Diallyl Trisulfide Is More Cytotoxic to Prostate Cancer Cells PC-3 than to Noncancerous Epithelial Cell Line PNT1A: A Possible Role of p66Shc signaling Axis

Diallyl trisulfide (DATS) is an organosulfur compound isolated from garlic, and has been shown to have anticancer activity both in vitro and in vivo. The aim of this study was to compare cytotoxic effects of DATS on prostate cancer cells PC-3 and noncancerous human prostate epithelial cells PNT1A. PC-3 prostate cancer and noncancerous human prostate epithelial cells PNT1A were used in the study. We observed that PNT1A cells had higher resistance to DATS-induced cell death than PC-3 cells. Investigating signaling pathways involved in the cell death we observed that p66Shc phosphorylation at serine 36 and extracellular signal-regulated kinase 1/2 activation induced by DATS, were significantly attenuated in PNT1A cells as compared to PC-3 cells. Moreover, DATS-induced Akt inactivation was also significantly reduced in PNT1A cells. In addition to that, DATS-induced reactive oxygen species generation was nearly completely abolished in PNT1A cells. Interestingly, DATS induced only slight decrease in the level of ferritin H, whereas ferritin L was elevated. These data suggest that cytotoxicity of DATS toward PNT1A cells is strongly reduced as opposed to PC-3 cancer cells, which corresponds to the lower activation of prodeath signaling pathway mediated by the adaptor protein p66Shc in the noncancerous PNT1A cells.     

Impact of JNK1, JNK2, and ligase Itch on reactive oxygen species formation and survival of prostate cancer cells treated with diallyl trisulfide

Purpose

In our previous study, we demonstrated that diallyl trisulfide (DATS) induced iron-dependent G2-M arrest of prostate cancer cell cycle. Moreover, ferritin degradation and an increase of labile iron pool has been linked to the activation of the JNK signaling axis. In the present work, we extended this study to determine which of the c-jun kinases is responsible for ferritin degradation and the role of iron in DATS-induced cell death. We hypothesized that JNK1 activates Itch ligase which will lead to ferritin ubiquitination, an increase in iron-dependent ROS formation and cell death.

Methods

PC-3 prostate cancer cells were used in this study. Cell viability, concentration of ROS, labile iron pool, and changes in ferritin and P-Itch and DNA damage were determined.

Results

We observed that DATS induced ferritin degradation through JNK, Itch signaling axis. DATS did not induce neither ROS formation nor increase the LIP in JNK1-DN transfected cells. We also observed that DATS increased JNK-dependent activating phosphorylation of E3ligase Itch. The cells transfected with inactive form of Itch were more resistant against cytotoxicity of DATS and showed lower DATS-induced ferritin degradation. Desferrioxamine a specific iron chelator had no effect neither on cell viability nor DNA damage evaluated by comet assay.

Conclusions

These results suggest that JNK1-dependent increase in LIP is mediated by Itch ubiquitin ligase.     

Functional relevance of d,l-sulforaphane-mediated induction of vimentin and plasminogen activator inhibitor-1 in human prostate cancer cells

Purpose

d,l-Sulforaphane (SFN) is a promising chemopreventive agent with in vivo efficacy against prostate cancer in experimental rodents. This study was undertaken to determine the role of vimentin and plasminogen activator inhibitor-1 (PAI-1) in anticancer effects of SFN.

Methods

Effect of SFN on levels of different proteins was determined by Western blotting or immunofluorescence microscopy. RNA interference of vimentin and PAI-1 was achieved by transient transfection. Apoptosis was quantified by flow cytometry. Transwell chambers were used to determine cell migration.

Results

Exposure of PC-3 and DU145 human prostate cancer cells to SFN resulted in induction of vimentin protein, which was accompanied by down-regulation of E-cadherin protein expression. The SFN-mediated induction of vimentin was also observed in a normal human prostate epithelial cell line. RNA interference of vimentin did not have any appreciable effect on early or late apoptosis resulting from SFN exposure. On the other hand, SFN-mediated inhibition of PC-3 and DU145 cell migration was significantly augmented by knockdown of the vimentin protein. Knockdown of vimentin itself was inhibitory against cell migration. The SFN-treated cells also exhibited induction of PAI-1, which is an endogenous inhibitor of urokinase-type plasminogen activator system. Similar to vimentin, PAI-1 knockdown resulted in a modest augmentation of PC-3 cell migration inhibition by SFN. Tumors from SFN-treated transgenic adenocarcinoma of mouse prostate mice showed a 1.7-fold increase in vimentin protein level compared with control tumors.

Conclusion

The present study indicates that vimentin and PAI-1 inductions confer modest protection against SFN-mediated inhibition of prostate cancer cell migration.     

Sulforaphane inhibits growth of phenotypically different breast cancer cells

Purpose

Cancer development and resistance to chemotherapy correlates with aberrant activity of mitogenic pathways. In breast cancers, pro-survival PI3K-AktmTOR-S6K1 signaling pathway is often hyperactive due to overexpression of genes coding for growth factors or estrogen receptors, constitutive activation of PI3K or Akt and loss of PTEN, a negative regulator of the pathway. Since epidemiologic as well as rodent tumor studies indicate that sulforaphane (SFN), a constituent of many edible cruciferous vegetables, might be a potent inhibitor of mammary carcinogenesis, we analyzed the response of four breast cancer cell lines representing different abnormalities in ErbB2/ER-PI3K-AktmTOR-S6K1 signaling pathway to this compound.

Methods

Four different breast cancer cell lines were used: MDA MB 231, MCF-7, SKBR-3 and MDA MB 468. Cell viability and ultrastructure, protein synthesis, autophagy induction and phosphorylation status of Akt and S6K1 kinases upon SFN treatment were determined.

Results

We observed that all four cell lines are similarly sensitive to SFN. SFN decreased phosphorylation of Akt and S6K1 kinases and at higher concentrations induced autophagy in all studied cell lines. Moreover, global protein synthesis was inhibited by SFN in investigated cell lines in a dose-dependent manner.

Conclusion

These results indicate that SFN is a potent inhibitor of the viability of breast cancer cells representing different activity of the ErbB2/ER-PI3K-AktmTOR-S6K1 pro-survival pathway and suggest that it targets downstream elements of the pathway.     

Diallyl trisulfide inhibits angiogenic features of human umbilical vein endothelial cells by causing Akt inactivation and down-regulation of VEGF and VEGF-R2

We have shown recently that diallyl trisulfide (DATS), a cancer-chemopreventive constituent of garlic, inactivates Akt to trigger mitochondrial translocation of proapoptotic protein BAD in human prostate cancer cells. Because Akt activation is implicated in the promotion of endothelial cell survival and angiogenesis, we hypothesized that DATS may inhibit angiogenesis. In the present study, we tested this hypothesis using human umbilical vein endothelial cells (HUVECs) as a model. Survival of HUVECs was reduced significantly in the presence of DATS in a concentration-dependent manner, with an IC50 of approximately 4 microM. The DATS-mediated suppression of HUVEC survival was associated with apoptosis induction characterized by accumulation of subdiploid cells, cytoplasmic histone-associated DNA fragmentation, and cleavage of caspase-3 and poly-(ADP-ribose)-polymerase. The DATS-induced DNA fragmentation was significantly attenuated in the presence of pan-caspase inhibitor zVAD-fmk and specific inhibitors of caspase-9 (zLEHD-fmk) and caspase-8 (zIETD-fmk). DATS treatment inhibited the formation of capillary-like tube structure and migration by HUVECs in association with suppression of vascular endothelial growth factor (VEGF) secretion and VEGF receptor-2 protein level and inactivation of Akt kinase. DATS treatment also caused activation of extracellular signal-regulated kinase 1/2 (ERK1/2) but not c-Jun NH2-terminal kinase (JNK) or p38 mitogen-activated protein kinase (p38MAPK).DATS-mediatedapoptosis induction and inhibition of HUVEC tube formation was partially but statistically significantly attenuated by pharmacologic inhibition of ERK1/2 but not JNK or p38MAPK. The present study demonstrates, for the first time, that DATS has the ability to inhibit angiogenic features of human endothelial cells.     

Neuroprotective effects of Hu-Yi-Neng,a diet supplement,On SH-SY5Y human neuroblastoma cells

Objectives

Oxidative stress is considered the potential risk to the development of dementia. Some medicines, vitamins, and diet supplements have been suggested to have possible benefits via the antioxidative effects to slow the decline of cognitive function in demented and non-demented individuals. However, few studies were conducted to examine their functions, especially in composite diet supplements. Hu-Yi-Neng is a composite diet supplement, including ginkgo biloba, extract of pine bark, phosphatidyl serine, docosahexaenoic acid, and folic acid, used extensively in Taiwan. Therefore, our aim is to investigate the potential protective effects of Hu-Yi-Neng on human neuron cells.

Materals&Methods

H₂O₂-induced neuronal toxicity was characterized in SH-SY5Y human neuroblastoma cells by the decrease of cell viability using PrestoBlue? assay and by the increase of intracellular reactive oxygen species (ROS) level using DCFH-DA (2′, 7′-dichlorodihydrofluorescin diacetate) assays. HO-1 mRNA expression was detected by real-time PCR. Akt and Erk 1/2 proteins were detected by western blotting.

Results

Pretreatment with Hu-Yi-Neng significantly reversed the decrease in cell viability induced by H₂O₂ in SH-SY5Y cells. Furthermore, Hu-Yi-Neng dose-dependently suppressed the elevation of intracellular reactive oxygen species (ROS) level. Hu-Yi-Neng protected SH-SY5Y cells from oxidative stress may via the increase in mRNA expression of heme oxygenase-1 (HO-1), an antioxidant enzyme. In addition, Hu-Yi-Neng inhibited H₂O₂-induced phosphorylation of Akt kinase but further increased the phosphorylation of Erk 1/2.

Conclusion

Our results suggest that Hu-Yi-Neng has protective effect against oxidative stress-induced neuron cell loss and it could be an ideal composite diet supplement for preventing neurodegenerative diseases.     

Effects of oxysterols on cell viability, inflammatory cytokines, VEGF, and reactive oxygen species production on human retinal cells: cytoprotective effects and prevention of VEGF secretion by resveratrol

Background and aims

Oxysterols are assumed to play important roles in age-related macular degeneration, a major cause of blindness. So we characterized the cytotoxic, oxidative, inflammatory, and angiogenic activities of oxysterols (7β-hydroxycholesterol (7β-OH), 7-ketocholesterol (7KC), 25-hydroxycholesterol (25-OH)) in human retinal ARPE-19 cells, and evaluated the protective effects of resveratrol (Rsv: 1 μM), a polyphenol from red wine.

Methods

ARPE-19 cells were treated with 7β-OH, 7KC, or 25-OH (5–40 μg/mL; 24–48 h) without or with Rsv. Cell viability was determined using trypan blue and the MTT assay. Cell death was characterized by electron microscopy and in situ detection of activated caspases with fluorochrome-labeled inhibitors of caspases. Reactive oxygen species (ROS) production was measured with hydroethidine. ELISA methods and a cytometric bead assay were used to quantify cytokines involved in inflammation (IL-8, IL-1β, IL-6, IL-10, IL-12p70, TNF-α, MCP-1) and VEGF.

Results

7β-OH and 7KC triggered a caspase-independent cell death process associated with the presence of multilamellar cytoplasmic structures evocating phospholipidosis, increased ROS production, and IL-8 secretion. 7β-OH enhanced VEGF secretion. No cytotoxic effects were identified with 25-OH, which highly stimulated ROS production, MCP-1, and VEGF secretion. With oxysterols, no IL-10, TNF-α, and IL-12p70 secretion were detected. 25-OH induced IL-8 secretion through the MEK/ERK½ signaling pathway, and Rsv showed cytoprotective activities and inhibited VEGF secretion.

Conclusion

7β-OH, 7KC, and 25-OH have cytotoxic, oxidative, inflammatory, and/or angiogenic activities on ARPE-19 cells. As Rsv has some protective effects against oxysterol-induced cell death and VEGF secretion it could be valuable in ARMD treatment.     

Aronia melanocarpa fruit extract exhibits anti-inflammatory activity in human aortic endothelial cells

Background

Altered expression of cell adhesion molecules (CAMs) has been implicated in a variety of chronic inflammatory conditions, including atherosclerosis. Regulation of adhesion molecule expression by specific redox-sensitive mechanisms has been reported. Additionally, it has been observed that the extract of Aronia melanocarpa (A. Melanocarpa) fruits, rich in polyphenols, exhibits potent anti-oxidant properties and displays cardioprotective activity.

Methods and results

Human aortic endothelial cells (HAECs) were pretreated with various concentrations (primarily 50?μg/mL) of Aronia Melanocarpa fruit extract prior to treatment with TNFα (10?ng/mL) for various periods of time. The surface protein and mRNA expression of ICAM-1 and VCAM-1 were determined using flow cytometry and real-time RT-PCR, respectively. Adhesion of peripheral blood mononuclear leucocytes (PBMLs) to TNFα-treated HAECs was evaluated by an adhesion assay. Activation of NF-κB was evaluated by measuring NF-κB p65 phosphorylation using flow cytometry. ROS production was determined by reduction in fluorescent 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Tested A. Melanocarpa extract significantly inhibited the expression of ICAM-1 and VCAM-1, attenuated the phosphorylation of NF-κB p65 and decreased intracellular ROS production in TNFα-treated HAECs.

Conclusion

We conclude that A. Melanocarpa fruit extract exhibits anti-inflammatory effects in HAECs by inhibiting the expression of endothelial CAMs, activation of NF-κB and production of ROS.     

Formononetin Induces Apoptosis in PC-3 Prostate Cancer Cells Through Enhancing the Bax/Bcl-2 Ratios and Regulating the p38/Akt Pathway

Formononetin (FN), a bioactive component extracted from the red clover (Trifolium pratense L.), has been long used for treating carcinomas in China. In the present study, we aim to investigate the potential therapeutical effects of FN on cell line of prostatic adenocarcinoma (PC-3) and human prostate epithelial cells (RWPE1). These findings indicated that FN significantly inhibited the cell growth of PC-3 in a dose-dependent manner, but no such effect was observed in RWPE1 cells. The apoptotic counts were effectively increased following the treatments as shown in flow cytometry. The results from Western blotting assay suggested that FN treatment contributed to the reduced Bcl-2 protein level and the elevated Bax expression in PC-3 cells, thereby resulting in the increasing Bax/Bcl-2 ratios. Furthermore, the phosphorylated level of p38 in PC-3 cells was activated through the FN treatment, whereas the endogenous Akt phosphorylation was blocked. Collectively, our findings demonstrate that FN exerts the anticarcinogenic effect on prostate cancer in vitro, in which the underlying mechanisms are associated with enhancing the Bax/Bcl-2 ratios and regulating the p38/Akt pathway, thus triggering apoptosis in tumor cells.     

Diallyl disulfide and diallyl trisulfide suppress oxidized LDL-induced vascular cell adhesion molecule and E-selectin expression through protein kinase A- and B-dependent signaling pathways

Uptake of oxidized LDL (ox-LDL) by vascular endothelial cells is a critical step in the initiation and development of atherosclerosis. Adhesion molecules are upregulated by ox-LDL and numerous inflammatory cytokines and play a pivotal role in atherogenesis. In this study, we examined whether diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), 3 major organosulfur compounds of garlic oil, reduce adhesion molecule expression induced by ox-LDL and, if so, through what mechanism. Human umbilical vein endothelial cells were preincubated with 1 mmol/L DAS, 200 mumol/L DADS, or 100 mumol/L DATS for 16 h and then with 40 mg/L ox-LDL for an additional 24 h. ox-LDL induction of cellular and cell surface expression of E-selectin and vascular cell adhesion molecule (VCAM)-1 was suppressed by garlic allyl sulfides in the order DATS > DADS > DAS. The adhesion of HL-60 cells to endothelial cells was inhibited 27 and 33% and the production of cellular peroxides was inhibited 43 and 50% by DADS and DATS, respectively (P < 0.05). ox-LDL alone dephosphorylated protein kinase B (PKB) and cAMP responsive element binding protein (CREB); such deactivation was reversed by DADS and DATS. Electrophoretic mobility shift assay showed that the activation of CREB binding to DNA was consistent with changes in CREB phosphorylation. The protein kinase A (PKA) inhibitor H89 reversed the suppression of VCAM-1 by DADS and DATS, but the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin had no effect. In contrast, wortmannin abolished DADS- and DATS-induced suppression of ox-LDL-induced E-selectin expression. These results suggest that the suppression of ox-LDL-induced E-selectin and VCAM-1 expression by DADS and DATS and, thus, monocyte adhesion to endothelial cells is likely dependent on the PI3K/PKB or PKA/CREB signaling pathway in an adhesion molecule-specific manner. To our knowledge, this is the first report that garlic modulates ox-LDL-mediated leukocyte adhesion to human endothelial cells through the PKB and PKA pathways.     

Lactobacillus GG-fermented milk prevents DSS-induced colitis and regulates intestinal epithelial homeostasis through activation of epidermal growth factor receptor

Background

Fermented milk is considered one of the best sources for efficient consumption of probiotic strains by hosts to promote good health. The purpose of this study was to investigate the effects of orally administering LGG-fermented milk (LGG milk) on intestinal inflammation and injury and to study the mechanisms of LGG milk’s action.

Methods

LGG milk and non-LGG-fermented milk (non-LGG milk) were administered through gavage to mice before and during dextran sodium sulfate (DSS)-induced intestinal injury and colitis. Inflammatory/injury score and colon length were assessed. Intestinal epithelial cells were treated with the soluble fraction of LGG milk to detect its effects on the epidermal growth factor receptor (EGFR) and its downstream target, Akt activation, cytokine-induced apoptosis, and hydrogen peroxide (H₂O₂)-induced disruption of tight junctions.

Results

LGG milk treatment significantly reduced DSS-induced colonic inflammation and injury, and colon shortening in mice, compared to that in non-LGG milk-treated and -untreated mice. The soluble fraction of LGG milk, but not non-LGG milk, stimulated the activation of EGFR and Akt in a concentration-dependent manner, suppressed cytokine-induced apoptosis, and attenuated H₂O₂-induced disruption of tight junction complex in the intestinal epithelial cells. These effects of LGG milk were blocked by the EGFR kinase inhibitor. LGG milk, but not non-LGG milk, contained two soluble proteins, p40 and p75, that have been reported to promote survival and growth of intestinal epithelial cells through the activation of EGFR. Depletion of p40 and p75 from LGG milk abolished the effects of LGG milk on prevention of cytokine-induced apoptosis and H₂O₂-induced disruption of tight junctions.

Conclusions

These results suggest that LGG milk may regulate intestinal epithelial homeostasis and potentially prevent intestinal inflammatory diseases through activation of EGFR by LGG-derived proteins.     

Antidiabetic activity of isoquercetin in diabetic KK -Ay mice

Background

Protective effects of omega-3 fatty acids against cellular damages of high glucose were studied on retinal pigmented epithelial (RPE) cells.

Methods

Retinal epithelial cells were incubated with omega-3 marine oils rich in EPA and DHA and then with high glucose (25 mM) for 48 hours. Cellular responses were compared to normal glucose (5 mM): intracellular redox status, reactive oxygen species (ROS), mitochondrial succinate deshydrogenase activity, inflammatory cytokines release and caveolin-1 expression were evaluated using microplate cytometry, ELISA and flow cytometry techniques. Fatty acids incorporation in retinal cell membranes was analysed using chromatography.

Results

Preincubation of the cells with fish oil decreased ROS overproduction, mitochondrial alterations and TNFα release. These protective effects could be attributed to an increase in caveolin-1 expression induced by marine oil.

Conclusion

Marine formulations rich in omega-3 fatty acids represent a promising therapeutic approach for diabetic retinopathy.     

Drug/diet synergy for managing malignant astrocytoma in mice: 2-deoxy-D-glucose and the restricted ketogenic diet

Background

Zinc has a wide spectrum of biological activities and its deficiency is related to various abnormalities of cell metabolism.

Methods

Wistar male rats, at age of 4 weeks, were fed a low-zinc diet for six weeks. The levels of bromodeoxyuridine incorporated into the prostatic DNA and the mRNA expression levels of prostate thymidylate synthase and thymidine kinase were examined.

Result

The low-zinc diet caused a marked reduction in the body growth and organ weights, resulted in a low hematopoiesis, hypo-albuminemia and hypocholesterolemia. Although there were few differences in plasma biochemical markers, plasma levels of luteinizing hormone and testosterone were reduced by the low-zinc diet. Bromodeoxyuridine-immunoreactive (S-phase) cells and mRNA expression levels of thymidylate synthase and thymidine kinase in the prostate cells were markedly affected by the low-zinc diet.

Conclusion

A low-zinc diet appears to reduce the body growth and organ weights including prostate, causing low plasma levels of luteinizing hormone and testosterone and reduction in prostate DNA replication in growing-rats.     

Fucoxanthin in association with Vitamin c acts as modulators of human neutrophil function

Introduction

Neutrophils provide the first line of defense of the innate immune system by phagocytosing, killing and digesting bacteria and fungi. During this process, neutrophils produce reactive oxygen species (ROS), which in excess, can damage the cells themselves and surrounding tissues. The carotenoid fucoxanthin (Fc) has been studied concerning its antioxidant and anti-inflammatory actions. Vitamin c (Vc) also demonstrates potent antioxidant action. This study aimed to evaluate the effect of Fc (2 μM) in association with Vc (100 μM) on functional parameters of human neutrophils in vitro.

Materials and methods

We evaluated the migration and phagocytic capacity, intracellular calcium mobilization, ROS production (O ₂ ^·? , H₂O₂, HOCl), myeloperoxidase activity, profile of antioxidant enzymes, phosphorylation of p38 MAPK and p65 NFκB subunit, GSH/GSSG ratio and release of pro-inflammatory cytokines (TNF-α and IL-6) in neutrophils under different stimuli.

Results

We verified an increase in phagocytic capacity for all treatments, together with an increase in intracellular calcium only in cells treated with Fc and Fc + Vc. ROS production was reduced by all treatments, although Vc was a better antioxidant than Fc. Phosphorylation of the p-65 subunit of NFκB was reduced in cells treated with Fc + Vc and release of TNF-α and IL-6 was reduced by all treatments. These findings indicate that the regulation of inflammatory cytokines by neutrophils is not exclusively under the control of the NFκB pathway. Fc reduced the activity of some antioxidant enzymes, whereas Vc increased GR activity and the GSH/GSSG ratio.

Conclusion

In conclusion, the results presented in this study clearly show an immunomodulatory effect of the carotenoid fc alone or in combination with Vc on the function of human neutrophils.     

Protective effects of olive oil phenolics and gallic acid on hydrogen peroxide-induced apoptosis

Purpose

Olive oil contains several phenolic compounds possessing antioxidant activity. The aim of this study was to investigate the protective effects of olive oil phenolic extract (OOPE) and one of its constituents, gallic acid (GA) against H₂O₂-induced oxidative stress and apoptotic cell death in HeLa cells, a model for human epithelial cells.

Methods

The cells were pretreated with nontoxic doses of OOPE or GA for 4, 24 and 48?h, and the intracellular reactive oxygen species (ROS) level was determined, before and after oxidative stress induction with H₂O₂. As an indicator of apoptosis, caspase 9 activity was measured.

Results

All pretreatments reduced ROS generation. Four hour incubation with OOPE or GA completely inhibited ROS generation. Increases in caspase 9 activity by OOPE and GA pretreatment under harsh stress conditions were inhibited 92 and 67.8%, respectively.

Conclusions

These results suggest that OOPE and GA act as powerful antioxidants against oxidative stress and exert anti-apoptotic effects.     

Diallyl Trisulfide Induces Apoptosis in Breast Ductal Carcinoma In Situ Derived and Minimally Invasive Breast Cancer Cells

Breast ductal carcinoma in situ (DCIS) is a localized form of breast cancer that can progress to invasive breast cancer. Diallyl trisulfide (DATS) is a bioactive compound from Allium vegetables reported to induce anticancer effects in several cancer models. The objective of this study was to characterize DATS-induced apoptosis in breast DCIS and minimally invasive breast cancer cells. Breast DCIS cells SUM 102PT (ductal carcinoma in situ with areas of micro-invasion) and SUM 225CWN (chest wall recurrence of ductal carcinoma in situ) were used in this study. DATS induced a dose-dependent reduction in the colony formation ability of breast DCIS cells. DATS inhibited DCIS cell growth by inducing apoptosis as shown by a dose-dependent increase in cytoplasmic histone-associated DNA fragmentation. Induction of apoptosis was more pronounced in SUM 102PT cells than in SUM 225CWN cells at similar concentrations of DATS. DATS-induced apoptosis was characterized by a dose-dependent increase in cleaved poly-ADP ribose polymerase (PARP). DATS treatment resulted in an increase in the cytochrome c levels and cleavage of caspases 3, 7, and 9. This study shows that DATS inhibits cell proliferation and induces apoptosis in breast DCIS derived and minimally invasive breast cancer cells, and supports further investigation of DATS as a potential chemopreventive agent for DCIS.     

Effect of docosahexaenoic acid on hypoxia/reoxygenation injury in human coronary arterial smooth muscle cells

Purpose

Hypoxia and reoxygenation (H/R) occur in a wide variety of important clinical conditions such as myocardial infarction. H/R injury is a complex phenomenon involving not only intracellular damage processes but also an injurious inflammatory response. Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, has long been proved to be protective against several types of cardiovascular disease. However, its beneficial effect during H/R is inconclusive. In this study, we employed an in vitro model to examine whether DHA is protective against H/R-induced cell damage in human coronary artery smooth muscle cells (HCASMCs).

Methods

HCASMCs in the absence or presence of DHA (1, 3, 10, and 30?μM) were subjected to control or H/R treatment using a modular incubator chamber to create hypoxic condition. Cell viability was evaluated by MTT assay. Spectrophotometric and spectrofluorometric assays were used to measure the generation of nitric oxide (NO) and reactive oxygen species (ROS), respectively. Inflammatory cytokines were determined by enzyme-linked immunosorbent assay. Intracellular calcium mobilization was estimated microfluorimetrically using calcium indicator dye, fura 2-acetomethyl ester.

Results

Hypoxia/reoxygenation caused significant injury in cultured HCASMCs. DHA at low concentrations (1, 3, and 10?μM) did not afford protection, whereas at 30?μM, it caused deleterious effects, presumably by enhancing the production of NO, ROS, IL-1β, and IL-6 and altering the intracellular calcium dynamics.

Conclusions

Our results do not support the protective function of DHA in H/R-injured coronary arterial smooth muscle cells.     

Aspalathin improves hyperglycemia and glucose intolerance in obese diabetic ob/ob mice

Purpose

Although several researches have demonstrated that rooibos extract has hypoglycemic effect, the role of aspalathin, a main polyphenol in the extract, remains unclear. Our aims were to find specific mechanisms for anti-diabetic action of aspalathin employing a rat skeletal muscle-derived cell line (L6 myocytes) and a rat-derived pancreatic β-cell line (RIN-5F cells) and to investigate its effect in type 2 diabetic model ob/ob mice.

Methods

We investigated in vitro the effect of aspalathin on the glucose metabolism through the studies on molecular mechanisms of glucose uptake using cultured L6 myotubes. We also measured the antioxidative ability of aspalathin against reactive oxygen species (ROS) generated by artificial advanced glycation end product (AGE) in RIN-5F cells. In vivo, ob/ob mice were fed 0.1 % aspalathin-containing diet for 5 weeks, and the effect of aspalathin on fasting blood glucose level, glucose intolerance, and hepatic gene expression was studied.

Results

Aspalathin dose dependently increased glucose uptake by L6 myotubes and promoted AMP-activated protein kinase (AMPK) phosphorylation. Aspalathin enhanced GLUT4 translocation to plasma membrane in L6 myoblasts and myotubes. In RIN-5F cells, aspalathin suppressed AGE-induced rises in ROS. In vivo, aspalathin significantly suppressed the increase in fasting blood glucose levels and improved glucose intolerance. Furthermore, aspalathin decreased expression of hepatic genes related to gluconeogenesis and lipogenesis.

Conclusions

Hypoglycemic effect of aspalathin is related to increased GLUT4 translocation to plasma membrane via AMPK activation. In addition, aspalathin reduces the gene expression of hepatic enzymes related to glucose production and lipogenesis. These results strongly suggest that aspalathin has anti-diabetic potential.