Taidong Qiao

Zinc ribbon domain-containing 1 (ZNRD1) mediates multidrug resistance of leukemia cells through regulation of P-glycoprotein and Bcl-2

Here, we investigated the role of zinc ribbon domain-containing 1 (ZNRD1) in multidrug resistance (MDR) of leukemia cells and the possible underlying mechanisms. ZNRD1 was found overexpressed in the vincristine-induced MDR leukemia cell HL-60/vincristine moreso than its parental cell HL-60. Up-regulation of ZNRD1 expression could confer resistance of both P-glycoprotein (P-gp)-related and P-gp-nonrelated drugs on HL-60 cells and suppress Adriamycin-induced apoptosis accompanied by decreased accumulation and increased releasing amount of Adriamycin. ZNRD1 could significantly up-regulate the expression of P-gp, Bcl-2, and the transcription of the MDR1 gene but not alter the expression of MDR-associated protein, glutathione S-transferase activity, or intracellular glutathione content in leukemia cells. In addition, inhibition of ZNRD1 expression by RNA interference or P-gp inhibitor could partially reverse ZNRD1-mediated MDR. The further study of the biological functions of ZNRD1 may be helpful for understanding the mechanisms of MDR of leukemia and developing possible strategies to treat leukemia. [Mol Cancer Ther 2005;4(12):1936–42]

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.

Overexpression of PrPC and Its Antiapoptosis Function in Gastric Cancer

Cellular prion protein (PrPC), a glycosylphosphatidylinositol-anchored membrane protein, was found in our lab to be widely expressed in gastric cancer cell lines. In order to evaluate its biological significance in human gastric cancer, we investigated its expression in a large series of gastric tissue samples (n = 124) by immuno histochemical staining with the monoclonal antibody 3F4. Compared with normal tissues, gastric adenocarcinoma showed increased PrPC expression, correlated with the histopathological differentiation (according to the WHO and Lauren classifications) and tumor progression (as documented by pTNM staging). To better understand the underlying mechanism, we introduced the PrPC and two pairs of RNAi into the poorly differentiated gastric cancer cell line AGS and found that PrPC suppressed ROS and slowed down apoptosis in transfected cells. Further study proved that the apoptosis-related protein Bcl-2 was upregulated whereas p53 and Bax were downregulated in the PrPC-transfected cells. A reverse effect was observed in PrPC siRNA-transfected cells. These results strongly suggested that PrPC might play a role as an effective antiapoptotic protein through Bcl-2-dependent apoptotic pathways in gastric cancer cells. Further study into the mechanism of these relationships might enrich the knowledge of PrP, better our understanding of the nature of gastric carcinoma, and further develop possible strategies to block or reverse the development of gastric carcinoma.

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.

Regulation of multidrug resistance by ribosomal protein L6 in gastric cancer cells

Ribosomal proteins (RP) L6 was previously identified as an up-regulated gene in multidrug-resistant gastric cancer cells SGC7901/ADR comparing to its parental cells SGC7901 by subtractive hybridization. The aim of this study was to explore the roles of RPL6 in multidrug resistance (MDR) in gastric cancer cells. Northern and Western blot analysis confirmed that RPL6 was overexpressed in SGC7901/ADR cells. By gene transfection, RPL6 was genetically up-regulated in SGC7901 or down-regulated in SGC7901/ADR cells. Up-regulation of RPL6 was associated with enhanced resistance to multiple anticancer drugs (adriamycin, vincristine, etoposide, 5-fludrouracil and cisplatin) and to adriamycin-induced apoptosis. Down-regulation of RPL6 reversed MDR and sensitized cells to adriamycin-induced apoptosis. Alteration of RPL6 showed no obvious influence on intracellular adriamycin accumulation, glutathione content and expression of glutathione S-transferase. RPL6 could up-regulate Bcl-2 and down-regulate Bax in cells. Together, this work demonstrates that RPL6 could regulate MDR in gastric cancer cells by suppressing drug-induced apoptosis.

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.

Reversal of multidrug resistance of vincristine-resistant gastric adenocarcinoma cells through up-regulation of DARPP-32

Here we investigated the roles of DARPP‐32 in multidrug resistance (MDR) of gastric cancer cells and the possible underlying mechanisms. We constructed the eukaryotic expression vector of DARPP‐32 and transfected it into human vincristine‐resistant gastric adenocarcinoma cell line SGC7901/VCR. Up‐regulation of DARPP‐32 could significantly enhance the sensitivity of SGC7901/VCR cells towards vincristine, adriamycin, 5‐fluorouracil and cisplatin, and could decrease the capacity of cells to efflux adriamycin. Whats more, the results of subrenal capsule assay confirmed that DARPP‐32 might play a certain role in MDR of gastric cancer. DARPP‐32 could significantly down‐regulate the expression of P‐gp and zinc ribbon domain‐containing 1 (ZNRD1), but not alter the expression of multidrug resistance‐associated protein (MRP) or the glutathione S‐transferase (GST). DARPP‐32 could also significantly decrease the anti‐apoptotic activity of SGC7901/VCR cells. Further study of the biological functions of DARPP‐32 might be helpful for understanding the mechanisms of MDR in gastric cancer.

Downregulated expression of CIAPIN1 may contribute to gastric carcinogenesis by accelerating cell proliferation and promoting cell cycle progression

Our previous studies revealed that cytokine induced apoptosis inhibitor 1 (CIAPIN1), which was reported to be essential in mouse definitive hematopoiesis, was related to multidrug resistance in gastric cancer cells and that the distribution of CIAPIN1 in normal human tissues was similar to the distribution of Ras. This study aimed to explore whether CIAPIN1 plays a role in gastric carcinogenesis. Expression of CIAPIN1 in normal, inflammatory gastric mucosa, gastric precancerous lesions and gastric adenocarcinoma was detected by immunohistochemistry and Western blotting and, influence of CIAPIN1 on the proliferation of gastric cancer cells was investigated by ectopic expression of CIAPIN1 and RNA interference (RNAi). Our immunohistochemical results demonstrated that the expression of CIAPIN1 in gastric antral mucosa was progressively reduced along the sequence of normal/inflammatory gastric mucosa-atrophy-intestinal metaplasia-dysplasia-adenocarcinoma. The downregulation of CIAPIN1 in cancerous tissues was further confirmed by Western blotting. No relationship between the expression level of CIAPIN1 and the clinicopathological parameters such as age, gender, differentiation, TNM stage and the existence of metastasis was found in gastric cancer patients. In in vitro cellular experiments, ectopic expression of CIAPIN1 by cDNA transfection resulted in suppression of cell proliferation and inhibition of cell cycle progression while knockdown of CIAPIN1 with siRNA accelerated cell proliferation and promoted cell cycle progression in SGC7901 and MKN28 gastric cancer cells. These results suggest that downregulated CIAPIN1 expression may contribute to gastric carcinogenesis by accelerating cell proliferation and promoting cell cycle progression.

RUNX3 inhibits growth of HCC cells and HCC xenografts in mice in combination with adriamycin

Here, we report that a loss or decrease of RUNX3 expression was found in 73 cases of HCCs as compared with that in normal liver tissues (P < 0.001). Various human HCC cell lines also exhibited loss or decrease of RUNX3 expression. The introduction of RUNX3 by an adenovirus vector into HCC cell lines which had decreased expressions of RUNX3 inhibited cell proliferation and cell cycle progression, decreased anchorage-independent growth, and inhibited tumorigenesis in nude mice. Exogenous expression of RUNX3 sensitized HCC cells to cytotoxic drugs and to apoptosis induced by chemotherapeutic drug adriamycin in vitro. Ectopic expression of RUNX3 in HCC cells enhanced caspase-8 and decreased Bcl-2 expression. Treatment of nude mice bearing subcutaneously established HCC tumors with a combination of an adenovirus expressing RUNX3 and adriamycin completely suppressed tumor growth. In conclusion, overexpression of RUNX3 might be a promising candidate as a treatment for HCC that would increase sensitivity to chemotherapeutic drugs.