Changcun Guo

Hypoxia‐inducible factor‐1α contributes to hypoxia‐induced chemoresistance in gastric cancer

Hypoxia induced drug resistance is a major obstacle in the development of effective cancer therapy. Our previous study revealed that hypoxia‐inducible factor‐1 (HIF‐1), the major transcriptional factor significantly activated by hypoxia, was overexpressed in gastric vincristine‐resistant cells SGC7901/vincristine (VCR) under normoxic conditions, which suggested that it was associated with drug resistance in gastric cancer cells. In the present study, a colony‐forming assay revealed that hypoxia and forced HIF‐1α expression increased maximal –8.9‐fold or –14.8‐fold of IC50 toward vincristine in gastric cancer cell lines SGC7901 and SGC7901/VCR, respectively (P < 0.01). Annexin‐V/propidium iodide staining analysis revealed hypoxia or forced HIF‐1α expression reduced apoptosis by 24% or 18% in SGC7901 cells (P < 0.05). Flow cytometry analysis of intracellular adriamycin revealed that hypoxia and forced expression of HIF‐1α increased –1.79‐fold or –2.36‐fold of the adriamycin releasing index, respectively (P < 0.05). However, resistance acquisition subject to hypoxia in vitro and in vivo was suppressed by blocking HIF‐1α expression with siRNA. We further demonstrated that HIF‐1 α overexpression showed a 1.85‐fold increased expression of Bcl‐2 and a 2.16‐fold decreased expression of Bax, and also showed significantly induced expression of p‐gp and MRP1, which indicated that HIF‐1α may confer hypoxia‐induced drug resistance via inhibition of drug‐induced apoptosis and decreases in intracellular drug accumulation. (Cancer Sci 2008; 99: 121–128)

MicroRNA Let-7f Inhibits Tumor Invasion and Metastasis by Targeting MYH9 in Human Gastric Cancer

Background MicroRNAs (miRNAs) are important regulators that play key roles in tumorigenesis and tumor progression. A previous report has shown that let-7 family members can act as tumor suppressors in many cancers. Through miRNA array, we found that let-7f was downregulated in the highly metastatic potential gastric cancer cell lines GC9811-P and SGC7901-M, when compared with their parental cell lines, GC9811 and SGC7901-NM; however, the mechanism was not clear. In this study, we investigate whether let-7f acts as a tumor suppressor to inhibit invasion and metastasis in gastric cancers. Methodology/Principal Real-time PCR showed decreased levels of let-7f expression in metastatic gastric cancer tissues and cell lines that are potentially highly metastatic. Cell invasion and migration were significantly impaired in GC9811-P and SGC7901-M cell lines after transfection with let-7f-mimics. Nude mice with xenograft models of gastric cancer confirmed that let-7f could inhibit gastric cancer metastasis in vivo after transfection by the lentivirus pGCsil-GFP- let-7f. Luciferase reporter assays demonstrated that let-7f directly binds to the 3′UTR of MYH9, which codes for myosin IIA, and real-time PCR and Western blotting further indicated that let-7f downregulated the expression of myosin IIA at the mRNA and protein levels. Conclusions/Significance Our study demonstrated that overexpression of let-7f in gastric cancer could inhibit invasion and migration of gastric cancer cells through directly targeting the tumor metastasis-associated gene MYH9. These data suggest that let-7f may be a novel therapeutic candidate for gastric cancer, given its ability to reduce cell invasion and metastasis.

Overexpression and significance of prion protein in gastric cancer and multidrug-resistant gastric carcinoma cell line SGC7901/ADR.

In our previous work, cellular prion protein (PrPc) was identified as an upregulated gene in adriamycin‐resistant gastric carcinoma cell line SGC7901/ADR compared to its parental cell line SGC7901. Here we investigate the expression of PrPc in gastric cancer and whether it was involved in multidrug resistance (MDR) of gastric cancer. We demonstrated that PrPc was ubiquitously expressed in gastric cancer cell lines and tissues. PrPc conferred resistance of both P‐glycoprotein (P‐gp)‐related and P‐gp‐nonrelated drugs on SGC7901, which was accompanied by decreased accumulation and increased releasing amount of adriamycin in PrPc‐overexpressing cell line. Inhibition of PrPc expression by antisense or RNAi technology could partially reverse multidrug‐resistant phenotype of SGC7901/ADR. PrPc significantly upregulated the expression of the classical MDR‐related molecule P‐gp but not multidrug resistance associated protein and glutathione S‐transferase pi. The PrPc‐induced MDR could be partially reversed by P‐gp inhibitor verapamil. PrPc could also suppress adriamycin‐induced apoptosis and alter the expression of Bcl‐2 and Bax, which might be another pathway contributing to PrPc‐related MDR. The further study of the biological functions of PrPc may be helpful for understanding the mechanisms of occurrence and development of clinical gastric carcinoma and PrPc‐related MDR and developing possible strategies to treat gastric cancer.

Cellular prion protein promotes proliferation and G1/S transition of human gastric cancer cells SGC7901 and AGS

The function of cellular prion protein (PrPC), the essential protein for the pathogenesis and transmission of prion diseases, is still largely unknown. The putative roles of PrPC are thought to be related to cell signaling, survival, and differentiation. In a previous study, we showed that PrPC was overexpressed in gastric cancer tissues. In the present report, we show that ectopic expression of PrPC could promote tumor‐igenesis, proliferation, and G1/S transition in gastric cancer cells. Furthermore, CyclinD1, a protein related to cell cycle, was shown to be significantly up‐regulated by PrPC at both mRNA and protein levels. PI3K/Akt pathway mediated above PrPC signal since PrPC increased the expression of phosphorylated Akt, and the specific inhibitor of Akt, LY294002, could markedly suppress growth of SGC7901 and transactivation of CyclinD1 induced by PrPC. Octapeptide repeat region played a vital role in this function, as deletion of this region abolished or reduced these effects. Collectively, this study demonstrates that overexpression of PrPC might promote the tumorigenesis and proliferation of gastric cancer cells at least partially through activation of PI3K/Akt pathway and subsequent transcriptional activation of CyclinD1 to regulate the G1/S phase transition, in which octapeptide repeat region might be an indispensable region.–Liang, J., Pan, Y., Zhang, D., Guo, C., Shi, Y., Wang, J., Chen, Y., Wang, X., Liu, J., Guo, X., Chen, Z., Qiao, T., Fan, D. Cellular prion protein promotes proliferation and G1/S transition of human gastric cancer cells SGC7901 and AGS. FASEB J. 21, 2247–2256 (2007)

Molecular basis of therapeutic approaches to gastric cancer

Gastric cancer is the top lethal cancer in Asia. As the majority of cases present with advanced disease, conventional therapies (surgery, chemotherapy, and radiotherapy) have limited efficacy to reduce mortality. Emerging modalities provide promise to combat this malignancy. Target‐protein‐based cancer therapy has become available in clinical practice. Numerous molecules have been shown potential to target specific pathways for tumor cell growth. Cyclooxygenase‐2 (COX‐2) is overexpressed in and correlated with gastric cancer, and knockdown of COX‐2 or administration of COX‐2 inhibitors suppresses tumor formation in models of gastric cancer. Induction of apoptosis, reduction of angiogenesis, and blocking of potassium ion channels may present new mechanisms of COX‐2 inhibition. Runt‐related transcription factor 3 (RUNX3) is a candidate tumor suppressor gene whose deficiency is causally related to gastric cancer. RUNX3 is downregulated in metastatic gastric cancer. RUNX3 activation inhibits angiogenesis in xenograft tumors in nude mice. Tumor microenvironment modulation also provides a powerful tool to inhibit cancer development and progress; details of the potential roles of angiopoietins are discussed in this review. Osteopontin is a secreted protein involved in stress response, inflammation, wound healing, and immune response. Inhibition of osteopontin by RNA interfering technique suppressed tumorigenesis as well as angiogenesis in gastric cancer. Immunotherapy remains another important choice of adjuvant therapy for cancer. A tumor‐specific antigen MG7‐Ag has been identified with great potential for inducing immune response in gastric cancer. Using HLA‐A‐matched allogeneic gastric cancer cells to induce tumor‐specific cytotoxic T lymphocytes appeared to be an alternative option of immunotherapy for gastric cancer.

Screening and identification of vascular-endothelial-cell-specific binding peptide in gastric cancer.

Antiangiogenesis therapy has become a hot field in cancer research. Blood vessels of tumor carry specific markers that are usually related to angiogenesis. Study of these heterogeneous molecules in different tumor vessels may be beneficial for promoting antiangiogenic therapy. In this study, we established an in vitro co-culture model of human umbilical vein endothelial cells (HUVECs) and gastric adenocarcinoma cell line SGC7901, screened the peptides binding specifically to the HUVECs co-cultured with gastric cancer cells (Co-HUVECs) using phage display peptides library, and studied the affinity of these peptides to gastric cancer vascular endothelial cells. After four rounds of panning, there was an obvious enrichment for the phages specifically binding to the Co-HUVECs, and the output/input ratio of Co-HUVECs increased about 590-fold (from 0.95×10−7 to 561.25×10−7). Five phage clones (M6, M3, M9, IN12, IN11), which could strongly bind to Co-HUVECs instead of wild-type HUVECs, were characterized by ELISA. In vitro cellular binding assay showed that phage IN11 preferably bound to Co-HUVECs rather than control HUVECs, and the number of the phage IN11 recovered from Co-HUVECs was 5.7- and 16.9-folds, respectively, as much as those from control HUVECs and GES cells. Immunocytochemical and immunohistochemical staining confirmed that phage IN11 could specifically bind to Co-HUVECs as well as vascular endothelial cells in gastric cancer tissue sections. Competitive and inhibitory assay revealed the synthetic peptide GEBP11 (CTKNSYLMC) displayed on phage IN11 could competitively inhibit binding of the phage IN11 to Co-HUVECs. Immunofluorescence microscopy showed that the fluorescence-labeled peptide GEBP11 was located at the membrane and perinuclear cytoplasm of Co-HUVECs. Meanwhile, GEBP11 was found to be able to target the gastric cancer vascular endothelial cells. Therefore, GEBP11 may be a potential candidate for targeted drug delivery in antivascular therapy and diagnosis of gastric cancer.

Tumor suppressor gene Runx3 sensitizes gastric cancer cells to chemotherapeutic drugs by downregulating Bcl‐2, MDR‐1 and MRP‐1

The Runx3 gene is a member of the runt domain family transcription factors, key regulators of development and differentiation in metazoan. Recently, Runx3 was identified as a tumor suppressor gene. Loss of Runx3 was found to be associated with genesis and progression of gastric cancer. In this study, we transfected the gastric cancer cell line SGC7901 with eukaryotic expression vector of Runx3. In vitro drug sensitivity assay suggested that SGC7901/Runx3 cells were more sensitive to various chemotherapeutic drugs. Blocking Runx3 expression in immortalized stomach mucosal cells (GES‐1) or gastric cancer cells (SGC7901) by Runx3‐specific small interfering RNA conferred the cells resistance to chemotherapeutic drugs. Flow cytometry examination suggested that expression of Runx3 in gastric cancer cells increased the intracellular accumulation and retention of adriamycin. Semiquantitative RT‐PCR and Western blot suggested that Runx3 downregulated expression of Bcl‐2, MDR‐1 (P‐gp) and MRP‐1. Binding of Runx3 to promoter sequences of Bcl‐2, MDR‐1 and MRP‐1 gene was detected by eletrophoretic mobility shift assay (EMSA) and supershift EMSA. We cloned the MDR‐1 and MRP‐1 gene promoters containing Runx binding sites and constructed the luciferase reporter vectors of these 2 promoters. Luciferase reporter assay suggested that Runx3 inhibited the promoter activity of the MDR‐1 and MRP‐1 promoter in SGC7901 cells. Taken together, our findings suggested that overexpression of Runx3 could sensitize gastric cancer cells to chemotherapeutic drugs by downregulating the Bcl‐2, 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.

CIAPIN1 inhibits the growth and proliferation of clear cell renal cell carcinoma

Our previous studies indicated a direct correlation with loss of CIAPIN1 and carcinogenesis of tumor in human gastric cancer. Here we presented that the expression of CIAPIN1 was absent or significantly decreased in 102 cases of clear cell renal cell carcinoma (CCRCC) tissues (P<0.05). Up-regulating CIAPIN1 by adenoviral vectors exhibited significant inhibition of CCRCC-derived cell growth in vitro and in vivo with G1 cell cycle arrest. Simultaneously, CIAPIN1-induced growth suppression was found partially to regulate various proteins, including inhibition of cyclinD1, cyclinE, cdk2, cdk4, p-Rb and VEGF, but up-regulation of p27Kip1 and Rb.

Hypoxia-mediated up-regulation of MGr1-Ag/37LRP in gastric cancers occurs via hypoxia-inducible-factor 1-dependent mechanism and contributes to drug resistance

Our previous study demonstrated hypoxia‐inducible factor‐1(HIF‐1) could prompt multidrug resistance (MDR) phenotype and MGr1‐Ag/37LRP, a novel drug‐resistance protein was reported by our labortary, associated with multidrug resistance in gastric cancer. Given this association, we hypothesized that MGr1‐Ag/37LRP contributed to HIF‐1‐dependent hypoxia‐induced MDR phenotype. Initial experiments revealed that blocking MGr1‐Ag/37LRP expression by siRNA in gastric cancer cells effectively reversed multidrug resistance phenotype induced by hypoxia. Subsequent analysis of MGr1‐Ag/37LRP mRNA and protein in gastric cancer cells revealed a time‐dependent manner increase with hypoxia. While the up‐regulation of MGr1‐Ag/37LRP was abolished by HIF‐1 inhibition with siRNA. Studies using luciferase promoter constructs revealed a significant increase in activity in cells subject to hypoxia and such hypoxia inducibility was lost in cells co‐transfected siRNA targeting HIF‐1. Analysis of the MGr1‐Ag/37LRP promoter revealed several potential binding sites for HIF‐1. Electrophoretic mobility shift assay and chromatin immunoprecipitation demonstrated a functional HIF‐1 binding site within MGr1‐Ag/37LRP gene regulatory sequence located at −16 to −11 relative to the transcriptional initiation point. These observations demonstrate that MGr1‐Ag/37LRP is actively engaged by hypoxia and represent a novel HIF‐1 target. Such results suggest hypoxia‐elicited MGr1‐Ag/37LRP expression as a pathway for resistance of gastric cancer to chemotherapeutics.