated the anti-proliferative effects of luteolin and apigenin, isolated from Ixeris sonchifolia Hance, on HepG2 human hepatocellular carcinoma cells. In MTT assay luteolin showed more efficient anti-proliferative effects on cells than apigenin did. According to propidium iodide staining and flow cytometry studies, we postulated that these effects might be a result of cell cyde arrest. Hence we examined the changes of protein expressions related to cell cycle arrest. Western blotting data demonstrated that the down-regulated expression of CDK4 was correlated to the increase of p53 and CDK inhibitor p21(WAF1/CIP1) protein. These data suggest that luteolin may have potential as an anti-cancer agent.

HS-1200, a synthetic chenodeoxycholic acid derivative, has cytotoxic activity in various human cancer cells. The present study was undertaken to examine whether HS-1200 sensitizes radiation-induced apoptosis in MCF-7 human breast carcinoma cells. Clonogenic assay elucidated that the combination treatment with HS-1200 and radiation induced more cytotoxic effects than the radiation treatment alone. Nuclear staining, DNA electrophoresis and Western blot analysis for poly(ADP-ribose) polymerase revealed that the increased cytotoxic effect by the combination treatment resulted from the augmentation of apoptosis. There was an increase in the expression level of Bax and its translocation onto the mitochondria, a reduction in the mitochondrial membrane potential in the earlier time-points, and the release of cytochrome c into the cytosol increased in the MCF-7 cells treated with radiation and HS-1200 compared to the cells treated only with radiation. Therefore, the synthetic bile acid derivative, HS-1200, could have the therapeutic potential as a radiosensitizer in MCF-7 cells.

We investigated the effects of ircinin-1, a lipid compound (a C25 sesterterpene tetronic acid) isolated from marine sponges (Sarcotragus sp.), on the modulation of cell cycle and induction of apoptosis in SK-MEL-2 human skin cancer cells (mutant p53). Ircinin-1 treatment on SK-MEL-2 cells resulted in a dose-dependent inhibition of cell growth and induced apoptotic cell death. Flow cytometric analysis revealed that ircinin-1 resulted in G1 arrest in cell cycle progression which was associated with a marked decrease in the protein expression of D-type cyclins and their activating partners Cdk 4 and 6 with concomitant inductions of p21WAF1/CIP1 and p27KIP1. The induction of p21WAF1/CIP1 appears to be transcriptionally upregulated and is p53-independent. In addition, ircinin-1 suppressed the phosphorylation of pRb protein and increased the co-association of pRb or proliferating cell nuclear antigen (PCNA) with p21WAF1/CIP1 in these cells. Ircinin-1 treatment also resulted in induction of apoptosis as determined by morphological changes, DNA fragmentation, alternated ratio of Bax/Bcl-2, cleavages of poly(ADP-ribose) polymerase and PLC-gamma1, and flow cytometric analysis. Ircinin-1 also induced cytochrome c release, cleavage activations of caspase-3 and -9, and upregulation of Fas and Fas-L. Even though the inhibitor of apoptosis protein (IAP) was expressed in ircinin-1-untreated or -treated SK-MEL-2 cells, only the level of cIAP-1, but not XIAP or cIAP-2, was decreased during ircinin-1-induced apoptosis at Western blot and RT-PCR studies. Taken together, these findings suggest that ircinin-1 has strong potential for development as an agent for prevention against skin cancer.

We previously reported that the synthetic chenodeoxycholic acid (CDCA) derivatives showed apoptosis-inducing activity on various cancer cells in vitro. This study was undertaken to explore whether synthetic CDCA derivatives, HS-1199 and HS-1200, had an anticancer effect on malignant glioblastoma cells. We administered them in culture to U-118MG, U-87MG, T98G, and U-373MG cells. The tested glioblastoma cells showed several lines of apoptotic manifestations, such as activation of caspase-3, degradation of DFF, production of poly(ADP-ribose) polymerase cleavage, nuclear condensation, inhibition of proteasome activity, reduction of mitochondrial membrane potential and the release of cytochrome c to cytosol and translocation of AIF to nuclei. Between the two synthetic derivatives, HS-1200 showed a stronger apoptosis-inducing effect than HS-1199. In vivo efficacy of HS-1200 was tested in U87MG cells inoculated into non-obese diabetic and severe combined immunodeficient (NOD/SCID) mice. The HS-1200 treatment significantly inhibited the increase of tumor size in NOD/SCID mice and prolonged the life spans. This study supports the possibility of synthetic CDCA derivatives as a potential chemotherapeutic agent.

Doxorubicin is known to be the most effective single cytotoxic drug against anaplastic thyroid carcinoma (ATC). Although doxorubicin has been shown to cause cell death, at least partly, by inducing apoptosis in ATC cells, the mechanism underlying its pharmacological efficacy has not been fully delineated. We, in this study, revealed that doxorubicin induced apoptosis in ATC cells by altering the acetylation state of histone. Doxorubicin reduced histone deacetylase activity and induced hyperacetylation of histone 3. Noticeably, ladder-like DNA fragments from their genomic DNA on agarose gel were not detected irrespective of several lines of evidence supporting the induction of apoptosis. Pulse field electrophoresis showed disintegration of nuclear DNA into giant fragments of 1-2 Mbp and high molecular-weight fragments of 100-1000 kbp. We next examined whether a histone deacetylase inhibitor trichostatin A (TsA) augmented doxorubicin-induced apoptosis in ATC cells. TSA potentiated doxorubicin-induced stage I apoptosis in ATC cells. Our study sheds light on the development of a new combination therapy strategy for more effective responses for ATC treatment.

Although inhibition of histone deacetylase has been demonstrated to induce apoptosis of various cancer cells, there is no report on its effect on mast cell demise to date. Here we studied whether a histone deacetylase inhibitor Trichostatin A (TSA) produces apoptosis in p815 mastocytoma cells. TSA prominently increased the amount of acetylated histones, H3, H4, H2A and H2B, in p815 mastocytoma cells. TSA reduced the viability of p815 mastocytoma cells, and many apoptotic manifestations such as generation of DNA fragmentation, activation of caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), and increase of DNA hypoploidy proved that the reduction of viability resulted from apoptosis. Whereas TSA treatment increased the expression level of Bad, it decreased the level of Bcl-2, Bcl-xL, and X-linked inhibitor of apoptosis protein. The reduction of mitochondrial membrane potential, the release of cytochrome c and Smac/DIABLO to cytosol, and mitochondrial localization of Bad were also shown. Taken together, TSA induces apoptosis on p815 mastocytoma cells in histone acetylation- and mitochondria-dependent fashion. Our data therefore provide the possibility that TSA could be considered as a novel therapeutic strategy for mastocytoma from its apoptosis-inducing activity.

As previously demonstrated, the synthetic bile acid derivatives mediate anti-proliferative properties in a variety of human cancer cells. In the present study, the effects of the synthetic derivatives of ursodeoxycholic acid (UDCA), HS-1030 and HS-1183, and chenodeoxycholic acid (CDCA), HS-1199 and HS-1200, on the proliferation of HT-29 human colon cancer cells were investigated. Whereas UDCA and CDCA had no effect on the growth of cells in the concentration ranges we have tested, HS-1199 and HS-1200 completely inhibited cell proliferation, while HS-1030 and HS-1183 showed weak inhibitory activities. Simultaneous estimation of cell cycle parameters and apoptosis by flow cytometry showed that the synthetic bile acid derivatives produced the arrest of cell cycle progression at the G1 phase and ensuing increase of sub-G1 fraction, which resulted in the induction of apoptosis. The induction of apoptosis was confirmed by observation of cleavages of poly(ADP-ribose) polymerase and DNA fragmentation. Furthermore, Western blot analysis showed decreased expression levels of cyclin Dl, cyclin E, cyclin A, Cdk2, Cdk4, and Cdk6 proteins. In addition, the synthetic bile acid derivatives markedly induced the level of Cdk inhibitor, p21 WAF1/C1P1 , in a p53-independent manner. Furthermore, the exposure of cells to the synthetic bile acid derivatives resulted in a decrease in the level of pRb and enhanced binding between pRb and E2F-1. Based on these data, these synthetic bile acid derivatives may serve as potential lead compounds in the treatment of colon cancer.

Three cDNA clones for the Machado–Joseph disease gene (MJD1) were isolated, two of which have a new exon sequence and a distinct 3′ terminal nucleotide sequence resulting in a new carboxyl terminal domain in the translated product. The nucleotide sequence of the other one is similar to the previously published one except for five polymorphisms, one of which is a single nucleotide substitution resulting in a change from the stop codon (TAA; allele A) to a tyrosine residue (TAC; allele C). Genetic analysis results suggest that Japanese MJD mutations are associated with allele A.