The autophagy inducer trehalose stimulates macropinocytosis in NF1-deficient glioblastoma cells.

BACKGROUND: Glioblastoma is a highly aggressive brain tumor. A big effort is required to find novel molecules which can cross the blood-brain barrier and efficiently kill these tumor cells. In this perspective, trehalose (α-glucopyranosyl-[1→1]-α-D-glucopyranoside), found in various dietary sources and used as a safe nutrient supplement, attracted our attention for its pleiotropic effects against tumor cells. METHODS: Human glioblastoma cell lines U373-MG and T98G were exposed to trehalose and analyzed at different time points. Cell proliferation was evaluated at medium term, and clonogenic capacity and cell morphology were evaluated at long term. Western blot was used to evaluate biochemical markers of autophagy (also measured in cells co-treated with EIPA or chloroquine), and mTOR, AMPK and ERK 1/2 signalling. Macropinocytosis was evaluated morphologically by bright-field microscopy; in cells loaded with the fluorescein-conjugated fluid-phase tracer dextran, macropinocytic vacuoles were also visualized by fluorescence microscopy, and the extent of macropinocytosis was quantified by flow cytometry. RESULTS: The long-term effect of trehalose on U373-MG and T98G cell lines was impressive, as indicated by a dramatic reduction in clonogenic efficiency. Mechanistically, trehalose proved to be an efficient autophagy inducer in macropinocytosis-deficient T98G cells and an efficient inducer of macropinocytosis and eventual cell death by methuosis in U373-MG glioblastoma cells, proved to be poorly responsive to induction of autophagy. These two processes appeared to act in a mutually exclusive manner; indeed, co-treatment of U373-MG cells with the macropinocytosis inhibitor, EIPA, significantly increased the autophagic response. mTOR activation and AMPK inhibition occurred in a similar way in the two trehalose-treated cell lines. Interestingly, ERK 1/2 was activated only in macropinocytosis-proficient U373-MG cells harbouring loss-of-function mutations in the negative RAS regulator, NF1, suggesting a key role of RAS signalling. CONCLUSIONS: Our results indicate that trehalose is worthy of further study as a candidate molecule for glioblastoma therapy, due to its capacity to induce a sustained autophagic response, ultimately leading to loss of clonogenic potential, and more interestingly, to force macropinocytosis, eventually leading to cell death by methuosis, particularly in tumor cells with RAS hyperactivity. As a further anticancer strategy, stimulation of macropinocytosis may be exploited to increase intracellular delivery of anticancer drugs.

Trehalose inhibits cell proliferation and amplifies long-term temozolomide- and radiation-induced cytotoxicity in melanoma cells: A role for autophagy and premature senescence.

Cutaneous melanomas frequently metastasize to the brain, with temozolomide (TMZ) plus radiotherapy (RT) offering little control of these lesions. We tested whether trehalose, a natural glucose disaccharide proved to induce autophagy, could enhance the effect of TMZ and ionizing radiation (IR). In two melanoma cell lines (A375 and SK-Mel-28), which greatly differ in chemosensitivity and radiosensitivity, trehalose significantly inhibited short-term cell proliferation and also enhanced IR-induced cytostasis. Interestingly, in TMZ-resistant SK-Mel-28 cells, trehalose was more effective than TMZ, and combined trehalose + TMZ further reduced cell proliferation. In long-term experiments, colony-forming capacity was dramatically reduced by trehalose, and even more by combined trehalose + TMZ or trehalose + IR. In resistant SK-Mel-28 cells, although growth was inhibited most with trehalose + TMZ + IR-6 Gy combined treatment, it is notable that trehalose + TMZ treatment was also very effective. Along with a direct antiproliferative effect, two further mechanisms may explain how trehalose potentiates TMZ- and IR-induced effects: the remarkable trehalose-stimulated autophagy in A375 cells, which were sensitive to TMZ- and IR-induced apoptosis; and the notable trehalose-stimulated premature senescence in SK-Mel-28 cells, which were resistant to apoptosis and less prone to autophagy. In normal melanocytes, trehalose induced a minor autophagy and cell proliferation inhibition, without affecting cell viability; moreover, when trehalose was used in combination with TMZ, the slight TMZ-induced cytotoxicity was not significantly reinforced. Together, our results suggest that trehalose, a safe nutrient supplement able to cross the blood-brain barrier, is a promising candidate, worthy to be further explored in vivo, to augment the therapeutic efficacy of TMZ and RT in melanoma brain metastases.

Calpains and cancer: friends or enemies?

Calpains are a complex family of ubiquitous or tissue-specific cysteine proteases that proteolyze a variety of substrates (leading to their degradation or functional modulation) and are implicated in several pathophysiological phenomena. In tumor cell biology, calpains are implicated in a triple way: they are involved in different processes crucial for tumor progression, including cell proliferation, apoptotic cell death, survival mechanisms, migration and invasiveness; they have aberrant expression in several human cancers; a variety of anticancer drugs induce cytotoxicity through activation of calpains or the latter can influence response to therapy. This review covers established and recent literature showing these diverse aspects in tumor cells.

The dark side of gamma-glutamyltransferase (GGT): Pathogenic effects of an 'antioxidant' enzyme.

Having long been regarded as just a member in the cellular antioxidant systems, as well as a clinical biomarker of hepatobiliary diseases and alcohol abuse, gamma-glutamyltransferase (GGT) enzyme activity has been highlighted by more recent research as a critical factor in modulation of redox equilibria within the cell and in its surroundings. Moreover, due to the prooxidant reactions which can originate during its metabolic function in selected conditions, experimental and clinical studies are increasingly involving GGT in the pathogenesis of several important disease conditions, such as atherosclerosis, cardiovascular diseases, cancer, lung inflammation, neuroinflammation and bone disorders. The present article is an overview of the laboratory findings that have prompted an evolution in interpretation of the significance of GGT in human pathophysiology.

Autoantibodies against the autophagic protein microtubule-associated light-chain 3 (LC3): Immunocharacterization of an atypical ANA pattern.

Autoantibodies to nuclear and cytoplasmic antigens are commonly detected by indirect immunofluorescence (IIF) on HEp-2 cells, and three major staining patterns (nuclear, cytoplasmic, and mitotic) are distinguished. Here, we report an atypical cytoplasmic pattern, not described so far, observed in the serum of a patient with a controversial diagnosis of systemic lupus erythematosus (SLE). Moreover, for the first time, we have revealed the presence of autoantibodies against the microtubule-associated light-chain 3 (LC3) protein, which plays a key role in the autophagic process. The target antigen has been identified in IIF by means of a competition test using purified anti-LC3 antibodies on HEp-2 cells, and confirmed by Western blot analysis using cellular or recombinant LC3 as antigen, immunoreacted with the patient's serum. The identification of this atypical pattern and the related autoantibody-antigen system sheds new light on autophagy, which is increasingly considered to be involved in the etiopathogenesis of autoimmune disorders, and could contribute to select more personalized therapies.

Cisplatin/hydrogel complex in cancer therapy.

Hydrogels containing alpha-amino acid residues (L-phenylalanine, L-histidine) were used to complex the chemotherapeutic agent cisplatin. The release of the drug in phosphate buffer solution showed an initial burst effect, followed by a near zero-order release phase over the seven days of reported period. Unlike the nonreleasing pattern of the hydrogel poly(N-acryloyl-L-phenylalanine-co-N-isopropylacrylamide) (CP2), the homopolymer poly(N-acryloyl-L-phenylalanine) (P9) hydrogel showed a released amount of cisplatin loaded from a water/DMSO mixture that was three times greater than that loaded from simple water. The hydrogel P9 formed with cisplatinum(II) complex species of well-defined stoichiometry; the drug species was released by a chemically controlled process. The Pt(II)/L (L is the monomeric unit of the polymer) stoichiometric molar ratio of 0.5, corresponding to two close carboxylate groups per Pt(II), was found by the viscometric data on the soluble polymer analogue. The platinum species released from cisplatin-loaded (from water) hydrogel retained its cytotoxic activity toward Me665/2/21 human melanoma cell line, in the same manner shown by the native cisplatin. On the contrary, the platinum species released from cisplatin-loaded (from water/DMSO) hydrogel was devoid of any cytotoxic effect.

Autophagy up-regulation by ulipristal acetate as a novel target mechanism in the treatment of uterine leiomyoma: an in vitro study.

OBJECTIVE: To assess the effect of ulipristal acetate (UPA) on the autophagic process of uterine leiomyoma cells. DESIGN: In vitro study in primary cultures of leiomyoma and myometrial cells isolated from biopsy specimen, and gene expression evaluation in biopsy material. SETTING: Cellular pathology laboratory. PATIENT(S): Premenopausal women (without hormonal treatment) undergoing myomectomy or hysterectomy for symptomatic leiomyomas. INTERVENTION(S): Surgical specimens collected from uterine leiomyomas and matched normal myometria. MAIN OUTCOME MEASURE(S): After treatment of myometrial and leiomyoma cells with UPA, autophagy was evaluated by Western blot analysis of the typical biochemical markers, LC3-II, LC3-II:LC3-I ratio, and p62/SQSTM1. The expression level of Atg7 and Atg4D proteins was also assessed by Western blot. RESULT(S): The increase of LC3-II protein, LC3-II:LC3-I ratio, and p62/SQSTM1 protein indicates that UPA treatment up-regulates the autophagic response in leiomyoma cells, whereas these markers were almost unchanged in myometrial cells. Consistently, an increased level of Atg7 and Atg4D proteins was observed only in UPA-treated leiomyoma cells. The autophagic machinery is put into motion selectively in these cells, despite that the basal messenger RNA levels of LC3, SQSTM1, and ATG7 in leiomyoma biopsy specimen were not significantly different from those found in normal myometrial biopsy material. CONCLUSION(S): In vitro UPA treatment stimulates the autophagic response selectively in leiomyoma cells, which adds a novel indication for the clinical use of this selective P receptor (PR) modulator. Autophagy up-regulation may potentially contribute to the leiomyoma shrinkage occurring in UPA-treated patients and warrants further study.

Potassium Ascorbate with Ribose: Promising Therapeutic Approach for Melanoma Treatment.

While surgery is the definitive treatment for early-stage melanoma, the current therapies against advanced melanoma do not yet provide an effective, long-lasting control of the lesions and a satisfactory impact on patient survival. Thus, research is also focused on novel treatments that could potentiate the current therapies. In the present study, we evaluated the effect of potassium ascorbate with ribose (PAR) treatment on the human melanoma cell line, A375, in 2D and 3D models. In the 2D model, in line with the current literature, the pharmacological treatment with PAR decreased cell proliferation and viability. In addition, an increase in Connexin 43 mRNA and protein was observed. This novel finding was confirmed in PAR-treated melanoma cells cultured in 3D, where an increase in functional gap junctions and a higher spheroid compactness were observed. Moreover, in the 3D model, a remarkable decrease in the size and volume of spheroids was observed, further supporting the treatment efficacy observed in the 2D model. In conclusion, our results suggest that PAR could be used as a safe adjuvant approach in support to conventional therapies for the treatment of melanoma.

Macrophage migration inhibitory factor protein and mRNA expression in cutaneous melanocytic tumours.

Macrophage migration inhibitory factor (MIF) is a widely expressed cytokine involved in various biological processes. Although MIF's functions in cancer have not been completely elucidated, its expression has usually been correlated with tumour progression and aggressiveness, and it is currently discussed as a new promising target for novel therapies. Recent studies seem to confirm its active role in melanoma pathobiology; however, its expression has not yet been extensively studied in melanocytic tumours. We evaluated MIF protein expression in 126 skin lesions, including benign and atypical nevi, melanoma and melanoma metastases. In 55 cases, we also assessed MIF mRNA expression by real-time RT-PCR. Benign nevi were subdivided into nevocytic and Spitz/blue types; and melanomas into the radial, and vertical growth phase. A strong cytoplasmic MIF positivity was found in most samples, although it was more heterogeneous in malignant tumours; MIF nuclear expression characterized Spitz/blue nevi, atypical nevi, melanomas and metastases. All samples expressed MIF mRNA but it was significantly lower in benign nevi vs atypical nevi, melanomas and metastases (p=0.001; p<0.0001; p=0.002, respectively). Our study shows a widespread distribution of MIF among melanocytic tumours. Whereas we observed a trend towards higher expression levels of mRNA in atypical and malignant tumours, MIF protein was highly expressed in all lesions, although limited to the cytoplasm in most benign nevi. These observations suggest differences in MIF protein storage, subcellular location and properties in most benign nevi vs atypical and malignant tumours that should be confirmed by further investigation and correlation with clinical outcome.

Suppressed translation and ULK1 degradation as potential mechanisms of autophagy limitation under prolonged starvation.

Macroautophagy/autophagy is a well-organized process of intracellular degradation, which is rapidly activated under starvation conditions. Recent data demonstrate a transcriptional upregulation of several autophagy genes as a mechanism that controls autophagy in response to starvation. Here we report that despite the significant upregulation of mRNA of the essential autophagy initiation gene ULK1, its protein level is rapidly reduced under starvation. Although both autophagic and proteasomal systems contribute to the degradation of ULK1, under prolonged nitrogen deprivation, its level was still reduced in ATG7 knockout cells, and only initially stabilized in cells treated with the lysosomal or proteasomal inhibitors. We demonstrate that under starvation, protein translation is rapidly diminished and, similar to treatments with the proteosynthesis inhibitors cycloheximide or anisomycin, is associated with a significant reduction of ULK1. Furthermore, it was found that inhibition of the mitochondrial respiratory complexes or the mitochondrial ATP synthase function that could also take place in the absence of substrates, promote upregulation of ULK1 mRNA and protein expression in an AMPK-dependent manner in U1810 lung cancer cells growing in complete culture medium. These inhibitors could also drastically increase the ULK1 protein in U1810 cells with knockout of ATG13, where the ULK1 expression is significantly diminished. However, such upregulation of ULK1 protein is negligible under starvation conditions, further signifying the contribution of translation and suggesting that transcriptional upregulation of ULK1 protein will be diminished under such conditions. Thus, we propose a model where inhibition of protein translation, together with the degradation systems, limit autophagy during starvation.

Antitubulinic effect of New Fluorazone Derivatives on Melanoma Cells.

Microtubules are composed by α- and ß-tubulin polypeptides. α-tubulin undergoes a reversible posttranslational modification whereby the C-terminal tyrosine residue is removed (Glu-tubulin) and re-added (Tyrtubulin). Recent studies have shown that α-tubulin tyrosine residues can be nitrated and the incorporation of NO2Tyr into the C-terminus of Glu-tubulin forms a complex that blocks the tyrosination/detyrosination cycle, an event that can compromise protein/enzyme functions, such as cell division. Since many studies demonstrated that Glu-tubulin levels increase in cancer, the aim of the present study was to investigate the effect of new drugs, fluorazone derivatives (K1-K2-K9-K10-K11), on the proliferation of melanoma cells. Our results demonstrated that these drugs, except for K2, were able to inhibit cellular proliferation without exhibiting cytotoxicity. The anti-proliferative effect was accompanied by the decrease of Glu-tubulin levels and the increase of its nitration. This effect seems to be a consequence of NO2 induction and NO2Tyr ligation to Glu-tubulin. Collectively, these results, showing that the fluorazone derivatives, by promoting NO2Tyr incorporation into α-tubulin, are able to arrest the cycle of detyrosination/tyrosination and to inhibit cell proliferation, offer new perspectives for the possible usage of these drugs, alone or in combination, as non-toxic, anti-proliferative agents in melanoma.

Redox modulation of protein kinase/phosphatase balance in melanoma cells: the role of endogenous and gamma-glutamyltransferase-dependent H2O2 production.

Alterations of protein kinase and protein phosphatase activities have been described in a number of tumors. Redox changes, such as in conditions of oxidant stress, have been reported to affect the cellular protein kinase/phosphatase balance. A basal production of reactive oxygen species (ROS), such as hydrogen peroxide (H(2)O(2)), exists in tumor cells, and the membrane-bound ecto-enzyme gamma-glutamyltransferase (GGT)-overexpressed in a variety of malignant tumors-is one of the mechanisms capable of promoting such a production. The present study was aimed to verify the interactions of GGT activity with protein phosphatase and kinase activities in Me665/2/60 melanoma cells, expressing high levels of this enzyme and exhibiting both basal and GGT-dependent production of hydrogen peroxide. An increase of total phosphatase as well as tyrosine phosphatase activities was observed after treatment of cells with both micromolar H(2)O(2) and GGT stimulation. Accordingly, stimulation of GGT resulted in decreased levels of phosphotyrosine. On the other hand, when serine/threonine phosphatase activities were selectively analyzed, both H(2)O(2) treatment and GGT stimulation caused their down-regulation.The data reported suggest that basal conditions of oxidant stress in melanoma may represent a factor contributing to the redox regulation of protein phosphorylation, and that GGT-mediated prooxidant reactions may participate in the process. As basal oxidant stress and expression of GGT activity are present in a variety of malignant tumors besides melanoma, these phenomena likely represent general mechanisms participating in the alteration of intracellular transduction during carcinogenesis.

Prooxidant reactions promoted by soluble and cell-bound gamma-glutamyltransferase activity.

Recent studies have provided evidence for the prooxidant roles played by molecular species originating during the catabolism of glutathione (GSH) effected by gamma-glutamyltransferase (GGT), an enzyme normally present in serum and on the outer surface of numerous cell types. The reduction of metal ions by GSH catabolites is capable of inducing the redox cycling processes, leading to the production of reactive oxygen species and other free radicals. Through the action of these reactive compounds, cell membrane GGT activity can ultimately produce oxidative modifications on a variety of molecular targets, involving oxidation and/or S-thiolation of protein thiol groups in the first place. This chapter is a survey of the procedures most suitable to reveal GGT-dependent prooxidant reactions and their effects at the cellular and extracellular level, including methods in histochemistry, cytochemistry, and biochemistry, with special reference to methods for the evaluation of protein thiol redox status.

Antiproliferative Effect of Rottlerin on Sk-Mel-28 Melanoma Cells.

Melanoma is the most aggressive and chemoresistant form of skin cancer. Mutated, constitutively active B-RAF is believed to play a crucial role, although the selective B-RAF inhibition has shown poor clinical success, since phenomena of resistance usually occur, likely arising from additional genetic aberrations, such as loss of function of p53 and PTEN, overexpression of cyclin D1, hyperactivation of NF-κB, and downregulation of p21/Cip1. Since all of them are present in the Sk-Mel-28 melanoma cells, this cell line could be an ideal, albeit hard to study, model to develop new therapeutic strategies. In the current study, we tested the cytostatic action of Rottlerin on Sk-Mel-28 melanoma cells, on the basis of the known Rottlerin effects on the main proliferative signaling pathways. We presented evidence that the drug inhibits cell growth by an Akt- and p21/Cip1-independent mechanism, involving the dual inhibition of ERK and NF-κB and downregulation of cyclin D1. In addition, we found that Rottlerin increases ERK phosphorylation, but, surprisingly, this resulted in decreased ERK activity. Pull-down experiments, using Rottlerin-CNBr-conjugated Sepharose beads, revealed that Rottlerin binds to ERK, independently from its phosphorylation status. This direct interaction could in part explain the paradoxical blockage of ERK downstream signaling and growth arrest. We would like to dedicate this paper to the memory of our friend and colleague, prematurely deceased, Claudia Torricelli, who actively contributed to this project.

Calpain-3 impairs cell proliferation and stimulates oxidative stress-mediated cell death in melanoma cells.

Calpain-3 is an intracellular cysteine protease, belonging to Calpain superfamily and predominantly expressed in skeletal muscle. In human melanoma cell lines and biopsies, we previously identified two novel splicing variants (hMp78 and hMp84) of Calpain-3 gene (CAPN3), which have a significant lower expression in vertical growth phase melanomas and, even lower, in metastases, compared to benign nevi. In the present study, in order to investigate the pathophysiological role played by the longer Calpain-3 variant, hMp84, in melanoma cells, we over-expressed it in A375 and HT-144 cells. In A375 cells, the enforced expression of hMp84 induces p53 stabilization, and modulates the expression of a few p53- and oxidative stress-related genes. Consistently, hMp84 increases the intracellular production of ROS (Reactive Oxygen Species), which lead to oxidative modification of phospholipids (formation of F2-isoprostanes) and DNA damage. Such events culminate in an adverse cell fate, as indicated by the decrease of cell proliferation and by cell death. To a different extent, either the antioxidant N-acetyl-cysteine or the p53 inhibitor, Pifithrin-α, recover cell viability and decrease ROS formation. Similarly to A375 cells, hMp84 over-expression causes inhibition of cell proliferation, cell death, and increase of both ROS levels and F2-isoprostanes also in HT-144 cells. However, in these cells no p53 accumulation occurs. In both cell lines, no significant change of cell proliferation and cell damage is observed in cells over-expressing the mutant hMp84C42S devoid of its enzymatic activity, suggesting that the catalytic activity of hMp84 is required for its detrimental effects. Since a more aggressive phenotype is expected to benefit from down-regulation of mechanisms impairing cell growth and survival, we envisage that Calpain-3 down-regulation can be regarded as a novel mechanism contributing to melanoma progression.

Autophagy is upregulated in ovarian endometriosis: a possible interplay with p53 and heme oxygenase-1.

OBJECTIVE: To evaluate the occurrence of the autophagic process in ovarian endometriomas compared with eutopic endometrium of affected women and with normal endometrium of healthy women. DESIGN: Biochemical and molecular study in tissue extracts. SETTING: University cellular pathology laboratory and university hospital. PATIENT(S): Patients with ovarian endometriosis (n = 13) and healthy women (n = 18). INTERVENTION(S): Specimens of endometrium were obtained by hysteroscopy from patients with endometriosis and from healthy control subjects; specimens of ovarian endometriomas were collected by laparoscopy. All patients underwent surgery after the end of menstrual bleeding, resulting in most of our patients (approximately 80% in each group) being in the proliferative phase. MAIN OUTCOME MEASURE(S): Autophagy was evaluated by Western blot analysis of biochemical markers (LC3-II, LC3-II/LC3-I ratio and p62) and by quantitative real-time polymerase chain reaction of autophagy-related genes (ATG14, BECN1, ATG7, and LC3B); apoptosis-related (p53 and Bcl-2) and oxidative stress-related (heme oxygenase-1) proteins were also evaluated by Western blot analysis. RESULT(S): All tested biochemical markers and messenger RNA levels of autophagy-related genes showed a significant up-regulation of autophagy in ovarian endometriomas compared with eutopic endometria of affected or healthy women. Moreover, a significant decrease of p53 protein and a significant increase of heme oxygenase-1 protein was also evident in endometriomas. CONCLUSION(S): The upregulated autophagic process observed in ovarian endometriomas can be regarded as an integral part of endometriosis pathogenesis, possibly contributing to survival of endometriotic cells in ectopic sites and to lesion maintenance. The decreased susceptibility to apoptosis and the persistent oxidative stress experienced by endometriotic cells could favor autophagy stimulation.

Glutathione catabolism as a signaling mechanism.

Glutathione (GSH) is the main intracellular thiol antioxidant, and as such participates in a number of cellular antitoxic and defensive functions. Nevertheless, non-antioxidant functions of GSH have also been described, e.g. in modulation of cell proliferation and immune response. Recent studies from our and other laboratories have provided evidence for a third functional aspect of GSH, i.e. the prooxidant roles played by molecular species originating during its catabolism by the membrane ectoenzyme gamma-glutamyl transpeptidase (GGT). The reduction of metal ions effected by GSH catabolites is capable to induce redox cycling processes leading to the production of reactive oxygen species (superoxide, hydrogen peroxide), as well as of other free radicals. Through the action of these reactive compounds, GSH catabolism can ultimately lead to oxidative modifications on a variety of molecular targets, involving oxidation and/or S-thiolation of protein thiol groups in the first place. Modulating effects of this kind have been observed on several important, redox-sensitive components of the signal transduction chains, such as cell surface receptors, protein phosphatase activities and transcription factors. Against this background, the prooxidant reactions induced by GSH catabolism appear to represent a novel, as yet unrecognized mechanism for modulation of cellular signal transduction.

Evaluation of MDR1, LRP, MRP, and topoisomerase IIalpha gene mRNA transcripts before and after interferon-alpha, and correlation with the mRNA expression level of the telomerase subunits hTERT and TEP1 in five unselected human melanoma cell lines.

Intrinsic and acquired multidrug-resistance (MDR) and the activity of the enzyme telomerase have been demonstrated in human melanoma. A direct regulation of the MDR pathways and of telomerase by interpheron-alpha (IFN-alpha), which is currently used in the therapy of advanced cutaneous melanoma, has also been hypothesized. In this study, we used five melanoma cell lines not selected in vitro for drug resistance (Me665/2/21, Me665/2/60, HT-144, SK-MEL-28, and SK-MEL-5), which in a previous study, had shown different responses to IFN-alpha in terms of proliferation, apoptosis, telomerase activity and expression of mRNA for the human telomerase reverse transcriptase (hTERT). We investigated the expression of the multidrug resistance (MDR1) gene, multidrug resistance protein (MRP), lung resistance protein (LRP), topoisomerase IIalpha (Topo IIalpha), hTERT, and telomerase-associated protein (TEP1), which is shared by telomerase and vault MDR proteins at the mRNA expression level, using the reverse transcription-PCR (RT-PCR). All cell lines showed an intrinsic expression of hTERT, TEP1, and MDR gene transcripts (only MDR1 mRNA was under the detection level in SK-MEL-28 cells). After IFN-alpha exposure, we observed either no effect, a trend towards a decrease of hTERT, MRP, and Topo IIalpha, or an increase of TEP1, MDR1, and LRP mRNA expression in some cell lines. Effects were usually temporary and not always significant. No correlation was found between hTERT and TEP1 mRNA expression, whereas significant positive correlations were found between TEP1 and MDR1 mRNA, and between TEP1 and LRP mRNA. IFN-alpha modulates differently MDR gene transcripts in human melanoma cell lines. Positive correlation between TEP1 and LRP also seems to identify them as common targets of IFN-alpha effects.

Cisplatin-induced apoptosis in melanoma cells: role of caspase-3 and caspase-7 in Apaf-1 proteolytic cleavage and in execution of the degradative phases.

Apoptosis protease-activating factor-1 (Apaf-1), which plays a central role in the formation of the apoptosome, is absent or poorly expressed (because of a transcriptional silencing by methylation) in a substantial percentage of metastatic melanomas and melanoma cell lines, which are unable to activate caspase-9 and execute the mitochondrial pathway of apoptosis. We studied cisplatin-induced apoptosis of the Apaf-1-positive human metastatic Me665/2/21 melanoma cells. Our results indicate that caspase-7 is already processed in still-adhering cells and such activation, contrary to the common view, precedes caspase-3 processing. As expected by the cytochrome c release into the cytosol, caspase-9 is processed to active forms (p37 and p35), along with a yet-unidentified p28. Interestingly, we also demonstrate a remarkable loss of Apaf-1 protein, along with the appearance of a related immunoreactive fragment of approximate, equals 26 kDa; such proteolytic degradation proves to be a caspase-3/-7-mediated event. Our data also indicate that the inhibition afforded by ac-DEVD-CHO on several components (i.e., caspase-3/-7 and caspase-9 activities), and Apaf-1 proteolytic degradation, does not significantly abrogate either the apoptotic morphology or the cleavage of canonical targets, such as poly(ADP-ribose) polymerase (PARP) and lamin B. These results suggest that caspase-3 and caspase-7 are dispensable for the execution of apoptosis and, in our cellular model, the point of no return could be out of the mitochondrial cascade.