Xiaoxuan Ning

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)

Hypoxia-inducible factor-1α induces Twist expression in tubular epithelial cells subjected to hypoxia, leading to epithelial-to-mesenchymal transition

Epithelial-to-mesenchymal transition (EMT) induced by chronic hypoxia is one of the critical causes of renal fibrosis. Twist, a basic helix-loop-helix transcription factor, is believed to be important in promoting EMT. We found that the expression of Twist was increased in human tubule cell lines (HK-2 and HKC) grown under hypoxic conditions. This was accompanied by reduced expression of the epithelial markers E-cadherin and ZO-1 and enhanced expression of the mesenchymal markers vimentin and alpha-smooth muscle actin. When Twist was overexpressed in these cells it induced a mesenchymal phenotype, whereas its knockdown by short interfering RNA (siRNA) effectively reversed hypoxia-induced EMT. We showed that transfection with siRNA to hypoxia-inducible factor-1alpha (HIF-1alpha), another basic helix-loop-helix transcription factor, reduced Twist expression. Twist promoters contain HIF1-alpha-binding sites and transfection of reporter constructs using the promoter showed increased transcription in cells subjected to hypoxia. Electrophoretic mobility shift and chromatin immunoprecipitation assays identified the presence of a functional HIF-1alpha-binding site within the proximal Twist gene promoter. In an in vivo assay using the rat remnant kidney we found that both Twist and HIF-1alpha were overexpressed in tubular epithelial cells showing EMT. These studies suggest that HIF-1alpha induces Twist expression in hypoxic tubular cells and that this plays a role in EMT during renal fibrogenesis.

Hypoxia-induced Bmi1 promotes renal tubular epithelial cell–mesenchymal transition and renal fibrosis via PI3K/Akt signal

In vitro and in vivo evidence shows that activation of HIF-1a/Twist-Bmi1 signaling in renal epithelial cells is associated with the development of chronic renal disease and may promote fibrogenesis via modulation of PI3K/Akt/Snail signaling by facilitating EMT.

Calcyclin-Binding Protein Inhibits Proliferation, Tumorigenicity, and Invasion of Gastric Cancer

Calcyclin-binding protein/Siah-1–interacting protein (CacyBP/SIP), a target protein of the S100 family, which includes S100A6, S100A1, S100A12, S100B, and S100P, has been identified as a component of a novel ubiquitinylation complex leading to β-catenin degradation. However, the function of CacyBP/SIP in gastric cancer has not been elucidated. In the present study, we prepared CacyBP/SIP overexpressing and knockdown cell lines of gastric cancer. Forced CacyBP/SIP expression inhibited the proliferation of gastric cancer cells, suppressed tumorigenicity in vitro, and prolonged the survival time of tumor-bearing nude mice. In addition, increased CacyBP/SIP repressed the invasive potential of gastric cancer cells. Conversely, the down-regulation of CacyBP/SIP by RNA interference showed the opposite effects. Further studies showed that depressed CacyBP/SIP increased the expression of total and nuclear β-catenin at the protein level and elevated the transcriptional activity of Tcf/LEF. Taken together, our results suggest that CacyBP/SIP may be a potential inhibitor of cell growth and invasion in the gastric cancer cell, at least in part through the effect on β-catenin protein expression and transcriptional activation of Tcf/LEF. (Mol Cancer Res 2007;5(12):1254–62)

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.

Multidrug-resistance-associated protein MGr1-Ag is identical to the human 37-kDa laminin receptor precursor.

Abstract. We report the isolation and functional characterization of the gene encoding MGr1-Ag, a multidrug-resistance-associated protein. A λgt11 cDNA library derived from colorectal carcinoma SW480 cells was screened with monoclonal antibody MGr1. DNA homology analysis of 22 positive clones (designated R1–R22) suggested human 37-kDa laminin receptor precursor (37LRP, R7/R9/R15/R16/R19/R20) and a novel gene (R22) as candidate genes encoding MGr1-Ag. Western blot analysis showed that anti-R20 serum reacted with a unique protein band that was consistent with MGr1-Ag, while anti-R22 serum could not react with MGr1-Ag. The coding gene for MGr1-Ag was amplified using reverse transcription-PCR. Sequence analysis revealed that the MGr1-Ag and 37LRP genes shared the same coding sequence. An in vitro drug sensitivity assay indicated that down-regulation of 37LRP by an antisense technique could significantly enhance the cytotoxicity of anticancer drugs to gastric cancer cells. Thus we draw the conclusion that MGr1-Ag is identical to 37LRP.

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.

Expression and clinical significance of Notch receptors in human renal cell carcinoma

Aims: The pathological roles of Notch receptors in renal cell carcinoma (RCC) are still unclear, although Notch receptors have been shown to have an effect on many malignant tumours. Therefore, in this study, we examined the patterns of expression and clinical significance of Notch receptors in RCC. Methods: Eighty‐four cases of renal cell carcinoma tissues were detected by immunohistochemistry. Eleven paired fresh surgical renal cell carcinoma and adjacent non‐neoplastic renal samples were analysed by Western blot and reverse transcriptase polymerase chain reaction. In addition, the expression of Notch receptors in renal cancer cell lines (A498 and 786‐O) and human normal kidney tubule epithelial cell lines (HK‐2 and HKC) were analysed by Western blot. Results: The expression levels of Notch1 and Notch4 were absent or significantly decreased in renal cell carcinoma tissues compared with the adjacent non‐neoplastic tissues [Notch1: 22.6% (19/84) versus 78.7% (59/75); Notch4: 27.4% (23/84) versus 73.3% (55/75); p < 0.05). Moreover, the levels of expression of Notch1 and Notch4 were also markedly down‐regulated in human renal cancer cell lines. Notch1 was negatively correlated with tumour stage, while Notch4 expression had no significant association with pathological parameters. The levels of expression of Notch2 and Notch3 were minimally detected in tumours and non‐neoplastic tissues. Conclusion: Our findings indicated that the expression of Notch receptors was deregulated and Notch signalling might play an important role in the progress of renal cell carcinoma.

MGr1-Ag is associated with multidrug-resistant phenotype of gastric cancer cells

Abstract.Background: MGr1-antigen (Ag) was previously reported as an upregulated protein in multidrug-resistant (MDR) gastric cancer cells. The aim of this study was to characterize the role of MGr1-Ag in the multidrug resistance of gastric cancer cells. Methods: Laser scanning confocal microscopy (LSCM), two-dimensional electrophoresis, and Western blot were used to detect MGr1-Ag in gastric cancer cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine the sensitivity of the MDR gastric cancer cells, SGC7901/VCR, to chemotherapeutic drugs. Adriamycin accumulation and retention in SGC7901/VCR cells were analyzed using flow cytometry. Results: LSCM showed that MGr1-Ag localized mainly on the membrane and partly in the cytoplasm of SGC7901/VCR cells. Western blot showed that the expression level of MGr1-Ag in SGC7901/VCR cells was higher than that in its parental cells, SGC7901, and that the apparent molecular weight and isoelectric point of MGr1-Ag were 42 kDa and pH 4.8, respectively. After incubation with MGr1 antibody, SGC7901/VCR cells showed significantly decreased IC50 values for adriamycin (from 0.887 ± 0.081 mg/l to 0.607 ± 0.084 mg/l; P , 0.05), vincristine (from 0.707 ± 0.055 mg/l to 0.557 ± 0.042 mg/l; P , 0.05), and 5-fluorouracil (from 4.367 ± 0.407 mg/l to 2.630 ± 0.644 mg/l; P , 0.05), as well as slightly increased IC50 values for mitomycin (from 0.183 ± 0.045 mg/l to 0.198 ± 0.048 mg/l; P . 0.05). In addition, incubation with MGr1 significantly enhanced adriamycin accumulation and retention in SGC7901/VCR cells. Conclusion: Overexpression of MGr1-Ag is associated with the MDR phenotype of gastric cancer cells.

S100A6 Protein Negatively Regulates CacyBP/SIP-Mediated Inhibition of Gastric Cancer Cell Proliferation and Tumorigenesis

Calcyclin-binding protein (CacyBP/SIP), identified on the basis of its ability to interact with S100 proteins in a calcium-dependent manner, was previously found to inhibit the proliferation and tumorigenesis of gastric cancer cells in our laboratory. Importantly, the effects of S100 proteins on the biological behavior of CacyBP/SIP in gastric cancer remain unclear. Herein, we report the construction of eukaryotic expression vectors for wild-type CacyBP/SIP and a truncated mutant lacking the S100 protein binding domain (CacyBP/SIPΔS100). The expressions of the wild-type and truncated recombinant proteins were demonstrated by transfection of MKN45 gastric cancer cells. Co-immunoprecipitation assays demonstrated interaction between S100A6 and wild-type CacyBP/SIP in MKN45 cells. Removal of the S100 protein binding domain dramatically reduced the affinity of CacyBP/SIP for S100 proteins as indicated by reduced co-immunoprecipitation of S100A6 by CacyBP/SIPΔS100. The MTT assay, FACS assay, clonogenic assay and tumor xenograft experiment were performed to assess the effect of CacyBP/SIP on cell growth and tumorigenesis in vitro and in vivo. Overexpression of CacyBP/SIP inhibited the proliferation and tumorigenesis of MKN45 gastric cancer cells; the proliferation and tumorigenesis rates were even further reduced by the expression of CacyBP/SIPΔS100. We also showed that S100 proteins negatively regulate CacyBP/SIP-mediated inhibition of gastric cancer cell proliferation, through an effect on β-catenin protein expression and transcriptional activation of Tcf/LEF. Although the underlying mechanism of action requires further investigation, this study provides new insight into the interaction between S100 proteins and CacyBP/SIP, which might enrich our knowledge of S100 proteins and be helpful for our understanding of the development of gastric cancer.