Aintzane Asumendi

The chemopreventive agent N-(4-hydroxyphenyl)retinamide induces apoptosis through a mitochondrial pathway regulated by proteins from the Bcl-2 family

N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) is a potent chemopreventive agent whose effect has been suggested to involve apoptosis induction. 4-HPR induces a loss of the mitochondrial transmembrane potential and the mitochondrial release of cytochrome c before caspase activation. Inhibition of mitochondrial membrane permeabilization (MMP) by transfection with Bcl-2 or the Cytomegalovirus UL37 gene product vMIA prevented caspase activation and cell death. In contrast to other retinoid derivatives, 4-HPR has no direct MMP-inducing effects when added to isolated mitochondria or when added to proteoliposomes containing the MMP-regulatory permeability transition pore complex (PTPC). Moreover, although reactive oxygen species (ROS) overproduction appears to be instrumental for 4-HPR-induced MMP and apoptosis, inhibition of the NF-κB or p53-mediated signal transduction pathways failed to modulate 4-HPR-induced apoptosis. 4-HPR was found to cause an antioxidant-inhibitable conformational change of both Bax and Bak, leading to the exposure of their N-termini and to the mitochondrial relocalization of Bax. Cells with a Bax−/− Bak−/− genotype were resistant against the 4-HPR-induced MMP, overproduction of ROS and cell death. Altogether, these data indicate that 4-HPR induces MMP through an ROS-mediated pathway that involves the obligatory contribution of the proapopotic Bcl-2 family members Bax and/or Bak.

Terfenadine induces apoptosis and autophagy in melanoma cells through ROS-dependent and -independent mechanisms

Previously we found that terfenadine, an H1 histamine receptor antagonist, acts as a potent apoptosis inducer in melanoma cells through modulation of Ca2+ homeostasis. In this report, focusing our attention on the apoptotic mechanisms activated by terfenadine, we show that this drug can potentially activate distinct intrinsic signaling pathways depending on culture conditions. Serum-deprived conditions enhance the cytotoxic effect of terfenadine and caspase-4 and -2 are activated upstream of caspase-9. Moreover, although we found an increase in ROS levels, the apoptosis was ROS independent. Conversely, terfenadine treatment in complete medium induced ROS-dependent apoptosis. Caspase-4, -2, and -9 were simultaneously activated and p73 and Noxa induction were involved. ROS inhibition prevented p73 and Noxa expression but not p53 and p21 expression, suggesting a role for Noxa in p53-independent apoptosis in melanoma cells. Finally, we found that terfenadine induced autophagy, that can promote apoptosis. These findings demonstrate the great potential of terfenadine to kill melanoma cells through different cellular signaling pathways and could contribute to define new therapeutic strategies in melanoma.

Histamine and Histamine Receptor Antagonists in Cancer Biology

Histamine has been demonstrated to be involved in cell proliferation, embryonic development, and tumour growth. These various biological effects are mediated through the activation of specific histamine receptors (H1, H2, H3, and H4) that differ in their tissue expression patterns and functions. Although many in vitro and in vivo studies of the modulatory roles of histamine in tumour development and metastasis have been reported, the effect of histamine in the progression of some types of tumours remains controversial; however, recent findings on the role of histamine in the immune system have shed new light on this question. This review focuses on the recent advances in understanding the roles of histamine and its receptors in tumour biology. We report our recent observations of the anti-tumoural effect of H1 histamine antagonists on experimental and human melanomas. We have found that in spite of exogenous histamine stimulated human melanoma cell proliferation, clonogenic ability and migration activity in a dose-dependent manner, the melanoma tumour growth was not modulated by in vivo histamine treatment. On the contrary, terfenadine-treatment in vitro induced melanoma cell death by apoptosis and in vivo terfenadine treatment significantly inhibited tumour growth in murine models. These observations increase our understanding of cancer biology and may inspire novel anticancer therapeutic strategies.

Bexarotene activates the p53/p73 pathway in human cutaneous T‐cell lymphoma

Background  Bexarotene is the first synthetic retinoid X receptor‐selective retinoid (rexinoid) approved for the treatment of cutaneous T‐cell lymphoma (CTCL). However, little is known about the signalling pathways by which it exerts its anticarcinogenic effect.

Dihydroceramide desaturase activity is modulated by oxidative stress

Oxidative stress has been implicated previously in the regulation of ceramide metabolism. In the present study, its effects on dihydroceramide desaturase were investigated. To stimulate oxidative stress, HEK (human embyronic kidney)-293, MCF7, A549 and SMS-KCNR cells were treated with H2O2, menadione or tert-butylhydroperoxide. In all cell lines, an increase in dihydroceramide was observed upon oxidative stress as measured by LC (liquid chromatography)/MS. In contrast, total ceramide levels were relatively unchanged. Mechanistically, dihydroceramide desaturase activity was measured by an in situ assay and decreased in a time- and dose-dependent fashion. Interestingly, no detectable changes in the protein levels were observed, suggesting that oxidative stress does not induce degradation of dihydroceramide desaturase. In summary, oxidative stress leads to potent inhibition of dihydroceramide desaturase resulting in significant elevation in dihydroceramide levels in vivo.

4-HPR-mediated leukemia cell cytotoxicity is triggered by ceramide-induced mitochondrial oxidative stress and is regulated downstream by Bcl-2

We have previously reported that, in leukemia cells, the cytotoxicity of the anticancer agent N-(4-hydroxyphenyl)retinamide (4-HPR) is mediated by mitochondria-derived reactive oxygen species (ROS) and cardiolipin peroxidation. Here, we have analyzed at greater depth the 4-HPR-triggered molecular events, demonstrating that 4-HPR induces an early (15 min) increase in ceramide levels by sphingomyelin hydrolysis and later (from 1 h) by de novo synthesis. Using specific inhibitors of both pathways, we demonstrate that ceramide accumulation is responsible for early ROS generation, which act as apoptotic signalling intermediates leading to conformational activation of Bak and Bax, loss of mitochondrial membrane potential (ΔΨm), mitochondrial membrane permeabilization (MMP) and cell death. Enforced expression of Bcl-2 has no effect on 4-HPR-induced oxidative stress, but notably prevents mitochondrial alterations and apoptosis, indicating that Bcl-2 functions by regulating events downstream of ROS generation. In conclusion, our study delineates for the fist time the sequence and timing of the principal events induced by 4-HPR in leukemia cells and points to the potential use of modulators of ceramide metabolism as enhancers in 4-HPR-based therapies.

Dihydroceramide accumulation and reactive oxygen species are distinct and nonessential events in 4-HPR-mediated leukemia cell death

4-(Hydroxyphenyl)retinamide (4-HPR) is a synthetic retinoid with a strong apoptotic effect towards different cancer cell lines in vitro, and it is currently tested in clinical trials. Increases of reactive oxygen species (ROS) and modulation of endogenous sphingolipid levels are well-described events observed upon 4-HPR treatment, but there is still a lack of understanding of their relationship and their contribution to cell death. LC-MS analysis of sphingolipids revealed that in human leukemia CCRF-CEM and Jurkat cells, 4-HPR induced dihydroceramide but not ceramide accumulation even at sublethal concentrations. Myriocin prevented the 4-HPR-induced dihydroceramide accumulation, but it did not prevent the loss of viability and increase of intracellular ROS production. On the other hand, ascorbic acid, Trolox, and vitamin E reversed 4-HPR effects on cell death but not dihydroceramide accumulation. NDGA, described as a lipoxygenase inhibitor, exerted a significantly higher antioxidant activity than vitamin E and abrogated 4-HPR-mediated ROS. It did not however rescue cellular viability. Taken together, this study demonstrates that early changes observed upon 4-HPR treatment, i.e., sphingolipid modulation and ROS production, are mechanistically independent events. Furthermore, the results indicate that 4-HPR-driven cell death may occur even in the absence of dihydroceramide or ROS accumulation. These observations should be taken into account for an improved design of drug combinations.

A Customized Pigmentation SNP Array Identifies a Novel SNP Associated with Melanoma Predisposition in the SLC45A2 Gene

As the incidence of Malignant Melanoma (MM) reflects an interaction between skin colour and UV exposure, variations in genes implicated in pigmentation and tanning response to UV may be associated with susceptibility to MM. In this study, 363 SNPs in 65 gene regions belonging to the pigmentation pathway have been successfully genotyped using a SNP array. Five hundred and ninety MM cases and 507 controls were analyzed in a discovery phase I. Ten candidate SNPs based on a p-value threshold of 0.01 were identified. Two of them, rs35414 (SLC45A2) and rs2069398 (SILV/CKD2), were statistically significant after conservative Bonferroni correction. The best six SNPs were further tested in an independent Spanish series (624 MM cases and 789 controls). A novel SNP located on the SLC45A2 gene (rs35414) was found to be significantly associated with melanoma in both phase I and phase II (P<0.0001). None of the other five SNPs were replicated in this second phase of the study. However, three SNPs in TYR, SILV/CDK2 and ADAMTS20 genes (rs17793678, rs2069398 and rs1510521 respectively) had an overall p-value<0.05 when considering the whole DNA collection (1214 MM cases and 1296 controls). Both the SLC45A2 and the SILV/CDK2 variants behave as protective alleles, while the TYR and ADAMTS20 variants seem to function as risk alleles. Cumulative effects were detected when these four variants were considered together. Furthermore, individuals carrying two or more mutations in MC1R, a well-known low penetrance melanoma-predisposing gene, had a decreased MM risk if concurrently bearing the SLC45A2 protective variant. To our knowledge, this is the largest study on Spanish sporadic MM cases to date.

Apoptosis of human T-cell acute lymphoblastic leukemia cells by diphenhydramine, an H1 histamine receptor antagonist.

Recently, it has been demonstrated that histamine plays an important role in the proliferation of normal and malignant cells. We have examined the effects of histamine, diphenhydramine, and cimetidine (H1 and H2 histamine receptor antagonists, respectively) on the in vitro proliferation of two human T-cell acute lymphoblastic leukemia cell lines, namely CCRF-CEM and Jurkat. Exogenous histamine did not alter the proliferation or viability of these cells. In contrast, diphenhydramine induced apoptosis in a dose- and time-dependent manner in both cell lines, whereas cimetidine failed to induce significant effects at similar concentrations. Diphenhydramine-induced apoptosis was evaluated in terms of morphology, flow cytometry, and the release of cytochrome c to the cytosol. The latter was partially mitigated by Bcl-2 overexpression. In human peripheral blood mononuclear cells, diphenhydramine inhibited cell proliferation without inducing apoptosis. Our findings indicate that endogenous histamine may be an important factor for the survival of CCRF-CEM, Jurkat, and peripheral blood mononuclear cells, and point to the potential application of H1 receptor antagonists as cytotoxic agents for the specific treatment of certain types of leukemia.

Cell-Centric View of Apoptosis and Apoptotic Cell Death-Inducing Antitumoral Strategies

Programmed cell death and especially apoptotic cell death, occurs under physiological conditions and is also desirable under pathological circumstances. However, the more we learn about cellular signaling cascades, the less plausible it becomes to find restricted and well-limited signaling pathways. In this context, an extensive description of pathway-connections is necessary in order to point out the main regulatory molecules as well as to select the most appropriate therapeutic targets. On the other hand, irregularities in programmed cell death pathways often lead to tumor development and cancer-related mortality is projected to continue increasing despite the effort to develop more active and selective antitumoral compounds. In fact, tumor cell plasticity represents a major challenge in chemotherapy and improvement on anticancer therapies seems to rely on appropriate drug combinations. An overview of the current status regarding apoptotic pathways as well as available chemotherapeutic compounds provides a new perspective of possible future anticancer strategies.