[Show abstract][Hide abstract] ABSTRACT: Background and objective: ST13, is the gene encoding the HSP70 interacting protein (HIP). Previous research has shown that ST13 mRNA and protein levels are down-regulated in colorectal cancer (CRC) tissues compared with adjacent normal tissues. This study aims at the role of ST13 in the proliferation and migration of CRC cells. Methods: The transcript level of ST13 in different CRC cell lines was evaluated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). ST13-overexpressed and ST13-knockdown CRC cells were constructed respectively by lentiviral transduction, followed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, plate colony formation, cell-cycle analysis, and migration assays to evaluate the influence of ST13 on proliferation and migration in vitro. Moreover, a mouse xenograft study was performed to test in vivo tumorigenicity of ST13-knockdown CRC cells. Results: Lentivirus-mediated overexpression of ST13 in CRC cells inhibited cell proliferation, colony formation, and cell migration in vitro. In contrast, down-regulation of ST13 by lentiviral-based short hairpin RNA (shRNA) interference in CRC cells significantly increased cell proliferation and cloning efficiency in vitro. In addition, down-regulation of ST13 expression significantly increased the tumorigenicity of CRC cells in vivo. Conclusions: ST13 gene is a proliferation regulator that inhibits tumor growth in CRC and may affect cell migration.
Journal of Zhejiang University SCIENCE B 11/2012; 13(11):884-93. DOI:10.1631/jzus.B1200037 · 1.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cancer-associated fibroblasts (CAFs) are one of the hallmarks of the cancer microenvironment. Recent evidence has indicated that CAFs are more competent in enhancing cancer cell growth and migration than normal fibroblasts. However, the unique protein expression of CAFs has not been fully elucidated. This study aims to investigate the characterizations of colon CAFs by comparing the differential protein expression between CAFs and normal fibroblasts.
Primary fibroblasts were isolated from surgical specimen of human colon cancer and matched normal colonic tissue. Purity of the cell population was verified through immunostain analysis. Total cell lysates and conditioned media from each group of cells were extracted, and protein expression analysis was conducted using the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) ProteinChip platform.
Most primary cells showed typical fibroblast-like features after two weeks. Increased proportion of α-smooth muscle actin-positive myofibroblasts was detected within the CAFs in four of the six pairs of primary cells. Fibroblast activation protein was weakly expressed in most cells without differences. Using SELDI-TOF-MS ProteinChip platform, four protein peaks mass over charge ratio (m/z) 1142, 3011, 4035, and 4945 were detected in the total cell lysates, and two protein peaks m/z 1368 and 1389 were detected in the conditioned media. The potential candidate proteins found in the Swiss-Prot database include morphogenetic neuropeptides, FMRFamide-related peptides, insulin-like growth factor II, thymosin β-4-like protein 3, and tight junction-associated protein 1.
Using the SELDI-ProteinChip platform, differential protein expressions were identified in colon CAFs compared with normal colonic stromal fibroblasts. The complex proteomic alternations in colon CAFs may play important roles related to the colon cancer microenvironment.
Journal of Zhejiang University SCIENCE B 03/2012; 13(3):159-67. DOI:10.1631/jzus.B1100266 · 1.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We aimed to perform a preliminary study of the association between induced pluripotent stem cell (iPS)-related genes and biological behavior of human colorectal cancer (CRC) cells, and the potential for developing anti-cancer drugs targeting these genes.
We used real-time reverse transcriptase polymerase chain reaction (RT-PCR) to evaluate the transcript levels of iPS-related genes NANOG, OCT4, SOX2, C-MYC and KLF4 in CRC cell lines and cancer stem cells (CSCs)-enriched tumor spheres. NANOG was knockdowned in CRC cell line SW620 by lentiviral transduction. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, plate colony formation, and a mouse xenograft model were used to evaluate alterations in biological behavior in NANOG-knockdown SW620 cells. Also, mock-knockdown and NANOG-knockdown cells were treated with 5-fluorouracil (5-FU) and survival rate was measured by MTT assay to evaluate drug sensitivity.
A significant difference in the transcript levels of iPS-related genes between tumor spheres and their parental bulky cells was observed. NANOG knockdown suppressed proliferation, colony formation, and in vivo tumorigenicity but increased the sensitivity to 5-FU of SW620 cells. 5-FU treatment greatly inhibited the expression of the major stemness-associated genes NANOG, OCT4, and SOX2.
These results collectively suggest an overlap between iPS-related genes and CSCs in CRC. Quenching a certain gene NANOG may truncate the aggressiveness of CRC cells.
Journal of Zhejiang University SCIENCE B 01/2012; 13(1):11-9. DOI:10.1631/jzus.B1100154 · 1.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To isolate an isogenic radioresistant cancer cell line after fractioned X-ray radiation and characterize the resistant cells.
D6 cells were exposed to repeated X-ray irradiation, and after a total dose of 5200 cGy in 8 fractions, a radioresistant monoclone D6-R was obtained. The radiosensitivity and drug sensitivity of the novel radioresistant D6-R cells, together with their parent D6 cells, were measured using clonogenic assay and MTT assay respectively. Cell cycle distribution was analyzed by flow cytometry. Fluorescence microscopy and flow cytometry were applied for apoptosis detection. Comet assay was used for the detection of DNA damage and repair.
D6-R cells showed higher and broader initial shoulder (D0=2.08 Gy, Dq=1.64 Gy, N=2.20) than the parent D6 cells (D0=1.84 Gy, Dq=0.34 Gy, N=1.20). They were 1.65-fold more radioresistant than D6 cells in terms of SF2 (63% vs 38%) and were more resistant to ADM (3.15-fold) and 5-FU (3.86-fold) as compared with the latter. It was found that D6-R cells had higher fractions of cells in S phase (53.4% vs 37.8%) and lower fractions of cells in G1 (44.1% vs 57.2%) and G2-M phase (2.5% vs 5%). There was no difference in radiation-induced apoptosis between D6-R and D6 cells. D6-R cells showed less initial DNA damage and increased capacity in DNA repair after irradiation, as compared with the parent cells.
D6-R cells have been isolated by exposing the parental D6 cells to repeated irradiation. The difference in cell cycle pattern together with the induction and repair of DNA damage might, at least partially, explain the mechanism of the radioresistance.
[Show abstract][Hide abstract] ABSTRACT: To identify the protein expression differences related to the CagA-induced ERK pathway activation in AGS cells.
Human AGS cells transfected with cagA and blank vector were treated with specific mitogen-activated protein kinase kinase (MEK) inhibitor. Total cell proteins were combined by strong anion exchange (SAX2) and weak cation exchange (CM10) ProteinChip arrays and analyzed using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) proteomics technology. Protein expression profiles were compared with those of inhibitor-untreated cagA transfectants. SwissProt/TrEMBL database searching for differentially expressed proteins was carried out using the TagIdent tool with the pI and mass information.
When a total of 16 proteins that showed expression differences in inhibitor-untreated cagA transfectants were compared with vector transfectants, three proteins with m/z 4229, 8162 and 9084 were found to have no expression differences after treatment with MEK inhibitor, while the other 13 maintained the same expression differences after inhibitor treatment. Seven pieces of meaningful matching information for the three proteins were obtained from database searching.
Biomarkers with m/z 4229, 8162 and 9084 are ERK1/2 phosphorylation dependent, and therefore are the downstream molecules of ERK1/2 in the ERK/MAPK signaling pathway. The three biomarkers may be important cancer-associated proteins according to SwissProt/TrEMBL database information.
World Journal of Gastroenterology 02/2008; 14(4):554-62. DOI:10.3748/wjg.14.554 · 2.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To characterize the CagA variable region of Helicobacter pylori isolates from Chinese patients.
DNA fragments in CagA variable region were amplified and sequenced respectively from genomic DNA of 19 isolates from patients with gastric cancer and 20 isolates from patients with chronic gastritis. The tendency of phosphorylation in tyrosine(s) of CagA proteins was evaluated subsequently by phosphorylation assay in vivo and in vitro respectively.
About 97.44% (38/39) H pylori isolates possessed CagA gene. CagA(+) strains contained 2-4 tandem five-amino-acid motifs EPIYA but only one EPIYA had repeated sequence in CagA variable region in different isolates. There was no significant difference between the number of EPIYA motifs in H pylori from patients with different diseases. However, only tyrosine site in EPIYA within repeated sequence could be phosphorylated by AGS cells in vivo although all tyrosine sites in EPIYA could be phosphorylated in vitro.
CagA in Chinese has no functional difference in perturbing cellular signal pathway among different H pylori isolates.
World Journal of Gastroenterology 03/2005; 11(6):880-4. DOI:10.3748/wjg.v11.i6.880 · 2.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To establish human colorectal crypt cell line.
Colorectal crypt cells were separated from human fetal gut by dispase I digestion, AKP-negative cells from fetal colorectal crypt were collected and cultured on Matrigel matrix. Subsequently the primary cultured cells were transfected with recombinant retrovirus containing human telomerase reverse transcriptase (hTERT) and simian virus 40 large T antigen (SV40 LT) in 48 h. The characterization of immortalized cells was identified after the transfection and cells were screened with antibiotics for 12 approximately 16 weeks and expanded.
Mucin, cytokeratin-pan, 8, 19 were presented in immortalized cells by immunohistochemical staining; ectopic expressions of both hTERT and SV40 LT were also found in immortalized cells by Western blotting. Agarose electrophoresis showed that the cells expressed Musashi-1 mRNA. No evidence of carcinogenesis was found in nude mouse experiment and soft-agarose cloning test.
The immortalized human colorectal crypt cells were characterized and the established cell line may be an ideal target for carcinogenesis study in vitro.
Zhejiang da xue xue bao. Yi xue ban = Journal of Zhejiang University. Medical sciences 10/2004; 33(5):379-84.
[Show abstract][Hide abstract] ABSTRACT: China is one of the countries with the highest incidence of H. pylori and more than 9090 isolates possessed the cagA gene. This study was to evaluate the biological activity of the H. pylori virulence factor cagA isolated from Chinese patients.
cagA DNA fragments were amplified from the genomic DNA and subsequently cloned into the mammalian expression vector for cell transfection and DNA sequencing. cagA protein, phosphorylated-tyrosine cagA and the complex of cagA precipitated with SHP-2 were identified respectively by western blot in the crude cell lysate from conditionally immortalized gastric epithelial cells at 48 hours after transfection with cagA DNA. In addition, the ability of induction of scattering phenotype was examined after transient expression of cagA in AGS cells.
The C-terminal half of cagA contained only one repeated sequence and three tandem five-amino-acid motifs glutamic acid-proline-isoleucine-tyrosine-alanine (EPIYA). Moreover, the amino acid sequence of D2 region in repeated sequence was aspartic acid-phenylanaline-aspartic acid (D-F-D) which was significantly distinguished from the three repeated sequences and aspartic acid-aspartic adid-leucine (D-D-L) in the western standard strain NCTC11637. Western blot revealed that cagA became phosphorylated in tyrosine site and bound with SHP-2 after transient expression of cagA DNA in gastric epithelial cells. Transient expression of cagA in AGS cells showed that cagA was able to induce the elongation phenotype although to a lesser extent than western strains.
cagA perturbs cell signaling pathways by binding with SHP-2. However, significant difference exists in amino acid sequence and biological function of cagA in Chinese compared with those of western countries.
Chinese medical journal 10/2004; 117(9):1330-3. · 1.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the distribution of H.pylori antigens in the gastric mucosa in patients with H.pylori infection, and the relationship between the distribution and gastric cancer.
Of 112 patients confirmed by pathological study to have chronic superficial gastritis, precancerous changes (chronic atrophic gastritis, intestinal metaplasia or atypical hyperplasia) and gastric cancer, 28 were H.pylori negative and 84 were H.pylori positive. H.pylori antigens in the gastric mucosa were detected by immunohistochemistry.
The H.pylori positive group, comprised 12 of 22 (50.0%) in the chronic superficial gastritis group, 22 of 25 (88.0%) in the precancerous changes group and 13 of 35 (37.1%) in the gastric cancer group. The positive rates of H.pylori antigens in the cytoplasm progressively increased, respectively at 0.0% (0/12), 63.6% (14/22) and 84.6% (11/13) for the same groups (chi(2)=19.76, P=0.000); H.pylori antigens were located in the mucus layer and above the neck of the mucosal gland in 9 of 12 (75.0%) cases with chronic superficial gastritis, at the neck of the mucosal gland and the isthmus in 12 of 22 (54.5%) cases with precancerous changes, below the isthmus in 9 of 13 (69.2%) cases with gastric cancer (chi(2)=25.30, P=0.000). In the H.pylori negative group, no H.pylori antigen was observed.
With the progression of chronic superficial gastritis-->precancerous changes-->gastric cancer, H.pylori antigens progressively migrated from the outer part to the inner part of the cell, and from the superficial to the deep gastric mucosa.
Journal of Zhejiang University SCIENCE 03/2004; 5(2):242-5. DOI:10.1631/jzus.2004.0242
[Show abstract][Hide abstract] ABSTRACT: To investigate the presence of H.pylori DNA within gastric epithelial cells in patients with H.pylori infection and its possible carcinogenic mechanism.
Total 112 patients, with pathologically confirmed chronic superficial gastritis, chronic atrophic gastritis, intestinal metaplasia, atypical hyperplasia or gastric cancer were studied. Among them, 28 were H.pylori negative and 84 H.pylori positive. H.pylori DNA in gastric epithelial cells was detected by GenPoint catalyzed signal amplification system for in situ hybridization.
In the H.pylori positive group, zero out of 24 chronic superficial gastritis (0.0%), four out of 25 precancerous changes (16.0%) and thirteen out of 35 gastric cancers (37.1%) showed H.pylori DNA in the nucleus of gastric epithelial cells, the positive rates of H.pylori DNA in the nucleus of gastric epithelial cells were progressively increased in chronic superficial gastritis, precancerous changes and gastric cancer groups (chi(2)=12.56, P=0.002); One out of 24 chronic superficial gastritis (4.2%), eleven out of 25 precancerous changes (44.0%) and thirteen out of 35 gastric cancers (37.1%) showed H.pylori DNA in the cytoplasm of gastric epithelial cells (chi(2)=10.86, P=0.004). In the H.pylori negative group, only one patient with gastric cancer was found H.pylori DNA in the nucleus of gastric epithelial cells; Only two patients, one patient with precancerous changes and another with gastric cancer, showed H.pylori DNA in the cytoplasm of gastric epithelial cells. Furthermore, H.pylori DNA must have been in the cytoplasm as long as it existed in the nucleus of gastric epithelial cells.
H.pylori DNA exists both in the nucleus and the cytoplasm of gastric epithelial cells in patients with H.pylori infections. The pathological progression from chronic superficial gastritis, precancerous changes to gastric cancer is associated with higher positive rates of H.pylori DNA presence in the nucleus of gastric epithelial cells.
World Journal of Gastroenterology 05/2002; 8(2):305-7. · 2.43 Impact Factor