Evaluation of polyamines as marker of melanoma cell proliferation and differentiation by an improved high-performance liquid chromatographic method.

The differentiation therapy is focused on the identification of new agents able to impair the proliferative and metastatic potential of cancer cells through the induction of differentiation. Although several markers of cell differentiation on tumor cells have been identified, their causal relationship with neoplastic competence has not been characterized in sufficient detail to propose their use as new pharmacological targets useful for the design of new differentiation agents. Polyamine level in cancer cells and in body fluids was proposed as potential marker of cell proliferation and differentiation. The main advantage of this marker is the possibility to evaluate the antineoplastic activity of new drugs able to induce cell differentiation and consequently to inhibit tumor growth and metastasis. The presented report shows a simply and highly reproducible reverse-phase high-performance liquid chromatographic (HPLC) method for the determination of ortho-phthalaldehyde (OPA) derivatives of polyamines: putrescine (PUT), cadaverine (CAD), spermidine (SPD) and spermine (SPM). The novelty of this method is the fluorescence response for OPA-derivate of SPM, generally low in other procedures, that has been significantly improved by the use of a fully endcapped packing material with minimal silanol interactions. The limits of detection for PUT, CAD, SPD and SPM were 0.6, 0.7, 0.8, and 0.4 pmol/mL, respectively. The analysis time was ≤ 20 min, and the relative recovery rate was of about 97%. To verify the usefulness of this method, it has been validated in a murine melanoma cell line (B16-F10) treated with two theophylline derivatives (namely 8-chlorotheophylline and 8-bromotheophylline). These two compounds increased the activity of tissue transglutaminase (TG2) and the synthesis of melanin, two recognized markers of melanoma cell differentiation, and significantly reduced the levels of intracellular polyamines.

Aloe-emodin exerts a potent anticancer and immunomodulatory activity on BRAF-mutated human melanoma cells.

Aim of this study was to extend the knowledge on the antineoplastic effect of aloe-emodin (AE), a natural hydroxyanthraquinone compound, both in metastatic human melanoma cell lines and in primary stem-like cells (melanospheres). Treatment with AE caused reduction of cell proliferation and induction of SK-MEL-28 and A375 cells differentiation, characterized by a marked increase of transamidating activity of transglutaminase whose expression remained unmodified. In vitro antimetastatic property of AE was evaluated by adhesion and Boyden chamber invasion assays. The effect of AE on melanoma cytokines/chemokines production was determined by a multiplex assay: interestingly AE showed an immunomodulatory activity through GM-CSF and IFN-γ production. We report also that AE significantly reduced the proliferation, stemness and invasive potential of melanospheres. Moreover, AE treatment significantly enhanced dabrafenib (a BRAF inhibitor) antiproliferative activity in BRAF mutant cell lines. Our results confirm that AE possesses remarkable antineoplastic properties against melanoma cells, indicating this anthraquinone as a promising agent for differentiation therapy of cancer, or as adjuvant in chemotherapy and targeted therapy. Further, its mechanisms of action support a potential efficacy of AE treatment to counteract resistance of BRAF-mutated melanoma cells to target therapy.

Inhibition of cell proliferation, migration and invasion of B16-F10 melanoma cells by α-mangostin.

In this study, we have evaluated the potential antineoplastic effects of α-mangostin (α-M), the most representative xanthone in Garcinia mangostana pericarp, on melanoma cell lines. This xanthone markedly inhibits the proliferation of high-metastatic B16-F10 melanoma cells. Furthermore, by deeply analyzing which steps in the metastatic process are influenced by xanthone it was observed that α-M strongly interferes with homotypic aggregation, adhesion, plasticity and invasion ability of B16-F10 cells, probably by the observed reduction of metalloproteinase-9 activity. The antiproliferative and antimetastatic properties of α-M have been established in human SK-MEL-28 and A375 melanoma cells. In order to identify pathways potentially involved in the antineoplastic properties of α-M, a comparative mass spectrometry proteomic approach was employed. These findings may improve our understanding of the molecular mechanisms underlying the anti-cancer effects of α-M on melanoma.

Antineoplastic activity of strawberry (Fragaria×ananassa Duch.) crude extracts on B16-F10 melanoma cells.

The antiproliferative and differentiation potential of anthocyanin-rich strawberry fruit crude extracts (SE) were investigated on B16-F10 murine melanoma cells. Treatment of melanoma cells with SE produced a remarkable reduction of cell proliferation, paralleled with both the lowering of the intracellular levels of polyamine, and the enhancement of tissue transglutaminase (TG2, EC 2.3.2.13) activity (used as a differentiation marker). To gain further insight into profiling altered protein expression as a potential biomarker of the SE action on melanoma cells, analysis of the proteomic profile was performed on the treated B16-F10 cells, compared to the control. Following SE treatment, 30 proteins resulted up-regulated, and 87 proteins were down-regulated. In particular proteins overexpressed in cancer cells, involved in tumor progression and metabolism, were down-regulated. The possibility that SE may affect the Warburg effect in B16-F10 melanoma cells is discussed.

Transglutaminases: key regulators of cancer metastasis.

The ability to metastasize represents the most important characteristic of malignant tumors. The biological details of the metastatic process remain somewhat unknown, due to difficulties in studying tumor cell behaviour with high spatial and temporal resolution in vivo. Several lines of evidence involve transglutaminases (TGs) in the key stages of tumor progression cascade, even though the molecular mechanisms remain controversial. TG expression and activity display a different role in the primary tumor or in metastatic cells. In fact, TG expression is low in the primary tumor mass, but augmented when cells acquire the metastatic phenotype. Nevertheless, in other cases, the use of inducers of TG transamidating activity seems to contrast tumor cell plasticity, migration and invasion. In the following review, the function of TGs in cancer cell migration into the extracellular matrix, adhesion to the capillary endothelium and its basement membrane, invasion and angiogenesis is discussed.

Aloin enhances cisplatin antineoplastic activity in B16-F10 melanoma cells by transglutaminase-induced differentiation.

Aloin, a natural anthracycline from aloe plant, is a hydroxyanthraquinone derivative shown to have antitumor properties. This study demonstrated that aloin exerted inhibition of cell proliferation, adhesion and invasion abilities of B16-F10 melanoma cells under non-cytotoxic concentrations. Furthermore, aloin induced melanoma cell differentiation through the enhancement of melanogenesis and transglutaminase activity. To improve the growth-inhibiting effect of anticancer agents, we found that the combined treatment of cells with aloin and low doses of cisplatin increases the antiproliferative activity of aloin. The results suggest that aloin possesses antineoplastic and antimetastatic properties, exerted likely through the induction of melanoma cell differentiation.

Tissue transglutaminase activity protects from cutaneous melanoma metastatic dissemination: an in vivo study.

The role of tissue transglutaminase (TG-2, TGase-2) in cancer development is still a fascinating field of research. The available reports do not elucidate fully its mechanism of action, due to the limitations of in vitro approaches. Therefore, to understand TG-2 role in cancer, we carried out an in vivo study with a more direct approach. TG-2 was in vivo overexpressed in a murine model of melanoma (intravenous injection of B16 melanoma cells in C57BL/6N mice) by means of a plasmid carrying the TG-2 cDNA. The evaluation of the frequency and size of the metastases indicated that the number of melanoma lung foci was more markedly reduced by TG-2 overexpression than the metastatic size. Then, TG-2 overexpressing mice showed a prolonged survival with respect to control mice. Further analyses were carried by means of proteomic analysis of melanoma cell lysates and meta-analysis of published transcriptomic datasets. Proteomic analysis of cell lysates from a human melanoma cell line compared to human keratinocytes showed significant differences in the expression of TG-2 substrates known to be involved in proliferation/differentiation and cancer progression. Taken together, these findings indicate a protective role of TG-2 enzymatic activity in melanoma progression in vivo.

Hippocampal polyamine levels and transglutaminase activity are paralleling spatial memory retrieval in the C57BL/6J mouse.

Although a potential role for polyamines and transglutaminases (TGs) in memory mechanisms have been proposed, hippocampal spermine (SPM) and spermidine (SPD) levels as well as transamidating activity of TG in spatial memory have not been addressed yet. It was therefore the aim of the study to assess hippocampal polyamines and TG activity at the probe trial in a spatial memory paradigm. C57BL/6J mice (20 animals per group) were used for the experiments and divided into a trained and a yoked (untrained) group. The Morris water maze (MWM) was selected as the memory test, animals were sacrificed within 5 min following the probe trial and hippocampi were taken for biochemical analysis. SPD and SPM levels were assessed by an analytical procedure according to Gismondi et al. Transamidating activity of TG was determined following the method described by Chung and Folk using [14C] methylamine as substrate. γ-(Glutamyl)-polyamine levels were evaluated by ion exchange chromatography according to Folk et al. Animals learned the task in the MWM as latencies and pathlengths were significantly reduced. At the probe trial mice showed significantly higher preference for the target quadrant. Free SPD and SPM levels were manifold decreased in the trained as compared to the yoked group. Transamidating activity of TG was fourfold increased in trained as compared to yoked controls. γ-(Glutamyl)-SPD was comparable while γ-(glutamyl)-SPM was significantly higher in the trained group. The findings show a potential role for polyamines, their derivative γ-(glutamyl)-SPM and transamidating activity of TG at memory retrieval or formation. Results from this study are extending and knowledge on polyamines and report for the first time involvement of γ-(glutamyl)-SPM and transamidating activity of TG that may form the basis for future neurochemical and pharmacological studies and indeed, modulation of polyamine and TG activity has been already proposed as a tentative therapeutical concept.

Preclinical evaluation of the antineoplastic efficacy of 7-(2-hydroxyethyl)theophylline on melanoma cancer cells.

Differentiation-based therapeutics are an underutilized but potentially a significant option for cancer treatment. The effect of methylxanthines on melanoma cell differentiation has been well documented. We report the in-vitro and in-vivo anticancer potential of a theophylline analogue, 7-(2-hydroxyethyl)theophylline (HET), on murine B16-F10 and human Sk-Mel 110 metastatic melanoma cell lines. The effects on cell proliferation were related to the induction of differentiation, demonstrated as increased intracellular transglutaminase activity. The involvement of this methylxanthine in the control of the in-vitro adhesion and in the in-vivo metastastic spread of melanoma cells was further investigated. HET oral administration of C57BL6/N mice intravenously injected with B16-F10 cells markedly reduced lung metastases frequency. The overall results demonstrated that HET possesses a remarkable in-vivo antimetastatic capability.

Evidences for a role of protein cross-links in transglutaminase-related disease.

Transglutaminases (TGs) are a large family of related and ubiquitous enzymes that catalyze the cross-linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate. Considerable and intense progress has been made in the understanding of the chemistry, molecular biology and cell biology of TGs. The knowledge that very different physiological and pathological processes are dependent on the presence of adequate levels of these cross-linking enzymes and on the amount of both free and protein-conjugated polyamines by TG, has generated an incredible amount of original research and review articles. It is clear that TG-mediated reactions are essential for some biological processes, such as blood coagulation, skin barrier formation and extracellular matrix assembly, but may also be involved in pathogenetic mechanisms responsible for several human diseases, such as cancer, AIDS, neurodegenerative disorders, celiac disease, and eye lens opacification. We present here a comprehensive review of recent insights into the pathophysiology of TGs related to their protein cross-linking activity.

Aloe-emodin as antiproliferative and differentiating agent on human U937 monoblastic leukemia cells.

AIMS: Aloe-emodin (AE), a plant derived anthraquinone, has been shown to have anticancer activity in various human cancer cell lines. We have recently reported that AE possesses a differentiative potential on melanoma cells. The purpose of this study was to investigate the possible modulation of defined markers of monocytic differentiation of AE on human U937 cell line. MAIN METHODS: U937 cells differentiation has been confirmed unequivocally by Griess and nitroblue tetrazolium reduction assays, protoporphyrin IX accumulation, expression of CD14 and CD11b surface antigens, phagocytic activity, migration and attachment ability. The effect on polyamine metabolism, apoptosis and cytokine production was also investigated. KEY FINDINGS: We showed that AE-treated U937 cells exhibit a noticeably rise in transglutaminase activity. This enhanced enzyme activity correlates with AE-induced growth arrest and differentiation to functionally mature monocytes. SIGNIFICANCE: Taken together, the results reported here show that AE can promote the macrophage differentiation of U937 cells, suggesting that this anthraquinone could be a potential candidate as a differentiation-inducing selective agent for therapeutic treatment of leukemia.

Spermidine delays eye lens opacification in vitro by suppressing transglutaminase-catalyzed crystallin cross-linking.

A Ca(2+)-dependent TG activity, identified in the eye lens of several mammalian species, has long been implicated in cataract formation. The precise mechanism of the involvement of this enzyme in this process remains unclear. The purpose of this work was to investigate the modulatory effect of polyamines on TG activity during rabbit eye lens in vitro opacification. We observed, in an in vitro Ca(2+)-induced cataract model, a rapid decrease of the endogenous levels of SPD with the progression of opacification, paralleled by an increase of crystallin cross-linking by bis(γ-glutamyl)SPD. This pattern was reversed adding exogenous SPD to the incubation medium. Indeed, endogenous SPD levels were restored and cross-linking by bis(γ-glutamyl)SPD were drastically reduced. Surprisingly, under this experimental condition, the loss of transparency of lens was delayed. We found that exogenous SPD incubation led to a remarkable increase of mono(γ-glutamyl)SPD, likely responsible of the inhibition of cross-linking of lens crystallins and of the transparency persistence.

Synergic effect of α-tocopherol and naringenin in transglutaminase-induced differentiation of human prostate cancer cells.

Prostate cancer is the second most common cancer in men worldwide. Its prevention and treatment remain a challenge to clinicians. Thus, there is an urgent need to discover novel, less toxic, and more effective therapies for patients. Many vitamins and related chemicals, including vitamin E, (tocopherols) have shown their anti-cancer activities as anti-oxidants, activators of transcription factors or factors influencing epigenetic events. Although laboratory tests including the use of animal models showed that this vitamin may have anticancer properties, whether it can effectively prevent the development and/or progression of prostate cancer in humans remains to be intensively studied. This review provides up-to-date information regarding the recent outcomes of laboratory, epidemiology and/or clinical trials on the effects of tocopherols on prostate cancer development, along with our last observations on a combined treatment of a prostate cancer cell line (PC-3) with two natural antineoplastic compounds, naringenin (NG) and α-tocopherol (α-TOC). We report the synergic effect of α-TOC and NG in transglutaminase-induced differentiation of human PC-3 prostate cancer cells. While our results are based on one histological class of tumor, the most significant implication of this observation is that establishes a new way in the screening for detecting new differentiative antineoplastic agents.

Polyamine concentration, transglutaminase activity and changes in protein synthesis during cryopreservation of shoot tips of apple variety Annurca.

Changes in metabolism and protein expression were analysed during cryopreservation of the ancient apple variety Annurca. Our experiments concerned transglutaminase activity, polyamine levels and protein expression associated with shoot tip dehydration. Cryopreserved shoot tips displayed 72% regrowth after treatment in liquid medium with 0.75 M sucrose for 1 day followed by dehydration to 19% moisture content (fresh weight basis). After dehydration, the concentration of polyamines putrescine and spermidine decreased compared with untreated controls, while spermine concentration remained unaffected. Transglutaminase activity was slightly reduced in treated samples, while post-thaw regrowth enzyme activity approached control values. We also detected significant changes in protein expression profiles and identified six proteins related with stress response or involved in the slowing down of the cell cycle. The relationship between biochemical parameters, protein synthesis and cryotolerance is discussed.

Antitumor activity of theophylline in combination with Paclitaxel: a preclinical study on melanoma experimental lung metastasis.

Melanoma is one of the most aggressive forms of tumor, being responsible for about 80% of skin cancer deaths. Much effort is being directed at obtaining less-toxic anticancer therapies, and the combination between low cytotoxic doses of chemotherapeutic drugs and natural differentiative compounds seems to be of particular importance. The present study was undertaken to examine the possible role of a combination therapy using paclitaxel (PTX) as chemotherapeutic molecule and theophylline (TH) as differentiative agent in the prevention of metastasis in B16-F10 melanoma-bearing C57BL6/N mice. In vitro proliferation studies demonstrated that TH enhanced the antiproliferative effect of PTX. In the in vivo experiments, a highly sensitive computerized image analysis method, performed on histological lung sections of mice injected with melanoma cells, was used to quantify the efficacy of the treatments. This study demonstrated that the simultaneous treatment of mice with TH and a low dose of PTX produced a similar anti-invasive effect than that caused by highly toxic PTX concentration.

Antitumor properties of aloe-emodin and induction of transglutaminase 2 activity in B16-F10 melanoma cells.

AIMS: Aloe-emodin (AE), a natural hydroxyanthraquinone compound, has been reported as a potential anticancer agent. We studied the antineoplastic properties of AE on highly metastatic B16-F10 melanoma murine cells. MAIN METHODS: Cell proliferation was assessed by cell counting and viability was investigated using MTT and Trypan Bleu exclusion tests. As a growth marker, we determined intracellular polyamine levels by high performance liquid chromatography. Then, we evaluated transglutaminase 2 (TG2) activity, protoporphyrin IX accumulation and melanin content as differentiative markers. Tyrosinase activity was checked by DOPA-staining assay. The antimetastatic effect of AE was evaluated by means of a series of in vitro metastatic assays, including aggregation, wound healing migration, adhesion, 3D-invasion, circular invasion and the Boyden chamber invasion assays. Gelatin zymography was performed to evaluate metalloproteinase activities. KEY FINDINGS: Our results demonstrated inhibitory effects of AE on melanoma cell proliferation and invasion power, accompanied by the stimulation of cell differentiation parameters. Cell differentiation correlated with a remarkable increase of the activity of the transamidating form of TG2, with a significative enhancement of cell adhesion and aggregation. Impaired invasion was paralleled by the decrease of the secretion of matrix metalloproteinase-9. SIGNIFICANCE: The overall data confirm a remarkable antiproliferative, antimetastatic and differentiative capability of this anthraquinone. Results suggest that AE appears particularly promising for its potential application in the newborn differentiation therapy of cancer.

Similar antineoplastic effects of nimesulide, a selective COX-2 inhibitor, and prostaglandin E1 on B16-F10 murine melanoma cells.

There is now increasing evidence that a constitutive expression of cyclooxygenase 2 (COX-2) plays a role in the development and progression of malignant ectodermal tumours. In this study, we investigated whether the selective inhibition of COX-2 would be beneficial in melanoma treatment. Nimesulide, a selective inhibitor of COX-2 that causes the breakdown of proinflammatory 2-series prostaglandins (PG2), adversely affected the growth of B16-F10 melanoma cells through the induction of differentiation. The intracellular levels of polyamine, as a proliferation marker, were reduced by the treatment; at the same time, transglutaminase activation and increase in melanin content, as differentiation indicators, were observed. The potential antimetastatic activity of the drug was further shown by means of the Boyden invasion assay and gelatin zymography for metalloproteinase activity. Comparable results were obtained after the treatment of cells with one of the 1-series PGs (PGE1). Therefore, our hypothesis is that the antineoplastic activity observed for nimesulide may be ascribed to intracellular changes in alterations in PG levels.

Phytochemicals and protein-polyamine conjugates by transglutaminase as chemopreventive and chemotherapeutic tools in cancer.

Identifying novel chemopreventive and chemotherapeutic agents and targeting them to patients at high risk of developing cancer or following curative treatment may go some way towards improving prognosis. This review examines current knowledge regarding the chemopreventive and chemotherapeutic potential of phytochemicals in cancer. Both in vitro and animal studies demonstrate that several phytochemicals increase the activity of intracellular transglutaminases, a family of enzymes involved in cell differentiation, through the covalent conjugation of polyamine to cellular protein, with promising anti-neoplastic properties. The substantial data available on certain plant secondary metabolites makes a strong case for integrating these safe and well-tolerated agents into clinical practice.

Anthraquinones danthron and quinizarin exert antiproliferative and antimetastatic activity on murine B16-F10 melanoma cells.

The anticancer potential of anthraquinones danthron and quinizarin on highly metastatic B16-F10 melanoma murine cells was investigated. Several parameters related to cell proliferation and differentiation, i.e. cell number, intracellular content of polyamines, transglutaminase (EC 2.3.2.13) activity and melanin synthesis were evaluated. Tumor cell growth was strongly reduced after treatment with danthron and quinizarin, as well as polyamine intracellular levels. Moreover, the induction of differentiation in treated cells was demonstrated by the increase of transglutaminase activity. In vitro analysis of adhesion, wound healing migration and invasion showed a remarkable antimetastatic activity of the anthraquinones. The overall results suggest that danthron and quinizarin possess significant antineoplastic properties, probably exerted through the induction of intracellular transglutaminase activity.

Post-translational modification of glutamine and lysine residues of HIV-1 aspartyl protease by transglutaminase increases its catalytic activity.

The human immunodeficiency virus type 1 aspartyl protease (HIV-1 PR) is a homodimeric aspartyl endopeptidase that is required for virus replication. HIV-1 PR was shown to act invitro as acyl-donor and -acceptor for both guinea pig liver transglutaminase (TG, EC 2.3.2.13) and human Factor XIIIa. These preliminary evidences suggested that the HIV-1 PR contains at least three TG-reactive glutaminyl and one lysyl residues. We report here that the incubation of HIV-1 PR with TG increases its catalytic activity. This increase is dependent upon the time of incubation, the concentration of TG and the presence of Ca2+. Identification of epsilon-(gamma-glutamyl)lysine in the proteolytic digest of the TG-modified HIV-1 PR suggested intramolecular covalent cross-linking of this protease which may promote a non-covalent dimerization and subsequent activation of this enzyme via a conformational change. This hypothesis is supported by the observation that the TG-catalyzed activation of HIV-1 PR was completely abolished by spermidine (SPD) which acts as a competitive inhibitor of epsilon-(gamma-glutamyl)lysine formation. Indeed, in the presence of 1mM SPD the formation of the isopeptide was decreased of about 80%. The main products of the TG-catalyzed modification of HIV-1 PR in the presence of SPD were N(1)-mono(gamma-glutamyl)SPD and N(8)-mono(gamma-glutamyl)SPD. Negligible amount of N(1),N(8)-bis(gamma-glutamyl)SPD were found. The significance of these results is discussed with respect to the activation of the protease by post-translational modification and design of potential inhibitors.