HDAC Inhibitors as Epigenetic Regulators

The cells were co-incubated at 37C in 5% CO2for 4 days. we found that TCS increased the percentage of effector T cells, particularly Interferon-gamma (IFN-) producing CD4+and CD8+T cells from tumor-bearing mice. TCS also promoted the vigorous proliferation of antigen-specific effector T cells, markedly increased Th1 cytokine secretion and elicited more memory T cells in tumor-bearing mice, consequently enhancing the anti-tumor response and inducing immune protection. Furthermore, we found that TCS upregulated the expression of tumor suppressor in lung cancer 1 (TSLC1) in 3LL tumor cells and the expression of its ligand, class I-restricted T cell-associated molecule (CRTAM), in effector T cells. Blocking TSLC1 expression with small interfering RNA (siRNA) significantly eliminated the effects of TCS on the proliferation and cytokine secretion of effector T cells, suggesting that TCS enhances anti-tumor immune response at least partially by boosting the interaction between TSLC1 and CRTAM. Collectively, our data demonstrate that TCS not only affects tumor cells directly, but also enhances anti-tumor immunityviathe interaction between TSLC1 and CRTAM. These findings may lead to the development of a novel approach for tumor regression. Keywords:anti-tumor immunity, CRTAM, immune protection, trichosanthin, TSLC1 == Introduction == Several dietary components found in plant-derived foods have been recognized for their anti-carcinogenic properties in recent years.1,2Notably, these natural agents have fewer side effects than do many synthetic agents.3 Trichosanthin (TCS), isolated from the root tubes ofTrichosanthes kirilowiiMax,4,5is a single-chain peptide with 247 amino acid residues.6,7It has a broad spectrum of biological and pharmacological activities, including the stimulation of abortion,8suppression of tumor growth9and inhibition of HIV activity.10TCS can inhibit tumor Lomeguatrib cell proliferationviasuppression of the PKC/MAPK signaling pathway11and induce apoptosis through downregulation of Bcl-2 and upregulation of Bax.12Additionally, TCS can induce apoptosis of JAR cellsviastimulating the production of reactive oxygen species.13 In addition to its direct effect on tumor cells, TCS shows immunomodulatory effects in naive mice. TCS has been found Lomeguatrib to be a T helper 2 (Th2)-type adjuvant that modulates the switching of immune responses to a Th2 pathway in a model of airway inflammation.14It can also induce the expression of Th2 cytokines, such as IL-4, IL-10 and tumor-growth factor- (TGF-).15In this way, TCS may induce Th2-type, rather than Th1-type, immunity in normal or inflammation conditions. However, the influence of TCS on immune response in tumors remains unknown. Recent studies have reported that TCS has a demethylation function and can restore the activity of the tumor suppressor in lung cancer 1 (TSLC1),SykandRASSF1Agenes, indicating a possible mechanism for TCS inhibition of Lomeguatrib tumor growth.3,16TSLC1 is a tumor suppressor gene that is widely expressed on stromal cells, but it is always lost because of promoter hypermethylation in tumor tissue. Its ligand, class I-restricted T cell-associated molecule (CRTAM), is only expressed on activated T cells, and the interaction between TSLC1 and CRTAM may promote the proliferation of activated T cells and their secretion of interferon (IFN)- secretion, thereby enhancing the anti-tumor effectiveness of T cells.17,18 In this study, we established an animal tumor model with the Lewis lung cancer cell line (3LL) in C57BL/6 mice in order to determine whether TCS is involved in the induction of anti-tumor immune response in tumor-bearing hosts. == Materials and methods == == Mice == Female C57BL/6 (H-2b) mice and nude mice aged at 46 weeks were purchased from Shanghai Experimental Center, Chinese Academy of Science, and housed in a pathogen-free environment in center of Laboratory Animal, Fudan University. All animal experiments were performed according to the Guide for the Care and Use of Medical Laboratory Animals (Ministry of Health, China, Lomeguatrib 1998) and with the ethical approval of the Shanghai Medical Laboratory Animal Care and Use Committee as well as the Ethical Committee of Fudan University. == Cell culture == The 3LL cell line (mouse Lewis lung cancer cell line) and FBL3 cell line (erythroleukemia cells) were purchased from Chinese Academy of Science. Tumor cell lines were cultured at 37 C under 5% CO2in a RPMI 1640 (Gibco, Grand Island, NY, USA) medium containing 10% heat-inactivated fetal bovine serum and supplemented with 2 mM glutamine, 100 IU/ml penicillin and 100 g/ml streptomycin sulfate. == Cell growth inhibitory activities == Cell growth-inhibitory activities of TCS on 3LL cells were evaluated by CCK-8 assay (Cell Counting Kit-8; Dojindo, Kumamoto, Japan). 3LL was seeded in 96-well plates (Corning, New York, USA) at a plating density of 1104/well, 24 h later, cells were exposed to TCS (Shanghai Jinshan Pharmaceutical Factory, Shanghai, China) at various doses (0, 25, 50 and 100 g/ml) in fresh RPMI 1640 medium. TCS was diluted by phosphate-buffered saline (PBS). Four replicate wells for each treatment dose were performed. The plate was placed at 37 C in 5% CO2for various time points (24, 48 Lomeguatrib and 72 h), and then the wells were added into 10 l CCK-8 reagent for Rabbit polyclonal to EARS2 appropriate time at 37 C, and measured at 450 nm by the Bio-Rad.