We studied the estrogenic activity of a component of Panax ginseng, ginsenoside-Rb1. The activity of ginsenoside-Rb1 was characterized in a transient transfection system, using estrogen receptor isoforms and estrogen-responsive luciferase plasmids, in COS monkey kidney cells. Ginsenoside-Rb1 activated both alpha and beta estrogen receptors in a dose-dependent manner with maximal activity observed at 100 microm, the highest concentration examined. Activation was inhibited by the estrogen receptor antagonist ICI 182,780, indicating that the effects were mediated through the estrogen receptor. Treatment with 17beta-estradiol or ginsenoside-Rb1 increased expression of the progesterone receptor, pS2, and estrogen receptor in MCF-7 cells and of AP-1-driven luciferase genes in COS cells. Although these data suggest that it is functionally very similar to 17beta-estradiol, ginsenoside-Rb1 failed to displace specific binding of [(3)H]17beta-estradiol from estrogen receptors in MCF-7 whole-cell ligand binding assays. Our results indicate that the estrogen-like activity of ginsenoside-Rb1 is independent of direct estrogen receptor association.

Interleukin (IL)-12 has been reported to induce cellular immune responses for protection against tumor formation. Here we investigate the utility of adenoviral delivery of IL-12 as an adjuvant for a human papillomavirus E7 subunit vaccine in a mouse tumor challenge model. Direct intratumoral injection of AdIL-12 resulted in a significant suppression of tumor growth compared to the control group. Injection of E7 protein into either a tumor site or the distance site along with AdIL-12 further enhanced antitumor effects significantly higher than either AdIL-12 or E7 injection alone. This combined injection resulted in complete regression of 9-mm-sized tumor in 40% of animals as well as lasting antitumor immunity against tumor recurrence. We also evaluated immune responses induced by these injections. AdIL-12 plus E7 enhanced E7-specific antibody responses significantly higher than AdIL-12 or E7 injection. In particular, the production level of interferon (IFN)-gamma from E7-specific CD4(+) T cells was similar between AdIL-12 group and AdIL-12 + E7 group. However, IFN-gamma production from E7-specific CD8(+) T cells was the most significant when injected with AdIL-12 + E7. This was consistent with intracellular IFN-gamma staining levels of CD8(+) T cells, suggesting that AdIL-12 + E7 injection enhances antitumor immunity in the human papillomavirus (HPV) 16 tumor model through increased expansion of the cytotoxic T-lymphocyte (CTL) subset. This enhanced protection appeared to be mediated by CD8(+) T cells, as determined by in vivo T-cell subset deletion. Thus, these studies demonstrate that E7 vaccines can induce CTL responses responsible for antitumor effects in the presence of IL-12 delivered via adenovirus vectors. This likely provides one additional approach for immune therapy against cervical cancers.

SDS3 is a key component of the histone deacetylase (HDAC)-dependent Sin3A co-repressor complex, serving to maintain its HDAC activity. Here, we report both exogenous and endogenous functional interaction between deubiquitinating enzyme USP17 and human SDS3 by MALDI-TOF-MS, co-immunoprecipitation assay, and GST pull-down assay. In this study, we demonstrated that SDS3 readily undergoes endogenous polyubiquitination, which is associated specifically with Lys-63-branched polyubiquitin chains and not with Lys-48-branched polyubiquitin chains. Further, we also demonstrated that USP17 specifically deubiquitinates Lys-63-linked ubiquitin chains from SDS3 and regulates its biological functions. The deubiquitinating activity of USP17 on SDS3 negatively regulates SDS3-associated HDAC activity. The constitutive expression of USP17 and its substrate SDS3 was involved in the inhibition of anchorage-independent tumor growth and blocks cell proliferation, leading to apoptosis in cervical carcinoma cells. Furthermore, we showed that USP17 and SDS3 mutually interact with each other to regulate cancer cell viability. These data support the possibility that SDS3, being a substrate of USP17, may play an important role in developing a novel therapeutic means to inhibit specific HDAC activities in cancer.Molecular processes such as phosphorylation, dephosphorylation, acetylation, deacetylation, ubiquitination, and deubiquitination of cellular proteins play an orchestrated role in coordinating homeostasis and contribute to the manifestation of physiological processes in health and disease. The process of ubiquitination is a well established event involving a complex of ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligase (E3) enzymes (1-4). Ubiquitination can be reversed by deubiquitinating enzymes (DUBs) 2 (3, 5). Most DUBs are cysteine proteases and consist of at least five known families: the ubiquitin C-terminal hydrolases, the ubiquitin-specific processing proteases (USP), Jab1/ Pab1/MPN domain-containing metallo-enzymes, Otu-domain ubiquitin aldehyde-binding proteins, and Ataxin-3/Josephin (3, 4, 6).USP17 was previously identified as a human ortholog of DUB-3 that is regulated by the IL-4 and IL-6 cytokines (7). Recent characterization of USP17 as a key regulator of cell proliferation has revealed its critical role in cell growth and survival (7-10). Previously, we showed that USP17 possesses two hyaluronan binding motifs (HABMs) in its C-terminal region and interacts with hyaluronan to regulate cell viability (11). However, the biological significance of these HABMs in USP17 remains to be fully understood.The Sin3 co-repressor complex has been linked to HDAC enzymatic activity by directly interacting with HDAC1 and HDAC2 within the Sin3-HDAC complex (12, 13). SDS3 is a subunit of the HDAC-dependent Sin3A co-repressor complex and exhibits a critical role in maintenance of mSin3-associated HDAC activity (14,15). It has a role in transcriptional repression, recruits HDAC activity, and enables...

SUMMARYWe previously reported that both E7 and CpG-oligodeoxynucleotide (ODN) are required for protecting animals from human papillomavirus (HPV) 16 E7-associated tumour challenge. Here we investigate dendritic cells (DC)-based approach in this protection. In the study, we isolated bone marrow-derived DC and stimulated DC with E7 and ODN. In vitro stimulation of DC with E7 plus ODN resulted in more production of interleukin-12, as compared to that with E7 or ODN alone. Further injection with E7+ODN-stimulated DC resulted in more significant tumour protection, as compared to stimulation with E7 or ODN alone. We further evaluated the levels of immune responses induced by DC stimulated with E7+ODN. We observed little enhancement of E7-specific antibody and T helper cell proliferative responses by E7+ODN stimulation, as compared to E7 stimulation. However, there was some enhancement of interferon-c (IFN-c) production from CD4 + T cells and a more significant production of IFN-c from CD8 + T cells by E7+ODN stimulation, as compared to E7 stimulation alone. This was consistent with intracellular IFN-c staining levels of CD8 + T cells. Tumour protection further appeared to be mediated by CD8 + T cells, as determined by in vivo T-cell depletion. Thus, these data suggest that upon ODN stimulation DC might function as a potent adjuvant for E7 protein delivery for induction of protective cellular immunity against HPV E7-associated tumour challenge.

Recent studies have suggested that photo-oxidative lesions produced by photodynamic therapy (PDT)-treated tumors are recognized by the host as altered self, (4) prompting a strong inflammatory and immune response.(5) In addition, the use of PDT vaccines has been studied to generate antitumor immunity and control tumor growth, suggesting that further improvements can be achieved in the optimization of the protocols for the generation of PDTgenerated cancer vaccines.(6,7) Because of the inflammatory/immune response triggered by PDT, this therapy has been shown to be particularly suitable for combination with a variety of immunotherapy based treatments, including angiogenic growth factors, matrix metalloproteinases, cytokines and adoptive transfer of immune cells. (8 -12) Overexpression of these molecules within PDTtargeted tissue can adversely affect tumor response. Therefore, experimental protocols combining PDT with procedures targeting these molecules are being examined in an effort to improve treatment efficacy.Oligodeoxynucleotide (ODN) containing unmethylated cytosine-phosphate-guanosine (CpG) motifs was originally isolated from components of bacterial DNA.(13) CpG-ODN immunotherapy has been studied as a strategy for tumor prevention as well as for treatment of immune disorders. (14,15) A variety of studies have shown that CpG-ODN can activate B cells, monocytes and natural killer cells, and induce a Th1-like pattern of cytokine production.(16-18) The CpG sequences drive macrophages to secrete interleukin-12, a potent inducer of interferon-γ (IFN-γ) production in vivo from natural killer cells. IFN-γ production drives Th1-type immune responses by inducing differentiation of type-1 Th cells, which see antigen in the presence of IFN-γ from the uncommitted T-cell pool. (19,20) Moreover, ODN enhances humoral responses and induces the development of enhanced cytotoxic T lymphocytes (CTL) activity. (17,21) ODN has been studied extensively as a strong immunomodulatory agent. (22)(23)(24) The aim of the present study was to further characterize the immunotherapeutic significance of the combination of CpGoligodeoxynucleotide and TC-1 tumor cell lysates induced by PDT. We examined the in vivo antitumor effect of PDT (or freezing/thawing)-generated cell lysates plus ODN injection and the immune response in a mouse model. Our results showed that PDT-cell lysates plus ODN showed a significant suppression of tumor growth at both prophylactic and therapeutic levels, compared to PDT (or F/T)-cell lysates or ODN alone. Materials and MethodsPreparation of PDT-generated tumor cell lysates. Radachlorin (25) was purchased from RADA-PHARMA group (RADA-PHARMA, Moscow, Russia). The light source was a diode laser with a 662 ± 3 nm wavelength (Won-PDT D662, Won Technology, Daejeon, Korea). The irradiation power in vitro was fixed to be 6.25 J/cm 2 at 20 mW/cm 2 for 5 min irradiation measured at a distance of 3 cm from the exit slit.To generate cell lysates by Radachlorin/PDT, TC-1 cells carrying human papillomavirus (HPV) 16 E7 (ATC...

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In this study, we modified cationic liposomes either by polyethylene glycol (PEG)-grafting or PEG-adding methods, and compared the physical properties of transfection complexes and transfection efficiency in-vitro and prolonged circulation in-vivo. The PEG-grafted transfection complexes were prepared by mixing plasmid DNA with PEG-grafted cationic liposomes, which were composed of DSPE-PEG 2000 and cationic lipids. The PEG-added transfection complexes were prepared by adding DSPE-PEG 2000 to the mixture of cationic liposomes and plasmid DNA. The particle sizes of the PEG-modified transfection complexes (approximately 200 nm) changed a little over 4 weeks compared with the conventional transfection complexes. In the presence of serum, the transfection efficiency of the conventional transfection complexes was lowered whereas the transfection efficiency of the PEG-modified transfection complexes was maintained. Moreover, the transfection efficiency of the conventional transfection complexes was significantly reduced when they were stored. However, the transfection efficiency was stable for the PEG-modified transfection complexes, even after two weeks of storage. Of the in-vitro transfection efficiencies, there was no difference between PEG-grafted and PEG-added transfection complexes. When the conventional, PEG-grafted, and PEG-added transfection complexes were administered into mice by the tail vein, the PEG-added transfection complexes showed a prolonged circulation of plasmid DNA compared with other transfection complexes. These results suggest that the PEG-added transfection complexes could be a useful non-viral vector because of their simplicity in preparation, enhanced stability and prolonged circulation compared with the conventional transfection complexes.

The initial aim of this study was to examine the expression profiles of P53 and its upstream genes, downstream genes, and cell cycle regulators to determine whether these markers are useful for making a differential diagnosis among the benign, borderline, and malignant ovarian epithelial tumors. Between borderline and malignant tumors, the increased expression levels of P53, Bax, Cyclin E, and cyclin-dependent kinase-2 as well as the decreased expression levels of growth arrest and DNA damage (GADD45) and murine double minute-2 (MDM2) were significantly associated with malignancy (P < 0.01, each). Using the receiver operating curve (ROC), the most reliable cutoff value of the added-up staining scores of those markers was 4.5 with 79% sensitivity and 89% specificity for malignancy. Between benign and borderline tumors, the P21 and Bax expression levels were significantly higher in borderline tumors, whereas the Bcl-2 expression level was much higher in benign tumors (P < 0.01, each). Using the ROC, the cutoff value of the added-up staining scores used to discriminate between the two groups was 2.5 with 70% sensitivity and 74% specificity for borderline tumors. Thus, for the differential diagnosis between borderline and malignant tumors, the cutoff value 4.5 of the cumulative staining scores can be used. However, the cutoff value 2.5 for discrimination between benign and borderline tumors may not be useful because of its relatively low sensitivity and specificity. In addition, the P53, GADD45, Cyclin E, and MDM2 expression levels in malignant ovarian tumors might be useful for determining the histologic grade and type.