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Song He,
Mudan Lu,
Wenqun Xue,
You Wang,
Yueming Zhao,
Shangfeng Gao,
Qing Ke,
Yonghua Liu, Peng Li,
Xiaopeng Cui,
Chun Cheng,
Aiguo Shen
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ABSTRACT: The p27(Kip1) cyclin-dependent kinase inhibitor is a negative regulator of cell cycle progression in G(1) phase; recent studies suggested that oncogenically activated kinase Akt/PKB can also phosphorylate p27(kip1) at T157 inducing its relocalization to the cytoplasm. To evaluate the significance of p-p27 Thr157 and PI3K pathway in hepatocellular carcinoma (HCC), we studied 51 hepatocellular carcinomas along with corresponding nontumoral tissue and the HCC cell lines. Immunohistochemistry and western blot analysis suggested that p-p27 Thr157 was overexpressed in HCC, which was positively correlated with proliferation marker Ki-67. Correlation analysis was performed among immunohistochemistry-assessed level of p-p27 Thr157, survival, and major clinical and pathological variables. Overexpressed p-p27 Thr157 was correlated with histological differentiation (P < 0.05). Univariate analysis showed that p-p27 Thr157 and Ki-67 expression were correlated with tumor-specific survival. In a multivariate analysis, p-p27 Thr157 and Ki-67 protein expression were proved to be an independent prognostic for HCC. While in vitro, treatment of LY294002 and transduction of mutant p27 (T157A) could diminish the expression of p-p27 Thr157 protein and arrest cells growth. Our results suggested that p-p27 Thr157 protein expression may be a favorable independent poor prognostic parameter for HCC. Gene therapeutic approaches aimed at PI3K or the pharmacologic inhibitors of PI3K and transduction of mutant p27 (T157A) to down-regulate p-p27 Thr157 expression could be developed for the management of HCC.
Medical Oncology 03/2011; 28(1):94-104. · 2.14 Impact Factor
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Qing Ke,
Juling Ji,
Chun Cheng,
Yixin Zhang,
Mudan Lu,
You Wang,
Li Zhang, Peng Li,
Xiaopeng Cui,
Li Chen,
Song He,
Aiguo Shen
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ABSTRACT: Spy1 is a novel cell cycle regulatory gene, which can control cell proliferation and survival through the atypical activation of cyclin-dependent kinases. Recent studies suggested that deregulation of Spy1 expression plays a key role in oncogenesis. To investigate the potential roles of Spy1 in hepatocellular carcinoma (HCC), expression of Spy1 was examined in human HCC samples.
Immunohistochemistry and Western blot analysis was performed for Spy1 in 61 hepatocellular carcinoma samples. The data were correlated with clinicopathological features. The univariate and multivariate survival analyses were also performed to determine their prognostic significance.
Spy1 was overexpressed in hepatocellular carcinoma as compared with the adjacent normal tissue. High expression of Spy1 was associated with histological grade and the level of alpha fetal protein (AFP) (P=0.009 and 0.003, respectively), and Spy1 was positively correlated with proliferation marker Ki-67 (P<0.001). Univariate analysis showed that Spy1 expression was associated with poor prognosis (P=0.03). Multivariate analysis indicated that Spy1 and Ki-67 protein expression was an independent prognostic marker for HCC (P=0.001 and 0.012, respectively). While in vitro, following release from serum starvation of HuH7 HCC cell, the expression of Spy1 was upregulated.
Our results suggested that Spy1 overexpression is involved in the pathogenesis of hepatocellular carcinoma, it may be a favorable independent poor prognostic parameter for hepatocellular carcinoma.
Experimental and Molecular Pathology 08/2009; 87(3):167-72. · 2.42 Impact Factor
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Mudan Lu,
Jianbo Ma,
Wenqun Xue,
Chun Cheng,
You Wang,
Yueming Zhao,
Qing Ke,
Haiou Liu,
Yonghua Liu, Peng Li,
Xiaopeng Cui,
Song He,
Aiguo Shen
[show abstract]
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ABSTRACT: The forkhead box proteins (FOXO proteins) comprise a large family of functionally diverse transcription factors involved in cellular proliferation, transformation, differentiation and longevity. Recently, ubiquitination and proteasome degradation of FOXO3a have been reported. In this study, we investigated the role of FOXO3a and Skp2 in human hepatocellular carcinoma progression. Immunohistochemical analysis was performed on formalin-fixed paraffin sections of 91 specimens. Furthermore in vitro, western-blot analysis and protein stabilization studies were used to study the relationship between FOXO3a and Skp2. We found that the expression of FOXO3a was negatively related with Skp2 expression (r = -0.583; p < 0.05) and FOXO3a expression correlated significantly with histological grade (p = 0.000), cirrhosis (p = 0.015), and tumor size (p = 0.043) while Skp2 expression correlated significantly with histological grade (p = 0.000) and tumor size (p = 0.005). Kaplan-Meier analysis revealed that survival curves of low versus high expressers of FOXO3a and Skp2 showed a highly significant separation in HCC (p < 0.01). Our results suggested that FOXO3a and Skp2 may be considered to be important prognosis in human hepatocellular carcinoma. In vitro studies suggested that the degradation of FOXO3a may dependent on the expression of Skp2 in the proliferated Huh7 cells.
Pathology & Oncology Research 04/2009; 15(4):679-87. · 1.37 Impact Factor
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Min Fei,
Mudan Lu,
You Wang,
Yueming Zhao,
Song He,
Shangfeng Gao,
Qing Ke,
Yonghua Liu, Peng Li,
Xiaopeng Cui,
Aiguo Shen,
Chun Cheng
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ABSTRACT: Human hepatocellular carcinoma (HCC) remains incurable with current therapies, and novel biologically based therapies are urgently needed. Arsenic agents have long been used as anticancer agents in traditional Chinese medicine. In this study, to evaluate the effect of As(2)O(3) on HCC cells, we investigate cell growth inhibition, cell cycle arrest, and the molecular mechanism after As(2)O(3) treatment in human HCC cells in vitro. We detected the proliferation of HCC cells by the Cell Counting Kit and FACS/Calibur Flow Cytometer and analyzed the expression and localization of FOXO3a by Western blotting Analysis and Cell Fractionation. Furthermore, we study the Akt activation after As(2)O(3) treatment and the HCC cells proliferation after combination of As(2)O(3) with PI3K inhibitor Wortmannin. As(2)O(3) significantly inhibited the proliferation of all the three HCC cell lines (SMMC7721, HepG2, Hep3B) tested in this study in a dose-dependent manner. Western blotting revealed that treatment HCC cells HepG2 with As(2)O(3) resulted in the increasing of FOXO3a expression and triggered phosphorylation of FOXO3a at the Thr(32) residue decrease. This FOXO3a accumulation correlated well with the As(2)O(3)-induced reduction of active Akt. Nuclear and cytoplasmic protein extracts isolated from the HCC cell line HepG2 revealed that the amount of nuclear FOXO3a was increased by treatment with As(2)O(3), whereas the amount of cytoplasmic FOXO3a was decreased. Both As(2)O(3) and PI3K/Akt inhibitor Wortmannin induced cell cycle arrest. However, compared with As(2)O(3) alone, PI3K inhibitor Wortmannin combined with As(2)O(3) enhanced the antitumor effect of As(2)O(3) through induction of apoptosis. These findings suggest that As(2)O(3) at a clinically safe concentration may be an effective chemotherapeutic agent, and that As(2)O(3) and PI3K/Akt inhibitor Wortmannin may synergize for HCC cells. Taken together, the present study may suggest a specific molecular mechanism by which HCC cell lines are susceptible to the As(2)O(3) therapy through FOXO3a expression and localization.
Medical Oncology 11/2008; 26(2):178-85. · 2.14 Impact Factor
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Mudan Lu,
Min Fei,
Chun Cheng,
You Wang,
Song He,
Yueming Zhao,
Shangfeng Gao,
Qing Ke, Peng Li,
Xiaopeng Cui,
Aiguo Shen
[show abstract]
[hide abstract]
ABSTRACT: The cyclin-dependent kinase (cdk) inhibitor p27(Kip1) is an important regulator of cell cycle progression as it negatively regulates G(0/1) progression and plays a major role in controlling the cell cycle. The screening of the p27(Kip1) sequence identified many potential phosphorylation sites. To investigate the effects of the overexpression of exogenous p27(Kip1) protein lacking the Thr157 sites on subcellular localization, cell cycle, and proliferation, a plasmid was constructed containing mutations of p27(Kip1) at Thr157 (T157A p27), and transfected into the SMMC7721 cell line with Lipofectamine. Wild-type and mutant p27 plasmids T157A were transfected separately as control groups.
We detected the proliferation of SMMC7721 cells by the Cell Counting Kit and FACS/Calibur Flow Cytometer and analyzed the expression and localization of p27(Kip1) by Western blotting analysis and cell fractionation. The cdk2 dependent kinase activity was determined by in vitro kinase assay.
Proliferation of SMMC7721 cells was greatly inhibited and cell cycle was arrested in G(0/1) phase after exogenous p27(Kip1) mutant expression much more than wild-type p27(Kip1). The expressed T157A p27(Kip1) proteins were translocated from the cytoplasm into nucleus much more compare with wild-type. Compared with pcDNA3.1-Myc control, transient transfection of T157A p27(Kip1) decreased expression of cyclin D1 and the phosphorylated form of retinoblastoma protein.
These findings support the potential effectiveness of a PI3K/Akt-resistant phosphorylated form of p27 in hepatocellular carcinoma gene therapy.
Archives of Medical Research 09/2008; 39(6):573-81. · 1.88 Impact Factor
