Flame Retardancy Properties and Rheological Behavior of PP/DiDOPO Conjugated Flame Retardant Composites.

A bridged 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide derivative (DiDOPO) with conjugated structure was utilized as a novel conjugated flame retardant, Polypropylene(PP)/DiDOPO conjugated flame retardant composites were papered by being melt-extruding with a twin-screw extruder. The flame retardant efficiency of PP/DiDOPO conjugated flame retardant composites were investigated by cone calorimetry, limiting oxygen index (LOI), vertical burning test (UL-94). Besides, the rheological behavior of PP/DiDOPO conjugated flame retardant composites are measured by ARES rheometer. The results showed that when the content of DiDOPO with conjugated structure was 16 wt%, the LOI values of PP/DiDOPO conjugated flame retardant composites was 24%, and PP/DiDOPO conjugated flame retardant composites reaches V-0 grade. The heat release rate (HRR), total heat release rate (THR) and CO2 of PP/DiDOPO conjugated flame retardant composites decreased, so PP/DiDOPO conjugated flame retardant composites had excellent flame retardant effect. Rheological analysis results indicated that DiDOPO with conjugated structure suppressed the melt dripping of PP/DiDOPO conjugated flame retardant composites by enhancing the melt stability. The results showed that the DiDOPO with conjugated structure can significantly enhance the flame retardancy effect of PP/DiDOPO conjugated flame retardant composites. In addition, the materials PP/DiDOPO might be with low conductivity and charge transport mobility.

Endoplasmic reticulum protein 29 (ERp29) confers radioresistance through the DNA repair gene, O(6)-methylguanine DNA-methyltransferase, in breast cancer cells.

Resistance of cancer cells to radiotherapy is a major clinical problem in cancer treatment. Therefore, understanding the molecular basis of cellular resistance to radiotherapy and identification of novel targets are essential for improving treatment efficacy for cancer patients. Our previous studies have demonstrated a significant role of ERp29 in breast cancer cell survival against doxorubicin-induced genotoxic stress. We here reported that ERp29 expression in the triple negative MDA-MB-231 breast cancer cells significantly increased cell survival against ionizing radiation. Methylation PCR array analysis identified that ERp29 expression increased promoter hypomethylation of the DNA repair gene, O(6)-methylguanine DNA-methyltransferase (MGMT), by downregulating DNA methyltransferase 1. Knockdown of MGMT in the ERp29-transfected cancer cells increased radiosensitivity, leading to a decreased post-irradiation survival. In addition, radiation treatment in the MGMT-knockdown cells elevated phosphorylation of γ-H2AX and cleavage of caspase 3, indicating that depletion of MGMT facilitates DNA double strands breaks and increases cell apoptosis. Hence, our studies prove a novel function of ERp29\MGMT in cancer cell survival against radiation. Targeting ERp29\MGMT axis may be useful for providing better treatment efficacy in combination with radiotherapy in breast cancer.

Friend or foe: Endoplasmic reticulum protein 29 (ERp29) in epithelial cancer.

The endoplasmic reticulum (ER) protein 29 (ERp29) is a molecular chaperone that plays a critical role in protein secretion from the ER in eukaryotic cells. Recent studies have also shown that ERp29 plays a role in cancer. It has been demonstrated that ERp29 is inversely associated with primary tumor development and functions as a tumor suppressor by inducing cell growth arrest in breast cancer. However, ERp29 has also been reported to promote epithelial cell morphogenesis, cell survival against genotoxic stress and distant metastasis. In this review, we summarize the current understanding on the biological and pathological functions of ERp29 in cancer and discuss the pivotal aspects of ERp29 as "friend or foe" in epithelial cancer.

Hyaluronic acid conjugated ß-cyclodextrin-oligoethylenimine star polymer for CD44-targeted gene delivery.

A new CD44-targeted gene delivery system, the star-shaped cationic polymer containing a ß-cyclodextrin (ß-CD) core and multiple branched oligoethylenimine (OEI) arms with conjugated oligomer of hyaluronic acid (HA), was synthesized by reductive amination between ß-CD-OEI star polymer and HA, and was characterized for pDNA condensation and nanoparticle formation, followed by evaluation for targeted gene delivery of luciferase reporter gene and wild type p53 gene in CD44-positive and CD44-negative cell lines. The ß-CD-OEI-HA polymer contained 6 arms of OEI (600 Da) and a short HA segment. It could fully condense pDNA to form nanoparticles with sizes ranging from 100 to 200 nm at N/P ratios of 8 or higher. The conjugation of HA reduced cytotoxicity of ß-CD-OEI-HA/pDNA polyplexes. It was found that CD44 receptor was highly expressed and localized at the membrane of MDA-MB-231 breast cancer cell line, while no CD44 was found at the membrane of MCF-7 epithelial cell line. Compared with PEI (25 kDa) and ß-CD-OEI star polymers, ß-CD-OEI-HA demonstrated significant increased gene transfection efficiency in MDA-MB-231 cells, while such effect was absent in MCF-7 cells. The targeted delivery of wild type p53 gene by ß-CD-OEI-HA in MDA-MB-231 cells resulted in an increased cell cycle arrest at sub-G1 phase.

Fucoidan induces cancer cell apoptosis by modulating the endoplasmic reticulum stress cascades.

BACKGROUND: Cancer metastasis is the main cause leading to disease recurrence and high mortality in cancer patients. Therefore, inhibiting metastasis process or killing metastatic cancer cells by inducing apoptosis is of clinical importance in improving cancer patient survival. Previous studies revealed that fucoidan, a fucose-rich polysaccharide isolated from marine brown alga, is a promising natural product with significant anti-cancer activity. However, little is known about the role of endoplasmic reticulum (ER) stress in fucoidan-induced cell apoptosis. PRINCIPAL FINDINGS: We reported that fucoidan treatment inhibits cell growth and induces apoptosis in cancer cells. Fucoidan treatments resulted in down-regulation of the glucose regulated protein 78 (GRP78) in the metastatic MDA-MB-231 breast cancer cells, and of the ER protein 29 (ERp29) in the metastatic HCT116 colon cancer cells. However, fucoidan treatment promoted ER Ca2+-dependent calmodulin-dependent kinase II (CaMKII) phosphorylation, Bcl-associated X protein (Bax) and caspase 12 expression in MDA-MB-231 cells, but not in HCT116 cells. In both types of cancer cells, fucoidan activated the phosphorylation of eukaryotic initiation factor 2 alpha (p-eIF2α)\CCAAT/enhancer binding protein homologous protein (CHOP) pro-apoptotic cascade and inhibited the phosphorylation of inositol-requiring kinase 1 (p-IRE-1)\X-box binding proteins 1 splicing (XBP-1s) pro-survival cascade. Furthermore, CHOP knockdown prevented DNA damage and cell death induced by fucoidan. CONCLUSION/SIGNIFICANCE: Fucoidan exerts its anti-tumor function by modulating ER stress cascades. Contribution of ER stress to the fucoidan-induced cell apoptosis augments our understanding of the molecular mechanisms underlying its anti-tumour activity and provides evidence for the therapeutic application of fucoidan in cancer.

miR-320a suppresses colorectal cancer progression by targeting Rac1.

MicroRNAs (miRNAs) have emerged as critical epigenetic regulators involved in cancer progression. miR-320a has been identified to be a novel tumour suppressive miRNA in colorectal cancer (CRC). However, the detailed molecular mechanisms are not fully understood. Here, we reported that miR-320a inversely associated with CRC aggressiveness in both cell lines and clinical specimens. Functional studies demonstrated that miR-320a significantly decreased the capability of cell migration/invasion and induced G0/G1 growth arrest in vitro and in vivo. Furthermore, Rac1 was identified as one of the direct downstream targets of miR-320a and miR-320a specifically binds to the conserved 8-mer at position 1140-1147 of Rac1 3'-untranslated region to regulate Rac1 protein expression. Over-expression of miR-320a in SW620 cells inhibited Rac1 expression, whereas reduction of miR-320a by anti-miR-320a in SW480 cells enhanced Rac1 expression. Re-expression of Rac1 in the SW620/miR-320a cells restored the cell migration/invasion inhibited by miR-320a, whereas knockdown of Rac1 in the SW480/anti-miR-320a cells repressed these cellular functions elevated by anti-miR-320a. Conclusively, our results demonstrate that miR-320a functions as a tumour-suppressive miRNA through targeting Rac1 in CRC.

The metastasis suppressor, N-myc downregulated gene 1 (NDRG1), is a prognostic biomarker for human colorectal cancer.

Metastasis remains to be one of the most prevalent causes leading to poor long-term survival of colorectal cancer (CRC) patients. The clinical significances of tumor metastatic suppressor, N-myc downregulated gene 1 (NDRG1), have been inconsistently reported in a variety of cancerous diseases. In this study with 240 CRC clinical specimens, we showed that NDRG1 expression was significantly decreased in most of CRC tissues compared to the paired non-tumor counterparts. Statistical analysis revealed a significant inverse correlation of NDRG1 expression with tumor stage, differentiation status and metastasis. Compared with NDRG1-negative group, NDRG1-positve group had better disease-free/overall survival (p = 0.000) over 5 years' follow-up. Furthermore, NDRG1 was considered to be an independent prognostic factor for overall survival (p = 0.001) and recurrence (p = 0.003). Our study concludes that NDRG1 is a novel favorable predictor for the prognosis in CRC patients.

Metastasis suppressor, NDRG1, mediates its activity through signaling pathways and molecular motors.

The metastasis suppressor, N-myc downstream regulated gene 1 (NDRG1), is negatively correlated with tumor progression in multiple neoplasms, being a promising new target for cancer treatment. However, the precise molecular effects of NDRG1 remain unclear. Herein, we summarize recent advances in understanding the impact of NDRG1 on cancer metastasis with emphasis on its interactions with the key oncogenic nuclear factor-kappaB, phosphatidylinositol-3 kinase/phosphorylated AKT/mammalian target of rapamycin and Ras/Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling pathways. Recent studies demonstrating the inhibitory effects of NDRG1 on the epithelial-mesenchymal transition, a key initial step in metastasis, TGF-ß pathway and the Wnt/ß-catenin pathway are also described. Furthermore, NDRG1 was also demonstrated to regulate molecular motors in cancer cells, leading to inhibition of F-actin polymerization, stress fiber formation and subsequent reduction of cancer cell migration. Collectively, this review summarizes the underlying molecular mechanisms of the antimetastatic effects of NDRG1 in cancer cells.

Holothurian glycosaminoglycan inhibits metastasis and thrombosis via targeting of nuclear factor-κB/tissue factor/Factor Xa pathway in melanoma B16F10 cells.

Holothurian glycosaminoglycan (hGAG) is a high-molecular-weight form of fucosylated chondroitin sulfate and has an antithrombotic effect. Our previous studies demonstrated that hGAG efficiently inhibited tumor cell metastasis. The interplays between thrombosis and tumor progression may have a major impact on hematogenous metastasis. In this study, we demonstrated that the mouse melanoma B16F10 cells treated with hGAG displayed a significant reduction of metastasis and coagulation capacity in vitro and in vivo. Mechanistic studies revealed that hGAG treatment in B16F10 cells remarkably inhibited the formation of fibrin through attenuating the generation of activated Factor Xa (FXa), without affecting the expression of urokinase (uPA) and plasminogen activator inhibitor 1 (PAI-1) that involved in fibrinolysis. Moreover, hGAG treatment downregulated the transcription and protein expression of tissue factor (TF). Promoter deletions, site mutations and functional studies identified that the nuclear transcription factor NF-κB binding region is responsible for hGAG-induced inhibition of TF expression. While the hGAG treatment of B16F10 cells was unable to inhibit NF-κB expression and phosphorylation, hGAG significantly prevented nuclear translocation of NF-κB from the cytosol, a potential mechanism underlying the transcriptional suppression of TF. Moreover, hGAG markedly suppressed the activation of p38MAPK and ERK1/2 signaling pathways, the central regulators for the expression of metastasis-related matrix metalloproteinases (MMPs). Consequently, hGAG exerts a dual function in the inhibition of metastasis and coagulation activity in mouse melanoma B16F10 cells. Our studies suggest hGAG to be a promising therapeutic agent for metastatic cancer treatment.

Targeting the metastasis suppressor, NDRG1, using novel iron chelators: regulation of stress fiber-mediated tumor cell migration via modulation of the ROCK1/pMLC2 signaling pathway.

The iron-regulated metastasis suppressor, N-myc downstream-regulated gene 1 (NDRG1), is up-regulated by cellular iron depletion mediated by iron chelators and can inhibit cancer cell migration. However, the mechanism of how NDRG1 achieves this effect remains unclear. In this study, we implemented established and newly constructed NDRG1 overexpression and knockdown models using the DU145, HT29, and HCT116 cancer cell lines to investigate the molecular basis by which NDRG1 exerts its inhibitory effect on cell migration. Using these models, we demonstrated that NDRG1 overexpression inhibits cell migration by preventing actin-filament polymerization, stress fiber assembly and formation. In contrast, NDRG1 knockdown had the opposite effect. Moreover, we identified that NDRG1 inhibited an important regulatory pathway mediated by the Rho-associated, coiled-coil containing protein kinase 1 (ROCK1)/phosphorylated myosin light chain 2 (pMLC2) pathway that modulates stress fiber assembly. The phosphorylation of MLC2 is a key process in inducing stress fiber contraction, and this was shown to be markedly decreased or increased by NDRG1 overexpression or knockdown, respectively. The mechanism involved in the inhibition of MLC2 phosphorylation by NDRG1 was mediated by a significant (P < 0.001) decrease in ROCK1 expression that is a key kinase involved in MLC2 phosphorylation. Considering that NDRG1 is up-regulated after cellular iron depletion, novel thiosemicarbazone iron chelators (e.g., di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone) were demonstrated to inhibit ROCK1/pMLC2-modulated actin-filament polymerization, stress fiber assembly, and formation via a mechanism involving NDRG1. These results highlight the role of the ROCK1/pMLC2 pathway in the NDRG1-mediated antimetastatic signaling network and the therapeutic potential of iron chelators at inhibiting metastasis.

The iron chelators Dp44mT and DFO inhibit TGF-ß-induced epithelial-mesenchymal transition via up-regulation of N-Myc downstream-regulated gene 1 (NDRG1).

The epithelial-mesenchymal transition (EMT) is a key step for cancer cell migration, invasion, and metastasis. Transforming growth factor-ß (TGF-ß) regulates the EMT and the metastasis suppressor gene, N-myc downstream-regulated gene-1 (NDRG1), could play a role in regulating the TGF-ß pathway. NDRG1 expression is markedly increased after chelator-mediated iron depletion via hypoxia-inducible factor 1α-dependent and independent pathways (Le, N. T. and Richardson, D. R. (2004) Blood 104, 2967-2975). Moreover, novel iron chelators show marked and selective anti-tumor activity and are a potential new class of anti-metabolites. Considering this, the current study investigated the relationship between NDRG1 and the EMT to examine if iron chelators can inhibit the EMT via NDRG1 up-regulation. We demonstrated that TGF-ß induces the EMT in HT29 and DU145 cells. Further, the chelators, desferrioxamine (DFO) and di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), inhibited the TGF-ß-induced EMT by maintaining E-cadherin and ß-catenin, at the cell membrane. We then established stable clones with NDRG1 overexpression and knock-down in HT29 and DU145 cells. These data showed that NDRG1 overexpression maintained membrane E-cadherin and ß-catenin and inhibited TGF-ß-stimulated cell migration and invasion. Conversely, NDRG1 knock-down caused morphological changes from an epithelial- to fibroblastic-like phenotype and also increased migration and invasion, demonstrating NDRG1 knockdown induced the EMT and enhanced TGF-ß effects. We also investigated the mechanisms involved and showed the TGF-ß/SMAD and Wnt pathways were implicated in NDRG1 regulation of E-cadherin and ß-catenin expression and translocation. This study demonstrates that chelators inhibit the TGF-ß-induced EMT via a process consistent with NDRG1 up-regulation and elucidates the mechanism of their activity.

ERp29 induces breast cancer cell growth arrest and survival through modulation of activation of p38 and upregulation of ER stress protein p58IPK.

Endoplasmic reticulum protein 29 (ERp29) is an ER luminal protein that has a role in protein unfolding and secretion, but its role in cancer is unclear. Recently, we reported that overexpression of ERp29 significantly inhibited cell proliferation and prevented tumorigenesis in highly proliferative MDA-MB-231 breast cancer cells. Here, we show that ERp29-induced cancer cell growth arrest is modulated by the interplay between the concomitant phosphorylation of p38 and upregulation of the inhibitor of the interferon-induced, double-stranded RNA-activated protein kinase, p58(IPK). In this cell model, ERp29 overexpression significantly downregulates modulators of cell proliferation, namely urokinase plasminogen activator receptor, ß(1)-integrin and epidermal growth factor receptor. Furthermore, ERp29 significantly (P<0.001) increases phosphorylation of p38 (p-p38) and reduces matrix metalloproteinase-9 secretion. The role of ERp29 in upregulating cyclin-dependent kinase inhibitors (p15 and p21) and in downregulating cyclin D(2) is demonstrated in slowly proliferating ERp29-overexpressing MDA-MB-231 cells, whereas the opposite response was observed in ERp29-knockdown MCF-7 cells. Pharmacological inhibition of p-p38 downregulates p15 and p21 and inhibits eIF2α phosphorylation, indicating a role for p-p38 in this process. Furthermore, p58(IPK) expression was increased in ERp29-overexpressing MDA-MB-231 cells and highly decreased in ERp29-knockdown MCF-7 cells. This upregulation of p58(IPK) by ERp29 suppresses the activation of p-p38/p-PERK/p-eIF2α by repressing eIF2α phosphorylation. In fact, reduction of p58(IPK) expression by RNA interference stimulated eIF2α phosphorylation. The repression of eIF2α phosphorylation by p58(IPK) prevents ERp29-transfected cells from undergoing ER-dependent apoptosis driven by the activation of ATF4/CHOP/caspase-3. Hence, the interplay between p38 phosphorylation and p58(IPK) upregulation has key roles in modulating ERp29-induced cell-growth arrest and survival.

The metastasis suppressor, N-myc downstream regulated gene 1 (NDRG1), upregulates p21 via p53-independent mechanisms.

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), has been shown to markedly reduce metastasis of numerous tumors. The current study was focused on further elucidating the molecular mechanisms behind the antitumor function of NDRG1. We have identified for the first time that NDRG1 upregulates the potent cyclin-dependent kinase inhibitor, p21. This effect was observed in three different cancer cell types, including PC3MM and DU145 prostate cancer cells and H1299 lung carcinoma cells, and occurred independently of p53. In addition, reducing NDRG1 expression using short hairpin RNA in PC3MM and DU145 cells resulted in significantly reduced p21 protein levels. Hence, p21 is closely correlated with NDRG1 expression in these latter cell types. Examining the mechanisms behind the effect of NDRG1 on p21 expression, we found that NDRG1 upregulated p21 via transcriptional and posttranscriptional mechanisms in prostate cancer cells, although its effect on H1299 cells was posttranscriptional only. Further studies identified two additional NDRG1 protein targets. The dominant-negative p63 isoform, ΔNp63, which has been found to inhibit p21 transcription, was downregulated by NDRG1. On the other hand, a truncated 50 kDa MDM2 isoform (p50(MDM2)), which may protect p21 from proteasomal degradation, was upregulated by NDRG1. The downregulation of ΔNp63 and upregulation of p50(MDM2) are potential mechanisms by which NDRG1 increases p21 expression in these cells. Additional functional studies identified that NDRG1 inhibits cancer cell migration, suggesting that p21 is a molecular player in its antimetastatic activity.

Endoplasmic reticulum protein 29 (ERp29): An emerging role in cancer.

The endoplasmic reticulum protein 29 (ERp29) is a molecule that facilitates processing and transport of proteins in the early secretory pathway. Structural and functional analyses have suggested a biological role as a putative chaperone in the endoplasmic reticulum. The N-terminal domain of ERp29 resembles the thioredoxin domain of protein disulfide isomerase, but lacks its redox-active function due to the absence of an active motif consisting of double cysteines. In the context of carcinogenesis, the role of ERp29 in cancer progression has not been fully elucidated. However, recent studies indicate that high expression of ERp29 inversely correlates to tumor progression. In addition, over-expression of ERp29 significantly inhibits proliferation and suppresses tumorigenesis by modulating ER stress signaling and the mesenchymal-epithelial transition in breast cancer cells. In this review, we summarize the biological properties of ERp29 and its novel function as a tumor suppressor.

Over-expression of ERp29 attenuates doxorubicin-induced cell apoptosis through up-regulation of Hsp27 in breast cancer cells.

The endoplasmic reticulum protein 29 (ERp29) has a critical role in regulating protein folding, maturation and secretion. However, its role in carcinogenesis remains elusive. Recently, we reported that ERp29 is a novel tumor suppressor and regulates mesenchymal-epithelial transition in MDA-MB-231 breast cancer cells. Here, we investigated whether ERp29 plays a role in the response of breast cancer cells to chemotherapeutic agents. We found that expression of ERp29 increased the resistance to doxorubicin, but not cisplatin and paclitaxel, and decreased the doxorubicin-induced cell apoptosis in MDA-MB-231 cells, whereas knockdown of ERp29 in MCF-7 cells increased the doxorubicin cytotoxicity. A proteomics study identified up-regulation of Hsp27 and down-regulation of stathmin-1, galectin and prohibitin in the doxorubicin-resistant, ERp29 over-expressing MDA-MB-231 cells. Further, we demonstrated that ERp29 up-regulated expression of Hsp27 by down-regulating eukaryotic translational initiation factor 2α (eIF2α). When Hsp27 was knocked down by siRNA in the doxorubicin-resistant, ERp29 over-expressing MDA-MB-231 cells and parental MCF-7 cells, cell viability was significantly decreased and doxorubicin-induced cell apoptosis was enhanced. These results indicate that Hsp27 is involved in the ERp29-mediated resistance to doxorubicin. Therefore, targeting of Hsp27, with a combination of other chemotherapeutic agents, is a rational strategy in treating doxorubicin-resistant cancer cells.

Silencing of the PP2A catalytic subunit causes HER-2/neu positive breast cancer cells to undergo apoptosis.

Protein phosphatase 2A (PP2A), in its activated form as a phosphatase, is a tumour suppressor. However, when PP2A is phosphorylated at the tyrosine residue (pY307), it loses its phosphatase activity and becomes inactivated. In our previous study, we found a higher expression of pY307-PP2A in HER-2/neu positive breast tumour samples and significantly correlated to tumour progression, and in this context, it could function as a proto-oncogene. The above and subsequent findings led us to postulate that the critical role of PP2A in maintaining the balance between cell survival and cell death may be linked to its phosphorylation status at its Y307 residue. Hence, we further investigated the effects of knocking down the PP2A catalytic subunit which contains the Y307 amino acid residue in two HER-2/neu positive breast cancer cell lines, BT474 and SKBR3. We showed that this causes the silenced HER-2/neu breast cancer cells to undergo apoptosis and furthermore, that such apoptosis is mediated by p38 MAPK-caspase 3/PARP activation. Understanding the role of PP2A in HER2/neu positive cells might thus provide insight into new targets for breast cancer therapy.

Overexpression of endoplasmic reticulum protein 29 regulates mesenchymal-epithelial transition and suppresses xenograft tumor growth of invasive breast cancer cells.

Endoplasmic reticulum protein 29 (ERp29) is a novel endoplasmic reticulum (ER) secretion factor that facilitates the transport of secretory proteins in the early secretory pathway. Recently, it was found to be overexpressed in several cancers; however, little is known regarding its function in breast cancer progression. In this study, we show that the expression of ERp29 was reduced with tumor progression in clinical specimens of breast cancer, and that overexpression of ERp29 resulted in G(0)/G(1) arrest and inhibited cell proliferation in MDA-MB-231 cells. Importantly, overexpression of ERp29 in MDA-MB-231 cells led to a phenotypic change and mesenchymal-epithelial transition (MET) characterized by cytoskeletal reorganization with loss of stress fibers, reduction of fibronectin (FN), reactivation of epithelial cell marker E-cadherin and loss of mesenchymal cell marker vimentin. Knockdown of ERp29 by shRNA in MCF-7 cells reduced E-cadherin, but increased vimentin expression. Furthermore, ERp29 overexpression in MDA-MB-231 and SKBr3 cells decreased cell migration/invasion and reduced cell transformation, whereas silencing of ERp29 in MCF-7 cells enhanced cell aggressive behavior. Significantly, expression of ERp29 in MDA-MB-231 cells suppressed tumor formation in nude mice by repressing the cell proliferative index (Ki-67 positivity). Transcriptional profiling analysis showed that ERp29 acts as a central regulator by upregulating a group of genes with tumor suppressive function, for example, E-cadherin (CDH1), cyclin-dependent kinase inhibitor (CDKN2B) and spleen tyrosine kinase (SYK), and by downregulating a group of genes that regulate cell proliferation (eg, FN, epidermal growth factor receptor (EGFR) and plasminogen activator receptor (uPAR)). It is noteworthy that ERp29 significantly attenuated the overall ERK cascade, whereas the ratio of p-ERK1 to p-ERK2 was highly increased. Taken together, our results showed that ERp29 is a novel regulator leading to cell growth arrest and cell transition from a proliferative to a quiescent state, and reprogramming molecular portraits to suppress the tumor growth of MDA--MB--231 breast cancer cells.

Tyrosine phosphorylation of PP2A is regulated by HER-2 signalling and correlates with breast cancer progression.

Activation of HER-2/neu leads to multiple signalling cascades and plays a vital role in cell survival and growth. We used a signal transduction antibody array to characterize the tyrosine phosphorylation profiles in heregulin (HRG alpha1)-treated BT474 breast cancer cells, and identified a group of 80 molecules in which tyrosine phosphorylation was highly regulated by HRG-enhanced HER-2/neu signalling. These phosphoproteins included many known HER-2/neu-regulated molecules (e.g., SHC, Akt, Syk and Stat1) and proteins that had not been previously linked to HER-2/neu signalling, such as Fas-associated death domain protein (FADD), apoptosis repressor with CARD domain (ARC), and the tumour suppressor, protein phosphatase type 2A (PP2A). Pharmacological inhibition with HER-2 inhibitor AG825, PI3K inhibitor LY294002, MEK1/2 inhibitor PD98095, and p38MAPK inhibitor SB203580 confirmed that PP2A phosphorylation was modulated by the complicated, HER-2/neu-driven downstream signal network, with the PI3K and MEK1/2 positively, while the p38MAPK negatively regulating its tyrosine phosphorylation. In breast tumour specimens, expression of tyrosine-phosphorylated PP2A (pY307-PP2A) was highly increased in the HER-2/neu positive breast tumours, and significantly correlated to tumour progression, thus enhancing its potential prognostic value. Our data provide meaningful information in the elucidation of the HER-2-driven tyrosine phosphorylation network, and in the development of phosphopeptide-related targets as prognostication indicators.

Cytokeratin 19 regulates endoplasmic reticulum stress and inhibits ERp29 expression via p38 MAPK/XBP-1 signaling in breast cancer cells.

Cytokeratin 19 (CK19) is widely used as a biomarker for the detection of disseminated tumor cells in blood and bone marrow, and its positivity is considered as an independent prognostication indicator in cancer patients. However, its role in breast cancer progression remains unknown. We had established a stable CK19-expressing clone in the CK19-negative BT549 human breast cancer cell line and found that CK19 expression in the BT549 cells caused cell cycle arrest, reduced cell motility and increased drug resistance. Further study revealed that CK19 expression regulated endoplasmic reticulum (ER) stress signaling by up-regulating p38/RNA-dependent protein kinase-like ER kinase (PERK)/p-eIF2alpha and 78 kDa glucose-regulated protein (Bip/GRP78), and down-regulating focal adhesion kinase (FAK). The level of ER protein 29 (ERp29) was shown to be decreased in the CK19-expressing BT549 cells by proteomic analyses and verified by Western blotting and RT-PCR. Pharmacological inhibition of p38 signaling by its specific inhibitor SB203580 or knockdown of p38 and transcription factor XBP-1 by siRNA in BT549/CK19 and MDA-MB-231 cells revealed that p38/XBP-1 signaling negatively regulated ERp29 expression. Our results indicated that CK19 modulates ER stress signaling and contributes to cell survival and dormancy in breast cancer cells.

Analysis of laser capture microdissected cells by 2-dimensional gel electrophoresis.

Laser capture microdissection (LCM) is a powerful tool for procuring near-pure populations of targeted cell types from specific microscopic regions of tissue sections, by overcoming problems due to tissue heterogeneity and minimizing intermixture and contamination by other cell types. The combination of LCM with various proteomic technologies has enabled high-throughput molecular analysis of human tumors, and provided critical tools in the search for novel disease markers and therapeutic targets. As an example, we describe the application of LCM in dissecting the tumor cells in breast cancer for macromolecular extraction and subsequent protein separation by 2-dimensional gel electrophoresis (2-D GE). The protocols and the key issues involved in preparing ethanol-fixed paraffin-embedded tissue blocks and microscopic sections, microdissecting the cells of interest using the PixCell II LCM system, extracting and separating the cellular proteins by 2-D GE, and preparing selective proteins for peptide mass analysis by mass spectrometry, are discussed. The aim is to provide a practical guide in performing high-throughput microdissection of target cells and gel-based proteomics, which can be adapted to research in cancer formation and growth.