Yanhui Yu

Identification of metastasis associated proteins in human lung squamous carcinoma using two-dimensional difference gel electrophoresis and laser capture microdissection

A quantitative proteomic approach was used to discover potential protein markers associated with lymph node metastasis (LNM) in human lung squamous carcinoma (LSC). Laser capture microdissection was performed to purify LSC cells with LNM (LNM LSC) and LSC without LNM (non-LNM LSC). The differentially expressed proteins between pooled microdissected non-LNM LSC and LNM LSC cells were identified by two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS). 14 proteins were found to be differentially expressed between non-LNM LSC and LNM LSC. Among these proteins, ten proteins were overexpressed in LNM LSC compared with non-LNM LSC, and four proteins were downregulated in LNM LSC. Some of these identified proteins (Annexin A2, HSP27, CK19, and 14-3-3sigma) were further confirmed by Western blotting and immunohistochemical analysis. These results show the value of LCM coupled with 2D-DIGE in identifying potential markers for lymph node metastasis of LSC, and also provide further insights into the prognosis of LSC.

Chemoresistance to 5-fluorouracil induces epithelial–mesenchymal transition via up-regulation of Snail in MCF7 human breast cancer cells

5-Fluorouracil (5-FU) is commonly used to treat breast cancer; however, it becomes increasingly ineffective with tumor progression. Epithelial-to-mesenchymal transition (EMT) is a process whereby cells acquire morphologic and molecular alterations facilitating tumor metastasis and progression. Emerging evidence associates chemoresistance with acquisition of EMT in cancer. However, it is not clear whether this phenomenon is involved in acquired resistance to 5-FU. Using a previously established in vitro cell model of 5-fluorouracil-resistant MCF7 cells (MCF7/5-FU), we assessed the cellular morphology, molecular changes, migration and invasion consistent with EMT. We found that silencing of Snail by stable RNA interference reversed the EMT and greatly abolished invasion behavior of MCF7/5-FU cells. We also showed that inhibition of Snail increased the sensitivity of 5-FU-resistant cells to 5-FU. Our study provided a new insight into EMT-like phenotypic changes associated with 5-FU resistance in MCF7 cells. We believed that down-regulation of Snail could be a potential novel therapeutic approach to overcoming chemoresistance and preventing metastasis during 5-FU chemotherapy.

Quantitative Proteomic Analysis of Formalin-fixed and Paraffin-embedded Nasopharyngeal Carcinoma Using iTRAQ Labeling, Two-dimensional Liquid Chromatography, and Tandem Mass Spectrometry

Formalin-fixed, paraffin-embedded (FFPE) tissue specimens represent a potentially valuable resource for protein biomarker investigations. In this study, proteins were extracted by a heat-induced antigen retrieval technique combined with a retrieval solution containing 2% SDS from FFPE tissues of normal nasopharyngeal epithelial tissues (NNET) and three histological types of nasopharyngeal carcinoma (NPC) with diverse differentiation degrees. Then two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins among the types of NPC FFPE tissues. Our study resulted in the identification of 730 unique proteins, the distributions of subcellular localizations and molecular functions of which were similar to those of the proteomic database of human NPC and NNET that we had set up based on the frozen tissues. Additionally, the relative expression levels of cathepsin D, keratin8, SFN, and stathmin1 identified and quantified in this report were consistent with the immunohistochemistry results acquired in our previous study. In conclusion, we have developed an effective approach to identifying protein changes in FFPE NPC tissues utilizing iTRAQ technology in conjunction with an economical and easily accessible sample preparation method.

Role of BCRP as a biomarker for predicting resistance to 5-fluorouracil in breast cancer

PurposeChemotherapy is not only important but also necessary for the patient of breast cancer. Breast cancer resistance protein (BCRP), an atypical drug efflux pump, mediates multidrug resistance in breast cancer. The aim of this study is to search new substrate of BCRP. The result will guide the drug selection of chemotherapy in BCRP-positive breast cancer.MethodsPA317/Tet-on/TRE-BCRP cell induced with doxycycline was used to screen the possible substrates of BCRP by MTT assay. The suspicious substrate [5-fluorouracil (5-Fu)] was further confirmed in PA317 and breast cancer cell MCF-7 by HLCP, apoptosis assay (staining and FACS) and RNAi technique.ResultsMitoxantrone, 5-Fu, adriamycin, Methotrexate, Pirarubicin, and Etoposide were identified as substrates of BCRP. However, Paclitaxel, Vincristine, Vindesine, Mitomycin C, and cisplatin were not mediated by BCRP. 5-Fu was identified as substrate of BCRP for the first time. The further study showed that the intracellular retention dose of 5-Fu and the 5-Fu induced cellular apoptosis all decreased when BCRP highly expressed. Furthermore, 5-Fu accumulation and 5-Fu induced DNA damage increased when BCRP was silenced by RNAi in breast cancer cells.Conclusions5-Fluorouracil may be a specific substrate which can be bound by BCRP. BCRP can predict the sensitivity of breast cancer to 5-Fu. And BCRP-targeted therapy will reverse the resistance of breast cancer to 5-Fu.

Up-regulation of breast cancer resistance protein plays a role in HER2-mediated chemoresistance through PI3K/Akt and nuclear factor-kappa B signaling pathways in MCF7 breast cancer cells

Human epidermal growth factor receptor 2 (HER2/neu, also known as ErbB2) overexpression is correlated with the poor prognosis and chemoresistance in cancer. Breast cancer resistance protein (BCRP and ABCG2) is a drug efflux pump responsible for multidrug resistance (MDR) in a variety of cancer cells. HER2 and BCRP are associated with poor treatment response in breast cancer patients, although the relationship between HER2 and BCRP expression is not clear. Here, we showed that transfection of HER2 into MCF7 breast cancer cells (MCF7/HER2) resulted in an up-regulation of BCRP via the phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-kappa B (NF-κB) signaling. Treatment of MCF/HER2 cells with the PI3K inhibitor LY294002, the IκB phosphorylation inhibitor Bay11-7082, and the dominant negative mutant of IκBα inhibited HER2-induced BCRP promoter activity. Furthermore, we found that HER2 overexpression led to an increased resistance of MCF7 cells to multiple antitumor drugs such as paclitaxel (Taxol), cisplatin (DDP), etoposide (VP-16), adriamycin (ADM), mitoxantrone (MX), and 5-fluorouracil (5-FU). Moreover, silencing the expression of BCRP or selectively inhibiting the activity of Akt or NF-κB sensitized the MCF7/HER2 cells to these chemotherapy agents at least in part. Taken together, up-regulation of BCRP through PI3K/AKT/NF-κB signaling pathway played an important role in HER2-mediated chemoresistance of MCF7 cells, and AKT, NF-κB, and BCRP pathways might serve as potential targets for therapeutic intervention.

Transcriptional suppression of breast cancer resistance protein (BCRP) by wild‐type p53 through the NF‐κB pathway in MCF‐7 cells

Breast cancer resistance protein (BCRP) has been shown to confer multidrug resistance, but the mechanisms of its regulation are poorly understood. Here, we investigate the effects of wild‐type and mutant p53, and nuclear factor kappa‐B (NF‐κB) (p50) on BCRP promoter activity in MCF‐7 cells. Our results demonstrated that wild‐type p53 markedly suppressed BCRP activity and enhanced the chemosensitivity of cells to mitoxantrone, whereas mutant p53 had little inhibitory effect. After inhibition of NF‐κB, similar results were obtained. Following knockdown of endogenous p53, BCRP and p50 expressions were increased, and the chemosensitivity of the cells to mitoxantrone was decreased. We conclude that wild‐type p53 acts as a negative regulator of BCRP gene transcription.

Identification of proteins responsible for the multiple drug resistance in 5-fluorouracil-induced breast cancer cell using proteomics analysis

PurposeThis study aimed to explore the mechanism of multi-drug resistance (MDR) in 5-fluorouracil (5-FU)-induced breast cancer cell MCF-7.MethodsMCF-7 cells were exposed in stepwise escalating concentration of 5-FU to develop the resistant cell line, MCF-7/5-FU. Biological and molecular characteristics of the cells were studied through MTT, flow cytometry, real-time PCR, western-blot, and the global protein profiles between MCF-7/5-FU and parental MCF-7 were compared using proteomic approach. Then some of the differentially expressed proteins were validated by western-blot. In addition, the role of 14-3-3σ was validated using gene transfection.ResultsDrug resistance of MCF-7/5-FU cells to 5-FU, MX, cDDP, ADM, TAXOL all increased significantly compared with MCF-7 cells and that maybe related to BCRP, but not MDR1 and MRP1. Differentially expressed proteins between MCF-7/5-FU and MCF-7 cells were identified; 12 proteins were up-regulated and 18 proteins were down-regulated in MCF-7/5-FU cells. Expressive levels of some proteins in western-blot validation were consistent with the results in proteomic analysis. Enforced 14-3-3σ expression can increase the sensitivity of MCF-7/5-FU cells to 5-FU and cDDP.ConclusionMDR of MCF-7/5-FU likely associated with differentially expressed proteins and 14-3-3σ may play a positive role in chemotherapy. These findings may provide theoretical support for the prediction of chemotherapeutic response and reverse of MDR.

TCRP1 promotes radioresistance of oral squamous cell carcinoma cells via Akt signal pathway

Tongue cancer resistance-associated protein 1 (TCRP1) is a novel gene located on human chromosome 11q13.4 which has been reported as a candidate related to chemotherapeutic resistance to cisplatin. Results suggest that TCRP also contribute to radioresistance in oral squamous cell carcinoma (OSCC) cells. We previously established exogenous overexpression of TCRP1 cell line Tca8113/TCRP1 and TCRP1 knockdown cell line Tca8113/PYM-siRNA and paired control cell lines, which provides a cell model system to investigate the roles and mechanisms of TCRP1-mediated radioresponse in OSCC. In this study, we first compared the radiosensitivity of up/down-regulating expression of TCRP1 cell lines and paired control cell lines by a clonogenic survival assay, Hoechst 33258 staining, cell growth assay, and comet assay. The results indicated that TCRP1 played a significant role in mediating OSCC radioresistance through decreased cells apoptosis and increased cellular proliferation and long-term survival. The further study found that TCRP1 function by up-regulating Akt activity and levels and then elevating the level of NF-κB. In summary, these results provided strong evidence for the linkage between TCRP1 and radiation sensitivity and may provide theoretical base of TCRP1 as a potential molecular mark of estimating the response for irradiation in OSCC.

Cloning and functional characterization of TCRP1, a novel gene mediating resistance to cisplatin in an oral squamous cell carcinoma cell line.

To explore the mechanisms of chemotherapy resistance, we previously established a multi‐drug resistant cell line, Tca8113/Pingyangmycin (Tca8113/PYM) and identified differential expression in known genes and ESTs using microarray analysis. From among those ESTs we have now identified a novel gene producing an mRNA of 1834 nucleotides translated into a protein having 235 amino acids. This gene was denominated as tongue cancer resistance‐associated protein 1 gene (TCRP1, accession number: EF363480). We further determined its functional characteristics. The results demonstrate that TCRP1 mediates a specific resistance to cisplatin in Tca8113 cells by reducing the cisplatin‐induced apoptosis. This suggests that TCRP1 might be a novel molecular target to develop agents to reverse cisplatin‐induced chemoresistance.

Reversal of BCRP-Mediated multidrug resistance by stable expression of small interfering RNAs

Breast cancer resistance protein (BCRP) is an ATP‐binding cassette multidrug transporter that confers resistance to various anticancer drugs like Mitoxantrone. Overexpression of BCRP confers multidrug resistance (MDR) in cancer cells and is a frequent impediment to successful chemotherapy. For stable reversal of BCRP‐depending MDR by RNA interference technology, a hU6‐RNA gene promoter‐driven expression vector encoding anti‐BCRP short hairpin RNA (shRNA) molecules was constructed. By treating endogenously and exogenously expresses high levels of BCRP cells with these constructs, expression of the targeted BCRP‐encoding mRNA, and transport protein was inhibited completely. Furthermore, the accumulation of mitoxantrone in the anti‐BCRP shRNA‐treated cells increased. And the sensitivity to mitoxantrone of anti‐BCRP shRNA‐treated cells is increased 14.6‐fold and 2.44‐fold respectively compared to their control (P < 0.05). These data indicated that stable shRNA‐mediated RNAi could be tremendously effective in reversing BCRP‐mediated MDR and showed promises in overcoming MDR by gene therapeutic applications. J. Cell. Biochem. 102: 75–81, 2007.