Rosmarinic acid influences collagen, MMPs, TIMPs, glycosylation and MUC1 in CRL-1739 gastric cancer cell line.

Rosmarinic acid (RA) is a natural phenylpropanoid with numerous pharmacological activities. Because of limited studies of the effects of RA action in gastric cancer cells we examined how 100 and 200 µM acid influences MMPs, TIMPs, collagen, MUC1 and specific sugar antigens in gastric adenocarcinoma CRL-1739 cells. We revealed inhibitory effect of RA on MMP-9 activity what was correlated with increased collagen type I expression, main ECM substrate degraded by MMPs. Tissue inhibitor of MMPs, TIMP-1 but not TIMP-2 was significantly decreased on the protein level and increased on mRNA level by RA action what can suggest TIMP-1 independent inhibitory action of an acid on MMP-9 activity. Glycosylation of gastric cancer proteins was also effected by RA. ELISA tests revealed inhibitory effect of an acid on Tn antigen in cell lysates and culture supernatant and on T antigen in cell lysates. RA inhibited also sialylated Tn antigen in protein of culture supernatant and sialyl T in cell lysates. Extracellular domain of MUC1 mucin, main carrier of Tn and T antigens was significantly inhibited by higher dose of RA. The data suggest potential usefulness of RA as a complementary agent supporting chemotherapy in cancer treatment.

The redox status of human breast cancer cell lines (MCF-7 and MDA-MB231) treated with novel dinuclear berenil-platinum(II) complexes.

This study compared the effects of cisplatinum and novel berenil-platinum(ll) complexes on the redox status of breast cancer cells that were estrogen receptor-positive (MCF-7) or estrogen receptor-negative (MDA-MB231). Both cell lines were treated with cisplatinum or the following berenil-platinum(ll) complexes: Pt2(isopropylamine)4(berenil)2, Pt2(piperidine)4(berenil)2, Pt2(2-picoline)4(berenil)2, Pt2(3-picoline)4(berenil)2, and Pt2(4-picoline)4(berenil)2. Changes in levels of reactive oxygen species, levels and activities of antioxidants, and lipid peroxidation products levels were measured. All investigated compounds enhanced ROS generation, reduced the activity of antioxidant enzymes (e.g., glutathione peroxidase and glutathione reductase), and decreased levels of small-molecule antioxidants (GSH, vitamins E and A). Such conditions are conducive to generating oxidative stress and phospholipids peroxidation. Cellular phospholipids in MCF-7 cells were most sensitive to the Pt2(isopropylamine)4(berenil)2 complex, whereas MDA-MB231 cells were not particularly sensitive to any berenil-platinum(ll) complex. These findings will facilitate future anticancer drug design strategy for breast cancer pharmacotherapy.

Effects of dinuclear berenil-platinum(II) complexes on fibroblasts redox status.

PURPOSE: Platinum(II) complex anticarcinogenic mechanisms are associated with changes in the cellular redox status of cancer as well as healthy cells. Therefore, the goal of the present study was to investigate oxidative modifications in cellular components following fibroblast exposure to novel dinuclear berenil-platinum(II) complexes. MATERIAL AND METHOD: ROS levels, antioxidant parameters level/activity, and damage to DNA, lipids, and proteins, including pro-apoptotic and anti-apoptotic factors in human skin fibroblasts following berenil-platinum(II) complex treatments i.e. Pt2(isopropylamine)4(berenil)2, Pt2(piperazine)4(berenil)4, Pt2(2-picoline)4(berenil)2, Pt2(3-picoline)4(berenil)2, and Pt2(4- picoline)4(berenil)2 were examined. RESULTS: Treatment of fibroblasts with platinum(II) complexes has shown that all compounds enhance total ROS and superoxide anion generation as well as change the activity of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase and decrease in the level of non-enzymatic antioxidants (GSH, vitamin C, E and A). Such a situation is conducive to oxidative stress formation and oxidative modifications of cellular macromolecules and to increase in the expression of proapoptotic proteins. Pt2(isopropylamine)4(berenil)2 elicited the most damage, which resulted in oxidative modification of cellular components. The therapeutic use of this complex would cause considerable side effects in patients, therefore the agent lacks drug potential; however Pt2(piperazine)4(berenil)2 and Pt2(2-picoline)4(berenil)2 exhibited reduced redox and increased apoptotic profiles compared to cisplatin. CONCLUSION: Results of this paper and preliminary data show that Pt2(2-picoline)4(berenil)2 is less dangers than cisplatin to fibroblasts and more disruptive than cisplatin to breast cancer cell metabolism, and therefore it is a promising candidate for use in future anticancer drug strategies.

Cytotoxicity evaluation of three light-cured dentin adhesive materials on human gingival fibroblasts, ex vivo.

PURPOSE: To evaluate the cytotoxic effects of three current light-cured dentin adhesives, in both uncured and post-cured conditions, on human gingival fibroblasts. MATERIAL AND METHODS: The materials tested were Heliobond, Adper Single Bond 2 and Xeno V, which are characterized by various compositions and application procedures. Each agent, in volumes of 5 and 10 µL, was tested after polymerization, and those unpolymerized were diluted in DMEM to 10-3 and 10-5. The cytotoxicity of the adhesives was assessed on the basis of a test of cell viability in a culture of human gingival fibroblasts, with the use of tetrazolic salt (MTT assay). RESULTS: The results showed that, among the adhesive/bonding systems tested, Xeno V was the least cytotoxic. There were statistically significant differences in cell survival between polymerized Xeno V, Adper Single Bond 2 and Heliobond in the amount of 5 µL as well as between the Xeno V and Adper Single Bond 2 in 10-5 dilutions. The tested adhesives were more toxic in the polymerized form than in the dilutions. Samples of 10 µL resulted in a lower survival percentage of fibroblasts compared to 5 µL. CONCLUSION: All the tested adhesives demonstrated cytopathic effects towards human gingival fibroblasts, but varied in their cytotoxicity. This has clinical implications. Dentists should follow the rules of adhesive application, precisely dose them and not allow direct contact with the gums as, even after polymerization, adhesive agents exhibit potential cytotoxic activity.

Human DNA topoisomerase inhibitors from Potentilla argentea and their cytotoxic effect against MCF-7.

Two polyphenolics, kaempferol 3-O-beta-D-(6"-E-p-coumaroyl)-glucopyranoside (tiliroside) (1) and methyl brevifolincarboxylate (2) isolated from aerial parts of Potentilla argentea L. (Rosaceae) were evaluated for their cytotoxicities against human breast carcionoma cell line (MCF-7) and their DNA-binding ability. The DNA-binding ability of these compounds was studied by means of the human DNA topoisomerase I and II inhibition assay and ethidium displacement assay using calf thymus DNA, poly(dA-dT)2 and poly(dG-dC)2. Compound 2 was much more active and showed a higher level of cytotoxic potency than compound 1, with IC50 values of 1.11 +/- 2 microM and 21.60 +/- 2 microM, respectively. In DNA topoisomerase I and II inhibition in vitro assays both investigated compounds 1 and 2 were more effective against topoisomerase II than I. The results of DNA binding studies reveal that methyl brevifolincarboxylate had a greater DNA binding affinity that tiliroside, which correlates with its greater potency as a topoisomerase I/II inhibitor.

Mitochondrially targeted ceramide LCL-30 inhibits colorectal cancer in mice.

The sphingolipid ceramide is intimately involved in the growth, differentiation, senescence, and death of normal and cancerous cells. Mitochondria are increasingly appreciated to play a key role in ceramide-induced cell death. Recent work showed the C16-pyridinium ceramide analogue LCL-30 to induce cell death in vitro by mitochondrial targeting. The aim of the current study was to translate these results to an in vivo model. We found that LCL-30 accumulated in mitochondria in the murine colorectal cancer cell line CT-26 and reduced cellular ATP content, leading to dose- and time-dependent cytotoxicity. Although the mitochondrial levels of sphingosine-1-phosphate (S1P) became elevated, transcription levels of ceramide-metabolising enzymes were not affected. In mice, LCL-30 was rapidly absorbed from the peritoneal cavity and cleared from the circulation within 24 h, but local peritoneal toxicity was dose-limiting. In a model of subcutaneous tumour inoculation, LCL-30 significantly reduced the proliferative activity and the growth rate of established tumours. Sphingolipid profiles in tumour tissue also showed increased levels of S1P. In summary, we present the first in vivo application of a long-chain pyridinium ceramide for the treatment of experimental metastatic colorectal cancer, together with its pharmacokinetic parameters. LCL-30 was an efficacious and safe agent. Future studies should identify an improved application route and effective partners for combination treatment.

Distribution and dynamic changes of sphingolipids in blood in response to platelet activation.

BACKGROUND: Sphingolipids are signaling molecules in a range of biological processes. While sphingosine-1-phosphate (S1P) is thought to be abundantly stored in platelets and released upon stimulation, knowledge about the distribution and function of other sphingolipids in blood is lacking. OBJECTIVES: To analyze the sphingolipid content of blood components with special emphasis on dynamic changes in platelets. METHODS: Blood components from mice and humans were prepared by gradient centrifugation and analyzed by liquid chromatography-mass spectrometry. Additionally, murine platelets were activated in vitro and in vivo. RESULTS: Isolated non-activated platelets of mice were devoid of S1P, but instead contained dihydrosphingosine-1-phosphate (dhS1P), along with a high concentration of ceramide. Activation of platelets in vitro led to a loss of dhS1P and an increase in sphingosine, accompanied by a reduction of ceramide content. Platelet activation in vivo led to an immediate and continuous rise of dhS1P in plasma, while S1P remained stable. The sphingolipid distribution of human blood was markedly different from mice. Human platelets contained dhS1P in addition to S1P. CONCLUSIONS: Mouse platelets contain dhS1P instead of S1P. Platelet activation causes loss of dhS1P and breakdown of ceramide, implying ceramidase activation. Release of dhS1P from activated platelets might be a novel signaling pathway. Finally, the sphingolipid composition of mouse and human blood shows large differences, which must be considered when studying sphingolipid biology.

Modulation of ceramide metabolism enhances viral protein apoptin's cytotoxicity in prostate cancer.

Despite local and systemic therapies, the National Cancer Institute estimates that prostate cancer will cause over 30,000 deaths in 2006. This suggests that additional therapeutic approaches are needed. The chicken anemia viral protein Apoptin causes tumor-selective apoptosis in human tumor lines independent of p53 and Bcl-2 status. Tet-regulated expression of Apoptin from an adenoviral vector showed cytotoxicity in DU145, PC-3, and LNCaP tumor cells regardless of expression of p53, Bcl-2, Bcl-xL, Bax, survivin, FLIP(S), XIAP, or CIAP. Apoptin expression caused an increase in the tumor suppressor lipid ceramide, which regulates the cellular stress response. Interestingly, 10 of 15 primary prostate cancers examined by Western blotting overexpressed acid ceramidase (AC), suggesting that ceramide deacylation might serve to negate elevated levels of ceramide, creating a more antiapoptotic phenotype. This was confirmed in AC-overexpressing cells in which we observed decreased sensitivity to apoptosis following treatment with Apoptin. Addition of the AC inhibitor LCL204, in combination with Apoptin, augmented cell killing. This effect was also demonstrated in vivo in that Apoptin and LCL204 cotreatment significantly reduced tumor growth in DU145 xenografts (P<0.05). Taken together, our data demonstrated that Apoptin is a promising therapeutic agent for prostate cancer and that its function is improved when combined with acid ceramidase inhibitors.

Combined therapeutic use of AdGFPFasL and small molecule inhibitors of ceramide metabolism in prostate and head and neck cancers: a status report.

As of January 2005, there were 1020 gene therapy clinical trials ongoing worldwide with 675 or 66.2% devoted to cancer gene therapy. The majority are occurring in the US and Europe (http://www.wiley.co.uk/genetherapy/clinical/). At the present time, to our knowledge there are no trials that employ gene delivery of Fas Ligand (FasL). As an important note, and in contrast to somatic cell therapy trials, there are no reported deaths due to therapeutic vector administration in any cancer gene therapy trial. That said, from our studies and from the published literature, the issue of gene delivery remains the major obstacle to successfully employing gene therapy for cancer treatment. Numerous laboratories are studying this with many different approaches. My co-workers and I have focused on the delivery issue by using various approaches that address tumor targeting and transgene expression. In addition, we are focusing on enhancing tumor cell killing via the bystander effect and through use of small molecules to enhance bystander activity.

Cytotoxicity and effect on collagen biosynthesis of proline analogue of melphalan as a prolidase-convertible prodrug in cultured human skin fibroblasts.

Proline analogue of melphalan (MEL-PRO) was synthesised as a prodrug susceptible to the action of ubiquitously distributed, cytosolic imidodipeptidase--prolidase [E.C.3.4.13.9]. Conjugation of melphalan (MEL) with proline (PRO) through an imido-bond resulted in formation of a good substrate for prolidase. The susceptibility of MEL-PRO to the action of prolidase was found to be similar, compared to glycyl-proline--the most abundant, endogenous substrate for prolidase and about 6-fold higher compared to its substrate--glycyl-hydroxyproline. We have compared the transport of MEL and its prodrug through cell membrane, their antimitotic activity, cytotoxicity and effect on collagen biosynthesis in cultured, normal human skin fibroblasts. The prodrug was found to be more effectively transported into the cells than the free drug. Moreover, a lower cytotoxicity, antimitotic activity and inhibitory effect on collagen biosynthesis of the prodrug, compared to the free drug were observed after 24 h of incubation. MEL and MEL-PRO at concentrations of 12 microM led to the decrease in cell viability in confluent human skin fibroblasts by about 40 and 20%, respectively, during 24 h of incubation. IC50 of MEL for DNA synthesis (measured by thymidine incorporation assay) was found at about 7 microM, while MEL-PRO used at this concentration produced about 35% reduction in thymidine incorporation. Similarly, MEL and MEL-PRO used at 7 microM concentrations inhibited collagen biosynthesis in fibroblasts cultured for 24 h to about 30 and 80% of control values, respectively. However, when the cells were cultured with the drugs for 72 h, similar effects of both drugs on DNA and collagen biosynthesis were observed. The data suggest that MEL-PRO may serve as a prolidase-convertible prodrug that evokes lower cytotoxicity, antimitotic activity, and lower inhibitory effect on collagen biosynthesis in fibroblast cultures, compared to the free drug.

Structural requirements of ceramide and sphingosine based inhibitors of mitochondrial ceramidase.

The effects of structural analogues of ceramide on rat brain mitochondrial ceramidase (mt-CDase) were investigated. Design of target compounds was mainly based on modifications of the key elements in ceramide and sphingosine, including stereochemistry, the primary and secondary hydroxyl groups, the trans double bond in the sphingosine backbone, and the amide bond. Mt-CDase was inhibited by (1) all stereoisomers of D-erythro-ceramide (D-e-Cer) with an IC50 of 0.11, 0.21, and 0.26 mol % for the L-threo, D-threo, and L-erythro isomers, respectively; (2) all stereoisomers of sphingosine with IC50 ranging from 0.04 to 0.14 mol %, N-methyl-D-erythro-sphingosine (N-Me-Sph, IC50 0.13 mol %); and (3) D-erythro-urea-C16-ceramide (C16-urea-Cer IC50 0.33 mol %). The enzyme was not inhibited by N-methyl ceramide (N-Me-C16-Cer), 1-O-methyl ceramide (1-O-Me-C16-Cer), 3-O-methyl ceramide (3-O-Me-C16-Cer), cis-D-erythro ceramide (cis-D-e-C16-Cer) and 3-O-methyl-D-erythro-sphingosine (3-O-Me-Sph). It was less potently inhibited by D-erythro-sphinganine (D-e-dh-Sph, IC50 0.20 mol %), D-erythro-dehydro sphingosine (D-e-deh-Sph, IC50 0.25 mol %), (2S)-3-keto-sphinganine (3-keto-dh-Sph, IC50 0.34 mol %), (2S) 3-keto-ceramide (3-keto-C16-Cer, IC50 0.60 mol %), and ceramine (C18-ceramine, IC50 0.62 mol %), 1-O-methyl-D-erythro-sphingosine (1-O-Me-Sph), cis-D-erythro-sphingosine (cis-D-e-Sph), (2S)-3-ketosphingosine (3-keto-Sph), (2S)-3-keto-dehyrosphingosine (3-keto-deh-Sph), and N,N-dimethyl-D-erythrosphingosine (N,N-diMe-Sph) were weak inhibitors whereas ceramide-1-phosphate (Cer-1-P) and sphingosine-1-phosphate (Sph-1-P) stimulated the enzyme. Thus, for inhibition, the enzyme requires the primary and secondary hydroxyl groups, the C4-C5 double bond, the trans configuration of this double bond, and the NH-protons from either the amide of ceramide or the amine of sphingosine. Therefore, these results provide important information on the requirements for ceramide-enzyme interaction, and they suggest that ligand interaction with the enzyme occurs in a high affinity low specificity manner, in contrast to catalysis which is highly specific for D-erythro-ceramide (D-e-Cer) but occurs with a lower affinity. In addition, this study identifies two competitive inhibitors of mt-CDase; urea-ceramide (C16-urea-Cer) and ceramine (C18-ceramine) that may be further developed and used to understand the mechanism of mt-CDase in vitro and in biologic responses.

Aromatic extended bisamidines: synthesis, inhibition of topoisomerases, and anticancer cytotoxicity in vitro.

A series of four aromatic extended bisamidines (12-15) differing in the nature of their terminal basic side chains were synthesized and evaluated for cytotoxic activity in MCF-7 cultured breast cancer cells. The concentrations of 12, 13, 14, and 15 needed to inhibit [3H]thymidine incorporation into DNA by 50% (IC50) were found to be 63 microM, 85 microM, 77 microM, and 97 microM, respectively. To test whether cytotoxic properties were related to DNA-binding and topoisomerase action, the bisamidines 12-15 were evaluated in a cell-free system. Data from the ethidium displacement assay showed that bisamidines 12-15 have significant affinity for DNA and show moderate specificity for AT base pairs. In the topoisomerase II assay, the relaxation of DNA was inhibited with all four drugs and the extent of inhibition was directly proportional to the drug concentration. This suggests that DNA binding may be implicated in the cytotoxicity of these bisamidines, possibly by inhibiting interactions between topoisomerase II and their DNA targets.

Synthesis, molecular modelling, and antiproliferative and cytotoxic effects of carbocyclic derivatives of distamycin with chlorambucil moiety.

New carbocylic analogues of distamycin and netropsin with chlorambucil moieties 5-8 have been synthesised. Data from the ethidium displacement assay showed that these compounds bind in the minor groove of DNA. The observed reduced affinity to AT pairs and increased affinity towards GC sequences of the carbocyclic lexitropsins with chlorambucil moiety 5-8 in comparison with netropsin and distamycin was observed and rationalised by means of molecular modelling techniques. All of the compounds 5-8 showed antiproliferative and cytotoxic effects in the standard cell line of the mammalian tumour MCF-7.

DNA-binding activity and cytotoxicity of the extended diphenylfuran bisamidines in breast cancer MCF-7 cells.

The DNA binding properties of three novel extended diphenylfuran bisamidines (1-3) possessing different dicationic terminal side chains were studied. The ultrafiltration assay showed that bisamidines 1-3 have significant affinity for DNA. The DNA-binding data for bisamidines 1-3 using homopolymers poly(dA-dT)- poly(dA-dT) and poly(dG-dC)- poly(dG-dC), indicated that these compounds show moderate specificity for AT base pairs. We studied the cytotoxicity effects of bisamidines 1-3, Hoechst 33258 and DAPI (4',6-diamidino-2-phenylindole) in cultured breast cancer MCF-7 cells. The bisamidines 1-3 showed comparable antitumour activity to Hoechst 33258, but were substantially more cytotoxic compared to DAPI. These data show that in broad terms the cytotoxic potency of bisamidines 1-3 in cultured breast cancer MCF-7 cells decreases with the size of the alkyl group substituent (cyclopropyl>isopropyl>cyclopentyl), in accord with their increases in DNA affinity, as shown by the binding constant values.

Decreased cytotoxicity and increased antimitotic activity of a proline analogue of chlorambucil as a prodrug susceptible to the action of fibroblast's prolidase.

We synthesized an proline analogue of chlorambucil (CH-pro) as a prodrug susceptible to the action of ubiquitously distributed, cytosolic imidopeptidase--prolidase [E.C.3.4.13.9]. A conjugation of chlorambucil (CH) with proline through an imido-bond resulted in the formation of a good substrate for prolidase. We have compared several aspects of biological actions of CH and its prodrug in cultured normal human skin fibroblasts. The prodrug was found to be more effectively transported into the cells than the free drug. Moreover, in opposition to CH, CH-pro had no inhibitory effect on fibroblast's prolidase activity against the endogenous substrate, glycyl-L-proline. Lower cytotoxicity and a higher antimitotic activity of the prodrug, compared to the free drug, was observed. CH and CH-pro at concentrations of 25 microM led to a 30% and 10%, decrease in cell viability in confluent human skin fibroblasts. IC50 values of CH and CH-pro for DNA synthesis was found to be 30 microM and 7 microM, suggesting higher antimitotic potency of the pro-drug compared to the free drug. CH-pro also evoked lower ability to inhibit collagen biosynthesis in cultured fibroblasts than the free drug. IC50 values of CH and CH-pro for collagen biosynthesis were found at about 15 microM and 30 microM, respectively. Targeting of prolidase as a prodrug-converting enzyme may serve as a novel strategy in pharmacotherapy of various diseases, leading to the increase in therapeutic efficacy and reduction in untoward side effects of antineoplastic agents.

Cloning and characterization of a novel human alkaline ceramidase. A mammalian enzyme that hydrolyzes phytoceramide.

Ceramidases are enzymes involved in regulating cellular levels of ceramides, sphingoid bases, and their phosphates. Based on sequence homology to the yeast alkaline ceramidases YPC1p (Mao, C., Xu, R., Bielawska, A., and Obeid, L. M. (2000) J. Biol. Chem. 275, 6876--6884) and YDC1p (Mao, C., Xu, R., Bielawska, A., Szulc, Z. M., and Obeid, L. M. (2000) J. Biol Chem. 275, 31369--31378), we report the identification and cloning of a cDNA encoding for a novel human alkaline ceramidase (aPHC) that hydrolyzes phytoceramide selectively. Northern blot analysis showed that aPHC was ubiquitously expressed, with the highest expression in placenta. Green fluorescent protein tagging showed that it was localized in both the Golgi apparatus and endoplasmic reticulum. Overexpression of aPHC in mammalian cells elevated in vitro ceramidase activity toward N-4-nitrobenz-2-oxa-1,3-diazole-C(12)-phytoceramide. Its expression in a yeast mutant strain devoid of any ceramidase activity restored the ceramidase activity and caused an increase in the hydrolysis of phytoceramide in yeast cells, thus leading to the decreased biosynthesis of sphingolipids. These data collectively suggest that, similar to the yeast phytoceramidase YPC1p, aPHC has phytoceramidase activity both in vitro and in cells; hence, it is a functional homolog of the yeast phytoceramidase YPC1p. However, in contrast to YPC1p, aPHC exhibited no reverse activity of ceramidase either in vitro or in cells. Biochemical characterization showed that aPHC had a pH optimum of 9.5, was activated by Ca(2+), but was inhibited by Zn(2+) and sphingosine. Substrate specificity showed that aPHC hydrolyzed phytoceramide preferentially. Together, these data demonstrate that aPHC is a novel human alkaline phytoceramidase, the first mammalian alkaline ceramidase to be identified as being specific for the hydrolysis of phytoceramide.

Biochemical characterization of the reverse activity of rat brain ceramidase. A CoA-independent and fumonisin B1-insensitive ceramide synthase.

We have previously purified a membrane-bound ceramidase from rat brain and recently cloned the human homologue. We also observed that the same enzyme is able to catalyze the reverse reaction of ceramide synthesis. To obtain insight into the biochemistry of this enzyme, we characterized in this study this reverse activity. Using sphingosine and palmitic acid as substrates, the enzyme exhibited Michaelis-Menten kinetics; however, the enzyme did not utilize palmitoyl-CoA as substrate. Also, the activity was not inhibited in vitro and in cells by fumonisin B1, an inhibitor of the CoA-dependent ceramide synthase. The enzyme showed a narrow pH optimum in the neutral range, and there was very low activity in the alkaline range. Substrate specificity studies were performed, and the enzyme showed the highest activity with d-erythro-sphingosine (Km of 0.16 mol %, and Vmax of 0.3 micromol/min/mg), but d-erythro-dihydrosphingosine and the three unnatural stereoisomers of sphingosine were poor substrates. The specificity for the fatty acid was also studied, and the highest activity was observed for myristic acid with a Km of 1.7 mol % and a Vmax of 0.63 micromol/min/mg. Kinetic studies were performed to investigate the mechanism of the reaction, and Lineweaver-Burk plots indicated a sequential mechanism. Two competitive inhibitors of the two substrates were identified, l-erythro-sphingosine and myristaldehyde, and inhibition studies indicated that the reaction followed a random sequential mechanism. The effect of lipids were also tested. Most of these lipids showed moderate inhibition, whereas the effects of phosphatidic acid and cardiolipin were more potent with total inhibition at around 2.5-5 mol %. Paradoxically, cardiolipin stimulated ceramidase activity. These results define the biochemical characteristics of this reverse activity. The results are discussed in view of a possible regulation of this enzyme by the intracellular pH or by an interaction with cardiolipin and/or phosphatidic acid.

Induction of apoptotic cell death and prevention of tumor growth by ceramide analogues in metastatic human colon cancer.

Dysfunction in the physiological pathways of programmed cell death may promote proliferation of malignant cells, and correction of such defects may selectively induce apoptosis in cancer cells. We measured the levels of ceramide, a candidate lipid mediator of apoptosis, in human metastatic colorectal cancer and tested in vitro and in vivo effects of various ceramide analogues in inducing apoptosis in metastatic colon cancer. Human colon cancer showed a > 50% decrease in the cellular content of ceramide when compared with normal colon mucosa. Application of ceramide analogues and ceramidase inhibitors induced rapid cell death through activation of various proapoptotic molecules, such as caspases and release of cytochrome c. Ceramidase inhibition increases the ceramide content of tumor cells, resulting in maximum activation of the apoptotic cascade. Normal liver cells were completely resistant to inhibitors of ceramidases. Treatment of nude mice with B13, the most potent ceramidase inhibitor, completely prevented tumor growth using two different aggressive human colon cancer cell lines metastatic to the liver. Therefore, B13 and related analogues of ceramide and inhibitors of ceramidases offer a promising therapeutic strategy with selective toxicity toward malignant but not normal cells. These studies also suggest that the ceramide content in cancer cells might be involved in the pathogenesis of tumor growth in vitro and in vivo.

Cytotoxicity activity of L-proline analogues of anthraquinone-2-carboxylic acid in breast cancer MCF-7 cells.

Although prolidase [E.C.3.4.13.9] is found in normal cells, substantially increased levels are found in some neoplastic tissues. Prolidase evokes the ability to hydrolyse the imido-bond of various low molecular weight compounds coupled to L-proline. The synthesis of three proline analogues of anthraquinone-2-carboxylic acid (1-3) has been performed. Treatment of these prodrugs with prolidase generated L-proline and the free drug, demonstrating their substrate susceptibility prolidase. The concentrations of 1, 2 and 3 needed to inhibit [1H]thymidine incorporation into DNA by 50% (IC50) in breast cancer MCF-7 cells were found to be 185 +/- 5 microM, 107 +/- 6 microM and 87 +/- 6 microM, respectively, suggesting a lower cytotoxic potency of these compounds compared to Hoechst 33228 (IC50 = 55 +/- 6 microM).