[Show abstract][Hide abstract] ABSTRACT: Cancer of the esophagus and gastroesophageal junction remains a virulent malignancy with poor prognosis. Rapid progresses were made in chemotherapeutic agents and the development of molecular markers allowed better identification of candidates for targeted therapy. This study aimed to identify the candidate peptides used for anti-angiogenic therapy of esophageal cancer by in vivo screening C7C peptide library for peptides binding specifically to blood vessels of human esophageal cancer.
The phage displayed C7C peptide library was injected intravenously into mice bearing human esophageal tumor xenografts under renal capsule. After 5 rounds of screening, 13 clones were picked up individually and sequenced. During each round of screening, titers of phage recovery were calculated from tumor xenograft and control tissues. Homing of these 9 peptides to tumor vessel was detected by calculating phage titers in the tumor xenograft and control tissues (lung and spleen) after each phage was injected into mice model, and compared with the distribution of phage M13 and VIII-related antigen in tumor xenograft by immunohistochemical staining. Comparisons among groups of data were made using one-way analysis of variance (ANOVA), followed by the Bonferroni multiple comparisons test.
The number of phage recovered from tumor tissue of each round increased gradually in tumor group while decreased in control groups (P < 0.01 in tumor and spleen, P < 0.05 in lung). Immunohistochemical staining showed similar staining pattern with M13 antibody or VIII-related antigen antibody, suggesting that phages displaying the selected peptides could home to blood vessel of human esophageal cancer. According to their DNA, 9 corresponding peptide sequences were deduced. And the homing ability to blood vessel of phages displaying the selected peptides was confirmed by comparing with their recovery in tumor and control tissues. Two motifs, YSXNXW and PXNXXN, were also obtained by analyzing the homology of these peptide sequences. The staining distribution of phage with the sequence of PNPNNST was similar to that of the blood vessel marker factor VIII-related antigen staining. After sequencing, each phage with the selected peptide of PNPNNST with 1.0 × 10(11) pfu/ml was injected intravenously into mice. The homing ability to tumor vessel of these 9 kinds of peptides in the xenograft was higher than control tissues (lung and spleen).
Nine peptides obtained from in vivo screening homed to the blood vessel of human esophageal cancer, and the two motifs of YSXNXW and PXNXXN are the possible biochemical recognition units binding to vascular endothelial cells of esophageal cancer.
Chinese medical journal 02/2011; 124(4):581-5. · 1.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To measure the neutralization activity in vitro of the antibodies induced by recombinant TGFbeta1 vaccine and to evaluate the vaccine's anti-liver fibrosis activity.
Balb/c mice were immunized with a fusion protein of the human TGFbeta1 epitope-inserted into a hepatitis B core antigen using a prokaryotic expression system. The antibody produced by the recombinant vaccine was determined using ELISA. The biological activity of the anti-TGFbeta1 antibody induced by the vaccine was measured by MTT using mink lung epithelial cell Mv-1-Lu as inhibiting cells. The fusion protein was used as a vaccine in a mice hepatic-fibrosis model.
A high titer of anti-TGFbeta1 antibody and a low of anti-HBc antibody were detected in the mice after the immunization. The serum antibodies induced combined with the fusion and antigenic peptide prevented the TGFbeta1 inhibiting activity in the Mv-1-Lu cell.
Recombinant fusion protein can be used as a cytokine vaccine to induce high titers of anti-TGFbeta1 antibodies. Our results show the potentiality of the fusion protein to be used as a vaccine for preventing liver fibrosis.
Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology 04/2006; 14(3):183-6.
[Show abstract][Hide abstract] ABSTRACT: To insert the constructed TGF-beta(1) epitope gene into the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-beta(1) antigenicity in its prokaryotic expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti-TGF-beta(1) vaccine.
The TGF-beta(1) encoding epitope gene (the mature TGF-beta(1) from 78-109 amino acid residues, TGF-beta(1)(32)) was amplified by polymerase chain reaction from the recombinant pGEM-7z/ TGF-beta(1)(32) vector. The HBcAg gene fragments (encoding HBcAg from 1-71 and 89-144 amino acid residues) were amplified from PYTA1-HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-beta(1)(32) and HBcAg89-144 into restrictive endonuclease enzyme and ligated with T(4) ligase. The fusion gene fragments HBcAg1-71-TGF-beta(1)(32)- HBcAg89-144 were recloned to pET28a(+) and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a (+)/CTC was transformed and expressed in E. coli BL21 (DE3) under induction of IPTG. After purification with Ni(+2)-NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope.
Enzyme digestion analysis and sequencing showed that TGF-beta(1) epitope gene was inserted into the el loop of C-terminus of truncated hepatitis B core antigen. SDS-PAGE analysis showed that relative molecular mass (Mr) of the expressed product by pET28a (+)/CTC was Mr 24,600. The output of the target recombinant protein was approximately 34.8% of the total bacterial protein, mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-beta(1) polyclonal IgG but not with anti-HBcAg. The purity of protein was about 90% and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-beta(1) antigenicity and could be used as anti-TGF-beta(1) vaccine.
A recombinant prokaryotic expression system with high expression efficiency of the target TGF-beta(1) epitope gene was successfully established. The fusion protein is in the form of self-assembling particles and HBcAg can increase the antigenicity of TGF-beta(1). The expressed TGF-beta(1) epitope gene shows good immunogenicity and antigenicity.
World Journal of Gastroenterology 11/2005; 11(40):6389-94. · 2.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Restoration of telomerase activity is essential for most of the malignancies. Telomerase reverse transcriptase (TERT) is the key component of telomerase. In this study, we designed a hammerhead ribozyme against human telomerase reverse transcriptase (hTERT) and observed its growth inhibition and pro-apoptosis effects on cancer cells. The efficiency of this ribozyme was verified in in vitro cleavage experiment. A recombinant retrovirus was constructed to transduce the ribozyme to telomerase positive colon carcinoma cell line SW480 and gastric carcinoma cell line SGC7901. We found that the ribozyme could strongly inhibit hTERT expression and telomerase activity, resulting in rapid apoptosis of cancer cells. Shortening of telomere and replicative senescence were not observed before cell death, indicating intensive inhibition of hTERT expression can induce apoptosis by some mechanism(s) except telomere shortening and replicative senescence. This study suggests that hTERT exerts a direct antiapoptotic function in cancer cells. Anti-hTERT ribozyme might be a potential means in the therapy of telomerase-positive malignancies.
[Show abstract][Hide abstract] ABSTRACT: To examine the expression and purification of the TGFbeta1 vaccine from prokaryotic expression system and to determine the antigenicity of the fusion protein of recombinant vector pET28a/ HBcAg1-71-TGFbeta132-HBcAg89-144.
The reconstructed vector pGEMEX-1/CTC was subcloned to pET28a and transformed into E. coli BL21 (DE3). The recombinant 6xHis- HBcAg1-71- TGFbeta132- HBcAg89-144 was to be expressed after induction by IPTG and purified with Ni-NTA-His affinity chromatography. The detection of the formation of core-like particles was done under an electron microscope and of their antigenity by using ELISA and Western blot.
A 2.46 x 10(4) protein was obtained by optimizing the conditions for both expression and purification. The protein had the TGFbeta1 antigenicity but not a HBc antigenity and the formed core-like particles were bigger than natural core particles.
The recombinant fusion protein in the prokaryotic expressed system can be used as an anti-TGFbeta1 vaccine to inhibit hepatic fibrosis.
Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology 09/2005; 13(8):582-5.
[Show abstract][Hide abstract] ABSTRACT: To investigate the correlation between expression of the osteopontin (OPN) and invasion and metastases in gastric cancer.
The expression of OPN, NF-kappaB p65 and matrix metallo-proteinase 9 (MMP-9) was detected by immunohistochemistry in non-cancer gastric tissue (n = 12 cases) and gastric cancer tissue (n = 72 cases).
(1) OPN, NF-kappaB p65 and MMP-9 were not expressed in 12 non-cancer gastric tissue samples(group A). Their expression rates were 43.3%, 40.0% and 46.7% respectively in 30 gastric cancer samples without lymph nodes metastasis (group B), but they increased to 76.9%, 73.1% and 80.8% in 26 gastric cancer samples with lymph nodes metastases (group C), and 87.5%, 81.3% and 93.8% respectively in 16 gastric cancer samples with lymph node and distant metastases (group D). (2) There were statistically significant differences in their expressions between group D and group B (P(a) = 0.004, P(c) = 0.007, P(e) = 0.002), and between group C and group B (P(b) = 0.011, P(d) = 0.013, P(f) = 0.009). (3) Despite some differences in positive expression rates, correlations existed between OPN and NF-kappaB p65, and between NF-kappaB p65 and MMP-9 (P(1) = 0.042, P(2) = 0.013; r(1)= 0.67, r(2)= 0.72).
Osteopondin espression is closely related to the invasion and metastases of gastric cancer. It may upregulate the expression of metastasis-related molecule MMP-9 by activating NF-kappaB pathway.
Zhonghua zhong liu za zhi [Chinese journal of oncology] 04/2005; 27(3):167-9.
[Show abstract][Hide abstract] ABSTRACT: Ion channels may play a role in carcinogenesis. Human ether-a-go-go-related gene (HERG) encoding one of the components of delayed rectifier potassium currents has been indicated to be involved in tumor cell growth and death. Our aim is to investigate the effects of cisapride, a specific blocker for HERG channel, on human gastric cancer cells.
The effects of cisapride on the proliferation, clonogenicity, cell cycle and apoptosis of gastric cancer cells were evaluated by MTT assay, clonogenicity assay, flow cytometry and transmission electron microscopy. The expression of HERG mRNA and protein in gastric cancer cells and tissues was measured by RT-PCR, Western blot and immunohistochemistry, respectively.
HERG mRNA and protein were exclusively expressed in gastric cancer cells. The HERG protein was localized in the cytoplasm and membrane of the gastric cancer cells. The proliferation of gastric cancer cells expressing HERG protein was inhibited in a time- and dose-dependent manner when treated with cisapride (P<0.05). The clonogenicity of gastric cancer cells treated with cisapride (100 nM) was reduced (P<0.05). Flow cytometric analysis indicated that cisapride tends to inhibit gastric cancer cells entering S phase from G(1) phase in the cell cycle (P<0.05). Apoptotic cells were found increased in gastric cancer cells treated with cisapride by both flow cytometry and electron microscopy.
As HERG channel blocker, cisapride, can inhibit the growth of gastric cancer cells by altering distribution of cell cycle and inducing apoptosis so as to be of potential value in the treatment of gastric cancer.
[Show abstract][Hide abstract] ABSTRACT: Antivascular therapy provides a promising method for anticancer therapy. But targeting to gastric cancer vessels is nonselective due in part to the lack of specific cell-surface receptors identified on target vascular cells. Herein we used in vivo screening of phage displayed peptide library to identify some peptides that bind selectively to endothelial cells of human gastric cancer rather than nonendothelial cells. After four rounds of selection, one phage was obtained with a cyclic 7-mer peptide CGNSNPKSC homing to human gastric adenocarcinoma . There was a 4.6 approximately 137.26-fold increase in the number of the selected phage in gastric cancer xenograft in comparision with control organs brain, heart, liver, spleen and kidney. Immunohistochemistry in mouse and human tissue showed that this phage peptide only bind to the endothelial cells of human gastric cancer. This peptide was observed only specific binding to HUVEC not to SGC-7901, Eca-109, LoVo and Hep-G2 by ELISA. The competitive and inhibitory result between the synthetic CGNSNPKSC peptide and the phage displaying the peptide CGNSNPKSC on HUVEC and in vivo was also confirmed its specific binding effect. This peptide may be a possible candidate for targeted drug delivery in antivascular therapy.
[Show abstract][Hide abstract] ABSTRACT: To design a hammerhead ribozyme targeting human telomerase reverse transcriptase (hTERT) and clone it's gene for future use in the study of tumor gene therapy.
Using the software RNA structure, the secondary structure of hTERT mRNA was predicted and the cleavage site of ribozyme was selected. A hammerhead ribozyme targeting this site was designed and bimolecular fold between the ribozyme and hTERT was predicted. The DNA encoding the ribozyme was synthesized and cloned into pGEMEX-1 and the sequence of the ribozyme gene was confirmed by DNA sequencing.
Triplet GUC at 1742 of hTERT mRNA was chosen as the cleavage site of the ribozyme. The designed ribozyme was comprised of 22nt catalytic core and 17nt flanking sequence. Computer-aided prediction suggested that the ribozyme and hTERT mRNA could cofold into a proper conformation. Endonuclease restriction and DNA sequencing confirmed the correct insertion of the ribozyme gene into the vector pGEMEX-1.
This fundamental work of successful designing and cloning of an anti-hTERT hammerhead ribozyme has paved the way for further study of inhibiting tumor cell growth by cleaving hTERT mRNA with ribozyme.
World Journal of Gastroenterology 02/2003; 9(1):104-7. · 2.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic liver diseases, such as fibrosis or cirrhosis, are more common in men than in women. This gender difference may be related to the effects of sex hormones on the liver. The aim of the present work was to investigate the effects of estrogen on CCL(4)-induced fibrosis of the liver in rats.
Liver fibrosis was induced in male, female and ovariectomized rats by CCL(4) administration. All the groups were treated with estradiol(1 mg/kg) twice weekly. And tamoxifen was given to male fibrosis model. At the end of 8 weeks, all the rats were killed to study serum indicators and the livers.
Estradiol treatment reduced aspartate aminotransferase(AST), alanine aminotransferase (ALT), hyaluronic acid(HA) and type IV collagen(CIV) in sera, suppressed hepatic collagen content, decreased the areas of hepatic stellate cells (HSC) positive for alpha-smooth muscle actin (alpha-SMA), and lowered the synthesis of hepatic type I collagen significantly in both sexes and ovariectomy fibrotic rats induced by CCL(4) administration. Whereas, tamoxifen had the opposite effect. The fibrotic response of the female liver to CCL(4) treatment was significantly weaker than that of male liver.
Estradiol reduces CCL(4)-induced hepatic fibrosis in rats. The antifibrogenic role of estrogen in the liver may be one reason for the sex associated differences in the progression from hepatic fibrosis to cirrhosis.
World Journal of Gastroenterology 11/2002; 8(5):883-7. · 2.43 Impact Factor