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.     

6‐Dehydrogingerdione,an active constituent of dietary ginger,induces cell cycle arrest and apoptosis through reactive oxygen species/c‐Jun N‐terminal kinase pathways in human breast cancer cells

This study is the first to investigate the anticancer effect of 6‐dehydrogingerdione (DGE), an active constituent of dietary ginger, in human breast cancer MDA‐MB‐231 and MCF‐7 cells. DGE exhibited effective cell growth inhibition by inducing cancer cells to undergo G2/M phase arrest and apoptosis. Blockade of cell cycle was associated with increased levels of p21, and reduced amounts of cyclin B1, cyclin A, Cdc2 and Cdc25C. DGE also enhanced the levels of inactivated phosphorylated Cdc2 and Cdc25C. DGE triggered the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl‐2 ratios, resulting in caspase‐9 activation. We also found the generation of reactive oxygen species is a critical mediator in DGE‐induced cell growth inhibition. DGE clearly increased the activation of apoptosis signal‐regulating kinase 1 and c‐Jun N‐terminal kinase (JNK), but not extracellular signal‐regulated kinase 1/2 (ERK1/2) and p38. In addition, antioxidants vitamin C and catalase significantly decreased DGE‐mediated JNK activation and apoptosis. Moreover, blocking JNK by specific inhibitors suppressed DGE‐triggered mitochondrial apoptotic pathway. Taken together, these findings suggest that a critical role for reactive oxygen species and JNK in DGE‐mediated apoptosis of human breast cancer.     

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     

Induction of apoptosis by tomato using space mutation breeding in human colon cancer SW480 and HT‐29 cells

BACKGROUND: As far as we know, there have been no reports concerning the functional characteristics of tomatoes using space mutation breeding. The aim of this study was to evaluate the anti‐colon cancer effect of tomatoes M1 and M2 using space mutation breeding. RESULTS: In the present study, obvious anti‐cancer activity was shown with tomato juice of M1 and M2 and their parent CK treatment in colon cancer cell lines SW480 and HT‐29 in cell growth inhibition. In addition, SW480 cells were more sensitive to M1 and M2 than HT‐29 cells in cell apoptosis. Furthermore, M1 and M2 induced cell cycle arrest both in G0–G1 and G2/M phases. CONCLUSION: These data suggest that consumption of tomato using space mutation breeding may provide benefits to inhibit growth of colon cancer cells. Therefore, tomato production using space mutation breeding may be a good candidate for development as a dietary supplement in drug therapy for colon cancer. Copyright © 2010 Society of Chemical Industry     

Asp238→Asn Creates a Novel Consensus N‐Glycosylation Site in Aspergillus awamori Glucoamylase

A single mutation, Asp238→Asn (D238N), of Aspergillus awamori glucoamylase (GA) was identified that increases extracellular production of the enzyme in Saccharomyces cerevisiae at 37 °C. The mutant was isolated as a suppressor of Gly396→Ser (G396S), a previously isolated temperature‐sensitive mutation that decreases the thermostability and extracellular production of GA expressed in S. cerevisiae. Culture supernatants of the double mutant G396S/D238N contained much more GA than supernatants of G396S at 33.5 and 37 °C but not at 30 °C. Additionally, culture supernatants of the D238N contained 1.5 to 2‐fold more GA than supernatants of wild‐type when grown at 37 °C but not at 30 or 33.5 °C. The D238N mutation creates a consensus N‐glycosylation site in GA. Mass spectrometry showed that the molecular weight of D238N was 2319 Da greater than that of the wild‐type GA and that of D238N/G396S was 3094 Da greater than that of G396S, suggesting the presence of an additional N‐linked glycan at residue 238. No difference in thermostability or activity was observed between the G396S and G396S/D238N mutants or between wild‐type and D238N GAs, and D238N did not affect intracellular GA levels at 30 or 37 °C.     

Phenethyl isothiocyanate inhibits STAT3 activation in prostate cancer cells

This study was undertaken to investigate the mechanism by which phenethyl isothiocyanate (PEITC), a natural compound from cruciferous vegetables, exhibits antitumor effect on prostate cancer cells. Cell proliferation, cell cycle, Western blot, gene transfer, and reporter assays were used to test the effects of PEITC on the growth and IL6/JAK/STAT3 pathway in prostate cancer. The result showed that PEITC significantly inhibited DU145 cell proliferation in a dose‐dependent manner and induced the cell arrest at G2‐M phase. PEITC inhibited both constitutive and IL‐6‐induced STAT3 activity in DU145 cells. IL‐6‐stimulated phosphorylation of JAK2, an STAT3 upstream kinase, was also attenuated by PEITC. Moreover, an antioxidant reagent, N‐acetyl‐L ‐cysteine (NAC) which suppresses reactive oxygen species (ROS) generation, reversed the early inhibitory effects of PEITC on cell proliferation, constitutive or IL‐6‐mediated JAK‐STAT3 phosphorylation in PCa cells. Taken together, our data demonstrated that PEITC can inhibit the activation of the JAK‐STAT3 signal‐cascade in prostate cancer cells and the underlying mechanism may be partially involved with blocking cellular ROS production during the early stage of the signaling activation by IL‐6.     

Identification of a structure specific Bcl-2 phosphorylating homoisoflavone molecule from Vietnamese coriander (Polygonatum odoratum) that induces apoptosis and G2/M cell cycle arrest in breast cancer cell lines

Bcl-2 is an anti-apoptotic protein which is over-expressed in many cancers. Modulating Bcl-2 expression is one of the most important strategies in the combat of cancer. We have isolated and identified a structure specific homoisoflavone from Vietnamese coriander (Polygonatum odoratum or Solomon seal) root which induces Bcl-2 phosphorylation, thereby causing mitotic arrest in breast cancer cells. Bioassay-directed fractionations resulted in a biologically active fraction for Bcl-2 phosphorylation. HPLC separation followed by mass spectrometry and NMR studies identified two compounds. We currently combine the chemistry and biological activity of Vietnamese coriander extracts by using bioassay directed fractionation to identify the structure specific Bcl-2 phosphorylating molecules, as well as their molecular mechanism of action. Only one molecule was responsible for Bcl-2 phosphorylation; it was identified as 2,3-dihydro3-[(15-hydroxyphenyl) methyl]-5,7-dihydroxy-6(8-dimethyl-4H-1-benzopyran-4-one) or 8-methyl-dihydrobenzopyranone (8-methyl-DBP, MW 314). The effect on Bcl-2 was structure specific, because 2,3 dihydro3-[(15-hydroxyphenyl) methyl]-5,7-dihydroxy-6-methyl-8-methoxy-4H-1-benzopyran-4-one or 8-methoxy-dihydrobenzopyranone (8-methoxy-DBP, MW 330), in contrast to 8-methyl-DBP, was not capable of Bcl-2 phosphorylation. Pure 8-methyl-DBP induced Bcl-2 phosphorylation in breast tumor cells, causes G2/M cell cycle arrest, up regulates the expression of p21 and p53 proteins and decreases cell viability demonstrated by a clonogenic assay. Therefore, these data demonstrate that Vietnamese coriander root contains 8-methyl-dihydrobenzopyrone (8-methyl-DBP, MW 314), which induces Bcl-2 phosphorylation, apoptosis, and G2/M cell cycle arrest in breast tumor cells.     

Solanum nigrum L. polyphenolic extract inhibits hepatocarcinoma cell growth by inducing G2/M phase arrest and apoptosis

BACKGROUND: Hepatocellular carcinoma (HCC) is a rapidly progressive cancer with poor prognosis. However, there have been no significant new developments in treating liver cancer. To search for an effective agent against HCC progression, we prepared a polyphenolic extract of Solanum nigrum L. (SNPE), a herbal plant indigenous to Southeast Asia and commonly used in oriental medicine, to evaluate its inhibitive effect on hepatocarcinoma cell growth. The growth inhibition of HepG2 cells in vitro and in vivo was determined in the presence of SNPE. RESULTS: We found 1 µg mL^?1 SNPE‐fed mice showed decreased tumor weight and tumor volume by 90%. Notably, 2 µg mL^?1 SNPE resulted in almost complete inhibition of tumor weight as well as tumor volume. In line with this notion, SNPE reduced the viability of HepG₂ cells in a dose‐dependent manner. HepG₂ cells were arrested in the G₂/M phase of the cell cycle; meanwhile, the protein levels of cell CDC25A, CDC25B, and CDC25C were clearly reduced. Moreover, sub‐G₁ phase accumulation and caspases‐3, 8, and 9 cleavages were induced by SNPE. CONCLUSION: This study shows that SNPE is a potent agent for HCC treatment through targeting G₂/M arrest and apoptosis induction, achieving cell growth inhibition. Copyright © 2010 Society of Chemical Industry     

Saccharomyces cerevisiae Gle2/Rae1 is involved in septin organization,essential for cell cycle progression

Gle2/Rae1 is highly conserved from yeast to humans and has been described as an mRNA export factor. Additionally, it is implicated in the anaphase‐promoting complex‐mediated cell cycle regulation in higher eukaryotes. Here we identify an involvement for Saccharomyces cerevisiae Gle2 in septin organization, which is crucial for cell cycle progression and cell division. Gle2 genetically and physically interacts with components of the septin ring. Importantly, deletion of GLE2 leads to elongated buds, severe defects in septin‐assembly and their cellular mislocalization. Septin‐ring formation is triggered by the septin‐regulating GTPase Cdc42, which establishes and maintains cell polarity. Additionally, activity of the master cell cycle regulator Cdc28 (Cdk1) is needed, which is, besides other functions, also required for G₂/M‐transition, and in yeast particularly responsible for initiating the apical–isotropic switch. We show genetic and physical interactions of Gle2 with both Cdc42 and Cdc28. Most importantly, we find that gle2? severely mislocalizes Cdc42, leading to defects in septin‐complex formation and cell division. Thus, our findings suggest that Gle2 participates in the efficient organization of the septin assembly network, where it might act as a scaffold protein. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.     

Anti‐inflammatory Potential of Quercetin‐3‐O‐β‐D‐(“2”‐galloyl)‐glucopyranoside and Quercetin Isolated from Diospyros kaki calyx via Suppression of MAP Signaling Molecules in LPS‐induced RAW 264.7 Macrophages

Diospyros kaki (DK) contains an abundance of flavonoids and has been used in folk medicine in Korea for centuries. Here, we report for the first time the anti‐inflammatory activities of Quercetin (QCT) and Quercetin 3‐O‐β‐(“2”‐galloyl)‐glucopyranoside (Q32G) isolated from DK. We have determine the no cytotoxicity of Q32G and QCT against RAW 264.7 cells up to concentration of 50 μM. QCT and Q32G demonstrated potent anti‐inflammatory activities by reducing expression of nitric oxide (NO), tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6 inducible NO synthase (iNOS), cyclooxygenase (COX)‐2, and mitogen‐activated protein kinase (MAPKs) in mouse RAW 264.7 macrophages activated with lipopolysaccharide (LPS). Both QCT or Q32G could decrease cellular protein levels of COX‐2 and iNOS as well as secreted protein levels of NO, PGE₂, and cytokines (TNF‐α, IL‐1β, and IL‐6) in culture medium of LPS‐stimulated RAW 264.7 macrophages. Immunoblot analysis showed that QCT and Q32G suppressed LPS‐induced MAP kinase pathway proteins p‐p38, ERK, and JNK. This study revealed that QCT and Q32G have anti‐inflammatory potential, however Q32G possess comparable activity as that of QCT and could be use as adjuvant to treat inflammatory diseases.     

Biphasic modulation of cell proliferation by sulforaphane at physiologically relevant exposure times in a human colon cancer cell line

Sulforaphane (SFN), a cancer chemopreventive compound derived from broccoli, is able to induce cell cycle arrest and apoptosis in various tumor cell lines. Here we show that cell growth inhibition by SFN follows a biphasic pattern: Transient exposure of 40-16 human colon carcinoma cells for up to 6 h resulted in reversible G(2)/M cell cycle arrest and cytostatic growth inhibition even at elevated concentrations, whereas a minimum continuous exposure time of 12 h was necessary for SFN to irreversibly arrest cells in G(2)/M phase and subsequently induce apoptosis. IC(50) values after 12 h of exposure followed by drug-free recovery up to 72 h (6.4-8.1 microM) were indistinguishable from those of chronic exposure for 24 to 72 h (5.4-6.6 microM). Low concentrations of SFN caused a transient decrease in glutathione (GSH) levels followed by GSH induction, which may be related to reversible G(2)/M arrest and cytostatic effects. Depletion of GSH does not seem to play a role in SFN-mediated apoptosis induction. Our data clearly contribute to a better understanding of the kinetics of antiproliferative activity of SFN.     

Constitutive Effects of Lead on Aryl Hydrocarbon Receptor Gene Battery and Protection by β‐carotene and Ascorbic Acid in Human HepG2 Cells

Lead (Pb) is an environmental pollutant that can get entry into human body through contaminated foods, drinks, and inhaled air leading to severe biological consequences, and has been responsible for many deaths worldwide. The objectives of this study were 1st to investigate the modulatory effects of environmentally relevant concentrations of Pb on AhR gene battery, which is controlling xenobiotics metabolism. 2nd, trials to reduce Pb‐induced adverse effects were done using some phytochemicals like β‐carotene or ascorbic acid. Human hepatoma (HepG2) cell lines were exposed to a wide range of Pb concentrations varying from physiological to toxic levels (0 to 10 mg/L) for 24 h. High Pb concentrations (1 to 10 mg/L) significantly reduced phase I (CYP1A1 and 1A2) and phase II (UGT1A6 and NQO1) xenobiotic metabolizing enzyme mRNA expression in a mechanistic manner through the AhR regulation pathway. Additionally, these Pb concentrations induced oxidative stress in HepG2 cells in terms of production of reactive oxygen species (ROS) and induced heme oxygenase‐1 mRNA expression in a concentration‐dependent phenomenon. Coexposure of HepG2 cells to physiological concentrations of some micronutrients, like β‐carotene (10 μM) or ascorbic acid (0.1 mM), along with Pb (1 mg/L) for 24 h significantly reduced the levels of ROS production and recovered AhR mRNA expression into the normal levels. Thus, consumption of foods rich in these micronutrients may help to reduce the adverse effects of lead in areas with high levels of pollution.     

君迁子叶杨梅苷诱导HepG2细胞凋亡及其作用机制

目的：探究君迁子叶杨梅苷诱导人肝癌细胞HepG2细胞凋亡及其作用机制。方法：噻唑蓝法测定杨梅苷对细胞存活率的影响；激光共聚焦显微镜结合Hoechst 33342染色观察杨梅苷对细胞形态的影响；流式细胞分析仪检测杨梅苷对细胞凋亡、周期阻滞、胞内活性氧（reactive oxygen species，ROS）水平和单丹璜酰戊二胺（monodansylcadaverine，MDC）荧光强度的影响；Western blot检测杨梅苷对细胞凋亡和自噬相关蛋白表达量的影响。结果：浓度为10～200 μmol/L的杨梅苷对人正常肝细胞L-02细胞无显著影响（P＞0.05），但可显著降低HepG2细胞的存活率（P＜0.05），促进其凋亡，提升ROS水平，增加MDC和细胞核荧光强度，将细胞阻滞在G2/M期；此外，可显著上调HepG2细胞中Bax、细胞色素c、Apaf-1、Caspase-9、Caspase-3、Beclin 1、Atg5和LC3-II的相对表达量（P＜0.05），下调Bcl-2和LC3-I的相对表达量（P＜0.05）。结论：杨梅苷具有抗肝癌作用，其机制与激活线粒体介导的凋亡通路、阻滞细胞周期、提升ROS水平和促进细胞自噬有关。实验可为杨梅苷作为天然抗肝癌药物的研究及应用提供参考。     

Electrospray MS‐based characterization of β‐carbolines – mutagenic constituents of thermally processed meat

The β‐carbolines 1‐methyl‐9H‐pyrido [3,4‐b]indole and 9H‐pyrido[3,4b]indole have been implicated as having causative roles in a number of human diseases, such as Parkinson's disease and cancer. As they can be formed during the heating of protein‐rich food, a number of analytical methodologies have been proposed for their detection and quantification in foodstuff. For this purpose, LC‐MS and LC‐MS/MS have emerged as the most specific analytical methods, and the quantification is based on the occurrence of unusual ions, such as [M+H‐(H^?)]⁺ and [M+H‐2H]⁺. In this study, we have investigated the formation of these ions by accurate‐mass electrospray MS/MS and demonstrated that these ions are formed from gas‐phase ion‐molecule reactions between water vapor present in the collision cell and the protonated molecule of 1‐methyl‐9H‐pyrido [3,4‐b]indole and 9H‐pyrido[3,4b]indole. Although this reaction has been previously described for heterocyclic amine ions, it has been overlooked in the most of recent LC‐MS and LC‐MS/MS studies, and no complete data of the fragmentation are reported. Our results demonstrate that additional attention should be given with respect to eliminating water vapor residues in the mass spectrometer when analysis of β‐carbolines is performed, as this residue may affect the reliability in the results of quantification.     

Sesamin attenuates intercellular cell adhesion molecule‐1 expression in vitro in TNF‐α‐treated human aortic endothelial cells and in vivo in apolipoprotein‐E‐deficient mice

Sesame lignans have antioxidative and anti‐inflammatory properties. We focused on the effects of the lignans sesamin and sesamol on the expression of endothelial‐leukocyte adhesion molecules in tumor necrosis factor‐α (TNF‐α)‐treated human aortic endothelial cells (HAECs). When HAECs were pretreated with sesamin (10 or 100 μM), the TNF‐α‐induced expression of intercellular cell adhesion molecule‐1 (ICAM‐1) was significantly reduced (35 or 70% decrease, respectively) by Western blotting. Sesamol was less effective at inhibiting ICAM‐1 expression (30% decrease at 100 μM). Sesamin and sesamol reduced the marked TNF‐α‐induced increase in human antigen R (HuR) translocation and the interaction between HuR and the 3'UTR of ICAM‐1 mRNA. Both significantly reduced the binding of monocytes to TNF‐α‐stimulated HAECs. Sesamin significantly attenuated TNF‐α‐induced ICAM‐1 expression and cell adhesion by downregulation of extracellular signal‐regulated kinase 1/2 and p38. Furthermore, in vivo, sesamin attenuated intimal thickening and ICAM‐1 expression seen in aortas of apolipoprotein‐E‐deficient mice. Taken together, these data suggest that sesamin inhibits TNF‐α‐induced extracellular signal‐regulated kinase/p38 phosphorylation, nuclear translocation of NF‐κB p65, cytoplasmic translocalization of HuR and thereby suppresses ICAM‐1 expression, resulting in reduced adhesion of leukocytes. These results also suggest that sesamin may prevent the development of atherosclerosis and inflammatory responses.     

Phosphorylations of Sds23/Psp1/Moc1 by stress‐activated kinase and cAMP‐dependent kinase are essential for regulating cell viability in prolonged stationary phase

Under nutritional deprivation caused by prolonged culture, actively growing cells prepare to enter stationary phase. We showed here that Sds23/Psp1/Moc1 was phosphorylated by both cAMP‐dependent kinase and stress‐activated MAP kinase Sty1 upon entry into stationary phase. Overexpression of the phosphorylation‐defective mutant Sds23/Psp1/Moc1 induced cell death in prolonged culture and blocked re‐entry into the cell division cycle. These phosphorylations are likely to be required for cell survival during stationary phase and for binding of Ufd2, a Schizosaccharomyces pombe homologue of multi‐ubiquitin chain assembly factor E4. Deletion of the Ufd2 gene and overexpression of Sds23/Psp1/Moc1 increased cell viability in prolonged stationary phase. These results suggested that Ufd2 induces cell death in prolonged nutrient deprivation, that Sds23/Psp1/Moc1 may be a target protein of the ubiquitin‐fusion degradation pathway for regulation of cell viability under this stress condition, and that Sty1 and PKA activity in stationary phase is essential for interaction between Sds23/Psp1/Moc1 and Ufd2. Copyright © 2013 John Wiley & Sons, Ltd.     

Anti‐inflammatory and anticancer activities of water‐soluble peptide extracts of buffalo and cow milk Cheddar cheeses

This article aimed to assess the anti‐inflammatory and anticancer potential of water‐soluble peptide (WSP) extracts from buffalo and cow milk Cheddar cheeses. Anti‐inflammatory activity was evaluated on the basis of nitric oxide (NO) production in lipopolysaccharide‐stimulated macrophage (RAW‐264.7) cells. A cell viability assay, cell cycle arrest and apoptosis were performed to explore anticancer activity in a colon cancer model (HT‐29). The WSP extracts of both Cheddar cheeses effectively inhibited NO production in activated macrophages. Maximum growth inhibition was observed in the HT‐29 cells at concentrations of 400 and 500 μg/mL. A significant increase in cell population at G₀/G₁ phase of the cell cycle was observed. Moreover, the WSP extracts also induced extensive apoptosis in colon cancer cells.