Minimal contribution of desmethyl-gefitinib, the major human plasma metabolite of gefitinib, to epidermal growth factor receptor (EGFR)-mediated tumour growth inhibition

Desmethyl-gefitinib is a major metabolite of gefitinib observed in human plasma at concentrations similar to those of gefitinib. The epidermal growth factor receptor (EGFR)-related inhibitory effects of gefitinib and desmethyl-gefitinib have been compared both in vitro, using enzyme kinase assays and tumour cell growth inhibition, and in vivo by assessment of tumour xenografts growth inhibition in the mouse. Both gefitinib (IC(50) = 0.022 microM) and its desmethyl metabolite (0.036 microM) inhibited subcellular EGFR tyrosine kinase activity with a similar potency and selectivity. However, desmethyl-gefitinib (IC(50) = 0.76 microM) was 15 times less active than gefitinib (0.049 microM) against EGF-stimulated KB cell growth in a whole cell assay. Following a preliminary pharmacokinetic study to compare apparent oral bioavailability, gefitinib (75 mg kg(-1)) and desmethyl-gefitinib (150 mg kg(-1)) were administered orally for 15 days to female nude mice bearing LoVo tumour xenografts. Tumour concentrations of gefitinib (AUC = 300 microg h g(-1)) were much higher than those of desmethyl-gefitinib (44.3 microg h g(-1)), although plasma concentrations of gefitinib (48.4 microg h ml(-1)) and desmethyl-gefitinib (39.0 microg h ml(-1)) were quite similar at these dose levels. Gefitinib produced significant tumour growth inhibition throughout the course of the study ultimately resulting in a 50% decrease (compared with controls) by day 15. In contrast, although present at comparable plasma levels, desmethyl-gefitinib had little effect on tumour growth and is, therefore, considered unlikely to contribute significantly to the therapeutic activity of gefitinib in the clinical situation.     

HL-37, a novel anthracene derivative, induces Ca(2+)-mediated apoptosis in human breast cancer cells

HL-37, a novel anthracene derivative, exhibited potent anticancer activity in many kinds of cancer cells. However, the exact mechanism and signaling pathway involved in HL-37-induced apoptosis have not been fully elucidated. Therefore, we explored the mechanisms of HL-37-mediated apoptosis in MCF-7 and MDA-MB-435 human breast cancer cells. When MCF-7 cells or MDA-MB-435 cells were co-incubated with HL-37, the percentage of apoptotic cell and S phase of cell cycle was markedly increased. In addition, a rise in intracellular calcium levels, ROS production, phosphorylation of JNK and activation of calpain were found in both MCF-7 cells and MDA-MB-435 cells after exposure to HL-37. With the HL-37-mediated reduction of mitochondrial membrane potential, cytochrome c was released from mitochondria to cytosol. Moreover, HL-37 strongly induced cleavage of caspase-4, caspase-9, as well as caspase-3 in MDA-MB-435 cells, whereas, activation of caspase-4, caspase-9 and caspase-7 but not caspase-3 was detected in MCF-7 cells. These results suggested that HL-37 induced MDA-MB-435 and MCF-7 cells apoptosis via oxidative stress and Ca(2+)/calpain/caspase-4 pathway.     

Molecular pharmacology of the human prostaglandin D2 receptor,CRTH2

1. The recombinant human prostaglandin D(2) (PGD(2)) receptor, hCRTH2, has been expressed in HEK293(EBNA) and characterized with respect to radioligand binding and signal transduction properties. High and low affinity binding sites for PGD(2) were identified in the CRTH2 receptor population by saturation analysis with respective equilibrium dissociation constants (K(D)) of 2.5 and 109 nM. This revealed that the affinity of PGD(2) for CRTH2 is eight times less than its affinity for the DP receptor. 2. Equilibrium competition binding assays revealed that of the compounds tested, only PGD(2) and several related metabolites bound with high affinity to CRTH2 (K(i) values ranging from 2.4 to 34.0 nM) with the following rank order of potency: PGD(2)>13,14-dihydro-15-keto PGD(2)>15-deoxy-Delta(12,14)-PGJ(2)>PGJ(2)>Delta(12)-PGJ(2)>15(S)-15 methyl-PGD(2). This is in sharp contrast with the rank order of potency obtained at DP : PGD(2)>PGJ(2)>Delta(12)-PGJ(2)>15-deoxy-Delta(12,14)-PGJ(2) >13,14-dihydro-15-keto-PGD(2). 3. Functional studies demonstrated that PGD(2) activation of recombinant CRTH2 results in decrease of intracellular cAMP in a pertussis toxin-sensitive manner. Therefore, we showed that CRTH2 can functionally couple to the G-protein G(alphai/o). PGD(2) and related metabolites were tested and their rank order of potency followed the results of the membrane binding assay. 4. By Northern blot analysis, we showed that, besides haemopoietic cells, CRTH2 is expressed in many other tissues such as brain, heart, thymus, spleen and various tissues of the digestive system. In addition, in situ hybridization studies revealed that CRTH2 mRNA is expressed in human eosinophils. Finally, radioligand binding studies demonstrated that two eosinophilic cell lines, butyric acid-differentiated HL-60 and AML 14.3D10, also endogenously express CRTH2.     

Effects of 15-deoxy-Delta12, 14 prostaglandin J2 and ciglitazone on human cancer cell cycle progression and death: the role of PPARgamma

The role of PPARgamma in ciglitazone and 15-d PGJ(2)-induced apoptosis and cell cycle arrest of Jurkat (before and after PPARgamma gene silencing), U937 (express high levels of PPARgamma) and HeLa (that express very low levels of PPARgamma) cells was investigated. PPARgamma gene silencing, per se, induced a G2/M cell arrest, loss of membrane integrity and DNA fragmentation of Jurkat cells, indicating that PPARgamma is important for this cell survival and proliferation. Ciglitazone-induced apoptosis was abolished after knockdown of PPARgamma suggesting a PPARgamma-dependent pro-apoptotic effect. However, ciglitazone treatment was toxic for U937 and HeLa cells regardless of the presence of PPARgamma. This treatment did not change the cell cycle distribution corroborating with a PPARgamma-independent mechanism. On the other hand, 15-d PGJ(2) induced apoptosis of the three cancer cell lines regardless of the expression of PPARgamma. These results suggest that PPARgamma plays an important role for death of malignant T lymphocytes (Jurkat cells) and PPARgamma agonists exert their effects through PPARgamma-dependent and -independent mechanisms depending on the drug and the cell type.     

The effect of prostaglandin E2 on histamine-stimulated calcium mobilization as a possible explanation for histamine tachyphylaxis in canine tracheal smooth muscle

Isolated strips of canine tracheal smooth muscle rapidly lost their responsiveness to histamine when placed in a zero calcium Krebs buffer. Responsiveness to acetylcholine, however, was not rapidly lost, and following 120 min of incubation in zero calcium buffer with frequent washes, 10% of the contractile response still remained. The kinetics of each loss of response suggest that primarily a loosely bound source of calcium is mobilized by histamine and a more tightly bound source is mobilized by acetylcholine. Consistent with these data were the effects of the calcium antagonist verapamil. In normal calcium Krebs solution, dose-response curves to histamine were markedly reduced by verapamil while acetylcholine responses were relatively unaffected. In calcium depleted tracheal strips, indomethacin potentiated the calcium dose-response curve, determined by incremental readdition of calcium in the presence of histamine (10(-4) M), with comparatively little effect on the calcium dose-response curve in the presence of acetylcholine (10(-6) M). Also, in indomethacin pretreated tracheal strips, a reduction in the histamine-calcium dose-response curve could be produced by exogenous addition of 2.8 X 10(-9) M and 2.8 X 10(-8) M PGE2. In the acetylcholine-calcium responses there was a significant reduction only at 2.8 X 10(-8) M PGE2. These data suggest that histamine mobilizes primarily a loosely bound, possibly extracellular source of calcium necessary for contraction, and this histamine-stimulated calcium mobilization is sensitive to the effects of PGE2.     

Riccardin D, a novel macrocyclic bisbibenzyl, induces apoptosis of human leukemia cells by targeting DNA topoisomerase II

We studied the effect of riccardin D, a macrocyclic bisbibenzyl, which was isolated from the Chinese liverwort plant, on human leukemia cells and the underlying molecular mechanism. Riccardin D had a significant antiproliferative effect on human leukemia cell lines HL-60, K562 and its multidrug resistant (MDR) counterpart K562/A02 cells, but showed no effect on the topoisomerase-II-deficient HL-60/MX2 cells, as measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The pBR322 DNA relaxation assay revealed that riccardin D selectively inhibited the activity of topoisomerase II (topo II). The suppression of topo II activity by riccardin D was stronger than that of etoposide, a known topo II inhibitor. After treatment with riccardin D, nuclear extracts of leukemia K562 and K562/A02 cells left the majority of pBR322 DNA in a supercoiled form. Further examination showed that riccardin D effectively induced HL-60, K562 and K562/A02 apoptosis as evidenced by externalization of phosphatidylserine and formation of DNA ladder fragments. The activation of cytochrome c, caspase-9, caspase-3 and cleaved poly ADP-ribose polymerase (PARP) was also enhanced, as estimated by Western blot analysis. By contrast, riccardin D was unable to induce apoptosis in the topoisomerase-II-deficient HL-60/MX2 cells, indicating that the induction of apoptosis by riccardin D was due to the inhibition of topo II activity. In addition, riccardin D was able to significantly decrease P-glycoprotein (P-gp) expression in K562/A02 cells. Taken together, our data demonstrate that riccardin D is a novel DNA topo II inhibitor which can induce apoptosis of human leukemia cells and that it has therapeutic potential for both regular and MDR strains of leukemia cells.     

Toxicity of the quinalphos metabolite 2-hydroxyquinoxaline: growth inhibition, induction of oxidative stress, and genotoxicity in test organisms

The quinalphos metabolite 2-hydroxyquinoxaline (HQO), previously shown to photocatalytically destroy antioxidant vitamins and biogenic amines in vitro, was tested for toxicity in several small aquatic organisms and for mutagenicity in Salmonella typhimurium. In the rotifer Philodina acuticornis, HQO caused the disappearance of large individuals and increased hydroperoxide concentration. The latter effect was not only observed in animals kept in a light/dark cycle, but also in constant darkness, indicating that HQO can assume a reactive state and/or form reactive intermediates under the influence of either light or redox-active metabolites, in particular, free radicals. Cell proliferation was inhibited in the ciliate Paramecium bursaria. In the dinoflagellate Lingulodinium polyedrum, which allows early detection of cellular stress on the basis of bioluminescence measurements, strong rises in light emission became apparent on the 2nd day of exposure to HQO and continued until cells died between 12 and 18 days of treatment. Oxidative damage of protein by HQO was demonstrated by measuring protein carbonyl in L. polyedrumin vivo as well as in light-exposed bovine serum albumin in vitro. In an Ames test of mutagenicity, HQO proved to be genotoxic in both light- and dark-exposed bacteria. HQO appears as a source of secondary quinalphos toxicity, which deserves further attention.     

The cyclo-oxygenase-dependent regulation of rabbit vein contraction: evidence for a prostaglandin E2-mediated relaxation

1. Arachidonic acid (0.01-1 microM) induced relaxation of precontracted rings of rabbit saphenous vein, which was counteracted by contraction at concentrations higher than 1 microM. Concentrations higher than 1 microM were required to induce dose-dependent contraction of vena cava and thoracic aorta from the same animals. 2. Pretreatment with a TP receptor antagonist (GR32191B or SQ29548, 3 microM) potentiated the relaxant effect in the saphenous vein, revealed a vasorelaxant component in the vena cava response and did not affect the response of the aorta. 3. Removal of the endothelium from the venous rings, caused a 10 fold rightward shift in the concentration-relaxation curves to arachidonic acid. Whether or not the endothelium was present, the arachidonic acid-induced relaxations were prevented by indomethacin (10 microM) pretreatment. 4. In the saphenous vein, PGE2 was respectively a 50 and 100 fold more potent relaxant prostaglandin than PGI2 and PGD2. Pretreatment with the EP4 receptor antagonist, AH23848B, shifted the concentration-relaxation curves of this tissue to arachidonic acid in a dose-dependent manner. 5. In the presence of 1 microM arachidonic acid, venous rings produced 8-10 fold more PGE2 than did aorta whereas 6keto-PGF1alpha and TXB2 productions remained comparable. 6. Intact rings of saphenous vein relaxed in response to A23187. Pretreatment with L-NAME (100 microM) or indomethacin (10 microM) reduced this response by 50% whereas concomitant pretreatment totally suppressed it. After endothelium removal, the remaining relaxing response to A23187 was prevented by indomethacin but not affected by L-NAME. 7. We conclude that stimulation of the cyclo-oxygenase pathway by arachidonic acid induced endothelium-dependent, PGE2/EP4 mediated relaxation of the rabbit saphenous vein. This process might participate in the A23187-induced relaxation of the saphenous vein and account for a relaxing component in the response of the vena cava to arachidonic acid. It was not observed in thoracic aorta because of the lack of a vasodilatory receptor and/or the poorer ability of this tissue than veins to produce PGE2.     

Continuous presence of H2O2 induces mitochondrial-mediated,MAPK- and caspase-independent growth inhibition and cytotoxicity in human gingival fibroblasts

The continuous generation of reactive oxygen species (ROS) is one of the most important events that occur during periodontal inflammation. Hydrogen peroxide (H₂O₂) is widely used in dental clinics. Many investigators have tried to elucidate the exact effect of H₂O₂ on human gingival fibroblasts (HGFs). These studies have shown that H₂O₂ induces growth inhibition and apoptosis in cells. However, the mechanisms involved in H₂O₂-induced cell death in HGFs are not completely understood. In this study, we examine how continuously generated H₂O₂ affects the viability and proliferation of HGFs using glucose oxidase (GO). We also explored the mechanisms by which the continuous presence of H₂O₂ induces cell death. GO treatment not only inhibited HGF growth and proliferation, but it also induced cell death in HGFs without typical apoptotic features such as nuclear DNA laddering. This GO-mediated cytotoxicity was proportional to the levels of intracellular ROS that were generated, rather than proportional to changes of cellular antioxidant activities. GO treatment also resulted in the loss of mitochondrial membrane potential and the relocation of mitochondrial apoptogenic factors. There was also an acute and severe depletion of cellular ATP levels. However, none of the pharmacological inhibitors specific for mitogen-activated protein kinases (MAPKs) or pancaspase prevented GO-induced cell death. Treatment with either catalase or acteoside significantly attenuated the GO-mediated cytotoxicity in the HGFs, thereby suggesting a protective effect of antioxidants against ROS-mediated gingival damage. Here we demonstrate that continuously generated H₂O₂ not only inhibits the viability and proliferation of HGFs, but also causes pyknotic/necrotic cell death through mitochondrial stress-mediated, MAPK- and caspase-independent pathways.     

Sulphasalazine is a potent inhibitor of prostaglandin 15-hydroxydehydrogenase: possible basis for therapeutic action in ulcerative colitis.

Sulphasalazine is a potent and selective inhibitor in vitro of prostaglandin 15-hydroxydehydrogenase in rabbit colon (ID50 = 50 micrometer) and in several other organs of different species, but does not inhibit prostaglandin delta-13 reductase or microsomal prostaglandin synthesis from arachidonic acid. It is suggested that this action may underly the therapeutic usefulness of sulphasalazine in ulcerative colitis for the prevention of relapse.     

天然二萜衍生物Jar-TTA双重抑制糖酵解及氧化磷酸化诱导食管癌细胞凋亡

目的研究二萜衍生物Jar-TTA对糖酵解/氧化磷酸化(OXPHOS)的双重抑制作用,并探讨其抗食管癌机制。方法MTT法检测Jar-TTA对食管癌细胞EC109、KYSE~(-1)50的抗增殖作用;荧光显微镜观察线粒体膜电位(MMP);流式细胞术定量分析细胞凋亡、MMP及葡萄糖摄取;能量代谢分析仪实时检测细胞糖酵解及OXPHOS; Western blot检测蛋白表达。结果 Jar-TTA明显抑制食管癌细胞的增殖,且呈浓度依赖性; 2、4、8μmol·L~(-1)Jar-TTA诱导EC109早期凋亡率分别为(27. 9±6. 1)%、(71. 1±9. 3)%和(65. 0±9. 5)%,与对照组相比,差异均具有显著性(P <0. 01); Jar-TTA明显抑制细胞的糖酵解和OXPHOS;此外,Jar-TTA诱导MMP下降,抑制葡萄糖摄取,同时下调葡萄糖转运蛋白4(GLUT4)及乳酸脱氢酶A(LDHA)表达。结论 Jar-TTA通过双重抑制糖酵解/OXPHOS诱导食管癌细胞凋亡,其机制可能与Jar-TTA降低MMP而干扰线粒体OXPHOS功能,以及下调GLUT4和LDHA而抑制葡萄糖摄取和糖酵解进程有关。     

Alpha-chaconine, a potato glycoalkaloid, induces apoptosis of HT-29 human colon cancer cells through caspase-3 activation and inhibition of ERK 1/2 phosphorylation.

Although alpha-chaconine, one of the two major potato trisaccharide glycoalkaloids, have shown cytotoxic effects on human cancer cells, the exact mechanism of this action of alpha-chaconine is not completely understood. In this study, we found that alpha-chaconine induced apoptosis of HT-29 cells in a time- and concentration-dependent manner by using flow cytometric analysis. We also found that caspase-3 activity and the active form of caspase-3 were increased 12 h after alpha-chaconine treatment. Caspase inhibitors, N-Ac-DEVD-CHO and Z-VAD-fmk, prevented alpha-chaconine-induced apoptosis, whereas alpha-chaconine-induced apoptosis was potentiated by PD98059, an extracellular signal-regulated kinase (ERK) inhibitor. However, pretreatment of the cells with LY294002 and SB203580, inhibitors of PI3K and p38, respectively, BAPTA-AM, an intracellular Ca(2+) chelator, and antioxidants such as N-acetylcysteine (NAC) and Trolox had no effect on the alpha-chaconine-induced cell death. In addition, phosphorylation of ERK was reduced by the treatment with alpha-chaconine. Moreover, alpha-chaconine-induced caspase-3 activity was further increased by the pretreatment with PD98059. Thus, the results indicate that alpha-chaconine induces apoptosis of HT-29 cells through inhibition of ERK and, in turn, activation of caspase-3.     

Testicular toxicity of 2-methoxyacetaldehyde, a possible metabolite of ethylene glycol monomethyl ether, in the rat

2-Methoxyacetaldehyde (MALD) was shown to produce specific cellular toxicity to pachytene spermatocytes in mixed testicular cell cultures as evidenced by morphological changes to these cells, an increase in germ-cell detachment and leakage of the pachytene spermatocyte marker enzyme lactate dehydrogenase-X. These effects occurred at concentrations where the known testicular toxicant, 2-methoxyacetic acid (MAA) was without effect (0.2 and 0.5 mM). In vivo, MALD also produced the characteristic testicular lesion reported previously for MAA and its parent compound ethylene glycol monomethyl ether (EGME). It is likely that MALD plays an important role in EGME-induced testicular toxicity.     

Irreversible inhibition of CYP2D6 by (-)-chloroephedrine, a possible impurity in methamphetamine.

(+)- and (-)-Chloroephedrine, and their respective aziridines, cis- and trans-1,2-dimethyl-3-phenylaziridine, have been reported present in clandestinely synthesized methamphetamine. Since methamphetamine and structurally related compounds are potential substrates for human liver CYP2D6, the possible interaction of the chloroephedrines with human liver CYP2D6 was evaluated. Computational methods (using Flexidock and HINT in SYBYL) were used to determine the feasibility of (+)- or (-)-chloroephedrine and cis- or trans-1,2-dimethyl-3-phenylaziridine binding in the active site of a three dimensional CYP2D6 molecular model. Although modeling indicates both (+)- and (-)-chloroephedrine would bind comparably to methamphetamine, the binding energies of cis- or trans-1,2-dimethyl-3-phenylaziridine products indicate a preference for trans-1,2-dimethyl-3-phenylaziridine, the product formed from (-)-chloroephedrine. The effects of (+)- and (-)-chloroephedrine on the metabolism of dextromethorphan in human liver microsomes were then experimentally evaluated. (+)-Chloroephedrine (50 micro M) had no effect on human CYP2D6. (-)-Chloroephedrine appeared to be selective for human CYP2D6 versus CYP1A2 and CYP3A4/5. The inhibition of CYP2D6 was time-dependent, not dependent on metabolic activation, and irreversible. It appeared to bind at the active site of CYP2D6 with an apparent K(i) of 226 micro M, with a k(int) of 0.039 min(-1), and a t(1/2) of 23 min. Due to the irreversible nature of this inhibition, this impurity in clandestinely synthesized methamphetamine may be important and warrant further study.     

Sulfur mustard induces apoptosis in cultured normal human airway epithelial cells: evidence of a dominant caspase-8-mediated pathway and differential cellular responses

We have shown that sulfur mustard (SM; bis-(2-chloroethyl) sulfide), an alkylating, vesicating chemical warfare agent, causes dermal toxicity, including skin microblisters, via the induction of both death receptor (DR) and mitochondrial pathways of apoptosis in human epidermal keratinocytes. While SM is known for its skin-vesicating properties, respiratory tract lesions are the main source of morbidity and mortality after inhalation exposure. We, therefore, investigated whether SM induces apoptotic cell death in normal human bronchial epithelial (NHBE) cells and small airway epithelial cells (SAEC) in vitro. Cells were exposed to various concentrations of SM (0, 50, 100, and 300 muM for 16 h) in the culture medium and then tested for the activation of apoptotic executioner caspase-3 and initiator caspases-8 and -9. Caspases-8 and -3 were activated by SM in both airway cell types, indicating the induction of a DR pathway of apoptosis in these cells; however, the levels of enzyme activation were different, depending on the cell type and the SM concentrations used. Consistent with enzyme activity results, immunoblot analyses revealed the proteolytic processing of the proenzymes to the active forms of caspases-8 and -3 in these cells after SM exposure. Interestingly, NHBE cells were found to be exquisitely sensitive to SM, compared to SAEC, with caspase-3 activities in SM-exposed NHBE cells approximately 2-fold higher and caspase-8 activities approximately 10-fold higher than in SAEC. Furthermore, SM activated caspase-9 in NHBE cells, but not in SAEC, indicating a possible role of the mitochondrial pathway only in the NHBE cells. The present study shows that both upper airway (NHBE cells) and deep lung (SAEC) epithelial cells undergo SM-induced apoptotic death in vitro, but distinct cell-type specific responses can be elicited, which may be attributed to intrinsic properties that characterize the response of these cells to SM. These findings need to be taken into consideration in the search for modulators of these pathways for the therapeutic intervention to reduce SM injury due to respiratory tract lesions.     

The biphasic effects of cyclopentenone prostaglandins, prostaglandin J(2) and 15-deoxy-Delta(12,14)-prostaglandin J(2) on proliferation and apoptosis in rat basophilic leukemia (RBL-2H3) cells

Mast cells produce chemical mediators, including histamine and arachidonate metabolites such as prostaglandin D(2) (PGD(2)) after antigen stimulation. Cyclopentenone prostaglandins of the J series, prostaglandin J(2) (PGJ(2)) and 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), are thought to be derivatives of PGD(2). In this study, the biphasic effects of the PGJ(2) and 15d-PGJ(2) on proliferation and apoptosis in rat basophilic leukemia cells (RBL-2H3), a tumor analog of mast cells, were examined. At low concentrations, 1 or 3 microM PGJ(2) and 15d-PGJ(2) induced cell proliferation, respectively. At high concentrations (10-30 microM) both the inhibition of viability and decrease in histamine content in RBL-2H3 cells were dose dependent. These effects were independent of the nuclear hormone receptor, peroxisome proliferator-activated receptor gamma (PPARgamma), since troglitazone, an agonist of PPARgamma did not cause any effects in RBL-2H3 cells. Cell death induced by PGJ(2) and 15d-PGJ(2) was the result of apoptotic processes, since RBL-2H3 cells treated with 30 microM of the prostaglandins had condensed nuclei, DNA fragmentation and increase in activities of caspase-3 and -9. Moreover, PGJ(2) or 15d-PGJ(2)-induced apoptotic effects were prevented by the caspase inhibitor, z-VAD-fmk. In conclusion, the PGJ(2) or 15d-PGJ(2)-induced apoptosis in RBL-2H3 cells occurs mainly via mitochondrial pathways instead of by PPARgamma-dependent mechanisms.