Influence of Polydatin on the Tumor Microenvironment In Vitro: Studies with a Colon Cancer Cell Model.

The tumor microenvironment of colon carcinoma, the site at which tumor cells and the host immune system interact, is influenced by signals from tumor cells, immunocompetent cells, and bacterial components, including LPS. A large amount of LPS is available in the colon, and this could promote inflammation and metastasis by enhancing tumor cell adhesion to the endothelium. Polydatin (PD), the 3-ß-D-glucoside of trans-resveratrol, is a polyphenol with anti-cancer, anti-inflammatory, and immunoregulatory effects. This study was designed to explore whether PD is able to produce antiproliferative effects on three colon cancer lines, to reduce the expression of adhesion molecules that are upregulated by LPS on endothelial cells, and to decrease the proinflammatory cytokines released in culture supernatants. Actually, we investigated the effects of PD on tumor growth in a coculture model with human mononuclear cells (MNCs) that mimics, at least in part, an in vitro tumor microenvironment. The results showed that PD alone or in combination with MNC exerts antiproliferative and proapoptotic effects on cancer cells, inhibits the production of the immunosuppressive cytokine IL-10 and of the proinflammatory cytokines upregulated by LPS, and reduces E-selectin and VCAM-1 on endothelial cells. These data provide preclinical support to the hypothesis that PD could be of potential benefit as a therapeutic adjuvant in colon cancer treatment and prevention.

Abscopal Effect and Drug-Induced Xenogenization: A Strategic Alliance in Cancer Treatment?

The current state of cancer treatment is still far from being satisfactory considering the strong impairment of patients' quality of life and the high lethality of malignant diseases. Therefore, it is critical for innovative approaches to be tested in the near future. In view of the crucial role that is played by tumor immunity, the present review provides essential information on the immune-mediated effects potentially generated by the interplay between ionizing radiation and cytotoxic antitumor agents when interacting with target malignant cells. Therefore, the radiation-dependent abscopal effect (i.e., a biological effect of ionizing radiation that occurs outside the irradiated field), the influence of cancer chemotherapy on the antigenic pattern of target neoplastic cells, and the immunogenic cell death (ICD) caused by anticancer agents are the main topics of this presentation. It is widely accepted that tumor immunity plays a fundamental role in generating an abscopal effect and that anticancer drugs can profoundly influence not only the host immune responses, but also the immunogenic pattern of malignant cells. Remarkably, several anticancer drugs impact both the abscopal effect and ICD. In addition, certain classes of anticancer agents are able to amplify already expressed tumor-associated antigens (TAA). More importantly, other drugs, especially triazenes, induce the appearance of new tumor neoantigens (TNA), a phenomenon that we termed drug-induced xenogenization (DIX). The adoption of the abscopal effect is proposed as a potential therapeutic modality when properly applied concomitantly with drug-induced increase in tumor cell immunogenicity and ICD. Although little to no preclinical or clinical studies are presently available on this subject, we discuss this issue in terms of potential mechanisms and therapeutic benefits. Upcoming investigations are aimed at evaluating how chemical anticancer drugs, radiation, and immunotherapies are interacting and cooperate in evoking the abscopal effect, tumor xenogenization and ICD, paving the way for new and possibly successful approaches in cancer therapy.

Fatty acid synthase inhibitor orlistat impairs cell growth and down-regulates PD-L1 expression of a human T-cell leukemia line.

Fatty Acid Synthase (FASN) is responsible for the de novo synthesis of fatty acids, which are involved in the preservation of biological membrane structure, energy storage and assembly of factors involved in signal transduction. FASN plays a critical role in supporting tumor cell growth, thus representing a potential target for anti-cancer therapies. Moreover, this enzyme has been recently associated with increased PD-L1 expression, suggesting a role for fatty acids in the impairment of the immune response in the tumor microenvironment. Orlistat, a tetrahydrolipstatin used for the treatment of obesity, has been reported to reduce FASN activity, while inducing a sensible reduction of the growth potential in different cancer models. We have analyzed the effect of orlistat on different features involved in the tumor cell biology of the T-ALL Jurkat cell line. In particular, we have observed that orlistat inhibits Jurkat cell growth and induces a perturbation of cell cycle along with a decline of FASN activity and protein levels. Moreover, the drug produces a remarkable impairment of PD-L1 expression. These findings suggest that orlistat interferes with different mechanisms involved in the control of tumor cell growth and can potentially contribute to decrease the tumor-associated immune-pathogenesis.

Inflammatory Microenvironment and Adipogenic Differentiation in Obesity: The Inhibitory Effect of Theobromine in a Model of Human Obesity In Vitro.

OBJECTIVE: Obesity is considered a clinic condition characterized by a state of chronic low-grade inflammation. The role of macrophages and adipocytokines in adipose tissue inflammation is in growing investigation. The physiopathological mechanisms involved in inflammatory state in obesity are not fully understood though the adipocytokines seem to characterize the biochemical link between obesity and inflammation. The aim of this work is to analyze the effect of theobromine, a methylxanthine present in the cocoa, on adipogenesis and on proinflammatory cytokines evaluated in a model of fat tissue inflammation in vitro. METHODS: In order to mimic in vitro this inflammatory condition, we investigated the interactions between human-like macrophages U937 and human adipocyte cell lines SGBS. The effect of theobromine on in vitro cell growth, cell cycle, adipogenesis, and cytokines release in the supernatants has been evaluated. RESULTS: Theobromine significantly inhibits the differentiation of preadipocytes in mature adipocytes and reduces the levels of proinflammatory cytokines as MCP-1 and IL-1ß in the supernatants obtained by the mature adipocytes and macrophages interaction. CONCLUSION: Theobromine reduces adipogenesis and proinflammatory cytokines; these data suggest its potential therapeutic effect for treating obesity by control of macrophages infiltration in adipose tissue and inflammation.

Beneficial and Detrimental Effects of Antiretroviral Therapy on HIV-Associated Immunosenescence.

Since the introduction of highly active antiretroviral therapy more than 2 decades ago, HIV-related deaths have dramatically decreased and HIV infection has become a chronic disease. Due to the inability of antiretroviral drugs to eradicate the virus, treatment of HIV infection requires a systemic lifelong therapy. However, even when successfully treated, HIV patients still show increased incidence of age-associated co-morbidities compared with uninfected individuals. Virus- induced immunosenescence, a process characterized by a progressive decline of immune system function, contributes to the premature ageing observed in HIV patients. Although antiretroviral therapy has significantly improved both the quality and length of patient lives, the life expectancy of treated patients is still shorter compared with that of uninfected individuals. In particular, while antiretroviral therapy can contrast some features of HIV-associated immunosenescence, several anti-HIV agents may themselves contribute to other aspects of immune ageing. Moreover, older HIV patients tend to have a worse immunological response to the antiviral therapy. In this review we will examine the available evidence on the role of antiretroviral therapy in the control of the main features regulating immunosenescence.

Drug-induced xenogenization of tumors: A possible role in the immune control of malignant cell growth in the brain?

In recent years, immune checkpoint inhibitors (ICpI) have provided the ground to bring tumor immunity back to life thanks to their capacity to afford a real clinical benefit in terms of patient's survival. Essential to ICpI success is the presence of tumor-associated neoantigens generated by non-synonymous mutations, since a direct relationship between mutation load of malignant cells and susceptibility to ICpI has been confidently established. However, it has been also suggested that high intratumor heterogeneity (ITH) associated with subclonal neoantigens could not elicit adequate immune responses. Several years ago we discovered that in vivo treatment of leukemic mice with triazene compounds (TZC) produces a marked increase of leukemia cell immunogenicity [a phenomenon termed Drug-Induced Xenogenization (DIX)] through point mutations able to generate strong tumor neoantigens (Drug-Induced Neoantigens, DIN). Immunogenic mutations are produced by TZC-dependent methylation of O6-guanine of DNA, that is suppressed by the DNA repair protein methyl-guaninemethyltransferase (MGMT). This minireview illustrates preclinical investigations conducted in animal models where DIN-positive murine leukemia cells were inoculated intracerebrally into histocompatible mice. The analysis of the literature indicates that the growth of xenogenized malignant cells is controlled by anti-DIN graft responses and by intra-cerebral or intravenous adoptive transfer of anti-DIN cytotoxic T lymphocytes. This survey reminds also that PARP inhibitors increase substantially the antitumor activity of TZC and can be administered with the intent of suppressing more efficiently tumor load and possibly reducing ITH through downsizing the polyclonality of xenogenized tumor cell population. Finally, the present report illustrates a hypothetical clinical protocol that could be considered as an example of future development of DIXbased tumor immuno-chemotherapy in brain malignancies. The protocol involves oral or intravenous administration of TZC along with loco-regional (i.e. intracerebral "wafer") treatment with agents able to increase tumor cell sensitivity to the cytotoxic and xenogenizing effects of TZC (i.e. MGMT and PARP inhibitors) without enhancing the systemic toxicity of these DNA methylating compounds.

Antitumour activity of resveratrol on human melanoma cells: A possible mechanism related to its interaction with malignant cell telomerase.

BACKGROUND: trans-Resveratrol (tRES) is a polyphenolic stilbene found in plant products which has attracted great attention because of its antioxidant, anti-inflammatory and anticancer properties. METHODS: The possible correlation between tRES-induced suppression of melanoma cell growth and its influence on telomerase expression has been investigated by biological assays. Moreover, in order to gain new knowledge about possible mechanisms of action of tRES as antineoplastic agent, its interaction with biologically relevant secondary structure-forming DNA sequences, its aggregation properties and copper-binding activity have been studied by CD, UV and fluorescence spectroscopies. RESULTS: Biological assays have confirmed that growth inhibitory properties of tRES well correlate with the reduction of telomerase activity and hTERT gene transcript levels in human melanoma cells. Biophysical studies in solution have proved that tRES binds all the studied DNA model systems with low affinity, however showing high ability to discriminate G-quadruplex vs. duplex DNA. In addition, tRES has shown no propensity to form aggregates in the explored concentration range and has been found able to bind Cu2+ ions with a 2:1 stoichiometry. CONCLUSIONS: From these biological and biophysical analyses it has emerged that tRES produces cytotoxic effects on human melanoma cells and, at a molecular level, is able to bind Cu2+ and cancer-involved G-quadruplexes, suggesting that multiple mechanisms of action could be involved in its antineoplastic activity. GENERAL SIGNIFICANCE: Expanding the knowledge on the putative mechanisms of action of tRES as antitumour agent can help to develop novel, effective tRES-based anticancer drugs.

Tumor immunotherapy: drug-induced neoantigens (xenogenization) and immune checkpoint inhibitors.

More than 40 years ago, we discovered that novel transplantation antigens can be induced in vivo or in vitro by treating murine leukemia with dacarbazine. Years later, this phenomenon that we called "Chemical Xenogenization" (CX) and more recently, "Drug-Induced Xenogenization" (DIX), was reproduced by Thierry Boon with a mutagenic/carcinogenic compound (i.e. N-methyl-N'-nitro-N-nitrosoguanidine). In both cases, the molecular bases of DIX rely on mutagenesis induced by methyl adducts to oxygen-6 of DNA guanine. In the present review we illustrate the main DIX-related immune-pharmacodynamic properties of triazene compounds of clinical use (i.e. dacarbazine and temozolomide).In recent years, tumor immunotherapy has come back to the stage with the discovery of immune checkpoint inhibitors (ICpI) that show an extraordinary immune-enhancing activity. Here we illustrate the salient biochemical features of some of the most interesting ICpI and the up-to-day status of their clinical use. Moreover, we illustrate the literature showing the direct relationship between somatic mutation burden and susceptibility of cancer cells to host's immune responses.When DIX was discovered, we were not able to satisfactorily exploit the possible presence of triazene-induced neoantigens in malignant cells since no device was available to adequately enhance host's immune responses in clinical settings. Today, ICpI show unprecedented efficacy in terms of survival times, especially when elevated mutation load is associated with cancer cells. Therefore, in the future, mutation-dependent neoantigens obtained by appropriate pharmacological intervention appear to disclose a novel approach for enhancing the therapeutic efficacy of ICpI in cancer patients.

Influence of fatty acid synthase inhibitor orlistat on the DNA repair enzyme O6-methylguanine-DNA methyltransferase in human normal or malignant cells in vitro.

Tetrahydrolipstatin (orlistat), an inhibitor of lipases and fatty acid synthase, is used orally for long-term treatment of obesity. Although the drug possesses striking antitumor activities in vitro against human cancer cells and in vitro and in vivo against animal tumors, it also induces precancerous lesions in rat colon. Therefore, we tested the in vitro effect of orlistat on the expression of O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme that plays an essential role in the control of mutagenesis and carcinogenesis. Western blot analysis demonstrated that 2-day continuous exposure to 40 µM orlistat did not affect MGMT levels in a human melanoma cell line, but downregulated the repair protein by 30-70% in human peripheral blood mononuclear cells, in two leukemia and two colon cancer cell lines. On the other hand, orlistat did not alter noticeably MGMT mRNA expression. Differently from lomeguatrib (a false substrate, strong inhibitor of MGMT) orlistat did not reduce substantially MGMT function after 2-h exposure of target cells to the agent, suggesting that this drug is not a competitive inhibitor of the repair protein. Combined treatment with orlistat and lomeguatrib showed additive reduction of MGMT levels. More importantly, orlistat-mediated downregulation of MGMT protein expression was markedly amplified when the drug was combined with a DNA methylating agent endowed with carcinogenic properties such as temozolomide. In conclusion, even if orlistat is scarcely absorbed by oral route, it is possible that this drug could reduce local MGMT-mediated protection against DNA damage provoked by DNA methylating compounds on gastrointestinal tract epithelial cells, thus favoring chemical carcinogenesis.

The antiretroviral agent saquinavir enhances hTERT expression and telomerase activity in human T leukaemia cells in vitro.

BACKGROUND: Saquinavir, a protease inhibitor utilized in HIV infection, shows antitumor activity in various experimental models. In previous studies performed in our laboratory the drug was found to induce a substantial increase of telomerase activity in normal peripheral blood mononuclear cells. Aim of the present investigation was to test whether saquinavir was able to increase telomerase activity and the expression of the catalytic subunit of telomerase, hTERT, in human malignant hematopoietic cells. METHODS: Human Jurkat CD4+ T cell leukaemia cell line was used throughout the present study. The antiproliferative effect of saquinavir was tested by the MTT assay. Telomerase activity was determined according to the telomeric repeat amplification protocol. The expression of hTERT mRNA was semi-quantitative evaluated by RT-PCR amplification and quantitative Real Time PCR. The binding of the transcription factor c-Myc to its specific E-Box DNA binding-site of hTERT promoter was analyzed by Electophoretic Mobility Shift Assay (EMSA). The amount of c-Myc in cytoplasm and nucleus of leukemia cells was determined by Western Blot analysis, and c-Myc down-regulation was obtained by siRNA transfection. RESULTS: Saquinavir produced a substantial increase of telomerase activity in Jurkat cells in vitro without increasing but rather reducing target cell proliferation rate. Telomerase up-regulation appeared to be the result of enhanced expression of hTERT. Saquinavir-mediated up-regulation of hTERT gene was the result of the increased binding of proteins to the E-Box sequence of the promoter. Moreover, saquinavir amplified the expression of c-Myc especially in the nuclear cell fraction. The direct influence of saquinavir on this transcription factor was also demonstrated by the antagonistic effect of the drug on siRNA induced c-Myc suppression. Since c-Myc is the main responsible for hTERT transcription, these findings suggest that the main mechanism underlying saquinavir-induced telomerase activation is mediated by c-Myc up-regulation. CONCLUSIONS: Saquinavir augments hTERT expression while inhibiting leukemic cell growth. Experimental evidences show that this effect is mediated by saquinavir-influenced increase of c-Myc levels. This could have relevance in terms of enhanced hTERT-dependent tumor cell immunogenicity and suggests new paharmacological approaches interfering with c-Myc dependent pathways.

Circulating tumor cells in colorectal cancer patients.

The availability of sensitive methods has allowed the detailed study of circulating tumor cells only recently. Evolving evidence support the prognostic and predictive role of these cells in patients affected by several solid tumors, including colorectal cancer. Ongoing studies are aimed at confirming that the molecular characterization of circulating tumor cells in peripheral blood and in bone marrow of patients is a powerful tool to improve the patient risk-stratification, to monitor activity of the drugs, to develop more appropriate targeted therapies and tailored treatments. In parallel, results from these correlative studies promise to gain a better biological understanding of the metastatic process. The clinical utility of the detection of circulating tumor cells in patients affected by colorectal cancer is still hampered by a number of specific hurdles. Improvement in sensitivity and specificity of the available methods of detection, standardization of these methods and functional characterization of circulating tumor cells in well designed and statistically well powered studies are the key steps to reach these ambitious objectives in colorectal cancer patients as well.

Molecular mechanisms involved in HIV-1-Tat mediated inhibition of telomerase activity in human CD4(+) T lymphocytes.

Human immunodeficiency virus type 1 (HIV-1) infection is characterized by a progressive decline of CD4(+) T cells and by other immune disorders that are similar to those observed during aging and which lead eventually to AIDS. One of the mechanisms involved in HIV-1 induced immunodeficiency may be the lack of telomerase induction and the consequent impairment of the potential required for CD4(+) T cell expansion. Telomerase compensates for the progressive telomere loss during cell division and preserves the replicative potential of T lymphocytes after repeated antigenic stimulation. The enzyme is activated by post-translational modifications, such as phosphorylation and also by the nuclear import of its catalytic subunit hTERT from the cytoplasm. In previous studies we found a reduction of telomerase activity in the nucleus of CD4(+) T cells infected with HIV-1 or non-infected but exposed to Tat protein. However, the mechanism for this loss of activity has not been elucidated yet. In the present study, we found that HIV-1 Tat inhibited telomerase activity in CD4(+) T cells by different mechanisms. First, it reduced nuclear levels of hTERT. Secondly, this protein perturbed the AKT pathway and the molecular interaction with the chaperones required for hTERT phosphorylation, nuclear import and activation. These results suggest that in addition to inducing direct cell death, HIV infection may also reduce the replicative potential of non-infected CD4(+) T cells and this may contribute to the overall immunodeficiency in AIDS patients.

NF-κB is activated in response to temozolomide in an AKT-dependent manner and confers protection against the growth suppressive effect of the drug.

BACKGROUND: Most DNA-damaging chemotherapeutic agents activate the transcription factor nuclear factor κB (NF-κB). However, NF-κB activation can either protect from or contribute to the growth suppressive effects of the agent. We previously showed that the DNA-methylating drug temozolomide (TMZ) activates AKT, a positive modulator of NF-κB, in a mismatch repair (MMR) system-dependent manner. Here we investigated whether NF-κB is activated by TMZ and whether AKT is involved in this molecular event. We also evaluated the functional consequence of inhibiting NF-κB on tumor cell response to TMZ. METHODS: AKT phosphorylation, NF-κB transcriptional activity, IκB-α degradation, NF-κB2/p52 generation, and RelA and NF-κB2/p52 nuclear translocation were investigated in TMZ-treated MMR-deficient (HCT116, 293TLα-) and/or MMR-proficient (HCT116/3-6, 293TLα+, M10) cells. AKT involvement in TMZ-induced activation of NF-κB was addressed in HCT116/3-6 and M10 cells transiently transfected with AKT1-targeting siRNA or using the isogenic MMR-proficient cell lines pUSE2 and KD12, expressing wild type or kinase-dead mutant AKT1. The effects of inhibiting NF-κB on sensitivity to TMZ were investigated in HCT116/3-6 and M10 cells using the NF-κB inhibitor NEMO-binding domain (NBD) peptide or an anti-RelA siRNA. RESULTS: TMZ enhanced NF-κB transcriptional activity, activated AKT, induced IκB-α degradation and RelA nuclear translocation in HCT116/3-6 and M10 but not in HCT116 cells. In M10 cells, TMZ promoted NF-κB2/p52 generation and nuclear translocation and enhanced the secretion of IL-8 and MCP-1. TMZ induced RelA nuclear translocation also in 293TLα+ but not in 293TLα- cells. AKT1 silencing inhibited TMZ-induced IκB-α degradation and NF-κB2/p52 generation. Up-regulation of NF-κB transcriptional activity and nuclear translocation of RelA and NF-κB2/p52 in response to TMZ were impaired in KD12 cells. RelA silencing in HCT116/3-6 and M10 cells increased TMZ-induced growth suppression. In M10 cells NBD peptide reduced basal NF-κB activity, abrogated TMZ-induced up-regulation of NF-κB activity and increased sensitivity to TMZ. In HCT116/3-6 cells, the combined treatment with NBD peptide and TMZ produced additive growth inhibitory effects. CONCLUSION: NF-κB is activated in response to TMZ in a MMR- and AKT-dependent manner and confers protection against drug-induced cell growth inhibition. Our findings suggest that a clinical benefit could be obtained by combining TMZ with NF-κB inhibitors.

Down-regulation of the PTTG1 proto-oncogene contributes to the melanoma suppressive effects of the cyclin-dependent kinase inhibitor PHA-848125.

We previously demonstrated that PHA-848125, a cyclin-dependent kinase inhibitor presently under Phase II clinical investigation, impairs melanoma cell growth. In this study, gene expression profiling showed that PHA-848125 significantly modulated the expression of 128 genes, predominantly involved in cell cycle control, in the highly drug-sensitive GL-Mel (p53 wild-type) melanoma cells. Up-regulation of 4 selected genes (PDCD4, SESN2, DDIT4, DEPDC6), and down-regulation of 6 selected genes (PTTG1, CDC25A, AURKA, AURKB, PLK1, BIRC5) was confirmed at protein levels. The same protein analysis performed in PHA-848125-treated M10 melanoma cells - p53 mutated and less sensitive to the drug than GL-Mel cells - revealed no DEPDC6 expression and no changes of PTTG1, PDCD4 and BIRC5 levels. Upon PHA-848125 treatment, a marked PTTG1 down-modulation was also observed in A375 cells (p53 wild-type) but not in CN-Mel cells (p53 mutated). PTTG1 silencing significantly inhibited melanoma cell proliferation and induced senescence, with effects less pronounced in p53 mutated cells. PTTG1 silencing increased PHA-848125 sensitivity of p53 mutated cells but not that of A375 or GL-Mel cells. Accordingly, in M10 but not in A375 cells a higher level of senescence was detected in PHA-848125-treated/PTTG1-silenced cells with respect to PHA-848125-treated controls. In A375 and GL-Mel cells, TP53 silencing attenuated PHA-848125-induced down-modulation of PTTG1 and decreased cell sensitivity to the drug. These findings indicate that PHA-848125-induced down-regulation of PTTG1 depends, at least in part, on p53 function and contributes to the antiproliferative activity of the drug. Our study provides further molecular insight into the antitumor mechanism of PHA-848125.

Exogenous control of the expression of Group I CD1 molecules competent for presentation of microbial nonpeptide antigens to human T lymphocytes.

Group I CD1 (CD1a, CD1b, and CD1c) glycoproteins expressed on immature and mature dendritic cells present nonpeptide antigens (i.e., lipid or glycolipid molecules mainly of microbial origin) to T cells. Cytotoxic CD1-restricted T lymphocytes recognizing mycobacterial lipid antigens were found in tuberculosis patients. However, thanks to a complex interplay between mycobacteria and CD1 system, M. tuberculosis possesses a successful tactic based, at least in part, on CD1 downregulation to evade CD1-dependent immunity. On the ground of these findings, it is reasonable to hypothesize that modulation of CD1 protein expression by chemical, biological, or infectious agents could influence host's immune reactivity against M. tuberculosis-associated lipids, possibly affecting antitubercular resistance. This scenario prompted us to perform a detailed analysis of the literature concerning the effect of external agents on Group I CD1 expression in order to obtain valuable information on the possible strategies to be adopted for driving properly CD1-dependent immune functions in human pathology and in particular, in human tuberculosis.

MicroRNA-155 targets the SKI gene in human melanoma cell lines.

The SKI protein is a transcriptional coregulator over-expressed in melanoma. Experimentally induced down-regulation of SKI inhibits melanoma cell growth in vitro and in vivo. MicroRNAs (miRNAs) negatively modulate gene expression and have been implicated in oncogenesis. We previously showed that microRNA-155 (miR-155) is down-regulated in melanoma cells as compared with normal melanocytes and that its ectopic expression impairs proliferation and induces apoptosis. Here, we investigated whether miR-155 could mediate melanoma growth inhibition via SKI gene silencing. Luciferase reporter assays demonstrated that miR-155 interacted with SKI 3'UTR and impaired gene expression. Transfection of melanoma cells with miR-155 reduced SKI levels, while inhibition of endogenous miR-155 up-regulated SKI expression. Specifically designed small interfering RNAs reduced SKI expression and inhibited proliferation. However, melanoma cells over-expressing a 3'UTR-deleted SKI were still susceptible to the antiproliferative effect of miR-155. Our data demonstrate for the first time that SKI is a target of miR-155 in melanoma. However, impairment of SKI expression is not the leading mechanism involved in the growth-suppressive effect of miR-155 found in this malignancy.

Detection of circulating tumor cells is improved by drug-induced antigen up-regulation: preclinical and clinical studies.

(51)Cr-prelabelled colon cancer cells (simulating 'circulating tumor cells', CTCs) were added to human peripheral blood and exposed to staurosporine (ST) to increase carcinoembryonic antigen (CEA) expression. CTCs were captured with immunomagnetic beads coated with Ber-EP4 monoclonal antibody, recognizing the common epithelial antigen present in the majority of cancer cells of epithelial origin, with capture efficiency of more than 80%. Moreover, ST treatment increased CEA expression without compromising Ber-EP4 capture efficiency. In a pilot clinical study on 37 patients, CTCs were captured using Ber-EP4 beads, and recognized by RT-PCR set for CEA or cytokeratin-19 (CK) mRNA detection. The results showed that: (a) the percentage of CEA-positive CTCs (CTC(CEA), 54.1%) was lower than that of CK-positive CTCs (CTC(CK), 70.3%); (b) in vitro ST treatment converted a significant number of CTC(CEA)-negative into CTC(CEA)-positive cases. Therefore, immunomagnetic capture combined with exposure to ST provides a feasible and sensitive technique for the detection of functionally-active CTCs responsive to ST-mediated CEA up-regulation.

Spindle cells from AIDS-associated Kaposi's sarcoma lesions express telomerase activity that is enhanced by Kaposi's sarcoma progression factors.

The activity of telomerase, a ribonucleoprotein maintaining the length of chromosome ends (telomeres) to levels allowing cells to replicate indefinitely, is undetectable in normal, differentiated cells, is present at low levels in some activated cell types (including endothelial cells) and it is highly expressed by tumor cells. Kaposi's sarcoma (KS), the most frequent tumor in Acquired Immune Deficiency Syndrome (AIDS) patients (AIDS-KS), arises as a disorder of new blood vessel formation (angiogenesis), but it may evolve into an aggressive cancer, characterized by the proliferation and invasion of spindle-shaped, endothelial-like cells (KS cells, KSC). Here we report that primary KSC express low telomerase levels which are strongly enhanced by KS initiation and progression factors including the inflammatory mediators interleukin (IL)-1beta, tumor necrosis factor (TNF)alpha and interferon (IFN)gamma, the angiogenic basic fibroblast growth factor (bFGF) and the Tat protein of Human Immunodeficiency Virus (HIV)-1. Noteworthy, the increase of telomerase activity promoted by these molecules parallels the induction of KSC growth and invasion. These preliminary in vitro findings encourage measuring telomerase activity in AIDS-KS lesions in order to survey the clinical progression of the disease.

The cyclin-dependent kinase inhibitor PHA-848125 suppresses the in vitro growth of human melanomas sensitive or resistant to temozolomide, and shows synergistic effects in combination with this triazene compound.

PHA-848125 is a novel cyclin-dependent kinase inhibitor under Phase I/II clinical investigation. In this study, we describe, for the first time, the effect of PHA-848125 on human melanoma cells in vitro. Seven melanoma cell lines with different sensitivity to temozolomide (TMZ) were exposed to PHA-848125 for 5 days and then assayed for cell growth. In all cases, including TMZ-resistant cells, PHA-848125 IC(50) values were significantly below the maximum plasma concentrations achievable in the clinic. In the most PHA-848125-sensitive cell line, the drug caused a concentration-dependent G(1) arrest. PHA-848125 also impaired phosphorylation of the retinoblastoma protein at CDK2 and CDK4 specific sites, decreased retinoblastoma protein and cyclin A levels, and increased p21(Cip1), p27(Kip1) and p53 expression. Combined treatment with fixed ratios of TMZ plus PHA-848125 was studied in three melanoma cell lines. PHA-848125 was added to the cells 48 h after TMZ and cell growth was evaluated after 3 additional days of culture. Parallel experiments were performed in the presence of O(6)-benzylguanine (BG), to prevent repair of methyl adducts at O(6)-guanine induced by TMZ. Drug combination of TMZ plus BG and PHA-848125 produced additive or synergistic effects on cell growth, depending on the cell line. In the absence of BG, the combination was still more active than the single agents in the cell line moderately sensitive to TMZ, but comparable to PHA-848125 alone in the two TMZ-resistant cell lines. When TMZ plus BG were used in combination with PHA-848125 against cultured normal melanocytes, neither synergistic nor additive antiproliferative effects were observed. Our results indicate that PHA-848125 can have a therapeutic potential in melanoma patients, alone or combined with TMZ. Moreover this agent appears to be particularly attractive on the bases of its effectiveness against TMZ-resistant melanoma cells.

Altered expression of selected microRNAs in melanoma: antiproliferative and proapoptotic activity of miRNA-155.

Altered expression of microRNAs (miRNAs) has been detected in cancer, suggesting that these small non-coding RNAs can act as oncogenes or tumor suppressor genes. In the present study, we investigated the expression of miRNA-17-5p, miRNA-18a, miRNA-20a, miRNA-92a, miRNA-146a, miRNA-146b and miRNA-155 by real-time quantitative RT-PCR in a panel of melanocyte cultures and melanoma cell lines and explored the possible role of miRNA-155 in melanoma cell proliferation and survival. The analyzed miRNAs were selected on the basis of previous studies strongly supporting their involvement in cancer development and/or progression. We found that miRNA-17-5p, miRNA-18a, miRNA-20a, and miRNA-92a were overexpressed, whereas miRNA-146a, miRNA-146b and miRNA-155 were down-regulated in the majority of melanoma cell lines with respect to melanocytes. Ectopic expression of miRNA-155 significantly inhibited proliferation in 12 of 13 melanoma cell lines with reduced levels of this miRNA and induced apoptosis in 4 out of 4 cell lines analyzed. In conclusion, our data further support the finding of altered miRNA expression in melanoma cells and establish for the first time that miRNA-155 is a negative regulator of melanoma cell proliferation and survival.