[New compound NL-608 (a nutlin analog) induces apoptosis in human breast cancer MCF-7 cells].

OBJECTIVE: To observe the effect of NL-608 (a nutlin analog) on apoptosis induction in human breast cancer MCF-7 cells in vitro, and investigate the relevant molecular mechanism. METHODS: The effect of NL-608 on proliferation of MCF-7 cells was determined by MTT assay. The apoptosis in MCF-7 cells was determined by flow cytometry with annexin V-FITC and PI. The activity of caspase 3, caspase 8 and caspase 9 was determined with caspase activity assay kit and Western blot, and the proteins of Fas and FasL were determined by Western blot. RESULTS: NL-608 showed a dose-dependent inhibitory effect on the proliferation of MCF-7 cells. It induced apoptosis in MCF-7 cells in a dose-dependent manner. The activity of caspase 3 and caspase 8 in MCF-7 cells was increased with the increasing concentration of NL-608, but caspase 9 had no changes. The proteins of Fas and FasL were increased in a dose-dependent manner. CONCLUSION: NL-608 induces apoptosis in MCF-7 cells in vitro through inducing caspase 3 activity and death receptor-mediated signal pathway.

Changes of constituents and activity to apoptosis and cell cycle during fermentation of tea.

Tea is believed to be beneficial for health, and the effects of the fermentation process on its contributions to apoptosis and cell cycle arrest of gastric cancer cells have not been completely investigated. In this study, the chemical components in green tea, black tea and pu-erh tea aqueous extracts were analyzed and compared. The polysaccharide and caffeine levels were substantially higher in the fermented black tea and pu-erh tea, while the polyphenol level was higher in the unfermented green tea. Hence, a treatment of tea aqueous extract and the components, which are emerging as promising anticancer agents, were pursued to determine whether this treatment could lead to enhance apoptosis and cell cycle arrest. In the human gastric cancer cell line SGC-7901, the cell viability and flow cytometry analysis for apoptotic cells indicated effects in a dose-dependent inhibition manner for the three tea treatment groups. The apoptosis rates were found to be elevated after 48 h of treatment with 31.2, 125, and 500 µg/mL of green tea extract, the higher catechins content may be involved in the mechanism. Cell cycle was arrested in S phase in the fermented black tea and pu-erh tea, and the populations were significantly decreased in G2/M phases, possibly due to the oxidation of tea polyphenols, which causes an increase of theabrownins. CCC-HEL-1 normal cells were not sensitive to tea extract. These findings suggest that the fermentation process causes changes of the compounds which might be involved in the changes of cell proliferation inhibition, apoptosis induction and cell cycle arrest.

Salidroside stimulated glucose uptake in skeletal muscle cells by activating AMP-activated protein kinase.

In the present study, we reported the metabolic effects of salidroside, one of the active components of Rhodiola Rosea, on skeletal muscle cells. Salidroside dose-dependently stimulated glucose uptake in differentiated L6 rat myoblast cells. Inhibitor of AMP-activated protein kinase (AMPK) by pretreating the cells with compound C potently reduced salidroside-stimulated glucose uptake, while inhibition of phosphatidylinositol 3-kinase (PI3K) by wortmannin exhibited no significant inhibitory effect on salidroside-mediated glucose transport activation. Western blotting analyses revealed that salidroside increased the phosphorylation level of AMPK and acetyl-CoA carboxylase (ACC). In addition, salidroside enhanced insulin-mediated Akt activation and glucose uptake, and such enhancement can be specifically inhibited by compound C. In summary, AMPK activation was involved in the effects of salidroside on glucose transport activation and insulin sensitivity. Salidroside can be further developed as potential compound for the anti-diabetic therapy.

B-type natriuretic peptide enhances mild hypoxia-induced apoptotic cell death in cardiomyocytes.

In the case of left ventricle remodeling after myocardial infarction, cardiomyocyte apoptosis is attributed to increased cardiac workload by the stimulus such as chronic hypoxia. B-Type natriuretic peptide, being known as a reliable prognostic of cardiovascular pathology, plays an important role in the myocardial infarction. However, the action of B-type natriuretic peptide on cardiomyocytes undergoing apoptosis is unclear. In the present study, B-type natriuretic peptide have exhibited the enhancive effects on the mild hypoxia-induced cardiomyocyte apoptosis with the manifestation of facilitating phosphatidylserine evagination and increasing typical fragmented nuclei. In addition, B-type natriuretic peptide aggravated the dissipation of delta psi(m), the depletion of intracellular ATP and the increase of caspase-3 activity. 8-Bromo-cGMP, which increased cGMP independent of B-type natriuretic peptide, could mimic B-type natriuretic peptide's effects; whereas cGMP-dependent protein kinase inhibitor, Rp-8-br-cGMP inhibited that. Further study revealed the enhancive effect of BNP on down-regulation of Bcl-2 mRNA expression in the presence of mild hypoxia. In conclusion, the present study demonstrated that B-type natriuretic peptide aggravated the cardiomyocyte apoptosis by influencing hypoxia-induced mitochondrial death pathway, which is true at least in this oxygen deprivation model; and this effect was partially realized through intracellular cGMP.

Inhibitory effects of saponins from Anemarrhena asphodeloides Bunge on the growth of vascular smooth muscle cells.

OBJECTIVE: To investigate the effects of saponins from Anemarrhena asphodeloides Bunge (SAaB) (Botanical Name: Anemarrhena Asphodeloidis Rhizoma) on the growth of vascular smooth muscle cells (VSMCs). METHODS: Cell proliferation was measured by a newly developed cell proliferation reagent, WST-1. Cell apoptosis was assayed by flow cytometry through detecting annexin V. Nitric oxide production was evaluated using confocal laser scanning microscopy with diaminofluorescein diacetate (DAF-2, DA). Cell aldose reductase (AR) activity, as well as the effect of Epalrestat and interleukin-1beta were also explored. RESULTS: WST assay showed that cell proliferation induced by serum was significantly inhibited by SAaB (P<0.01). Flow cytometry analysis revealed that SAaB could enhance apoptotic rate of VSMCs (P<0.01). Nitric oxide production was significantly enhanced after administration of SAaB and interleukin-1beta. Moreover, AR activity of VSMCs was also remarkably inhibited by both SAaB and Epalrestat (P<0.01). CONCLUSION: SAaB can inhibit proliferation and enhance apoptosis of VSMCs. It may protect vascular cells by inhibiting VSMC proliferation and augmenting apoptotic rate of VSMCs via NO-dependent pathway.

Astragaloside IV improved barrier dysfunction induced by acute high glucose in human umbilical vein endothelial cells.

The purpose of the present study was to examine the effects of astragaloside IV, a saponin isolated from Astragalus membranaceus (Fisch) Bge, on the impairment of barrier function induced by acute high glucose in cultured human vein endothelial cells. High glucose (27.8 mM) induced a decrease in transendothelial electrical impedance and an increase in cell monolayer permeability in human umbilical vein endothelial cells. Endothelial barrier dysfunction stimulated by high glucose was accompanied by translocation and activation of protein kinase C (PKC), the redistribution of F-actin and formation of intercellular gaps, suggesting that increases in PKC activity and rearrangement of F-actin could be associated with endothelial barrier dysfunction induced by acute high glucose. Application of astragaloside IV inhibited high glucose-induced endothelial barrier dysfunction in a dose-dependent manner, which is compatible with inhibition of PKC translocation and improvement of F-actin rearrangements. Western blot analysis revealed that high glucose-induced PKC alpha and beta2 overexpression in the membrane fraction were significantly reduced by astragaloside IV. These findings indicate that astragaloside IV protected endothelial cells from high glucose-induced barrier impairment by inhibiting PKC activation, as well as improving cytoskeleton remodeling.

Intracellular-free calcium dynamics and F-actin alteration in the formation of macrophage foam cells.

The formation of macrophage foam cells, which is the key event in atherosclerosis, occurs by the uptake of oxidized low-density lipoprotein (Ox-LDL) via the scavenger receptor (CD36) pathway. Ca(2+) plays an important role in atherosclerosis. However, in the spatiotemporal view, the correlation between kinetic changes of intracellular-free calcium ([Ca(2+)](i)) and the cellular dysfunctions in the formation of macrophage foam cells has not yet been studied in detail. By the use of confocal laser scanning microscope and flow cytometer, we have detected Ca(2+) dynamics, the assembly of F-actin, and the expression of CD36 under the exposure of U937-derived macrophages to Ox-LDL. The uptake of Ox-LDL significantly increased [Ca(2+)](i) in U937-derived macrophages in both acute and chronic treatments (P<0.01). In particular, the increases of the induced [Ca(2+)](i) were different in the presence or absence of extracellular Ca(2+) under acute exposure. A time-dependent rise in F-actin assembly and CD36 expression at 12 and 24h was induced, respectively, by Ox-LDL. The spatiotemporal increases of [Ca(2+)](i) induced by Ox-LDL probably have the key effect on the early phrase in the formation of macrophage foam cells.

Regulative effects of hawthorn leave flavonoids on cytotoxicity, NO and Ca2+ in hypoxia-treated human umbilical vein endothelial cells.

OBJECTIVE: To evaluate the potential effect of HLF (Hawthorn leave flavonoids, w/w, 80% flavonoids) against thrombus formation, effect of HLF on hypoxia-treated human umbilical vein endothelial cell (HUVECs) was studied. METHOD: The levels of cytotoxicity and NO upon HUVECs were studied by flow cytometry. Moreover, the level of calcium ion in HUVECs was examined through laser scanning confocal microscopy. RESULT: Data from this study showed that HLF at concentrations of 5 micrograms/ml and 10 micrograms/ml decreased the cytotoxicity of hypoxia to HUVECs (P<0.05, P<0.01). The intracellular levels of NO and calcium ion were downregulated by HLF at concentrations of 5 micrograms/ml (P<0.01; P<0.01) and 10 micrograms/ml (vs control, P<0.01; P<0.01) too. CONCLUSION: Results observed suggest that HLF protect HUVECs from hypoxia partly through its regulative effect on NO and calcium ion levels.