Yi-Te Yo

Licorice and Licochalcone-A Induce Autophagy in LNCaP Prostate Cancer Cells by Suppression of Bcl-2 Expression and the mTOR Pathway

Licorice is a common Chinese medicinal herb with antitumor activity. Some components in licorice root have been shown to induce cell cycle arrest or apoptosis in cancer cells. This paper demonstrates for the first time that licorice Glycyrrhiza glabra and its component licochalcone-A (LA) can induce autophagy in addition to apoptosis in human LNCaP prostate cancer cells. Exposure of cells to licorice or LA resulted in several confirmed characteristics of autophagy, including the appearance of autophagic vacuoles revealed by monodansylcadaverine (MDC) staining, formation of acidic vesicular organelles (AVOs), and autophagosome membrane association of microtubule-associated protein 1 light chain 3 (LC3) characterized by cleavage of LC3 and its punctuate redistribution, as well as ultrastructural observation of autophagic vacuoles by transmission electron microscopy. Autophagy induction was accompanied by down-regulation of Bcl-2 and inhibition of the mammalian target of rapamycin (mTOR) pathway. In summary, licorice can induce caspase-dependent and autophagy-related cell death in LNCaP cells.

Intra-articular lentivirus-mediated delivery of galectin-3 shRNA and galectin-1 gene ameliorates collagen-induced arthritis

Different members of the galectin family may have inhibitory or stimulatory roles in controlling immune responses and regulating inflammatory reactions in autoimmune diseases such as rheumatoid arthritis (RA). A hypothetical model of a cross talk between galectin-1 and galectin-3 has been established in the circumstance of rheumatoid joints. As galectin-3 is a positive regulator and galectin-1 is a negative regulator of inflammation and autoimmune responses, in this study we evaluated the effects of local knockdown of galectin-3 or overexpression of galectin-1 on ameliorating collagen-induced arthritis (CIA) in rats. Lentiviral vectors encoding galectin-3 small hairpin RNA (shRNA) and galectin-1, as well as two control vectors expressing luciferase shRNA and green fluorescent protein, were individually injected intra-articularly into the ankle joints of rats with CIA, and their treatment responses were monitored by measuring the clinical, radiological and histological changes. Our results show that both knockdown of galectin-3 and overexpression of galectin-1 induced higher percentages of antigen-induced T-cell death in the lymph node cells from arthritic rats. Furthermore, these treatments significantly reduced articular index scores, radiographic scores and histological scores, accompanied with decreased T-cell infiltrates and reduced microvessel density in the ankle joints. Our findings implicate galectin-3 and galectin-1 as potential therapeutic targets for the treatment of RA.

Amelioration of collagen-induced arthritis in rats by adenovirus-mediated PTEN gene transfer.

OBJECTIVE The phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway is known to be activated in rheumatoid arthritis (RA) synovial tissue, which impacts cell growth, proliferation, survival, and migration. Phosphatase and tensin homolog deleted from chromosome 10 (PTEN) functions as a negative regulator of PI 3-kinase signaling, thus blocking Akt activation. The aim of this study was to examine the effect of PTEN gene transfer in rats with collagen-induced arthritis (CIA). METHODS Adenoviral vectors encoding human PTEN (AdPTEN) or beta-galactosidase (AdLacZ) were injected intraarticularly into rats with CIA, and their treatment responses were monitored by measures of clinical, radiographic, and histologic changes. The expression of phosphorylated Akt, total Akt, vascular endothelial growth factor (VEGF), proinflammatory cytokines, and chemokines, as well as the extent of microvessel density in the ankle joints were determined. RESULTS AdPTEN treatment reduced Akt phosphorylation and decreased VEGF production in human RA synovial fibroblasts. Compared with AdLacZ treatment of the rats with CIA, AdPTEN treatment significantly reduced ankle circumference, articular index scores, radiography scores, and histology scores, and also decreased microvessel density and levels of VEGF and interleukin-1beta. Furthermore, PTEN gene transfer led to down-regulation of Akt activation and increased apoptosis in the ankle joints. CONCLUSION This study is the first to demonstrate the in vivo effect of intraarticular gene delivery of PTEN on amelioration of arthritis symptoms in rats with CIA, which involved antiangiogenic, antiproliferative, and antiinflammatory effects of PTEN via inhibition of the PI 3-kinase/Akt signaling pathway. Our findings also implicate the PI 3-kinase/Akt pathway as a therapeutic target for the treatment of RA or other inflammatory diseases.

Low-Dose Etoposide Enhances Telomerase-Dependent Adenovirus-Mediated Cytosine Deaminase Gene Therapy through Augmentation of Adenoviral Infection and Transgene Expression in a Syngeneic Bladder Tumor Model

The human telomerase reverse transcriptase (hTERT) promoter can selectively drive transgene expression in many telomerase-positive human cancer cells. Here we evaluated combination therapy of adenoviral vector Ad-hTERT-CD encoding E. coli cytosine deaminase (CD) driven by the hTERT promoter and low-dose etoposide (0.1 microg/mL) for treating bladder cancer. Ad-hTERT-CD conferred sensitivity to 5-fluorocytosine (5-FC) in bladder cancer cells, which could be enhanced by etoposide treatment, but not in normal cells. Such effect was correlated with up-regulation of hypoxia-inducible factor (HIF)-1alpha expression. By contrast, etoposide activated p53 and down-regulated hTERT promoter activity in normal cells. Etoposide also increased adenoviral infection via enhancement of coxsackie-adenovirus receptor expression on bladder cancer and normal cells. Combination index analysis revealed that combined therapy of Ad-hTERT-CD (10(9) plaque-forming units)/5-FC (200 mg/kg) with etoposide (2 mg/kg) synergistically suppressed tumor growth and prolonged survival in mice bearing syngeneic MBT-2 bladder tumors. This combination therapy regimen induced complete tumor regression and generated antitumor immunity in 75% of tumor-bearing mice. Furthermore, increased infiltrating CD4(+) and CD8(+) T cells and necrosis within tumors were found in mice receiving combination therapy of Ad-hTERT-CD and etoposide compared with those treated with either treatment alone. Thus, the potential high therapeutic index of the combination therapy may be an appealing therapeutic intervention for bladder cancer. Furthermore, because a majority of human tumors exhibit high telomerase activity, adenovirus-mediated CD gene therapy driven by the hTERT promoter in combination with low-dose etoposide may be applicable to a broad spectrum of cancers.

Tumor-Selective Replication of an Oncolytic Adenovirus Carrying Oct-3/4 Response Elements in Murine Metastatic Bladder Cancer Models

Purpose: Oncolytic adenoviruses are attractive therapeutics for cancer because they selectively replicate in tumors. However, targeting tumor metastasis remains a major challenge for current virotherapy for cancer. Oct-3/4 is specifically expressed in embryonic stem cells and tumor cells. Oct-3/4 highly expressed in cancer cells may be a potential target for cancer therapy. We developed an E1B-55 kDa–deleted adenovirus, designated Ad.9OC, driven by nine copies of Oct-3/4 response element for treating Oct-3/4–expressing metastatic bladder cancer. Experimental Design: We examined the expression of Oct-3/4 in human bladder tumor tissues and bladder cancer cell lines. We also evaluated the cytolytic and antitumor effects of Ad.9OC on bladder cancer cells in vitro and in vivo. Results: Oct-3/4 expression was detected in bladder cancer cell lines, as well as in human bladder tumor tissues. Notably, Oct-3/4 expression was higher in metastatic compared with nonmetastatic bladder cancer cells. Ad.9OC induced higher cytolytic activity in metastatic bladder cancer cells than in their nonmetastatic counterparts, whereas it did not cause cytotoxicity in normal cells. Pharmacologic and short hairpin RNA–mediated Oct-3/4 inhibition rendered bladder cancer cells more resistant to Ad.9OC-induced cytolysis. Replication of Ad.9OC was detected in murine bladder cancer cells and bladder tumor tissues. We also showed the effectiveness of Ad.9OC for treating bladder cancer in subcutaneous, as well as metastatic, bladder tumor models. Conclusions: Ad.9OC may have therapeutic potential for treating Oct-3/4–expressing tumors. Especially, metastatic bladder tumors are good target for Ad.9OC treatment. Because Oct-3/4 is expressed in a broad spectrum of cancers, Ad.9OC may be broadly applicable.

Amelioration of Rat Collagen-Induced Arthritis Through CD4+ T Cells Apoptosis and Synovial Interleukin-17 Reduction by Indoleamine 2,3-Dioxygenase Gene Therapy

Indoleamine 2,3-dioxygenase (IDO) has been known as an emerging therapeutic target in autoimmunity-related arthritis. The treatment responses of adenoviral vectors encoding IDO (AdIDO) gene therapy in rat collagen-induced arthritis (CIA) were examined in this study. The therapeutic effects on ankle circumference, articular index, and radiographic and histological scores were evaluated in AdIDO-injected ankle joints. We further determined CD4+ T-cell numbers and their apoptotic status, CD68(+) macrophage numbers, kynurenine (a downstream tryptophan metabolite) concentrations, interleukin-17 (IL-17) levels, and retinoic acid-related orphan receptor γt (RORγt) expression in synovial tissues of CIA rats receiving AdIDO treatment. Reduction of ankle circumference, articular index, and radiographic and histological scores were noted in AdIDO-treated ankles, as compared with those receiving injection of control vectors. Furthermore, IDO gene transfer led to decreased infiltrating CD4+ T cells with enhanced apoptosis, reduced CD68+ macrophage numbers, increased kynurenine levels, lower IL-17 concentrations, and decreased RORγt expression within the ankle joints. In addition, such a therapy diminished type II collagen-specific IL-17 production and RORγt expression in CD4+ T cells from draining lymph nodes of CIA rats. Our results demonstrate for the first time that intra-articular delivery of IDO gene ameliorated ankle arthritis of CIA rats by induction of CD4+ T-cell apoptosis and reduction of synovial IL-17 production through the supplement of kynurenine. Taken together, these findings implicate the novel strategy of using IDO gene as a therapeutic approach in treating patients with rheumatoid arthritis.

Mitochondrial ribosomal protein S36 delays cell cycle progression in association with p53 modification and p21WAF1/CIP1 expression

Ribosomal biogenesis is correlated with cell cycle, cell proliferation, cell growth and tumorigenesis. Some oncogenes and tumor suppressors are involved in regulating the formation of mature ribosome and affecting the ribosomal biogenesis. In previous studies, the mitochondrial ribosomal protein L41 was reported to be involved in cell proliferation regulating through p21WAF1/CIP1 and p53 pathway. In this report, we have identified a mitochondrial ribosomal protein S36 (mMRPS36), which is localized in the mitochondria, and demonstrated that overexpression of mMRPS36 in cells retards the cell proliferation and delays cell cycle progression. In addition, the mMRPS36 overexpression induces p21WAF1/CIP1 expression, and regulates the expression and phosphorylation of p53. Our result also indicate that overexpression of mMRPS36 affects the mitochondrial function. These results suggest that mMRPS36 plays an important role in mitochondrial ribosomal biogenesis, which may cause nucleolar stress, thereby leading to cell cycle delay. J. Cell. Biochem. 100: 981–990, 2007.

Coexpression of Flt3 ligand and GM-CSF genes modulates immune responses induced by HER2/neu DNA vaccine.

DNA vaccine and dendritic cells (DCs)-based vaccine have emerged as promising strategies for cancer immunotherapy. Fms-like tyrosine kinase 3-ligand (Flt3L) and granulocyte–macrophage-colony-stimulating factor (GM-CSF) have been exploited for the expansion of DC. It was reported previously that combination of plasmid encoding GM-CSF with HER2/neu DNA vaccine induced predominantly CD4+ T-cell-mediated antitumor immune response. In this study, we investigated the modulation of immune responses by murine Flt3L and GM-CSF, which acted as genetic adjuvants in the forms of bicistronic (pFLAG) and monocistronic (pFL and pGM) plasmids for HER2/neu DNA vaccine (pN-neu). Coexpression of Flt3L and GM-CSF significantly enhanced maturation and antigen-presentation abilities of splenic DC. Increased numbers of infiltrating DC at the immunization site, higher interferon-γ production, and enhanced cytolytic activities by splenocytes were prominent in mice vaccinated with pN-neu in conjunction with pFLAG. Importantly, a potent CD8+ T-cell-mediated antitumor immunity against bladder tumors naturally overexpressing HER2/neu was induced in the vaccinated mice. Collectively, our results indicate that murine Flt3L and GM-CSF genes coexpressed by a bicistronic plasmid modulate the class of immune responses and may be superior to those codelivered by two separate monocistronic plasmids as the genetic adjuvants for HER2/neu DNA vaccine.

579. Generation of potent cellular immune responses induced by intradermal delivery of plasmid DNA coexpressing Flt3 ligand and GM-CSF

The protective efficacy of DNA vaccine has been demonstrated in murine models against infectious diseases or tumor growth. Recently, Fms-like tyrosine kinase 3-ligand (Flt3L) has been identified as an important cytokine for the generation of professional antigen-presenting cells (APCs), particularly dendritic cells (DCs). Administration of Flt3L protein or recombinant DNA encoding Flt3L into mice results in the expansion of the DC population in vivo. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is also critical for DC survival and differentiation in vitro. Previous studies have shown that codelivery of Flt3L-encoding and GM-CSF-encoding plasmids with antigen results in increase in DC numbers in comparison with codelivery of either plasmid alone. In this study, we constructed a bicistronic plasmid vector, Flt3L-IRES-GM-CSF plasmid, coexpressing murine Flt3L lacking intracellular domain and GM-CSF by using an internal ribosome entry site (IRES). Expressions of GM-CSF and Flt3L could be detected in COS-7 cells transfected with the plasmid. The immune stimulatory activity of plasmid vector coexpressing Flt3L and GM-CSF (coexpressed DNA vaccine) in comparison with codelivery of Flt3L and GM-CSF plasmids (codelivered DNA vaccine) was studied in mice. We first immunized mice by intradermal injection of various plasmid vectors, including 40 μg Flt3L-IRES-GM-CSF plasmid, 40 μg Flt3L plasmid plus 40 μg GM-CSF plasmid, 40 μg Flt3L plasmid alone, 40 μg GM-CSF plasmid alone, or 40 μg control plasmid. Expressions of Flt3L and GM-CSF were not detected in the sera of mice receiving intradermal injection of the plasmids. Next, we showed the enhanced recruitment of DCs to the immunization site and proliferation of DCs and T cells in the spleen and lymph node. The populations of DC, CD4+, and CD8+ T cells were increased significantly by approximately 50% to 60% in mice receiving coexpressed DNA vaccine compared with those in mice receiving control plasmid. Compared to mice treated with codelivered DNA vaccine, there were 15% and 41% increases in the populations of DC and CD4+ T cells, respectively. The CD4+ but not CD4+CD25+ T cells were found to be the major group in the increased T-cell population in the spleen and lymph node. We will investigate the function of the DCs harvested from mice receiving coexpressed DNA plasmid by mixed lymphocyte reaction and antigen-specific T cell proliferation. In addition, we found that there was an increase, ranging from 20% to 40%, in the populations of MHC class II+, CD4+, and CD8+ T cells in the spleen of mice bearing syngeneic bladder tumor that received coexpressed DNA vaccine compared with those that received codelivered DNA vaccine or control plasmid. Immunization of mice with coexpressed DNA vaccine delayed bladder tumor growth. Taken together, regarding the application of Flt3L and GM-CSF, our results indicate that coexpressed DNA vaccine induces at least equal or higher immune stimulatory response compared with codelivered DNA vaccine, while only half the amount of DNA is needed. Currently, we are using the coexpressed DNA vaccine in combination with plasmid DNA encoding tumor epitopes or tumor-specific oncolytic virus for immune-gene therapy of cancer.

599. Low-dose etoposide enhances tumor-specific adenovirus-mediated cytosine deaminase gene therapy through induction of integrin |[alpha]|v|[beta]|3 and |[alpha]|v|[beta]|5 overexpression and upregulation of human telomerase reverse transcriptase promoter

Purpose: The human telomerase reverse transcriptase (hTERT) promoter is used to restrict adenoviral expression of suicide or proapoptotic genes in telomerase-positive cancer cells. Etoposide can enhance intratumoral transgene expression in mice immunized with adenoviral vectors. The aim of this study is to evaluate whether greater therapeutic benefit can be achieved by combination treatment of low-dose etoposide and replication-incompetent adenoviral vectors encoding cytosine deaminase (CD) driven by the hTERT promoter for murine bladder carcinoma.