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This study is to investigate the protective effect of astaxanthin against injured hepatocyte L-02 cells induced by sodium azide (NaN3) and reveal the possible mechanisms. Hepatocyte L-02 cells were exposed to 100 mmol.L-1 NaN3 with various concentrations of astaxanthin pre-incubated, then the cell viability was measured by MTT method; The level of reactive oxygen species (ROS) was determined by DCFH-DA method; The changes of mitochondrial membrane potential (MMP) and apoptosis ratio were detected by JC-1 method and Annexin V-FITC/PI double stain method, respectively. Results showed that after cells were exposed to 100 mmol.L-1 NaN3 for 3 hours, the cell viability significantly decreased; ROS level and the percentage of late phase apoptosis increased obviously; MMP was also declined. When cells were pretreated with astaxanthin, the cell damage and late phase apoptosis ratio reduced and MMP was maintained. However, the level of ROS showed insignificant decrease (P>0.05). The beneficial concentration of astaxanthin in improving cell viability and MMP was not in a dose dependent manner and the most effective of which was 0.10 nmol.L-1 (P<0.01). In order to reveal its possible non-antioxidant mechanism, mitochondrial membrane was imitated and H+ transferring function of astaxanthin was also detected by bilayer lipid membrane (BLM) method. Results showed that 2.0% astaxanthin could transfer H+ efficiently. These suggested the mechanisms of astaxanthin in protection of hepatocyte L-02 cells not via its ROS quenching capability but via its H+ transferring function, which improved the mitochondrial function and had the sequence biology effects.

Novel anti-angiogenesis activity of fascaplysin via VEGF blockage was recently revealed by our previous study in addition to the reported cyclin-dependent kinase 4 (CDK4) selective inhibition. To uncover more details of this pharmacologically prospective property, this study further investigated whether fascaplysin had direct anti-proliferation effects on human umbilical vein endothelial cells (HUVEC), which might be contributing to anti-angiogenesis. The results showed that G1 cell cycle arrest was induced by 2.6 μM fascaplysin in a time-dependent manner, and exhibited more sensitive than hepatocarcinoma cells BeL-7402 and Hela cells. Approximately 56.09 ± 2.63% of the cells were arrested at the G1 phase after 24h, and 64.94 ± 2.07% after 36 h, comparing to the 22.82 ± 1.2% in methanol treated cells. Apoptosis of HUVEC cells was induced by 1.3 μM fascaplysin and indicated by the sub-G1, Hoechst staining, terminal deoxynucleotidyl transferase dUTP-mediated nicked end labeling (TUNEL) assay, and annexin-V and propidium (PI) label. This apoptosis response was further confirmed by the detection of active caspase-3 and by western blotting using antibodies against Bax, Bcl-2, procaspase-8, and Bid, indicating that apoptosis in HUVEC cells may involve a mitochondria pathway, by the demonstration of an increase in the Bax/Bcl-2 ratio. Together, our results suggest that the anti-angiogenesis activity of fascaplysin is through the direct effects of cell cycle arrest and apoptosis on HUVEC., (Copyright © 2009 Elsevier Masson SAS. All rights reserved.)

Previous studies have shown that highly sulfated lambda-carrageenan oligosaccharides (lambda-CO) possess an anti-angiogenetic effect, while high concentrations of lambda-CO present a cytotoxic effect towards human umbilical vein endothelial cells (HUVECs). The aim of this study was to explore the underlying mechanism of lambda-CO on inhibiting cell proliferation. lambda-CO elicited reactive oxygen species (ROS) production with concentrations at 0.8 and 1 mg/ml, and this event was accompanied by the increase of early apoptotic cells, nuclear morphology changes and cell cycle arrest at the S and G2/M phases. However, prevention of oxidative stress by N-acetyl-L-cysteine (NAC) could abolish the effect of lambda-CO on these events. Yet, lambda-CO induced a depolarization of mitochondrial transmembrane potential. At 0.8 mg/ml, lambda-CO induced up-regulation of p53 and Bax, down-regulation of Bcl-2 and activation of caspase-9 and -3. These results suggest that exposure to a high concentration of lambda-CO activates the mitochondrial-mediated apoptotic pathway and cell cycle arrest by generation of ROS.

This study is to investigate the effect of fascaplysin on human cervical cancer cells (HeLa) in order to provide insights into the mechanisms of growth suppression involved in fascaplysin-mediated apoptosis. Cytotoxic activity of fascaplysin on HeLa cells was determined using MTT assay, cell cycle analysis, and apoptosis (Annexin V-FITC and PI double staining) studies. The role of the molecules in cell cycle regulation and apoptosis was analyzed by Western blotting and flow cytometry. Fascaplysin markedly inhibited HeLa cells proliferation in a dose-dependent manner, however, did not provoke G1 phase arrest in HeLa cells with downregulation of CDK4, cyclin D1 and CDK4-specific Ser795 pRb phosphorylation. Furthermore, fascaplysin induced significantly apoptosis evidenced by sub-G1 peak and Annexin V-FITC and PI double staining. The molecular mechanism of fascaplysin-induced apoptosis was characterized with the activation of caspase-3, -8, and -9, truncation of Bid, release of cytochrome c into cytosol, and down-regulation of Bcl-2 level. Fascaplysin exhibits anti-proliferation effect towards human cervical cancer HeLa cells through induction of apoptosis via extrinsic death pathway and mitochondrial pathway, but not arresting cell cycle progression at G1 phase. All together, these data sustain our contention that fascaplysin has anticancer properties and merits further investigation as a potential therapeutic agent.

Antineoplaston A10 (3-phenylacetylamino-2,6-piperidinedion) is a naturally occurring substance and was the first antineoplaston in the human body to be chemically identified. The effect of antineoplaston A10 on human hepatocellular carcinoma cell lines HepG2 and HLE has been examined. Antineoplaston A10 displayed anti-proliferative action inhibiting cell growth in a dose- and time-dependent manner in vitro as measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays. Incubation with antineoplaston A10 for 48 h induced apoptotic events such as a typical apoptotic morphology, formation of a characteristic ladder pattern of DNA migration and accumulation of sub-G1 phase cells. Next, hepatoma xenografts in nude mice were employed to study the antitumor effects of antineoplaston A10 in vivo. Oral administration of antineoplaston A10 delayed the growth of HepG2 and HLE cells in the mice without a reduction in body weight. A higher proportion of apoptotic cells in xenografts was observed by means of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. In addition, the level of expression of apoptotic marker p53 increased while that of anti-apoptotic protein bcl-2 decreased, as evaluated with immunohistochemical staining in the xenografts. These results suggested that antineoplaston A10 may inhibit the growth of human hepatoma cells through the induction of apoptosis.