Graft-versus-host disease is one of the major transplant-related complications in allogeneic hematopoietic stem cell transplantation. Continued efforts have been made to prevent the occurrence of severe graft-versus-host disease by eliminating or suppressing donor-derived effector T cells. Conventional immunosuppression does not adequately prevent graft-versus-host disease, especially in mismatched transplants. Unfortunately, elimination of donor-derived T cells impairs stem cell engraftment, and delays immunologic reconstitution, rendering the recipient susceptible to post-transplant infections and disease relapse, with potentially lethal consequences. In this review, we discuss the role of dynamic immune regulation in controlling graft-versus-host disease, and how cell-based therapies are being developed using regulatory T cells and other tolerogenic cells for the prevention and treatment of graft-versus-host disease. In addition, advances in the design of cytoreductive conditioning regimens to selectively target graft-versus-host disease-inducing donor-derived T cells that have improved the safety of allogeneic stem cell transplantation are reviewed. Finally, we discuss advances in our understanding of the tolerogenic facilitating cell population, a phenotypically and functionally distinct population of bone marrow-derived cells which promote hematopoietic stem cell engraftment while reducing the risk of graft-versus-host disease. Graft-versus-host disease (GVHD) remains a major obstacle for the clinical application of hematopoietic stem cell (HSC) transplantation . GVHD is+ T cell and CD8+ T cell)-mediated GVHD are multifactorial and include activation of macrophages and antigen-presenting cells (APC) by transplantation conditioning regimens to damage host tissue, releasing soluble cytokines such as TNF-α and IL-1; alloreactive T cell activation, proliferation and differentiation in response to host or donor APC; and alloreactive T cell infiltration and release of pro-inflammatory cytokines which leads to damage of the target tissue [+/CD25+/forkhead/winged helix transcription factor + (FoxP3+), CD8+/CD28-, T/natural killer (NK) cells, and TCR+/CD4-/CD8-, most studies have concentrated on CD4+/CD25+/FoxP3+ T cells [+ T cell population, CD4+/CD25+/FoxP3+ regulatory T cells (Treg) have been demonstrated to suppress a variety of immune responses dependent on effector T cells.The mechanisms of donor T cell  production, direct cell-cell contact has also been postulated to be required as a mechanism of action. Natural Treg are generated in the thymus and are nonspecific in their suppressive capability [reg must encounter antigens to exert their suppressive effects, once activated they suppress in an antigen-nonspecific manner, presumably through the release of immunosuppressive cytokines such as IL-10 and TGF-β [reg are inducible and need to be activated through their TCR in order to mediate their suppressive activities. The expression of receptors of chemokines, such as C-C chemokine receptor type 5 (CCR5) and CXC chemokine receptor 3 (CXCR3), on antigen-specific Treg support a role for proper trafficking of Treg to target tissue in the prevention of acute GVHD in murine models [reg to secondary lymphoid tissues and sites of inflammation may play an important role in the control of GVHD.CD4 of GVHD  and, to  of GVHD -8. Althopability ,9. Althoe models ,12. A rereg can suppress proliferative activity of conventional CD4+ T cells and CD8+ T cells to alloantigenic stimulation in vitro and induce transplantation tolerance and reduce acute GVHD occurrence in vivo [+/CD25+ Treg isolated from the spleen or bone marrow of C57BL/6 mice can suppress lethal GVHD induced by transplanted donor CD4+/CD25- T effector cells after allogeneic T cell-depleted bone marrow transplantation (BMT) [+/CD25+ Treg does not abrogate the beneficial effect of donor T cell-mediated GVL [et al. recently evaluated the impact of early infusion of freshly isolated human donor CD4+/CD25+ Treg followed by conventional T cells in human leukocyte antigen (HLA)-haploidentical HSC transplantation [reg prevented acute as well as chronic GVHD in the absence of immunosuppressive drug therapy after transplantation. Moreover, Treg promoted immune reconstitution of CD4+ T cells, CD8+ T cells, B cells, and NK cells, and improved protective immunity against cytomegalovirus infection.Several studies in experimental murine models have shown that T in vivo -15. Donoon (BMT) . Importaated GVL . Ianni eantation . Adoptivreg-based therapy to prevent GVHD is obtaining sufficient numbers of antigen-specific Treg and maintaining their regulatory properties after infusion [ex vivo expansion of mouse recipient-specific CD4+/CD25+ Treg can be obtained using donor Treg stimulation with allogeneic recipient APC [Ex vivo expansion of Treg resulted in specific tolerance for recipient-type alloantigens, but not for third-party antigens. Moreover, the Treg effectively controlled GVHD while favoring immune reconstitution and maintaining GVL in a murine model [reg from human CD4+/CD25-/CD45RA+ cells exhibit a potent suppressive function in vitro compared with natural Treg, and suppress acute GVHD in a xenogeneic NOD/SCID mouse model [reg for the prevention and/or treatment of GVHD have recently been described. Ianni et al. reported that the adoptive transfer of freshly isolated, donor-derived natural Treg followed by conventional donor-derived T cells (Tcon) at the time of full-haplotype-mismatched adult HSC transplantation in patients being treated for hematologic malignancies prevented acute or chronic GVHD while favoring Tcon-mediated post-transplantation immune reconstitution [et al. detailed a 'first-in-human' phase 1 clinical trial of nonspecific (CD3/CD28 bead)-based ex vivo expanded umbilical cord blood-derived Treg in patients after umbilical cord blood transplantation and noted a reduced incidence of severe acute GVHD [The major limitation for the clinical application of Tinfusion . Studiesient APC ,19. Ex vne model . Anotherse model . Clinicatitution . Brunsteute GVHD .reg as diagnostic and prognostic cellular biomarkers for acute GVHD have been investigated in mouse and rat models and human HSC transplantation [+ Treg in skin biopsies from GVHD patients was significantly higher in patients responding to GVHD treatment and with a lower grade of GVHD compared with those with more severe GVHD [reg can regulate immune responses and induce tolerance to alloantigen after HSC transplantation. These findings provide evidence for the use of Treg as a useful biologic to improve GVHD diagnosis and treatment.Tantation -25. Studantation , suggestere GVHD . Taken tIn GVHD, functional immune cells in the donor bone marrow recognize the recipient as foreign and mount an immunologic attack on the patient's organs and tissues, impairing their ability to function . Strict et al. recently reported the outcomes of 68 patients with poor-risk hematologic malignancies who were conditioned with fludarabine, cyclophosphamide and 2 Gy total body irradiation prior to receiving T cell-replete bone marrow from HLA-haploidentical, first-degree relatives [The major clinical rationale for HLA-haploidentical BMT is to extend the potential benefits of HSC transplantation and the graft-versus-tumor effect to patients who lack an HLA-matched donor. The major challenge is to reduce the incidence of fatal graft rejection, severe GVHD and treatment-related mortality while promoting engraftment. Luznik elatives . Donors elatives . Recoverelatives . One yeaBased upon the results of Luznik and Fuchs ,36,39, w+/TCR- facilitating cells (FC) capable of promoting allogeneic HSC engraftment and establishment of mixed chimerism [T cell depletion (TCD) of allogeneic bone marrow is extremely effective at reducing the risk of GVHD, but greatly increases the risk of engraftment failure. Three possible reasons for TCD-associated-engraftment failure have been hypothesized. First, HSC can engraft across MHC-matched but minor disparate barriers, but fail to engraft in allogeneic recipients because they are harmed or removed during TCD. Second, T cells act as essential partner cells to promote allogeneic HSC engraftment. Third, an additional bone marrow population that shares some T cell markers protects the HSC from rejection or 'facilitates' HSC engraftment, but is inadvertently removed as an 'innocent bystander' during TCD. Investigations in different laboratories have provided strong evidence in support of the latter hypothesis. Initial studies in mice using different cell subsets sorted from donor bone marrow identified a distinct population of CD8himerism . The addhimerism . Transpl+/TCR- FC population shares phenotypic characteristics with CD8α plasmacytoid precursor dendritic cells (p-preDC) and that total FC are heterogeneous in morphology [et al. [+/TCR- FC were positive for CD11c+ (65% to 70%) and B220+ (75% to 88%) expression. While only 15% of the B220+ population was composed of B cells, 55% co-expressed CD11c (CD11c+/B220+), suggestive of a dendritic cell component. Further analysis into this CD11c+ dendritic subset revealed that a p-preDC phenotype (CD11dim/B220+/CD11b-) is the predominant cell type (93% to 95%) within this CD11c+ subset , and secondary transplantation of the Treg with donor HSC plus third-party HSC resulted in engraftment of only the donor HSC, demonstrating antigen-specificity.It was believed that p-preDC were the primary activators of naive CD4 as well . The Tay T cells . The las T cells . Recent  sources . The B7 ficiency . CD86 isntiation . More reIn recent years there have been significant advances in understanding the mechanism of FC function, which have provided additional support for the promising potential for FC to be used in the clinic to generate tolerance after HSC transplantation. Data collected from recent studies have helped to distinguish FC from other hematopoietic cells such as p-preDC. However, there are questions that remain to be answered regarding the role of FC in inducing tolerance, preventing GVHD and promoting chimerism following BMT. A phase 2 clinical study approved by the Food and Drug Administration is currently underway to test the use of FC-based HSC transplantation in recipients of living donor renal allografts. Preliminary findings have demonstrated that high levels of donor macrochimerism without GVHD can be established in mismatched recipients conditioned with fludarabine, cyclophosphamide and 200 cGy total body irradiation .The use of cell-based therapies in the prevention and treatment of GVHD is now being applied in a number of clinical protocols, with promising results. Cell-based therapies have the potential to promote engraftment, preserve immunocompetence and prevent GVHD, thereby avoiding the toxicity of broad acting nonspecific immunosuppressive agents.reg: regulatory T cell; TNF-α: tumor necrosis factors-α; UCB: umbilical cord blood.APC: antigen-presenting cells; BMT: bone marrow transplantation; CCR5: C-C chemokine receptor; CXCR3: CXC chemokine receptor 3; DC: dendritic cells; FC: facilitating cells; FoxP3: forkhead/winged helix transcription factor; GVHD: graft-versus-host disease; GVL: graft-versus-leukemia; HLA: human leukocyte antigen; HSC: hematopoietic stem cell; IL-1: interleukin-1; MHC: major histocompatibility complex; NK: natural killer; p-preDC: plasmacytoid precursor dendritic cells; TCD: T cell depletion; Tcon: conventional donor-derived T cells; TCR: T cell receptor; TGF-β: transforming growth factor beta; TSTI has significant equity interest in Regenerex, LLC, a start-up biotechnology company based on the facilitating cell technology. All other authors have no conflict of interest to declare.reg cells in GVHD prevention; HX: studies on role of cyclophosphamide; IG: review of FC mechanism of action; STI: overall editing and preparation of review. All authors have read and approved the final manuscript.JL: overall preparation and editing of review; YH: studies on role of TThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/10/48/prepub


A dichotomy between immunologically active and quiescent cancer phenotypes has been demonstrated for several types of cancer. Central to such dichotomy is the master regulator of the acute inflammatory process interferon regulatory factor (IRF)-1. Although the relevance of IRF-1 to the immune biology of cancer is emerging, it remains unknown whether the responsiveness of IRF-1 to immune cytokines is able to point out different cancer behaviours. Here, we explored the significance of the IFN-γ- and TNF-α-induced activation of IRF-1 in 15 melanoma cell lines. We measured IRF-1 activation in 15 melanoma cell lines at basal level and after treatment with IFN-γ, TNF-α and a combination of both using ImageStream technique (Amnis Corp). Microarray analysis was applied to compare transcriptional patterns between cell lines characterised by high and low IRF-1 activation. Functional interpretation analysis was performed using the Ingenuity Pathway Analysis system (IPA) tools 3.0. We observed a strong positive correlation between IRF-1 activation at basal level and after IFN-γ and TNF-α treatment  suggesting that both the cytokines have a strong effect in inducing IRF-1 activation. Given that different cell lines have different amplitude of response to cytokine stimulation, we next asked whether the inducibility of IRF-1 by IFN-γ and TNF-α was able to distinguish a parallel different behaviour of cancer cells. Toward this aim, we measured the difference of nuclear translocation score between controls and stimulated cells and further selected 3 cell lines with low and 3 cell lines with high delta IRF-1 nuclear translocation. Microarray demonstrated that the 3 cell lines with low and the 3 with high IRF-1 inducible translocation scores differed in the expression of 597 annotated transcripts. Functional interpretation analysis showed mTOR and Wnt/β-cathenin as the top upregulated pathways in the cell lines with low inducible IRF-1 activation, suggesting that a low IRF-1 inducibility recapitulates a cancer phenotype already described in literature characterised by poor prognosis. In conclusion, our findings suggest that the responsiveness of IRF-1 to IFN-γ and TNF-α is able to point out different cancer phenotypes and support the central role of this transcription factor in influencing different tumor behaviours.


Chemokines have been implicated in tumor progression and metastasis. In melanoma, chemokine receptors have been implicated in organ selective metastasis by regulating processes such as chemoattraction, adhesion and survival.In this study we have analyzed, using flow cytometry, the systems formed by the chemokine receptors CXCR3, CXCR4, CXCR7, CCR7 and CCR10 and their ligands in thirteen human melanoma cell lines (five established from primary tumors and eight established from metastasis from different tissues). WM-115 and WM-266.4 melanoma cell lines (obtained from a primary and a metastatic melanoma respectively) were xenografted in nude mice and the tumors and cell lines derived from them were also analyzed.Our results show that the melanoma cell lines do not express or express in a low degree the chemokine receptors on their cell surface. However, melanoma cell lines show intracellular expression of all the aforementioned receptors and most of their respective ligands. When analyzing the xenografts and the cell lines obtained from them we found variations in the intracellular expression of chemokines and chemokine receptors that differed between the primary and metastatic cell lines. However, as well as in the original cell lines, minute or no expression of the chemokine receptors was observed at the cell surface.Coexpression of chemokine receptors and their ligands was found in human melanoma cell lines. However, this expression is intracellular and receptors are not found at the cell membrane nor chemokines are secreted to the cell medium. The levels of expressed chemokine receptors and their ligands show dynamic variations after xenotransplantation that differ depending on the origin of the cell line (from primary tumor or from metastasis). Chemokines are chemoattractant cytokines which were initially characterized by their important role in leukocyte recruitment during inflammatory and immune responses. They acChemokines and their receptors are of great importance in the melanoma tumor progression. The expCancer metastasis is the complex process by which primary tumors spread to a distant location and is the main cause of death for most cancer patients. ChemokiThe aim of this work was to characterize the secretion and the intracellular expression of the chemokines CXCL9, CXCL10, CXCL11, CXCL12, CCL19, CCL21, CCL27 and CCL28 and the surface and intracellular expression of their chemokine receptors CXCR3, CXCR4, CXCR7, CCR7 and CCR10 in human melanoma cell lines, and the effect of xenotransplantation on the chemokine/chemokine receptor expression. We have included cell lines of primary and metastatic origin, as differences in chemokine receptors expression have been reported in uveal melanoma cell lines depending on their origin.2 using RPMI 1640 medium, supplemented with 10% fetal bovine serum inactivated by heat, 2 mM L-glutamine, 100 UI/mL penicillin and 100 μg/mL streptomycin sulfate .The selected cell lines included cells of primary and metastatic origin. Thirteen human melanoma cell lines were used Table , five es6 cells, and due to different growth rates of tumors, the animals inoculated with WM-266.4 were sacrificed 35 days after inoculation and the ones inoculated with WM-115 were sacrificed 77 days after inoculation. All procedures were performed according to the institutional recommendations and guidelines for the good care of laboratory animals, and were approved by the Research Ethical Committee of the University Clinic Hospital-INCLIVA, Valencia.The primary WM-115 and the metastatic WM-266.4 cell lines, established from the same patient, were inoculated into 4–6 weeks old BALB/c athymic nude mice . Each mouse was inoculated with 200 μl containing 2×10Tumors were extracted and disaggregated using 0.02% collagenase type II . Cells were resuspended in culture medium and part of them were immediately used in flow cytometry experiments (hereafter referred as xenografts WM-115-X and WM-266-X) and some cells were maintained in culture to establish new cell lines after xenotransplantation. Five cell lines were obtained from different xenografts of WM-115 and six cell lines were derived from distinct xenografts of WM-266.4. These modified cell lines were named WM-115-CX and WM-266-CX (CX stands for cultured xenografts).Cells were grown in Lab-Tek chamber slides . After washing with PBS, the cells were fixed with cold methanol-acetone for 5 min. Mouse monoclonal antibodies (mAb) against human CXCR3, CXCR4, CXCR7, CCR7, CXCL12, CCL19, CCL27 were from R&D Systems  and mouse mAb against human CXCL10 was from BD Biosciences . Goat anti-human CCR10 polyclonal antibody (pAb) was from Abcam , goat anti-human CXCL9 and CCL21 pAbs were from R&D Systems and rabbit anti-human CXCL11 pAb was from Peprotech . The cells reacted with each of these primary antibodies for 1 h at room temperature. The attached antibodies were visualized by the avidin-biotin-peroxidase procedure .All mAbs against chemokine receptors were conjugated to phycoerytrin (PE). mAbs against CXCR3, CXCR4, CCR7 and correspondent isotypic controls were purchased from BD Biosciences. mAbs against CXCR7 and CCR10 and correspondent isotypic controls were purchased from R&D Systems. All chemokine ligands  were detected using primary mouse-anti-human mAbs from R&D Systems with a secondary goat anti-mouse antibody labeled with FITC (R&D Systems). The DNA intercalating fluorochrome 7-Aminoactinomycin D  was used for dead cells staining.6cells/ml . Subsequently, 100 μl of this cell suspension were incubated on ice for 30 min with the chemokine receptors mAbs and correspondent isotypic controls. After incubation, cells were washed with ice-cold PBS and resuspended in 500 μl of culture medium for flow cytometric analysis. To determine dead cells 3 μg/ml 7-ADD were added prior to cytometric analysis. The mean percentage (%) of cells which expressed chemokine receptors at the cell surface, as well as the mean fluorescence intensity (MFI) calculated as the ratio between the mean fluorescence of the positive population in the samples stained with the mAb anti-receptor and the correspondent isotypic control were determined.Cells at subconfluency (50–70%) were detached with 2 mM EDTA in PBS, washed and resuspended in ice-cold culture medium at 1×106cells/ml. Then, 100 μl volumes of this cell suspension were incubated on ice for 30 min with the chemokine receptors mAb and correspondent isotypic controls. After incubation, cells were washed with ice-cold 1%BSA/PBS and resuspended in 500 μl of ice-cold 1%BSA/PBS for flow cytometric analysis. The mean percentage (%) of cells which expressed chemokines intracellularly, as well as the MFI calculated as the ratio between the mean fluorescence of samples stained with the mAb anti-receptor and the correspondent isotypic control were determined.Cells at subconfluency (50–70%) were detached with 2 mM EDTA in PBS, washed, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton/PBS adjusting cell suspension to 1×106cells/ml. Subsequently, 100 μl volumes of this cell suspension were incubated on ice for 30 min with the chemokine unconjugated mAbs. Afterwards, cells were washed twice with ice-cold 1%BSA/PBS and incubated on ice for 30 min with the secondary FITC-conjugated antibody. An aliquot of 100 μl of cell suspension incubated only with the secondary FITC-conjugated antibody was used as a control for all the chemokine ligands. Finally, cells were washed twice and resuspended in 500 μl of ice-cold 1%BSA/PBS for flow cytometric analysis. The mean percentage (%) of cells which expressed chemokine receptors intracellularly, as well as the MFI calculated as the ratio between the mean fluorescence of samples stained with the mAb anti-receptor and the correspondent isotypic control were determined.Cells at subconfluency (50–70%) were detached with 2 mM EDTA in PBS, washed, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton/PBS adjusting cell suspension to 1×10The quantification of cell surface expression of chemokine receptors and the intracellular expression of chemokines and their receptors was always performed in the same day, 24 h after sub-culturing, using cells with identical culture conditions.All the analyses were performed in a FC500 MCL flow cytometer  with an air-cooled argon ion laser . This standard instrument is equipped with two light scatter detectors that measure the forward scatter (an estimation of cell size) and the side scatter (an estimation of intracellular complexity), and five photomultiplier tubes that detect the appropriately filtered light. FITC fluorescence was collected at 525 ± 20 nm, PE at 575 ± 20 nm and 7-AAD at 675 ± 20 nm. When determining dead cells (for quantification of the cell surface expression of receptors) all measurements were restricted to live cells by gating the cells that excluded 7-AAD. In all other cases, the population was selected based on forward and side scatter parameters.MILLIPLEX™ Multi-Analyte Profiling (MAP)  according to manufactures indications. Furthermore, as a positive control the secretion of IL-8 and Gro were also quantified. Cells were grown in 10 ml of culture medium and after 24 hours of sub-culturing reached approximately 70% confluency. The processed samples were subsequently analyzed using Luminex 100™ System .The quantification of chemokine secretion levels was performed in cell culture medium collected 24 hours after sub-culturing the cells using the commercial multiplex kits All measurements in cell lines were made in triplicate. For flow cytometry experiments, the number of positive cells stained with the different antibodies was compared with the number of positive cells in the correspondent negative controls (isotype or secondary antibody) and the differences were analyzed using Student’s t-test and considered significant when p < 0.05. For chemokine secretion experiments, the concentration obtained in each sample was compared to the lowest standard concentration of the standard curve and the differences were analyzed using Student’s t-test, and considered significant when p < 0.05. The comparison between the expression of chemokines and their receptors between the original cell lines WM-115 and WM-266.4 and the tumors  and cell lines  obtained after xenotransplantation was analyzed using Student’s t-test and considered significant when p < 0.05.We found that melanoma cell lines did not express or express in a low degree . The chemokines exhibiting lower levels of intracellular expression were CXCL10 and CCL28 Table . RepreseCXCL10 was the only chemokine secreted in the studied melanoma cell lines. This chemokine was secreted in low concentrations by A375 and SK-Mel2 . All melanoma cell lines secreted the control chemokines IL-8 and Gro . The results confirm the results obtained by flow cytometry, with staining of all receptors and chemokines, with the exception of CCL10. Some representative examples are shown in Figure Tumors obtained from the xenotransplanted WM-266.4 cell line grew faster than those obtained from the WM-115 cell line. The growth of the former was noticed after 10 days of inoculation while the growth of the latter was noticed after 30 days of inoculation. After tumor disaggregation, a subset of cells was used directly for quantification of the expression of chemokines and their receptors by flow cytometry and another subset was cultured for a few passages for posterior quantification, in order to compare them with the original cell lines. When cultured, the cells exhibited some variability in the morphology and were slightly different from the original cell lines.When compared with the original cell lines, the WM-115-X and WM-266-X tumors obtained after xenotransplantation, as well as the cell lines derived from them (WM-115-CX and WM-266-CX) had similar patterns of cell surface expression of receptors, that is, minute or no expression was observed, without significant differences with the original cell lines.When compared with the original cell line, the WM-115-X xenografts, as well as the derived WM-115-CX cell lines, showed a substantial increase in the expression of CCR7 and CCR10, a considerable decrease in the expression of CXCR4 and a slight but significant decrease in the expression of CXCR3. CXCR7 showed an increased expression in the WM-115-X xenografts that was not observed in the derived WM-115-CX cell lines that were not significantly different from the original cell line . Our results differ from a study that describes functional plasma membrane CXCR4 in the cell lines MeWo and A375[All cell lines significantly expressed all the receptors intracellularly, although there was significant variability in the pattern of expression between the different cell lines. The intracellular expression of chemokine receptors, in other tumors, has been shown to be correlated with metastasis directed to lymph nodes and with a bad prognosis , CXCL11 (CXCR3 and CXCR7), CXCL12 (CXCR4 and CXCR7), CCL19, CCL21 (CCR7) and CCL27, CCL28 (CCR10). We found a certain pattern in their expression. Most chemokines were expressed in all cell lines. However, chemokines CXCL10 and CCL28 had a low or null expression in most of the cell lines (Table We also analyzed the secretion of chemokines in the culture medium of all cell lines. From all the chemokines studied, the only chemokine secreted by the melanoma cell lines was CXCL10. This chemokine was secreted in low concentrations by A375 and SK-Mel-2. As a positive control we also quantified the secretion of the IL-8 and Gro chemokines that are produced by human melanoma[There is evidence, based on experiments designed to avoid HIV-1 infection,39, thatin vivo environment and stimuli to these established melanoma cell lines we xenografted the primary cell line WM-115 and the metastatic cell line WM-266.4 that were initially derived from the same patient[Tumor microenvironment originates in the interactions between malignant and non-transformed cells. Intercellular communication is driven by a complex network of cytokines, chemokines, growth factors, and inflammatory and matrix enzymes. Chemoki patient, into nuA comparative analysis of global gene expression has been performed between human melanoma cell lines with different metastatic capacity and the xenografts obtained by their subcutaneous injection into immunocompromised mice, demonstIn conclusion, we find coexpression of chemokine receptors and their ligands in human melanoma cell lines. However, this expression is intracellular and receptors are not found at the cell membrane nor chemokines are secreted to the cell medium. The levels of intracellularly expressed chemokine receptors and their ligands show dynamic variations after xenotransplantation that differ depending on the origin of the cell line. These changes could affect cell trafficking assays that correlate in vitro and in vivo data. These results could also have implications in the studies that analyze chemokines or chemokine receptors expression in melanomas that do not ascertain the cell membrane location of chemokine receptors or the secretion of chemokines to the extracellular medium.The authors declare that they have no competing interests.SP, AMR, RGB, CM and RCC designed the study. SP carried out the flow cytometry and chemokine secretion experiments, did the statistical analysis and drafted the manuscript. RGB, TSN, LT carried out the immunocytochemistry assays and performed the xenografts. All authors interpreted the results, read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/118/prepub


Rhabdoid tumors (RTs) are aggressive pediatric malignancies with poor prognosis. N-(4-hydroxy phenyl) retinamide (4-HPR or fenretinide) is a potential chemotherapeutic for RTs with activity correlated to its ability to down-modulate Cyclin D1. Previously, we synthesized novel halogen-substituted and peptidomimetic-derivatives of 4-HPR that retained activity in MON RT cells. Here we analyzed the effect of 4-HPR in inhibiting the growth of several RT, glioma, and breast cancer cell lines and tested their effect on cell cycle, apoptosis and Cyclin D1 expression.Effect of compounds on RT cell cycle profiles, and cell death were assessed by MTS cell survival assays and FACS analysis. The effects of treatment on Cyclin D1 expression were determined by immunoblotting. The efficacy of these compounds on glioma and breast cancer cell lines was also determined using MTS assays.50 values in all cell lines tested and did not cause cell death. In MON RT cells, the iodo-substituted compounds were more active than 4-HPR in inducing cell cycle arrest and apoptosis. Additionally, the activity of the compounds correlated with their ability to down-modulate Cyclin D1: while active compounds reduced Cyclin D1 levels, inactive ATRA did not. In glioma and breast cancer cell lines, 4-HPR and 4-HPR derivatives showed variable efficacy.Low micromolar concentrations of 4-HPR derivatives inhibited cell survival of all RT cells tested. The 4-HPR derivatives altered RT cell cycle profiles and induced high levels of cell death that was correlated with their potency. ATRA exhibited high IC50s in RT cells. These derivatives are of general interest, as conjugation of halogen radioisotopes such as 18F, 124I, or 131I to 4-HPR will allow us to combine chemotherapy and radiotherapy with a single drug, and to perform PET/SPECT imaging studies in the future.Here we demonstrate, for the first time, that the inhibitory activities of novel halogen-substituted and peptidomimetic derivatives of 4-HPR are correlated to their ability to induce cell death and down-modulate Cyclin D1. These 4-HPR derivatives showed varied potencies in breast cancer and glioma cell lines. These data indicate that further studies are warranted on these derivatives of 4-HPR due to their low IC Furthermore, we have found that 4-HPR effectively inhibits RT growth and that the ability of 4-HPR to inhibit RTs is correlated to down-modulation of Cyclin D1 .4-HPR: N-(4-hydroxyphenyl)retinamide; ANOVA: analysis of variance; ATRA: All-trans retinoic acid; DMEM: Dulbecco's modified Eagle's medium; FACS: Fluorescence activated cell sorting; FDA: Food and Drug Administration; IC50: half maximal inhibitory concentration; MTS: methyl-tetrazolium salt; PET: positron emission tomography; RAR: Retinoic acid receptor; RPMI: Roswell Park Memorial Institute culture medium; RT(s): Rhabdoid Tumor(s); SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis.The authors declare that they have no competing interests.MES participated in the design of the study, performed all experiments, analyzed and interpreted the data, and drafted the manuscript. BCD synthesized the 4-HPR derivatives used for this study. GVK conceived of the study, designed the experiments, analyzed and interpreted data, and corrected the manuscript. All authors read and approved the final manuscript.


Nuclear hormone receptors (NHRs) are transcription factors that regulate carbohydrate and lipid metabolism, immune responses, and inflammation. Although several NHRs, including peroxisome proliferator-activated receptor-γ (PPARγ) and PPARα, demonstrate a renoprotective effect in the context of diabetic nephropathy (DN), the expression and role of other NHRs in the kidney are still unrecognized. To investigate potential roles of NHRs in the biology of the kidney, we used quantitative real-time polymerase chain reaction to profile the expression of all 49 members of the mouse NHR superfamily in mouse kidney tissue (C57BL/6 and db/m), and cell lines of mesangial (MES13), podocyte (MPC), proximal tubular epithelial (mProx24) and collecting duct (mIMCD3) origins in both normal and high-glucose conditions. In C57BL/6 mouse kidney cells, hepatocyte nuclear factor 4α, chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) and COUP-TFIII were highly expressed. During hyperglycemia, the expression of the NHR 4A subgroup including neuron-derived clone 77 (Nur77), nuclear receptor-related factor 1, and neuron-derived orphan receptor 1 significantly increased in diabetic C57BL/6 and db/db mice. In renal cell lines, PPARδ was highly expressed in mesangial and proximal tubular epithelial cells, while COUP-TFs were highly expressed in podocytes, proximal tubular epithelial cells, and collecting duct cells. High-glucose conditions increased the expression of Nur77 in mesangial and collecting duct cells, and liver x receptor α in podocytes. These data demonstrate NHR expression in mouse kidney cells and cultured renal cell lines and suggest potential therapeutic targets in the kidney for the treatment of DN. Diabetic nephropathy (DN) is a major microvascular complication in patients with diabetes mellitus, ultimately leading to end-stage renal diseases In humans, 48 members of the nuclear hormone receptor (NHR) superfamily of transcription factors have been identified (49 in mice). Several modulators of NHRs have been developed as oral drugs for the treatment of diabetes and dyslipidemia. Synthetic agonists for peroxisome proliferator-activated receptor-γ (PPARγ) and PPARα, such as thiazolidinediones and fibrates, improve glycemic control in type 2 diabetic patients and lower serum triglyceride levels in hyperlipidemic patients There is increasing evidence of NHR expression in peripheral tissues including adipose tissue db/db mice (BKS.Cg-db/leprdblepr) and male non-diabetic db/m mice (BKS.Cg-db/lepr+) were purchased from CLEA Japan . Db/db mice and db/m mice were euthanized at 9 weeks of age. All mice were maintained under a 12-h light/12-h dark cycle with free access to food and tap water. Animal care and procedures were performed according to the Guidelines for Animal Experimentation at Okayama University, the Japanese Government Animal Protection and Management Law, and the Japanese Government Notification on Feeding and Safekeeping of Animals. The experimental protocol was approved by the Animal Ethics Review Committee of Okayama University (OKU-2011326). All surgery was performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering.Eight-week-old male C57BL/6J mice were purchased from Charles River . Diabetes was induced by peritoneal injection of 200 mg/kg streptozotocin  in citrate buffer (pH 4.5). C57BL/6J mice were euthanized at 8 weeks after the induction of diabetes. Six-week-old male diabetic Murine mesangial (MES13), podocyte (MPC), proximal tubular epithelial (mProx24), and collecting duct (mIMCD3) cells were cultured as previously described RNA was isolated from kidney cortex samples or cultured cells using an RNeasy Mini kit . Single-strand cDNA was synthesized from the extracted RNA using a real-time polymerase chain reaction (RT-PCR) kit . To evaluate the mRNA expression of each NHR, quantitative RT-PCR (qPCR) was performed using TaqMan® Array Plates and StepOnePlus™  and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems). Primers were purchased from Applied Biosystems. Each sample was analyzed in quadruplicate and normalized for S18 mRNA expression. Primer sequences for mouse genes are provided in Immunofluorescent staining was performed as described previously The expression of NHRs in mouse kidney tissue (C57/BL6 and db/m) and renal  cell lines was determined by qPCR analysis.The composition and rank order of NHR expression in C57/BL6 mouse kidney are shown in The composition of NHR expression in C57/BL6 mouse kidney is shown in The relative mRNA levels of NHRs expressed in C57BL/6 mouse kidney are shown in rank order in In C57BL/6 mouse kidney, the most abundant receptor was HNF4α and other abundant receptors were chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) and COUP-TFIII, VDR, PPARα, FXRβ, and ERRα. In contrast, COUP-TFII was the most abundant NHR in db/m mouse kidney, and the mRNA levels of HNF4α, COUP-TFIII, and VDR were lower in db/m mice than in C57BL/6 mice.A similar survey of NHR transcripts in renal cell lines revealed significant differences compared with the receptor expression pattern observed in mouse kidney. COUP-TFII was the most abundant NHR in the mProx24 and mIMCD3 cell lines, and the second most abundant receptor in the MPC cell line. In contrast, PPARδ was the most highly expressed receptor in the MES13 cell line, and the second most abundant NHR in the mProx24 cell line. In addition to PPARδ, thyroid hormone receptor α (TRα), COUP-TFII, COUP-TFIII, glucocorticoid receptor (GR), RXRα, and LXRβ were found to be abundant receptors in the MES13 cell line. In the MPC cell line, COUP-TFII, COUP-TFIII, RXRα, LXRβ, TRα, and GR were highly expressed, while COUP-TFII, PPARδ, COUP-TFIII, and TRα were abundant in the mProx24 cell line. In the mIMCD3 cell line, the expression pattern of NHRs was similar to that in the MPC cell line. Furthermore, retinoic acid receptor α (RARα), reverse-ErbAβ (Rev-ERBβ), and PPARδ were highly expressed NHRs in the mIMCD3 cell line.The expression of endocrine receptors was compared for all renal cell lines . AndrogeThe expression of adopted orphan receptors in the renal cell lines was also compared . LXRα anWe also compared the expression of orphan receptors in the renal cell lines . ERRα, HTo determine differential expression of NHRs in response to hyperglycemia, we compared the expression of NHRs in the kidneys of streptozotocin-induced type 1 diabetic C57BL/6 mice and control C57BL/6 mice . SimilarSignificantly increased RNA levels were observed for neuron-derived orphan receptor 1 (NOR1) and nuclear receptor-related factor 1 (NURR1), members of the NHR 4A (NR4A) subgroup, and ERβ and ERRβ, members of the NR3A subgroup, in the kidneys of streptozotocin-induced diabetic C57BL/6 mice . A similA similar evaluation of glucose-mediated changes in gene expression in renal cell lines was performed . In the Since the RNA level of NOR1 was the highest in the kidneys of streptozotocin-induced diabetic C57BL/6 mice, and the third highest in diabetic db/db mice , we confNHRs are one of the largest transcription factor families in the mammalian genome. A total of 48 human and 49 mouse genes that encode NHRs have been identified In this study, we identified 25 NHRs that are expressed in C57BL/6 mouse kidney. The receptors detected in abundance, including HNF4α, VDR, PPARα, FXR, and ERRα, have already been reported to play a role in the pathogenesis of DN The analysis of NHR expression in renal cell lines showed that most are expressed mainly in proximal tubular epithelial cells and mesangial cells. However, we showed that PPARα and LXRα are more highly expressed in mesangial cells than in the other renal cell types, which is consistent with previous reports that PPARα and LXRα are expressed in mesangial cells ERRs are highly expressed in the kidney and renal cell lines. Intriguingly, ERRα is the only NHR that is highly expressed in podocytes, while ERRγ is the only NHR that is highly expressed in collecting duct cells. It is reported that ERRα is a key regulator of interferon-γ-induced mitochondrial reactive oxygen species production and host defense The most interesting and novel finding in this study is that the members of the NR4A subgroup, including NOR1, NURR1, and Nur77, are highly induced in the kidneys of diabetic C57BL/6 and db/db mice. All three members of the NR4A subgroup are expressed in energy-dependent tissues such as skeletal muscle, brain, adipose tissues, heart, and liver suggesting a possible role in energy metabolism We were surprised that the expression profile of NHRs in the kidneys of diabetic C57BL/6J and db/db mice is partially overlapping like the members of the NR4A subgroup, but mostly different as shown in In summary, we have analyzed the expression of all NHRs present in normal and high-glucose conditions in mouse kidney tissue and renal cell lines. The functions of most of these NHRs are still unknown; however, our data provide the basis for further studies to elucidate potential therapeutic targets in the pathogenesis of DN.Table S1Primers used for quantitative real-time PCR.(DOC)Click here for additional data file.


Cervical cancer continues to be an important worldwide health problem for women. Up to 35% of patients who are diagnosed with and appropriately treated for cervical cancer will recur and treatment results are poor for recurrent disease. Given these sobering statistics, development of novel therapies for cervical cancer remains a high priority. We evaluated the expression of Tissue Factor (TF) in cervical cancer and the potential of hI-con1, an antibody-like-molecule targeted against TF, as a novel form of immunotherapy against multiple primary cervical carcinoma cell lines with squamous- and adenocarcinoma histology.in vitro expression of TF in cervical carcinoma cell lines by immunohistochemistry (IHC), real time-PCR (qRT-PCR) and flow cytometry. Sensitivity to hI-con1-dependent cell-mediated-cytotoxicity (IDCC) was evaluated in 5-hrs-51chromium-release-assays against cervical cancer cell lines in vitro.Because TF is a transmembrane receptor for coagulation factor VII/VIIa (fVII), in this study we evaluated the p = 0.0023 qRT-PCR; p = 0.0042 flow cytometry). All primary cervical cancer cell lines tested overexpressing TF, regardless of their histology, were highly sensitive to IDCC , while negligible cytotoxicity was seen in the absence of hI-con1 or in the presence of rituximab-control-antibody. Low doses of interleukin-2 further increased the cytotoxic effect induced by hI-con1 (p = 0.025) while human serum did not significantly decrease IDCC against cervical cancer cell lines (p = 0.597).Cytoplasmic and/or membrane TF expression was observed in 8 out of 8 (100%) of the tumor tissues tested by IHC and in 100% (11 out of 11) of the cervical carcinoma cell lines tested by real-time-PCR and flow cytometry but not in normal cervical keratinocytes (TF is highly expressed in squamous and adenocarcinoma of the uterine cervix. hI-con1 induces strong cytotoxicity against primary cervical cancer cell lines overexpressing TF and may represent a novel therapeutic agent for the treatment of cervical cancer refractory to standard treatment modalities. Despite aggressive screening programs, cervical cancer remains an important public health issue. In the United States about 12,200 new cases of cervical cancer as well as 4,210 deaths from cervical cancer are estimated for 2010 . A deepeAngiogenesis, the formation of new vessels from pre-existing vasculature, is known to represent a critical step in the development, progression and metastatic process of human solid tumors. Tissue factor (TF), a transmembrane receptor for coagulation factor VII/VIIa (fVII), is aberrantly expressed in human cancers and on endothelial cells within the tumor vasculature ,5. Impor1 Fc as the effector domain; the two chains are held together by the disulfide bonds normally present in IgG. The hI-con1 is designed to bind to TF with far higher affinity and specificity than can be achieved with an anti-TF antibody. Indeed, the hI-con1 has several important advantages over monoclonal antibodies for targeting TF including: 1) The Kd for fVII domain binding to TF is about 10-12 M [d in the range of 10-8 to 10-9 M for TF [c domain of the hI-con1 may thus potentially activate powerful cytolytic responses mediated by antibody-dependent cell-mediated cytotoxicity (ADCC) against both TF-expressing tumor cells and tumor vascular endothelial cells that bind the hI-con1 molecule.hI-con1™  is a previously characterized immuno-conjugate molecule developed against TF -12. It i 10-12 M , in contM for TF , and 2) M for TF . The humin vitro potential of hI-con1 as a novel immunotherapeutic agent against biologically aggressive cervical cancer cell lines overexpressing TF.To our knowledge, no studies have specifically described the expression of tissue factor in cervical cancer. Therefore, in this report we investigated the expression of TF at mRNA and protein levels in multiple primary cervical tumor cell lines and evaluated for the first time the Primary cervical tumor cell lines from eleven patients harboring cervical cancer were obtained from fresh tumor biopsies collected at the time of primary surgery or surgery for cancer recurrence under approval of the Institutional Review Board. Tumors were staged according to the International Federation of Gynecologists and Obstetricians staging system. Source-patient characteristics of these eleven cell lines are described in Table CT) method (Applied Biosystems) was used to determine gene expression in each sample relative to the value observed in the lowest nonmalignant cervical keratinocyte cell sample, using glyceraldehyde-3-phosphate dehydrogenase (Assay ID Hs99999905_m1) RNA as internal control.RNA isolation from 11 primary cervical cancer cell lines (Table 2O2. Steam and high pH (pH 9) were used for antigen retrieval. The slides were then incubated overnight at 4°C with monoclonal anti-TF antibody . EnVisionTM system  was used for secondary detection and the reactions were visualized with diaminobenzidine. Appropriate positive and negative controls were used with each case. Both cytoplasmic and membranous immunoreactivity was considered positive. Immunostaining was assessed using a semi-quantitative scoring system, as follows: 0, negative (0% to 5% staining); 1+, weakly positive (10% to 20%); 2+, moderately positive (20% to 50%); 3+ and 4+, strongly positive , as previously described [Formalin-fixed, paraffin-embedded tissue blocks obtained from 5 stage IB patients, 1 stage IIA and 2 stage III B patients  were retrieved from a separate set of 8 patients from the surgical pathology files at Yale University. All tumor samples and eight normal cervical tissue specimens used as controls were reviewed by a surgical pathologist (NB). The level of TF expression was then evaluated on the most representative block by standard immunohistochemical (IHC) staining. For immunohistochemistry, 4 μm sections were cut from the formalin-fixed paraffin-embedded blocks. Following deparaffinization and rehydration, endogenous peroxidase was blocked in 3% Hescribed .2, 25 mM arginine, 0.01% Tween 80, pH 7.4 buffer and sterile-filtered. It was provided in aliquots of 80 μL at a stock concentration of 350 μg/ml which were used for our flow cytometry and hI-con1-dependent cell-mediated cytotoxicity (IDCC) studies. Briefly, cervical cancer cell lines and phytohemagglutinin (PHA)-stimulated control peripheral blood lymphocytes (PBL) were stained with hI-con1 at a concentration of 30 μg/ml for 30 minutes on ice or with a commercially available FITC-conjugated mouse anti-human TF immunoglobulin . After hI-con1 staining, cells were washed twice with the same buffer and secondary mouse-anti-human antibody  was added for a further 30 minutes. Analysis was conducted with a FACSCalibur instrument using Cell Quest software .Clinical grade hI-con1 was produced for Iconic Therapeutics Inc.  by Laureate Pharma,  by cultivation of BHK cells transfected with a vector containing the gene sequence originally described by Hu and Garen -12. The 51Cr) release assay was performed to measure the cytotoxic reactivity of Ficoll-Hypaque-separated PBL from several healthy donors in combination with hI-con1 against cervical cancer cell lines. The release of 51Cr from the target cells was measured as previously described [®; Genentech) was used as control in all bioassays. IDCC was calculated as the percentage of killing of target cells observed with mAb plus effector cells as compared with 51Cr release from target cells incubated alone.A standard 5-hours chromium (escribed  as evideTo investigate the effect of interleukin-2 (IL-2) on IDCC, effector PBL were incubated for 5 hours at 37°C at a final concentration of IL-2  ranging from 50 to 100 IU/ml in 96-well microtiter plates. Target cells were primary cervical cancer cell lines exposed to hI-con1 (concentrations ranging from 1 to 80 μg/ml), whereas controls included the incubation of target cells alone or with PBL in the presence or absence of IL-2 or mAb, respectively. Rituximab was used as a control mAb. IDCC was calculated as the percentage of killing of target cells observed with mAb plus effector PBL, as compared with target cells incubated alone. Each experiment was performed with PBL collected from multiple normal donors, with results from a representative donor presented.51Cr) release assay identical to the previous IDCC assays was used, except that human serum in a dilution of 1:2 was added to the effector cells. This human serum was used as a source of complement to test for complement-mediated target cell lysis. To test for the possible inhibition of IDCC against a chemoresistant squamous cervical cancer cell line by physiological human serum concentrations of γ-globulin, human serum was diluted 1:2 before being added in the presence or absence of effector PBL. Experiments were also performed using heat-inactivated human serum  in the presence of effector PBL. Controls included the incubation of target cells alone or with either PBL or mAb separately. Rituxan was used as a control mAb.A standard five-hour chromium  were calculated by computing them on the ΔCTs and then reverse-transforming the results to obtain means (95% CI) of relative copy numbers. Differences in TF expression by IHC and flow cytometry were analyzed by the unpaired t-test. Kruskal-Wallis test and chi-square analysis were used to evaluate differences in hI-con1-dependent cell-mediated cytotoxicity levels in primary tumor cell lines. Statistical analysis was performed using SPSS version 17 . A p-value of < 0.05 was considered statistically significant.For qRT-PCR data, the right-skewing was removed by taking copy-number ratios relative to the lowest-expressing normal cervical keratinocyte sample ("relative copy numbers"), logp = 0.023). Of the eleven tumors tested, nine showed a high mRNA copy number  .Surface TF receptor expression was evaluated by FACS analysis on all eleven primary cervical cancer cell lines using hI-con1 and an anti-human TF control mAb. As negative controls, several PHA-stimulated PBL established from healthy donors or the same cervical cancer patients from which the tumor cell lines had been established were also studied. Reactivity against TF was found by flow cytometry in all cervical cancer cell lines tested, with ADX-1 ARK-1 and ADX-3 ARK-3 showing lower expression levels when compared to all the remaining cells lines  ratios of 25:1 and 50:1 . Similarly, primary cervical cancer cell lines incubated with rituximab (2.5 μg/ml) control antibody displayed no significant cytotoxicity (range: 0.5% - 13.03%). In contrast, as representatively shown in Figure p < 0.001). Importantly, high levels of hI-con1-induced cytotoxicity were also detected against CVX-1 ARK-1, a squamous cervical cancer cell line established from a patient experiencing recurrent disease after chemo-radiation resistant tumor , and CVX-1 ARK-1 and CVX-7 ARK-7 , were tested for their sensitivity to natural killer cell (NK) cytotoxicity when challenged with heterologous PBL, collected from several healthy donors, in standard 5-hours p = 0.025) whereas no significant increase in cytotoxicity was detected after 5 hours of IL-2 treatment in the absence of hI-con1 or in the presence of rituximab control mAb  on IDCC against primary cervical cancer cell lines, PBL from healthy donors were incubated for 5 hours in the presence of 50 to 100 IU/ml of IL-2. As representatively shown in Figure b Figure . These r51Cr release assays. The addition of untreated or heat inactivated human serum diluted 1:2 did not significantly alter the cytotoxic effect of hI-con1 against CVX-7 ARK-7 cell line when compared to hI-con1-mediated killing without serum  was added to a representative squamous cervical cancer cell line (CVX-7 ARK-7) during standard 5-hours The management of disseminated carcinoma of the cervix that is no longer amenable to control with surgery or radiation therapy has not improved significantly with the advent of modern chemotherapy ,3. The oin vitro activity of hI-con1, a previously characterized immuno-conjugate molecule developed against TF [in vitro. We found both squamous- and adenocarcinoma cell lines to overexpress TF when compared to normal cervical keratinocytes. We speculate that TF expression may be a common and important event in malignant transformation of the cervix into biologically aggressive squamous and adenocarcinoma cervical cancers. Furthermore, both cell lines available to this study established from recurrent/metastatic tumors were found to express extremely high levels of TF. These data are consistent with previous reports in other human solid tumors showing that a high production of TF and vascular endothelial growth factor (VEGF), generate solid tumors characterized by highly aggressive biologic behavior [We have evaluated Tissue Factor (TF) expression by qRT-PCR, IHC and flow cytometry in cervical tissue samples and multiple primary cervical cancer cell lines with squamous- and adenocarcinoma histology, some of which were derived from recurrent chemo-radiation resistant tumors. In addition, we have tested the ainst TF -12, as abehavior . In thisbehavior ,6. Althobehavior ,6. Thus,behavior -12,18.in vitro.To our knowledge, our results are the first to describe self-production of TF-coagulation factor VII complex by primary cervical cancer cell lines. These data are however consistent with multiple previous reports demonstrating an association between cervical cancer and deep-vein thrombosis and/or pulmonary thromboembolism  -22, a pain vitro, suggesting that TF-mediated enhancement of tumor growth requires a factor present in vivo that is not present when cells are grown in vitro [in vivo leading to activation of type-2 proteinase activated receptor (PAR2) dependent signaling [in vivo.TF is known to play an important role in tumor metastatic process, possibly by inducing the coating of the tumor cell with fibrin that would trap the cells in the microvasculature, thereby aiding metastases -26. Receignaling . These f51Cr release assays. Taken together, these in vitro results strongly suggest that TF may provide a novel target for the treatment of resistant/residual cervical disease and the destruction of their tumor vasculature that should result in hI-con1-induced lysis of tumor cells as well as tumor endothelial cells in vivo. Consistent with this hypothesis and the central role of angiogenesis in cervical cancer development and progression, bevacizumab, a humanized antibody able to bind and inactivate VEGF, has been recently reported to shrink cervical tumors and delay progression without appreciable toxicity. On the basis of these encouraging results, bevacizumab is currently being studied in a Gynecologic Oncology Group (GOG) phase III trial [The potential cytotoxic activity of hI-con1 against human melanoma and prostate tumor cells has previously been demonstrated by Hu et al. -12. In aII trial .in vivo, even in the presence of high concentrations of human IgG. In this study we showed that hI-con1-dependent cytotoxicity against cervical cancer cell lines was not inhibited by high concentrations (up to 50%) of human serum. These data, therefore, suggest that in the presence of effector PBL, human serum may not significantly decrease IDCC against cervical cancer cell lines. Moreover, these results indicate that the binding of hI-con1 to the Fc receptor on mononuclear effector cells is likely to occur in vivo.For effective cytotoxicity, the effector cells must be able to interact with the immune-conjugate at the target site in vitro and in vivo is known to be mainly dependent on the number and function of NK cells [cγRIII receptors, which are activator molecules, and not FcγRIIb receptors, which inhibit ADCC [in vitro experiments reveal a significant increase of hI-con1-induced cytotoxicity after the brief incubation of PBL and tumor cells with IL-2 compared to the cytotoxicity induced by hI-con1 in the absence of IL-2. IL-2 seems to therefore enhance the cytotoxic potential of the effector cells. The administration of low doses of IL-2 might therefore be a valid therapeutic option in order to increase IDCC in heavily pretreated cervical carcinoma patients.The degree of ADCC NK cells . NK cellbit ADCC . NK cellbit ADCC -31. Therbit ADCC ,31. Thesbit ADCC . This anbit ADCC , has beebit ADCC ,35. ImpoIn conclusion, high expression of TF in a large number of cervical carcinomas makes hI-con1 an attractive agent for immunotherapy of recurrent/refractory cervical cancer. In this study, we have demonstrated significant hI-con1-mediated killing of multiple primary cervical cancer cell lines regardless of their squamous or adenocarcinoma histology. hI-con1 might therefore represent a novel treatment of this aggressive disease and, potentially, multiple other human tumors overexpressing TF.TF: tissue factor; fVII: Factor VII; hI-con1: human immuno-conjugate molecule; IDCC: hI-con1-dependent cell-mediated cytotoxicity; FBS: fetal bovine serum; IHC: immunohistochemistry; mAb: monoclonal antibody; NK cells: natural killer cells; PBL: peripheral blood lymphocytes; qRT-PCR: quantitative real-time-polymerase chain reaction; CVX: cervical squamous cell carcinoma; ADX: cervical adenocarcinoma.The authors declare that they have no competing interests.EC, JV, SB, MG, MB, PT, and LC carried out the molecular in vitro studies including RT-PCR, flow cytometry and IDCC assays, as well as statistical analysis. NB carried out the IHC studies on the tissue samples. DS, MA, PES, TJR, SP, CJL, and AS participated in the design of the study and drafted the manuscript. AS conceived the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/263/prepub


Human T cell leukemia virus type 1 (HTLV-1) is an etiological agent of adult T-cell leukemia (ATL) and its viral oncoprotein Tax1 plays critical roles in T cell transformation and ATL development. Tax1 converts a T-cell line CTLL-2 from IL-2 dependent growth into IL-2-independent one, and the conversion requires inhibition of apoptosis. In this study, we investigated the involvement of Bim, an apoptosis inducer after cytokine deprivation, in Tax1-induced transformation. Bim expression is strongly induced in CTLL-2 cells after IL-2 starvation, whereas such induction is markedly reduced in CTLL-2 cells transformed by Tax1. A Tax1 mutant defective of NF-kB2 activation transformed CTLL-2 less efficiently than wild type Tax, but this low transforming activity was rescued by knockdown of Bim in CTLL-2, suggesting that activation of noncanonical NF-kB2 pathway is involved in Tax1-mediated Bim repression. Furthermore, the expression of Bim was lower in HTLV-1 transformed and ATL cell lines than HTLV-1-negative T-cell lines. These results suggest that the Bim repression by Tax1 is crucial for the survival of HTLV-1 infected cells and may play a role in ATL development.


We investigated the oxidative modifications of lipids, proteins and DNA, potential molecular targets of oxidative stress, in two lymphoblastoid cell lines: B95-8 and Raji, after EBV lytic cycle induction. Conjugated dienes level was measured as biomarker of lipid peroxidation. Malondialdehyde adduct and protein carbonyl levels, as well as protein thiol levels were measured as biomarkers of protein oxidation. DNA fragmentation was evaluated as biomarker of DNA oxidation.After 48 h (peak of lytic cycle), a significant increase in conjugated dienes level was observed in B95-8 and Raji cell lines (p = 0.0001 and p = 0.019 respectively). Malondialdehyde adduct, protein carbonyl levels were increased in B95-8 and Raji cell lines after EBV lytic cycle induction as compared to controls . Proteins thiol levels were decreased by induction in B95-8 and Raji cell lines . DNA fragmentation was also detected in B95-8 and Raji cell lines after EBV lytic cycle induction as compared to controls.The results of this study demonstrate the presence of increased combined oxidative modifications in lipids, proteins in B95-8 and Raji cells lines after EBV lytic cycle induction. These results suggest that lipid peroxidation, protein oxidation and DNA fragmentation are generally induced during EBV lytic cycle induction and probably contribute to the cytopathic effect of EBV. Epstein Barr virus (EBV) is a ubiquitous gammaherpesvirus that infects more than 90% of the human population. The EBV can infect its target cells in both a latent and a lytic mode. The latent cycle is characterized by the expression of six Epstein-Barr nuclear antigens (EBNAs), three latent membrane proteins LMP1, LMP2A, and LMP2B, two untranslated RNAs (EBER), and a family of transcripts from the BMH1A region of the genome . The latEBV is associated with a spectrum of malignancies of lymphoid and epithelial cell origin, such as Burkitt's lymphoma , nasophaA lot of evidences demonstrated that EBV is implicated in the genesis of oxidative damages in vitro or in vivo and that could participate in the pathogenicity of the EBV. An excessive production of reactive oxygen specie (ROS) was highlighted in EBV positive lymphoblastoid cell lines transformed in vitro  or derivOxygen free radicals or, more generally, ROS, are products of normal cellular metabolism. ROS are well recognised for playing a dual role as both deleterious and beneficial species, since they can be either harmful or beneficial to living systems . Damage However, there is no adequate knowledge in the literature about protein and DNA modifications in cells after EBV lytic cycle induction. Many different types of protein oxidative modifications can be induced by free radicals ,31. ProtThis study sought the effect of virus replication on lipids, proteins and DNA oxidation of two lymphoblastoid cells lines B95-8 and Raji. The induction of the lytic cycle was done by TPA. The lipids, proteins and DNA damages were then determined after 48 h, the peak of lytic cycle , by measCD level was measured 48 h after induction of the lytic cycle to evaluate lipid peroxidation. The study was performed in parallel cultures with or without TPA treatment. Our data show a significant rise in CD level in B958 and Raji cell lines after EBV lytic cycle induction (p = 0.0001 and p = 0.019 respectively) .MDA adduct level was monitored in B958 and Raji cell lines after 48 h of lytic cycle induction. MDA adduct level was low in B958 and Raji cell line without TPA treatment. Our data show a significant rise in MDA adduct level in B958 and Raji cell lines after EBV lytic cycle induction (p = 0.008 and p = 0.006) Figure .The deviation between basal level and post induction level of MDA adduct was higher in B958 than in Raji cell line. (p = 0.08).PC and SH levels were assessed in B958 and Raji cells after 48 h of lytic cycle induction to determine proteins oxidation. A significant rise in PC level was observed in B958 and Raji cell lines, 48 h after EBV lytic cycle induction as compared to controls (p = 0.0030 and p = 0.0039 respectively). Protein thiol level was decreased by induction in B95-8 and Raji cell lines  .To investigate the DNA fragmentation, B95-8 and Raji cells were treated with 8 nM TPA, and DNA was isolated and analyzed by agarose gel electrophoresis. These experiments demonstrated that DNA from B95-8 and Raji cells after EBV lytic cycle induction was fragmented  , and thiA significant rise in CD levels was observed in Raji and B958 cell lines 48 h after EBV lytic cycle induction. We observed an increase in CD level in the two cell lines, indicating the presence of oxidative stress as a consequence of induction of the lytic cycle.Many different types of protein oxidative modifications can be induced by free radicals. Their oxidative damage can be evaluated by different ways, such as protein carbonyls, MDA adduct as well as SH level.Protein carbonyls determination has been particularly proposed as a marker of oxidative stress, since they form early and they are generally stable .Our data have show a significant rise in PC and MDA adduct levels as well as a decrease in SH level in Raji and B958 cells line, 48 h after EBV lytic cycle induction. Taken together, these results demonstrate protein oxidation during lytic cycle induction. Oxidized proteins cause major physiological perturbations including loss of structure and function . BesidesOur data demonstrated that oxidative damage was higher in B958 as compared to Raji cell line. Interesting, B958 cell line can produce active EBV viral particles , contrarDNA fragmentation was detected in B95-8 and Raji cell lines after EBV lytic cycle induction as compared to controls. In fact, DNA damage is described in biopsies nasopharyngeal carcinoma patients , where E202 and FeSO4 induce BZLF-1 gene expression [In our study, oxidative stress causes lipid peroxidation and protein oxidation. In addition, it is well known that ROS induce DNA strand breaks in human lymphocytes . For thepression . HoweverIt is well know that cytokines were among those first reported to generate ROS in nonphagocytic cells. It is generally accepted that ROS generated by these ligand/receptor-initiated pathways can function as true second messengers and mediate important cellular functions such as proliferation and programmed cell death . HoweverIn lymphoblastoid cell line, several pathways could be a source of ROS production such as mitochondria or NADPH oxydase. In fact, Lajeunesse et al reported that BZLF-1 gene expression in human cell lines results in a dramatic reorganization of mitochondria accompanied by a significant alteration of mitochondrial membrane potential, which can be a sign of an increase in ROS production caused by the damage of the mitochondrial electron transport system .Martin et al. reported that infection of neutrophils by EBV in vitro, rapidly reduces their survival, as confirmed by DNA staining with propiduim iodide and Hoechst 33342 and by DNA analysis . LarocheIn lyphoblastoid cell line, we described an oxidative stress status, 48 h after EBV activation, which correspond of a peak of EBV lytic cycle induction and we don't observe death of cells.Hitochi et al. reported that treatment of Raji cells with flavonoids, quercetin resulted in effective inhibition of EA induction by TPA. None of the other flavonoids such as rutin, catechin and fi-naphthoflavone affected the induction of EBV-EA by TPA .We reported that EBV replication induced oxidative stress in lymphoblastoid cell lines. In a previous study we have demonstrated that oxidative stress caused EBV lytic cycle induction as demonstrated by BZLF-1 gene expression. Thus, a vicious cycle may be initiated, whereby replication of EBV and ROS production amplify one another. This may lead to an increase in the number of EBV-infected cells and thus favor the development of EBV associated diseases, especially in immunodeficient individuals. The use of therapeutic drugs such as chemotherapy or immunosuppressive drug, which are known to induct oxidative stress may up-regulate the expression of BZLF-1, and may be a risk factor for EBV reactivation ,61. InhiIn summary, EBV lytic cycle induction was able to damage lipids, proteins and DNA, crucial biomolecules in living cells. Same damages were observed in EBV associated malignancies such as DNA damage in nasopharyngeal carcinoma biopsies patients, and proteins modification in autoimmunes diseases. This investigation may provide important clues to the mechanisms involved in the development of cancer and autoimmunes deseases.Cell lines and culture conditions• Raji is a human Burkitt's lymphoma-derived cell line, harboring the latent form of EBV cycle .• B95-8 is a lymphoblastoid cell line established from peripheral blood lymphocytes of a cotton-top marmoset (Saguinus Oedipus) following in vitro infection with EBV . A smallAll the cell lines were grown in RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum (FCS) and 2 mM L-glutamine in tissue culture flasks (Nunc). They were passaged twice a week and kept at 37°C in a humidified atmosphere of 95% air and 5% CO2.6 cells were stimulated with 8 nM TPA for 2 h, when the cells were in logarithmic phase growth, usually 48 h after placing them in culture. The cells were washed two times with Phosphate buffer saline (PBS) and further incubated for 48 h in fresh culture medium [For the induction of the lytic cycle, 3 × 10e medium .Cells were centrifuged at 3,000 rpm for 10 min. The pellet was resuspended in 500 μl of deionized water and lysed by five cycles of sonication during 20 s at 37% .Proteins were determined using the Protein Assay Kit from Bio-Rad (France) and bovine serum albumin served as the standard.Conjugated diene level was evaluated as described by Kurien and Scofield  with modProtein thiols were quantified spectrophotometrically using 5,5-dithionitrobenzoic acid (DTNB); 250 ml of freshly prepared 10 mM DTNB in 0.05 M phosphate buffer pH 8, were added to 50 ml of cell lysat in 1200 ml of 0.05 M phosphate buffer. After incubation in the dark for 15 min at room temperature, the release of 5-thiobenzoic acid was quantified by measuring the absorbance at 412 nm and converted to absolute values using N-acetyl cysteine as standard (0-0.1 mM). A correlation coefficient of r2 = 0.999 was obtained. The absorbance of samples lacking DTNB was subtracted to account for the background absorbance at 412 nm. Samples were analysed in duplicate.The MDA content was assessed by colorimetric assay, as described by Palacio et al . This asPC levels were measured according to the method based on spectrophotometric detection of the reaction of 2,4-dinitrophenylhydrazine (DNPH) with protein carbonyl to form protein hydrazones . BrieflyAfter EBV lytic cycle induction, B958 and Raji cells were lysed in a buffer containing 10 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA and 0.5% Triton X-100 for 30 min on ice. Lysates were vortexed and cleared by centrifugation at 10 000 g for 20 min. Fragmented DNA in the supernatant was extracted with an equal volume of neutral phenol chloroform isoamyl alcohol mixture (25:24:1) and analyzed electrophoretically on 2% agarose gels containing 0.1 mg/ml of ethidium bromide .Statistical analysis was carried out by Student's t-test value, to assess the statistical significance of the obtained differences between treated and non-treated cells. A p < 0.05 was considered to be statistically significant.2O2: Hydrogen peroxide; PBS: phosphate buffer saline.EBV: Epstein Barr virus; TPA: 12- 0-tetradecanoylphorbol-13-acetate; TBA: thiobarbituric acid reactivity; MDA: malondialdehyde; SOD: superoxide dismutase; CAT: catalase; LPDs: lymphoproliferative diseases; PTLD: posttransplant lymphoproliferative disorder; SH: thiol; ROS: Reactive oxygen species; DNPH: 2,4-dinitrophenylhydrazine; CD: Conjugated dienes; OD: Optical density; TBA: Thiobarbituric acid; DTPA: Tris-cacodylic acid- diethylenetriaminepenta-acetic acid; HThe authors declare that they have no competing interests.BG and RN prepared the study design, carried out all the biological studies, analyzed and discussion of the data, and drafted the manuscript. MMJ helped with chemical analysis of the extract and correction of the manuscript. RBM carried out some biological assays and helped with the manuscript preparation. SL participated in the study design, discussion the data and helped to draft and correction of the manuscript. All authors have read and approved the final manuscript.


Stachys laxa Boiss. and Buhse. from Siah-bishe in Mazandaran province, Stachys trinervis Aitch. and Hemsl. from Karaj in Alborz province, Stachys subaphylla Rech. F. and Stachys turcomanica Trautv. from Golestan province have been collected in May 2008. Total extracts were obtained through MeOH/H2O (80/20) and then partitioned between CHCl3, EtOAc and MeOH. These fractions and total extracts have been investigated for in-vitro cytotoxic activity against the colon carcinoma (HT-29), colorectal adenocarcinoma (Caco-2), breast ductal carcinoma (T47D) and Swiss mouse embryo fibroblast (NIH 3T3) cell lines using MTT assay -2,5-di phenyltetrazolium bromide). At each cell line, doses of 3.125, 6.25, 12.5, 25, 100, 200, 400 and 800 µg/mL in 1% (v/v) DMSO of all samples were tested. Ethyl acetate and chloroform fractions of Stachys laxa against proliferation of T47D and HT-29 cell lines and chloroform fraction of Stachys subaphylla and Stachys subaphylla ethyl acetate fraction toward T47D cell line exhibited highest cytotoxic activity (IC50 < 50 µg/mL). Ethyl acetate and chloroform fractions of Stachys turcomanica against HT-29 cell line, except methanol fraction of Stachys subaphylla, the other extrcts on T47D cell line, represented moderate cytotoxic activity (IC50 < 70 µg/mL). All fractions of S. trinervis demonstrated no effective cytotoxic activity. IC50 values confirmed that the growth and proliferation of HT-29 and T47D cells were most affected by chloroform and ethyl acetate fractions of Stachys laxa and Stachys turcomanica due to their nonpolar compounds.The aerial parts of  Stachys belongs to the plant family of Lamiaceae. The most species of this genus has been previously analyzed in numerous studies concerning their chemical composition, pharmacological properties and therapeutic uses. This family is well represented in the flora of Iran, at least with 200-300 species in the world , 7-epi-á-selinene (10.5 %), β-elemene (9.2 %) and β-pinene (8.6 %) (S. trinervis oil were identified as α-pinene (42.68 %), δ-2-carene (31.90 %), 1,8-cineole (7.03 %), limonene (4.39 %) and (Z)-β-ocimene (4.21 %). S. subaphylla oil major constituents were δ-2-carene (23.93 %), α-pinene (19.29 %), sabinene (19.11 %), δ-3-carene (9.22 %) and (Z)-β- ocimene (5.90 %) (S. laxa have been identified and the major constituents were germacrene D (40.1%), β-caryophylene (16.7%), β-phellandrene (5.5%), caryophyllene oxide (4.6%), linalool (3.2%) and α-cadinol (2.6%)  . Major c(5.90 %) . Thirty inene 19. %, sabin2-carene .90 %, 1,Plant materialS. laxa Boiss. and Buhse., from Siah-bishe in Mazandaran province, S. trinervis Aitch. and Hemsl from Karaj in Alborz province, and S. subaphylla Rech. F. and S. turcomanica Trautv. from Golestan province were collected in May 2008. The plants have been identified and deposited at the Herbarium of Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.The aerial parts of ExtractionStachys were cleaned and shade dried. These parts were coarse powdered in a hand mill and stored at room temperature. Two hundred grams of powdered plants were extracted through perculation method with 80% aq. MeOH three times at room temperature. The extract was evaporated using rotary evaporator and consequently partitioned between CHCl3, EtOAc and MeOH. Each fraction evaporated with rotary evaporator and has been stored at refrigerator for the investigation of cytotoxic activity.Freshly collected aerial parts of four species of Cytotoxicity assayThe colon carcinoma (HT-29), colorectal adenocarcinoma (Caco-2) and ductal carcinoma (T47D) cell lines were mentioned as exponentially growing cultures in RPMI 1640 cell culture medium , supplemented with 10% fetal bovine serum , for HT-29 cells and 15% FBS for Caco-2 and T47D cells. The Swiss mouse embryo fibroblast (NIH 3T3) cell line was kept in Dulbecco’s modified Eagle’s medium  supplemented with 10% FBS. 100 IU/mL penicillin and 100 µg/mL streptomycin  were added to the media. All the cell lines were cultured at 37°C in air /carbon dioxide (95:5) atmosphere.4 cells/well were plated in 96-well plates  and incubated for 24 h before the addition of drugs. After 96 h of incubation in Caco2 cells and 48 h of incubation in HT-29, NIH/3T3 and T47D cells, 20 µL of MTT  reagent (5 mg/mL) in phosphate buffered saline (PBS) was added to each well. The plates were incubated at 37°C for 4 h. The medium was discharged and the formazan blue, which had been formed in the cells, were dissolved with 100 µL dimethyl sulphoxide (DMSO). After the incubation at 37°C for 10 min, absorbance at 570 nm at the dissolved solutions was detected using a micro plate reader . The cell viability in MTT assay was calculated as the percentage of control value. Methotrexate was used as the positive control. Cytotoxicity was expressed as the concentration of extract inhibiting cell growth with 50% (IC50 ± SD). All tests and analysis were run in triplicate.Cytotoxic activity was measured using modified MTT assay . 1×104 cStatistical analysis50 (the median growth inhibitory concentration) values were calculated from the IC50 of dose-response curve in the sigma plot 11 software. Data representative of three independent experiments with similar results were presented as mean ± SD.IC50 ± SD are reported in S. laxa Boiss. showed high cytotoxicity on T47D, HT-29 (IC50 < 50 µg/mL). The cytotoxicity of ethyl acetate fraction of S. turcomanica Trautv. and chloroform fraction of S. subaphylla were better than the other fractions on T47D cell line (IC50 < 50 µg/mL). Total extract and fractions of S. trinervis did not affect the cell lines.The effects of these plant extracts on the proliferative response of the HT-29, Caco-2 and T47D cell lines have been analyzed by treating the cells with different concentrations of the extracts and significant decrease in cell lines proliferation were observed. ICS. laxa exhibited greatest cytotoxicities on T47D and HT-29 cell lines compared with polar fraction and total extract. According to the data, the cytotoxic activity of chloroform and ethyl acetate fractions on HT-29 and T47D cell lines were much stronger than that of Caco-2. It indicated that chloroform and ethyl acetate fractions of S. laxa had potential cytotoxic selectivity on T47D cell line. There was a report about the antioxidation and total phenol content of some Stachys spp. The research implied that total phenol content and FRAP value of methanolic extract are in this order: S. laxa > S. turcomanica > S. subaphylla > S. trinervis  since its pharmacological active compounds are not pure and further researches are needed for defining potential component as cytotoxic natural medicines.Among all the samples, nonpolar (chloroform and ethyl acetate fractions) fractions of rinervis . Except ica ssp. , 35, but


Lycopene, a major carotenoid component of tomato, has a potential anticancer activity in many types of cancer. Epidemiological and clinical trials rarely provide evidence for mechanisms of the compound’s action, and studies on its effect on cancer of different cell origins are now being done. The aim of the present study was to determine the effect of lycopene on cell cycle and cell viability in eight human cancer cell lines.Human cell lines were treated with lycopene (1–5 μM) for 48 and 96 h. Cell viability was monitored using the method of MTT. The cell cycle was analyzed by flow cytometry, and apoptotic cells were identified by terminal deoxynucleotidyl transferase-mediated dUTP nick labeling (TUNEL) and by DAPI.Our data showed a significant decrease in the number of viable cells in three cancer cells lines  after 48 h treatment with lycopene, and changes in the fraction of cells retained in different cell cycle phases. Lycopene promoted also cell cycle arrest followed by decreased cell viability in majority of cell lines after 96 h, as compared to controls. Furthermore, an increase in apoptosis was observed in four cell lines  when cells were treated with lycopene.Our findings show the capacity of lycopene to inhibit cell proliferation, arrest cell cycle in different phases and increase apoptosis, mainly in breast, colon and prostate lines after 96 h. These observations suggest that lycopene may alter cell cycle regulatory proteins depending on the type of cancer and the dose of lycopene administration. Taken together, these data indicated that the antiproliferative effect of lycopene was cellular type, time and dose-dependent. Diets high in fruits and vegetables are associated with reduced rates of cancer and coronary heart disease. Lycopene, a major carotenoid component of tomato, exhibited potential anticancer activity in many types of cancer ,2. EpideDespite epidemiological and small clinical trial evidence suggesting a possible protective effect of lycopene, the mechanism of its action including cell cycle arrest and induction of apoptosis, remain poorly understood ,7. LycopLycopene has antiproliferative effect on prostate and breast cancer cell lines. In breast cancer models, reduced expression of cell cycle regulatory proteins, such as cyclins D1 and E and the cyclin-dependent kinases 2 and 4, as well as suppression of insulin-like growth factor (IGF-I) action have been correlated with lycopene’s effects on proliferation ,14. OtheDeregulated cell cycle is one of the major hallmarks of cancer cells. These cells may lose the ability to regulate the cell cycle and control their rate of proliferation. A rate-limiting step in the cell cycle that is often disturbed in cancer is the progression of cells through the first gap (G1) phase . RecentlAlthough, the role of lycopene in prevention of prostate cancer has been studied more extensively, human studies with tomatoes and lycopene on cancer affecting other organs are now being undertaken. The majority of studies on cell cycle were carried out in prostate and breast cancer cell lines after 48 h of lycopene treatment. The aim of the present study was to determine the effect of lycopene on cell cycle and cell proliferation in eight different human cancer cell lines at different time points.All cells lines were shown to have normal growth characteristics expected under standard in vitro culture conditions. Previous studies reported that lycopene formulated as 10% WS granules was not toxic. Hereafter, all the results refer to lycopene with this vehicle, already used in our previous studies in vitro .The plating of cancer cells lines was followed by 24 h recovery, and cell were subsequently incubated with 1, 3 or 5 μM lycopene during 24, 48 and 96 h. Lycopene was internalized into all cell lines, and reached the highest intracellular concentrations in HT-29 and T-84 lines. We tested the effect of lycopene on viability of the same cell lines in higher concentrations, but the results showed the same characteristics of inhibition from 5 to 40 micromolar. The mean intracellular lycopene concentration was 0.2-1.5 fmol/L, no increase was observed from 24 to 48 h, and only a slight increase was noted after 96 hours (data not shown).We used the MTT assay to monitor the cell viability. After treatment for 48 h, a significant decrease in the number of viable cells was observed in three cancer cells lines as compared to controls, reaching 30% reduction in HT-29 and T84 cells and 10% in MCF-7 cells. A discreet effect was observed in DU 145 and HeLa cells only with 5 μM lycopene. No effect was seen in the other cells . Moreover, treatment with lycopene (3 and 5 μM) for 48 h in MCF-7 cells resulted in G0/G1 arrest, showed by the accumulation of cells in G0/G1 with concomitant decrease in in the G2/M phase (p < 0.05) . A549 cells showed a small increase of cells in G2/M phase (p < 0.05), with no significant difference among the different concentrations of lycopene.The DU145 cell line showed an increase in cells retained in the G2/M phase, followed by a decrease of cells in G0/G1 phase when treated with 3 and 5 μM of lycopene (p < 0.01). This same was observed in Hep-G2 line at all concentrations, being more pronounced with higher concentrations (3–5 μM) of lycopene, coupled with a decrease in the percentage of cells in the phase S (p < 0.05). Similar to the data on cell viability, lycopene did not change the growth profile of two cell lines (Hela and Hep-2) (p > 0.05).Quantification of apoptosis can be a useful measure of cancer cell kinetics. Alteration of the balance between proliferation and apoptosis is associated with cancer. The cancer cell lines were incubated with lycopene 3 μM) for 96 h. An increase in apoptosis was observed in four cell lines  treated with lycopene  reached more rapidly higher intracellular concentrations observed in other cells at 48 h. It is to be noted that these two cell lines responded earlier to lycopene, reaching maximal decrease of viability already at 48 h. The other four cell lines that responded to lycopene reached the same level of cell number decrease only after 96 h. Colon cells normally have access to alimentary lycopene, and may be provided by molecular pathways that accelerate lycopene internalization. However, the fact that other cell types that are sensitive to lycopene reached the same inhibition levels after 48 h indicated that the lycopene uptake rate is apparently not limiting for its anticancer activity, provided the equilibrium with lycopene present in the external environment. This suggests that maintaining the equilibrium of lycopene input and output may be relevant for its effect on cancer, recommending a permanent minimal supply of alimentary lycopene.In the present study, the cell lines that have active steroid receptors (estrogen or androgen) and are sensitive to this signaling pathway were also sensitive to lycopene-mediated inhibition of cell proliferation. Interference in this pathway that controls cell proliferation in association with retinoid receptor was associated with apoptosis and inhibition of the cell cycle progression ,28. FurtMany studies in mammary ,21 and pProstate cancer is the second most common cause of cancer death in American men . EpidemiIn breast cancer MCF-7 cells lycopene inhibited cell proliferation as monitored by MTT. Lycopene at micromolar concentrations inhibited MCF-7 cell growth in a dose-dependent manner and the minimal inhibiting concentration was 3.5 μM by MTT and 5 μM by BrdU assays at 48 hours. ,25 In ouBurgues et al.  observedPrevious studies ,18,35 reHwang and Bowen  demonstrIn our study, lycopene was able to promote cell cycle arrest, resulting in an increase of cells in G0/G1 phase or G2/M, depending upon the tumor cell type. Whilst arrest in G0/G1 can be reverted and cells will proceed the proliferation after interruption of the treatment, the G2/M arrest leads potentially to apoptosis . LycopenIncreased resistance to apoptosis is a hallmark of many tumor cells. The functional inhibition of specific anti-apoptotic factors may provide a rational basis for the development of novel therapeutic strategies. Therefore, apoptosis deficiency is considered to be a major cause of the therapeutic resistance of tumors in the clinic, since many chemo- and radiotherapeutic agents act through the induction of apoptosis. Our results showed a significant increase in apoptosis that followed arrest cell cycle in four cancer cell lines . This may indicate that lycopene could be proposed as adjuvant in cancer chemotherapy.Other reports demonstrated that lycopene can inhibit the proliferation of cancer cells through the induction of apoptosis in different cell lines ,30, but We demonstrated the capacity of lycopene to inhibit cell proliferation, arrest cell cycle in different phases and increase apoptosis, mainly in breast, colon and prostate cell lines after 96 h. These observations suggest that lycopene may alter cell cycle-regulatory proteins depending on the type of cancer and the dose of lycopene administration. Taken together, these data indicated that the potential anticancer effect of lycopene was cellular type, time and dose-dependent.All-trans lycopene was purchased from Sigma Chemical Company . Water-soluble (WS) lycopene (10%) was provided by Roche . Dulbecco’s cell culture medium and bovine serum albumin were obtained from Sigma, and fetal bovine serum (FBS) from Laborclin . Tissue culture flasks and cell scrapers were obtained from Nunc . All the chemicals were of analytical grade.2 tissue culture flasks, 5.0 × 106 cells / flask, and maintained routinely in the Dulbecco’s medium supplemented (DMEM) with 10% fetal bovine serum (FBS) and 2 g/L HEPES buffer, pH 7.4, under 5% CO2 atmosphere. Human colon carcinoma cells (T-84) and human lung adenocarcinoma-derived cells (A549) were maintained in DMEM:HAM-F12 containing 10% FBS and 100 U/ml penicillin. Cells were passaged at 70–80% confluence, about twice a week by trypsinization. For each experiment, all the cells were seeded at 104 cell/cm2 in 6 and 96 multiwell plates for cell cycle and cell proliferation analyses, respectively. After 24 hours, the culture medium was changed and each concentration of lycopene (WS) dissolved in water at 50 °C within a range from 1 to 5 μM. The controls were included on each plate. The cells were then incubated for 48 and 96 hours with daily medium replacement [All the cells lines were obtained from the Rio de Janeiro Cell Bank that has certified their identity and quality . Human prostate cancer cells (DU145), human colon adenocarcinoma cells (HT-29), human cervical cancer cell line (Hela), breast cancer cell line (MCF-7), human liver carcinoma cells (Hep-G2) and human laryngeal carcinoma (Hep-2) cells were plated 25 cmlacement .4 cells/cm2 with DMEM, plated in 96-well plates  at 200 μL/well and then incubated for 12 h according to routine procedure. After being treated with lycopene (1–5 μM) and incubated for 48 h and 96 h (5 wells for each sample), 20 μL/well MTT (5 g/L) was added to each well. The medium was then removed after 4 h incubation and 100 μL/well sodium dodecyl sulfate (SDS) was added to dissolve the reduced formazan product. Finally, the plate was read in an enzyme-linked immunosorbent microplate reader  at 490 nm. The cellular proliferation inhibition rate (CPIR) was calculated using the following formula: CPIR =  × 100%.The status of cancer cell lines viability was determined by MTT assay . Exponentially growing HSC were adjusted to 1.0 × 106 cells were resuspended in 1.0 mL ice-cold VindeLov solution [Cells were rinsed briefly with calcium- and magnesium-free phosphate-buffered saline (PBS) and detached with trypsin at room temperature. After centrifugation, as the cells were washed twice with PBS, 1 × 10solution , contain4 cells/cm2) were treated with lycopene at a concentration of 3 μM in 96-well plates. After 96 h of incubation, cell death was detected by TdT-mediated dUTP terminal nick-end labeling kit . Nuclei were counterstained with 1 μg/mL DAPI during 3 min. Slides were examined in a fluorescence microscope  and images were acquired with AxioCam Hrc digital camera. Apoptotic data are reported as percentage apoptosis, obtained by determining the numbers of apoptotic cells versus the total number of cells. For each sample, a minimum of 3 counts involving a minimum of 100–200 cells/count were scored. Apoptotic data are presented as the mean ± SD for three independently performed experiments.Human cancer cells lines . Statistical comparisons were carried out by ANOVA and post hoc Tukey’s test using Graph Pad Prism 4.0 and Statistical 6.0 program. The differences were considered significant when The authors declare that they have no competing interests.TAJ, OFL, MNB, MGA and BR performed experiments and summarized the data; TTA, MRB and BR designed experiments; TAJ, MRB and BR wrote the paper; all authors have read and approved the final manuscript.