A possible interplay between HR-HPV and stemness in tumor development: an in vivo investigation of CD133 as a putative marker of cancer stem cell in HPV18-infected KB cell line.

High-risk HPVs (HR-HPVs) are DNA viruses considered as primary etiologic factors in malignancies of the low female genital tract. Their presence has also been documented in oropharyngeal and laryngeal cancers. However, HPV infection is considered a necessary but not sufficient cause of tumoral development; meantime, increasing evidences on the tumorigenic role of cancer stem cells (CSCs) have been documented in the literature. CSCs represent a small subpopulation of neoplastic cells with self-renewal potential, capable of maintaining tumor growth and cell differentiation, also involved in metastatic process, recurrence, and resistance to chemotherapeutic agents. In the present study, performed on KB cell lines, we evaluated the tumor forming potential of CSCs, and their relationship with the HPV infection status. We started our study by identifying the most aggressive cell line on the minimal number of cells being able of growth in vivo in a model of athymic nude mice (BALB/c nu/nu). We used an oral-derived KB cell line separated in the KB-CD133+ and KB-CD133- populations, by using immunomagnetic beads and fluorescence-activated cell sorting (FACS). The separated populations were injected in athymic nude mice (BALB/c nu/nu). Xenograft tumors have been analyzed for tumor size, CD133 expression by immunohistochemistry (IHC) and for DNA HR-HPV integration by in situ hybridization (ISH), comparing CD133-enriched xenograft tumors versus the CD133 non-enriched ones. On standard conditions, the KB cell line has a poor population of glycosylated CD133 marker (<5.0%) when investigated with antibodies versus CD133, and more specifically its glycosylated epitope (AC133). Enriched CD133 KB cells possess a higher capacity of tumor growth in xenograft models of nude mice when compared to KB CD133-negative cells. We observed that the AC133 epitope, extensively used to purifying hematopoietic stem cells, is able to select an epithelial subpopulation of cancer stem cells with aggressive behavior. We retain that CD133 may be a useful target in anticancer strategies including pharmacological and immunological therapies.

Resveratrol (3,5,4'-trihydroxystilbene) and its properties in oral diseases.

Health promotion strategies and lifestyle changes are important in disease prevention. Oral health has received a large amount of attention previously as it is a fundamental component of general health and it contributes to the quality of life. Therefore, the study of associations between diet, health and the presence of bioactive compounds in food is receiving a substantial amount of attention. In the present review the effects and targets of a natural polyohenolic stilbenoid compound; resveratrol (3,5,4'-trihydroxystilbene; RSV) is assessed, and the future prospects for RSV in promoting oral health are considered. RSV is a phytoalexin, synthesized by a wide range of plants and abundantly extracted in grape skin, it has been purported to exert a multiplicity of anti-inflammatory, anti-viral, anti-microbial, estrogenic, anticancer, cardioprotective, neuroprotective and immunomodulatory functions. In this review, following an introduction documenting the biochemistry of RSV and RSV glucosides, the bioavailability and pharmacokinetics of RSV are described. Considering its multiple properties, the present review has focused on the potential benefits of RSV as an antioxidant and chemopreventive agent.

Synthesis and Enantiomeric Separation of a Novel Spiroketal Derivative: A Potent Human Telomerase Inhibitor with High in Vitro Anticancer Activity.

The synthesis, the enantiomeric separation, and the characterization of new simple spiroketal derivatives have been performed. The synthesized compounds have shown a very high anticancer activity. Cell proliferation assay showed that they induce a remarkable inhibition of cell proliferation in all cell lines treated, depending on culture time and concentration. The compounds have also shown a potent nanomolar human telomerase inhibition activity and apoptosis induction. CD melting experiments demonstrate that spiroketal does not affect the G-quadruplex (G4) thermal stability. Docking studies showed that telomerase inhibition could be determined by a spiroketal interaction with the telomerase enzyme.

Downregulation of proinflammatory cytokines in HTLV-1-infected T cells by Resveratrol.

BACKGROUND: Human T-cell leukemia virus (HTLV-1) is a lymphotropic retrovirus associated to adult T cell leukemia (ATL) and to non-neoplastic inflammatory conditions affecting the central nervous system, lung or skin. The inflammatory disorders associated to HTLV-1 are mediated by different proinflammatory cytokines as IL-1α, IL-6, TNF-α. The release and the role of IL-17 is still debated. Aims of this study were to analyze IL-17 induction by HTLV-1 infection and to determine whether resveratrol (RES) is able to down regulate the pathway of cytokines production either in HTLV-1 chronically infected MT-2 cell line or in human CD4+ cells infected in vitro with HTLV-1. METHODS: MT-2 and HTLV-1 infected CD4+ cells were analyzed for proinflammatory cytokine production before or after RES treatment. The concentrations of IL-17, IL-1α, IL-6, and TNF-α were measured in cell culture supernatants by ELISA and SearchLight™ technology. The IL-17 mRNA expression was evaluated by RT-PCR. NF-kB activation was detected by non-radioactive, Electro Mobility Shift Assay (EMSA). HTLV-1 RNA expression was detected by Real-time-PCR (RQ-PCR). RESULTS: We found that RES is capable of inducing a dose-dependent inhibition of IL-1α, IL-6 and TNF-α production in vitro and can down regulate the expression of IL-17 at both mRNA and protein levels in HTLV-1 infected cells. This effect was associated with a dose-dependent inhibition of the of the nuclear factor kappa-B (NF-kB) activity. Conversely, RES did not apparently affect HTLV-1 proliferation. CONCLUSIONS: These results support the anti-inflammatory properties of RES, suggesting that it might be a useful therapeutic agent for the treatment of HTLV-1 related inflammatory diseases.

Interferon-Beta combined with interleukin-2 restores human natural cytotoxicity impaired in vitro by ionizing radiations.

It is well known that ionizing radiations induce a marked downregulation of antigen-dependent and natural immunity for a prolonged period of time. This is due, at least in part, to radiation-induced apoptosis of different lymphocyte subpopulations, including natural killer (NK) cells. Aim of this study was to investigate the capability of Beta Interferon (ß-IFN) and Interleukin-2 (IL2), alone or in combination, to restore the functional activity of the natural immune system. Mononuclear cells (MNCs) obtained from intact or in vitro irradiated human peripheral blood were treated in vitro with ß-IFN immediately before or at the end of the 4-day treatment with IL2. Time-course analysis was performed on the NK activity, the total number and the apoptotic fraction of CD16+ and CD56+ cells, the 2 main NK effector cell subpopulations. The results indicate that radiation-induced impairment of natural cytotoxicity of MNC could be successfully antagonized by the ß-IFN+IL2 combination, mainly when exposure to ß-IFN preceded IL2 treatment. This radioprotective effect is paralleled by lower levels of radiation-induced apoptosis and increased expression of the antiapoptotic Bcl-2 protein. Since natural immunity can play a significant role in antitumor host's resistance, these results could provide the rational basis for a cytokine-based pharmacological strategy able to restore immune responsiveness and to afford possible therapeutic benefits in cancer patients undergoing radiotherapy.

Evaluation of antigen specific recognition and cell mediated cytotoxicity by a modified lysispot assay in a rat colon carcinoma model.

BACKGROUND: Antigen-specific CD8+ cytotoxic T lymphocytes represent potent effector cells of the adaptive immune response against viruses as well as tumours. Therefore assays capable at exploring the generation and function of cytotoxic T lymphocytes represent an important objective for both clinical and experimental settings. METHODS: Here we show a simple and reproducible assay for the evaluation of antigen-specific CD8+ cytotoxic T lymphocytes based on a LysiSpot technique for the simultaneous determination of antigen-specific IFN-γ production and assessment of tumor cytolysis. The assay was developed within an experimental model of colorectal carcinoma, induced by the colorectal tumor cell line DHD-K12 that induces tumors in BDIX rats and, in turn, elicits a tumor- specific immune response. RESULTS: Using DHD-K12 cells transfected to express Escherichia coli ß-galactosidase as target cells, and by the fine setting of spot colours detection, we have developed an in vitro assay that allows the recognition of cytotoxic T lymphocytes induced in BDIX rats as well as the assessment of anti-tumour cytotoxicity. The method highlighted that in the present experimental model the tumour antigen-specific immune response was bound to killing target cells in the proportion of 55%, while 45% of activated cells were not cytotoxic but released IFN-γ. Moreover in this model by an ELISPOT assay we demonstrated the specific recognition of a nonapeptide epitope called CSH-275 constitutionally express in DHD-K12 cells. CONCLUSIONS: The assay proved to be highly sensitive and specific, detecting even low frequencies of cytotoxic/activated cells and providing the evaluation of cytokine-expressing T cells as well as the extent of cytotoxicity against the target cells as independent functions. This assay may represent an important tool to be adopted in experimental settings including the development of vaccines or immune therapeutic strategies.

Anti-inflammatory effect of resveratrol and polydatin by in vitro IL-17 modulation.

Interleukin-17 (IL-17) is a proinflammatory cytokine produced, although not exclusively, by T helper 17 recently identified as a distinct T helper lineage mediating tissue inflammation. IL-17 is known to be involved in a number of chronic disorders although the mechanisms regulating its production in inflammatory disease are still unclear. The beneficial properties of the polyphenolic compound resveratrol including its anti-inflammatory, antioxidant, and antitumor effects, its role in the aging process and in the prevention of heart and neurodegenerative diseases are well-known. In addition, derivatives of resveratrol, including glucosylated molecules as polydatin have been linked to similar beneficial effects. We have investigated the effects of resveratrol and polydatin on the in vitro production of IL-17 in a model of inflammation in vitro. The results obtained by activated human peripheral blood mononuclear cells, stimulated with anti-CD3/anti-CD28 monoclonal antibodies and treated with these polyphenolic compounds at different concentrations show that both decrease IL-17 production in a concentration-dependent manner. This study confirms the anti-inflammatory activity of resveratrol and its derivatives and suggests a potential clinical relevance in the therapy of inflammatory diseases.

Resveratrol down-regulates the growth and telomerase activity of breast cancer cells in vitro.

A number of previous studies investigated the in vitro effects of resveratrol on malignant human breast epithelial cell replication. The aim of the present study was to evaluate the activity of resveratrol on human metastatic breast cancer cells. The study was performed on the MCF-7 tumor cell line. Cell growth, cell cycle perturbation and apoptosis were evaluated by trypan blue dye exclusion assay, flow cytometric analysis and confocal fluorescence microscopy. TRAP assay and Western blot analysis respectively detected levels of telomerase activity and levels of hTERT in intracellular compartments of MCF-7 cells treated with resveratrol. Resveratrol has a direct inhibitory effect on cell proliferation. The results demonstrate that the drug induces apoptosis in MCF-7 cells, in a time- and concentration-related manner. Our results also show that the growth-inhibitory effect of resveratrol on malignant cells is mainly due to its ability to induce S-phase arrest and apoptosis in association with reduced levels of telomerase activity. In particular, TRAP assay and Western blot analysis respectively showed that resveratrol treatment down-regulates the telomerase activity of target cells and the nuclear levels of hTERT, the reverse transcriptase subunit of the telomerase complex. In our experimental model of breast cancer, resveratrol shows direct antiproliferative and pro-apoptotic effects. Studies on telomerase function and intracellular hTERT distribution point out that this agent is endowed with additional suppressive functions on critical tumor biological properties. These results speak in favor of a potential role of resveratrol in chemoprevention/chemotherapy of breast cancer.

A novel telomerase-based approach to detect natural cell-mediated cytotoxic activity against tumor cells in vitro.

This study was designed to develop a novel technical approach based on tumor-associated telomerase activity to detect cytotoxic activity of effector cells of the natural immune system against neoplastic cells. Human K562, DAUDI or Raji leukemia cells were co-cultured with NK or LAK effector cells at 37 degrees C for 4 h. Target cell killing was evaluated by 51Cr-release assay (CRA) or reduction of telomerase activity (R-TRAPCTX) of the target after exposure to effector cells. NK and LAK effector cells tested against K562 target cells at effector/target ratio of 50:1 showed cytotoxicity of 65% and 78%, respectively, with CRA and 51% and 74%, respectively, with R-TRAPCTX. Incorrect results were obtained with CRA when target cells were admixed with normal fibroblasts, whereas R-TRAPCTX was not influenced by the presence of normal cells. Control experiments performed with telomerase-negative cells showed that telomerase activity of effector cells was not altered during the cytolytic reaction. Moreover, supernatants obtained from effector-target cell co-cultures did not influence telomerase activity of targets. This novel R-TRAPCTX method to assay anti-tumor natural and possibly antigen-dependent cell-mediated cytotoxicity appears to provide sensible advantages over classical CRA or gamma-interferon release by effector cells in presence of target cells (ELISPOT), since (a) it furnishes reliable data on effector cell killing against neoplastic cells, even when malignant cells are admixed with normal cells, as frequently occurs in tumor biopsies, not manageable with CRA; (b) it provides an actual measure of target cell killing, not furnished by ELISPOT technique.