Hydrolytic pathway protects against ceramide-induced apoptosis in keratinocytes exposed to UVB.

Although ceramides (Cers) are key constituents of the epidermal permeability barrier, they also function as apoptogenic signals for UVB irradiation-induced apoptosis in epidermal keratinocytes. As epidermis is continuously exposed to UV irradiation, we hypothesized that Cer hydrolysis protects keratinocytes from UVB-induced apoptosis by attenuating Cer levels. Both low-dose UVB (L-UVB) (< 35 mJ cm(-2)) and high-dose UVB (H-UVB) (> or = 45 mJ cm(-2)) irradiation inhibited DNA synthesis in cultured human keratinocytes, but apoptosis occurred only after H-UVB. Whereas Cer production increased after both L- and H-UVB, it normalized only in L-UVB-exposed keratinocytes, but remained elevated after H-UVB. Both acidic ceramidase (aCDase) and neutral ceramidase (nCDase) activities declined after L- and H-UVB, but returned to normal only in L-UVB cells, with decreased CDase activities or mRNA or protein levels being sustained in H-UVB cells. Inhibition of CDase using either a CDase inhibitor, N-oleoylethanolamine, or small interfering RNA (siRNA) (either to a- and/or n-CDase(s)) sensitized keratinocytes to L-UVB-induced apoptosis in parallel with further Cer accumulation. Blockade of sphingosine kinase 1 (SPHK1) (but not SPHK2) by siRNA also increased apoptosis in L-UVB keratinocytes, revealing that conversion of sphingosine to sphingosine-1-phosphate (S1P) further protects keratinocytes from UVB-induced cell death. Thus, Cer → sphingosine → S1Pmetabolic conversion protects against UVB-induced, Cer-mediated apoptosis in keratinocytes, but excessive UVB overwhelms this mechanism, thereby leading to keratinocyte apoptosis.

New antitumor imidazo[2,1-b]thiazole guanylhydrazones and analogues.

The synthesis of new antitumor 6-substituted imidazothiazole guanylhydrazones is described. Moreover, a series of compounds with a different basic chain at the 5 position were prepared. Finally, the replacement of the thiazole ring in the imidazothiazole system was also considered. All the new compounds prepared were submitted to the NCI cell line screen for evaluation of their antitumor activity. A few selected compounds were submitted to additional biological studies concerning effects on the cell cycle, apoptosis, and mitochondria.

Meta-analysis of anticancer drug structures--significance of their polar allylic moieties.

This meta-analysis examines a wide range of small molecule anticancer drugs to search for a structure common to all. Although they encompass a very wide range of structures, nearly all reveal the presence of an allylic O, N, or S atom. In some, the allylic oxygen is a carbonyl group, or an alcohol group, which can be substituted (ester, lactone, glycoside, ether) or replaced by an amino or imino nitrogen Some antineoplastic drugs do not exhibit this moiety but are converted in vivo to allylic derivatives. An allylic hydroxyl is also present in most sphingolipids, ubiquitous body components that control proliferative and anti-proliferative cell functions. Ceramide, the precursor of all the allylic sphingolipids, seems to be a general inducer of apoptosis in cancer cells. Further examination of sphingolipids and anticancer drugs shows the frequent occurrence of [i] double bonds conjugated to the allylic bond, (ii) two or more allylic moieties in each molecule, (iii) lipophilic features, especially linear chains, and (iv) attachment of an O, N, or S atom to a carbon atom of the allylic double bond, e.g., -CH(2)-C(OMe)=CH-CH(OH)-CH(2)-. Suggested mechanisms of action: (a) allylic ketone drugs undergo a Michael condensation with tumor thiols or other reactive groups; (b) allylic OH drugs undergo oxidation to an allylic ketone, generating reactive oxygen; (c) some interfere with mitochondrial ubiquinone, blocking ATP production; (d) some act as a ceramide mimic (inhibitor or agonist) in ceramide-controlled kinases, phosphatases, and proteases; (e) many antineoplastic drugs stimulate ceramide-forming processes.

Allylic structures in cancer drugs and body metabolites that control cell life and death.

This review presents data supporting the hypothesis that the anticancer activity of ceramide and many antineoplastic drugs is due to a 3-carbon allylic moiety (-C = C-C-) containing oxygen or nitrogen. The polar atom appears as an alcohol, ether, ester, amide, ketone, amine or imino group. Some drugs lack the allylic moiety, but metabolic oxidation or oxygenation in patients introduces the moiety. The allylic compounds kill cancer cells by: i) interference with ubiquinone in mitochondria, generating reactive oxygen species (ROS); ii) activation of enzymatic hydrolysis of sphingomyelin by the ROS, forming ceramide, which initiates mitochondrial destruction and apoptosis; iii) activation of the phosphorylation and dephosphorylation of proteins involved in apoptosis by ceramide and some allylic drugs and iv) activation of certain proteases, such as cathepsin D, by ceramide.

Preventing the binding of pathogens to the host by controlling sphingolipid metabolism.

The binding of many pathogens and toxins to human cells can be inhibited by (1) depleting host cells of their surface glycosphingolipids; (2) coating the binding sites on pathogens (adhesins) with glycosphingolipid-like substances (decoys); (3) coating the host's glycosphingolipids with substances that compete with the pathogen for binding. Details of using these methods are described.

Sphingolipids as coenzymes in anion transfer and tumor death.

Many kinds of natural sphingolipids and their analogs stimulate or inhibit a wide assortment of biochemical phenomena and enzymes. The puzzle considered here is: how can these lipids control so many different kinds of processes? In almost every study in which a structural comparison was made, an allylic alcohol moiety [-CH=CH-CH(OH)-] was found to be an essential feature of the sphingolipid. Many of those stimulations lead to cell death, emphasizing the importance of allylic sphingolipid structure in the design of chemotherapeutic agents. The proposal offered here is that these lipids function as coenzymes, in which the allylic moiety acts as an anion transferring agent, forming transient phosphate or acyl or peptidyl esters for the synthesis or hydrolysis of phosphoproteins, proteins, and phospholipids. Sphingolipids that inhibit these reactions may simply displace the active sphingolipids from their sites in the enzymes' active regions, or bind to the enzymes' allosteric region. This kind of competition could act as a major homeostatic control mechanism. Some of the allylic sphingolipids also generate reactive oxygen, possibly by oxidation of the allylic alcohol group. This explains the need to control redox-controlling metabolites in sphingolipid-controlled processes (e.g., glutathione). Many anticancer drugs that produce apoptosis in tumors possess an allylic alcohol residue, affect protein phosphorylation, and produce reactive oxygen species. They may be therapeutically useful because they control the action of sphingolipids as anion transfer agonists or inhibitors.

Poly-drug cancer therapy based on ceramide.

Thousands of research studies have reported that many kinds of cancer cells and tumors can be killed by treatments that increase the concentration of a simple cellular sphingolipid, ceramide (Cer). While there are many ways to elevate tumor Cer levels, this approach is complicated by the central, complex role of Cer in cell homeostasis: Cer is readily metabolized to form other sphingolipids that increase the tumor's growth rate, metastasis, and resistance to the patient's immune system. This review points out the need to prevent this metabolic conversion while simultaneously stimulating the enzymes that increase the formation of Cer. I describe here many of the enzymes that need stimulation or inhibition, and drugs or metabolites or dietary components that modify each of the enzymes. The review also points to the importance of the allylic alcohol group in Cer and in many cancer drugs, suggesting that the hydroxyl group participates in phosphate transfer to and from proteins by forming a temporary phosphate ester. The allylic hydroxyl may also reduce the ketone moieties in mitochondrial ubiquinone, with formation of reactive oxygen species and apoptogenic breakdown. The level of Cer in tumors can be increased by: (1) direct administration of Cer or a Cer analogue, and (2) stimulation of Cer synthesis from its elementary precursors, or from (3) sphingomyelin by hydrolysis, or from (4) the glucosphingolipids by hydrolysis, or (5) by acylation of sphingosine. In addition, Cer concentration can be raised by slowing its conversion to (6) sphingomyelin, (7) glucosylCer, (8) Cer phosphate, and (9) sphingosine + fatty acid by hydrolysis. Therapeutic radiation stimulates the de novo synthesis of Cer in tumors. Conversion of sphingosine (from Cer) to sphingosine phosphate probably also ought to be blocked.

Designing anticancer drugs via the achilles heel: ceramide, allylic ketones, and mitochondria.

Published reports are reviewed as the basis of a proposal that an effective antineoplastic drug should contain several features: (a) resemblance to the natural lipid, ceramide; (b) an allylic alcohol and/or allylic ketone moiety; (c) a hydroxyl and/or a nitrogen atom near the allylic group; (d) conjugated double bonds as part of the allylic region. The drug should produce reactive oxygen species in tumor mitochondria, stimulate the generation of ceramide in the tumor, and condense with mitochondrial glutathione. It is pointed out that some antibiotics with these features are also active against cancer cells; perhaps anticancer drugs with these features will prove useful as antibiotics. Common problems in working with lipoidal substances are discussed.

Killing tumours by ceramide-induced apoptosis: a critique of available drugs.

Over 1000 research papers have described the production of programmed cell death (apoptosis) by interventions that elevate the cell content of ceramide (Cer). Other interventions, which lower cellular Cer, have been found to interfere with apoptosis induced by other agents. Some studies have shown that slowing the formation of proliferation-stimulating sphingolipids also induces apoptosis. These relationships are due to the two different aspects of Cer: Cer itself produces apoptosis, but metabolic conversion of Cer into either sphingosine 1-phosphate or glucosphingolipids leads to cell proliferation. The balance between these two aspects is missing in cancer cells, and yet intervention by stimulating or blocking only one or two of the pathways in Cer metabolism is very likely to fail. This results from two properties of cancer cells: their high mutation rate and the preferential survival of the most malignant cells. Tumours treated with only one or two drugs that elevate Cer can adjust the uncontrolled processes to either maintain or to 'aggravate' the excessive growth, angiogenesis and metastasis characteristics of tumours. These treatments might simply elevate the production of growth factors, receptors and other substances that reduce the effectiveness of Cer. Tumour cells that do not adapt in this way undergo apoptosis, leaving the adapted cells free to grow and, ultimately, to 'subdue' their host. Thus it is important to kill every type of cancer cell present in the tumour rapidly and simultaneously, using as many different agents to control as many pathways as possible. To aid this approach, this article catalogues many of the drugs that act on different aspects of Cer metabolism. The techniques described here may lead to the development of practical chemotherapy for cancer and other diseases of excess proliferation.