Molecular lipidomics of exosomes released by PC-3 prostate cancer cells

The molecular lipid composition of exosomes is largely unknown. In this study, sophisticated shotgun and targeted molecular lipidomic assays were performed for in-depth analysis of the lipidomes of the metastatic prostate cancer cell line, PC-3, and their released exosomes. This study, based in the quantification of approximately 280 molecular lipid species, provides the most extensive lipid analysis of cells and exosomes to date. Interestingly, major differences were found in the lipid composition of exosomes compared to parent cells. Exosomes show a remarkable enrichment of distinct lipids, demonstrating an extraordinary discrimination of lipids sorted into these microvesicles. In particular, exosomes are highly enriched in glycosphingolipids, sphingomyelin, cholesterol, and phosphatidylserine (mol% of total lipids). Furthermore, lipid species, even of classes not enriched in exosomes, were selectively included in exosomes. Finally, it was found that there is an 8.4-fold enrichment of lipids per mg of protein in exosomes. The detailed lipid composition provided in this study may be useful to understand the mechanism of exosome formation, release and function. Several of the lipids enriched in exosomes could potentially be used as cancer biomarkers.     

Proteomic analysis of conditioned media from the PC3, LNCaP, and 22Rv1 prostate cancer cell lines: discovery and validation of candidate prostate cancer biomarkers

Early detection of prostate cancer is problematic due to the lack of a marker that has high diagnostic sensitivity and specificity. The prostate specific antigen (PSA) test, in combination with digital rectal examination, is the gold standard for prostate cancer diagnosis. However, this modality suffers from low specificity. Therefore, specific markers for clinically relevant prostate cancer are needed. Our objective was to proteomically characterize the conditioned media from three human prostate cancer cell lines of differing origin [PC3 (bone metastasis), LNCaP (lymph node metastasis), and 22Rv1 (localized to prostate)] to identify secreted proteins that could serve as novel prostate cancer biomarkers. Each cell line was cultured in triplicate, followed by a bottom-up analysis of the peptides by two-dimensional chromatography and tandem mass spectrometry. Approximately, 12% (329) of the proteins identified were classified as extracellular and 18% (504) as membrane-bound among which were known prostate cancer biomarkers such as PSA and KLK2. To select the most promising candidates for further investigation, tissue specificity, biological function, disease association based on literature searches, and comparison of protein overlap with the proteome of seminal plasma and serum were examined. On the basis of this, four novel candidates, follistatin, chemokine (C-X-C motif) ligand 16, pentraxin 3 and spondin 2, were validated in the serum of patients with and without prostate cancer. The proteins presented in this study represent a comprehensive sampling of the secreted and shed proteins expressed by prostate cancer cells, which may be useful as diagnostic, prognostic or predictive serological markers for prostate cancer.     

前列腺癌组织及PC-3细胞中STAT3及磷酸化STAT3的表达

目的　研究前列腺癌组织及前列腺癌细胞株PC- 3 中STAT3 蛋白及磷酸化STAT3 蛋白的表达。方法　常规石蜡包埋切片SABC免疫组化法检测45例前列腺癌组织、20例前列腺增生组织中STAT3 及磷酸化STAT3 表达, 细胞免疫化学法检测前列腺癌细胞株PC 3细胞STAT3及磷酸化STAT3表达。结果　STAT3在前列腺癌及前列腺增生组织表达阳性率分别为77. 8%和50. 0%, 两者间具有显著差异; 磷酸化STAT3在前列腺癌及前列腺增生组织表达阳性率分别为68. 9%和35. 0%, 两者间具显著差异(P<0 .05); PC- 3细胞中STAT3及磷酸化STAT3表达阳性。结论　STAT3蛋白在前列腺癌中高表达且持续激活, 可能与前列腺癌的发生具有密切联系。     

Primary functional identification of gene TMSG-1

TMSG-1 (Tumor metastasis suppressor gene-1) is a cancer metastasis-related gene cloned by means of mRNA differential display from human prostate cancer cell lines with different metas-tatic potential[1], which has higher expression in non-metastatic cell line, whereas lower expres-sion in highly metastatic cell line. In samples of primary gastric carcinoma, the TMSG-1 expres-sion markedly decreased in gastric carcinoma with lymph node metastases. It was found that protein encoded by TMS…     

Profiling of microRNAs in exosomes released from PC-3 prostate cancer cells

Exosomes are small extracellular vesicles released to the extracellular milieu through fusion of multivesicular bodies with the plasma membrane. These vesicles contain microRNAs and might therefore be vehicles transferring genetic information between cells. The aim of this study was to investigate whether there was a sorting of microRNAs into exosomes in the prostate cancer cell line PC-3. In addition, microRNAs in PC-3 cells and in the non-cancerous prostate cell line RWPE-1 were compared. Exosomes were isolated from the conditioned media from PC-3 cells by ultracentrifugation and inspected by electron microscopy. Total RNA was isolated and microRNAs were analyzed by microarray analysis and real time RT-PCR. MicroRNA microarray analysis revealed that the microRNA profile of PC-3 released exosomes was similar to the profile of the corresponding parent cells. Nevertheless, a sorting of certain microRNAs into exosomes was observed, and low number microRNAs (microRNAs with a low number in their name) were found to be underrepresented in these vesicles. Moreover, the miRNA profile of PC-3 cells resembled the miRNA profile of RWPE-1 cells, though some miRNAs were found to be differently expressed in these cell lines. These results show that exosomes from PC-3 cells, in agreement with previous reports from other cell types, contain microRNAs. Furthermore, this study supports the idea that there is a sorting of microRNAs into exosomes and adds a new perspective by pointing at the underrepresentation of low number miRNAs in PC-3 released exosomes.     

Characterization of membrane-shed microvesicles from cytokine-stimulated β-cells using proteomics strategies

Microparticles and exosomes are two of the most well characterized membrane-derived microvesicles released either directly from the plasma membrane or released through the fusion of intracellular multivesicular bodies with the plasma membrane, respectively. They are thought to be involved in many significant biological processes such as cell to cell communication, rescue from apoptosis, and immunological responses. Here we report for the first time a quantitative study of proteins from β-cell-derived microvesicles generated after cytokine induced apoptosis using stable isotope labeled amino acids in cell culture combined with mass spectrometry. We identified and quantified a large number of β-cell-specific proteins and proteins previously described in microvesicles from other cell types in addition to new proteins located to these vesicles. In addition, we quantified specific sites of protein phosphorylation and N-linked sialylation in proteins associated with microvesicles from β-cells. Using pathway analysis software, we were able to map the most distinctive changes between microvesicles generated during growth and after cytokine stimulation to several cell death and cell signaling molecules including tumor necrosis factor receptor superfamily member 1A, tumor necrosis factor, α-induced protein 3, tumor necrosis factor-interacting kinase receptor-interacting serine-threonine kinase 1, and intercellular adhesion molecule 1.     

Interaction of Pyk2 and PTP-PEST with leupaxin in prostate cancer cells

We have identified the presence of leupaxin (LPXN), which belongs to the paxillin extended family of focal adhesion-associated adaptor proteins, in prostate cancer cells. Previous studies have demonstrated that LPXN is a component of the podosomal signaling complex found in osteoclasts, where LPXN was found to associate with the protein tyrosine kinases Pyk2 and c-Src and the cytosolic protein tyrosine phosphatase-proline-, glutamate-, serine-, and threonine-rich sequence (PTP-PEST). In the current study, LPXN was detectable as a 50-kDa protein in PC-3 cells, a bone-derived metastatic prostate cancer cell line. In PC-3 cells, LPXN was also found to associate with Pyk2, c-Src, and PTP-PEST. A siRNA-mediated inhibition of LPXN resulted in decreased in vitro PC-3 cell migration. A recombinant adenoviral-mediated overexpression of LPXN resulted in an increased association of Pyk2 with LPXN, whereas a similar adenoviral-mediated overexpression of PTP-PEST resulted in decreased association of Pyk2 and c-Src with LPXN. The overexpression of LPXN in PC-3 cells resulted in increased migration, as assessed by in vitro Transwell migration assays. On the contrary, the overexpression of PTP-PEST in PC-3 cells resulted in decreased migration. The overexpression of LPXN resulted in increased activity of Rho GTPase, which was decreased in PTP-PEST-overexpressing cells. The increase in Rho GTPase activity following overexpression of LPXN was inhibited in the presence of Y27632, a selective inhibitor of Rho GTPase. In conclusion, our data demonstrate that LPXN forms a signaling complex with Pyk2, c-Src, and PTP-PEST to regulate migration of prostate cancer cells. PC-3; protein tyrosine phosphatase-proline-, glutamate-, serine-, and threonine-rich sequence; c-Src; migration     

Calcium signals in prostate cancer cells: specific activation by bone-matrix proteins

Cancer of the prostate commonly metastasizes to bony sites where cells acquire an aggressive, rapidly proliferating, androgen-independent phenotype. The interaction between bone and prostate, thus, becomes a key factor in disease progression. Fluctuations in intracellular ionized Ca2+ [Ca2+]i are rapid, regulated signal transduction events often associated with cell proliferation. Hence, Ca2+ signals provide a convenient measure of early events in cancer cell growth. This study developed single cell fluorescent imaging techniques to visualize Ca2+ signals in Fura-2 loaded prostatic cancer cell lines of various metastatic phenotypes. Solubilized bone fractions containing extracellular matrix and associated proteins were tested for the ability to trigger Ca2+ signals in prostate cancer cell lines. Fractions representing the complete repertoire of non-collagenous proteins present in mineralized bone were tested. Results demonstrated that two bone fractions termed D3b- and D4a-triggered Ca2+ signals in prostate cancer cells derived from bone (PC-3), but not brain (DU-145) metastases of prostate cancer. Lymph-node derived LNCaP cells also did not produce a Ca2+ signal in response to addition of soluble bone matrix. No other bone fractions produced a Ca2+ signal in PC-3 cells. It is of interest that bone fractions D3b and D4a contain a number of non-collagenous matrix proteins including osteonectin (SPARC) and osteopontin (OPN), as well as prothrombin. Moreover, antibody LM609 that recognizes the alpha v beta 3 integrin, blocks the ability of OPN to trigger a Ca2+ transient in PC-3 cells. These studies support a conclusion that bone-matrix proteins play a role in the growth and progression of metastatic prostate cancer, and that prior growth in bone may be associated with development of a bone-matrix-responsive phenotype.     

Lycopene increases urokinase receptor and fails to inhibit growth or connexin expression in a metastatically passaged prostate cancer cell line: a brief communication

The carotenoid lycopene, found in tomatoes, has been associated with decreasing prostate cancer risk. Potential mechanisms for this risk reduction include lycopene's status as a potent antioxidant, its inhibitory effect on cell proliferation, and its ability to increase intercellular gap junctional communication. Presently, in the United States, almost 200,000 men are diagnosed with prostate cancer and approximately 30,000 succumb to its metastatic effects. Therefore, novel treatment strategies are needed for patients who currently have the disease, especially those in advanced, i.e., metastatic status. In this study, we sought to determine if lycopene's inhibitory properties on premalignancy could be extended to advanced prostate cancer by assessing effects on a cell line derived through metastatic passage, the PC-3MM2. We report that in this cell line, lycopene has a potentially unwanted effect of upregulating expression of the urokinase plasminogen activator receptor and facilitating invasion while failing to significantly inhibit proliferation or to induce detectable levels of the gap junctional protein connexin 43 expression. Our results indicate that some caution should be taken with regard to use of lycopene to treat potentially advanced and metastatic prostate cancers.     

Sulforaphane, a cruciferous vegetable-derived isothiocyanate, inhibits protein synthesis in human prostate cancer cells

Sulforaphane (SFN) is a compound derived from cruciferous plants. Its anticancer properties have been demonstrated both, in cancer cell lines as well as tumors in animal models. It has been shown that SFN inhibits cell proliferation, induces apoptosis, autophagy, and sensitizes cancer cells to therapies. As induction of catabolic processes is often related to perturbation in protein synthesis we aimed to investigate the impact of SFN on this process in PC-3 human prostate cancer cells. In the present study we show that SFN inhibits protein synthesis in PC-3 cells in a dose- and time-dependent manner which is accompanied by a decreased phosphorylation of mTOR substrates. Translation inhibition is independent of mitochondria-derived ROS as it is observed in PC-3 derivatives devoid of functional mitochondrial respiratory chain (Rho0 cells). Although SFN affects mitochondria and slightly decreases glycolysis, the ATP level is maintained on the level characteristic for control cells. Inhibition of protein synthesis might be a protective response of prostate cancer cells to save energy. However, translation inhibition contributes to the death of PC-3 cells due to decreased level of a short-lived protein, survivin. Overexpression of this anti-apoptotic factor protects PC-3 cells against SFN cytotoxicity. Protein synthesis inhibition by SFN is not restricted to prostate cancer cells as we observed similar effect in SKBR-3 breast cancer cell line.     

The Role of Adhesion Molecules as Biomarkers for the Aggressive Prostate Cancer Phenotype

Background

Currently available methods for diagnosis and staging of prostate cancer lack the sensitivity to distinguish between patients with indolent prostate cancer and those requiring radical treatment. Alterations in key adherens (AJ) and tight junction (TJ) components have been hailed as potential biomarkers for prostate cancer progression but the majority of research has been carried out on individual molecules.

Objective

To elucidate a panel of biomarkers that may help distinguish dormant prostate cancer from aggressive metastatic disease.

Methods

We analysed the expression of 7 well known AJ and TJ components in cell lines derived from normal prostate epithelial tissue (PNT2), non-invasive (CAHPV-10) and invasive prostate cancer (LNCaP, DU145, PC-3) using gene expression, western blotting and immunofluorescence techniques.

Results

Claudin 7, α –catenin and β-catenin protein expression were not significantly different between CAHPV-10 cells and PNT2 cells. However, in PC-3 cells, protein levels for claudin 7, α –catenin were significantly down regulated (−1.5 fold, p = <.001) or undetectable respectively. Immunofluoresence showed β-catenin localisation in PC-3 cells to be cytoplasmic as opposed to membraneous.

Conclusion

These results suggest aberrant Claudin 7, α – and β-catenin expression and/or localisation patterns may be putative markers for distinguishing localised prostate cancer from aggressive metastatic disease when used collectively.     

Primary functional identification of gene <Emphasis Type="Italic">TMSG-1</Emphasis>

TMSG-1 was a tumor metastasis-related gene identified using mRNA differential display, whose expression level was lower in cancer cell lines with higher metastatic potential and in tumor tissue with metastasis. TMSG-1 was transfected to prostate cancer cell line (PC-3M-1E8) with high metastatic potential to observe the effects of increased expression of TMSG-1 on V-ATPase activity, intracellular pH and cell apoptosis. Subcellular localization of the encoded protein of TMSG-1 was determined by using GFP. Results showed that there were no differences of V-ATPase activity among parental PC-3M-1E8 cell line, pcDNA3 transfectant and anti-TMSG-1 transfectant, whereas the V-ATPase activity was significantly higher in TMSG-1 transfectant than that in parental PC-3M-1E8 cell line, pcDNA3 transfectant and Anti-TMSG-1 transfectant (p<0.001). Intracellular pH (pHi) was detected by using the pH-dependent fluorescence probe BECEF. Results showed the pHi was significantly increased in TMSG-1 transfectant. Cell apoptosis assay demonstrated cell apoptosis was significantly higher in -1 transfectant (p<0.01) and BCL2 expression was down regulated. Subcellular localization of TMSG-1 protein showed TMSG-1 was a transmembrane protein, which predicted TMSG-1 protein was located in cytoplasm system, such as endoplasmic reticulum and mitochondrial. These results indicated TMSG-1 up regulation in prostate cancer cell line could promote V-ATPase activity, increase pHi and cell apoptosis, and inhibit the expression of BCL2.     

Cholesterol regulates prostasome release from secretory lysosomes in PC-3 human prostate cancer cells

Prostasomes are vesicles secreted by epithelial cells of the prostate gland. However, little is known about the mechanism and the regulation of prostasome secretion. Since endocytic organelles may be involved in prostasome release, PC-3-derived prostasomes were investigated by Western blot analysis for the presence of marker proteins normally associated with these organelles. Prostasomes secreted by PC-3 cells contain clathrin, Tsg101, Hrs, Rab11, Rab5, LAMP-1, LAMP-2, LAMP-3/CD63, and annexin II. Moreover, electron microscopy of PC-3 cells revealed the presence of characteristic multivesicular body-like secretory lysosomes containing vesicles with the same size-distribution as released prostasomes. Ultrastructural immunogold labelling showed that LAMP-1, LAMP-2 and LAMP-3/CD63 were associated with these vesicles. In addition, we have investigated whether cholesterol plays a role in prostasome release by the human prostate cancer cell line PC-3. Interestingly, prostasome release was significantly increased when the cholesterol levels of PC-3 cells were reduced by the cholesterol-sequestering agent methyl-beta-cyclodextrin (MBCD), or by treatment with lovastatin and mevalonate. In conclusion, these studies indicate that cholesterol plays an important role in the release of prostasomes by the human prostate cancer PC-3 cells, and suggest that prostasomes may be released after fusion of secretory lysosomes with the plasma membrane.     

Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells

Most clinically approved biomarkers of cancer are glycoproteins, and those residing on the cell surface are of particular interest in biotherapeutics. We report a method for selective labeling, affinity enrichment, and identification of cell-surface glycoproteins. PC-3 cells and primary human prostate cancer tissue were treated with peracetylated N-azidoacetylgalactosamine, resulting in metabolic labeling of cell surface glycans with the azidosugar. We used mass spectrometry to identify over 70 cell surface glycoproteins and biochemically validated CD146 and integrin beta-4, both of which are known to promote metastatic behavior. These results establish cell-surface glycoproteomics as an effective technique for discovery of cancer biomarkers.     

The proteomics of prostate cancer exosomes

Exosomes and other microvesicles are emerging as rich reservoirs of tumor-specific proteins and biomarkers for cancer detection and progression. For prostate cancer, exosomes secreted by the prostate can be isolated from prostatic secretions, seminal fluid, tissue, urine or blood for further proteomic analysis. Structurally, prostate-derived exosomes are distinct in size, membrane composition and specific prostate protein content, potentially providing a novel and easily isolatable source of biomarkers from clinical biofluids. The key to these isolation strategies will be the targeting of specific prostatic proteins expressed in these exosomes, thus requiring detailed proteomic characterizations. A summary of ongoing efforts to characterize the proteome of these unique prostate cancer-associated exosomes and their potential applications for use in biomarker assays is presented.     

Regulation of circGOLPH3 and its binding protein CBX7 on the proliferation and apoptosis of prostate cancer cells

To clarify the mechanism of circGOLPH3 regulation on prostate cancer cells, we performed an overexpression and interference circGOLPH3 assay in prostate cancer cells PC-3 and then evaluated cellular viability, proliferation, cell cycle, and apoptosis of prostate cancer cells by MTT, CCK8, Edu stain, TUNEL stain, and flow cytometry. Binding proteins of CircGOLPH3 were identified by RNA pull-down, mass spectrometry, and RNA-binding protein immunoprecipitation (RIP) assays. The expressions of CircGOLPH3 and CBX7 were measured by qRT-PCR. The results showed that after overexpression of circGOLPH3, the proliferative capacity and the viability of PC-3cells were significantly improved, whereas apoptosis was inhibited. CircGOLPH3 could bind to the CBX7 protein that was highly expressed in the PC-3 cell. Additionally, a functional test on CBX7 showed that the CBX7 overexpression notably improved the proliferative capacity and the viability of PC-3 cells and decreased cellular apoptosis, which was consistent with the effects of circGOLPH3. The validated the present study that circGOLPH3 and its binding protein CBX7 can promote prostate cancer cell proliferation and inhibit apoptosis.     

Novel MMP-9 substrates in cancer cells revealed by a label-free quantitative proteomics approach

Matrix metalloproteinase-9 (MMP-9) is implicated in tumor metastasis as well as a variety of inflammatory and pathological processes. Although many substrates for MMP-9, including components of the extracellular matrix, soluble mediators such as chemokines, and cell surface molecules have been identified, we undertook a more comprehensive proteomics-based approach to identify new substrates to further understand how MMP-9 might contribute to tumor metastasis. Previous proteomics approaches to identify protease substrates have depended upon differential labeling of each sample. Instead we used a label-free quantitative proteomics approach based on ultraperformance LC-ESI-high/low collision energy MS. Conditioned medium from a human metastatic prostate cancer cell line, PC-3ML, in which MMP-9 had been down-regulated by RNA interference was compared with that from the parental cells. From more than 200 proteins identified, 69 showed significant alteration in levels after depletion of the protease (>+/-2-fold), suggesting that they might be candidate substrates. Levels of six of these (amyloid-beta precursor protein, collagen VI, leukemia inhibitory factor, neuropilin-1, prostate cancer cell-derived growth factor (PCDGF), and protease nexin-1 (PN-1)) were tested in the conditioned media by immunoblotting. There was a strong correlation between results by ultraperformance LC-ESI-high/low collision energy MS and by immunoblotting giving credence to the label-free approach. Further information about MMP-9 cleavage was obtained by comparison of the peptide coverage of collagen VI in the presence and absence of MMP-9 showing increased sensitivity of the C- and N-terminal globular regions over the helical regions. Susceptibility of PN-1 and leukemia inhibitory factor to MMP-9 degradation was confirmed by in vitro incubation of the recombinant proteins with recombinant MMP-9. The MMP-9 cleavage sites in PN-1 were sequenced. This study provides a new label-free method for degradomics cell-based screening leading to the identification of a series of proteins whose levels are affected by MMP-9, some of which are clearly direct substrates for MMP-9 and become candidates for involvement in metastasis.     

前列腺癌细胞系中环状RNA circRNA-1565的表达及鉴定

目的:探讨前列腺癌细胞系中的环状RNA circRNA-1565的表达及鉴定。方法:培养前列腺正常上皮细胞(RWPE-1)、4种前列腺癌细胞(22RV1、LNCap、PC-3、DU145),提取总RNA,采用RT-PCR检测不同细胞系中circRNA-1565的表达,并对其进行成环性验证及细胞内亚定位实验,进行差异比较。结果:circRNA-1565在正常前列腺细胞系(RWPE-1)中表达量极低,在转移性前列腺癌细胞系中高表达,在非转移性前列腺癌细胞系中低表达。且circRNA-1565是一条主要定位于细胞质内的具有有效环状结构的RNA分子。结论:circRNA-1565在不同前列腺癌细胞系中存在差异表达,可能会通过mi RNA sponge途径发挥生物学作用。