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ABSTRACT: Our previous studies indicate that Twist plays important roles in hypoxia-induced tubular epithelial-mesenchymal transition and the development of kidney fibrosis in cellular and animal models. However, the expression and clinical significance of Twist in patients with chronic kidney disease are not clear.
We analyzed the degree of expression and localization of Twist in renal biopsies from a wide variety of hypoxic kidney diseases and correlated their immunostaining scores with clinical and histologic parameters. In particular, we also retrospectively analyzed whether the degree of expression of Twist in the renal interstitium was correlated with renal survival.
Activated Twist was strongly expressed in tubular epithelial cell nuclei from the kidneys of patients with chronic kidney diseases, while little positive staining for Twist was found in the renal tubules of normal kidneys (p = 0.001). Twist protein in the tubulointerstitium was inversely correlated with estimated glomerular filtration rate (eGFR; r = -0.468, p = 0.029) and positively correlated with serum creatinine (r = 0.44, p = 0.045) and the percentage of tubulointerstitial fibrosis (r = 0.551, p = 0.000). Moreover, a high level of Twist was correlated with activation of HIF-1α expression and E-cadherin repression across all disease groups (p = 0.000, p = 0.000, respectively). By multivariate analysis, the experimental data show that the factors influencing renal survival were eGFR [relative risk (RR) 4.39 (95%CI 1.342, 14.393), p = 0.014] and the degree of expression of Twist [RR 3.43 (95% CI 1.098, 10.684), p = 0.034].
Our results raise the possibility that Twist activation is a common mechanism in the pathophysiology of a wide range of chronic hypoxic renal diseases and that Twist staining in renal biopsy specimens might provide a valuable histologic index of progression.
American Journal of Nephrology 01/2012; 35(2):141-51. · 2.54 Impact Factor
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ABSTRACT: 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.
PLoS ONE 01/2012; 7(1):e30185. · 4.09 Impact Factor
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Xueyan Guo,
Yongquan Shi,
Yawen Gou,
Jipeng Li,
Shuang Han,
Yanqi Zhang,
Jianhua Huo, Xiaoxuan Ning,
Li Sun,
Yu Chen,
Shiren Sun,
Daiming Fan
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ABSTRACT: Our previous works revealed that human ribosomal protein S13 (RPS13) was up-regulated in multidrug-resistant gastric cancer cells and overexpression of RPS13 could protect gastric cancer cells from drug-induced apoptosis. The present study was designed to explore the role of RPS13 in tumorigenesis and development of gastric cancer. The expression of RPS13 in gastric cancer tissues and normal gastric mucosa was evaluated by immunohistochemical staining and Western blot analysis. It was found RPS13 was expressed at a higher level in gastric cancer tissues than that in normal gastric mucosa. RPS13 was then genetically overexpressed in gastric cancer cells or knocked down by RNA interference. It was demonstrated that up-regulation of RPS13 accelerated the growth, enhanced in vitro colony forming and soft agar cologenic ability and promoted in vivo tumour formation potential of gastric cancer cells. Meanwhile, down-regulation of RPS13 in gastric cancer cells resulted in complete opposite effects. Moreover, overexpression of RPS13 could promote G1 to S phase transition whereas knocking down of RPS13 led to G1 arrest of gastric cancer cells. It was further demonstrated that RPS13 down-regulated p27(kip1) expression and CDK2 kinase activity but did not change the expression of cyclin D, cyclin E, CDK2, CDK4 and p16(INK4A). Taken together, these data indicate that RPS13 could promote the growth and cell cycle progression of gastric cancer cells at least through inhibiting p27(kip1) expression.
Journal of Cellular and Molecular Medicine 11/2009; 15(2):296-306. · 4.13 Impact Factor
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Kidney International 07/2009; 76(4):461-462. · 6.61 Impact Factor
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ABSTRACT: 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.
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.
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.
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.
Pathology 06/2009; 41(4):335-41. · 2.38 Impact Factor
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Shiren Sun, Xiaoxuan Ning,
Yanqi Zhang,
Yuanyuan Lu,
Yongzhan Nie,
Shuang Han,
Lili Liu,
Rui Du,
Lin Xia,
Lijie He,
Daiming Fan
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ABSTRACT: 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.
Kidney International 04/2009; 75(12):1278-87. · 6.61 Impact Factor
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ABSTRACT: 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.
International Journal of Cancer 12/2008; 124(7):1707-15. · 5.44 Impact Factor
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Lili Liu, Xiaoxuan Ning,
Shuang Han,
Hongbo Zhang,
Li Sun,
Yongquan Shi,
Shiren Sun,
Changcun Guo,
Fang Yin,
Taidong Qiao,
Kaichun Wu,
Daiming Fan
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ABSTRACT: Hypoxia is a common environmental stress that influences signaling pathways and cell function, which through initiates intracellular
signaling pathways and hence leads to the activation of the transcription factor hypoxia-inducible factor 1 (HIF-1). In this
study, we initially confirm that hypoxia activates HIF-1α protein expression in a time-dependent manner with a maximum reached
at 60 min in vitro and 4h in vivo in gastric mucosa epithelial cells. The expression of HIF-1α is correlated with the activation
of HIF-1 DNA binding and transcriptional activity. Hypoxia does not affect HIF-1α mRNA transcription but regulates HIF-1α
protein expression through a translation-dependent pathway to regulate protein synthesis. Hypoxia could induce phosphorylation
of Akt, MAPK (ERK), and a target of p70S6K1. PI3K and MAPK inhibitor and LY294002 and U0126 could inhibit hypoxia-induced
HIF-1 and VEGF expression. We also investigated the role of reactive oxygen species (ROS) involved in HIF-1 and VEGF expression.
Exogenous addition of H2O2 was sufficient to activate Akt and ERK, scavengers of H2O2 significantly inhibited hypoxia-induced Akt and ERK, and subsequent HIF-1α expression and transcriptional activity. In conclusion,
our data suggested that hypoxia-PI3K signaling through Akt and ERK kinases regulated ROS-dependent, hypoxia-induced HIF-1
activation and VEGF expression in gastric mucosa epithelial cells.
Molecular Biology 05/2008; 42(3):403-412. · 0.66 Impact Factor
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Lili Liu, Xiaoxuan Ning,
Li Sun,
Hongbo Zhang,
Yongquan Shi,
Changcun Guo,
Shuang Han,
Jie Liu,
Shiren Sun,
Zheyi Han,
Kaichun Wu,
Daiming Fan
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ABSTRACT: 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-1 alpha expression increased maximal -8.9-fold or -14.8-fold of IC(50) 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-1 alpha 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-1 alpha 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-1 alpha expression with siRNA. We further demonstrated that HIF-1 alpha 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-1 alpha may confer hypoxia-induced drug resistance via inhibition of drug-induced apoptosis and decreases in intracellular drug accumulation.
Cancer Science 01/2008; 99(1):121-8. · 3.33 Impact Factor
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Xiaoxuan Ning,
Shiren Sun,
Liu Hong,
Jie Liang,
Lili Liu,
Shuang Han,
Zhiguo Liu,
Yongquan Shi,
Yuan Li,
Weiqin Gong,
Shanhong Zhang,
Yu Chen,
Xueyan Guo,
Yi Cheng,
Kaichun Wu,
Daiming Fan
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ABSTRACT: 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 beta-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 beta-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 beta-catenin protein expression and transcriptional activation of Tcf/LEF.
Molecular Cancer Research 01/2008; 5(12):1254-62. · 4.29 Impact Factor
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Lili Liu, Xiaoxuan Ning,
Li Sun,
Yongquan Shi,
Shuang Han,
Changcun Guo,
Yu Chen,
Shiren Sun,
Fang Yin,
Kaichun Wu,
Daiming Fan
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ABSTRACT: Drug resistance is a major obstacle in the development of effective cancer therapy. It was reported that many chemotherapeutic drugs such as vincristine (VCR), a potent anti-tumor agent that associates with microtubules and disrupts the microtubular system, was found in acquisition of drug-resistance associated with an increase of HIF-1 expression via activating the NF-gammaB signal pathway. However, the multifactorial mechanism responsible for VCR increased HIF-1alpha expression remains to be fully elucidated. MGr1-Ag was previously reported by our laboratory as an upregulated protein in VCR-resistant cell lines SGC7901/VCR. In our study, detection of HIF-1 expression in SGC7901 cells and SGC7901/VCR cell or VCR-treated SGC7901cells showed that VCR could induce a significant expression of HIF-1alpha and VCR-resistant SGC7901/VCR cells had much higher expression of HIF-1alpha. Under nonhypoxic condition, VCR could enhance DNA binding activity and transcriptional activity of HIF-1alpha by 5.42- and 9.42-fold, respectively. Further study showed that forced expression of MGr1-Ag/37LRP upregulated HIF-1alpha protein expression and transcriptional activity in gastric cancer cell under nonhypoxic condition whereas siRNA targeting MGr1-Ag showed a markedly decreased VCR-induced HIF-1alpha expression and transcriptional activity (P < 0.05). SiRNA targeting FAK or inhibitors of phosphatidylinositol 3-kinase (PI3K) and MAPK could inhibit VCR-induced HIF-1alpha expression, suggesting FAK-PI3K and p42/44MAPK (Erk1/2) may be the major signaling molecules in MGr1-Ag/37LRP-induced HIF-1alpha expression and activity. These data support a model in which MGr1-Ag was a focal point for the convergence of VCR-mediated signaling events leading to HIF-1Alpha induction, thus revealing a novel aspect of HIF-1alpha regulation.
Molecular and Cellular Biochemistry 10/2007; 303(1-2):151-60. · 2.06 Impact Factor
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Jie Liang,
Guanhong Luo, Xiaoxuan Ning,
Yongquan Shi,
Huihong Zhai,
Shiren Sun,
Haifeng Jin,
Zhenxiong Liu,
Faming Zhang,
Yuanyuan Lu,
Yunping Zhao,
Xiong Chen,
Hongbo Zhang,
Xuegang Guo,
Kaichun Wu,
Daiming Fan
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ABSTRACT: The prion protein (PrPC) has a primary role in the pathogenesis of transmissible spongiform encephalopathies, which causes prion disorders partially due to Ca2+ dysregulation. In our previous work, we found that overexpressed PrPC in gastric cancer was involved in apoptosis, cell proliferation, and metastasis of gastric cancer. To better understand how PrPC acts in gastric cancer, a human microarray was performed to select differentially regulated genes that correlate with the biological function of PrPC. The microarray data were analyzed and revealed 3798 genes whose expression increased at least 2-fold in gastric cancer cells transfected with PrPC. These genes encode proteins involved in several aspects of cell biology, among which, we specially detected molecules related to calcium, especially the S100 calcium-binding proteins, and found that PrPC upregulates S100A1, S100A6, S100B, and S100P but downregulates CacyBP in gastric cancer cells. We also found that intracellular Ca2+ levels in cells transfected with PrPC increased, whereas these levels decreased in knockdowns of these cells. Taken together, PrPC might increase intracellular Ca2+, partially through calcium-binding proteins, or PrPC might upregulate the expression of S100 proteins, partially through stimulating the intracellular calcium level in gastric cancer. Though the underlying mechanisms need further exploration, this study provides a new insight into the role of PrPC in gastric cancer and enriches our knowledge of prion protein.
Biochemistry and Cell Biology 07/2007; 85(3):375-83. · 2.67 Impact Factor
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ABSTRACT: Calcyclin-binding protein/Siah-1-interacting protein (CacyBP/SIP), a target protein of S100, has been identified as a component of a novel ubiquitinylation complex leading to beta-catenin degradation, which was found to be related to the malignant phenotypes of gastric cancer. However, the roles of CacyBP/SIP in renal cell carcinoma still remain unclear. In the present study, we had analyzed the expression of the CacyBP/SIP protein in human renal cancer cells and clinical tissue samples. The possible roles of CacyBP/SIP in regulating the malignant phenotype of renal cancer cells were also investigated. The results demonstrated that the expression of CacyBP/SIP was markedly down-regulated in renal cell carcinoma tissues and cell lines. Ectopic overexpression of CacyBP/SIP in A498 cells inhibited the proliferation of this cell and delayed cell cycle progression significantly, which might be related to the down-regulation of Cyclin D1 through reducing beta-catenin protein. CacyBP/SIP also suppressed colony formation in soft agar and its tumorigenicity in nude mice. Taken together, our work showed that CacyBP/SIP, as a novel down-regulated gene in renal cell carcinoma, suppressed proliferation and tumorigenesis of renal cancer cells.
Biochemical and Biophysical Research Communications 05/2007; 356(4):864-71. · 2.48 Impact Factor
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Jun Wang,
Kaichun Wu,
Dexin Zhang,
Hongwei Tang,
Huahong Xie,
Liu Hong,
Yanglin Pan,
Mei Lan,
Shengjuan Hu, Xiaoxuan Ning,
Daiming Fan
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ABSTRACT: The roles of angiopoietins in gastric cancer progression are still not fully understood. In this study the expressions of angiopoietin-1 (Ang-1), -2 (Ang-2) were compared by immunohistochemistry in 53 gastric cancer and 23 normal gastric mucosa samples. Results revealed that Ang-2 expression was significantly increased in gastric cancer tissues (74%) and was correlated with higher TNM stage, lymph node metastasis as well as distance metastasis. The expression of Ang-1 was also elevated in cancerous tissues (66%) and significantly associated with differentiation degree. In addition, Ang-2 as well as its receptor Tie2 expressions were higher in 12 pairs of gastric cancer tissue samples than those in corresponding adjacent samples by Western blot, while Ang-1 expression showed great heterogeneity. Furthermore, the expressions of Ang-1 and Ang-2 were almost positive in eight gastric cancer cell lines. Among them, AGS expressed both Ang-2 and a relatively moderate amount of Ang-2(443), a novel splice form of Ang-2, while others showed only Ang-2 mRNA expression.
Biochemical and Biophysical Research Communications 12/2005; 337(1):386-93. · 2.48 Impact Factor
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ABSTRACT: Hypoxia-inducible factor 1 (HIF-1) plays a critical role in controlling oxygen delivery and metabolic adaptation to hypoxic conditions in hypoxic tumor cells. HIF-1 activation is initiated by several factors including mitogen-activated protein kinase (MAPK) superfamily. We have previously reported that mitogen-activated protein kinase phosphatase DUSP1 (MKP-1) was implicated in the negative regulation of HIF-1alpha subunit phosphorylation and HIF-1 activity. However, the molecular basis by which MKP-1 influences HIF-1 activity is not clarified. In this paper, we show that hypoxia transcriptionally induces MKP-1 expression in a time-dependent manner. Meanwhile, hypoxia also activates extracellular signal-regulated kinase (ERK) whose activity is enhanced or reduced by MKP-1 suppression or MKP-1 overexpression, respectively. We also show that suppression of MKP-1 expression facilitates the interaction between HIF-1alpha subunit and p300, a co-activator of HIF-1. Moreover, MKP-1 suppression leads to enhanced HIF-1 activity, which can be counteracted by PD98059, an ERK kinase inhibitor. Taken together, the results presented here suggest that hypoxia-induced MKP-1 protects overactivation of HIF-1 activation through inhibiting ERK kinase activity.
Experimental Cell Research 11/2005; 309(2):410-8. · 3.58 Impact Factor
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ABSTRACT: Gastric cancer is one of the most common malignant tumors in China. This paper focuses on the development of a DNA vaccine containing four mimotopes of MG7Ag for gastric cancer (multi-epitope vaccine). By inoculating BALB/c mice, the vaccine was characterized and compared with a similar vaccine containing only one mimotope (mono-epitope vaccine) and other controls. Cellular ELISA indicated that serum titer of antibody against MG7Ag was significantly higher in mice immunized with the multi-epitope vaccine than that in the group immunized with the mono-epitope vaccine (0.8627 vs 0.6754, P < 0.05). And ELISPOT assay showed that the number of INF-gamma spots induced by multi-epitope vaccine was significantly larger than that of the group immunized with mono-epitope vaccine(93.3 vs 70.7, P < 0.05). Two weeks after tumor challenge, the weight of tumor in each mouse was evaluated, and the tumor masses formed in the mice immunized with multi-epitope vaccine were markedly smaller than those formed in the mice immunized with mono-epitope vaccine. These studies demonstrated that both humoral and cellular response were induced by the two vaccines and the efficiency of multi-epitope vaccine is stronger than that of the mono-epitope vaccine.
Cancer biology & therapy 03/2005; 4(3):308-12. · 2.64 Impact Factor
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ABSTRACT: CpG oligodeoxynucleotides (CpG ODN) have been shown to have potent adjuvant activity for a wide range of antigens. Of particular interest is their improved activity when closely associated with the antigen. The purpose of this study is to construct a nanovaccine coencapsulated with a gastric cancer specific antigen MG7 mimotope peptide and adjuvant CpG ODN 1645 using new nanotechnology as nanoemulsion and evaluate its immunocompetence. Nanoemulsion vaccine was prepared using magnetic ultrasound methods. BALB/c mice were immunized and the in vivo effectiveness was evaluated using tumor challenge assay. It was shown that the tumor masses formed in the mice immunized with coencapsulated nanovaccine (0.0825 g) markedly smaller (P < 0.01) than those formed in the mice immunized with nanovaccine encapsulated with antigen peptide alone (0.4465 g). A tumor inhibiting rate as high as 82.5% of the coencapsulated nanovaccine was obtained, while nanovaccine encapsulated with peptide only could not achieve the same effect (28.5%) (P < 0.01). Enzyme-linked immunospot assay (ELISPOT) showed that immunization using MG7 mimotope peptide coencapsulated with CpG ODN within the same nanoemulsion enhanced the frequency of splenocytes secreting IFN-gamma significantly (P < 0.01) when compared with immunization using MG7 peptide encapsulated in nanoemulsion alone (197spots/1 x 10(6) vs. 73 spots/1 x 10(6)). Cellular ELISA indicated that serum titer of antibody against MG7-Ag was significantly higher (P < 0.01) in mice immunized with coencapsulation form nanovaccine (0.7884) than that in the group immunized with nanovaccine encapsulated with MG7 peptide alone (0.3616). Using intracellular flow cytometric analysis, it was found that the IFN-gamma response was contributed by CD4+ T-cells. Our experiments suggest that a vaccinal approach using nano-delivery system carrying in tumoral epitope and CpG ODN as adjuvant may have important implications for cancer therapy.
Cancer biology & therapy 02/2005; 4(2):218-24. · 2.64 Impact Factor
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ABSTRACT: To study the mechanisms of multidrug resistance (MDR) mediated by human 67 000 laminin receptor (LR) with a relative molecular mas of 67 000 in gastric cancer cells.
Antisense RNA expression vector corresponding to LR precursor (LRP) was constructed by DNA recombinant technique, and transferred into gastric cancer MDR cells SGC7901/VCR with Lipofect AMINE. Western blot was employed to determine the LR expression level in gastric cancer cells. The sensitivity of gastric cancer cells to chemotherapeutic drugs was evaluated with MTT assay. Flow cytometry was used to analyze the cell cycle and to assess the mean fluorescence intensity of intracellular adriamycin in gastric cancer cells.
Western blotting analysis demonstrated a decreased expression level of LR in SGC7901/VCR cells transfected with LRP antisense RNA expression vector. In comparison with the gastric cancer cells with out transfection or transfected with invalid vector, LR down-regulated transfectants (SGC7901/VCR-anLRP) showed higher sensitivity to vincristine, adriamycin, 5-fluodrouracil and cisplatin, and increased accumulation and retention of adriamycin. Cell cycle analysis suggested G1 block and spontaneous apoptosis of SGC7901/VCR-anLRP cells.
LR might take part in mediation of MDR in gastric cancer cells through interfering with drug accumulation and cell apoptosis.
Zhonghua yi xue za zhi 08/2002; 82(14):986-9.
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ABSTRACT: 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.
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.
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 IC(50) 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 IC(50) 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.
Overexpression of MGr1-Ag is associated with the MDR phenotype of gastric cancer cells.
Gastric Cancer 02/2002; 5(3):154-9. · 2.42 Impact Factor
