Induction of apoptosis by Myagropsis myagroides extracts is associated with a caspase dependent pathway and reactive oxygen species generation in human cancer cells

The purpose of this study was to elucidate the mechanisms underlying apoptosis induced by an ethanol extracts from Myagropsis myagroides (ME) in HeLa, U937, and PC-3 cells. ME treatment for 24 h significantly inhibited cell viability in a dose-dependent manner, and induced apoptosis. Moreover, ME treatment triggered the cleavage of caspase-8, ?9, ?3, and poly(ADP-ribose) polymerase (PARP). A general caspase inhibitor (z-VAD-fmk) inhibited ME-induced activation of caspase-3, PARP cleavage, and cell death. ME treatment also triggered the release of cytochrome c from the mitochondria to the cytosol and stimulated the cleavage of Bid, up-regulation of Bax, and down-regulation of Bcl-2. Furthermore, ME treatment caused reactive oxygen species (ROS) generation. An antioxidant N-acetylcysteine (NAC) blocked MEinduced activation of caspase-3, PARP cleavage, and cell death. Overall, these results suggest that ME-induced apoptosis is mediated by a caspase dependent pathway and ROS generation in HeLa, U937, and PC-3 cells.     

Kaempferol induced apoptosis via endoplasmic reticulum stress and mitochondria‐dependent pathway in human osteosarcoma U‐2 OS cells

Kaempferol is a natural flavonoid. Previous studies have reported that kaempferol has anti‐proliferation activities and induces apoptosis in many cancer cell lines. However, there are no reports on human osteosarcoma. In this study, we investigate the anti‐cancer effects and molecular mechanisms of kaempferol in human osteosarcoma cells. Our results demonstrate that kaempferol significantly reduces cell viabilities of U‐2 OS, HOB and 143B cells, especially U‐2 OS cells in a dose‐dependent manner, but exerts low cytotoxicity on human fetal osteoblast progenitor hFOB cells. Comet assay, DAPI staining and DNA gel electrophoresis confirm the effects of DNA damage and apoptosis in U‐2 OS cells. Flow cytometry detects the increase of cytoplasmic Ca²⁺ levels and the decrease of mitochondria membrane potential. Western blotting and fluorogenic enzymatic assay show that kaempferol treatment influences the time‐dependent expression of proteins involved in the endoplasmic reticulum stress pathway and mitochondrial signaling pathway. In addition, pretreating cells with caspase inhibitors, BAPTA or calpeptin before exposure to kaempferol increases cell viabilities. The anti‐cancer effects of kaempferol in vivo are evaluated in BALB/c^nu/nu mice inoculated with U‐2 OS cells, and the results indicate inhibition of tumor growth. In conclusion, kaempferol inhibits human osteosarcoma cells in vivo and in vitro.     

Lunasin induces apoptosis and modifies the expression of genes associated with extracellular matrix and cell adhesion in human metastatic colon cancer cells

Scope: Lunasin is an arginine‐glycine‐aspartic acid (RGD) cancer preventive peptide. The objective was to evaluate the potential of lunasin to induce apoptosis in human colon cancer cells and their oxaliplatin‐resistant (OxR) variants, and its effect on the expression of human extracellular matrix and adhesion genes. Methods and results: Various human colon cancer cell lines which underwent metastasis were evaluated in vitro using cell flow cytometry and fluorescence microscopy. Lunasin cytotoxicity to different colon cancer cells correlated with the expression of α₅b₁ integrin, being most potent to KM12L4 cells (IC₅₀ = 13 μM). Lunasin arrested cell cycle at G2/M phase with concomitant increase in the expression of cyclin‐dependent kinase inhibitors p21 and p27. Lunasin (5–25 μM) activated the apoptotic mitochondrial pathway as evidenced by changes in the expressions of Bcl‐2, Bax, nuclear clusterin, cytochrome c and caspase‐3 in KM12L4 and KM12L4‐OxR. Lunasin increased the activity of initiator caspase‐9 leading to the activation of caspase‐3 and also modified the expression of human extracellular matrix and adhesion genes, downregulating integrin α₅, SELE, MMP10, integrin β₂ and COL6A1 by 5.01‐, 6.53‐, 7.71‐, 8.19‐ and 10.10‐fold, respectively, while upregulating COL12A1 by 11.61‐fold. Conclusion: Lunasin can be used in cases where resistance to chemotherapy developed.     

The flavonoid tangeretin activates the unfolded protein response and synergizes with imatinib in the erythroleukemia cell line K562

We explored the mechanism of cell death of the polymethoxyflavone tangeretin (TAN) in K562 breakpoint cluster region‐abelson murine leukemia (Bcr‐Abl+) cells. Flow cytometric analysis showed that TAN arrested the cells in the G₂/M phase and stimulated an accumulation of the cells in the sub‐G₀ phase. TAN‐induced cell death was evidenced by poly(ADP)‐ribose polymerase cleavage, DNA laddering fragmentation, activation of the caspase cascade and downregulation of the antiapoptotic proteins Mcl‐1 and Bcl‐x_L. Pretreatment with the pancaspase inhibitor Z‐VAD‐FMK_blocked caspase activation and cell cycle arrest but did not inhibit apoptosis which suggest that other cell killing mechanisms like endoplasmic reticulum (ER)‐associated cell death pathways could be involved. We demonstrated that TAN‐induced apoptosis was preceded by a rapid activation of the proapoptotic arm of the unfolded protein response, namely PKR‐like ER kinase. This was accompanied by enhanced levels of glucose‐regulated protein of 78 kDa and of spliced X‐box binding protein 1. Furthermore, TAN sensitized K562 cells to the cell killing effects of imatinib via an apoptotic mechanism. In conclusion, our results suggest that TAN is able to induce apoptosis in Bcr‐Abl+ cells via cell cycle arrest and the induction of the unfolded protein response, and has synergistic cytotoxicity with imatinib.     

Red mold dioscorea‐induced G2/M arrest and apoptosis in human oral cancer cells

BACKGROUND: Monascus‐fermented products are among the most commonly used traditional food supplements. Dioscorea is known to exhibit anticancer properties. In this study the effects of the ethanol extract of red mold dioscorea (RMDE) on cell proliferation, cell cycle and apoptosis in human oral cancer cells were investigated. RESULTS: RMDE exercised growth inhibition on squamous cell carcinoma‐25 (SCC‐25) cells. RMDE‐mediated G2/M phase arrest was associated with the down‐regulation of NF‐κB, resulting in the inhibition of cyclin B1 and CDK1 expression; this may be the mechanism by which RMDE inhibits cancer cells. Furthermore, the proapoptotic activity of RMDE was revealed by the Annexin V‐FITC/PI double‐staining assay. In addition, the proapoptotic effect of RMDE was evident by the inhibition of Bax expression in the mitochondria, resulting in the activation of caspase‐9 and caspase‐3 and subsequent triggering of the mitochondrial apoptotic pathway. RMDE also enhanced caspase‐8 activity, indicating the involvement of the death receptor pathway in RMDE‐mediated SCC‐25 cell apoptosis. CONCLUSION: RMDE treatment inhibited the growth of SCC‐25 cells by arresting cell cycle at the G2/M phase and induced apoptosis in a time‐ and dose‐dependent manner. Therefore RMDE may be a good candidate for development as a dietary supplement against oral cancer. Copyright © 2010 Society of Chemical Industry     

Mechanism of Hericium erinaceus (Yamabushitake) mushroom-induced apoptosis of U937 human monocytic leukemia cells

Phytochemicals in some foods are a potential source of bioactive safe compounds for cancer chemoprevention and suppression of tumor initiation, promotion, and metastasis. In the present study, we evaluated hot water (HWE), microwaved 50% ethanol (MWE), acidic (ACE), and alkaline (AKE) extracts of the fruitbody (sporocarp) of Hericium erinaceus (Yamabushitake, Lion's Mane) mushrooms for their ability to induce apoptosis (programmed cell death) in U937 human monocytic leukemia cells. Cell culture, cell viability, cytotoxicity, flow cytometry, chromosomal DNA integrity, mitochondrial membrane potential, expression of pro- and anti-apoptotic proteins, and activation and inhibition of caspase assays were carried out to help define the mechanism of observed apoptosis. The aqueous and aqueous/ethanolic extracts were active in all assays, whereas the acidic and alkaline extracts with the similar proximate compositions were both inactive. The results of the bioassays with the active extracts are consistent with an apoptosis mechanism governing suppression of the cell proliferation pathway that involves activation of mitochondria-mediated caspase-3 and caspase-9 but not caspase-8. Proximate analysis of the freeze-dried mushroom powder showed that it contains high amounts of proteins, carbohydrates, and minerals. The results indicate that H. erinaceus mushrooms may have therapeutic potential against human leukemia.     

Eupafolin,a flavonoid isolated from Artemisia princeps,induced apoptosis in human cervical adenocarcinoma HeLa cells

Although eupafolin, a flavone found in Artemisia princeps Pampanini, has been shown to inhibit the growth of several human cancer cells, its mode of action is poorly understood. In this study, we investigated the pro‐apoptotic activities of eupafolin in human cervical carcinoma HeLa cells. It was found that eupafolin induced apoptosis in a dose‐dependent manner, as evidenced by DNA fragmentation and the accumulation of positive cells for annexin V. In addition, eupafolin triggered the activations of caspases‐3, ‐6, ‐7, ‐8, and ‐9 and the cleavages of their substrates, such as, poly (ADP‐ribose) polymerase and lamin A/C. Furthermore, treatment with eupafolin resulted in a loss of mitochondrial membrane potential (ΔΨ_m), increased the release of cytochrome c to the cytosol, and altered the expression levels of B‐cell lymphoma 2 (Bcl‐2) family proteins. Interestingly, caspase‐8, an initiator caspase, was activated after the loss of ΔΨ_m and the activations of caspases‐3 and ‐9. Moreover, treatment with z‐DEVD‐fmk (a specific caspase‐3 inhibitor) and the overexpression of Bcl‐2 prevented eupafolin‐stimulated caspase‐8 activation. Altogether, these results suggest that the eupafolin‐induced apoptosis in HeLa cells is mediated by caspase‐dependent pathways, involving caspases‐3, ‐9, and ‐8, which are initiated by the Bcl‐2‐dependent loss of ΔΨ_m.     

Induction of apoptosis in U937 human leukaemia cells by the hexane fraction of an extract of immature Citrus grandis Osbeck fruits

The antiproliferative effect of an immature Citrus grandis Osbeck fruit extract was investigated using U937 human leukaemia cells. Maximum cytotoxicity was observed using the hexane fraction (HF) of the extract. Cell death was dose-dependent (IC₅₀ = ca. 60 μg/ml) and was characterised by chromatin condensation, apoptotic body formation, and DNA fragmentation. The induction of apoptosis was confirmed by caspase-3 activity assays and by immunoblotting using antibodies against Bcl-2, Bax, poly(ADP-ribose) polymerase (PARP), caspase-9, and caspase-3. The molecular mechanism underlying HF-induced apoptosis in U937 cells may involve a mitochondria-mediated signalling pathway, as demonstrated by an increase in the Bax/Bcl-2 expression ratio. Analyses of the HF by gas chromatography (GC) and GC-mass spectrometry (MS) tentatively identified 19 compounds, including γ-sitosterol (17.5%), 7-methoxy-8-(2-oxo-3-methylbutyl) coumarin (6.8%), stigmasterol (3.8%), and campesterol (3.4%). Together, our results provide the first evidence that the HF of an immature C. grandis Osbeck fruit extract induces apoptosis in U937 cells.     

Chrysophanol induces necrosis through the production of ROS and alteration of ATP levels in J5 human liver cancer cells

Anthraquinone compounds have been shown to induce apoptosis in different cancer cell types. Effects of chrysophanol, an anthraquinone compound, on cancer cell death have not been well studied. The goal of this study was to examine if chrysophanol had cytotoxic effects and if such effects involved apoptosis or necrosis in J5 human liver cancer cells. Chrysophanol induced necrosis in J5 cells in a dose‐ and time‐dependent manner. Non‐apoptotic cell death was induced by chrysophanol in J5 cells and was characterized by caspase independence, delayed externalization of phosphatidylserine and plasma membrane disruption. Blockage of apoptotic induction by a general caspase inhibitor (z‐VAD‐fmk) failed to protect cells against chrysophanol‐induced cell death. The levels of reactive oxygen species production and loss of mitochondrial membrane potential (ΔΨ_m) were also determined to assess the effects of chrysophanol. However, reductions in adenosine triphosphate levels and increases in lactate dehydrogenase activity indicated that chrysophanol stimulated necrotic cell death. In summary, human liver cancer cells treated with chrysophanol exhibited a cellular pattern associated with necrosis and not apoptosis.     

Apoptotic Activity of Lactobacillus plantarum DGK‐17‐Fermented Soybean Seed Extract in Human Colon Cancer Cells via ROS–JNK Signaling Pathway

Fermented food has been always possesses upper hand compared to normal food due to its antibacterial, antioxidant, and anticancer properties. Soybeans, which have high nutritional value, are widely consumed in Korea. In this study, soybean seed powder fermented with Lactobacillus plantarum DGK‐17, which was previously isolated from kimchi, showed anticancer potential. Fermented soybean extract (FSE) resulted in morphological changes, reduction of cancer cell colony formation and apoptotic cell death of HCT‐116 colon cancer cells in a dose‐dependent manner, and IC₅₀ value of 111 μg. FSE treatment caused reduction of cell growth in a dose‐dependent manner via release of lactate dehydrogenase. FSE treatment induced HCT‐116 apoptotic cell death as confirmed by the presence of fragmented nuclei, oxidative burst, and reduced mitochondrial membrane potential (ΔΨ_m). Further, FSE treatment sensitized cells to ER stress via IRE1‐α induction. FSE treatment also resulted in JNK activation, subsequently causing activation of Bax and downregulation of BCl2. Weakened mitochondrial membrane potential (ΔΨ_m) also caused release of Cyto C, further activating caspase‐mediated cell death. Therefore, this study reveals the apoptotic role of DGK‐17‐fermented soybean seed extract in human colon cancer HCT‐116 cells.     

The p53-, Bax- and p21-dependent inhibition of colon cancer cell growth by 5-hydroxy polymethoxyflavones

Scope: Previously, we reported that 5‐hydroxy polymethoxyflavones (5OH‐PMFs) isolated from orange, namely 5‐hydroxy‐6,7,8,3′,4′‐pentamethoxyflavone, 5‐hydroxy‐3,6,7,8,3′,4′‐hexamethoxyflavone (5HHMF) and 5‐hydroxy‐6,7,8,4′‐tetramethoxyflavone (5HTMF), potently induced apoptosis and cell‐cycle arrest in multiple human colon cancer cells. Herein, using isogenic variants of HCT116 human colon cancer cells, we investigated the effects of p53, Bax and p21 on the apoptosis and cell‐cycle arrest induced by different 5OH‐PMFs. Methods and results: Annexin V/PI co‐staining assay demonstrated that 5HHMF and 5HTMF significantly induced apoptosis in HCT116 (p53^+/+) cells but not in HCT116 (p53^?/?) cells. Furthermore, 5HHMF and 5HTMF significantly induced apoptosis in HCT116 (Bax^+/?) cells, whereas their pro‐apoptotic effects on HCT116 (Bax^?/?) cells were marginal. All three 5OH‐PMFs increased G0/G1 cell population of HCT116 (p53^+/+) cells, and these effects were abolished in HCT116 (p53^?/?) and HCT116 (p21^?/?) cells. Immunoblotting analysis showed that 5HHMF and 5HTMF increased the levels of cleaved caspase‐3, cleaved PARP in both HCT116 (p53^+/+) and HCT116 (Bax^+/?) cells and these effects were much weaker in HCT116 (p53^?/?) and HCT116 (Bax^?/?) cells. Conclusion: Our results demonstrated that 5OH‐PMFs, especially 5HHMF and 5HTMF, induce apoptosis and cell‐cycle arrest by p53‐, Bax‐ and p21‐dependent mechanism.     

Aqueous and Ethanol Extracts of Daylily Flower (Hemerocallis fulva L.) Protect HUVE Cells Against High Glucose

The content of several phenolic acids and flavonoids in aqueous extract (AE) and ethanol extract (EE) of daylily flower (Hemerocallis fulva L.) was analyzed. The effects of AE or EE at 0.5%, 1%, or 2% in HUVE cells against high glucose‐induced cell death, oxidative, and inflammatory damage were examined. Results showed that seven phenolic acids and seven flavonoids could be detected in AE or EE, in the range of 29 to 205 and 41 to 273 mg/100 g, respectively. Compared with the control groups, high glucose raised the activity of caspase‐3 and caspase‐8; suppressed Bcl‐2 mRNA expression and increased Bax mRNA expression; and induced HUVE cells apoptosis. The pretreatments from AE or EE at 1% or 2% reduced caspase‐3 activity and Bax mRNA expression, and enhanced cell viability. High glucose decreased glutathione content; stimulated the production of reactive oxygen species, interleukin‐6, tumor necrosis factor‐alpha, and prostaglandin E₂; raised the activity of cyclooxygenase‐2 and nuclear factor kappa B p50/65 binding; and reduced the activity of glutathione peroxidase, glutathione reductase, and catalase in HUVE cells. AE pretreatments at 1% and 2% reversed these changes. These novel findings suggested that daylily flower was rich in phytochemicals, and could be viewed as a potent functional food against diabetes.     

Cocoa‐rich diet attenuates beta cell mass loss and function in young Zucker diabetic fatty rats by preventing oxidative stress and beta cell apoptosis

We have recently shown that cocoa flavanols may have anti‐diabetic potential by promoting survival and function of pancreatic beta‐cells in vitro. In this work, we investigated if a cocoa‐rich diet is able to preserve beta‐cell mass and function in an animal model of type 2 diabetes and the mechanisms involved. Our results showed that cocoa feeding during the prediabetic state attenuates hyperglycaemia, reduces insulin resistant, and increases beta cell mass and function in obese Zucker diabetic rats. At the molecular level, cocoa‐rich diet prevented beta‐cell apoptosis by increasing the levels of Bcl‐xL and decreasing Bax levels and caspase‐3 activity. Cocoa diet enhanced the activity of endogenous antioxidant defenses, mainly glutathione peroxidase, preventing thus oxidative injury induced by the pre‐diabetic condition and leading to apoptosis prevention. These findings provide the first in vivo evidence that a cocoa‐rich diet may delay the loss of functional beta‐cell mass and delay the progression of diabetes by preventing oxidative stress and beta‐cell apoptosis.     

Free Radical Scavenging and Leukemia Cell Growth Inhibitory Properties of Onion Powders Treated by Different Heating Processes

ABSTRACT: Methanol extracts of onion powder dried by hot air (60 °C), vacuum (35 °C), and lyophilization (35 °C) were used to study the effects of drying method on the quercetin composition and the subsequent antioxi-dative changes. It was found that hot air-dried onion had higher radical scavenging activities in both DPPH and peroxide radicals than those of the freeze- and vacuum-dried onions. HPLC analyses showed that freeze- and vacuum-dried onions contained more quercetin glycosides, whereas hot air-dried onion dominated in aglycone. A strong cell proliferation inhibition activity in hot air-dried onion was observed for leukemia cell lines CEM and U937, whereas freeze- and vacuum-dried onions gave comparatively moderate inhibition. Low cell proliferation inhibition was obtained in 3 dried onions for leukemia cell lines K562, P3HR-1, and Raji.     

Shallot and licorice constituent isoliquiritigenin arrests cell cycle progression and induces apoptosis through the induction of ATM/p53 and initiation of the mitochondrial system in human cervical carcinoma HeLa cells

This study is the first to investigate the anticancer effect of isoliquiritigenin (ISL) in human cervical carcinoma HeLa cells. The results reveal that ISL inhibits HeLa cells by blocking cell cycle progression in the G2/M phase and inducing apoptosis. Blockade of cell cycle is associated with increased activation of ataxia telangiectasia‐mutated (ATM). Activation of ATM by ISL phosphorylated p53 at Serine15, resulting in increased stability of p53 by decreasing p53 and murine double minute‐2 (MDM2) interaction. In addition, ISL‐mediated G2/M phase arrest was also associated with decreases in the amounts of cyclin B, cyclin A, cdc2, and cdc25C, and increases in the phosphorylation of Chk2, cdc25C, and cdc2. The specific ATM inhibitor caffeine significantly decreased ISL‐mediated G2/M arrest by inhibiting the phosphorylation of p53 (Serine15) and Chk2. ISL induced apoptotic cell death is associated with changes in the expression of Bax and Bak, decreasing levels of Bcl‐2 and Bcl‐X_L, and subsequently triggering mitochondrial apoptotic pathway. In addition, pretreatment of cells with caspase‐9 inhibitor blocked ISL‐induced apoptosis, indicating that caspase‐9 activation is involved in ISL‐mediated HeLa cell apoptosis. These findings suggest that ISL may be a promising chemopreventive agent against human uterine cervical cancer.     

Isoangustone A present in hexane/ethanol extract of Glycyrrhiza uralensis induces apoptosis in DU145 human prostate cancer cells via the activation of DR4 and intrinsic apoptosis pathway

Glycyrrhiza uralensis (licorice) is one of the most frequently prescribed ingredients in Oriental medicine, and licorice extract has been shown to exert anti‐carcinogenic effects. However, its use as a cancer chemopreventive agent is rather limited, due to the fact that its principal component, glycyrrhizin, is known to induce hypertension. This study determined the effects of a hexane/ethanol extract of G. uralensis (HEGU), which contains undetectable amounts of glycyrrhizin, on the apoptosis of androgen‐insensitive DU145 cells. HEGU induced apoptosis and increased the levels of cleaved caspase‐9, caspase‐7, caspase‐3 and poly (ADP‐ribose) polymerase (PARP). HEGU also induced mitochondrial membrane depolarization and cytochrome c release to the cytosol. HEGU increased the levels of Fas, death receptor 4 (DR4), cleaved caspase‐8, Mcl‐1S, and truncated Bid proteins. A caspase‐8 inhibitor suppressed HEGU‐induced apoptosis. An active fraction of HEGU was separated via column chromatography and the structure of the active compound isoangustone A was identified via ¹H‐NMR and ¹³C‐NMR. Isoangustone A increased apoptotic cells, the cleavage of PARP and caspases, and the levels of DR4 and Mcl‐1S. Transfection with DR4 small interfering RNA attenuated HEGU‐ and isoangustone A‐induced apoptosis. These results demonstrate that the activation of DR4 contributes to HEGU‐ and isoangustone A‐induced apoptosis of DU145 cells.     

Antioxidant,immunomodulatory and antiproliferative effects of gelatin hydrolysates from seabass (Lates calcarifer) skins

This study investigated the antioxidant, immunomodulatory and antiproliferative potentials of gelatin hydrolysates from seabass skins in cell model systems. Gelatin hydrolysates were extracted from seabass skins using different processes and enzyme concentrations. The ability of the hydrolysates to protect against H₂O₂‐induced DNA damage was assessed on U937 cells using the Comet assay, and one of the samples showed DNA protective effects. All samples showed immunomodulatory potential by significantly (P < 0.05) reducing interleukin‐6 (IL‐6) and IL‐1β production in lipopolysaccharide (LPS)‐stimulated RAW 264.7 macrophage cells. Antiproliferative activities of seabass skin hydrolysates were measured using human colon cancer (Caco‐2) and liver cancer (HepG2) cell lines as the model cell cultures. The inhibition of cell proliferation of Caco‐2 and HepG2 cancer cells occurred in a dose‐dependent manner at concentrations of 1–25 mg mL^?1. Therefore, seabass skin hydrolysates prepared using an appropriate process could serve as a potential functional food ingredient with various health benefits.     

银杏中银杏酸诱导人白血病细胞U937凋亡的研究

目的:研究银杏酸体外对U937细胞生长的抑制作用,探讨其作用机理。方法:采用MTT法检测银杏酸对U937细胞增殖的影响,激光共聚焦显微镜观察细胞形态的变化,DNA琼脂糖电泳检测其生化特征的改变,流式细胞仪分析细胞凋亡率。结果:U937细胞经银杏酸作用24～48h,细胞的生长明显被抑制;银杏酸作用28h出现了细胞皱缩、核浓缩、体积缩小等明显的凋亡的形态学特征;细胞DNA经琼脂糖电泳可见典型的梯形条带;10.0μg/ml的银杏酸作用40h后,细胞凋亡率为14%。结论:银杏酸对U937细胞具有明显的抗肿瘤活性,其作用机理与诱导细胞凋亡有关。