Zhiming Hao

The potent inhibitory effects of cisapride, a specific blocker for human ether-a-go-go-related gene (HERG) channel, on gastric cancer cells

Background: Ion channels may play a role in carcinogenesis. Human ether-go-go-related gene (HERG) encoding one of the components of delayed rectifier potassium currents has been indicated with involvement 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. Methods: The effects of cisapride on the proliferation, clonogenicity, cell cycle andapoptosis 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. Results: 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

Characterization of a specific phage-displayed Peptide binding to vasculature of human gastric cancer.

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 4 rounds of selection, one phage was obtained with a cyclic 7-mer peptide CGNSNPKSC homing to human gastric adenocarcinoma .There was a 4.6~137.26 –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.

CIAPIN1 confers multidrug resistance by upregulating the expression of MDR-1 and MRP-1 in gastric cancer cells.

In a previous study, we observed that cytokine-induced apoptosis inhibitor 1 (CIAPIN1), a newly identified apoptosis inhibitor, was upregulated at the mRNA level in a multidrug-resistant gastric cancer cell line SGC7901/VCR. The aim of this study was to explore the role of CIAPIN1 in the development of multidrug resistance (MDR) in gastric cancer cells. Upregulation of CIAPIN1 in MDR gastric cancer cells was confirmed by semiquantitative RT-PCR and Western blotting. Using cDNA transfection and RNA interference, we successfully established stable transfectants with up-regulation (i.e. SGC7901-pCIAPIN1) or down-regulation (i.e. SGC7901-pSiCIAPIN1 and SGC7901/ADR-pSiCIAPIN1) of CIAPIN1 expression, respectively. In vitro drug sensitivity assay demonstrated that overexpression of CIAPIN1 conferred MDR in SGC7901 cells whereas downregulation of CIAPIN1 sensitized SGC7901 and SGC7901/ADR cells to anticancer drugs. CIAPIN1 protected both SGC7901 and SGC7901/ADR cells from ADR-induced apoptosis and reduced intracellular accumulation and retention of adriamycin. Moreover, expression of P-glycoprotein (P-gp or MDR-1, a product of MDR-1 gene) and MDR-related protein-1 (MRP-1) was upregulated by CIAPIN1. In addition, Western blotting revealed that CIAPIN1 decreased the expression of Bcl-2, Bax and p53. Therefore, it is concluded that CIAPIN1 confers MDR in gastric cancer cells, likely by upregulating MDR-1 and MRP-1.

Expression of delayed rectifier potassium channels and their possible roles in proliferation of human gastric cancer cells.

Voltage-gated potassium (Kv) channels have been reported to be involved in the proliferation of many types of cells, including tumor cells. The overexpression of the Kv channels and related channel activity are involved in the neoplastic process. Our previous study has shown the existence of delayed rectifier potassium (IK) current in gastric cancer cells SGC7901. However, the expression and function of most delayed rectifier potassium (KD) channel subunits in gastric cancer cells are not completely resolved. Here we examine expression of KD channel subunits in Kv1-Kv3 families in immortalized gastric epithelial cells GES and various gastric cancer cells (including AGS, KATO¢Û, MKN28, MKN45, MGC803, SGC7901, SGC7901/ADR, and SGC7901/VCR), and their roles in cell proliferation. RT-PCR analysis reveals that all cell lines examined express Kv1.3, Kv1.5, Kv1.6, Kv2.1, and Kv2.2. However, Kv1.2 and Kv3.2 genes are barely detectable in any given cancer cell lines. Kv1.5 protein, high mRNA levels in all cell lines examined, is also expressed in some cancer cells lines and more frequently detected in gastric cancer tissues. Down-regulation of the expression of Kv1.5 in SGC7901 with RNA interference significantly inhibited the proliferation and tumorigenicity of SGC7901 cells. Moreover, in Ca2+-containing rather than Ca2+-free medium, KCl (50mM) stimulated a rapid increase in the concentration of cytosolic calcium in empty vector transfected cells that was blocked by verapamil. Likewise, decrease the expression of Kv1.5 with short interfering RNA also blocked the depolarization-induced influx of Ca2+. This finding suggests that more than one kind of KD channel subunits are expressed in various gastric cancer cell lines. Kv1.5 may involved in tumor cells proliferation by controlling Ca2+ entry, and the interference of KD channels expression and/or activity could provide a novel strategy to reverse the malignant phenotype of gastric cancer cells.

Attenuation of CCl4-Induced Hepatic Fibrosis in Mice by Vaccinating against TGF-β1

Transforming growth factor β1 (TGF-β1) is the pivotal pro-fibrogenic cytokine in hepatic fibrosis. Reducing the over-produced expression of TGF-β1 or blocking its signaling pathways is considered to be a promising therapeutic strategy for hepatic fibrosis. In this study, we evaluated the feasibility of attenuating hepatic fibrosis by vaccination against TGF-β1 with TGF-β1 kinoids. Two TGF-β1 kinoid vaccines were prepared by cross-linking TGF-β1-derived polypeptides (TGF-β125–[41-65] and TGF-β130–[83-112]) to keyhole limpet hemocyanin (KLH). Immunization with the two TGF-β1 kinoids efficiently elicited the production of high-levels of TGF-β1-specific antibodies against in BALB/c mice as tested by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The antisera neutralized TGF-β1-induced growth-inhibition on mink lung epithelial cells (Mv1Lu) and attenuated TGF-β1-induced Smad2/3 phosphorylation, α-SMA, collagen type 1 alpha 2 (COL1A2), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression in the rat hepatic stellate cell (HSC) line, HSC-T6. Vaccination against TGF-β1 with the kinoids significantly suppressed CCl4-induced collagen deposition and the expression of α-SMA and desmin, attenuated hepatocyte apoptosis and accelerated hepatocyte proliferation in BALB/c mice. These results demonstrated that immunization with the TGF-β1 kinoids efficiently attenuated CCl4-induced hepatic fibrosis and liver injury. Our study suggests that vaccination against TGF-β1 might be developed into a feasible therapeutic approach for the treatment of chronic fibrotic liver diseases.

The effect of somatostatin and SSTR3 on proliferation and apoptosis of gastric cancer cells

In the present study, we detected the expression of SSTR3 protein in 40 patients with gastric adenocarcinoma and 40 cases of normal gastric mucosa by immunoperoxidased staining. SSTR3 mRNA and protein were also examined in gastric cancer cell lines and eternal gastric epithelial cell line by RT-PCR, immunofluorescence and Western blot. The effect of octreotide on the growth of gastric cancer cells was examined by MTT test, and the apoptosis by flow cytometry. Competitive protein binding method was also used to evaluate the role of SSTR3. The results were: (1) SSTR3 protein existed in the membrane of gastric cancer cells. In normal gastric mucosa, SSTR3 protein distributed to the cellular membrane and cytoplasm or interstitial tissue in submucosa. The expression of SSTR3 protein was significantly lower in gastric cancer compared with normal mucosa. Moreover, the poor-differentiated adenocarcinoma was lower than the well-differentiated adenocarcinoma, and the similar result in cell lines. (2) Octreotide could inhibit the growth and induce the apoptosis of gastric cancer and normal epithelial cells that expressed SSTR3, but didn’t affect the cells with no or weakly expression of SSTR3. (3) When the cells were administrated octreotide in combination of SSTR3 antibody, the effect of octreotide decreased dramatically. The preliminary study suggested that SSTR3 might play a role in the growth and apoptosis of gastric cancer. In those gastric cancers that expressed SSTR3, octreotide could be effective in inhibiting cell growth and inducing cell apoptosis through mediation of SSTR3.

CIAPIN1 inhibits gastric cancer cell proliferation and cell cycle progression by downregulating cyclyinD1 and upregulating p27

CIAPIN1, a newly identified apoptosis-related molecule, was found to be downregulated in human gastric cancer tissues as compared to the matched adjacent nonneoplastic tissues. In this study, we investigated the effect of CIAPIN1 on in vitro and in vivo proliferation of gastric cancer cells. Ectopic expression and knockdown of CIAPIN1 in gastric cancer cells SGC7901 and MKN45 were achieved by transduction with recombinant adenoviruses expressing human CIAPIN1 (Ad-CIAPIN1) and human CIAPIN1-specific small interference RNA (Ad-CIAPIN1siRNA). Gastric cancer cells with upregulated expression of CIAPIN1 exhibited significantly decreased proliferation, while cells with downregulated CIAPIN1 expression showed significantly quicker growth rate as compared with their respective controls in both in vitro and in vivo tests. Also, CIAPIN1 suppressed colony formation of SGC7901 and MKN45 cells in soft agar cloning test. Furthermore, cell cycle distribution analysis revealed that CIAPIN1 induced cell cycle arrest at G1/S phase. Downregulation of CyclyinD1 and upregulation of P27 might contribute, at least in part, to this altered cell cycle distribution. This study demonstrates that CIAPIN1 is a suppressor of gastric cancer cell proliferation and suggests CIAPIN1 might play an important role in the development of human gastric cancer.

Distribution of CIAPIN1 in Normal Fetal and Adult Human Tissues

CIAPIN1, a newly identified antiapoptotic molecule that plays an essential role in mouse definitive hematopoiesis, is considered a downstream effector of the receptor tyrosine kinase-Ras signaling pathway. Our previous studies have indicated that CIAPIN1 is involved in the development of multidrug resistance (MDR) in gastric cancer cells. However, the mechanism of CIAPIN1-mediated antiapoptosis and MDR has not been fully elucidated. To reveal the possible physiological role of CIAPIN1, we examined the expression and distribution of CIAPIN1 in fetal and adult human tissues using immunohistochemistry. We found that CIAPIN1 was ubiquitously distributed in fetal and adult tissues, and was localized in both the cytoplasm and the nucleus. The expression patterns of CIAPIN1 were similar in fetal and adult tissues, and was correlated with the previously described expression pattern of p21ras. These observations suggest that CIAPIN1 expression appears to be involved in cell differentiation, and that it might exert universal and possibly important physiological functions under the regulation of Ras in humans.

Regulation of vascular endothelial growth factor (VEGF) production and angiogenesis by tissue Factor (TF) in SGC-7901 gastric cancer cells.

Tissue factor (TF), an initiator of the extrinsic coagulation cascade, is expressed in a wide range of cancer cells and plays important roles in cancer progression and metastasis. We demonstrated between TF and vascular endothelial growth factor (VEGF) production differences in 4 human gastric cell lines. One of these cell lines, SGC-7901, a high TF and VEGF producer, was grown subcutaneously in severe combined immuno-deficient (SCID) mice. The SCID mice generated solid tumors characterized by intense vascularity. In contrast, SGC-7901 cells transfected with antisense TF cDNA generated relatively avascular tumors in SCID mice, as determined by immunohistochemical staining of tumor vascular endothelial cells with anti-VIII factor antibody. To investigate the structure-function relationship between TF and VEGF, the SGC-7901 cell line was transfected with antisense a full-length TF cDNA, a cytoplasmic deletion mutant lacking the distal three serine residues (potential substrates for protein kinase C), or an extracellular domain mutant, which has markedly diminished function for activation of factor X. Cells transfected with the full-length antisense TF sequence produced decreased levels of both TF and VEGF. Transfectants with the extracellular domain mutant produced high levels of VEGF mRNA. However, cells transfected with the cytoplasmic deletion mutant construct produced increased levels of TF, but little or no VEGF. Thus, the cytoplasmic tail of TF may signal VEGF expression in some tumor cells.

Subcellular Localization of CIAPIN1

Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) is a newly identified anti-apoptotic molecule. Our previous studies have demonstrated that CIAPIN1 is ubiquitously expressed in normal fetal and adult human tissues and confers multidrug resistance in gastric cancer cells, possibly by upregulating the expression of multidrug resistance gene 1 and multidrug resistance-related protein 1. However, fundamental biological functions of CIAPIN1 have not been elucidated. In this study, we first predicted the subcellular localization of CIAPIN1 with bioinformatic approaches and then characterized the intracellular localization of CIAPIN1 in both human and mouse cells by a combination of techniques including (a) immunohistochemistry and immunofluorescence, (b) His-tagged CIAPIN1 expression, and (c) sub-cellular fractionation and analysis of CIAPIN1 in the fractions by Western blotting. All methods produced consistent results; CIAPIN1 was localized in both the cytoplasm and the nucleus and was accumulated in the nucleolus. Bioinformatic prediction disclosed a putative nuclear localization signal and a putative nuclear export signal within both human and mouse CIAPIN1. These findings suggest that CIAPIN1 may undergo a cytoplasm-nucleus-nucleolus translocation.