α-Pinene, a Main Component of Pinus Essential Oils, Enhances the Expression of Insulin-Sensitive Glucose Transporter Type 4 in Murine Skeletal Muscle Cells.

Glucose transporter-4 (GLUT4) represents the major glucose transporter isoform responsible for glucose uptake into insulin-sensitive cells, primarily in skeletal muscle and adipose tissues. In insulin-resistant conditions, such as type 2 diabetes mellitus, GLUT4 expression and/or translocation to the cell plasma membrane is reduced, compromising cell energy metabolism. Therefore, the use of synthetic or naturally occurring molecules able to stimulate GLUT4 expression represents a good tool for alternative treatments of insulin resistance. The present study aimed to investigate the effects of essential oils (EOs) derived from Pinus spp. (P. nigra and P. radiata) and of their main terpenoid constituents (α- and ß-pinene) on the expression/translocation of GLUT4 in myoblast C2C12 murine cells. For this purpose, the chemical profiles of the EOs were first analyzed through gas chromatography-mass spectrometry (GC-MS). Cell viability was assessed by MTT assay, and GLUT4 expression/translocation was evaluated through RT-qPCR and flow cytometry analyses. The results showed that only the P. nigra essential oil (PnEO) and α-pinene can increase the transcription of the Glut4/Scl2a4 gene, resulting in a subsequent increase in the amount of GLUT4 produced and its plasma membrane localization. Moreover, the PnEO or α-pinene can induce Glut4 expression both during myogenesis and in myotubes. In summary, the PnEO and α-pinene emulate insulin's effect on the GLUT4 transporter expression and its translocation to the muscle cell surface.

Cytotoxicity of Isoxazole Curcumin Analogs on Chronic Myeloid Leukemia-Derived K562 Cell Lines Sensitive and Resistant to Imatinib.

Despite curcumin (CUR) inhibiting cell proliferation in vitro by activating apoptotic cell death, its use in pharmacological therapy is hampered by poor solubility, low stability in biological fluids, and rapid removal from the body. Therefore, CUR-derivatives with better biological and chemical-physical characteristics are needed. The bis-ketone moiety of CUR strongly influences its stability in slightly alkaline solutions such as plasma. Here, we considered its replacement with isoxazole, beta-enamine, or oxime groups to obtain more stable derivatives. The evaluation of the chemical-physical characteristics showed that only of the isoxazole derivatives 2 and 22 had better potential than CUR in terms of bioavailability. The UV-visible spectrum analysis showed that derivatives 2 and 22 had better stability than CUR in solutions mimicking the biological fluids. When tested on a panel of cell lines, derivatives 2 and 22 had marked cytotoxicity (IC50 = 0.5 µM) compared with CUR only (IC50 = 17 µM) in the chronic myeloid leukemia (CML)-derived K562 cell line. The derivative 22 was the more selective for CML cells. When administered at the average concentration found for CUR in the blood of patients, derivatives 2 and 22 had potent effects on cell cycle progression and apoptosis initiation, while CUR was ineffective. The apoptotic effect of derivatives 2 and 22 was associated with low necrosis. In addition, derivative 22 was able to reverse drug resistance in K562 cells resistant to imatinib (IM), the reference drug used in CML therapy. The cytotoxicity of derivative 22 on IM-sensitive and resistant cells was associated with upregulation of FOXN3 and CDKN1A expression, G2/M arrest, and triggering of apoptosis. In conclusion, derivative 22 has chemical-physical characteristics and biological effects superior to CUR, which allow us to hypothesize its future use in the therapy of CML and CML forms resistant to IM, either alone or in combination with this drug.

Phytochemicals as Immunomodulatory Agents in Melanoma.

Cutaneous melanoma is an immunogenic highly heterogenic tumor characterized by poor outcomes when it is diagnosed late. Therefore, immunotherapy in combination with other anti-proliferative approaches is among the most effective weapons to control its growth and metastatic dissemination. Recently, a large amount of published reports indicate the interest of researchers and clinicians about plant secondary metabolites as potentially useful therapeutic tools due to their lower presence of side effects coupled with their high potency and efficacy. Published evidence was reported in most cases through in vitro studies but also, with a growing body of evidence, through in vivo investigations. Our aim was, therefore, to review the published studies focused on the most interesting phytochemicals whose immunomodulatory activities and/or mechanisms of actions were demonstrated and applied to melanoma models.

A Central Contribution of TG2 Activity to the Antiproliferative and Pro-Apoptotic Effects of Caffeic Acid in K562 Cells of Human Chronic Myeloid Leukemia.

Caffeic acid (CA) has shown antitumor activity in numerous solid and blood cancers. We have recently reported that CA is active in reducing proliferation and triggering apoptosis in both Imatinib-sensitive and resistant Chronic Myeloid Leukemia (CML) cells. Tissue transglutaminase type 2 (TG2) enzyme is involved in cell proliferation and apoptosis of numerous types of cancer. However, its activity has different effects depending on the type of tumor. This work investigated the possible involvement of TG2 activation in the triggering of CA-dependent anticancer effects on the K562 cell line, which was studied as a model of CML. CA-dependent changes in TG2 activity were compared with the effects on cell proliferation and apoptosis. The use of N-acetylcysteine (NAC), an antioxidant molecule, suggested that the antiproliferative effect of CA was due to the increase in reactive oxygen species (ROS). The use of a TG2 inhibitor showed that TG2 activity was responsible for the increase in ROS generated by CA and reduced both caspase activation and triggering of CA-dependent apoptosis. The knocking-down of TGM2 transcripts confirmed the crucial involvement of TG2 activation in CML cell death. In conclusion, the data reported, in addition to ascertaining the important role of TG2 activation in the antiproliferative and pro-apoptotic mechanism of CA allowed us to hypothesize a possible therapeutic utility of the molecules capable of triggering the activation pathways of TG2 in the treatment of CML.

Reduction of oxidative stress and ornithine decarboxylase expression in a human prostate cancer cell line PC-3 by a combined treatment with α-tocopherol and naringenin.

Differentiation of a human aggressive PC-3 cancer cell line was obtained, in a previous investigation, by the synergic effect of α-tocopherol (α-TOC) and naringenin (NG). This combined treatment induced apoptosis and subsequent reduction of the PC-3 cell proliferation and invasion, by a pro-differentiating action. Since one of the peculiar characteristics of NG and α-TOC is their strong antioxidant activity, this study aimed to investigate their potential effect on the activity of the main enzymes involved in the antioxidant mechanism in prostate cancer cells. NG and α-TOC administered singularly or combined in the PC-3 cell line, affected the activity of several enzymes biomarkers of the cellular antioxidant activity, as well as the concentration of total glutathione (GSH + GSSG) and thiobarbituric acid reactive substances (TBARS). The combined treatment increased the TBARS levels and superoxide dismutase (SOD) activity, while decreased the glutathione S-transferase (GST), glutathione reductase (GR), and glyoxalase I (GI) activities. The results obtained indicate that a combined treatment with these natural compounds mitigated the oxidative stress in the human PC-3 cell line. In addition, a significant reduction of both ornithine decarboxylase (ODC) expression and intracellular levels of polyamines, both well-known positive regulators of cell proliferation, accompanied the reduction of oxidative stress observed in the combined α-TOC and NG treatment. Considering the established role of polyamines in cell differentiation, the synergism with NG makes α-TOC a potential drug for further study on the differentiation therapy in prostate cancer patients.

Caffeic Acid Enhances the Anti-Leukemic Effect of Imatinib on Chronic Myeloid Leukemia Cells and Triggers Apoptosis in Cells Sensitive and Resistant to Imatinib.

Among the phenolic acids tested on the K562 cell line, a model of chronic myeloid leukemia (CML), caffeic acid (CA) was biologically active on sensitive and imatinib (IM)-resistant cells at micro-molar concentration, either in terms of reduction of cell proliferation or triggering of apoptosis. The CA treatment provoked mitochondrial membrane depolarization, genomic DNA fragmentation and phosphatidylserine exposure, hallmarks of apoptosis. Cell cycle analysis following the treatment with comparable cytotoxic concentrations of IM or CA showed marked differences in the distribution profiles. The reduction of cell proliferation by CA administration was associated with increased expression of two cell cycle repressor genes, CDKN1A and CHES1, while IM at a cytotoxic concentration increased the CHES1 but not the CDKN1A expression. In addition, CA treatment affected the proliferation and triggered the apoptosis in IM-resistant cells. Taken together, these data suggested that CA induced the anti-proliferative effect and triggered apoptosis of CML cells by a different mechanism than IM. Finally, the combined administration of IM and CA at suboptimal concentrations evidenced a synergy of action in determining the anti-proliferative effect and triggering apoptosis. The ability of CA to potentiate the anti-leukemic effect of IM highlighted the nutraceutical potential of CA in CML.

Flavonoids: A Myth or a Reality for Cancer Therapy?

Nutraceuticals are biologically active molecules present in foods; they can have beneficial effects on health, but they are not available in large enough quantities to perform this function. Plant metabolites, such as polyphenols, are widely diffused in the plant kingdom, where they play fundamental roles in plant development and interactions with the environment. Among these, flavonoids are of particular interest as they have significant effects on human health. In vitro and/or in vivo studies described flavonoids as essential nutrients for preventing several diseases. They display broad and promising bioactivities to fight cancer, inflammation, bacterial infections, as well as to reduce the severity of neurodegenerative and cardiovascular diseases or diabetes. Therefore, it is not surprising that interest in flavonoids has sharply increased in recent years. More than 23,000 scientific publications on flavonoids have described the potential anticancer activity of these natural molecules in the last decade. Studies, in vitro and in vivo, show that flavonoids exhibit anticancer properties, and many epidemiological studies confirm that dietary intake of flavonoids leads to a reduced risk of cancer. This review provides a glimpse of the mechanisms of action of flavonoids on cancer cells.

Targeting Melanoma-Initiating Cells by Caffeine: In Silico and In Vitro Approaches.

The beneficial effects of coffee on human diseases are well documented, but the molecular mechanisms of its bioactive compounds on cancer are not completely elucidated. This is likely due to the large heterogeneity of coffee preparations and different coffee-based beverages, but also to the choice of experimental models where proliferation, differentiation and immune responses are differently affected. The aim of the present study was to investigate the effects of one of the most interesting bioactive compounds in coffee, i.e., caffeine, using a cellular model of melanoma at a defined differentiation level. A preliminary in silico analysis carried out on public gene-expression databases identified genes potentially involved in caffeine's effects and suggested some specific molecular targets, including tyrosinase. Proliferation was investigated in vitro on human melanoma initiating cells (MICs) and cytokine expression was measured in conditioned media. Tyrosinase was revealed as a key player in caffeine's mechanisms of action, suggesting a crucial role in immunomodulation through the reduction in IL-1ß, IP-10, MIP-1α, MIP-1ß and RANTES secretion onto MICs conditioned media. The potent antiproliferative effects of caffeine on MICs are likely to occur by promoting melanin production and reducing inflammatory signals' secretion. These data suggest tyrosinase as a key player mediating the effects of caffeine on melanoma.

Polyamine Oxidase Is Involved in Spermidine Reduction of Transglutaminase Type 2-Catalyzed ßH-Crystallins Polymerization in Calcium-Induced Experimental Cataract.

In an in vitro Ca2+-induced cataract model, the progression of opacification is paralleled by a rapid decrease of the endogenous levels of spermidine (SPD) and an increase of transglutaminase type 2 (TG2, EC 2.3.2.13)-catalyzed lens crystallins cross-linking by protein-bound N1-N8-bis(γ-glutamyl) SPD. This pattern was reversed adding exogenous SPD to the incubation resulting in a delayed loss of transparency of the rabbit lens. The present report shows evidence on the main incorporation of SPD by the catalytic activity of TG2, toward ßH-crystallins and in particular to the ßB2- and mostly in ßB3-crystallins. The increase of endogenous SPD in the cultured rabbit lens showed the activation of a flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAO EC 1.5.3.11). As it is known that FAD-PAO degrades the N8-terminal reactive portion of N1-mono(γ-glutamyl) SPD, the protein-bound N8-mono(γ-glutamyl) SPD was found the mainly available derivative for the potential formation of ßB3-crystallins cross-links by protein-bound N1-N8-bis(γ-glutamyl)SPD. In conclusion, FAD-PAO degradation of the N8-terminal reactive residue of the crystallins bound N1-mono(γ-glutamyl)SPD together with the increased concentration of exogenous SPD, leading to saturation of glutamine residues on the substrate proteins, drastically reduces N1-N8-bis(γ-glutamyl)SPD crosslinks formation, preventing crystallins polymerization and avoiding rabbit lens opacification. The ability of SPD and MDL 72527 to modulate the activities of TG2 and FAD-PAO involved in the mechanism of lens opacification suggests a potential strategy for the prevention of senile cataract.

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.

Chemical Composition of Essential Oils from Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis, and Their Effects on the HIV-1 Tat Protein Function.

New drugs would be beneficial to fight resistant HIV strains, in particular those capable of interfering with essential viral functions other than those targeted by highly active antiretroviral therapy drugs. Despite the central role played by Tat protein in HIV transcription, a search for vegetable extracts able to hamper this important viral function was never carried out. In this work, we evaluated the chemical composition and possible interference of essential oil from Thymus vulgaris, Cananga odorata, Cymbopogon citratus, and Rosmarinus officinalis with the Tat/TAR-RNA interaction and with Tat-induced HIV-1 LTR transcription. GC/MS Analysis demonstrated the biodiversity of herbal species translated into essential oils composed of different blends of terpenes. In all of them, 4 - 6 constituents represent from 81.63% to 95.19% of the total terpenes. Essential oils of Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis were active in interfering with Tat functions, encouraging further studies to identify single terpenes responsible for the antiviral activity. In view of the quite different composition of these essential oils, we concluded that their interference on Tat function depends on specific terpene or a characteristic blend.

Biological Implications and Functional Significance of Transglutaminase Type 2 in Nervous System Tumors.

Transglutaminase type 2 (TG2) is the most ubiquitously expressed member of the transglutaminase family. TG2 catalyzes the transamidation reaction leading to several protein post-translational modifications and it is also implicated in signal transduction thanks to its GTP binding/hydrolyzing activity. In the nervous system, TG2 regulates multiple physiological processes, such as development, neuronal cell death and differentiation, and synaptic plasticity. Given its different enzymatic activities, aberrant expression or activity of TG2 can contribute to tumorigenesis, including in peripheral and central nervous system tumors. Indeed, TG2 dysregulation has been reported in meningiomas, medulloblastomas, neuroblastomas, glioblastomas, and other adult-type diffuse gliomas. The aim of this review is to provide an overview of the biological and functional relevance of TG2 in the pathogenesis of nervous system tumors, highlighting its involvement in survival, tumor inflammation, differentiation, and in the resistance to standard therapies.

The side chain of glutamine 13 is the acyl-donor amino acid modified by type 2 transglutaminase in subunit T of the native rabbit skeletal muscle troponin complex.

Subunit T of the native muscle troponin complex is a recognised substrate of transglutaminase both in vitro and in situ with formation of isopeptide bonds. Using a proteomic approach, we have now determined the precise site of in vitro labelling of the protein. A preparation of troponin purified from ether powder from mixed rabbit skeletal muscles was employed as transglutaminase substrate. The only isoform TnT2F present in our preparation was recognised as acyl-substrate by human type 2 transglutaminase which specifically modified glutamine 13 in the N-terminal region. During the reaction, the troponin protein complex was polymerized. Results are discussed in relation to the structure of the troponin T subunit, in the light of the role of troponins in skeletal and cardiac muscle diseases, and to the rules governing glutamine side chain selection by tissue transglutaminase.

Accumulation of central fat correlates with an adverse oxidative balance in non-obese postmenopausal women.

The aim of the present study was to investigate whether accumulation of central fat is correlated with systemic oxidative stress (OxS) in non-obese apparently healthy postmenopausal women. Serum parameters of OxS (hydroperoxides and non-enzymatic antioxidants) along with body fat distribution, as assessed by dual-energy-X-ray absorptiometry (DXA), were evaluated in 134 non-obese postmenopausal women. Multiple regression analysis showed that central (trunk) fat significantly correlated with both markers of OxS independently of confounding factors (i.e. BMI, smoking, age, hypertension, legs and arms fat mass). In specific, the standardized regression coefficient was positive for hydroperoxides (ß = 0.324, p < 0.05) and negative for antioxidants (ß = -0.495, p < 0.01) level. In conclusion, the current data showed that the increase in central fat is an independent predictor of OxS condition among non-obese women in postmenopausal status. The possible pro-oxidant effects of the excess in central adiposity might be more harmful among post- than among pre-menopausal women, due to the postulated ability of E2 to contrast oxidative challenge and the related diseases.

Characterization of Stable Pyrazole Derivatives of Curcumin with Improved Cytotoxicity on Osteosarcoma Cell Lines.

Curcumin (CUR) is a natural molecule that is unstable due to the presence of a bis-ketone. To obtain more stable derivatives in biological fluids, the bis-ketone was replaced with pyrazole or O-substituted oximes. Their stability in solution was studied by UV-visible spectrophotometry. The effects on proliferation were studied by MTT assay and/or clonogenicity assay. Induction of apoptosis was evaluated by annexin V staining and Western blot analysis. The bioavailability was obtained from the analysis of the molecular chemical-physical characteristics. The replacement of the bis-ketone with a pyrazole ring or O-substituted oximes improved the stability of all the CUR-derivative molecules. These derivatives were more stable than CUR in solution and were generally cytotoxic on a panel of cancer cell lines tested, and they promoted caspase-dependent apoptosis. Derivative 1 was the most potent in the osteosarcoma (OS) lines. With respect to CUR, this derivative showed cytotoxicity at least three times higher in the MTT assay. In addition, in the clonogenic assay, 1 maintained the activity in conditions of long treatment presumably by virtue of its improved stability in biological fluids. Notably, 1 should have improved chemical-physical characteristics of bioavailability with respect to CUR, which should allow for reaching higher blood levels than those observed in the CUR trials. In conclusion, 1 should be considered in future clinical studies on the treatment of OS, either alone or in combination with other medications currently in use.

Effects of the regulatory ligands calcium and GTP on the thermal stability of tissue transglutaminase.

Tissue transglutaminase undergoes thermal inactivation with first-order kinetics at moderate temperatures, in a process which is affected in opposite way by the regulatory ligands calcium and GTP, which stabilize different conformations. We have explored the processes of inactivation and of unfolding of transglutaminase and the effects of ligands thereon, combining approaches of differential scanning calorimetry (DSC) and of thermal analysis coupled to fluorescence spectroscopy and small angle scattering. At low temperature (38-45°C), calcium promotes and GTP protects from inactivation, which occurs without detectable disruption of the protein structure but only local perturbations at the active site. Only at higher temperatures (52-56°C), the protein structure undergoes major rearrangements with alterations in the interactions between the N- and C-terminal domain pairs. Experiments by DSC and fluorescence spectroscopy clearly indicate reinforced and weakened interactions of the domains in the presence of GTP and of calcium, and different patterns of unfolding. Small angle scattering experiments confirm different pathways of unfolding, with attainment of limiting values of gyration radius of 52, 60 and 90 Å in the absence of ligands and in the presence of GTP and calcium. Data by X-rays scattering indicate that ligands influence retention of a relatively compact structure in the protein even after denaturation at 70°C. These results suggest that the complex regulation of the enzyme by ligands involves both short- and long-range effects which might be relevant for understanding the turnover of the protein in vivo.

Reduction in Fatigue Symptoms Following the Administration of Nutritional Supplements in Patients with Multiple Sclerosis.

Despite recent advances in immune-modulatory drugs, pharmacological therapies have been proven ineffective in severe presentations of multiple sclerosis (MS), including secondary progressive MS. At present, therapeutic interventions' performance is primarily focused on ameliorating symptoms to improve the patient's quality of life (QOL). Among complementary treatments, nutrition has been considered a decisive factor to control symptoms and enhance the wellness of MS patients. Although no special diets are associated with MS, the impact of diet and dietary supplements on the course of progressive forms of the disease has been studied during the last few years. Fatigue is among the most common and disabling symptoms reported by MS patients. Fatigue has been defined in the Multiple Sclerosis Council for Clinical Practice Guidelines (MSCCPG, 1998) as a "subjective lack of physical and/or mental energy that the individual perceives as an interference with habitual and desired activities". This study aimed to compare the psychometric functioning of the "Fatigue Severity Scale" (FSS) and the "Modified Fatigue Impact Scale" (MFIS) in our sample of people with MS. Specifically, during chronic treatment, the change in these two parameters with two vitamin-rich dietary supplements (Citozym® and Ergozym®) was evaluated. The impact of these nutritional supplements revealed differences in antioxidant and anti-inflammatory parameters among the volunteers in the treatment group, with a subsequent improvement in fatigue. In conclusion, the results obtained have confirmed the effectiveness of complementary nutritional therapies, evaluated essentially based on hematological biomarkers, through which it is possible to act on disability to improve the QOL of MS patients.

Thermodynamics of binding of regulatory ligands to tissue transglutaminase.

The transamidating activity of tissue transglutaminase is regulated by the ligands calcium and GTP, via conformational changes which facilitate or interfere with interaction with the peptidyl-glutamine substrate. We have analysed binding of these ligands by calorimetric and computational approaches. In the case of GTP we have detected a single high affinity site (K (D) approximately 1 microM), with moderate thermal effects suggestive that binding GTP involves replacement of GDP, normally bound to the protein. On line with this possibility no significant binding was observed during titration with GDP and computational studies support this view. Titration with calcium at a high cation molar excess yielded a complex binding isotherm with a number of "apparent binding sites" in large excess over those detectable by equilibrium dialysis (6 sites). This binding pattern is ascribed to occurrence of additional thermal contributions, beyond those of binding, due to the occurrence of conformational changes and to catalysis itself (with protein self-crosslinking). In contrast only one site for binding calcium with high affinity (K (D) approximately 0.15 microM) is observed with samples of enzyme inactivated by alkylation at the active site (to prevent enzyme crosslinkage and thermal effects of catalysis). These results indicate an intrinsic ability of tissue transglutaminase to bind calcium with high affinity and the necessity of careful reassessment of the enzyme regulatory pattern in relation to the concentrations of ligands in living cells, taking also in account effects of ligands on protein subcellular compartimentation.

The Role of Tissue Transglutaminase in Cancer Cell Initiation, Survival and Progression.

Tissue transglutaminase (transglutaminase type 2; TG2) is the most ubiquitously expressed member of the transglutaminase family (EC 2.3.2.13) that catalyzes specific post-translational modifications of proteins through a calcium-dependent acyl-transfer reaction (transamidation). In addition, this enzyme displays multiple additional enzymatic activities, such as guanine nucleotide binding and hydrolysis, protein kinase, disulfide isomerase activities, and is involved in cell adhesion. Transglutaminase 2 has been reported as one of key enzymes that is involved in all stages of carcinogenesis; the molecular mechanisms of action and physiopathological effects depend on its expression or activities, cellular localization, and specific cancer model. Since it has been reported as both a potential tumor suppressor and a tumor-promoting factor, the role of this enzyme in cancer is still controversial. Indeed, TG2 overexpression has been frequently associated with cancer stem cells' survival, inflammation, metastatic spread, and drug resistance. On the other hand, the use of inducers of TG2 transamidating activity seems to inhibit tumor cell plasticity and invasion. This review covers the extensive and rapidly growing field of the role of TG2 in cancer stem cells survival and epithelial⁻mesenchymal transition, apoptosis and differentiation, and formation of aggressive metastatic phenotypes.

Differential effects of chemotherapeutic drugs versus the MDM-2 antagonist nutlin-3 on cell cycle progression and induction of apoptosis in SKW6.4 lymphoblastoid B-cells.

We have compared the cytotoxic/cytostatic responses of the SKW6.4 lymphoblastoid B-cells to the alkylating agent chlorambucil, the purine analog fludarabine, the non-genotoxic activator of the p53 pathway, Nutlin-3, used alone or in association with the death-inducing ligand recombinant TRAIL. Exposure to chlorambucil, fludarabine, and Nutlin-3 induced p53 accumulation and variably affected cell cycle progression in SKW6.4 lymphoblastoid cells. In particular, chlorambucil induced cell cycle accumulation at the G2/M checkpoint; Nutlin-3 induced early cell cycle arrest at the G1/S checkpoint, while fludarabine showed an intermediate behavior. On the other hand, recombinant TRAIL alone did not affect cell cycle progression but induced a rapid increase of apoptosis. Analysis of the gene expression profile of the p53-transcriptional targets showed distinct features between chlorambucil, Nutlin-3 and fludarabine, which likely account for their differential effect on cell cycle in SKW6.4 cells. In particular, chlorambucil upregulated the steady-state mRNA expression of SFN/14-3-3sigma, a gene involved in G2/M cell cycle arrest. Of note, all agonists upregulated TRAIL-R2 expression in SKW6.4 cells both at the mRNA and protein levels. Consistently, pretreatment with chlorambucil, fludarabine and Nutlin-3 enhanced SKW6.4 sensitivity to TRAIL-mediated apoptosis.