Gorka Pérez-Yarza

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.

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.

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.

A structural analysis of cytoskeleton components during the execution phase of apoptosis

SummaryDuring the execution phase of apoptosis, the cell undergoes a set of morphological changes which reveal the activation of a complex machinery leading the cell to its disruption into small, spherical, membrane-bounded fragments called apoptotic bodies. In the present study, we have focused on the implications of the micro-filament network in the early stages of the active phase of apoptosis. By using confocal microscopy, we have analysed the location of the actin microfilaments and two actin-binding proteins, α-actinin and myosin, in F9 embryonal carcinoma cells undergoing apoptosis during the stages previous to their fragmentation. Our results show that these proteins locate in the centre of the disrupting cell and form a three-dimensional structure which suggests the existence of a fully functional contractile system involved in the fragmentation of the cell and the formation of apoptotic bodies.

Laminin surface binding sites and metastatic potential of 3LL tumor cells, increased by indomethacin

The level of laminin receptor expression on tumor cell surface has been correlated with the capacity of tumor cells to metastasize. In the present work we show that indomethacin treatment of a low metastatic 3LL tumor cells increases the ability of these cells to form lung metastasis and the binding of [125I] laminin on their cell surface. Scatchard analysis showed that the incubation with indomethacin (10(-7) M) for 48 h induced a specific increase of laminin binding sites on 3LL cell surface (1.5 fold per cell), presenting both a high and low affinity class of binding sites. On the other hand, indomethacin treatment (2 mg/kg weight) of tumor bearing mice increased the number of spontaneous metastatic nodules on the lung surface. Likewise, when 3LL tumor cells were incubated with indomethacin (10(-7) M) for 48 h, we observed an enhancement of lung metastatic nodules after intravenous injection of tumor cells. This last effect was partially reversed by peptides DPGYIGSR or YIGSR, corresponding to the active site at the B1 chain of laminin, with ability to bind the 67-kD laminin cell surface receptors. In summary, our results show that the increased attachment of 3LL tumor cells to laminin mediated by indomethacin is directly correlated with the metastatic activity of these cells, and suggests that the indomethacin effect on the metastatic potential could involve a modulation of laminin receptors on tumor cell surface.

Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

BackgroundN-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) is a synthetic retinoid with potent pro-apoptotic activity against several types of cancer, but little is known regarding mechanisms leading to chemoresistance. Ceramide and, more recently, other sphingolipid species (e.g., dihydroceramide and dihydrosphingosine) have been implicated in 4-HPR-mediated tumor cell death. Because sphingolipid metabolism has been reported to be altered in drug-resistant tumor cells, we studied the implication of sphingolipids in acquired resistance to 4-HPR based on an acute lymphoblastic leukemia model.MethodsCCRF-CEM cell lines resistant to 4-HPR were obtained by gradual selection. Endogenous sphingolipid profiles and in situ enzymatic activities were determined by LC/MS, and resistance to 4-HPR or to alternative treatments was measured using the XTT viability assay and annexin V-FITC/propidium iodide labeling.ResultsNo major crossresistance was observed against other antitumoral compounds (i.e. paclitaxel, cisplatin, doxorubicin hydrochloride) or agents (i.e. ultra violet C, hydrogen peroxide) also described as sphingolipid modulators. CCRF-CEM cell lines resistant to 4-HPR exhibited a distinctive endogenous sphingolipid profile that correlated with inhibition of dihydroceramide desaturase. Cells maintained acquired resistance to 4-HPR after the removal of 4-HPR though the sphingolipid profile returned to control levels. On the other hand, combined treatment with sphingosine kinase inhibitors (unnatural (dihydro)sphingosines ((dh)Sph)) and glucosylceramide synthase inhibitor (PPMP) in the presence or absence of 4-HPR increased cellular (dh)Sph (but not ceramide) levels and were highly toxic for both parental and resistant cells.ConclusionsIn the leukemia model, acquired resistance to 4-HPR is selective and persists in the absence of sphingolipid profile alteration. Therapeutically, the data demonstrate that alternative sphingolipid-modulating antitumoral strategies are suitable for both 4-HPR-resistant and sensitive leukemia cells. Thus, whereas sphingolipids may not be critical for maintaining resistance to 4-HPR, manipulation of cytotoxic sphingolipids should be considered a viable approach for overcoming resistance.

Vitronectin and dermcidin serum levels predict the metastatic progression of AJCC I-II early-stage melanoma.

Like many cancers, an early diagnosis of melanoma is fundamental to ensure a good prognosis, although an important proportion of stage I–II patients may still develop metastasis during follow‐up. The aim of this work was to discover serum biomarkers in patients diagnosed with primary melanoma that identify those at a high risk of developing metastasis during the follow‐up period. Proteomic and mass spectrophotometry analysis was performed on serum obtained from patients who developed metastasis during the first years after surgery for primary tumors and compared with that from patients who remained disease‐free for more than 10 years after surgery. Five proteins were selected for validation as prognostic factors in 348 melanoma patients and 100 controls by ELISA: serum amyloid A and clusterin; immune system proteins; the cell adhesion molecules plakoglobin and vitronectin and the antimicrobial protein dermcidin. Compared to healthy controls, melanoma patients have high serum levels of these proteins at the moment of melanoma diagnosis, although the specific values were not related to the histopathological stage of the tumors. However, an analysis based on classification together with multivariate statistics showed that tumor stage, vitronectin and dermcidin levels were associated with the metastatic progression of patients with early‐stage melanoma. Although melanoma patients have increased serum dermcidin levels, the REPTree classifier showed that levels of dermcidin <2.98 μg/ml predict metastasis in AJCC stage II patients. These data suggest that vitronectin and dermcidin are potent biomarkers of prognosis, which may help to improve the personalized medical care of melanoma patients and their survival.