Proteomic analysis of cancer stem cells in human prostate cancer cells

Results from recent studies support the hypothesis that cancer stem cells (CSCs) are responsible for tumor initiation and formation. Here, we applied a proteome profiling approach to investigate the mechanisms of CSCs and to identify potential biomarkers in the prostate cancer cell line DU145. Using MACS, the DU145 prostate cancer cell line was isolated into CD44+ or CD44− cells. In sphere culture, CD44+ cells possessed stem cell characteristics and highly expressed genes known to be important in stem cell maintenance. In addition, they showed strong tumorigenic potential in the clonogenic assay and soft agar colony formation assay. We then analyzed and identified proteins that were differentially expressed between CD44+ and CD44− using two-dimensional gel electrophoresis and LC-MS/MS. Cofilin and Annexin A5, which are associated with proliferation or metastasis in cancer, were found to be positively correlated with CD44 expression. These results provide information that will be important to the development of new cancer diagnostic tools and understanding the mechanisms of CSCs although a more detailed study is necessary to investigate the roles of Cofilin and Annexin A5 in CSCs.     

Proteomic analysis of gemcitabine-induced drug resistance in pancreatic cancer cells

Currently, the most effective agent against pancreatic cancer is gemcitabine (GEM), which inhibits tumor growth by interfering with DNA replication and blocking DNA synthesis. However, GEM-induced drug resistance in pancreatic cancer compromises the therapeutic efficacy of GEM. To investigate the molecular mechanisms associated with GEM-induced resistance, 2D-DIGE and MALDI-TOF mass spectrometry were performed to compare the proteomic alterations of a panel of differential GEM-resistant PANC-1 cells with GEM-sensitive pancreatic cells. The proteomic results demonstrated that 33 proteins were differentially expressed between GEM-sensitive and GEM-resistant pancreatic cells. Of these, 22 proteins were shown to be resistance-specific and dose-dependent in the regulation of GEM. Proteomic analysis also revealed that proteins involved in biosynthesis and detoxification are significantly over-expressed in GEM-resistant PANC-1 cells. In contrast, proteins involved in vascular transport, bimolecular decomposition, and calcium-dependent signal regulation are significantly over-expressed in GEM-sensitive PANC-1 cells. Notably, both protein-protein interaction of the identified proteins with bioinformatic analysis and immunoblotting results showed that the GEM-induced pancreatic cell resistance might interplay with tumor suppressor protein p53. Our approach has been shown here to be useful for confidently detecting pancreatic proteins with differential resistance to GEM. Such proteins may be functionally involved in the mechanism of chemotherapy-induced resistance.     

Proteomic analysis of proteins responsible for the development of doxorubicin resistance in human uterine cancer cells

Drug resistance is a common cause of failure in cancer chemotherapy treatments. In this study, we used a pair of uterine sarcoma cancer lines, MES-SA, and the doxorubicin-resistant MES-SA/Dx5 as a model system to examine resistance-dependent cellular responses and to identify potential therapeutic targets. We used two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to examine the global protein expression changes induced by doxorubicin treatment and doxorubicin resistance. A proteomic study revealed that doxorubicin-exposure altered the expression of 87 proteins in MES-SA cells, while no significant response occurred in similarly treated MES-SA/Dx5 cells, associating these proteins with drug specific resistance. By contrast, 37 proteins showed differential expression between MES-SA and MES-SA/Dx5, indicating baseline resistance. Further studies have used RNA interference, cell viability analysis, and analysis of apoptosis against asparagine synthetase (ASNS) and membrane-associated progesterone receptor component 1 (mPR) proteins, to monitor and evaluate their potency on the formation of doxorubicin resistance. The proteomic approach allowed us to identify numerous proteins, including ASNS and mPR, involved in various drug-resistance-forming mechanisms. Our results provide useful diagnostic markers and therapeutic candidates for the treatment of doxorubicin-resistant uterine cancer.     

Proteomic analysis of microvesicles derived from human colorectal cancer cells

Microvesicles (MV) are membrane vesicles secreted from the plasma and endosomal membrane compartment by various cell types such as hematopoietic, epithelial, and tumor cells. Actively growing tumor cells shed MV, and the rate of shedding increases in malignant tumors. Although recent progress in this area has revealed that tumor-derived MV play multiple roles in tumor growth and metastasis via immune escape, tumor invasion, and angiogenesis, the mechanism of vesicle formation and the biological roles of tumor-derived MV are not understood. Here, we report the first global proteomic analysis of highly purified MV from human colorectal cancer cells. Using 1D SDS gel electrophoresis and nano-LC-MS/MS analyses, we identified a total of 547 microvesicular proteins from three independent experiments with high confidence; 416 proteins were identified at least in two trials, including 181 as yet unreported proteins. We identified 49 proteins involved in the biogenesis of MV, including annexins, ADP-ribosylation factors, and Rab proteins. We also identified 28 proteins that may function in tumorigenesis via promotion of migration, invasion, and growth of tumor cells, immune modulation, metastasis, and angiogenesis. Taken together with previously reported results, our observations suggest that tumor-derived MV may act as communicasomes, that is, extracellular organelles that play diverse roles in intercellular communication. This information will help elucidate the biogenesis and functions of tumor-derived MV, and aid in the development of effective vaccines for various cancers, including colorectal cancer.     

Proteomic analysis of human lysosomes: application to monocytic and breast cancer cells

To date, about fifty lysosomal hydrolases have been identified, and most of them are targeted towards the lysosomes through a specific mannose-6-phosphate (M-6-P) tag. As more lysosomal hydrolases were expected to be discovered, we performed a proteomic study of soluble lysosomal proteins. Human cells were induced to secrete M-6-P proteins which were affinity purified on immobilized M-6-P receptor. The purified proteins were resolved by two-dimensional electrophoresis and analyzed by mass spectrometry. Twenty-two proteins were identified, among which 16 were well-known lysosomal hydrolases. The remaining species distributed as follows: epididymis-specific alpha-mannosidase is a new mannosidase homolog, cystatin F and CREG (cellular repressor of E1A-stimulated genes) were previously identified as M-6-P proteins (Journet et al., Electrophoresis 2000, 21, 3411-3419), and the last three, which are not hydrolases, were up to now considered as nonlysosomal. This two-dimensional reference map of human U937 M-6-P proteins was afterwards used for comparison with M-6-P proteins purified either from U937 differentiated into macrophage-like cells, or from human breast cancer MCF7 cells. Phorbol ester induced differentiation of U937 cells led to limited proteolytic cleavage or maturation of a discrete number of hydrolases. Five additional lysosomal hydrolases were identified from MCF7 samples. These results prove the usefulness of such a procedure to analyze the lysosomal content of various cell lines, to discover new M-6-P proteins, as well as to point towards unknown biological processes.     

Proteomic analysis of cisplatin resistance in human ovarian cancer using 2-DE method

Platinum-based chemotherapy, such as cisplatin, is the primary treatment for human ovarian cancer. However, overcoming drug resistance has become an important issue in cancer chemotherapy. In this study, we performed 2-DE and ESI-Q-TOF MS/MS analysis to identify differential proteins expression between cisplatin-sensitive (A2780S) and cisplatin-resistant (A2780-CP) ovarian cancer cell lines. Of the 14 spots identified as differentially expressed (±over twofold, P < 0.05) between the two cell lines, ten spots (corresponding to ten unique proteins) were positively identified by ESI-Q-TOF MS/MS analysis. These proteins include capsid glycoprotein, fructose-bisphosphate aldolase C, heterogeneous nuclear ribonucleoproteins A2/B1, putative RNA-binding protein 3, Ran-specific GTPase-activating protein, ubiquitin carboxyl-terminal hydrolase isozyme L1, stathmin, ATPSH protein, chromobox protein homolog3 and phosphoglycerate kinase 1. The proteins identified in this study would be useful in revealing the mechanisms underlying cisplatin resistance and also provide some clues for further research.     

Proteomic analysis of the response of human lung cells to uranium

The industrial use of uranium and particularly of depleted uranium, has pinpointed the need to review its chemical impact on human health. A proteomic approach was used to evaluate the response of a human lung cell line (A549) to uranium. We established the first 2-D reference map of the A549 cell line, identifying 87 spots corresponding to 81 major proteins. Uranium treatment triggered differential expression of 18 spots, of which 14 corresponded to fragments of cytokeratin 8 (CK8) and cytokeratin (CK18) and 1 to peroxiredoxin 1. We probed several hypotheses regarding CK cleavage, and observed that it did not result from caspase or calpain activity. Furthermore, we showed that the fragments are recognised by an anti-ubiquitin antibody (KM691). These results suggest a regulatory pathway involving CK ubiquitinylation or dysfunction in the proteasome-ubiquitin system in response to uranium exposure in human lung cells.     

Proteomic analysis of apoptosis induction in human lung cancer cells by recombinant MVL

Lung cancer is still difficult to treat by current chemotherapeutic procedures. We recently found that MVL, an anti-HIV lectin from blue-green algae Microcystis viridis, also has antitumor activity. The objective of this study was to investigate apoptosis-inducing activity of recombinant MVL (R-MVL) and proteomic changes in A549 cells, and to identify the molecular pathways responsible for the anti-cancer action of R-MVL. We found that R-MVL induces A549 cells apoptosis in a dose-dependent manner by using MTT assay, fluorescent microscope (FM) and flow cytometry (FCM), and the IC50 was calculated to be 24.12 μg/ml. Subsequently, 7 altered proteins in R-MVL-treated A549 cells were identified, including upregulated aldehyde dehydrogenase 1 and β-actin, and five downregulated proteins: heat shock protein 90, heat shock 60, plastin 3, tropomyosin 3, and β-tubulin. Further bioinformatics analysis predicted the potential pathways for R-MVL to induce apoptosis of A549 cells. In conclusion, this is the first report to investigate anti-cancer activity of R-MVL and its mechanism of action by proteomics analysis. Our observations provide potential therapeutic targets for lung cancer inhibitor intervention and implicated the development of novel anti-cancer therapeutic strategies.     

Proteomic profiling of endothelial cells in human lung cancer

Genomic and proteomic analysis of normal and diseased tissues have yielded an abundance of molecular information for diagnostic and potential therapeutic targets. Changing the target of analysis from poorly accessible cells within tissues to easily accessible vascular endothelium has theoretical advantages in tissue-specific targeting. In this study, we sought to map a large-scale proteome of microvascular endothelium in human non-small cell lung cancer (NSCLC) and normal lung tissues, and identify lung cancer-related endothelial cell (EC)-selective proteins. Endothelial cells were isolated within NSCLC tissues and adjacent-normal lung tissue of lung cancer patients by using CD31-immunomagnetic beads. The complex proteins from the ECs were separated by one-dimensional gel electrophoresis, and the proteins in each gel band were digested by trypsin. Peptides were separated by online reverse-phase liquid-chromatography and analyzed by electrospray ionization (ESI) ion trap tandem mass spectrometry. Approximately 600-1000 proteins were identified in each individual sample. Five patient cases of paired individual data, extracted from the protein identification data sets of both normal- and cancer-derived ECs, were analyzed by subtractive proteomics. An average of 300 proteins was specifically identified from each lung cancer-derived EC isolate, compared to normal lung-derived ECs. With the use of several comparative analyses, we identified among those 300 proteins, 16 common candidate proteins that were detected in at least 3 of 5 cases specific to lung cancer-derived ECs. Proteins selectively identified in cancer-derived ECs, including coatomer protein complex, subunit gamma (COPG), and peroxiredoxin 4 (PRDX4), were validated by Western blot analysis. In an additional experiment in which 16 cancer samples were analyzed by immunohistochemistry, PRDX4, thymopoietin (TMPO), and COPG were confirmed to be abundantly expressed in lung cancer-derived ECs and in cancerous lung cells. Further ongoing analysis of these 16 candidate proteins will determine their potential applicability to NSCLC-specific diagnosis and therapeutics.     

Proteomic analysis of cellular response to microcystin in human amnion FL cells

Microcystins (MC), the potent inhibitor of protein phosphatase 1 and 2A, are hepatotoxins of increasing importance due to its high acute toxicity and potent tumor promoting activity. So far, the exact mechanisms of MC-induced hepatotoxicity and tumor promoting activity have not been fully elucidated. To better understand the mechanisms underlying microcystin-RR (MC-RR) induced toxicity as well as provide the possibility for the establishment of biomarkers for MC-RR exposure, differential proteome analysis on human amnion FL cells treated by MC-RR was carried out using two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Image analysis of silver-stained 2-dimensional gels revealed that 89 proteins showed significant differential expression in MC-RR treated cells compared with control, and 8 proteins were unique to MC-RR treated cells and 8 proteins were only detected in control cells. Sixty-six proteins were further identified with high confidence by peptide mass fingerprinting. Some of the identified differentially expressed proteins have clearly relationship with the process of apoptosis, signal transduction, and cytoskeleton alteration which are consistent with the literature. The functional implications of alterations in the levels of these proteins were discussed. However, most of which have not been reported previously to be involved in cellular processes responded to MC-RR. Therefore, this work will provide new insight into the mechanism of MC-RR toxicity.     

Proteomic analysis of iron overload in human hepatoma cells

Iron-mediated organ damage is common in patients with iron overload diseases, namely, hereditary hemochromatosis. Massive iron deposition in parenchymal organs, particularly in the liver, causes organ dysfunction, fibrosis, cirrhosis, and also hepatocellular carcinoma. To obtain deeper insight into the poorly understood and complex cellular response to iron overload and consequent oxidative stress, we studied iron overload in liver-derived HepG2 cells. Human hepatoma HepG2 cells were exposed to a high concentration of iron for 3 days, and protein expression changes initiated by the iron overload were studied by two-dimensional electrophoresis and mass spectrometry. From a total of 1,060 spots observed, 21 spots were differentially expressed by iron overload. We identified 19 of them; 11 identified proteins were upregulated, whereas 8 identified proteins showed a decline in response to iron overload. The differentially expressed proteins are involved in iron storage, stress response and protection against oxidative stress, protein folding, energy metabolism, gene expression, cell cycle regulation, and other processes. Many of these molecules have not been previously suggested to be involved in the response to iron overload and the consequent oxidative stress.     

Proteomic and redox-proteomic analysis of berberine-induced cytotoxicity in breast cancer cells

Berberine is a natural product isolated from herbal plants such as Rhizoma coptidis which has been shown to have anti-neoplastic properties. However, the effects of berberine on the behavior of breast cancers are largely unknown. To determine if berberine might be useful in the treatment of breast cancer and its cytotoxic mechanism, we analyzed the impact of berberine treatment on differential protein expression and redox regulation in human breast cancer cell line MCF-7 using lysine- and cysteine-labeling two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry (MS). This study demonstrated that 96 and 22 protein features were significantly changed in protein expression and thiol reactivity, respectively and revealed that berberine-induced cytotoxicity in breast cancer cells involves dysregulation of protein folding, proteolysis, redox regulation, protein trafficking, cell signaling, electron transport, metabolism and centrosomal structure. Our work shows that this combined proteomic strategy provides a rapid method to study the molecular mechanisms of berberine-induced cytotoxicity in breast cancer cells. The identified targets may be useful for further evaluation as potential targets in breast cancer therapy.     

Proteomic and functional analysis of Argonaute-containing mRNA-protein complexes in human cells

Members of the Argonaute (Ago) protein family associate with small RNAs and have important roles in RNA silencing. Here, we analysed Ago1- and Ago2-containing protein complexes in human cells. Separation of Ago-associated messenger ribonucleoproteins (mRNPs) showed that Ago1 and Ago2 reside in three complexes with distinct Dicer and RNA-induced silencing complex activities. A comprehensive proteomic analysis of Ago-containing mRNPs identified a large number of proteins involved in RNA metabolism. By using co-immunoprecipitation experiments followed by RNase treatment, we biochemically mapped interactions within Ago mRNPs. Using reporter assays and knockdown experiments, we showed that the putative RNA-binding protein RBM4 is required for microRNA-guided gene regulation.     

Proteomic analysis of isolated membrane fractions from superinvasive cancer cells

The superinvasive phenotype exhibited by paclitaxel-selected variants of an in vitro invasive clonal population of the human cancer cell line, MDA-MB-435S were examined using DIGE (Fluorescence 2-D Difference Gel Electrophoresis) and mass spectrometry. Isolation of membrane proteins from the MDA-MB-435S-F/Taxol-10p4p and parental populations was performed by temperature-dependent phase partitioning using the detergent Triton X-114. Subsequent DIGE-generated data analysed using Decyder software showed many differentially-expressed proteins in the membrane fraction. 16 proteins showing statistically significant upregulation in the superinvasive cells were identified by MALDI-ToF. Proteins upregulated in the superinvasive population include Galectin-3, Cofilin, ATP synthase beta subunit, voltage-dependent anion channel 1, voltage dependent anion channel 2, ER-60 protein, MHC class II antigen DR52, Beta actin, TOMM40 protein, Enolase 1, Prohibitin, Guanine nucleotide-binding protein, Annexin II, Heat shock 70 kDa protein, Stomatin-like protein 2 and Chaperonin. Many of these proteins are associated with inhibition of apoptosis, the progression of cancer, tumourigenicity, metastasis, actin remodelling at the leading edge of cells, polarized cell growth, endocytosis, phagocytosis, cellular activation, cytokinesis, and pathogen intracellular motility. These results suggest a correlation between the increased abundance of these proteins with the superinvasive phenotype of the paclitaxel-selected MDA-MB-435S-F/Taxol-10p4p population.     

Proteomic analysis of microvesicles derived from human mesenchymal stem cells

Mesenchymal stem cells (MSCs) have emerged as a promising means for treating degenerative or incurable diseases. Recent studies have shown that microvesicles (MVs) from MSCs (MSC-MVs) contribute to recovery of damaged tissues in animal disease models. Here, we profiled the MSC-MV proteome to investigate their therapeutic effects. LC-MS/MS analysis of MSC-MVs identified 730 MV proteins. The MSC-MV proteome included five positive and two variable known markers of MSCs, but no negative marker, as well as 43 surface receptors and signaling molecules controlling self-renewal and differentiation of MSCs. Functional enrichment analysis showed that cellular processes represented by the MSC-MV proteins include cell proliferation, adhesion, migration, and morphogenesis. Integration of MSC's self-renewal and differentiation-related genes and the proteome of MSC-conditioned media (MSC-CM) with the MSC-MV proteome revealed potential MV protein candidates that can be associated with the therapeutic effects of MSC-MVs: (1) surface receptors (PDGFRB, EGFR, and PLAUR); (2) signaling molecules (RRAS/NRAS, MAPK1, GNA13/GNG12, CDC42, and VAV2); (3) cell adhesion (FN1, EZR, IQGAP1, CD47, integrins, and LGALS1/LGALS3); and (4) MSC-associated antigens (CD9, CD63, CD81, CD109, CD151, CD248, and CD276). Therefore, the MSC-MV proteome provides a comprehensive basis for understanding the potential of MSC-MVs to affect tissue repair and regeneration.     

Proteomic analysis of lipid microdomains from lipopolysaccharide-activated human endothelial cells

The endothelium plays a critical role in orchestrating the inflammatory response seen during sepsis. Many of the inflammatory effects of Gram-negative sepsis are elicited by lipopolysaccharide (LPS), a glycolipid component of bacterial cell walls. Lipid-rich microdomains have been shown to concentrate components of the LPS signaling system. However, much remains to be learned about which proteins are constituents of lipid microdomains, and how these are regulated following cell activation. Progress in this area would be accelerated by employing global proteomic analyses, but the hydrophobicity of membrane proteins presents an analytical barrier to the effective application of such approaches. Herein, we describe a method to isolate detergent-resistant membranes from endothelial cells, and prepare these samples for proteomic analysis in a way that is compatible with subsequent separations and mass spectrometric (MS) analysis. In the application of these sample preparation and MS analyses, 358 proteins from the lipid-rich microdomains of LPS-activated endothelial cell membranes have been identified of which half are classified as membrane proteins by Gene Ontology. We also demonstrate that the sample preparation method used for solubilization and trypsin digestion of lipid-rich microdomains renders the membrane spanning sequences of transmembrane proteins accessible for endoproteolytic hydrolysis. This analysis sets the analytical foundation for an in-depth probing of LPS signaling in endothelial cells.     

Proteomic analysis of cervical cancer cells treated with suberonylanilide hydroxamic acid

Suberonylanilide hydroxamic acid (SAHA) is an orally administered histone deacetylase inhibitor (HDACI) that has shown significant antitumour activity in a variety of tumour cells. To identify proteins involved in its antitumour activity, we utilized a proteomic approach to reveal protein expression changes in the human cervical cancer cell line HeLa following SAHA treatment. Protein expression profiles were analysed by 2-dimensional polyacrylamide gel electrophoresis (2-DE) and protein identification was performed on a MALDI-Q-TOF MS/MS instrument. As a result, a total of nine differentially expressed proteins were visualized by 2-DE and Coomassie brilliant blue (CBB) staining. Further, all the changed proteins were positively identified via mass spectrometry (MS)/MS analysis. Of these, PGAM1 was significantly downregulated in HeLa cells after treatment with SAHA. Moreover, PGAM1 has been proven to be downregulated in another cervical cancer cell line (CaSki) by western blot analysis. Together, using proteomic tools, we identified several differentially expressed proteins that underwent SAHA-induced apoptosis. These changed proteins may provide some clues to a better understanding of the molecular mechanisms underlying SAHA-induced apoptosis in cervical cancer.