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      Plumbagin elicits differential proteomic responses mainly involving cell cycle, apoptosis, autophagy, and epithelial-to-mesenchymal transition pathways in human prostate cancer PC-3 and DU145 cells

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          Abstract

          Plumbagin (PLB) has exhibited a potent anticancer effect in preclinical studies, but the molecular interactome remains elusive. This study aimed to compare the quantitative proteomic responses to PLB treatment in human prostate cancer PC-3 and DU145 cells using the approach of stable-isotope labeling by amino acids in cell culture (SILAC). The data were finally validated using Western blot assay. First, the bioinformatic analysis predicted that PLB could interact with 78 proteins that were involved in cell proliferation and apoptosis, immunity, and signal transduction. Our quantitative proteomic study using SILAC revealed that there were at least 1,225 and 267 proteins interacting with PLB and there were 341 and 107 signaling pathways and cellular functions potentially regulated by PLB in PC-3 and DU145 cells, respectively. These proteins and pathways played a critical role in the regulation of cell cycle, apoptosis, autophagy, epithelial to mesenchymal transition (EMT), and reactive oxygen species generation. The proteomic study showed substantial differences in response to PLB treatment between PC-3 and DU145 cells. PLB treatment significantly modulated the expression of critical proteins that regulate cell cycle, apoptosis, and EMT signaling pathways in PC-3 cells but not in DU145 cells. Consistently, our Western blotting analysis validated the bioinformatic and proteomic data and confirmed the modulating effects of PLB on important proteins that regulated cell cycle, apoptosis, autophagy, and EMT in PC-3 and DU145 cells. The data from the Western blot assay could not display significant differences between PC-3 and DU145 cells. These findings indicate that PLB elicits different proteomic responses in PC-3 and DU145 cells involving proteins and pathways that regulate cell cycle, apoptosis, autophagy, reactive oxygen species production, and antioxidation/oxidation homeostasis. This is the first systematic study with integrated computational, proteomic, and functional analyses revealing the networks of signaling pathways and differential proteomic responses to PLB treatment in prostate cancer cells. Quantitative proteomic analysis using SILAC represents an efficient and highly sensitive approach to identify the target networks of anticancer drugs like PLB, and the data may be used to discriminate the molecular and clinical subtypes, and to identify new therapeutic targets and biomarkers, for prostate cancer. Further studies are warranted to explore the potential of quantitative proteomic analysis in the identification of new targets and biomarkers for prostate cancer.

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          Most cited references 74

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          Establishment and characterization of a human prostatic carcinoma cell line (PC-3).

          The establishment, characterization, and tumorigenicity of a new epithelial cell line (PC-3) from a human prostatic adenocarcinoma metastatic to bone is reported. The cultured cells show anchorage-independent growth in both monolayers and in soft agar suspension and produce subcutaneous tumors in nude mice. Culture of the transplanted tumor yielded a human cell line with characteristics identical to those used initially to produce the tumor. PC-3 has a greatly reduced dependence upon serum for growth when compared to normal prostatic epithelial cells and does not respond to androgens, glucocorticoids, or epidermal or fibroblast gowth factors. Karyotypic analysis by quinacrine banding revealed the cells to be completely aneuploid with a modal chromosome number in the hypotriploid range. At least 10 distinctive marker chromosomes were identified. The overall karyotype as well as the marker chromosomes are distinct from those of the HeLa cell. Electron microscopic studies revealed many features common to neoplastic cells of epithelial origin including numerous microvilli, junctional complexes, abnormal nuclei and nucleoli, abnormal mitochondria, annulate lamellae, and lipoidal bodies. Overall, the functional and morphologic characteristics of PC-3 are those of a poorly-differentiated adenocarcinoma. These cells should be useful in investigating the biochemical changes in advanced prostatic cancer cells and in assessing their response to chemotherapeutic agents.
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            A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC).

            Stable isotope labeling by amino acids in cell culture (SILAC) is a simple, robust, yet powerful approach in mass spectrometry (MS)-based quantitative proteomics. SILAC labels cellular proteomes through normal metabolic processes, incorporating non-radioactive, stable isotope-containing amino acids in newly synthesized proteins. Growth medium is prepared where natural ("light") amino acids are replaced by "heavy" SILAC amino acids. Cells grown in this medium incorporate the heavy amino acids after five cell doublings and SILAC amino acids have no effect on cell morphology or growth rates. When light and heavy cell populations are mixed, they remain distinguishable by MS, and protein abundances are determined from the relative MS signal intensities. SILAC provides accurate relative quantification without any chemical derivatization or manipulation and enables development of elegant functional assays in proteomics. In this protocol, we describe how to apply SILAC and the use of nano-scale liquid chromatography coupled to electrospray ionization mass spectrometry for protein identification and quantification. This procedure can be completed in 8 days.
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              Functional and quantitative proteomics using SILAC.

               Matthias Mann (2006)
              Researchers in many biological areas now routinely characterize proteins by mass spectrometry. Among the many formats for quantitative proteomics, stable-isotope labelling by amino acids in cell culture (SILAC) has emerged as a simple and powerful one. SILAC removes false positives in protein-interaction studies, reveals large-scale kinetics of proteomes and - as a quantitative phosphoproteomics technology - directly uncovers important points in the signalling pathways that control cellular decisions.
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                Author and article information

                Journal
                Drug Des Devel Ther
                Drug Des Devel Ther
                Drug Design, Development and Therapy
                Drug Design, Development and Therapy
                Dove Medical Press
                1177-8881
                2015
                07 January 2015
                : 9
                : 349-417
                Affiliations
                [1 ]School of Public Health, Wuhan University, Wuhan, Hubei, People’s Republic of China
                [2 ]Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China
                [3 ]Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA
                [4 ]Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, Guizhou, People’s Republic of China
                [5 ]Center for Traditional Chinese Medicine, Sarasota, FL, USA
                [6 ]Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China
                [7 ]Bio-X Institutes, Key Laboratory for the Genetics of Development and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai, People’s Republic of China
                Author notes
                Correspondence: Zong-Fu Mao, School of Public Health, Wuhan University, 185 Donghu Road, Wuchang District, Wuhan 430071, Hubei, People’s Republic of China, Tel +86 27 6875 9609, Email zfmao@ 123456126.com
                Shu-Feng Zhou, Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, 12901 Bruce B Downs Blvd., MDC 30, Tampa, FL 33612, USA, Tel +1 813 974 6276, Fax +1 813 905 9885, Email szhou@ 123456health.usf.edu
                Article
                dddt-9-349
                10.2147/DDDT.S71677
                4294653
                © 2015 Qiu et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0) License

                The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed.

                Categories
                Original Research

                Pharmacology & Pharmaceutical medicine

                emt, proteomics, silac

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