Dian-Chun Fang

Ascl2 Knockdown Results in Tumor Growth Arrest by miRNA-302b-Related Inhibition of Colon Cancer Progenitor Cells

Background Achaete scute-like 2 (Ascl2), a basic helix-loop-helix (bHLH) transcription factor, controls the fate of intestinal stem cells. However, the role of Ascl2 in colon cancer progenitor cells remains unknown. The cell line HT-29 (47.5–95% of CD133+ population) and LS174T (0.45% of CD133+ population) were chosen for functional evaluation of Ascl2 in colon cancer progenitor cells after gene knockdown by RNA interference. Methodology/Principal Findings Immunohistochemistry demonstrated that Ascl2 was significantly increased in colorectal adenocarcinomas. Downregulation of Ascl2 using RNA interference in cultured colonic adenocarcinoma HT-29 and LS174T cells reduced cellular proliferation, colony-forming ability, invasion and migration in vitro, and resulted in the growth arrest of tumor xenografts in vivo. The Ascl2 protein level in CD133+ HT-29 cells was significantly higher than in CD133− HT-29 cells. Ascl2 blockade via shRNA interference in HT-29 cells (shRNA-Ascl2/HT-29 cells) resulted in 26.2% of cells staining CD133+ compared with 54.7% in control shRNA-Ctr/HT-29 cells. The levels of ‘stemness’ associated genes, such as CD133, Sox2, Oct4, Lgr5, Bmi1, and C-myc, were significantly decreased in shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells in vitro as well as in the corresponding tumor xenograft (CD133 was not performed in shRNA-Ascl2/LS174T cells). The shRNA-Ascl2/HT-29 cells had inhibited abilities to form tumorspheres compared with control. The microRNA (miRNAs) microarrays, identified 26 up-regulated miRNAs and 58 down-regulated miRNAs in shRNA-Ascl2/HT-29 cells. Expression levels of let-7b, miRNA-124, miRNA-125b, miRNA-17, miRNA-20a and miRNA-302b, involved in the regulation of ‘stemness’, were quantified with qPCR, which confirmed their identities. Restoration of miRNA-302b, via its mimic, led to the restoration of shRNA-Ascl2/HT-29 ‘stemness’ characteristics, including tumorsphere formation and ‘stemness’ associated genes levels, and the recovery of cellular behaviors, including colony-forming ability, invasion and migration in vitro. Conclusions/Significance Ascl2 may be a potential target for the inhibition of colon cancer progenitor cells, and functions through a miR-302b-related mechanism.

Loss of heterozygosity and loss of expression of the DCC gene in gastric cancer.

AIM: To investigate the role of DCC gene inactivation in the development and progression of gastric cancer. METHODS: Loss of heterozygosity and loss of expression of the DCC gene was studied in 51 surgical specimens of gastric cancer using detection based on polymerase chain reaction. RESULTS: Loss of heterozygosity was found in 35.3% (18 of 51) of specimens and was detected more often in stage III and IV (50%) than in stage I and II cancers (14.3%) (p < 0.05). Occurrence of loss of heterozygosity was not correlated with histological type, tumour size, depth of invasion, or lymph node metastasis. Loss of expression was found in 49% of cases (25 of 51). Loss of expression was not significantly correlated with any clinicopathological variable. CONCLUSIONS: Loss of heterozygosity and loss of expression of the DCC gene are often encountered in gastric cancer. Loss of heterozygosity of the DCC gene is a late event and associated with malignant progression.

SOX2 in Gastric Carcinoma, but not Hath1, is Related to Patients’ Clinicopathological Features and Prognosis

BackgroundSOX2 and Hath1 are transcription factors that are critical for the control of terminal cell differentiation in the gastrointestinal mucosa. This study investigated the correlations between SOX2 and Hath1 expression in gastric carcinoma and patients’ clinicopathological features and prognosis.MethodsHath1 and SOX2 were detected by immunohistochemistry in gastric carcinoma (n = 50). Probability of survival of patients after surgery was estimated by the Kaplan–Meier method and compared using Log-rank test.ResultsHath1 and SOX2 were inversely expressed in gastric carcinoma. Patients with strong SOX2 expression (++ to +++) showed lower incidences of lymph node metastasis (p = 0.007), deeper invasion (p = 0.010), and III–IV clinical stages (p = 0.011) compared to patients with low SOX2 expression (− to +). There was no significant difference in SOX2 and Hath1 expression in the cancerous tissues of the patients with and without Helicobacter pylori infection (p > 0.05). The patients with strong expression of SOX2 in their cancerous tissues (++ to +++) had a better prognosis than those with low expression of SOX2 (− to +; p = 0.005). There was no correlation between Hath1 expression level and prognosis (p = 0.676).ConclusionsSOX2 and Hath1 are inversely expressed in gastric carcinoma. SOX2 provides a survival advantage to patients of gastric carcinoma and appears to be associated with metastasis and clinical stages.

Infrequent loss of heterozygosity of APC/MCC and DCC genes in gastric cancer showing DNA microsatellite instability.

AIM: To investigate the role of DNA microsatellite instability (MSI) in gastric carcinogenesis by studying associations between MSI status, clinicopathological features, and loss of genetic loci. METHODS: Six microsatellite loci and loss of heterozygosity at APC, DCC, and MCC were analysed by polymerase chain reaction based methods in 53 cases of advanced gastric cancer. RESULTS: MSI was observed in 32.1% of gastric carcinomas (17/53) and 20% of foci of intestinal metaplasia (3/15). Seven gastric carcinomas (13.7%) were MSI-high (MSI-H) (three loci or more) and 10 (18.9%) were MSI-low (MSI-L) (one or two loci). The frequency of MSI-H was higher in intestinal (25.0%) than in diffuse carcinomas (3.7%) (p < 0.05). None of the MSI-H tumours showed loss of heterozygosity at APC, MCC, or DCC loci. CONCLUSIONS: MSI may have an important and early role in a subset of gastric cancers, particularly the intestinal type. The MSI-H subset of gastric cancer has features in common with its colorectal counterpart, whereas MSI-L and microsatellite stable cancers appear to develop through the loss of heterozygosity pathway.

Alterations of telomerase activity and terminal restriction fragment in gastric cancer and its premalignant lesions

Aims: In order to explore the role of alterations of telomerase activity and terminal restriction fragment (TRF) length in the development and progression of gastric cancer.

Detection of telomerase activity in biopsy samples of colorectal cancer

Background: Telomerase is a ribonucleoprotein that synthesizes telomeric DNA onto chromosomal ends. The expression of telomerase is thought to be required for cellular immortality and oncogenesis.

Programmed Cell Death 4 (PDCD4) Enhances the Sensitivity of Gastric Cancer Cells to TRAIL-Induced Apoptosis by Inhibiting the PI3K/Akt Signaling Pathway

AbstractObjective: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is thought to be a promising anti-neoplastic agent because of its ability to selectively induce apoptosis in cancer cells. However, some cancer cells are resistant to TRAIL. The mechanisms underlying this resistance are unclear. The aim of this study was to explore the role of programmed cell death 4 (PDCD4) in regulating TRAIL sensitivity in gastric cancer cells. Methods: PDCD4 complementary DNA and PDCD4-specific short-hairpin RNA (shRNA) fragments were transfected into TRAIL-sensitive and -resistant gastric cancer cells. Expression of PDCD4 and Akt was detected via western blot. Cell survival and apoptosis were measured using 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry (FCM) assays. Results: We found that upregulation of PDCD4 enhanced TRAIL sensitivity in gastric cancer cells. Downregulation of PDCD4 decreased TRAIL sensitivity. Inhibition of Akt by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 induced PDCD4 activity and enhanced TRAIL sensitivity in TRAIL-resistant gastric cancer cells. Conclusion: We demonstrated that PDCD4 regulates TRAIL sensitivity in gastric cancer cells by inhibiting the PI3K/Akt signaling pathway.

H-2Kb–Restricted CTL Epitopes from Mouse Heparanase Elicit an Antitumor Immune Response In vivo

The identification of CTL epitopes from tumor antigens is very important for the development of peptide-based, cancer-specific immunotherapy. Heparanase is broadly expressed in various advanced tumors and can serve as a universal tumor-associated antigen. Although several epitopes of heparanase antigen are known in humans, the corresponding knowledge in mice is still rather limited. The present study was designed to predict and identify the CTL epitopes in the mouse heparanase protein. For this purpose, H-2K(b)-restricted CTL epitopes were identified by using the following four-step procedure: (a) a computer-based epitope prediction from the amino acid sequence of mouse heparanase, (b) a peptide-binding assay to determine the affinity of the predicted epitopes with the H-2K(b) molecule, (c) the testing of the induction of CTLs toward various carcinoma cells expressing heparanase antigens and H-2K(b), and (d) the induction of immunoprotection and immunotherapy in vivo. The results showed that, of the tested peptides, effectors induced by peptides of mouse heparanase at residue positions 398 to 405 (LSLLFKKL; mHpa398) and 519 to 526 (FSYGFFVI; mHpa519) lysed three kinds of carcinoma cells expressing both heparanase and H-2K(b) (B16 melanoma cells, EL-4 lymphoma cells, and Lewis lung cancer cells). In vivo experiments indicated that mHpa398 and mHpa519 peptides offered the possibility of not only immunizing against tumors but also treating tumor-bearing hosts successfully. Our results suggest that the mHpa398 and mHpa519 peptides are novel H-2K(b)-restricted CTL epitopes capable of inducing heparanase-specific CTLs in vitro and in vivo. These epitopes may serve as valuable tools for the preclinical evaluation of vaccination strategies.

hTERT-based therapy: a universal anticancer approach (Review).

Human telomerase reverse transcriptase (hTERT) has been identified as a major protein involved in aberrant cell proliferation, immortalization, metastasis and stemness maintenance in a majority of tumors, yet it has little or no expression in normal somatic cells. During the past few years, the development of hTERT-based therapies such as immunotherapy, suicide gene therapy and small-molecule interfering therapy have become critical and specific for eradicating all types of cancer. Here, current knowledge regarding hTERT and its involvement in various cancers and its role as a target of cancer therapies are reviewed. Additionally, hurdles to new cancer therapy development and new therapeutic opportunities are described, along with areas that require further investigation.

Effects of Helicobacter pylori infection on mucin expression in gastric carcinoma and pericancerous tissues

Background:  Helicobacter pylori infection is one of the major causes of human gastric carcinoma and can disturb the gastric mucosa barrier. Mucins have not only lubricating and protecting functions, but are also related to signal transduction, turnover of gastric epithelium and carcinogenesis of gastric mucosa. The aim of this study was to investigate the relationship between H. pylori infection and aberrant mucin expression in patients with gastric carcinoma.