Shujuan Shao

Gadd45a Interacts with Aurora-A and Inhibits Its Kinase Activity

Centrosome stability is required for successful mitosis in mammalian cells. Amplification of the centrosome leads to chromosomal missegregation and generation of aneuploidy, which are closely associated with cell transformation and tumorigenesis (Doxsey, S. J. (2001) Nat. Cell Biol. 3, E105-E108; Hinchcliffe, E. H., and Sluder, G. (2001) Genes Dev. 15, 1167-1181; Pihan, G. A., Purohit, A., Wallace, J., Malhotra, R., Liotta, L., and Doxsey, S. J. (2001) Cancer Res. 61, 2212-2219). However, there are currently limited insights into mechanism(s) for this critical biological event. Here we show that Gadd45a, a DNA damage-inducible protein that is regulated by tumor suppressors p53 and BRCA1, participates in the maintenance of centrosome stability. Mouse embryonic fibroblasts derived from gadd45a knock-out mice exhibit centrosome amplification (designated as increased centrosome numbers). Introduction of exogenous Gadd45a into mouse embryonic fibroblasts isolated from gadd45a-null mice substantially restored the normal centrosome profile. In contrast to p21waf1/cip1, which ensures coordinated initiation of centrosome, Gadd45a had no significant effect on centrosome duplication in S phase. Interestingly Gadd45a was found to physically associate with Aurora-A protein kinase, whose deregulated expression results in centrosome abnormality. Furthermore Gadd45a was demonstrated to strongly inhibit Aurora-A kinase activity and to antagonize Aurora-A-induced centrosome amplification. These findings identify a novel mechanism for Gadd45a in the maintenance of centrosome stability and broaden understandings of p53- and BRCA1-regulated signaling pathways in maintaining genomic fidelity.

Fructose-Bisphosphate Aldolase A Is a Potential Metastasis-Associated Marker of Lung Squamous Cell Carcinoma and Promotes Lung Cell Tumorigenesis and Migration

Fructose-bisphosphate aldolase A (ALDOA) is a key enzyme in glycolysis and is responsible for catalyzing the reversible conversion of fructose-1,6-bisphosphate to glyceraldehydes-3-phosphate and dihydroxyacetone phosphate. ALDOA contributes to various cellular functions such as muscle maintenance, regulation of cell shape and mobility, striated muscle contraction, actin filament organization and ATP biosynthetic process. Here, we reported that ALDOA is a highly expressed in lung squamous cell carcinoma (LSCC) and its expression level is correlated with LSCC metastasis, grades, differentiation status and poor prognosis. Depletion of ALDOA expression in the lung squamous carcinoma NCI-H520 cells reduces the capabilities of cell motility and tumorigenesis. These data suggest that ALDOA could be a potential marker for LSCC metastasis and a therapeutic target for drug development.

Centrosomal Nlp is an oncogenic protein that is gene-amplified in human tumors and causes spontaneous tumorigenesis in transgenic mice

Disruption of mitotic events contributes greatly to genomic instability and results in mutator phenotypes. Indeed, abnormalities of mitotic components are closely associated with malignant transformation and tumorigenesis. Here we show that ninein-like protein (Nlp), a recently identified BRCA1-associated centrosomal protein involved in microtubule nucleation and spindle formation, is an oncogenic protein. Nlp was found to be overexpressed in approximately 80% of human breast and lung carcinomas analyzed. In human lung cancers, this deregulated expression was associated with NLP gene amplification. Further analysis revealed that Nlp exhibited strong oncogenic properties; for example, it conferred to NIH3T3 rodent fibroblasts the capacity for anchorage-independent growth in vitro and tumor formation in nude mice. Consistent with these data, transgenic mice overexpressing Nlp displayed spontaneous tumorigenesis in the breast, ovary, and testicle within 60 weeks. In addition, Nlp overexpression induced more rapid onset of radiation-induced lymphoma. Furthermore, mouse embryonic fibroblasts (MEFs) derived from Nlp transgenic mice showed centrosome amplification, suggesting that Nlp overexpression mimics BRCA1 loss. These findings demonstrate that Nlp abnormalities may contribute to genomic instability and tumorigenesis and suggest that Nlp might serve as a potential biomarker for clinical diagnosis and therapeutic target.

Hepatocyte Growth Factor Induces Invasion and Migration of Ovarian Cancer Cells by Decreasing the Expression of E-cadherin, β-catenin, and Caveolin-1

Distant metastases are unusual occurrences at presentation and during the progression of epithelial ovarian cancer. There are no good clinical predictors of this phenomenon. In this study, we examined the role of hepatocyte growth factor (HGF) in cell metastasis in ovarian cancer HO8910 cells. We found that HGF has functions in biological processes essential to metastasis, including morphological remodeling, invasion and migration (P = 0.000, P = 0.001). Western blotting showed that in HGF treated group, the expression of E‐cadherin, β‐catenin, and caveolin‐1 was decreased as compared with the control group (P = 0.000, P = 0.002, P = 0.000). Immunofluorescence staining demonstrated that the population of E‐cadherin, β‐catenin, and caveolin‐1 at the cell membrane was downregulated. Reverse transcriptase polymerase chain reaction analysis revealed the decreased expression of E‐cadherin, β‐catenin, and caveolin‐1 at the mRNA level. Our data indicated that HGF leads to downregulation of E‐cadherin, β‐catenin, and caveolin‐1, disassembly of cell–cell contacts, and invasion and migration enhancement in human ovarian cancer cells. Anat Rec 293:1125–1133, 2010.

Caveolin-1 knockdown is associated with the metastasis and proliferation of human lung cancer cell line NCI-H460

Caveolin-1 (CAV-1), one component of caveolae, involves in multiple cellular processes and signal transductions. We previously showed that the expression of CAV-1 gene in NCI-H446 cells inhibited cell proliferation and promoted cell metastasis. Here we explore the function of CAV-1 on tumor growth and metastasis by using NCI-H460 in vitro. First, we established NCI-H460 cell line, which CAV-1 was stably knockdown. Then we investigated the effects of CAV-1 on the morphology, proliferation, cell cycle and metastasis potential for NCI-H460 cell by crystal violet stains, CCK-8, colony formation, flow cytometry, scratch-wound assay and transwell assay. Western blot was used to examine the expression changes of cyclin D1, PCNA, E-cadherin and β-catenin. Our results showed stable knockdown of CAV-1 inhibited the proliferation of NCI-H460 cells. Cell cycle of the transfected cells was arrested in G1/S phase and the expressions of cyclin D1 and PCNA protein were downregulated. Downregulation of CAV-1 promoted the migration and invasion abilities of NCI-H460 cells in vitro. The expression of β-catenin increased and the level of E-cadherin decreased. In summary, our findings provide experimental evidence that CAV-1 may function as a proproliferative and antimetastatic gene in NCI-H460 cell line.

Anatomical View of the Protein Composition and Protein Characteristics for Gloydius shedaoensis Snake Venom via Proteomics Approach

The bioactive protein components from snake venom complexes have been utilized for studies of enzymology, structural biology, and pharmacology. The Gloydius shedaoensis snake (GSS) is the only snake species found exclusively at the Chinese Shedao (snake) Island in Dalian. To investigate the protein components of Chinese GSS venom (GSSV), we initialized a proteomic assay for GSSV by the combination of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis with high‐performance liquid chromatography (HPLC)‐nanoelectrospray ionization tandem mass spectrometry (nESI‐MS/MS). Thirty gel bands visualized by Coomassie blue staining were excised and digested by trypsin. The tryptic‐digested peptides were separated by HPLC and subsequently sequenced by nESI‐MS/MS. Twenty‐four types of proteins were identified by searching the mass spectrometry data against NCBInr database through TurboSequest Bioworks. The most abundant proteins are phospholipase A2, metalloproteinase, L‐amino acid oxidase (LAAO), serine protease/thrombin‐like enzyme. Except for 20 types of known snake venom proteins, the homolog peptides of hypothetical protein PFLC2230, LOC495267 protein, DEAD/DEAH box helicase‐like, and pancreatic trypsin 1 from other organisms are matched for GSSV protein components. Mass spectrometric data also indicated that (i) dimerization happens to PLA2s as monomer and dimer of PLA2s coexist in GSSV and (ii) truncation or hydrolysis might happen to LAAOs as three molecular‐weight‐ranged LAAO species are present in GSSV. The results provide an “anatomical” view of the protein composition and important information for protein characteristics of GSSV. Anat Rec, 2011.

Structural modifications due to interface chemistry at metal-nitride interfaces

Based on accurate first principles density functional theory (DFT) calculations, an unusual phenomenon of interfacial structural modifications, due to the interface chemistry influence is identified at two metal-nitride interfaces with strong metal-nitrogen affinity, Al/TiN {111} and Al/VN {111} interfaces. It is shown that at such interfaces, a faulted stacking structure is energetically preferred on the Al side of the interface. And both intrinsic and extrinsic stacking fault energies in the vicinity Al layers are negligibly small. However, such phenomenon does not occur in Pt/TiN and Pt/VN interfaces because of the weak Pt-N affinity. Corresponding to structural energies of metal-nitride interfaces, the linear elasticity analysis predicts characteristics of interfacial misfit dislocations at metal-nitride interfaces.

Elevated GAPDH expression is associated with the proliferation and invasion of lung and esophageal squamous cell carcinomas.

Glyceraldehyde‐3‐phosphate dehydrogenase, is one of the most investigated housekeeping genes and widely used as an internal control in analysis of gene expression levels. The present study was designed to assess whether GAPDH is associated with cancer cell growth and progression and, therefore may not be a good internal control in cancer research. Our results from clinical tissue studies showed that the levels of GAPDH protein were significantly up‐regulated in lung squamous cell carcinoma tissues, compared with the adjacent normal lung tissues, and this was confirmed by western blotting and immunohistochemistry. GAPDH knockdown by siRNA resulted in significant reductions in proliferation, migration, and invasion of lung squamous carcinoma cells in vitro. In a nude mouse cancer xenograft model, GAPDH knockdown significantly inhibited the cell proliferation and migration/invasion in vivo. In summary, GAPDH may not be an appropriate internal control for gene expression studies, especially in cancer research. The role of GAPDH in cancer development and progression should be further examined in pre‐clinical and clinical studies.

Higher expression of Caveolin-1 inhibits human small cell lung cancer (SCLC) apoptosis in vitro.

Small cell lung cancer (SCLC) is the most aggressive type of lung cancer, and its treatment is closely associated with apoptosis. Caveolin-1 plays an important role in the development of a variety of human cancers. This study sought to investigate the influence of Caveolin-1 on the apoptosis of SCLC in vitro. We demonstrate that higher expression of Caveolin-1 leads to inhibition of cisplatin and Ultraviolet Radiation (UVR)-induced apoptosis in SCLC cells; and also could decrease caspase-3 activity and increase the stability of Bcl-2 at the protein level. Our findings illuminate a potential molecular mechanism regarding CAV-1s role as anti-apoptosis protein.

Reversal effect of Raf-1/Mdr-1 siRNAs co-transfection on multidrug resistance in KBv200 cell line

Multidrug resistance (MDR) is a major barrier for chemotherapy of many cancers. Mdr-1 plays a key role in the development of MDR as extensively verified. However, the role of Raf-1 overexpression in the development of multidrug resistance in human squamous carcinoma (KBv200) cells remains largely unknown. The aim of this study was to investigate the correlation of Raf-1 overexpression with the development of multidrug resistance in KBv200 cells. Furthermore, we explored the reversal effect of Raf-1 siRNA transfection and Raf-1/Mdr-1 siRNAs co-transfection on the multidrug resistance of KBv200 cells and potential mechanism of reversing the multidrug resistance. MTT and flow cytometry assay were used to investigate the reversal effect of single transfection with either Raf-1 or Mdr-1 siRNA and double transfection with Raf-1/Mdr-1 siRNAs to vincristine of KBv200 cells. RT-PCR, immunofluorescence and Western Blot were used to detect mRNA and protein expression of Raf-1 and multidrug-resistant gene Mdr-1. The results of gene detection showed that the expression levels of both Raf-1 and Mdr-1 were greatly decreased upon Raf-1 silencing alone or in combination with Mdr-1 silencing. Raf-1 or Mdr-1 siRNA single transfection could reverse the multidrug resistance of KBv200 cells effectively. Compared with single transfection, Raf-1/Mdr-1 siRNAs co-transfection can significantly reduce IC(50) values and increase the apoptotic rates of KBv200 cells. The above results suggested that Raf-1 gene may be a novel target for reversing the multidrug resistance of human squamous carcinoma cells. Raf-1/Mdr-1 siRNAs co-transfection might be a promising approach to abrogate the multidrug resistance of cancer cells. The potential mechanism may be via inhibiting the multidrug-resistant gene Mdr-1 expression efficiently.