Evidence on the operation of ATP-induced capacitative calcium entry in breast cancer cells and its blockade by 17beta-estradiol

Little is known about the regulation of cytosolic calcium Ca(2+) levels ([Ca(2+)](i)) in breast cancer cells. We investigated the existence of capacitative calcium entry (CCE) in the tumorigenic cell line MCF-7 and its responsiveness to ATP. MCF-7 cells express purinergic receptors as well as estrogen receptors (ER). Depletion of calcium stores with thapsigargin (TG, 500 nM) or ATP (10 microM) in the absence of extracellular Ca(2+), resulted in a rapid and transient elevation in [Ca(2+)](i). After recovery of basal levels, Ca(2+) readmission (1.5 mM) to the medium increased Ca(2+) influx (twofold over basal), reflecting pre-activation of a CCE pathway. Cells pretreated with TG were unable to respond to ATP, thus indicating that the same Ca(2+) store is involved in their response. Moreover, IP(3)-dependent ATP-induced calcium mobilization and CCE were completely blocked using compound U-73122, an inhibitor of phospholipase C. Compound 2-APB (75 microM) and Gd(3+) (10 microM), antagonists of the CCE pathway, completely prevented ATP-stimulated capacitative Ca(2+) entry. CCE in MCF-7 cells was highly permeable to Mn(2+) and to the Ca(2+) surrogate Sr(2+). Mn(2+) entry sensitivity to Gd(3+) matched that of the Ca(2+) entry pathway. 17Beta-estradiol blocked ATP-induced CCE, but was without effect on TG-induced CCE. Besides, the estrogen blockade of the ATP-induced CCE was completely abolished by preincubation of the cells with an ER monoclonal antibody. ER alpha immunoreactivity could also be detected in a purified plasma membrane fraction of MCF-7 cells. These results represent the first evidence on the operation of a ATP-responsive CCE pathway in MCF-7 cells and also indicate that 17beta-estradiol interferes with this mechanism by acting at the cell surface level.     

The registration of nitric oxide production in mammals using a water-soluble complex of N-methyl-D,L-glucamine dithiocarbamate with Fe3+

The level of nitric oxide production in the intact rabbit organism was studied using the water-soluble complex of Fe3+ with MGD as a selective spin trap for nitric oxide. The Fe(3+)-MGD3 complex was injected intravenously. It was shown by the EPR method that this injection resulted in the formation of paramagnetic complexes in the urine, as Cu(2+)-MGD2, and nitric oxide spin adducts: nitric oxide-Fe(2+)-MGD2 and nitric oxide-Fe(3+)-MGD2. The level of nitric oxide production was estimated by the ratio of the total amount of these adducts to the nitric oxide-Fe(2+)-MGD2 level, formed after the addition of excessive S-nitrosoglutathione. This value for intact animals was 1.33 +/- 0.13%.     

Growth,morphological and biochemical changes in oxa-spermine derivative-treated MCF-7 human breast cancer cells

The growth inhibitory properties of two oxa-spermine derivatives named compound 1 and compound 2, representatives of a novel type of polyamine derivatives, were studied. Dose-response growth inhibitory curves obtained after 48h drug exposure demonstrated the much higher cytotoxic activity of compound 1 towards MCF-7 human breast cancer cells. Further experiments with compound 1 showed that this oxa-spermine derivative exhibited considerable cytotoxicity with IC(50) values of 3.74 microM and 2.93 microM after 24h and 48h drug exposure respectively. In MCF-7 cells, after 8h drug (10 microM) exposure it caused shrinkage, chromatin condensation and nuclear fragmentation. However, no clear DNA laddering was detected in treated cells. Drug treatment provoked an increase in polyamine oxidase (PAO) activity. This enzyme is able to produce cytotoxic H(2)O(2) and 3-acetamidopropanal, catalyzing the oxidative deamination of N(1)-acetylated derivatives of spermine and spermidine to spermidine and putrescine respectively. Taken together these data demonstrate that the novel oxa-polyamine derivative compound 1 has considerable cytotoxic activity towards MCF-7 cells and indicate that an induction of PAO may be involved in its cytotoxic and apoptotic effects.     

Anti-microbial action against verotoxigenic Escherichia coli O157:H7 of nitric oxide derived from sodium nitrite

The levels of verotoxin-1 and verotoxin-2 released by verotoxigenic Escherichia coli O157:H7 treated in vitro with sodium nitrite, sodium chloride and several antibiotics were evaluated. Of the three strains of E. coli O157:H7 used in this study, two strains produced both verotoxin-1 and verotoxin-2, and one strain produced only verotoxin-2. Treatment of E. coli O157:H7 with sodium nitrite (6000 mg/l, minimum inhibitory concentration) did not increase the levels of verotoxin-1 and verotoxin-2 compared with a treatment by sodium chloride or antibiotics. When the electron paramagnetic resonance spectrum of sodium nitrite-treated bacterial cells was examined at 77 K to clarify the mechanism for the anti-bacterial activity of nitric oxide derived from sodium nitrite, electron paramagnetic resonance signals with g-values of 2.035 and 2.010 were observed. These were identified as being derived from iron-nitric oxide complexes. It appears that the dinitrosyl iron complexes in the E. coli O157:H7 cells were generated from the reaction of iron-sulfur proteins (enzymes) with nitric oxide formed by the reduction of sodium nitrite. The amount of ATP was decreased by the presence of sodium nitrite in the cell suspension. These findings indicate that nitric oxide derived from sodium nitrite penetrated the cells and inactivated enzymes related to the respiratory chain.     

Acute toxicity of cadmium and copper in hepatopancreas cells from the Roman snail (Helix pomatia)

The toxic effects of cadmium (Cd) and copper (Cu) on cellular metabolism and cell morphology were investigated in isolated hepatopancreas cells from the Roman snail (Helix pomatia). Cell viability was unaffected during 1 h of incubation with 100 microM Cd, but was significantly reduced from 93% in controls to 87% and 85% with 100 microM Cu and 500 microM Cd, respectively. The adverse effect of 500 microM Cd on cell viability was not observed in cells isolated from Cd pretreated snails. Oxygen consumption remained constant in the presence of 100 microM Cu but was inhibited by 38% after 1 h of exposure to 500 microM Cd. Hepatopancreas cells showed enhanced formation of reactive oxygen species when exposed to 100 microM Cu, but not in the presence of Cd. Morphologically, an increase in cell volume of Cd-exposed cells was noted, while cell membrane bleb formation was induced by both metals. The latter may have been induced by metal effects on the actin filamentous network of the cells which showed distinct actin-staining within the blebs at the cell surface. Overall, our data indicate that both Cd and Cu are acutely toxic for hepatopancreas cells form the Roman snail with Cu being more toxic than Cd.     

Why does iron abrogate the cytotoxic effect of S-nitrosothiols on human and animal cultured cells?

It was found that dinitrosyl iron complexes (DNIC) with thiol-containing ligands (cysteine or glutathione) of concentrations up to 1 mM produce no cytotoxic effect on cultured cells from human milk gland carcinoma (MCF-7). The cytotoxic action on MCF-7 cells was produced by S-nitrosocysteine: at a concentration of 1 mM, it induced the death of 50% cells. A more stable S-nitrosothiol, S-nitrosoglutathione, did not produce any cytotoxic effect at the same concentration. It is assumed that the negative action of nitrosocysteine is due to its rapid degradation, which results in the accumulation of large amounts of free NO molecules followed by their oxidation by superoxide ions to peroxynitrite, an efficient inhibitor of metabolic processes. These processes seem to be not characteristic of the more stable S-nitrosoglutathione. The cytotoxic effect of nitrosocysteine was completlly abrogated by the addition of 0.2 mM ferrous citrate complex to the medium. When S-nitrosoglutathione NO (0.5 mM) or S-nitrosoglutathione (0.5 mM) + Fe(2+)-citrate (0.2 mM) were added to the medium, protein-bound dinitrosyl iron complexes formed with the involvement of endogenous or exogenous iron were detected in cells. The amount of the complexes in the presence of exogenous iron increased four times, reaching the value of 1.6 nmole/5 x 10(6) cells. Therefore, it was proposed that the blockade of the cytotoxic action of S-nitrosoglutathione by iron complexes is due to Cys-NO transformation of S-nitrosocysteine into dinitrosyl iron complexes. The high stability of these complexes ensures only a gradual accumulation of nitric oxide in cells.     

Copper modifies liver microsomal UDP-glucuronyltransferase activity through different and opposite mechanisms

Treatment of hepatic microsomes with Fe(3+)/ascorbate activates UDP-glucuronyltransferase (UGT), a phenomenon totally prevented and reversed by reducing agents. At microM concentrations, iron and copper ions catalyze the formation of ROS through Fenton and/or Haber-Weiss reactions. Unlike iron ions, indiscriminate binding of copper ions to thiol groups of proteins different from the specialized copper-binding proteins may occur. Thus, we hypothesize that incubation of hepatic microsomes with the Cu(2+)/ascorbate system will lead to both UGT oxidative activation and Cu(2+)-binding induced inhibition, simultaneously. We studied the effects of Cu(2+) alone and in the presence of ascorbate on rat liver microsomal UGT activity. Our results show that the effects of both copper alone and in the presence of ascorbate were copper ion concentration- and incubation time-dependent. At very low Cu(2+) (25nM), this ion did not modify UGT activity. In the presence of ascorbate, however, UGT activity was increased. At higher copper concentrations (10 and 50microM), this ion led to UGT activity inhibition. In the presence of ascorbate, 10microM Cu(2+) activated UGT at short incubation periods but inhibited this enzyme at longer incubation times; 50microM Cu(2+) only inhibited UGT activity. Thiol reducing agent 2,4-dithiothreitol prevented and reversed UGT activation while EDTA prevented both, UGT activation and inhibition. Our results are consistent with a model in which Cu(2+)-induced oxidation of UGT leads to the activation of the enzyme, while Cu(2+)-binding leads to its inhibition. We discuss physiological and pathological implications of these findings.     

Effect of timosaponin A-III,from Anemarrhenae asphodeloides Bunge (Liliaceae), on calcium mobilization in vascular endothelial and smooth muscle cells and on vascular tension

The effects of timosaponin A-III (TA-III), from Rhizoma Anemarrhenae, on Ca(2+) mobilization in vascular endothelial cells and smooth muscle cells and on vascular tension have been explored. TA-III increased intracellular Ca(2+) concentrations ([Ca(2+)](i)) in endothelials cells at a concentration larger than 5 microM with an EC(50) of 15 microM, and increased [Ca(2+)](i) in smooth muscle cells at a concentration larger than 1 microM with an EC(50) of 8 microM. Within 5 min, the [Ca(2+)](i) signal was composed of a gradual rise, and the speed of rising depended on the concentration of TA-III. The [Ca(2+)](i) signal was abolished by removing extracellular Ca(2+) and was recovered after reintroduction of Ca(2+). The TA-III-induced [Ca(2+)](i) increases in smooth muscle cells were partly inhibited by 10 microM nifedipine or 50 microM La(3+), but was insensitive to 10 microM verapamil and diltiazem. TA-III (10-100 microM) inhibited 0.3 microM phenylephrine-induced vascular contraction, which was abolished by pretreatment with 100 microM N(omega)-nitro-L-arginine (L-NNA) or by denuding the aorta. TA-III also increased [Ca(2+)](i) in renal tubular cells with an EC(50) of 8 microM. Collectively, the results show for the first time that TA-III causes [Ca(2+)](i) increases in the vascular system. TA-III acted by causing Ca(2+) influx without releasing intracellular Ca(2+). TA-III induced relaxation of phenylephrine-induced vascular contraction via inducing release of nitric oxide from endothelial cells.     

Mechanism of non-enzymic transamination reaction between histidine and α-oxoglutaric acid

Non-enzymic transamination reactions at 85 degrees between various amino acids and alpha-oxoglutaric acid are catalysed by metal ions, e.g. Al(3+), Fe(2+), Cu(2+) and Fe(3+). The reaction is optimum at pH4.0. Of the 14 amino acids studied histidine is the most active. In the presence of Al(3+) histidine transaminates with alpha-oxoglutaric acid, forming glutamic acid and Al(3+)-imidazolylpyruvic acid complex as the end products. However, in the presence of Fe(2+) or Cu(2+) the products are glutamic acid and a 1:2 metal ion-imidazolylpyruvic acid chelate. The greater effectiveness of histidine in these reactions is attributed to the presence of the tertiary imidazole nitrogen atom, which is involved in the formation of stable sparingly soluble metal ion-imidazolylpyruvic acid complexes or chelates as end products of these reactions. Of the metal ions studied only Al(3+), Fe(2+), Fe(3+) and Cu(2+) are effective catalysts for the transamination reactions, and EDTA addition completely inhibits the catalytic effect of the Al(3+). Spectrophotometric evidence is presented to demonstrate the presence of metal ion complexes of Schiff bases of histidine as intermediates in the transamination reactions. These results may contribute to understanding the role of histidine in enzyme catalysis.     

Metal toxicity in Chlamydomonas reinhardtii. Effect on sulfate and nitrate assimilation

Cadmium (Cd(2+)) or copper (Cu(2+)) ions are toxic for Chlamydomonas reinhardtii growth, at 300 microM, and the alga may accumulate about 0.90+/-0.02 and 0.64+/-0.02% of its dry weight, respectively. Metal contamination changes the elemental composition of dried alga biomass, which indicates the possibility to use C. reinhardtii as biosensor and bioremediator of the aquatic contamination by heavy metals. Either, Cd(2+) or Cu(2+), inhibits about 20% of the nitrate consumption rate by the cells, while only Cd(2+) increases about 40% the sulfate consumption rate. The presence of 1 mM calcium (Ca(2+)) in the culture medium increases the C. reinhardtii productivity (about 50%), the nitrate uptake rate (about 20%) and the sulfate uptake rate (about 30%). In addition, Ca(2+) overcomes the Cd(2+) (300 microM) toxicity by decreasing (about 35%) the intracellular accumulation of metal. Sulfur-starvation induces in C. reinhardtii the expression of serine acetyltransferase and O-acetylserine(thiol)lyase activities, but decreases 50% the consumption rate of nitrate by the cells. Sulfate is also required for the full expression of the nitrate reductase (NR), nitrite reductase (NiR) and glutamate synthase activities.     

The regulation of oestrone sulphate formation in breast cancer cells.

The formation of oestrone sulphate has been examined in MCF-7 (oestrogen receptor positive, ER+) and MDA-MB-231 (ER negative, ER-) breast cancer cells. Using intact cell monolayers and a physiological substrate concentration, progesterone (1 microM) and dexamethasone (1 microM) both increased oestrone sulphate formation in MCF-7 cells. In MDA-MB-231 cells, dexamethasone, but not progesterone, increased conjugate formation. A number of growth factors, cytokines and human serum albumin (HSA), which have previously been found to regulate oestrogen synthesis, were also examined for their ability to regulate oestrone sulphate formation. In MCF-7 cells epidermal growth factor, acidic and basic fibroblast growth factors, insulin-like growth factor-type I and insulin all stimulated oestrone sulphate formation. The cytokines, tumour necrosis factor alpha (TNFalpha) and interleukin-1beta also increased conjugate formation in the ER+ cells, as did HSA. In contrast, in MDA-MB-231 cells TNFalpha was without effect and HSA inhibited oestrone sulphate formation. The ability to modulate oestrone sulphate formation in ER+ cells may be an important mechanism to limit the availability of oestrogen to interact with the ER.     

The influence of opioids on matrix metalloproteinase-2 and -9 secretion and mRNA levels in MCF-7 breast cancer cell line

Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in degradation of extracellular matrix, a process that initiates uncontrolled spread of proliferating cancer cells and therefore plays a crucial role in cancer invasion and metastasis. Compounds able to modulate MMP activity may become important tools in cancer research. In the present study we examined the effect of two μ-selective opioids, morphine and endomorphin-2 (EM-2) on the production of MMP-2 and MMP-9 in MCF-7 cells. We report that both opioids time- and concentration-dependently inhibited the expression and secretion of these MMPs. The observed effect was not reversed by naloxone (Nal). Further experiments showed that morphine and EM-2 decreased endothelial nitric oxide synthase (eNOS) mRNA level and nitric oxide (NO) secretion in MCF-7 cells. These findings indicate that attenuation of MMP secretion by opioids was not mediated by opioid receptors but was under the control of nitric oxide system.     

Store-operated calcium entry in vascular endothelial cells is inhibited by cGMP via a protein kinase G-dependent mechanism

Store-operated Ca(2+) entry in vascular endothelial cells not only serves to refill the intracellular Ca(2+) stores, but also acts to stimulate the synthesis of nitric oxide, a key vasodilatory factor. In this study, we examined the role of cGMP in regulating the store-operated Ca(2+) entry in aortic endothelial cells. Cyclopiazonic acid (CPA) and thapsigargin, two selective inhibitors of endoplasmic reticulum Ca(2+)-ATPase, were used to induce store-operated Ca(2+) entry. 8-Bromo-cGMP, an activator of protein kinase G, inhibited the CPA- or thapsigargin-induced Ca(2+) entry in a concentration-dependent manner. An inhibitor of protein kinase G, KT5823 (1 microM) or H-8 (10 microM), abolished the inhibitory action of 8-bromo-cGMP and resumed Ca(2+) entry. Addition of S-nitroso-N-acetylpenicillamine (a nitric oxide donor) or dipyridamole (a cGMP phosphodiesterase inhibitor) during CPA treatment elevated cellular cGMP levels, stimulated protein kinase G activity, and at the same time reduced Ca(2+) influx due to CPA. Patch clamp study confirmed the existence of a CPA-activated Ca(2+)-permeable channel sensitive to cGMP inhibition. These results suggest that cGMP via a protein kinase G-dependent mechanism may play a key role in the regulation of the store-operated Ca(2+) entry in vascular endothelial cells.     

NO对金丝桃悬浮细胞生长及金丝桃素生物合成的促进作用研究

一氧化氮 (NO)是近年来发现的一种新型植物信号分子。以硝普钠 (Sodiumnitroprusside ,SNP)为一氧化氮 (NO)的供体 ,研究外源NO对金丝桃悬浮细胞生长及金丝桃素生物合成的影响。试验结果表明 ,金丝桃悬浮细胞在含 0 5和 15 0mmol LSNP的培养基中培养 2 0d后 ,细胞的干重分别为对照组的 140%和50% ;细胞中金丝桃素的含量分别为对照组的 98%和210%。试验结果表明 ,低浓度SNP处理有利于金丝桃悬浮细胞生长 ,而高浓度SNP可以促进金丝桃素的合成。在细胞培养初期 (0d)加入 0.5mmol LSNP并在指数生长后期 (14d)加入15.0mmol LSNP的金丝桃悬浮细胞在培养 2.5d后 ,细胞的干重和金丝桃素的含量分别为对照组的1.4和1.8倍 ,金丝桃素的产量达15.2mg/L ,比对照高3.2倍。SNP对金丝桃悬浮细胞生长及金丝桃素含量的影响可以被NO专一性淬灭剂CPITO(2-4-carboxyphenyl-4 ,4 ,5 ,5-tetramethylimidazoline-1-oxyl-3-oxide)所抑制,说明SNP是通过其分解产物NO影响细胞生长和金丝桃素的合成。试验结果同时表明,在15.0mmol/L的SNP处理下,金丝桃悬浮细胞中的苯丙氨酸解氨酶(PAL)的活性显著升高,推测NO可能通过触发金丝桃悬浮细胞的防卫反应,激活了细胞中金丝桃素的生物合成途径。     

Cyclooxygenase-2 directly regulates gene expression of P450 Cyp19 aromatase promoter regions pII, pI.3 and pI.7 and estradiol production in human breast tumor cells

The present studies evaluated the direct effects of the presence of human cyclooxygenase-2 (Cox-2) on gene expression of specific promoter regions of the P450 Cyp19 enzyme aromatase enzyme and its product, estradiol, in Cox-2 null estrogen-dependent MCF-7 breast tumor cells and in a stable clone of MCF-7 cells containing transfected Cox-2 cDNA, designated as MCF-7/Cox-2 Clone 10. Clone 10 human breast tumor cells have significantly increased gene expression of total mRNA of the P450 Cyp19 enzyme aromatase, with high levels of gene expression of specific aromatase promoter (p) regions pII, pI.3, and p1.7, with no significant change in mRNA levels of p1.4. Clone 10 human breast tumor cells produced significantly increased amounts of both prostaglandin E2 (PGE2) derived from Cox-2 enzyme activity and estradiol derived from aromatase enzyme activity (p<0.01), compared to MCF-7/vector control cells. The greatest inhibition of PGE2 or estradiol production was observed by the combination of the selective Cox-2 inhibitor celecoxib (25 microM) and the aromatase inhibitor, formestane (10nM) (p<0.01). The greatest anti-proliferative effect in Cox-2 null MCF-7/vector control cells was observed with the combination of 25 microM celecoxib and 10nM formestane but not with 10 microM celecoxib, suggesting that there are Cox-2-independent mechanisms involved in the anti-proliferative effect of this agent at doses greater than 10 microM. Celecoxib (25 microM) also significantly inhibited proliferation of MCF-7/Cox-2 Clone 10 human breast tumor cells, with no further anti-proliferative activity with the addition of 10 nM formestane observed at either 24 or 48 h of treatment. These studies demonstrate that Cox-2 directly regulates gene expression of specific aromatase promoter regions and regulates aromatase enzyme activity. Agents that inhibit Cox-2 or block the biological effects of PGE2 may be useful in significantly limiting aromatase activity and proliferation of human breast tumor cells regardless of the presence of Cox-2. In addition, the unique human breast tumor cell model used in these studies may be a useful tool in identifying the spectrum of activities of agents that block the biological effects of PGE2 and estradiol.     

Heregulinbeta activates store-operated Ca2+ channels through c-erbB2 receptor level-dependent pathway in human breast cancer cells

The heregulinbeta (HRGbeta) is a ligand to activate c-erbB2/c-erbB3 interaction and can subsequently increases cytosolic [Ca(2+)](i). In the two human breast cancer cell lines, MCF-7 shows a low c-erbB2 expression level, whereas SK-BR-3 overexpress c-erbB2 receptor. In this article, we have found that in MCF-7, HRGbeta induced Ca(2+) release from the endoplasmic reticulums (ER) and subsequently activated Ca(2+) entry via store-operated Ca(2+) channel (SOC). However, in SK-BR-3, HRGbeta failed to induce Ca(2+) release and Ca(2+)entry. RNA interference to decrease c-erbB2 level in SK-BR-3 resulted in reactivation of HRGbeta-evoked Ca(2+) release and Ca(2+) entry via SOC, which was similar to that of MCF-7. In addition, in the absence of HRGbeta, a constitutive activation of SOC was observed in SK-BR-3 rather than in MCF-7 and c-erbB2-siRNA treated SK-BR-3. Compared to the cells with low c-erbB2 level, c-erbB2 might tend to interact with c-erbB3 in the resting state in the cells with high c-erbB2 level, which resulted in different [Ca(2+)](i) responses to HRGbeta. In SK-BR-3, the Ca(2+) mobilization in the presence or in the absence of HRGbeta was completely blocked by PLC inhibitor U73122. In summary, our results indicate that HRGbeta-induced SOC was regulated by c-erbB2 level and dependent on activation of PLC in human breast cancer cells.     

Ionomycin, a carboxylic acid ionophore, transports Pb(2+) with high selectivity

Studies utilizing phospholipid vesicle loaded with chelator/indicators for polyvalent cations show that ionomycin transports divalent cations with the selectivity sequence Pb(2+) > Cd(2+) > Zn(2+) > Mn(2+) > Ca(2+) > Cu(2+) > Co(2+) > Ni(2+) > Sr(2+). The selectivity of this ionophore for Pb(2+) is in contrast to that observed for A23178 and 4-BrA23187, which transport Pb(2+) at efficiencies that are intermediate between those of other cations. When the selectivity difference of ionomycin for Pb(2+) versus Ca(2+) was calculated from relative rates of transport, with either cation present individually and all other conditions held constant, a value of approximately 450 was obtained. This rose to approximately 3200 when both cations were present and transported simultaneously. 1 microM Pb(2+) inhibited the transport of 1 mM Ca(2+) by approximately 50%, whereas the rate of Pb(2+) transport approached a maximum at a concentration of 10 microM Pb(2+) when 1 mM Ca(2+) was also present. Plots of log rate versus log ionomycin or log Pb(2+) concentration indicated that the transporting species is of 1:1 stoichiometry, ionophore to Pb(2+), but that complexes containing an additional Pb(2+) may occur. The species transporting Pb(2+) may include H.IPb.OH, wherein ionomycin is ionized once and the presence of OH(-) maintains charge neutrality. Ionomycin retained a high efficiency for Pb(2+) transport in A20 B lymphoma cells loaded with Indo-1. Both Pb(2+) entry and efflux were observed. Ionomycin should be considered primarily as an ionophore for Pb(2+), rather than Ca(2+), of possible value for the investigation and treatment of Pb(2+) intoxication.     

Differential effect of copper (II) on the cytochrome P450 enzymes and NADPH-cytochrome P450 reductase: inhibition of cytochrome P450-catalyzed reactions by copper (II) ion

Inhibitory effects of Cu(2+) on the cytochrome P450 (P450)-catalyzed reactions of liver microsomes and reconstituted systems containing purified P450 and NADPH-P450 reductase (NPR) were seen. However, Zn(2+), Mg(2+), Mn(2+), Ca(2+), and Co(2+) had no apparent effects on the activities of microsomal P450s. Cu(2+) inhibited the reactions catalyzed by purified P450s 1A2 and 3A4 with IC(50) values of 5.7 and 8.4 microM, respectively. Cu(2+) also inhibited reduction of cytochrome c by NPR (IC(50) value of 5.8 microM). Copper caused a decrease in semiquinone levels of NPR, although it did not disturb the rate of formation of semiquinone. P450 reactions supported by an oxygen surrogate, tert-butyl hydroperoxide, instead of NPR and NADPH, were inhibited by the presence of Cu(2+). The results indicate that Cu(2+) inhibits the P450-catalyzed reactions by affecting both P450s and NPR. It was also found that the inhibition of catalytic activities of P450s by Cu(2+) involves overall conformational changes of P450s and NPR, investigated by CD and intrinsic fluorescence spectroscopy. These results suggest that the inhibitory effect of Cu(2+) on the P450-catalyzed reactions may come from the inability of an efficient electron transfer from NPR to P450 and also the dysfunctional conformation of NPR and P450.     

Nordihydroguaiaretic acid (NDGA), an inhibitor of the HER2 and IGF-1 receptor tyrosine kinases, blocks the growth of HER2-overexpressing human breast cancer cells

We have reported that nordihydroguaiaretic acid (NDGA) inhibits the tyrosine kinase activities of the IGF-1 receptor (IGF-1R) and the HER2 receptor in breast cancer cells. Herein, we studied the effects of NDGA on the growth of estrogen receptor (ER) positive MCF-7 cells engineered to overexpress HER2 (MCF-7/HER2-18). These cells are an in vitro model of HER2-driven, ER positive, tamoxifen resistant breast cancer. NDGA was equally effective at inhibiting the growth of both parental MCF-7 and MCF-7/HER2-18 cells. Half maximal effects for both cell lines were in the 10-15 microM range. The growth inhibitory effects of NDGA were associated with an S phase arrest in the cell cycle and the induction of apoptosis. NDGA inhibited both IGF-1R and HER2 kinase activities in these breast cancer cells. In contrast, Gefitinib, an epidermal growth factor receptor inhibitor but not an IGF-1R inhibitor, was more effective in MCF-7/HER2-18 cells than in the parental MCF-7 cells and IGF binding protein-3 (IGFBP-3) was more effective against MCF-7 cells compared to MCF-7/HER2-18. MCF-7/HER2-18 cells are known to be resistant to the effects of the estrogen receptor inhibitor, tamoxifen. Interestingly, NDGA not only inhibited the growth of MCF-7/HER2-18 on its own, but it also demonstrated additive growth inhibitory effects when combined with tamoxifen. These studies suggest that NDGA may have therapeutic benefits in HER2-positive, tamoxifen resistant, breast cancers in humans.