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.     

s‐Ethyl Cysteine and s‐Methyl Cysteine Protect Human Bronchial Epithelial Cells Against Hydrogen Peroxide Induced Injury

Protective effects and actions from s‐ethyl cysteine (SEC) and s‐methyl cysteine (SMC) for BEAS‐2B cells were examined. BEAS‐2B cells were pretreated with SEC or SMC at 4, 8, or 16 μmol/L, and followed by hydrogen peroxide (H₂O₂) treatment. Data showed that H₂O₂ enhanced Bax, caspase‐3 and caspase‐8 expression, and declined Bcl‐2 expression. However, SEC or SMC dose‐dependently decreased caspase‐3 expression and reserved Bcl‐2 expression. H₂O₂ increased reactive oxygen species (ROS) production, and lowered glutathione level, glutathione peroxide, and glutathione reductase activities in BEAS‐2B cells. SEC or SMC pretreatments reduced ROS generation, and maintained glutathione redox cycle in those cells. H₂O₂ upregulated the expression of both p47^phox and gp91^phox. SEC and SMC downregulated p47^phox expression. SEC or SMC at 8 and 16 μmol/L decreased H₂O₂‐induced release of inflammatory cytokines. H₂O₂ stimulated the activation of nuclear factor‐κB (NF‐κB) and mitogen‐activated protein kinase. SEC and SMC pretreatments dose‐dependently downregulated NF‐κB p65 and p‐p38 expression. Pyrrolidine dithiocarbamate or SB203580 inhibited NF‐κB activation and p38 phosphorylation; thus, SEC or SMC pretreatments failed to affect protein expression of these factors. These novel findings suggest that SEC or SMC could protect bronchial cells and benefit respiratory epithelia stability and functions.     

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.     

Quercetin‐induced apoptotic cascade in cancer cells: Antioxidant versus estrogen receptor α‐dependent mechanisms

The flavonol quercetin, especially abundant in apple, wine, and onions, is reported to have anti‐proliferative effects in many cancer cell lines. Antioxidant or pro‐oxidant activities and kinase inhibition have been proposed as molecular mechanisms for these effects. In addition, an estrogenic activity has been observed but, at the present, it is poorly understood whether this latter activity plays a role in the quercetin‐induced anti‐proliferative effects. Here, we studied the molecular mechanisms of quercetin committed to the generation of an apoptotic cascade in cancer cells devoid or containing transfected estrogen receptor α (ERα; i.e., human cervix epitheloid carcinoma HeLa cells). Although none of tested quercetin concentrations increase reactive oxygen species (ROS) generation in HeLa cells, quercetin stimulation prevents the H₂O₂‐induced ROS production both in the presence and in the absence of ERα. However, this flavonoid induces the activation of p38/MAPK, leading to the pro‐apoptotic caspase‐3 activation and to the poly(ADP‐ribose) polymerase cleavage only in the presence of ERα. Notably, no down‐regulation of survival kinases (i.e., AKT and ERK) was reported. Taken together, these findings suggest that quercetin results in HeLa cell death through an ERα‐dependent mechanism involving caspase‐ and p38 kinase activation. These findings indicate new potential chemopreventive actions of flavonoids on cancer growth.     

Novel transporters from Kluyveromyces marxianus and Pichia guilliermondii expressed in Saccharomyces cerevisiae enable growth on l‐arabinose and d‐xylose

Genes encoding l ‐arabinose transporters in Kluyveromyces marxianus and Pichia guilliermondii were identified by functional complementation of Saccharomyces cerevisiae whose growth on l ‐arabinose was dependent on a functioning l ‐arabinose transporter, or by screening a differential display library, respectively. These transporters also transport d ‐xylose and were designated KmAXT1 (arabinose–xylose transporter) and PgAXT1, respectively. Transport assays using l ‐arabinose showed that KmAxt1p has K_m 263 mm and V_max 57 nm /mg/min, and PgAxt1p has K_m 0.13 mm and V_max 18 nm /mg/min. Glucose, galactose and xylose significantly inhibit l ‐arabinose transport by both transporters. Transport assays using d ‐xylose showed that KmAxt1p has K_m 27 mm and V_max 3.8 nm /mg/min, and PgAxt1p has K_m 65 mm and V_max 8.7 nm /mg/min. Neither transporter is capable of recovering growth on glucose or galactose in a S. cerevisiae strain deleted for hexose and galactose transporters. Transport kinetics of S. cerevisiae Gal2p showed K_m 371 mm and V_max 341 nm /mg/min for l ‐arabinose, and K_m 25 mm and V_max 76 nm /mg/min for galactose. Due to the ability of Gal2p and these two newly characterized transporters to transport both l ‐arabinose and d ‐xylose, one scenario for the complete usage of biomass‐derived pentose sugars would require only the low‐affinity, high‐throughput transporter Gal2p and one additional high‐affinity general pentose transporter, rather than dedicated d ‐xylose or l ‐arabinose transporters. Additionally, alignment of these transporters with other characterized pentose transporters provides potential targets for substrate recognition engineering. Accession Nos: KmAXT1: GZ791039; PgAXT1: GZ791040 Copyright © 2015 John Wiley & Sons, Ltd.     

Aminopeptidase from jumbo squid (Dosidicus gigas) hepatopancreas: purification,characterisation, and casein hydrolysis

An aminopeptidase (AP) was partially purified from jumbo squid (Dosidicus gigas) hepatopancreas with 154.24‐fold and yield of 6.15%. The purification procedure consisted of ammonium sulphate fractionation and DEAE‐Sephacel chromatography. The enzyme was approximately 48–53 kDa as estimated by SDS‐PAGE. With l ‐leu‐p‐NA, it had optimum activity at pH 8.0 and 30 °C. The K_m and V_max/K_m values of the enzymes for l ‐leu‐p‐NA were 0.326 mm and 2787 at 37 °C, respectively. Activation energy (E_a) of the enzyme was 53.50 kJ M^?1.The AP showed activity against seven synthetic substrates: l ‐proline>l ‐methionine>Ac. l ‐γ‐glutamic>l ‐glycine>l ‐leucine>l ‐alanine>l ‐lysine‐p‐NA. The enzyme was strongly inhibited by Bestatin, partially inhibited by a metal‐chelating agent and by PCMB, a cystein protease inhibitor. Zn²⁺ and (or) Ca²⁺ seemed to be its metal cofactor(s). Incubation of casein with the partially purified AP resulted in a degree of hydrolysis of 6%.     

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.     

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.     

PURIFICATION AND CHARACTERIZATION OF TRYPSIN FROM PYLORIC CAECA OF BIGEYE SNAPPER (PRICANTHUS MACRACANTHUS)

Trypsin from the pyloric caeca of bigeye snapper was purified and characterized. Trypsin had an apparent molecular weight of 23.8 kDa when analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and substrate‐gel electrophoresis. The trypsin fraction consisted of three isoforms as evidenced by the appearance of three different bands on native‐PAGE. Optimal activity was observed at 55C and pH range of 8–11. The activity of trypsin fraction was completely inhibited by soybean trypsin inhibitor and was partially inhibited by E‐64 and ethylenediaminetetraacetic acid. CaCl₂ partially protected the trypsin fraction from activity loss at 40C, while NaCl (0–20%) decreased the activity in a concentration‐dependent manner. The apparent Michaelis–Menten constant (K_m) and catalytic constant (k_cat) were 0.312 mM and 1.06 s, respectively when Nα‐Benzoyl‐dl ‐arginine ρ‐nitroanilide was used as a substrate. Trypsin from the pyloric caeca of bigeye snapper generally showed similar characteristics to other fish trypsins.     

Purification,characterisation and application of α‐l‐rhamnosidase from Penicillium citrinum MTCC‐8897

An α‐l ‐rhamnosidase secreted by Penicillium citrinum MTCC‐8897 has been purified to homogeneity from the culture filtrate of the fungal strain using ammonium sulphate precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The sodium dodecyl sulphate/polyacrylamide gel electrophoresis analysis of the purified enzyme gave a single protein band corresponding to the molecular mass 51.0 kDa. The native polyacrylamide gel electrophoresis also gave a single protein band confirming the enzyme purity. The K_m and V_max values of the enzyme for p‐nitrophenyl α‐l ‐rhamnopyranoside were 0.36 mm and 22.54 μmole min^?1 mg^?1, respectively, and k_cat value was 17.1 s^?1 giving k_cat/K_m value of 4.75 × 10⁴ m ^?1 s^?1. The pH and temperature optima of the enzyme were 7.0 and 60 °C, respectively. The purified enzyme liberated l ‐rhamnose from naringin, rutin, hesperidin and wine, indicating that it has biotechnological application potential for the preparation of l ‐rhamnose and other pharmaceutically important compounds from natural glycosides containing terminal α‐l ‐rhamnose and also in the enhancement of wine aroma.     

Dicaffeoylquinic acids in Yerba mate (Ilex paraguariensis St. Hilaire) inhibit NF-κB nucleus translocation in macrophages and induce apoptosis by activating caspases-8 and -3 in human colon cancer cells

Scope : The biological functions of caffeoylquinic acid (CQA) derivatives from various plant sources have been partially elucidated. The objectives were to isolate and purify diCQAs from Yerba mate tea leaves and assess their anti‐inflammatory and anti‐cancer capabilities in vitro and explore their mechanism of action. Methods and results : Methanol extracts of dried mate leaves were resolved by flash chromatography and further purified resulting in two fractions one containing 3,4‐ and 3,5‐diCQAs and the other 4,5‐diCQA with NMR‐confirmed structures. Both fractions inhibited LPS‐induced RAW 264.7 macrophage inflammation by suppressing nitric oxide/inducible nitric oxide and prostaglandin E₂/cyclooxygenase‐2 pathways through inhibiting nucleus translocation of Nuclear factor κB subunits, p50 and p65. The diCQA fractions inhibited Human colon cancer cells CRL‐2577 (RKO) and HT‐29 cell proliferation by inducing apoptosis in a time‐ and concentration‐dependent manner, but did not affect the protein levels of p21, p27, p53, and Bax:Bcl‐2 ratio in RKO cells. In HT‐29 cells, however, the diCQA fractions increased Bax:Bcl‐2 ratio. The diCQA fractions increased the activation of caspase‐8 leading to cleavage of caspase‐3 in both RKO and HT‐29 colon cancer cells. Conclusion : The results suggest that diCQAs in Yerba mate could be potential anti‐cancer agents and could mitigate other diseases also associated with inflammation.     

Role of acidic sphingomyelinase in thymol‐mediated dendritic cell death

Scope : Thymol is a component of several plants with antimicrobial activity. Little is known about the effects of thymol on immune cells of the host. This study addressed the effects of thymol on dendritic cells (DCs), regulators of innate and adaptive immunity. Methods and results : Immunohistochemistry, Western blotting and fluorescence‐activated cell sorting analysis were performed in bone marrow‐derived DCs either from wild‐type mice or from mice lacking acid sphingomyelinase (ASM^?/?) treated and untreated for 24 h with thymol (2–100 μg/mL). Thymol treatment resulted in activation of ASM, stimulation of ceramide formation, downregulation of anti‐apoptotic Bcl‐2 and Bcl‐xL proteins, activation of caspase 3 and caspase 8, DNA fragmentation as well as cell membrane scrambling. The effects were dependent on the presence of ASM and were lacking in ASM^?/? mice or in wild‐type DCs treated with sphingomyelinase inhibitor amitriptyline. Conclusion : Thymol triggers suicidal DC death, an effect mediated by and requiring activation of ASM.     

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.     

Disodium Guanylate Alleviates Acute Hepatic Injury Induced by Carbon Tetrachloride Via Antioxidative Stress and Antiapoptosis In Vivo and In Vitro

Hepatic injury is one of the most common digestive system diseases worldwide in clinic. Guanylic acid or guanosine monophosphate (GMP) was an important component of nucleotides, which is mainly in the form of sodium salt (disodium guanylate, GMP‐Na₂). However, its effect on hepatic injury has not yet been investigated. This study is to investigate the protective effects of GMP‐Na₂ on acute hepatic injury induced by carbon tetrachloride (CCl₄), and to explore its mechanism. The hepatic injury models of mice and HL‐7702 cells were induced by CCl₄. The alanine transaminase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), glutathione peroxidase (GSH‐Px), malondialdehyde (MDA), total antioxidant capacity (T‐AOC) were determined by biochemical method. Hematoxylin–eosin staining were used to determine the morphological changes on liver tissue in mice. The mRNA and protein expressions of caspase‐3, Bax, and Bcl‐2 were detected by RT‐PCR and Western blot analysis. Our results show that GMP‐Na₂ treatment significantly decreased the activities of ALT and AST, and the levels of MDA as well as increased the levels of SOD, GSH‐Px, and T‐AOC. Importantly, GMP‐Na₂ effectively enhanced the antiapoptosis function by upregulating Bcl‐2 expression and downregulating caspase‐3 and Bax expressions in vivo and in vitro. Moreover, the histopathological changes of liver tissue were obviously improved after GMP‐Na₂ treatment. These findings suggest that GMP‐Na₂ has protective effects on hepatic injury, and its mechanisms may be associated with antioxidative stress and antiapoptosis.     

Purification and functional characterisation of an α‐l‐rhamnosidase from Penicillium citrinum MTCC‐3565

An extracellular α‐l ‐rhamnosidase from Penicillium citrinum MTCC‐3565 has purified to homogeneity from its culture filtrate using ethanol precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The purified enzyme gave a single protein band corresponding to molecular mass of 45.0 kDa in SDS‐PAGE analysis showing the purity of the enzyme preparation. The native PAGE analysis showed the monomeric nature of the purified enzyme. Using p‐nitrophenyl α‐l ‐rhamnopyranoside as substrate, K_m and V_max values of the enzyme were 0.30 mm and 27.0 μm min mg^?1, respectively. The k_cat value was 20.1 s giving k_cat/K_m value of 67.0 mm s^?1 for the same substrate. The pH and temperature optima of the enzyme were 8.5 and 50 °C, respectively. The activation energy for the thermal denaturation of the enzyme was 29.9 KJ mol^?1. The α‐l ‐rhamnosidase was able to hydrolyse naringin, rutin and hesperidin and liberated l ‐rhamnose, indicating that the purified enzyme can be used for the preparation of α‐l ‐rhamnose and pharmaceutically important compounds by derhamnosylation of natural glycosides containing terminal α‐l ‐rhamnose. The α‐l ‐rhamnosidase was active at the level of ethanol concentration present in wine, indicating that it can be used for improving wine aroma.     

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.     

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     

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.     

An α‐l‐rhamnosidase from Aspergillus awamori MTCC‐2879 and its role in debittering of orange juice

The extracellular α‐l ‐rhamnosidase has been purified by growing a new fungal strain Aspergillus awamori MTCC‐2879 in the liquid culture growth medium containing orange peel. The purification procedure involved ultrafiltration using PM‐10 membrane and anion‐exchange chromatography on diethyl amino ethyl cellulose. The purified enzyme gave single protein band in SDS‐PAGE analysis corresponding to molecular mass 75.0 kDa. The native PAGE analysis of the purified enzyme also gave a single protein band, confirming the purity of the enzyme. The K_m and V_max values of the enzyme for p‐nitrophenyl‐α‐l ‐rhamnopyranoside were 0.62 mm and 27.06 μmole min^?1 mg^?1, respectively, yielding k_cat and k_cat/k_m values 39.90 s^?1 and 54.70 mm ^?1 s^?1, respectively. The enzyme had an optimum pH of 7.0 and optimum temperature of 60 °C. The activation energy for the thermal denaturation of the enzyme was 35.65 kJ^?1 mol^?1 K^?1. The purified enzyme can be used for specifically cleaving terminal α‐l ‐rhamnose from the natural glycosides, thereby contributing to the preparation of pharmaceutically important compounds like prunin and l ‐rhamnose.