The organosulfur compound ajoene, a constitutent of garlic, has been shown to induce apoptosis in a leukemic cell line as well as in blood cells of a leukemic patient. The mechanisms of action of ajoene, however, are unknown. The present study aims to characterize the molecular events leading to ajoenetriggered apoptosis. We show here that ajoene (20 M) leads to a time-dependent activation of caspase-3-like activity as well as to the proteolytic processing of procaspase-3 and -8. Activation of caspases was necessary for ajoene-induced apoptosis since the broad-range caspase inhibitor zVAD-fmk completely abrogated ajoene-mediated DNA fragmentation. Although the initiator caspase-8 was activated, the CD95 death receptor was not involved in death signaling since the HL-60 clone used was shown to express a functionally inactive CD95 receptor. Furthermore, ajoene induced the release of cytochrome c, which was not inhibited by zVAD-fmk indicating that cytochrome c release precedes caspase activation. Ajoene also led to a dissipation of the mitochondrial transmembrane potential. Overexpression of Bcl-x L clearly diminished ajoeneinduced caspase activation as well as apoptosis. These results indicate that apoptosis in leukemia cells triggered by ajoene is based on the activation of a mitochondria-dependent caspase cascade which includes also the activation of the initiator caspase-8. Leukemia (2002) 16, 74-83.
Treatment of human promyeloleukemic HL-60 cells with the experimental antileukemic drug ajoene induces the activation of the mitogen-activated protein kinases (MAPKs) c-Jun NH 2 -terminal kinase (JNK), p38 and extracellular signal-regulated kinases (ERK) 1/2 as well as the survival kinase Akt. JNK activation occurred in HL-60/neo, HL-60/bcl-x L , and in HL-60 cells pretreated with the pan-caspase inhibitor zVAD-fmk, indicating that JNK activation is not dependent on ajoene-induced mitochondria perturbation and subsequent caspase activation. Cells overexpressing a dominant-negative JNK showed no altered sensitivity towards ajoene suggesting that the activation of JNK is not necessary for ajoeneinduced cell death. Inhibition of p38 MAPK by SB 203580 had no influence on ajoene-mediated apoptosis. In contrast, inhibition of ERK1/2 vastly enhanced ajoeneinduced cell death. The survival kinase Akt, in contrast, did not participate in ajoene-induced death signaling as shown by the use of the phosphatidylinositol-3-kinase inhibitor wortmannin. Thus in contrast to the previous findings regarding stress-induced cell death, ajoenemediated activation of JNK and p38 has no impact on ajoene-induced apoptosis in HL-60 cells. Blockade of ERK1/2 but not Akt pathways leads to sensitization of cells against ajoene-mediated apoptosis supporting the view that inhibition of ERK1/2 is a valuable strategy to increase the sensitivity of promyeloleukemic cells towards ajoene.
2, 5-Dialkylfuran and tetrahydrofuran compounds as structural elements of Annonaceae acetogenins like Asitrocinwere synthesized starting from furfural by Grignard reactions, lithiation of the furan ring and addition of aliphatic aldehydes. Hydrogenation of the furan rings over Pd-catalyst gave the corresponding tetrahydrofurans. All resulting compounds showed no or rather less antimicrobial activity against grampositive, gram-negative bacteria and fungi compared to tetracycline or clotrimazol but high cytotoxic activity against HL 60 cell line determined in the MTT assay.
.2,5-Dialkylfuran and tetrahydrofuran compounds as structural elements of Annonaceae acetogenins like Asitrocin were synthesized starting from furfural by Grignard reactions, lithiation of the furan ring and addition of aliphatic aldehydes. Hydrogenation of the furan rings over Pd-catalyst gave the corresponding tetrahydrofurans. All resulting compounds showed no or rather less antimicrobial activity against grampositive, gram-negative bacteria and fungi compared to tetracycline or clotrimazol but high cytotoxic activity against HL 60 cell line determined in the MTT assay. . So more simple compounds with the same or even higher potency are interesting for the development of new anticancer drugs. One of the discussed mechanisms of action is a complexation of mitochondrial iron by the dihydroxyalkyltetrahydrofuran core of the acetogenins [1]. So this partial structure of the natural compounds was the target of our synthesis. On the other hand some furancarbinols as 4-ipomeanol are well known cytotoxic compounds. 4-Ipomeanol is after bioactivation in lung tissue (clara cells) by cytochrom P 450 an alkylating agent [7]. It is tested in phase II studies against human lung cancer. So bisalkylfuran compounds might be interesting for the development of new anticancer drugs too. In addition, the products should also be tested for their antimicrobial activity (Scheme 1). Keywords Scheme 1. ChemistryCommercially available furan-2-carbaldehyde was reacted in a Grignard reaction with dodecylmagnesium bromide to give the alcohol 1 in good yield [3,4]. The furan ring of 1 was hydrogenated over Pd/C-catalyst under normal pressure to the tetrahydrofuran derivative 2 (mixture of diastereomers). On the other hand the furan ring of 1 was lithiated with n-BuLi, (a second equivalent of n-BuLi was consumed for deprotonation of the hydroxy group) and the resulting dianion was reacted with pent-4-enal to give the dialkylfuran 3 as an inseparable mixture of diastereomers (seperation was impossible by column chromatography). 3 was also hydrogenated with Pd/C in methanol to give the tetrahydrofuran 4 as a diastereomeric mixture. In the course of this hydrogenation the double bond was also reduced (Scheme 2).In the same manner homologues were prepared by reaction of furan-2-carbaldehyde with pent-4-enylmagnesiumbromide to the alcohol 5, followed by lithiation with two equivalents n-BuLi and addition of tridecanal.The diol 6 was obtained only in low yield. Hydrogenation of 6 gave the disubstituted tetrahydrofuran 7 (Scheme 3).It was impossible for us to separate the diastereomeric mixtures by flash column chromatography on silica gel or by preparative high-pressure liquid chromatography. Pharmacological results and discussionThe compounds described above were tested in the agar diffusion assay against E. coli, S. hominis, P. antimicrobia and the yeasts Y. lipolytica, C. glabrata and the filamentou fungi A. niger. All compounds showed only minor or no antimicrobial activity, only 2, 3, 4, 5 and 6 showed a moderate activity (Table 1). The cytot...
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