Phenformin Induces Cell Cycle Change,Apoptosis, and Mesenchymal-Epithelial Transition and Regulates the AMPK/mTOR/p70s6k and MAPK/ERK Pathways in Breast Cancer Cells

Breast cancer remains a world-wide challenge, and additional anti-cancer therapies are still urgently needed. Emerging evidence has demonstrated the potent anti-tumor effect of biguanides, among which phenformin was reported to potentially be a more active anti-cancer agent than metformin. However, little attention has been given to the role of phenformin in breast cancer. In this study, we reveal the role of phenformin in cell death of the MCF7, ZR-75-1, MDA-MB-231 and SUM1315 breast cancer cell lines. The respective IC₅₀ values of phenformin in MCF7, ZR-75-1, MDA-MB-231 and SUM1315 cells were 1.184±0.045 mM, 0.665±0.007 mM, 2.347±0.010 mM and 1.885±0.015 mM (mean± standard error). Phenformin induced cell cycle change and apoptosis in breast cancer cells via the AMPK/mTOR/p70s6k and MAPK/ERK pathways. Interestingly, phenformin induced MET (mesenchymal-epithelial transition) and decreased the migration rate in breast cancer cell lines. Furthermore, our results suggest that phenformin inhibits breast cancer cell metastasis after intracardiac injection into nude mice. Taken together, our study further confirms the potential benefit of phenformin in breast cancer treatment and provides novel mechanistic insight into its anti-cancer activity in breast cancer.     

Effect of amidine derivatives on nitric oxide production by Leishmania amazonensis promastigotes and axenic amastigotes.

The effects of pentamidine isethionate (reference drug) and N,N'-diphenyl-4-methoxy-benzamidine (test compound) on NO. production by Leishmania amazonensis promastigotes and axenic amastigotes were investigated by measuring nitrite, a by-product of nitric oxide released into culture supernatants. The NO. production by infective promastigotes was inhibited by OCH(3)-amidine in about 23.53% and by pentamidine in only 3.78%. In axenic amastigotes, the inhibition of NO. production by OCH(3)-amidine was significantly higher (52.94%; p=0.01) than that by pentamidine, which inhibited this radical production nonsignificantly (25.29%; p=0.1). The mechanism of amidine derivatives, as an antimicrobial agent, is unknown. However, other amidines, such as a diamidine (pentamidine), contain chemical structures shared by the guanidino group of the nitric oxide synthase substrate L-arginine, suggesting the possibility of an interaction with this enzyme or electronic factors (substituent constant) that alter physical and chemical properties significant for biological activity.     

Purification and characterisation of glutathione transferase from the giant African snail, Archachatina marginata.

1. Glutathione-S-transferase has been purified from the hepatopancreas of Archachatina marginata to homogeneity. 2. The enzyme was found to be a dimer with a molecular weight of 44,000. The subunits sizes were 22,500 and 23,500 respectively. The isoelectric points of the enzyme were 8.35, 7.95 and 4. The enzyme was most stable at temperature below 40 degrees C. Upon denaturation by 4 M urea, only 56% of the activity could be recovered. 3. The Kms for glutathione and 1-chloro-2,4-dinitrobenze (CDNB) were 0.23 mM and 0.4 mM respectively. The specific activity of the enzyme with CDNB and p-nitrophylacetate as substrates were 47 mumol/mg and 38 mumol/mg respectively. 4. Inhibition studies showed that S-hexylglutathione, Rose Bengal, iodoacetamide, sodium azide and Procion Blue H-B were good inhibitors with I50 values ranging from 18.5 microM to 299 mM. 5. The amino acid composition showed that the enzyme had a relatively high content of hydrophobic and acidic amino acid residues. The peptide maps of the tryptic digests of the native and performic acid-oxidised enzyme indicated that there might be about two disulphide bridges per molecule of the enzyme.     

Production and partial characterization of high molecular weight extracellular alpha-amylase from Thermoactinomyces vulgaris isolated from Egyptian soil

Optimizing production of alpha-amylase production by Thermoactinomyces vulgaris isolated from Egyptian soil was studied. The optimum incubation period, temperature and initial pH of medium for organism growth and enzyme yield were around 24 h, 55 degrees C and 7.0, respectively. Maximum alpha-amylase activity was observed in a medium containing starch as carbon source. The other tested carbohydrates (cellulose, glucose, galactose, xylose, arabinose, lactose and maltose) inhibited the enzyme production. Adding tryptone as a nitrogen source exhibited a maximum activity of alpha-amylase. Bactopeptone and yeast extract gave also high activity comparing to the other nitrogen sources (NH4CI, NH4NO3, NaNO3, KNO3, CH3CO2NH4). Electrophoresis profile of the produced two alpha-amylase isozymes indicated that the same pattern at about 135-145 kDa under different conditions. The optimum pH and temperature of the enzyme activity were 8.0 and 60 degrees C, respectively and enzyme was stable at 50 degrees C over 6 hours. The enzyme was significantly inhibited by the addition of metal ions (Na+, Co2+ and Ca2+) whereas CI- seemed to act as activator. The enzyme was not affected by 0.1 mM EDTA while higher concentration (10 mM EDTA) totally inactivated the enzyme.     

Inhibitory effects of fluorobenzaldehydes on the activity of mushroom tyrosinase

The effects of fluorobenzaldehydes (2-,3- and 4-fluorobenzaldehyde) on the activity of mushroom tyrosinase have been studied. The results show that fluorobenzaldehydes can strongly inhibit both monophenolase activity and diphenolase activity of the enzyme and the inhibition is reversible. The IC₅₀ values were estimated as 1.62 mM, 1.06 mM and 0.16 mM for diphenolase activity and as 1.35 mM, 1.18 mM and 1.05 mM for monophenolase activity, respectively. The lag time of the monophenolase was obviously lengthened by these three fluorobenzaldehydes. When the concentration of inhibitors reached 2.0 mM, the lag time was lengthened from 33 s to 142 s, 168 s and 190 s, respectively. Kinetic analyses show that the inhibition mechanism of 2-fluorobenzaldehyde on the diphenolase was competitive inhibition of the diphenolase activity, and that of 3-fluorobenzaldehyde and 4-fluorobenzaldehyde were of a mixed-type. The inhibition constants for these three fluorobenzaldehydes on the diphenolase were determined and compared.     

[3H]proline incorporation and hydroxyproline concentration in articular cartilage during the development of osteoarthritis caused by immobilization. A study in vivo with rabbits.

1. Activation of glucose 6-phosphate is one of the unique properties of pyruvate kinase from Mycobacterium smegmatis. 2. Pyruvate kinase, partially purified from ultrasonic extracts of the mycobacteria by (NH4)2SO4 fractionation, exhibited sigmoidal kinetics at various concentrations of phosphoenolpyruvate, with a high degree of co-operativity (Hill coefficient, h = 3.7) and S0.5 value of 1.0 mM. 3. In the presence of glucose 6-phosphate, the degree of co-operativity shown by the phosphoenolpyruvate saturation curve was decreased to h = 2.33 and the S0.5 value was lowered to 0.47 mM. 4. The enzyme was activated by AMP and ribose 5-phosphate also, but the activation constant was lowest with glucose 6-phosphate (0.24 mM). 5. The enzyme was strongly inhibited by ATP at all phosphoenolpyruvate concentrations. The concentrations of ATP required to produce half-maximal inhibition of enzyme activity at non-saturating (0.2 mM) and saturating (2 mM) phosphoenolpyruvate concentrations were 1.1 mM and 3 mM respectively. 6. The inhibition of ATP was partially relieved by glucose 6-phosphate. 7. The enzyme exhibited Michaelis-Menten kinetics with ADP as the variable substrate, with an apparent Km of 0.66 mM. 8. The enzyme required Mg2+ or Mn2+ ions for activity. It was not activated by univalent cations. 9. The kinetic data indicate that under physiological conditions glucose 6-phosphate probably plays a significant role in the regulation of pyruvate kinase activity.     

Metformin and phenformin activate AMP-activated protein kinase in the heart by increasing cytosolic AMP concentration

AMP-activated protein kinase (AMPK) acts as a cellular energy sensor: it responds to an increase in AMP concentration ([AMP]) or the AMP-to-ATP ratio (AMP/ATP). Metformin and phenformin, which are biguanides, have been reported to increase AMPK activity without increasing AMP/ATP. This study tests the hypothesis that these biguanides increase AMPK activity in the heart by increasing cytosolic [AMP]. Groups of isolated rat hearts (n = 5-7 each) were perfused with Krebs-Henseleit buffer with or without 0.2 mM phenformin or 10 mM metformin, and (31)P-NMR-measured phosphocreatine, ATP, and intracellular pH were used to calculate cytosolic [AMP]. At various times, hearts were freeze-clamped and assayed for AMPK activity, phosphorylation of Thr(172) on AMPK-alpha, and phosphorylation of Ser(79) on acetyl-CoA carboxylase, an AMPK target. In hearts treated with phenformin for 18 min and then perfused for 20 min with Krebs-Henseleit buffer, [AMP] began to increase at 26 min and AMPK activity was elevated at 36 min. In hearts treated with metformin, [AMP] was increased at 50 min and AMPK activity, phosphorylated AMPK, and phosphorylated acetyl-CoA carboxylase were elevated at 61 min. In metformin-treated hearts, HPLC-measured total AMP content and total AMP/ATP did not increase. In summary, phenformin and metformin increase AMPK activity and phosphorylation in the isolated heart. The increase in AMPK activity was always preceded by and correlated with increased cytosolic [AMP]. Total AMP content and total AMP/ATP did not change. Cytosolic [AMP] reported metabolically active AMP, which triggered increased AMPK activity, but measures of total AMP did not.     

Inhibitory effects of fluorobenzaldehydes on the activity of mushroom tyrosinase

The effects of fluorobenzaldehydes (2-,3- and 4-fluorobenzaldehyde) on the activity of mushroom tyrosinase have been studied. The results show that fluorobenzaldehydes can strongly inhibit both monophenolase activity and diphenolase activity of the enzyme and the inhibition is reversible. The IC50 values were estimated as 1.62 mM, 1.06 mM and 0.16 mM for diphenolase activity and as 1.35 mM, 1.18 mM and 1.05 mM for monophenolase activity, respectively. The lag time of the monophenolase was obviously lengthened by these three fluorobenzaldehydes. When the concentration of inhibitors reached 2.0 mM, the lag time was lengthened from 33 s to 142 s, 168 s and 190 s, respectively. Kinetic analyses show that the inhibition mechanism of 2-fluorobenzaldehyde on the diphenolase was competitive inhibition of the diphenolase activity, and that of 3-fluorobenzaldehyde and 4-fluorobenzaldehyde were of a mixed-type. The inhibition constants for these three fluorobenzaldehydes on the diphenolase were determined and compared.     

Purification and characterization of 3-phosphoglycerate kinase from Ehrlich ascites carcinoma cells

3-Phosphoglycerate kinase (3-PGK) has been purified to apparent homogeneity from Ehrlich ascites carcinoma (EAC) cells by (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. The enzyme has been partially characterized and compared with the characteristics of this enzyme of other normal and malignant cells. The EAC cell 3-PGK is composed of a single subunit of 47 kDa. It has a broad pH optimum (pH 6.0-7.5) for its enzymatic activity. The apparent Km values of 3-phosphoglycerate (3-PGA) and ATP for 3-PGK have been found out to be 0.25 mM and 0.1 mM respectively. Similar to 3-PGK of other cells, the EAC enzyme requires either Mg2+ or Mn2+ for full activity; the optimum concentrations of Mg2+ and Mn2+ are 0.8 mM and 0.5 mM respectively. When ATP and 3-PGA act as substrates, ADP, the reaction product of 3-PGK-catalyzed reaction has been found to inhibit this enzyme. Kinetic studies were made on the inhibition of ADP in presence of the substrates ATP and 3-PGA. Attempts to hybridize 3-PGK and glyceraldehyde-3-phosphate dehydrogenase of EAC cells by NAD or glutaraldehyde were unsuccessful.     

Trypanothione reductase activity is prominent in metacyclic promastigotes and axenic amastigotes of Leishmania amazonesis. Evaluation of its potential as a therapeutic target

The activity of trypanothione reductase in Leishmania amazonensis was evaluated and it was demonstrated that TR is expressed in the soluble fractions of infective promastigotes and amastigotes, while non-infective promastigotes expressed the enzyme at basal levels. This data allows an association of enzyme activity and the infective capacity of the parasite. We have also previously demonstrated that amidine compounds (N, N'-diphenyl-4-methoxy-benzamidine and pentamidine) were active against this parasite. Here, experiments concerning the effect of these compounds on TR activity, showed that both compounds significantly inhibited the enzyme. However, against glutathione reductase, only pentamidine showed a significant inhibitory action, suggesting an association with the toxic effects of this drug used in the clinic for the treatment of leishmaniasis.     

Phosphoenolpyruvate carboxylase from soybean nodule cytosol. Evidence for isoenzymes and kinetics of the most active component

Phosphoenolpyruvate carboxylase (orthophosphate:oxaloacetate carboxylase (phosphorylating), EC 4.1.1.31) from plant cells of soybean nodules was studied to assess its role in providing carbon skeletons for aspartate and asparagine synthesis. The enzyme was purified 119-fold by (NH4)2SO4 fractionation and DEAE-cellulose, BioGel A-1.5m, and hydroxyapatite chromatography. Five activity bands were resolved with discontinuous polyacrylamide gel electrophoresis. A small quantity of enzyme from the most active band was separated from the others by preparative electrophoresis. The apparent Michaelis constants of this enzyme for phosphoenolpyruvate and HCO3- were 9.4.10(-2) and 4.1.10(-1) mM, respectively. A series of metabolite tested at 1 mM had no significant effect on enzyme activity. These experiments indicate that the major factors directly controlling phosphoenolpyruvate carboxylase activity in vivo are phosphoenolpypyruvate and HCO3- concentrations.     

4-Aminobutyrate:2-oxoglutarate aminotransferase from Candida. Purification and properties

An enzyme which catalyzes the transamination of 4-aminobutyrate with 2-oxoglutarate was purified 588-fold to homogeneity from Candida guilliermondii var. membranaefaciens, grown with 4-aminobutyrate as sole source of nitrogen. An apparent relative molecular mass of 107,000 was estimated by gel filtration. The enzyme was found to be a dimer made up of two subunits identical in molecular mass (Mr 55,000). The enzyme has a maximum activity in the pH range 7.8-8.0 and a temperature optimum of 45 degrees C. 2-Oxoglutarate protects the enzyme from heat inactivation better than pyridoxal 5'-phosphate. The absorption spectrum of the enzyme exhibits two maxima at 412 nm and 330 nm. The purified enzyme catalyzes the transamination of omega-amino acids; 4-aminobutyrate is the best amino donor and low activity is observed with beta-alanine. The Michaelis constants are 1.5 mM for 2-oxoglutarate and 2.3 mM for 4-aminobutyrate. Several amino acids, such as alpha,beta-alanine and 2-aminobutyrate, are inhibitors (Ki = 38.7 mM, Ki = 35.5 mM and Ki = 33.2 mM respectively). Propionic and butyric acids are also inhibitors (Ki = 3 mM and Ki = 2 mM).     

Purification and characterization of streptomycin 6-kinase, an enzyme implicated in self-protection of a streptomycin-producing micro-organism

Streptomycin 6-kinase of the streptomycin-producing strain Streptomyces griseus HUT 6037 was purified by fractionation with (NH4)2SO4 and chromatography on DEAE-Sephadex A-25, hydroxyapatite and Sephadex G-100. After PAGE of the final fraction, a protein band corresponding to streptomycin 6-kinase was detected, together with a less intense band having no enzyme activity. Molecular weights determined by SDS-PAGE and by Sephadex G-100 chromatography were about 36000 and 38000, respectively, suggesting that the enzyme was a monomer. The isoelectric point of the enzyme was pH 6.6. Among the nucleoside 5'-triphosphates tested, ATP was the preferred phosphoryl donor. The Km values for streptomycin and ATP were 3.5 mM and 0.4 mM, respectively. The enzyme activity was strongly inhibited by EDTA and AgNO3. It was shown by using an in vitro protein-synthesizing system that purified streptomycin 6-kinase could protect polyphenylalanine synthesis of the streptomycin-susceptible S. griseus strain KSN from inhibition by streptomycin.     

Purification and some kinetic properties of carbonic anhydrase from rainbow trout (Oncorhynchus mykiss) liver and metal inhibition

In the present study, carbonic anhydrase (CA) enzyme was purified from rainbow trout (RT) liver with a specific activity of 4318 EUxmg(-1) and a yield of 38% using Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The overall purification was approximately 2260-fold. To check the purity and determine subunit molecular weight of enzyme, SDS-polyacrylamide gel electrophoresis was performed, which showed a single band and MW of approx. 29.4 kDa. The molecular weight of native enzyme was estimated to be approx. 31 kDa by Sephadex-G 200 gel filtration chromatography. Optimum and stable pH were determined as 9.0 in 1 M Tris-SO(4) buffer and 8.5 in 1 M Tris-SO(4) buffer at 4 degrees C, respectively. The optimum temperature, activation energy (E(a)), activation enthalpy ((DeltaH) and Q(10) from Arrhenius plot for the RT liver CA were 40 degrees C, 2.88 kcal/mol, 2.288 kcal/mol and 1.53, respectively. The purified enzyme had an apparent K(m) and V(max) of 0.66 mM and 0.126 micromol x min(-1) for 4-nitrophenylacetate, respectively. K(cat) of the CA was found to be 32.8 s(-1). The inhibitory effects of low concentrations of different metals (Co(II), Cu(II), Zn(II) and Ag(I)) on CA activity were determined using the esterase method under in vitro conditions. The obtained IC(50) values, 50% inhibition of in vitro enzyme activity, were 0.03 mM for cobalt, 30 mM for copper, 47.1 mM for zinc and 0.01 mM for silver. K(i) values for these substances were also calculated from Linewaever-Burk plots as 0.050 mM for cobalt, 1.950 mM for copper, 7.035 mM for zinc and 2.190 mM for silver respectively and determined that cobalt and zinc inhibit the enzyme a competitive manner and copper and silver inhibit the enzyme in an uncompetitive manner.     

Purification and properties of human serum dopamine-β -hydroxylase

1. Two different molecular forms of dopamine-beta-hydroxylase were isolated from human serum; a major component (Peak I enzyme) with a molecular weight of 368000 and with a higher specific activity and a minor component (Peak II enzyme) with a molecular weight of 188000 and with a lower specific activity. 2. Both forms require ascorbic acid for the activity, and are stimulated by fumarate. Addition of N-ethylmaleimide or copper also increased the activity. The optimal pH of both forms in the presence of 20mM tyramine as substrate is 5.0. 3. Km values toward tyramine of Peak I enzyme and Peak II enzyme were 1.67 mM and 14.2 mM respectively. 4. Both Peak I enzyme and Peak II enzyme are glycoprotein.     

In vitro inhibition of stable 1,3-beta-D-glucan synthase activity from Neurospora crassa.

Glucan synthase activity of Neurospora crassa was isolated by treatment of protoplast lysates with 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, containing 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 200 mM inorganic phosphate, 10 microM GTP, 1 mM DTT, 10 mM sodium fluoride, and 600 mM glycerol. Resulting activity was partially purified by sucrose gradient density sedimentation. Approximately 70% of enzyme activity in the sucrose gradient peak fraction was soluble and enzyme activity was purified 7.3-fold. Partially purified enzyme activity had a half-life of several weeks at 4 degrees C, and a Km(app) of 1.66 +/- 0.28 mM. Inhibitors (Cilofungin, papulacandin B, aculeacin A, echinocandin B, sorbose and UDP) of 1,3-beta-D-glucan synthase activity were tested against crude particulate and detergent treated enzyme fractions and the Ki(app) of each inhibitor determined. It seems likely that this stable preparation of glucan synthase activity may be useful for in vitro enzyme screens for new glucan synthase inhibitors.     

Guanylate cyclase from human blood platelets

Human blood platelets were disrupted by ultrasonication, and the guanylate cyclase activity was determined in the 105,000 g supernatant. The guanylate cyclase preparation obtained in the absence of dithiothreitol (DTT) was characterized by a nonlinear dynamics of cGMP synthesis during incubation at 37 degrees C. The use of 0.2 mM DTT during platelet ultrasonication stabilized the guanylate cyclase reaction and did not influence the enzyme activity. With a rise in DTT concentration up to 2 mM the guanylate cyclase activity diminished. Sodium nitroprusside stimulated the enzyme; this effect was enhanced in the presence of DTT. The maximum guanylate cyclase activity was revealed at 4 mM Mn2+ or Mg2+ and with 1 mM GTP. In the presence of Mn2+ the enzyme activity was higher than with Mg2+. The apparent Km values for GTP in the presence of 4 mM Mn2+ and Mg2+ was 30 and 200 microM, respectively. At GTP/cation ratio of 1:4 the Km values for Mn2+ and Mg2+ were nearly the same (249 and 208 microM, respectively). It was assumed that besides being involved in the formation of the GTP-substrate complex, Mn2+ exerts a strong influence on guanylate cyclase by oxidizing the SH-groups of the enzyme.     

Trypanothione Reductase Activity is Prominent in Metacyclic Promastigotes and Axenic Amastigotes of Leishmania amazonesis. Evaluation of its Potential as a Therapeutic Target

The activity of trypanothione reductase in Leishmania amazonensis was evaluated and it was demonstrated that TR is expressed in the soluble fractions of infective promastigotes and amastigotes, while non-infective promastigotes expressed the enzyme at basal levels. This data allows an association of enzyme activity and the infective capacity of the parasite. We have also previously demonstrated that amidine compounds (N, N′-diphenyl-4-methoxy-benzamidine and pentamidine) were active against this parasite. Here, experiments concerning the effect of these compounds on TR activity, showed that both compounds significantly inhibited the enzyme. However, against glutathione reductase, only pentamidine showed a significant inhibitory action, suggesting an association with the toxic effects of this drug used in the clinic for the treatment of leishmaniasis.     

Tyrosylprotein sulfotransferase in rat submandibular salivary glands.

1. The transfer of sulfate ester group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to poly-(Glu6, Ala3, Tyr1) (EAY; Mr 47 kDa) in rat submandibular salivary gland has been investigated. The highest tyrosylprotein sulfotransferase activity was obtained in the Golgi-enriched fraction in the presence of 2 mM 5'AMP, 20 mM MnCl2 and 50 mM NaF at pH 6.2. 2. The apparent Km values for EAY and PAPS were 1.6 x 10(-6) and 1.9 x 10(-6) M, respectively. 3. Inclusion of NaCl, EDTA, NEM and DTT was inhibitory for the enzyme activity. The enzyme was 28 times less susceptible to 2,6-dichloro-4-nitrophenol inhibition than to phenol sulfotransferase inhibition. 4. This study is the first report characterizing a sulfotransferase activity specific for tyrosylprotein in rat submandibular salivary glands.     

Purification and characterization of intestinal alkaline phosphatase from harp seal

1. Alkaline phosphatase (EC 3.1.3.1.) from harp seal (Phagophilus groenlandicus) has been purified by concanavalin A-Sepharose chromatography to homogeneity with a specific activity of 1200-1500 units/mg of protein. 2. The mol. wt of the enzyme and its subunits were estimated as 260,000 and 70,000, respectively. By chromatofocusing the isoelectric point of this enzyme is 5.5. 3. With p-nitrophenylphosphate, pH-optimum and KM for the enzyme are 9.8 and 0.9 mM, respectively. 4. The enzyme was strongly inhibited by Sn4+, Fe3+ and Zn2+, whereas Mg2+ and Mn2+ were effective activators of the enzyme. Seal alkaline phosphatase was slightly inhibited by high concentrations of Ca2+ and Cr3+. 5. The enzyme activity reached a maximum at 55-60 degrees C. It was shown that the heat stability of seal and calf intestinal alkaline phosphatases were equal at 37 and 56 degrees C.