Show-Li Chen

Overexpression of SPARC gene in human gastric carcinoma and its clinic–pathologic significance

Gastric cancer is the second most common cancer in the world and the fifth leading cause of cancer-related death in Taiwan. To improve the survival of gastric cancer patients, biomarkers for early detection and effective anticancer therapy are required. An essential first step is to profile gene expression in gastric cancer and identify genes that are aberrantly expressed, and to do this cDNA microarrays were performed. The clinic–pathologic correlation and prognostic significance of the aberrantly expressed genes were evaluated to identify novel biomarkers of gastric cancer. Fresh surgical samples of tumour tissue and matching noncancerous mucosa were obtained immediately after gastric resection in 43 patients. Secreted Protein, Acidic and Rich in Cysteine (SPARC) (Osteonectins), one of the most highly expressed genes in both intestinal and diffuse gastric cancers in our microarray results, was selected for further study. The overexpression of SPARC was verified using real-time quantitative-reverse transcription–polymerase chain reaction (Q-RT–PCR), Northern blot and immunohistochemical staining. The expression of SPARC in tumour tissues was, on average, 4.27-fold increased (95% CI 2.68–5.85) compared to adjacent noncancerous mucosa (P<0.001). The expression of SPARC was higher in advanced (T2, T3 and T4) cancer compared to the early (T1) cancer (P=0.048) with regard to depth of wall invasion. Higher expression of SPARC was significantly associated with lymph node metastasis (P<0.001), lymphatic invasion (P=0.004) and perineural invasion (P=0.047). Expression of SPARC in patients in stage II and above was significantly higher than those in stage I (P=0.017). The 3-year survival of patients with lower expression of SPARC was significantly better than those with a higher expression (log rank P=0.047). These data indicate the potential of SPARC as a prognostic marker for gastric cancer.

Induction of CD8 T Cells by Vaccination with Recombinant Adenovirus Expressing Human Papillomavirus Type 16 E5 Gene Reduces Tumor Growth

ABSTRACT The potential of the E5 protein as a tumor vaccine candidate has not been explored yet. In this study, we evaluate the human papillomavirus type 16 (HPV-16) E5 protein delivered by an adenovirus vector as a tumor vaccine for cervical lesions. The results demonstrate that a single intramuscular injection of a recombinant adenovirus carrying the HPV-16 E5 gene into syngeneic animals can reduce the growth of tumors which contain E5 gene expression. Moreover, the E5 vaccine-induced tumor protection occurs through CD8 T cells but not through CD4 T cells in in vitro assays. In addition, our studies using knockout mice with distinct T-cell deficiencies confirm that cytotoxic T-lymphocyte-induced tumor protection is CD8 dependent but CD4 independent. Hence, HPV-16 E5 can be regarded as a tumor rejection antigen.

Retinal Protection from Acute Glaucoma-Induced Ischemia-Reperfusion Injury through Pharmacologic Induction of Heme Oxygenase-1

PURPOSE To investigate the protective effects of cobalt protoporphyrin (CoPP), a potent heme oxygenase (HO)-1 inducer, in a rat model of ischemia-reperfusion injury and to document the possible antiapoptotic and anti-inflammatory mechanisms underlying the protection. METHODS Rats pretreated with intraperitoneal injection of CoPP (5 mg/kg) were subjected to retinal ischemia by increases in intraocular pressure to 130 mm Hg for 60 minutes. The protective effects of CoPP were evaluated by determining the morphology of the retina, counting the survival of retinal ganglion cells (RGCs), and measuring apoptosis in retinal layers. In addition, expressions of HO-1, caspase-3, p53, Bcl-xL, monocyte chemoattractant protein (MCP)-1, and inducible nitric oxide synthase (iNOS) were documented by Western blot analysis. Detection of HO-1, NF-kappaB, and CD68 protein in the retina was performed by immunohistochemistry or immunofluorescence. RESULTS Pharmacologic induction of HO-1 by CoPP led to HO-1 expression in the full retinal layer. HO-1 overexpression alleviated apoptosis in the retina, preserved RGCs, and attenuated the reduction of inner retinal thickness after ischemia-reperfusion injury. Concurrently, overexpression of HO-1 was associated with inhibition of caspase-3, p53, NF-kappaB, and iNOS and with increased expression of Bcl-xL. Meanwhile, the anti-inflammatory effect of HO-1 was related to reduction in the recruitment of macrophage infiltration in the retina through the suppression of MCP-1. These beneficial effects of HO-1 induced by CoPP were diminished by the HO-1 inhibitor ZnPP. CONCLUSIONS Overexpression of HO-1 by pharmacologic induction protected the retina from subsequent cellular damage caused by ischemia-reperfusion injury through antiapoptotic and anti-inflammatory effects.

HLA-A*0201 T-cell epitopes in severe acute respiratory syndrome (SARS) coronavirus nucleocapsid and spike proteins.

Abstract The immunogenicity of HLA-A∗0201-restricted cytotoxic T lymphocyte (CTL) peptide in severe acute respiratory syndrome coronavirus (SARS-CoV) nuclear capsid (N) and spike (S) proteins was determined by testing the proteins’ ability to elicit a specific cellular immune response after immunization of HLA-A2.1 transgenic mice and in vitro vaccination of HLA-A2.1 positive human peripheral blood mononuclearcytes (PBMCs). First, we screened SARS N and S amino acid sequences for allele-specific motif matching those in human HLA-A2.1 MHC-I molecules. From HLA peptide binding predictions (http://thr.cit.nih.gov/molbio/hla_bind/), ten each potential N- and S-specific HLA-A2.1-binding peptides were synthesized. The high affinity HLA-A2.1 peptides were validated by T2-cell stabilization assays, with immunogenicity assays revealing peptides N223–231, N227–235, and N317–325 to be the first identified HLA-A∗0201-restricted CTL epitopes of SARS-CoV N protein. In addition, previous reports identified three HLA-A∗0201-restricted CTL epitopes of S protein (S978–986, S1203–1211, and S1167–1175), here we found two novel peptides S787–795 and S1042–1050 as S-specific CTL epitopes. Moreover, our identified N317–325 and S1042–1050 CTL epitopes could induce recall responses when IFN-γ stimulation of blood CD8+ T-cells revealed significant difference between normal healthy donors and SARS-recovered patients after those PBMCs were in vitro vaccinated with their cognate antigen. Our results would provide a new insight into the development of therapeutic vaccine in SARS.

15-deoxy-delta (12,14)-prostaglandin j2 induces vascular endothelial cell apoptosis through the sequential activation of mapks and p53

15-Deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) is a potent anti-angiogenic factor and induces endothelial cell apoptosis, although the mechanism remains unclear. In this study, 15d-PGJ2 was found to increase p53 levels of the human umbilical vein endothelial cells by stabilizing p53. Both 15d-PGJ2-induced apoptosis and the induction of p21Waf1 and Bax can be abolished by p53 small interfering RNA but not by peroxisome proliferator-activated receptor γ inhibitors. Moreover, 15d-PGJ2 activated JNK and p38 MAPK while inducing p53 phosphorylation at sites responsible for p53 activity. JNK inhibitor (SP600125) or p38 MAPK inhibitor (SB203580) pretreatment attenuated 15d-PGJ2-mediated apoptosis and suppressed the p21Waf1 and Bax expressions without affecting p53 protein accumulation. Pretreatment with SP600125 partially prevented the phosphorylation of p53 at serines 33 and 392 induced by 15d-PGJ2. 15d-PGJ2 was also found to induce reactive oxygen species generation and partially blocked nuclear factor-κB activity. Pretreatment with antioxidant N-acetylcysteine prevented the p53 accumulation, the phosphorylations of JNK and p38 MAPK, the inhibition of NF-κB activity, as well as the apoptosis induced by 15d-PGJ2. Using a mouse model of corneal neovascularization, it was demonstrated in vivo that 15d-PGJ2 induced reactive oxygen species generation, activated JNK and p38 MAPK, induced p53 accumulation/phosphorylation, and induced vascular endothelial cell apoptosis, which could be abolished by N-acetylcysteine, SP600125, SB203580, or a virus-derived amphipathic peptides-based p53 small interfering RNA. This is the first study that 15d-PGJ2 induces vascular endothelial cell apoptosis through the signaling of JNK and p38 MAPK-mediated p53 activation both in vitro and in vivo, further establishing the potential of 15d-PGJ2 as an anti-angiogenesis agent.

Pigment Epithelium-derived Factor (PEDF) Promotes Tumor Cell Death by Inducing Macrophage Membrane Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)

Pigment epithelium-derived factor (PEDF) is an intrinsic anti-angiogenic factor and a potential anti-tumor agent. The tumoricidal mechanism of PEDF, however, has not been fully elucidated. Here we report that PEDF induces the apoptosis of TC-1 and SK-Hep-1 tumor cells when they are cocultured with bone marrow-derived macrophages (BMDMs). This macrophage-mediated tumor killing is prevented by blockage of TNF-related apoptosis-inducing ligand (TRAIL) following treatment with the soluble TRAIL receptor. PEDF also increases the amount of membrane-bound TRAIL on cultured mouse BMDMs and on macrophages surrounding subcutaneous tumors. PEDF-induced tumor killing and TRAIL induction are abrogated by peroxisome proliferator-activated receptor γ (PPARγ) antagonists or small interfering RNAs targeting PPARγ. PEDF also induces PPARγ in BMDMs. Furthermore, the activity of the TRAIL promoter in human macrophages is increased by PEDF stimulation. Chromatin immunoprecipitation and DNA pull-down assays confirmed that endogenous PPARγ binds to a functional PPAR-response element (PPRE) in the TRAIL promoter, and mutation of this PPRE abolishes the binding of the PPARγ-RXRα heterodimer. Also, PPARγ-dependent transactivation and PPARγ-RXRα binding to this PPRE are prevented by PPARγ antagonists. Our results provide a novel mechanism for the tumoricidal activity of PEDF, which involves tumor cell killing via PPARγ-mediated TRAIL induction in macrophages.

Nuclear Receptor Interaction Protein (NRIP) expression assay using human tissue microarray and immunohistochemistry technology confirming nuclear localization

BackgroundA novel human nuclear receptor interaction protein (NRIP) has recently been discovered by Chen SL et al, which may play a role in enhancing the transcriptional activity of steroid nuclear receptors in prostate (LNCaP) and cervical (C33A) cancer cell lines. However, knowledge about the biological functions and clinical implications of NRIP, is still incomplete. Our aim was to determine the distribution of NRIP expression and to delineate the cell types that express NRIP in various malignant tumors and healthy non-pathological tissues. This information will significantly affect the exploration of its physiological roles in healthy and tumor cells.MethodsBy using tissue microarray (TMA) technology and an anti-NRIP monoclonal antibody immunohistochemical (IHC) survey, NRIP expression was examined in 48 types of tumors and in a control group of 48 matched or unmatched healthy non-neoplastic tissues.ResultsOur survey results showed that ten cases were revealed to express the NRIP in six malignancies (esophageal, colon, breast, ovarian, skin, and pancreatic cancers), but not all of these specific tumor types consistently showed positive NRIP expression. Moreover, malignant tumors of the stomach, prostate, liver, lung, kidney, uterine cervix, urinary bladder, lymph node, testis, and tongue revealed no NRIP expression. Among the control group of 48 matched and unmatched non-neoplastic tissues, all of them demonstrated IHC scores less than the cut-off threshold of 3. In addition, ten cores out of thirty-six carcinomatous tissues revealed positive NRIP expression, which indicated that NRIP expression increases significantly in carcinoma tissue cores, comparing to the matched controlled healthy tissues.ConclusionThis is the first study to use a human TMA and IHC to validate the nuclear localization for this newly identified NRIP expression. In considering the use of NRIP as a potential diagnostic tool for human malignancies survey, it is important to note that NRIP expression carries a sensitivity of only 23%, but has a specificity of 100%. There is also a significant difference in positive NRIP expression between primary carcinomatous tissues and matched controlled healthy tissues. Although further large-scale studies will merit to be conducted to evaluate its role as a potential adjunct for cancer diagnosis, data from this study provides valuable references for the future investigation of the biological functions of NRIP in humans.

Pigment Epithelium-Derived Factor Is an Intrinsic Antifibrosis Factor Targeting Hepatic Stellate Cells

The liver is the major site of pigment epithelium-derived factor (PEDF) synthesis. Recent evidence suggests a protective role of PEDF in liver cirrhosis. In the present study, immunohistochemical analyses revealed lower PEDF levels in liver tissues of patients with cirrhosis and in animals with chemically induced liver fibrosis. Delivery of the PEDF gene into liver cells produced local PEDF synthesis and ameliorated liver fibrosis in animals treated with either carbon tetrachloride or thioacetamide. In addition, suppression of peroxisome proliferator-activated receptor gamma expression, as well as nuclear translocation of nuclear factor-kappa B was found in hepatic stellate cells (HSCs) from fibrotic livers, and both changes were reversed by PEDF gene delivery. In culture-activated HSCs, PEDF, through the induction of peroxisome proliferator-activated receptor gamma, reduced the activity of nuclear factor-kappa B and prevented the nuclear localization of JunD. In conclusion, our observations that PEDF levels are reduced during liver cirrhosis and that PEDF gene delivery ameliorates cirrhosis suggest that PEDF is an intrinsic protector against liver cirrhosis. Direct inactivation of HSCs and the induction of apoptosis of activated HSCs may be two of the mechanisms by which PEDF suppresses liver cirrhosis.

Breast Cancer Amplified Sequence 2, a Novel Negative Regulator of the p53 Tumor Suppressor

Breast cancer amplified sequence 2 (BCAS2) was reported previously as a transcriptional coactivator of estrogen receptor. Here, we report that BCAS2 directly interacts with p53 to reduce p53 transcriptional activity by mildly but consistently decreasing p53 protein in the absence of DNA damage. However, in the presence of DNA damage, BCAS2 prominently reduces p53 protein and provides protection against chemotherapeutic agent such as doxorubicin. Deprivation of BCAS2 induces apoptosis in p53 wild-type cells but causes G(2)-M arrest in p53-null or p53 mutant cells. There are at least two apoptosis mechanisms induced by silencing BCAS2 in wild-type p53-containing cells. Firstly, it increases p53 retention in nucleus that triggers the expression of apoptosis-related genes. Secondly, it increases p53 transcriptional activity by raising p53 phosphorylation at Ser(46) and decreases p53 protein degradation by reducing p53 phosphorylation at Ser(315). We show for the first time that BCAS2, a small nuclear protein (26 kDa), is a novel negative regulator of p53 and hence a potential molecular target for cancer therapy.

PEDF promotes self‐renewal of limbal stem cell and accelerates corneal epithelial wound healing

Limbal epithelial stem cell (LSC) transplantation is a prevalent therapeutic method for patients with LSC deficiency. The maintenance of stem cell characteristics in the process of culture expansion is critical for the success of ocular surface reconstruction. Pigment epithelial‐derived factor (PEDF) increased the numbers of holoclone in LSC monolayer culture and preserved the stemness of LSC in suspension culture by evidence of ΔNp63α, Bmi‐1, and ABCG2 expression. BrdU pulse‐labeling assay also demonstrated that PEDF stimulated LSCs proliferation. In air‐lift culture of limbal equivalent, PEDF was capable of increasing the numbers of ΔNp63α‐positive cells. The mitogenic effect of PEDF was found to be mediated by the phosphorylations of p38 MAPK and STAT3 in LSCs. Synthetic 44‐mer PEDF (residues 78–121) was as effective as the full length PEDF in LSC expansion in suspension culture and limbal equivalent formation, as well as the activation of p38 MAPK and STAT3. In mice subjecting to mechanical removal of cornea epithelium, 44‐mer PEDF facilitated corneal wound healing. Microscopically, 44‐mer PEDF advanced the early proliferative response in limbus, increased the proliferation of ΔNp63α‐positive cells both in limbus and in epithelial healing front, and assisted the repopulation of limbus in the late phase of wound healing. In conclusion, the capability of expanding LSC in cell culture and in animal indicates the potential of PEDF and its fragment (e.g., 44‐mer PEDF) in ameliorating limbal stem cell deficiency; and their uses as therapeutics for treating corneal wound. STEM Cells 2013;31:1775–1784