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Fulltext Unlocking the mystery behind the activation phenomenon of T1 lipase: a molecular dynamics simulations approach

Abdul Rahman MZ, Salleh AB, Abdul Rahman RN, Abdul Rahman MB, Basri M, Leow TC

Protein Sci, 2012 Aug;21(8):1210-21.
PMID: 22692819 DOI: 10.1002/pro.2108

The activation of lipases has been postulated to proceed by interfacial activation, temperature switch activation, or aqueous activation. Recently, based on molecular dynamics (MD) simulation experiments, the T1 lipase activation mechanism was proposed to involve aqueous activation in addition to a double-flap mechanism. Because the open conformation structure is still unavailable, it is difficult to validate the proposed theory unambiguously to understand the behavior of the enzyme. In this study, we try to validate the previous reports and uncover the mystery behind the activation process using structural analysis and MD simulations. To investigate the effects of temperature and environmental conditions on the activation process, MD simulations in different solvent environments (water and water-octane interface) and temperatures (20, 50, 70, 80, and 100°C) were performed. Based on the structural analysis of the lipases in the same family of T1 lipase (I.5 lipase family), we proposed that the lid domain comprises α6 and α7 helices connected by a loop, thus forming a helix-loop-helix motif involved in interfacial activation. Throughout the MD simulations experiments, lid displacements were only observed in the water-octane interface, not in the aqueous environment with respect to the temperature effect, suggesting that the activation process is governed by interfacial activation coupled with temperature switch activation. Examining the activation process in detail revealed that the large structural rearrangement of the lid domain was caused by the interaction between the hydrophobic residues of the lid with octane, a nonpolar solvent, and this conformation was found to be thermodynamically favorable.

Matched MeSH terms: Enzyme Activation
Fulltext Anti- and pro-lipase activity of selected medicinal, herbal and aquatic plants, and structure elucidation of an anti-lipase compound

Ado MA, Abas F, Mohammed AS, Ghazali HM

Molecules, 2013;18(12):14651-69.
PMID: 24287996 DOI: 10.3390/molecules181214651

Plants that help in slowing down the digestion of triacylglycerols (TAGs) in the pancreas and small intestine of humans play an important role in the reduction of obesity. On the other hand, there may be plants or plant parts that stimulate intestinal lipolytic activity, thus contributing to greater TAG assimilation. The aim of this study was to evaluate the aqueous methanolic extracts of ninety eight (98) medicinal, herbal and aquatic plant materials from Malaysia for their effect on porcine pancreatic lipase (PPL) activity and to identify the structure of an anti-lipase compound from one of the sources. The degree of inhibition was also quantified as relative to orlistat activity against PPL (orlistat equivalents). Results revealed that while 19.4% of the extracts were found to have anti-lipase activity ≥80%, 12% were actually found to promote PPL activity. Twenty two percent (22.4%) exhibited moderate inhibition (41%-80%) and 2% were neutral toward PPL activity. The ripe fruit of Averrhoa carambola and the leaves of Archidendron jiringa (Jack) I.C Nielsen L. (jering), Cynometra cauliflora (nam-nam) and Aleurites moluccana (L.) Willd (candle nut/buah keras) had the highest (100%) anti-lipase activity and are equivalent to 0.11 µg orlistat/mL. Plants that stimulated lipase activity included Pimpinella anisum L. (aniseed/jintan manis), activating the enzyme by 186.5%. Kaempferol 3-O-rhamnoside was isolated from the ethyl acetate fraction of C. cauliflora leaves and found to be an active lipase inhibitor. The structure was elucidated using 1H-NMR, 13C-NMR and 2D-NMR analyses.

Matched MeSH terms: Enzyme Activation/drug effects
Effects of novel diarylpentanoid analogues of curcumin on secretory phospholipase A2 , cyclooxygenases, lipo-oxygenase, and microsomal prostaglandin E synthase-1

Ahmad W, Kumolosasi E, Jantan I, Bukhari SN, Jasamai M

Chem Biol Drug Des, 2014 Jun;83(6):670-81.
PMID: 24406103 DOI: 10.1111/cbdd.12280

Arachidonic acid and its metabolites have generated a heightened interest due to their significant role in inflammation. Inhibiting the enzymes involved in arachidonic acid metabolism has been considered as the synergistic anti-inflammatory effect. A series of novel curcumin diarylpentanoid analogues were synthesized and evaluated for their inhibitory effects on activity of secretory phospholipase A2 , cyclooxygenases, soybean lipo-oxygenase as well as microsomal prostaglandin E synthase-1. Among the curcumin analogues, compounds 3, 6, 9, 12, and 17 exhibited strong inhibition of secretory phospholipase A2 activity, with IC50 values ranging from 5.89 to 11.02 μm. Seven curcumin analogues 1, 3, 6, 7, 9, 11, and 12 showed inhibition of cyclooxygenases-2 with IC50 values in the range of 46.11 to 94.86 μm, which were lower than that of curcumin. Compounds 3, 6, 7, 12, and 17 showed strong inhibition of lipo-oxygenase enzyme activity. Preliminary screening of diarylpentanoid curcumin analogues for microsomal prostaglandin E synthase-1 activity revealed that four diarylpentanoid curcumin analogues 5, 6, 7, and 13 demonstrated higher inhibition of microsomal prostaglandin E synthase-1 activity with IC50 ranging from 2.41 to 4.48 μm, which was less than that of curcumin. The present results suggest that some of these diarylpentanoid analogues were able to inhibit the activity of these enzymes. This raises the possibility that diarylpentanoid analogues of curcumin might serve as useful starting point for the design of improved anti-inflammatory agents.

Matched MeSH terms: Enzyme Activation/drug effects
Fulltext In Vitro Evaluation of the Anti-Diabetic Potential of Aqueous Acetone Helichrysum petiolare Extract (AAHPE) with Molecular Docking Relevance in Diabetes Mellitus

Akinyede KA, Oyewusi HA, Hughes GD, Ekpo OE, Oguntibeju OO

Molecules, 2021 Dec 28;27(1).
PMID: 35011387 DOI: 10.3390/molecules27010155

Diabetes mellitus (DM) is a chronic metabolic condition that can lead to significant complications and a high fatality rate worldwide. Efforts are ramping up to find and develop novel α-glucosidase and α-amylase inhibitors that are both effective and potentially safe. Traditional methodologies are being replaced with new techniques that are less complicated and less time demanding; yet, both the experimental and computational strategies are viable and complementary in drug discovery and development. As a result, this study was conducted to investigate the in vitro anti-diabetic potential of aqueous acetone Helichrysum petiolare and B.L Burtt extract (AAHPE) using a 2-NBDG, 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxy-d-glucose uptake assay. In addition, we performed molecular docking of the flavonoid constituents identified and quantified by liquid chromatography-mass spectrometry (LC-MS) from AAHPE with the potential to serve as effective and safe α-amylase and α-glucosidase inhibitors, which are important in drug discovery and development. The results showed that AAHPE is a potential inhibitor of both α-amylase and α-glucosidase, with IC50 values of 46.50 ± 6.17 (µg/mL) and 37.81 ± 5.15 (µg/mL), respectively. This is demonstrated by a significant increase in the glucose uptake activity percentage in a concentration-dependent manner compared to the control, with the highest AAHPE concentration of 75 µg/mL of glucose uptake activity being higher than metformin, a standard anti-diabetic drug, in the insulin-resistant HepG2 cell line. The molecular docking results displayed that the constituents strongly bind α-amylase and α-glucosidase while achieving better binding affinities that ranged from ΔG = -7.2 to -9.6 kcal/mol (compared with acarbose ΔG = -6.1 kcal/mol) for α-amylase, and ΔG = -7.3 to -9.0 kcal/mol (compared with acarbose ΔG = -6.3 kcal/mol) for α-glucosidase. This study revealed the potential use of the H. petiolare plant extract and its phytochemicals, which could be explored to develop potent and safe α-amylase and α-glucosidase inhibitors to treat postprandial glycemic levels in diabetic patients.

Matched MeSH terms: Enzyme Activation
Goniothalamin selectively induces apoptosis on human hepatoblastoma cells through caspase-3 activation

Al-Qubaisi M, Rosli R, Subramani T, Omar AR, Yeap SK, Ali AM, et al.

Nat Prod Res, 2013;27(23):2216-8.
PMID: 23767409 DOI: 10.1080/14786419.2013.800979

Goniothalamin is a biologically active styrylpyrone derivative isolated from various Goniothalamus species. The ability of goniothalamin to induce apoptosis via caspase-3 activation against hepatoblastoma (HepG2) and normal liver cells (Chang cells) was studied using morphological and biochemical evaluations. HepG2 and Chang cells were treated with goniothalamin for 72 h and analysed by TUNEL and Annexin-V/PI staining. Furthermore, the post-mitochondrial caspase-3 was quantified using ELISA. In view of our results, goniothalamin induced apoptosis on treated cells via alteration of cellular membrane integrity and cleavage of DNA. On the other hand, post-mitochondrial caspase-3 activity was significantly elevated in HepG2 cells treated with goniothalamin after 72 h. These findings suggest that goniothalamin induced apoptosis on HepG2 liver cancer cells via induction of caspase-3 with less sensitivity on the cell line of Chang cells.

Matched MeSH terms: Enzyme Activation
Production of biodiesel by lipase-catalyzed transesterification of vegetable oils: a kinetics study

Al-Zuhair S

Biotechnol Prog, 2005 Sep-Oct;21(5):1442-8.
PMID: 16209548

Kinetics of production of biodiesel by enzymatic methanolysis of vegetable oils using lipase has been investigated. A mathematical model taking into account the mechanism of the methanolysis reaction starting from the vegetable oil as substrate, rather than the free fatty acids, has been developed. The kinetic parameters were estimated by fitting the experimental data of the enzymatic reaction of sunflower oil by two types of lipases, namely, Rhizomucor miehei lipase (RM) immobilized on ion-exchange resins and Thermomyces lanuginosa lipase (TL) immobilized on silica gel. There was a good agreement between the experimental results of the initial rate of reaction and those predicted by the proposed model equations, for both enzymes. From the proposed model equations, the regions where the effect of alcohol inhibition fades, at different substrate concentrations, were identified. The proposed model equation can be used to predict the rate of methanolysis of vegetable oils in a batch or a continuous reactor and to determine the optimal conditions for biodiesel production.

Matched MeSH terms: Enzyme Activation
Fulltext Antioxidant, Anti-Inflammatory, and Inhibition of Acetylcholinesterase Potentials of Cassia timoriensis DC. Flowers

Alhawarri MB, Dianita R, Razak KNA, Mohamad S, Nogawa T, Wahab HA

Molecules, 2021 Apr 29;26(9).
PMID: 33946788 DOI: 10.3390/molecules26092594

Despite being widely used traditionally as a general tonic, especially in South East Asia, scientific research on Cassia timoriensis, remains scarce. In this study, the aim was to evaluate the in vitro activities for acetylcholinesterase (AChE) inhibitory potential, radical scavenging ability, and the anti-inflammatory properties of different extracts of C. timoriensis flowers using Ellman's assay, a DPPH assay, and an albumin denaturation assay, respectively. With the exception of the acetylcholinesterase activity, to the best of our knowledge, these activities were reported for the first time for C. timoriensis flowers. The phytochemical analysis confirmed the existence of tannins, flavonoids, saponins, terpenoids, and steroids in the C. timoriensis flower extracts. The ethyl acetate extract possessed the highest phenolic and flavonoid contents (527.43 ± 5.83 mg GAE/g DW and 851.83 ± 10.08 mg QE/g DW, respectively) as compared to the other extracts. In addition, the ethyl acetate and methanol extracts exhibited the highest antioxidant (IC50 20.12 ± 0.12 and 34.48 ± 0.07 µg/mL, respectively), anti-inflammatory (92.50 ± 1.38 and 92.22 ± 1.09, respectively), and anti-AChE (IC50 6.91 ± 0.38 and 6.40 ± 0.27 µg/mL, respectively) activities. These results suggest that ethyl acetate and methanol extracts may contain bioactive compounds that can control neurodegenerative disorders, including Alzheimer's disease, through high antioxidant, anti-inflammatory, and anti-AChE activities.

Matched MeSH terms: Enzyme Activation/drug effects
Fulltext An expedient synthesis, acetylcholinesterase inhibitory activity, and molecular modeling study of highly functionalized hexahydro-1,6-naphthyridines

Almansour AI, Kumar RS, Arumugam N, Basiri A, Kia Y, Ali MA

Biomed Res Int, 2015;2015:965987.
PMID: 25710037 DOI: 10.1155/2015/965987

A series of hexahydro-1,6-naphthyridines were synthesized in good yields by the reaction of 3,5-bis[(E)-arylmethylidene]tetrahydro-4(1H)-pyridinones with cyanoacetamide in the presence of sodium ethoxide under simple mixing at ambient temperature for 6-10 minutes and were assayed for their acetylcholinesterase (AChE) inhibitory activity using colorimetric Ellman's method. Compound 4e with methoxy substituent at ortho-position of the phenyl rings displayed the maximum inhibitory activity with IC50 value of 2.12 μM. Molecular modeling simulation of 4e was performed using three-dimensional structure of Torpedo californica AChE (TcAChE) enzyme to disclose binding interaction and orientation of this molecule into the active site gorge of the receptor.

Matched MeSH terms: Enzyme Activation
Fulltext Purification and characterization of alkaline-thermostable protease enzyme from Pitaya (Hylocereus polyrhizus) waste: a potential low cost of the enzyme

Amid M, Manap MY, Zohdi NK

Biomed Res Int, 2014;2014:259238.
PMID: 25328883 DOI: 10.1155/2014/259238

The thermoalkaline protease enzyme from pitaya (Hylocereus polyrhizus) waste was purified by a factor of 221.2 with 71.3% recovery using ammonium sulphate precipitation, gel filtration, and cation exchange chromatography. Gel filtration chromatography together with sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) revealed that the enzyme is monomeric with a molecular weight of 26.7 kDa. The apparent K m and V max of the protease were 2.8 mg/mL and 31.20 u/min, respectively. The optimum pH and temperature were 8.0 and 70°C. The enzyme was highly active and stable over a wide pH range (from pH 3.0 to pH 11.0 with the optimum activity at pH 8.0). The protease has broad specificity toward azocasein, casein, hemoglobin, and gelatine. Activity of the enzyme was inhibited by Fe(2+) and Zn(2+), while protease activity was increased in the presence of Ca(2+) and Mg(2+) and Cu(2+) by factors of 125%, 110%, and 105%, respectively. The alkaline protease showed extreme stability toward surfactants and oxidizing agent. The purified protease exhibited extreme stability in the presence of organic solvents and inhibitors. In addition, the enzyme was relativity stable toward organic solvents and chelating agents, such as ethylenediaminetetraacetic acid (EDTA). The enzyme, derived from pitaya peel, possesses unique characteristics and could be used in various industrial and biotechnological applications.

Matched MeSH terms: Enzyme Activation
Fulltext Optimization of processing parameters for extraction of amylase enzyme from dragon (Hylocereus polyrhizus) peel using response surface methodology

Amid M, Manap MY, Zohdi N

ScientificWorldJournal, 2014;2014:640949.
PMID: 25050403 DOI: 10.1155/2014/640949

The main goal of this study was to investigate the effect of extraction conditions on the enzymatic properties of thermoacidic amylase enzyme derived from dragon peel. The studied extraction variables were the buffer-to-sample (B/S) ratio (1:2 to 1:6, w/w), temperature (-18°C to 25°), mixing time (60 to 180 seconds), and the pH of the buffer (2.0 to 8.0). The results indicate that the enzyme extraction conditions exhibited the least significant (P < 0.05) effect on temperature stability. Conversely, the extraction conditions had the most significant (P < 0.05) effect on the specific activity and pH stability. The results also reveal that the main effect of the B/S ratio, followed by its interaction with the pH of the buffer, was significant (P < 0.05) among most of the response variables studied. The optimum extraction condition caused the amylase to achieve high enzyme activity (648.4 U), specific activity (14.2 U/mg), temperature stability (88.4%), pH stability (85.2%), surfactant agent stability (87.2%), and storage stability (90.3%).

Matched MeSH terms: Enzyme Activation/drug effects
Fulltext Microencapsulation of purified amylase enzyme from pitaya (Hylocereus polyrhizus) peel in Arabic gum-chitosan using freeze drying

Amid M, Manap Y, Zohdi NK

Molecules, 2014;19(3):3731-43.
PMID: 24662085 DOI: 10.3390/molecules19033731

Amylase is one of the most important enzymes in the world due to its wide application in various industries and biotechnological processes. In this study, amylase enzyme from Hylocereus polyrhizus was encapsulated for the first time in an Arabic gum-chitosan matrix using freeze drying. The encapsulated amylase retained complete biocatalytic activity and exhibited a shift in the optimum temperature and considerable increase in the pH and temperature stabilities compared to the free enzyme. Encapsulation of the enzyme protected the activity in the presence of ionic and non-ionic surfactants and oxidizing agents (H₂O₂) and enhanced the shelf life. The storage stability of amylase is found to markedly increase after immobilization and the freeze dried amylase exhibited maximum encapsulation efficiency value (96.2%) after the encapsulation process. Therefore, the present study demonstrated that the encapsulation of the enzyme in a coating agent using freeze drying is an efficient method to keep the enzyme active and stable until required in industry.

Matched MeSH terms: Enzyme Activation/drug effects
Fulltext A Novel Aqueous Two Phase System Composed of Surfactant and Xylitol for the Purification of Lipase from Pumpkin (Cucurbita moschata) Seeds and Recycling of Phase Components

Amid M, Manap MY, Hussin M, Mustafa S

Molecules, 2015 Jun 17;20(6):11184-201.
PMID: 26091076 DOI: 10.3390/molecules200611184

Lipase is one of the more important enzymes used in various industries such as the food, detergent, pharmaceutical, textile, and pulp and paper sectors. A novel aqueous two-phase system composed of surfactant and xylitol was employed for the first time to purify lipase from Cucurbita moschata. The influence of different parameters such as type and concentration of surfactants, and the composition of the surfactant/xylitol mixtures on the partitioning behavior and recovery of lipase was investigated. Moreover, the effect of system pH and crude load on the degree of purification and yield of the purified lipase were studied. The results indicated that the lipase was partitioned into the top surfactant rich phase while the impurities partitioned into the bottom xylitol-rich phase using an aqueous two phase system composed of 24% (w/w) Triton X-100 and 20% (w/w) xylitol, at 56.2% of tie line length (TLL), (TTL is one of the important parameters in this study and it is determined from a bimodal curve in which the tie-line connects two nodes on the bimodal, that represent concentration of phase components in the top and bottom phases) and a crude load of 25% (w/w) at pH 8.0. Recovery and recycling of components was also measured in each successive step process. The enzyme was successfully recovered by the proposed method with a high purification factor of 16.4 and yield of 97.4% while over 97% of the phase components were also recovered and recycled. This study demonstrated that the proposed novel aqueous two phase system method is more efficient and economical than the traditional aqueous two phase system method for the purification and recovery of the valuable enzyme lipase.

Matched MeSH terms: Enzyme Activation
α-Glucosidase activity of oleanolic acid and its oxidative metabolites: DFT and Docking studies

Anouar el H, Zakaria NS, Alsalme A, Shah SA

Mini Rev Med Chem, 2015;15(14):1148-58.
PMID: 26205959

A natural pentacyclic triterpenoid oleanolic acid 1 and its biotransformed metabolites 2-3 are potential α-glucosidase inhibitors. To elucidate the inhibitory mechanism of compounds 1, 2 and 3 against α-glucosidase, we calculated (i) their electronic and optical properties using DFT and TD-DFT at the B3LYP/6-31G(d) level in gas and IEF-PCM solvent; and (ii) their binding energies to α-glucosidase via docking study. DFT results showed that the α-glucosidase inhibtion is mainly depend on the polarity parameters of the studied compounds. Docking results revealed that the activity increased with binding energies (i.e. the stability of ligand-receptor complex). The specroscopic data of oleanolic acid 1 and its metabolites 2 and 3 are well predicetd for 13C NMR chemical shifts (R2=99%) and 1H NMR chemical shifts (R2=90%); and for (ii) UV/vis spectra. The assignments and interpretation of NMR chemical shifts and bathochromic shift of λMAX absorption bands are discussed.

Matched MeSH terms: Enzyme Activation/drug effects
Lipase-catalyzed esterification of palm-based 9,10-dihydroxystearic acid and 1-octanol in hexane--a kinetic study

Awang R, Basri M, Ahmad S, Salleh AB

Biotechnol Lett, 2004 Jan;26(1):11-4.
PMID: 15005144

The esterification of palm-based 9,10-dihydroxystearic acid (DHSA) and 1-octanol in hexane as catalyzed by lipase from Rhizomucor meihei (Lipozyme IM) followed Michaelis-Menten kinetics. The esterification reaction follows a Ping-Pong, Bi-Bi mechanism. The maximum rate was estimated to be 1 micromol min(-1) mg(-1) catalyst in hexane at 50 degrees C, and the Michaelis-Menten constants for DHSA and 1-octanol were 1.3 M and 0.7 M, respectively.

Matched MeSH terms: Enzyme Activation
Synthesis and cholinesterase inhibitory activity study of new piperidone grafted spiropyrrolidines

Basiri A, Abd Razik BM, Ezzat MO, Kia Y, Kumar RS, Almansour AI, et al.

Bioorg Chem, 2017 12;75:210-216.
PMID: 28987876 DOI: 10.1016/j.bioorg.2017.09.019

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder, which affected 35 million people in the world. The most practiced approach to improve the life expectancy of AD patients is to increase acetylcholine neurotransmitter level at cholinergic synapses by inhibition of cholinesterase enzymes. A series of unreported piperidone grafted spiropyrrolidines 8(a-p) were synthesized and evaluated in vitro for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities. Therein, compounds 8h and 8l displayed more potent AChE enzyme inhibition than standard drug with IC50 values of 1.88 and 1.37 µM, respectively. Molecular docking simulations for 8l possessing the most potent AChE inhibitory activities, disclosed its interesting binding templates to the active site channel of AChE enzymes. These compounds are remarkable AChE inhibitors and have potential as AD drugs.

Matched MeSH terms: Enzyme Activation/drug effects
One-pot microwave assisted stereoselective synthesis of novel dihydro-2'H-spiro[indene-2,1'-pyrrolo-[3,4-c]pyrrole]-tetraones and evaluation of their antimycobacterial activity and inhibition of AChE

Bharkavi C, Vivek Kumar S, Ashraf Ali M, Osman H, Muthusubramanian S, Perumal S

Bioorg Med Chem Lett, 2017 Jul 15;27(14):3071-3075.
PMID: 28552337 DOI: 10.1016/j.bmcl.2017.05.050

An efficient one-pot microwave assisted stereoselective synthesis of novel dihydro-2'H-spiro[indene-2,1'-pyrrolo[3,4-c]pyrrole]-tetraone derivatives through three-component 1,3-dipolar cycloaddition of azomethine ylides generated in situ from ninhydrin and sarcosine with a series of 1-aryl-1H-pyrrole-2,5-diones is described. The synthesised compounds were screened for their antimycobacterial and AChE inhibition activities. Compound 4b (IC50 1.30µM) has been found to display twelve fold antimycobacterial activity compared to cycloserine and it is thirty seven times more active than pyrimethamine. Compound 4h displays maximum AchE inhibitory activity with IC50 value of 0.78±0.01µmol/L.

Matched MeSH terms: Enzyme Activation/drug effects
Fulltext Aqueous fraction of Nephelium ramboutan-ake rind induces mitochondrial-mediated apoptosis in HT-29 human colorectal adenocarcinoma cells

Chan CK, Goh BH, Kamarudin MN, Kadir HA

Molecules, 2012 May 31;17(6):6633-57.
PMID: 22728359 DOI: 10.3390/molecules17066633

The aim of this study was to investigate the cytotoxic and apoptotic effects of Nephelium ramboutan-ake (pulasan) rind in selected human cancer cell lines. The crude ethanol extract and fractions (ethyl acetate and aqueous) of N. ramboutan-ake inhibited the growth of HT-29, HCT-116, MDA-MB-231, Ca Ski cells according to MTT assays. The N. ramboutan-ake aqueous fraction (NRAF) was found to exert the greatest cytotoxic effect against HT-29 in a dose-dependent manner. Evidence of apoptotic cell death was revealed by features such as chromatin condensation, nuclear fragmentation and apoptotic body formation. The result from a TUNEL assay strongly suggested that NRAF brings about DNA fragmentation in HT-29 cells. Phosphatidylserine (PS) externalization on the outer leaflet of plasma membranes was detected with annexin V-FITC/PI binding, confirming the early stage of apoptosis. The mitochondrial permeability transition is an important step in the induction of cellular apoptosis, and the results clearly suggested that NRAF led to collapse of mitochondrial transmembrane potential in HT-29 cells. This attenuation of mitochondrial membrane potential (Δψm) was accompanied by increased production of ROS and depletion of GSH, an increase of Bax protein expression, and induced-activation of caspase-3/7 and caspase-9. These combined results suggest that NRAF induces mitochondrial-mediated apoptosis.

Matched MeSH terms: Enzyme Activation/drug effects
Fulltext Goniothalamin induces coronary artery smooth muscle cells apoptosis: the p53-dependent caspase-2 activation pathway

Chan KM, Rajab NF, Siegel D, Din LB, Ross D, Inayat-Hussain SH

Toxicol. Sci., 2010 Aug;116(2):533-48.
PMID: 20498002 DOI: 10.1093/toxsci/kfq151

Goniothalamin (GN), a styryl-lactone isolated from Goniothalamus andersonii, has been demonstrated to possess antirestenostic properties by inducing apoptosis on coronary artery smooth muscle cells (CASMCs). In this study, the molecular mechanisms of GN-induced CASMCs apoptosis were further elucidated. Apoptosis assessment based on the externalization of phosphatidylserine demonstrated that GN induces CASMCs apoptosis in a concentration-dependent manner. The GN-induced DNA damage occurred with concomitant elevation of p53 as early as 2 h, demonstrating an upstream signal for apoptosis. However, the p53 elevation in GN-treated CASMCs was independent of NAD(P)H: quinone oxidoreductase 1 and Mdm-2 expression. An increase in hydrogen peroxide and reduction in free thiols confirmed the role for oxidative stress in GN treatment. Pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-FMK) that significantly abrogated GN-induced CASMCs apoptosis suggested the involvement of caspase(s). The role of apical caspase-2, -8, and -9 was then investigated, and sequential activation of caspase-2 and -9 but not caspase-8 leading to downstream caspase-3 cleavage was observed in GN-treated CASMCs. Reduction of ATP level and decrease in oxygen consumption further confirmed the role of mitochondria in GN-induced apoptosis in CASMCs. The mitochondrial release of cytochrome c was seen without mitochondrial membrane potential loss and was independent of cardiolipin. These data provide insight into the mechanisms of GN-induced apoptosis, which may have important implications in the development of drug-eluting stents.

Matched MeSH terms: Enzyme Activation
The pyranoxanthone inophyllin A induces oxidative stress mediated-apoptosis in Jurkat T lymphoblastic leukemia cells

Chan KM, Hamzah R, Rahaman AA, Jong VY, Khong HY, Rajab NF, et al.

Food Chem Toxicol, 2012 Aug;50(8):2916-22.
PMID: 22613213 DOI: 10.1016/j.fct.2012.04.048

Inophyllin A (INO-A), a pyranoxanthone isolated from the roots of Calophyllum inophyllum represents a new xanthone with potential chemotherapeutic activity. In this study, the molecular mechanism of INO-A-induced cell death was investigated in Jurkat T lymphoblastic leukemia cells. Assessment of phosphatidylserine exposure confirmed apoptosis as the primary mode of cell death in INO-A-treated Jurkat cells. INO-A treatment for only 30 min resulted in a significant increase of tail moment which suggests that DNA damage is an early apoptotic signal. Further flow cytometric assessment of the superoxide anion level confirmed that INO-A induced DNA damage was mediated with a concomitant generation of reactive oxygen species (ROS). Investigation on the thiols revealed an early decrease of free thiols in 30 min after 50 μM INO-A treatment. Using tetramethylrhodamine ethyl ester, a potentiometric dye, the loss of mitochondrial membrane potential (MPP) was observed in INO-A-treated cells as early as 30 min. The INO-A-induced apoptosis progressed with the simultaneous activation of caspases-2 and -9 which then led to the processing of caspase-3. Taken together, these data demonstrate that INO-A induced early oxidative stress, DNA damage and loss of MMP which subsequently led to the activation of an intrinsic pathway of apoptosis in Jurkat cells.

Matched MeSH terms: Enzyme Activation
Telomerase activation and human papillomavirus infection in invasive uterine cervical carcinoma in a set of Malaysian patients

Cheah PL, Looi LM, Ng MH, Sivanesaratnam V

J Clin Pathol, 2002 Jan;55(1):22-6.
PMID: 11825919

AIM: Telomerase activity was studied in invasive uterine cervical carcinoma to assess whether it was activated during cervical malignant transformation and to look for a possible association with human papillomavirus (HPV) infection in a set of Malaysian patients.
METHODS: Histologically confirmed invasive cervical carcinoma and benign cervices were assayed for telomerase activity using a commercial telomerase polymerase chain reaction (PCR) enzyme linked immunosorbent assay kit. The same cases were subjected to PCR detection of HPV using type specific (HPV types 6b, 11, 16, and 18) followed by L1 open reading frame (ORF) consensus primers.
RESULTS: HPV was detected in 18 (13 HPV-16, one HPV-6b, four only L1 ORF) of 20 invasive cervical carcinoma and one (only L1 ORF) of 19 benign cervices. Raised telomerase activity (A(450 nm) > 0.215) was detected in 11 cervical carcinomas, with A(450 nm) ranging between 0.238 and 21.790 (mean, 3.952) in positive squamous carcinomas, whereas A(450 nm) was only 0.222 in the one positive adenosquamous carcinoma. Five of 11 cervical carcinomas in stage I, three of six in stage II, both in stage III, and the only case in stage IV showed telomerase activation. Increased telomerase activity was noted in five of the 12 lymph node negative, five of the seven lymph node status unknown cases, and the one case with presumed lymph node metastasis. Ten of 18 HPV positive and one of two HPV negative cervical carcinomas showed telomerase upregulation.
CONCLUSIONS: Telomerase is activated in invasive cervical carcinoma. Although larger studies are needed, there seems to be no clear association between telomerase upregulation and HPV status, although there is a suggestion of increased telomerase activity in squamous carcinomas and late stage disease.

Matched MeSH terms: Enzyme Activation

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