Does reductive metabolism predict response to tirapazamine (SR 4233) in human non-small-cell lung cancer cell lines?
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The bioreductive drug tirapazamine (TPZ, SR 4233, WIN 59075) is a lead compound in a series of potent cytotoxins that selectively kill hypoxic rodent and human solid tumour cells in vitro and in vivo. Phases II and III trials have demonstrated its efficacy in combination with both fractionated radiotherapy and some chemotherapy. We have evaluated the generality of an enzyme-directed approach to TPZ toxicity by examining the importance of the one-electron reducing enzyme NADPH:cytochrome P450 reductase (P450R) in the metabolism and toxicity of this lead prodrug in a panel of seven human non-small-cell lung cancer cell lines. We relate our findings on TPZ sensitivity in these lung lines with our previously published results on TPZ sensitivity in six human breast cancer cell lines (Patterson et al (1995) Br J Cancer 72: 1144-1150) and with the sensitivity of all these cell types to eight unrelated cancer chemotherapeutic agents with diverse modes of action. Our results demonstrate that P450R plays a significant role in the activation of TPZ in this panel of lung lines, which is consistent with previous observations in a panel of breast cancer cell lines (Patterson et al (1995) Br J Cancer 72: 1144-1150; Patterson et al (1997) Br J Cancer 76: 1338-1347). However, in the lung lines it is likely that it is the inherent ability of these cells to respond to multiple forms of DNA damage, including that arising from P450R-dependent TPZ metabolism, that underlies the ultimate expression of toxicity. (+info)
Afferent arteriolar adenosine A2a receptors are coupled to KATP in in vitro perfused hydronephrotic rat kidney.
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Adenosine is known to exert dual actions on the afferent arteriole, eliciting vasoconstriction, by activating A1 receptors, and vasodilation at higher concentrations, by activating lower-affinity A2 receptors. We could demonstrate both of these known adenosine responses in the in vitro perfused hydronephrotic rat kidney. Thus, 1.0 microM adenosine elicited a transient vasoconstriction blocked by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and 10-30 microM adenosine reversed KCl-induced vasoconstriction. However, when we examined the effects of adenosine on pressure-induced afferent arteriolar vasoconstriction, we observed a third action. In this setting, a high-affinity adenosine vasodilatory response was observed at concentrations of 10-300 nM. This response was blocked by both 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3, 5]triazin-5-yl-amino]ethyl)phenol (ZM-241385) and glibenclamide and was mimicked by 2-phenylaminoadenosine (CV-1808) (IC50 of 100 nM), implicating adenosine A2a receptors coupled to ATP-sensitive K channels (KATP). Like adenosine, 5'-N-ethylcarboxamidoadenosine (NECA) elicited both glibenclamide-sensitive and glibenclamide-insensitive vasodilatory responses. The order of potency for the glibenclamide-sensitive component was NECA > adenosine = CV-1808. Our findings suggest that, in addition to the previously described adenosine A1 and low-affinity A2b receptors, the renal microvasculature is also capable of expressing high-affinity adenosine A2a receptors. This renal adenosine receptor elicits afferent arteriolar vasodilation at submicromolar adenosine levels by activating KATP. (+info)
Cross talk between A(1) and A(2A) adenosine receptors in the hippocampus and cortex of young adult and old rats.
(35/1501)
Adenosine modulates synaptic transmission by acting on inhibitory A(1) and facilitatory A(2A) receptors, the densities of which are modified in aged animals. We investigated how A(2A) receptor activation influences A(1) receptor function and whether this interaction is modified in aged rats. In hippocampal and cortical nerve terminals from young adult (6 wk), but not old rats (24 mo), the A(2A) receptor agonist, 2-[4-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680; 30 nM) decreased the binding affinity of a selective A(1) receptor agonist, cyclopentyladenosine (CPA), an effect prevented by the A(2A) antagonist, (4-(2-[7-amino-2-(2-furyl (1,2,4)-triazolo(2,3-a (1,3,5)triazin-5-yl-aminoethyl)phenol (ZM 241385, 20 nM). This effect of CGS 21680 required intact nerve terminals and was also observed in the absence of Ca(2+). This A(2A)-induced "desensitization" of A(1) receptors was prevented by the protein kinase C inhibitor, chelerythrine (6 microM), and was not detected in the presence of the protein kinase C activator, phorbol-12,13-didecanoate (250 nM), which itself caused a reduction in binding affinity for CPA. The protein kinase A inhibitor, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (10 microM), and the protein kinase A activator, 8-Br-cAMP (1 mM), had no effects on the A(2A)-induced A(1) receptor desensitization. This A(2A)-induced A(1) receptor desensitization had a functional correlation because CGS 21680 (10 nM) attenuated by 40% the inhibition caused by CPA (10 nM) on CA1 area population spike amplitude in hippocampal slices. This A(2A)/A(1) interaction may explain the attenuation by adenosine deaminase (2 U/ml), which removes tonic A(1) inhibition, of the facilitatory effect of CGS 21680 on synaptic transmission. The requirement of tonic A(1) receptor activation for CGS 21680 to induce facilitation of synaptic transmission was reinforced by the observation that the A(1) receptor antagonist, 1, 3-dipropyl-8-cyclopentylxanthine (20 nM) prevented CGS 21680 (10 nM) facilitation of population spike amplitude. The present results show the ability of A(2A) receptors to control A(1) receptor function in a manner mediated by protein kinase C, but not protein kinase A, in young adult but not in aged rats. (+info)
The hypoxic cell: a target for selective cancer therapy--eighteenth Bruce F. Cain Memorial Award lecture.
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It has been appreciated for more than 50 years that very low levels of oxygenation, or hypoxia, both protect cells from killing by X-irradiation and are present in solid tumors but not in normal tissues. Until recently, however, there has been no definitive proof that hypoxia in human tumors contributes to radiotherapy treatment failure. We now know that hypoxia in solid tumors is not only a major problem for radiation therapy but also leads to resistance to most anticancer drugs and, importantly, appears to accelerate malignant progression and increase metastasis. To date, efforts to overcome the problem of hypoxia have had only limited success. However, the recent development of new drugs that are nontoxic until they are activated in the hypoxic cell opens a new era. The first of these new drugs to be tested clinically, tirapazamine, a drug that is highly toxic to hypoxic but not aerobic cells, has already demonstrated efficacy in selective potentiation of cisplatin in randomized Phase III trials with non-small cell lung cancer. The unique presence of hypoxic cells in human tumors provides an important target for selective cancer therapy. (+info)
Use of the triazolotriazine [3H]ZM 241385 as a radioligand at recombinant human A2B adenosine receptors.
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Radiolabeled ZM 241385 (4-(2-[7-amino-2- inverted question markfuryl inverted question mark inverted question mark1,2,4 inverted question marktriazolo inverted question mark2,3-a inverted question mark inverted question mark1,3,5 inverted question marktriazin-5-ylaminoethyl)p henol), has previously been used as a high affinity radioligand for the labeling of A2A adenosine receptors in cell membranes. Another subtype, the A2B receptor, is the least well-defined subtype of adenosine receptors and lacks selective pharmacological probes. In the present study, we have used [3H]ZM 241385 as a radioligand to label recombinant human A2B adenosine receptors in HEK-293 cell membranes, that do not express A2A adenosine receptors, and found that the pharmacological profile is consistent with the SAR of A2B receptors. Saturable, specific binding (Kd 33.6 nM, Bmax 4.48 pmol/mg protein) that was best described by a one-site model was found, and specific binding was approximately 75% of total binding. [3H]ZM 241385 binding was displaceable by a large number of compounds known to interact with A2B receptors; thus, this method has promise as a tool in the search for agonists and antagonists selective for this subtype. Xanthine analogs, which are antagonists, proved to be the most potent displacers. The Ki of XAC, xanthine amine congener, was 12.3 nM, while CPX (8-cyclopentyl-1,3-dipropylxanthine) was less potent. The non-selective triazoloquinazoline antagonist CGS 15943 (Ki 16.4 nM), which is similar in structure to ZM 241385, was slightly less potent than XAC. The non-xanthine A2B-antagonist alloxazine displaced [3H]ZM 241385-binding with a Ki of 462 nM, similar to its affinity in functional assays. Adenosine derivatives known to activate this receptor subtype, such as NECA (5'-N-ethylcarboxamidoadenosine) and R-PIA (N6-phenylisopropyladenosine), were considerably less potent than the 8-substituted xanthines examined. (+info)
Identification and assay of lamotrigine in human milk with gas chromatography and densitometry.
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Conditions necessary for isolation and assay of lamotrigine in the human milk with gas chromatography in the range of concentrations 2-20 micrograms/ml, and densitometry in the range of 2-10 micrograms/ml are discussed. Lamotrigine was extracted with methylene chloride from the alkaline solution. In gas chromatography capillary column DB-17, flame-ionizing detector, and octacozanole as the internal standard have been used. Densitometric measurements have been performed at 217 nm. (+info)
Influence of tension time on muscle fiber sarcolemmal injury in rat diaphragm.
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We hypothesized that the amount of sarcolemmal injury is directly related to the total tension time (TT(tot)), calculated as mean tension x total stimulation time. Diaphragm strips from Sprague-Dawley rats were superfused at optimal muscle length with Krebs containing procion orange to identify sarcolemmal injury. TT(tot) was induced by stimulation with 100 Hz for 3 min at duty cycles of 0.02, 0.15, 0.3, and 0.6, or with continuous contractions at 0.2, 0.4, 0.6, and 1.0 of maximal tension. A significant positive correlation between TT(tot) and the percentage of fibers with injured sarcolemma (r(2) = 0.63, P < 0.05) is seen. Stimulation (at 100 Hz, duty cycle = 1) resulted in fast fatigue with low injury, likely caused by altered membrane conductivity. Stimulations inducing the largest injury are those showing progressive force loss and high TT(tot), where injury may be due to activation of membrane degradative enzymes. The maximal tension measured at 20 min poststimulation was inversely related to the number of fibers injured, suggesting loss of force is caused by cellular injury. (+info)
Role of adenosine A(2B) receptors in vasodilation of rat pial artery and cerebral blood flow autoregulation.
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This study was aimed to investigate the underlying mechanism of vasodilation induced by the activation of A(2B) adenosine receptors in relation to cerebral blood flow (CBF) autoregulation. Changes in pial arterial diameters were observed directly through a closed cranial window. N(omega)-nitro-L-arginine methyl ester (L-NAME, nitric oxide synthase inhibitor) significantly suppressed the concentration-dependent vasodilations induced by adenosine and 5'-N-ethylcarboxamido-adenosine (NECA) but not the vasodilation by CGS-21680 (A(2A)-receptor agonist). Moreover, NECA-induced vasodilation was suppressed by alloxazine (1 micromol/l) but not by ZM-241385 (1 micromol/l, A(2A) antagonist), which suggests mediation by A(2B)- receptor activation. Otherwise, the level of nitrite/nitrate was concentration dependently increased in the artificial cerebrospinal fluid (CSF) when adenosine and NECA were suffused over the cortical surface. L-NAME and alloxazine, but not ZM-241385, largely inhibited their releases. The lower limit of CBF autoregulation was little affected following pretreatment with L-NAME or alloxazine. Thus it is suggested that adenosine-induced vasodilation via activation of A(2B)-adenosine receptors of the rat pial artery is coupled to the production of nitric oxide, which contributes little to CBF autoregulation. (+info)