Effect of Hibiscus anthocyanins-rich extract induces apoptosis of proliferating smooth muscle cell via activation of P38 MAPK and p53 pathway

Hibiscus sabdariffa L. (Malvaceae), an attractive plant believed to be native to Africa, is cultivated in Sudan and in eastern Taiwan. It has been reported to contain a number of protocatechuic acid and anthocyanins. In vitro experimental studies have shown that anthocyanins administration of the extract produces anti-inflammation and chemoprevention effects. In spite of the wide use of Hibiscus sabdariffa L. in folk medicine for treating various diseases, our previous study indicated a potency of Hibiscus sabdariffa extract (HSE) in anti-atherosclerosis. The mechanisms of anthocyanins administration of the extract produce from Hibiscus sabdariffa L. to attenuate atherosclerosis were not clarified. In this study, we found that Hibiscus anthocyanins (HAs) could inhibit the serum-stimulated proliferation of smooth muscle cell (SMC) and result in cell apoptosis. The HAs inducing cell apoptosis was dose dependent. We further used SB203580 (p38 inhibitor) to block cellular apoptosis and evaluate its effect on the HAs-inducing SMC death via some apoptosis criteria including DNA fragmentation and flow cytometry. We suggested that the mechanisms of the inhibitory effect of HAs on atherosclerosis could be via inhibiting the proliferation of SMC. HAs induces apoptosis via (i) activating p38 MAP kinase that subsequently phosphorylates target protein c-Jun and transduces the signal to further activate the apoptotic protein cascades that contain Fas-mediated signaling (Fas/caspase-8 signaling module) and (ii) activating p53 and inducing bax expression. As an outcome of the events, cytochrome c releases from the mitochondria, leading to cell apoptosis. In these experiments, HAs showed strong potential to induce SMC cell apoptosis via p38 and p53 pathway. In consequence, the rate of atherosclerotic formation is slowed down, and the progress is suppressed.     

Bilberry and blueberry anthocyanins act as powerful intracellular antioxidants in mammalian cells

Berry anthocyanins have pronounced health effects, even though they have a low bioavailability. The common mechanism underlying health protection is believed to relate to antioxidant activity. Berry extracts, chemically characterised for their phenolic content, were prepared from bilberries (Vaccinium myrtillus L.) and blueberries (Vaccinium corymbosum L.); the bilberry extract was further purified to obtain the anthocyanin fraction. The antioxidant activity of each extract was examined at the cellular level. For this purpose a specific assay, known as cellular antioxidant activity assay (CAA), was implemented in different cell lines: human colon cancer (Caco-2), human hepatocarcinoma (HepG2), human endothelial (EA.hy926) and rat vascular smooth muscle (A7r5). Here we show for the first time that anthocyanins had intracellular antioxidant activity if applied at very low concentrations (<1 μg/l; nM range), thereby providing a long-sought rationale for their health protecting effects in spite of their unfavorable pharmacokinetic properties.     

Dietary calcium decreases plasma cholesterol by down‐regulation of intestinal Niemann–Pick C1 like 1 and microsomal triacylglycerol transport protein and up‐regulation of CYP7A1 and ABCG 5/8 in hamsters

Scope: It has been shown that calcium supplementation favorably modifies plasma lipoprotein profile in postmenopausal women. The present study investigated the interaction of dietary calcium with genes of transporters, receptors and enzymes involved in cholesterol metabolism. Methods and results: Forty‐eight ovariectomized hamsters were fed one of the four diets containing 0, 2, 6 and 8 g calcium per kg. Plasma total cholesterol (TC), triacylglycerols (TG), and non‐high density lipoprotein cholesterol were dose‐dependently decreased, whereas high‐density lipoprotein cholesterol (HDL‐C) was dose‐dependently increased with the increasing dietary calcium levels. Dietary calcium had no effect on protein mass of hepatic sterol regulatory element binding protein‐2 (SREBP), liver X receptor‐alpha (LXR), 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMGR), LDL receptor (LDLR) and cholesterol‐7α‐hydroxylase (CYP7A1). However, dietary calcium up‐regulated the mRNA levels of hepatic CYP7A1 and intestinal ATP binding cassette transporters (ABCG5/8) whereas it down‐regulated the intestinal Niemann‐Pick C1 like 1 (NPC1L1) and microsomal triacylglycerol transport protein (MTP). In addition, dietary calcium increased the activity of intestinal acyl coenzyme A: cholesterol acyltransferase 2, while it decreased plasma cholesteryl ester transport protein (CETP). Conclusion: Beneficial modification of lipoprotein profile by dietary calcium was mediated by sequestering bile acid absorption and enhancing excretion of fecal cholesterol, via up‐regulation of mRNA CYP7A1 and intestinal ABCG 5/8 with down‐regulation of mRNA NPC1L1 and MTP.     

Naringenin up‐regulates the expression of death receptor 5 and enhances TRAIL‐induced apoptosis in human lung cancer A549 cells

Scope: While TRAIL is relatively non‐toxic to normal cells, it can selectively induce apoptosis in many types of transformed cells. Nevertheless, some non‐small cell lung cancer (NSCLC) cells are particularly resistant to the effects of TRAIL. Here, we report that in combination with naringenin exposure to TRAIL induced apoptosis in TRAIL‐resistant NSCLC A549 cells with no detectable inhibitory effects on cell proliferation of normal lung fibroblast cells. Methods and results: Cytotoxicity was evaluated by MTT assay. Apoptosis was detected using DAPI staining, and flow cytometry. The protein levels were determined by Western blot analysis. Caspase activity was measured using a colorimetric assay. For knockdown of Bid and DR5 expression, Bid and DR5 siRNAs were transfected into cells via lipofection. We could show that following exposure to naringenin, DR5 proteins were up‐regulated and knockdown of DR5 expression by siRNA attenuated naringenin plus TRAIL‐induced apoptosis. Naringenin and TRAIL effectively induced Bid cleavage and siRNA‐mediated silencing of Bid reduced the sensitizing effect of naringenin. Furthermore, co‐treatment with naringenin and TRAIL resulted in reduction of the clonogenic capacity of A549 cells, and surviving clones could be re‐sensitized for repeated TRAIL treatment. Conclusion: Our results indicate that treatment with a combination of TRAIL and naringenin may be a safe strategy for treatment of resistant NSCLC.     

A novel mechanism underlying phytate‐mediated biological action‐phytate hydrolysates induce intracellular calcium signaling by a Gαq protein‐coupled receptor and phospholipase C‐dependent mechanism in colorectal cancer cells

Phytate (inositol hexa‐phosphate, IP6) possesses multiple biological functions including anticancer activity. IP6 is converted to inositol di‐, tri‐, and tetra‐phosphates (IP2, IP3, and IP4) by phytase in large intestinal microbes; however, their contribution to the IP6‐mediated functions has not been investigated. We have developed the preparations of IP2–4 and IP3‐rich phytate hydrolysate (IP3‐RPH) by IP6 digestion using microbial phytase, and examined the induction of intracellular Ca²⁺ signaling in response to the preparations in colorectal cancer cells. IP2–4, but not inositol (IP0) and IP6, induced increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in Caco‐2 cells with the following rank order: IP3>IP2=IP4. Inositol tri‐phosphate (IP3)‐RPH induced increases in [Ca²⁺]_i in both undifferentiated Caco‐2 and HT‐29 cells, but not in differentiated Caco‐2. The IP3‐RPH‐induced [Ca²⁺]_i increase was resistant to extracellular Ca²⁺ depletion, however, it was impaired by inhibitors of phospholipase C, inositol 1, 4, 5 tri‐phosphate receptor, ryanodine receptor, and Gαq protein. These results show that the putative G protein‐coupled receptor on the plasma membrane senses the IP6 hydrolysates and activates phospholipase Cβ, resulting in Ca²⁺ mobilization through Ca²⁺ channels coupled with the inositol 1, 4, 5 tri‐phosphate and ryanodine receptors on the sarco‐endoplasmic reticulum Ca²⁺ store in colorectal cancer cells.