Chengchao Shou

Expression of hiwi gene in human gastric cancer was associated with proliferation of cancer cells

Stem cell genetics research may be critical to our understanding of carcinogenesis, as both stem cells and cancer cells possess the ability to self‐renew. Recent discoveries have indicated that the piwi family of genes plays an essential role in stem cell self‐renewal in diverse organisms. The hiwi gene, the human homolog of the piwi family, participates in germ cell proliferation and its overexpression may cause the development of germ cell malignancy, but its expression and function in epithelial solid cancers have not been explored. In the present study, we investigated whether there was an association between hiwi expression and human gastric cancer and its potential mechanism. RT‐PCR findings demonstrated that hiwi was expressed in different gastric cancer cell lines. To identify the HIWI protein in gastric cancer, we developed a specific monoclonal antibody against HIWI and immunohistochemistry was performed on various gastric tissues. We found that the expression ratio of hiwi in normal gastric tissues, atrophic gastritis, intestinal metaplasia and gastric cancers was 10% (5/50), 36% (18/50), 36% (18/50) and 76% (38/50), respectively, which was consistent with precancerous development. Notably, the expression pattern of hiwi in gastric cancer tissues was similar to that of Ki67, which was used as a marker of proliferation. Moreover, the suppression of hiwi by antisense or RNAi inhibited the growth of gastric cancer cells and induced cell cycle arrest in G2/M phase. These results suggest that hiwi may be involved in the development of gastric cancer and is a potential target for cancer therapy.

The association of the expression level of protein tyrosine phosphatase PRL-3 protein with liver metastasis and prognosis of patients with colorectal cancer

Purpose To investigate PRL-3 protein expression in normal colorectal epithelia and colorectal cancers with monoclonal antibody (MAb) against PRL-3.Methods MAb against PRL-3 was prepared with the hybridoma technique, and its specificity was confirmed with ELISA and Western blotting assays. The expression of PRL-3 protein in normal colorectal epithelia and colorectal cancers was examined by immunohistochemistry assay. Logistic regression and survival analysis were performed to determine the clinical significance of PRL-3 expression.Results MAb 3B6 against PRL-3 was obtained and showed high specificity. PRL-3 protein was expressed in two of 28 (7.1%) normal colorectal epithelia, 21 of 88 (23.9%) primary colorectal cancers, 22 of 41 (53.7%) metastatic lymph nodes and eight of 12 (66.7%) liver metastases, respectively. The PRL-3 expression rates of metastases were significantly higher than those of primary colorectal cancers and normal colorectal epithelia (P <0.05). PRL-3 expression was significantly associated with the liver metastasis of colorectal cancer (P = 0.004) and tended to shorten survival time (P = 0.0145).Conclusions This is the first study demonstrating that PRL-3 is a potential marker for liver metastasis of colorectal cancer and negatively influences the prognosis of colorectal cancer patients.

PRL-3 promotes the motility, invasion, and metastasis of LoVo colon cancer cells through PRL-3-integrin β1-ERK1/2 and-MMP2 signaling

BackgroundPhosphatase of regenerating liver-3 (PRL-3) plays a causative role in tumor metastasis, but the underlying mechanisms are not well understood. In our previous study, we observed that PRL-3 could decrease tyrosine phosphorylation of integrin β1 and enhance activation of ERK1/2 in HEK293 cells. Herein we aim to explore the association of PRL-3 with integrin β1 signaling and its functional implications in motility, invasion, and metastasis of colon cancer cell LoVo.MethodsTranswell chamber assay and nude mouse model were used to study motility and invasion, and metastsis of LoVo colon cancer cells, respectively. Knockdown of integrin β1 by siRNA or lentivirus were detected with Western blot and RT-PCR. The effect of PRL-3 on integrin β1, ERK1/2, and MMPs that mediate motility, invasion, and metastasis were measured by Western blot, immunofluorencence, co-immunoprecipitation and zymographic assays.ResultsWe demonstrated that PRL-3 associated with integrin β1 and its expression was positively correlated with ERK1/2 phosphorylation in colon cancer tissues. Depletion of integrin β1 with siRNA, not only abrogated the activation of ERK1/2 stimulated by PRL-3, but also abolished PRL-3-induced motility and invasion of LoVo cells in vitro. Similarly, inhibition of ERK1/2 phosphorylation with U0126 or MMP activity with GM6001 also impaired PRL-3-induced invasion. In addition, PRL-3 promoted gelatinolytic activity of MMP2, and this stimulation correlated with decreased TIMP2 expression. Moreover, PRL-3-stimulated lung metastasis of LoVo cells in a nude mouse model was inhibited when integrin β1 expression was interfered with shRNA.ConclusionOur results suggest that PRL-3s roles in motility, invasion, and metastasis in colon cancer are critically controlled by the integrin β1-ERK1/2-MMP2 signaling.

p37 from Mycoplasma hyorhinis promotes cancer cell invasiveness and metastasis through activation of MMP-2 and followed by phosphorylation of EGFR

High Mycoplasma infection in gastric cancer tissues suggests a possible association between Mycoplasma infection and tumorigenesis. By using human gastric cancer cells AGS and mouse melanoma cells B16F10 stably expressing p37, the major immunogen of Mycoplasma hyorhinis, we found that p37 enhanced cell motility, migration, and invasion in vitro. With experimental metastasis model in C57BL/6 mice, p37 adenovirus-infected B16F10 cells formed more metastasis lesions in the lung. Furthermore, p37 promoted the phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase and the activity of matrix metalloproteinase-2 (MMP-2). Inhibitor of MMPs significantly blocked p37-induced EGFR but has little effect on extracellular signal-regulated kinase phosphorylation, whereas the p37-induced MMP-2 activation was only partially suppressed by inhibitor of MEK1/2 or by inhibitor of EGFR. However, all these inhibitors significantly reduced the p37-induced invasiveness of AGS cells. These results suggest that p37 may stimulate invasion by increasing the activity of MMP-2, thereby inducing EGFR phosphorylation and contributing to tumor metastasis on M. hyorhinis infection. p37 and its regulated molecules could be the potential targets for cancer therapy. [Mol Cancer Ther 2008;7(3):530–7]

Diagnosis of gastric cancer using decision tree classification of mass spectral data

Although gastric cancer is the second leading cause of cancer death worldwide, specific and sensitive biomarkers that can be used for its diagnosis are still unavailable. Attempting to improve on current approaches to the serological diagnosis of gastric cancer, we subjected serum samples from 245 individuals (including 127 gastric cancer patients, 100 age‐ and sex‐matched healthy individuals, nine benign gastric lesion patients and nine colorectal cancer patients) for analysis by surface‐enhanced laser desorption/ionization (SELDI) mass spectrometry. Peaks were detected with Ciphergen SELDI software version 3.1.1 and analyzed with Biomarker Patterns’ software 5.0. We developed a classifier for separating the gastric cancer groups from the healthy groups. Three protein masses with 1468, 3935 and 7560 m/z were selected as a potential ‘fingerprint’ for the detection of gastric cancer. It was able to distinguish the gastric cancer patients from the health volunteers with a sensitivity of 95.6% and a specificity of 92.0% in the training set. In the blinding set, it was capable of differentiating the gastric cancer samples from the others with a specificity of 88.0%, a sensitivity of 85.3%, and an accuracy of 86.4%. These values were all higher than those achieved in a parallel analysis by measuring serum carcinoembryonic antigen (CEA) and carbohydrate antigen (CA)19‐9 together. Therefore, the decision tree analysis of serum proteomic patterns has the potential to be used in gastric cancer diagnosis. (Cancer Sci 2007; 98: 37–43)

Suppression of tumor growth and metastasis by a VEGFR-1 antagonizing peptide identified from a phage display library

Although the VEGF‐Flk‐1‐pathway has been known as the major driving force of angiogenesis, new evidence has shown that VEGFR‐1/Flt‐1 plays important roles during the neovascularization under pathological conditions including tumor, atherosclerosis and arthritis. In search of Flt‐1 receptor antagonizing peptides, we screened a phage display 12‐mer‐peptide library with recombinant Flt‐1 protein. Seven candidate peptides were identified that specifically bound to VEGF receptor Flt‐1, of which peptide F56 (WHSDMEWWYLLG) almost abolished VEGF binding to receptor Flt‐1 in vitro. In vivo, F56 fused with DHFR (DHFR‐F56) inhibited angiogenesis in a CAM assay. Moreover, DHFR‐F56 significantly inhibited the growth of nodules of human gastric cancer cell line MGC‐803 in BALB/c nude mice. Histological analyses showed that necrosis of the implanted tumor was markedly enhanced following treatment with DHFR‐F56. In the severe combined immunodeficiency disease (SCID) mouse model for studying metastasis of the human breast cancer cell line BICR‐H1, synthetic peptide F56 significantly inhibited tumor growth and lung metastases. Taken together, our results have demonstrated that peptide F56, as a Flt‐1 receptor antagonist, fulfilled the antiangiogenic and antimetastatic effects by specifically interfering with the interaction between VEGF and receptor Flt‐1. Thus, short peptide F56 may have clinical potential in tumor therapy.

Neuronal protein synuclein γ predicts poor clinical outcome in breast cancer

Synuclein γ (SNCG), previously identified as a breast cancer‐specific gene (BCSG1), is highly expressed in breast carcinomas but not in normal epithelium. SNCG regulates many pathways in growth and progression of breast cancer. To determine if SNCG is a biomarker for clinical prognosis of breast cancer, we generated a panel of murine monoclonal antibodies (mAbs) against human SNCG and correlated SNCG protein expression in 358 clinical breast cancer specimens with clinical outcome. A panel of 14 mAbs was characterized by ELISA, immunoprecipitation (IP), Western blot, immunocytochemistry and immunohistochemistry. SNCG protein expression was determined in 438 clinical breast specimens by immunohistochemical analysis using mAb 5C5. Expression of SNCG was strongly correlated with the stage, lymph node involvement, metastasis, tumor size and Her‐2 status, but its expression was not associated with ER and PR expression status. While 71.4% of advanced breast cancers were positive for SNCG expression, only 26.8% of Stage I/II breast cancers were positive for SNCG expression and 5.2% of benign hyperplasia expressed SNCG. SNCG protein was not detectable in normal tissue adjacent to breast cancer. After a median follow‐up of 64 months, patients with an SNCG‐positive tumor had a significantly shorter disease‐free survival and overall survival and a high probability of death compared no expression of SNCG. Multivariate analysis demonstrated that SNCG was a strong independent prognostic variable. SNCG is a new unfavorable prognostic marker for breast cancer progression and a potential target for breast cancer treatment.

Chemical Constituents from the Rhizomes of Smilax glabra and Their Antimicrobial Activity

Six new phenolic compounds, named smiglabrone A (1), smiglabrone B (2), smilachromanone (3), smiglastilbene (4), smiglactone (5), smiglabrol (6), together with fifty-seven known ones 7–63were isolated from the rhizomes of Smilax glabra. Their structures were elucidated on the basis of extensive spectroscopic analyses, as well as by comparison with literature data. Twenty-seven of these compounds were obtained from and identified in the genus Smilax for the first time. The absolute configuration of (2S)-1,2-O-di-trans-p-coumaroylglycerol (43) was determined for the first time using the exciton-coupled circular dichroism (ECCD) method. Thirty isolated compounds were evaluated for their antimicrobial activity against three Gram-negative bacteria, three Gram-positive bacteria and one fungus, and the corresponding structure-activity relationships were also discussed. Eighteen compounds were found to be antimicrobial against the microorganisms tested and the minimum inhibitory concentrations (MIC) were in the range of 0.0794–3.09 mM. Among them, compound 1 showed antimicrobial activity against Canidia albicans with MIC value of 0.146 mM, which was stronger than cinchonain Ia with an MIC of 0.332 mM. Compounds 3 and 4 exhibited inhibitory activity against Staphylococcus aureus with MIC values of 0.303 and 0.205 mM, respectively. The results indicated that these antimicrobial constituents of this crude drug might be responsible for its clinical antimicrobial effect.

Generation of novel monoclonal antibodies and their application for detecting ARD1 expression in colorectal cancer

Arrest defective 1 (ARD1) is an acetyltransferase involved in cell cycle control in yeast. ARD1 interacts with human N-acetyltransferase (NATH) to form a functional N-terminal acetyltransferase complex. Recently it had been linked with proliferation and apoptosis in mammalian cells, but its function in cancer development remains unclear. To evaluate significance of ARD1 expression in human colorectal cancer, we generated a panel of monoclonal antibodies (mAbs) with high specificity and sensitivity against ARD1. All of the 10 different clones could be used in ELISA and Western blot, and clone 10C12, 13G2, and 4D10 can interact with ARD1 in eukaryotic cells by immunoprecipitation (IP). Clones of 14D4 and 10C12 were strongly reacted to ARD1 in immunocytochemistry (ICH) and immunohistochemistry (IHC). ARD1 expression was evaluated in human colorectal cancer and colitis tissues by immunohistochemical analysis with mAb 14D4. Forty-one were ARD1-positive in 50 colorectal cancer tissues and only 12 were weak positive in the 50 matched normal tissues (P < 0.001). Moreover, ARD1 expression was not detectable in 20 cases of colitis tissue (P < 0.001). Furthermore, all of the six human colorectal cancer cell lines we examined were also ARD1-positive at mRNA and protein levels. Taken together, the novel mAbs against ARD1 we generated could be good tools for both basic and clinical studies, and ARD1 could be a potential biomarker in colorectal cancer.

Prognostic significance of phosphatase of regenerating liver-3 expression in ovarian cancer.

Phosphatase of regenerating liver-3 (PRL-3) is overexpressed in several human cancers and associated with tumor progression, invasion and metastasis. However, the correlation between PRL-3 expression and clinical outcome in ovarian cancer has not been studied. In the present study, we investigated the expression of PRL-3 in 119 ovarian cancers and 30 normal ovarian tissues by immunohistochemistry with an anti-PRL-3 mouse monoclonal antibody 3B6, and analyzed its relationship with clinicopathologic factors and survival. The results demonstrated that PRL-3 expression was significantly higher in ovarian cancers compared to normal ovarian tissues (P < 0.001). PRL-3 expression is not correlated with patient age, menstruation, tumor size, histological type, residual tumor, or other clinical findings. The patients with PRL-3-positive tumors had a significant poor prognosis than those with PRL-3-negative tumors. Univariate analysis identified PRL-3 expression as a poor outcome predictor (HR 1.925, 95% CI, 1.046–3.544, P = 0.035). Multivariate analysis indicated that PRL-3 expression was an independent prognostic factor of borderline significance (HR 1.695, 95% CI, 0.914–3.145, P = 0.094). Our results suggest that PRL-3 may serve as a valuable marker for diagnosis of ovarian cancer and as a potential independent prognostic factor for ovarian cancer.