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In microarray data analysis, we are often required to combine several dependent partial test results. To overcome this, many suggestions have been made in previous literature; Tippett’s test and Fisher’s omnibus test are most popular. Both tests have known null distributions when the partial tests are independent. However, for dependent tests, their (even, asymptotic) null distributions are unknown and additional numerical procedures are required. In this paper, we revisited Stouffer’s test based on z-scores and showed its advantage over the two aforementioned methods in the analysis of large-scale microarray data. The combined statistic in Stouffer’s test has a normal distribution with mean 0 from the normality of the z-scores. Its variance can be estimated from the scores of genes in the experiment without an additional numerical procedure. We numerically compared the errors of Stouffer’s test and the two p-value based methods, Tippett’s test and Fisher’s omnibus test. We also analyzed our microarray data to find differentially expressed genes by non-genotoxic and genotoxic carcinogen compounds. Both numerical study and the real application showed that Stouffer’s test performed better than Tippett’s method and Fisher’s omnibus method with additional permutation steps. [ABSTRACT FROM AUTHOR]

Cancer is the second most common cause of death in the USA. Among the known classes of anticancer agents, the microtubule-targeted antimitotic drugs are considered to be one of the most important. They are usually classified into microtubule-destabilizing (e.g., Vinca alkaloids) and microtubule-stabilizing (e.g., paclitaxel) agents. Combretastatin A4 (CA-4), which is a natural stilbene isolated from Combretum caffrum, is a microtubule-destabilizing agent that binds to the colchicine domain on β-tubulin and exhibits a lower toxicity profile than paclitaxel or the Vinca alkaloids. In this paper, we describe the docking study, synthesis, antiproliferative activity and selectivity index of the N-acylhydrazone derivatives (5a–r) designed as CA-4 analogues. The essential structural requirements for molecular recognition by the colchicine binding site of β-tubulin were recognized, and several compounds with moderate to high antiproliferative potency (IC50 values ≤18 µM and ≥4 nM) were identified. Among these active compounds, LASSBio-1586 (5b) emerged as a simple antitumor drug candidate, which is capable of inhibiting microtubule polymerization and possesses a broad in vitro and in vivo antiproliferative profile, as well as a better selectivity index than the prototype CA-4, indicating improved selective cytotoxicity toward cancer cells. [ABSTRACT FROM AUTHOR]

The regulation of chromosome separation during mitosis is not fully understood yet. Microtubules forming mitotic spindles are targets of treatment strategies which are aimed at (i) the triggering of the apoptosis or (ii) the interruption of uncontrolled cell division. Despite these facts, only few physical models relating to the dynamics of mitotic spindles exist up to now. In this paper, we present the first electromechanical model which enables calculation of the electromagnetic field coupled to acoustic vibrations of the mitotic spindle. This electromagnetic field originates from the electrical polarity of microtubules which form the mitotic spindle. The model is based on the approximation of resonantly vibrating microtubules by a network of oscillating electric dipoles. Our computational results predict the existence of a rapidly changing electric field which is generated by either driven or endogenous vibrations of the mitotic spindle. For certain values of parameters, the intensity of the electric field and its gradient reach values which may exert a not-inconsiderable force on chromosomes which are aligned in the spindle midzone. Our model may describe possible mechanisms of the effects of ultra-short electrical and mechanical pulses on dividing cells—a strategy used in novel methods for cancer treatment. [ABSTRACT FROM AUTHOR]

Lung cancer is one of the most common types of cancer and causes 1.38 million deaths annually, as of 2008 worldwide. Identifying natural anti-lung cancer agents has become very important. Gambogenic acid (GNA) is one of the active compounds of Gamboge, a traditional medicine that was used as a drastic purgative, emetic, or vermifuge for treating tapeworm. Recently, increasing evidence has indicated that GNA exerts promising anti-tumor effects; however, the underlying mechanism remains unclear. In the present paper, we found that GNA could induce the formation of vacuoles, which was linked with autophagy in A549 and HeLa cells. Further studies revealed that GNA triggers the initiation of autophagy based on the results of MDC staining, AO staining, accumulation of LC3 II, activation of Beclin 1 and phosphorylation of P70S6K. However, degradation of p62 was disrupted and free GFP could not be released in GNA treated cells, which indicated a block in the autophagy flux. Further studies demonstrated that GNA blocks the fusion between autophagosomes and lysosomes by inhibiting acidification in lysosomes. This dysfunctional autophagy plays a pro-death role in GNA-treated cells by activating p53, Bax and cleaved caspase-3 while decreasing Bcl-2. Beclin 1 knockdown greatly decreased GNA-induced cell death and the effects on p53, Bax, cleaved caspase-3 and Bcl-2. Similar results were obtained using a xenograft model. Our findings show, for the first time, that GNA can cause aberrant autophagy to induce cell death and may suggest the potential application of GNA as a tool or viable drug in anticancer therapies. [ABSTRACT FROM AUTHOR]

Involved in many diseases such as cancer, diabetes, neurodegenerative, inflammatory and respiratory disorders, G-protein-coupled receptors (GPCRs) are among the most frequent targets of therapeutic drugs. It is time-consuming and expensive to determine whether a drug and a GPCR are to interact with each other in a cellular network purely by means of experimental techniques. Although some computational methods were developed in this regard based on the knowledge of the 3D (dimensional) structure of protein, unfortunately their usage is quite limited because the 3D structures for most GPCRs are still unknown. To overcome the situation, a sequence-based classifier, called “iGPCR-drug”, was developed to predict the interactions between GPCRs and drugs in cellular networking. In the predictor, the drug compound is formulated by a 2D (dimensional) fingerprint via a 256D vector, GPCR by the PseAAC (pseudo amino acid composition) generated with the grey model theory, and the prediction engine is operated by the fuzzy K-nearest neighbour algorithm. Moreover, a user-friendly web-server for iGPCR-drug was established at http://www.jci-bioinfo.cn/iGPCR-Drug/. For the convenience of most experimental scientists, a step-by-step guide is provided on how to use the web-server to get the desired results without the need to follow the complicated math equations presented in this paper just for its integrity. The overall success rate achieved by iGPCR-drug via the jackknife test was 85.5%, which is remarkably higher than the rate by the existing peer method developed in 2010 although no web server was ever established for it. It is anticipated that iGPCR-Drug may become a useful high throughput tool for both basic research and drug development, and that the approach presented here can also be extended to study other drug – target interaction networks. [ABSTRACT FROM AUTHOR]

Background: Carcinomas make up the majority of cancers. Their accurate and specific diagnoses are of great significance for the improvement of patients' curability. Methodology/Principal Findings: In this paper, we report an effectual example of the in vivo fluorescence molecular imaging of carcinomas with extremely high specificity based on whole cell-SELEX aptamers. Firstly, S6, an aptamer against A549 lung carcinoma cells, was adopted and labeled with Cy5 to serve as a molecular imaging probe. Flow cytometry assays revealed that Cy5-S6 could not only specifically label in vitro cultured A549 cells in buffer, but also successfully achieve the detection of ex vivo cultured target cells in serum. When applied to in vivo imaging, Cy5-S6 was demonstrated to possess high specificity in identifying A549 carcinoma through a systematic comparison investigation. Particularly, after Cy5-S6 was intravenously injected into nude mice which were simultaneously grafted with A549 lung carcinoma and Tca8113 tongue carcinoma, a much longer retention time of Cy5-S6 in A549 tumor was observed and a clear targeted cancer imaging result was presented. On this basis, to further promote the application to imaging other carcinomas, LS2 and ZY8, which are two aptamers selected by our group against Bel-7404 and SMMC-7721 liver carcinoma cells respectively, were tested in a similar way, both in vitro and in vivo. Results showed that these aptamers were even effective in differentiating liver carcinomas of different subtypes in the same body. Conclusions/Significance: This work might greatly advance the application of whole cell-SELEX aptamers to carcinomas-related in vivo researches. [ABSTRACT FROM AUTHOR]

Background: Human papillomavirus 16 (HPV16) is a high-risk DNA tumour virus, which is a major causative agent of cervical cancer. Cellular transformation is associated with deregulated expression of the E6 and E7 oncogenes. E7 has been shown to bind a number of cellular proteins, including the cell cycle control protein pRb. In this study, RNA aptamers (small, single-stranded oligonucleotides selected for high-affinity binding) to HPV16 E7 were employed as molecular tools to further investigate these protein-protein interactions. Methodology/Principal Findings: This study is focused on one aptamer (termed A2). Transfection of this molecule into HPV16-transformed cells resulted in inhibition of cell proliferation (shown using real-time cell electronic sensing and MTT assays) due to the induction of apoptosis (as demonstrated by Annexin V/propidium iodide staining). GST-pull down and bead binding assays were used to demonstrate that the binding of A2 required N-terminal residues of E7 known to be involved in interaction with the cell cycle control protein, pRb. Using a similar approach, A2 was shown to disrupt the interaction between E7 and pRb in vitro. Furthermore, transfection of HPV16-transformed cells with A2 appeared to result in the loss of E7 and rise in pRb levels, as observed by immunoblotting. Conclusions/Significance: This paper includes the first characterisation of the effects of an E7 RNA aptamer in a cell line derived from a cervical carcinoma. Transfection of cells with A2 was correlated with the loss of E7 and the induction of apoptosis. Aptamers specific for a number of cellular and viral proteins have been documented previously; one aptamer (Macugen) is approved for clinical use and several others are in clinical trials. In addition to its role as a molecular tool, A2 could have further applications in the future. [ABSTRACT FROM AUTHOR]

Among the many identified androgen-regulated genes, sGCα1 (soluble guanylyl cyclase α1) appears to play a pivotal role in mediating the pro-cancer effects of androgens and androgen receptor. The classical role for sGCα1 is to heterodimerize with the sGCβ1 subunit, forming sGC, the enzyme that mediates nitric oxide signaling by catalyzing the synthesis of cyclic guanosine monophosphate. Our published data show that sGCα1 can drive prostate cancer cell proliferation independent of hormone and provide cancer cells a pro-survival function, via a novel mechanism for p53 inhibition, both of which are independent of sGCβ1, NO, and cGMP. All of these properties make sGCα1 an important novel target for prostate cancer therapy. Thus, peptides were designed targeting sGCα1 with the aim of disrupting this protein’s pro-cancer activities. One peptide (A-8R) was determined to be strongly cytotoxic to prostate cancer cells, rapidly inducing apoptosis. Cytotoxicity was observed in both hormone-dependent and, significantly, hormone-refractory prostate cancer cells, opening the possibility that this peptide can be used to treat the usually lethal castration-resistant prostate cancer. In mouse xenograft studies, Peptide A-8R was able to stop tumor growth of not only hormone-dependent cells, but most importantly from hormone-independent cells. In addition, the mechanism of Peptide A cytotoxicity is generation of reactive oxygen species, which recently have been recognized as a major mode of action of important cancer drugs. Thus, this paper provides strong evidence that targeting an important AR-regulated gene is a new paradigm for effective prostate cancer therapy. [ABSTRACT FROM AUTHOR]

In this manuscript, we use genetic data to provide a three-faceted analysis on the links between molecular subclasses of glioblastoma, epithelial-to-mesenchymal transition (EMT) and CD133 cell surface protein. The contribution of this paper is three-fold: First, we use a newly identified signature for epithelial-to-mesenchymal transition in human mammary epithelial cells, and demonstrate that genes in this signature have significant overlap with genes differentially expressed in all known GBM subtypes. However, the overlap between genes up regulated in the mesenchymal subtype of GBM and in the EMT signature was more significant than other GBM subtypes. Second, we provide evidence that there is a negative correlation between the genetic signature of EMT and that of CD133 cell surface protein, a putative marker for neural stem cells. Third, we study the correlation between GBM molecular subtypes and the genetic signature of CD133 cell surface protein. We demonstrate that the mesenchymal and neural subtypes of GBM have the strongest correlations with the CD133 genetic signature. While the mesenchymal subtype of GBM displays similarity with the signatures of both EMT and CD133, it also exhibits some differences with each of these signatures that are partly due to the fact that the signatures of EMT and CD133 are inversely related to each other. Taken together these data shed light on the role of the mesenchymal transition and neural stem cells, and their mutual interaction, in molecular subtypes of glioblastoma multiforme. [ABSTRACT FROM AUTHOR]

Aims: DDX3 is an RNA helicase that has antiapoptotic properties, and promotes proliferation and transformation. In addition, DDX3 was shown to be a direct downstream target of HIF-1α (the master regulatory of the hypoxia response) in breast cancer cell lines. However, the relation between DDX3 and hypoxia has not been addressed in human tumors. In this paper, we studied the relation between DDX3 and the hypoxic responsive proteins in human breast cancer. Methods and Results: DDX3 expression was investigated by immunohistochemistry in breast cancer in comparison with hypoxia related proteins HIF-1α, GLUT1, CAIX, EGFR, HER2, Akt1, FOXO4, p53, ERα, COMMD1, FER kinase, PIN1, E-cadherin, p21, p27, Transferrin receptor, FOXO3A, c-Met and Notch1. DDX3 was overexpressed in 127 of 366 breast cancer patients, and was correlated with overexpression of HIF-1α and its downstream genes CAIX and GLUT1. Moreover, DDX3 expression correlated with hypoxia-related proteins EGFR, HER2, FOXO4, ERα and c-Met in a HIF-1α dependent fashion, and with COMMD1, FER kinase, Akt1, E-cadherin, TfR and FOXO3A independent of HIF-1α. Conclusions: In invasive breast cancer, expression of DDX3 was correlated with overexpression of HIF-1α and many other hypoxia related proteins, pointing to a distinct role for DDX3 under hypoxic conditions and supporting the oncogenic role of DDX3 which could have clinical implication for current development of DDX3 inhibitors. [ABSTRACT FROM AUTHOR]

Receptor Tyrosine Kinases (RTKs) are involved in many cellular processes and play a major role in the control of cell fate. For these reasons, RTK activation is maintained under tight control. Met is an essential RTK that induces proliferation, differentiation, migration, survival and branching morphogenesis. Deregulation of Met by overexpression, amplification or lack of effective degradation leads to cancer and metastasis. We have shown that Met relies on CD44v6 for its activation and for signaling in several cancer cell lines and also in primary cells. In this paper, we show that internalization of Met is dependent on CD44v6 and the binding of Ezrin to the CD44v6 cytoplasmic domain. Both CD44v6 and Met are co-internalized upon Hepatocyte Growth Factor induction suggesting that Met-induced signaling from the endosomes relies on its collaboration with CD44v6 and the link to the cytoskeleton provided by ERM proteins. [ABSTRACT FROM AUTHOR]

Background: Our previous research results showed that Type II cGMP dependent protein kinase (PKG II) could block the activation of epidermal growth factor receptor (EGFR) and consequently inhibit the proliferation and the related MAPK/ERK-mediated signal transduction of gastric cancer cell line BGC-823, suggesting that PKG II might inhibit other EGFR-triggered signal transduction pathways and related biological activities of gastric cancer cells. This paper was designed to investigate the potential inhibition of PKG II on EGF/EGFR-induced migration activity and the related signal transduction pathways. Methodology/Principal Findings: In gastric cancer cell line AGS, expression and activity of PKG II were increased by infecting the cells with adenoviral construct encoding PKG II cDNA (Ad-PKG II) and treating the cells with cGMP analogue 8-pCPT-cGMP. Phosphorylation of proteins was detected by Western Blotting and active small G protein Ras and Rac1 was measured by “Pull-down” method. Cell migration activity was detected with trans-well equipment. Binding between PKG II and EGFR was detected with Co-IP. The results showed EGF stimulated migration of AGS cell and the effect was related to PLCγ1 and ERK-mediated signal transduction pathways. PKG II inhibited EGF-induced migration activity and blocked EGF-initiated signal transduction of PLCγ1 and MAPK/ERK-mediated pathways through preventing EGF-induced Tyr 992 and Tyr 1068 phosphorylation of EGFR. PKG II bound with EGFR and caused threonine phosphorylation of it. Conclusion/Significance: Our results systemically confirms the inhibition of PKG II on EGF-induced migration and related signal transduction of PLCγ1 and MAPK/ERK-mediated pathways, indicating that PKG II has a fargoing inhibition on EGF/EGFR related signal transduction and biological activities of gastric cancer cells through phosphorylating EGFR and blocking the activation of it. [ABSTRACT FROM AUTHOR]

The incidence of male breast cancer (MBC) is rising. Current treatment regimens for MBC are extrapolated from female breast cancer (FBC), based on the assumption that FBC prognostic features and therapeutic targets can be extrapolated to MBC. However, there is yet little evidence that prognostic features that have been developed and established in FBC are applicable to MBC as well. In a recent study on FBC, a combination of mitotic index and Bcl2 expression proved to be of strong prognostic value. Previous papers on Bcl2 expression in MBC were equivocal, and the prognostic value of Bcl2 combined with mitotic index has not been studied in MBC. The aim of the present study was therefore to investigate the prognostic value of Bcl2 in combination with mitotic index in MBC. Immunohistochemical staining for Bcl2 was performed on tissue microarrays of a total of 151 male breast cancer cases. Mitotic index was scored. The prognostic value of Bcl2 expression and Bcl2/mitotic index combinations was evaluated studying their correlations with clinicopathologic features and their prediction of survival. The vast majority of MBC (94%) showed Bcl2 expression, more frequently than previously described for FBC. Bcl2 expression had no significant associations with clinicopathologic features such as tumor size, mitotic count and grade. In univariate survival analysis, Bcl2 had no prognostic value, and showed no additional prognostic value to tumor size and histological grade in Cox regression. In addition, the Bcl2/mitotic index combination as opposed to FBC did not predict survival in MBC. In conclusion, Bcl2 expression is common in MBC, but is not associated with major clinicopathologic features and, in contrast to FBC, does not seem to have prognostic value, also when combined with mitotic index. [ABSTRACT FROM AUTHOR]

Background: Carcinomas make up the majority of cancers. Their accurate and specific diagnoses are of great significance for the improvement of patients' curability. Methodology/Principal Findings: In this paper, we report an effectual example of the in vivo fluorescence molecular imaging of carcinomas with extremely high specificity based on whole cell-SELEX aptamers. Firstly, S6, an aptamer against A549 lung carcinoma cells, was adopted and labeled with Cy5 to serve as a molecular imaging probe. Flow cytometry assays revealed that Cy5-S6 could not only specifically label in vitro cultured A549 cells in buffer, but also successfully achieve the detection of ex vivo cultured target cells in serum. When applied to in vivo imaging, Cy5-S6 was demonstrated to possess high specificity in identifying A549 carcinoma through a systematic comparison investigation. Particularly, after Cy5-S6 was intravenously injected into nude mice which were simultaneously grafted with A549 lung carcinoma and Tca8113 tongue carcinoma, a much longer retention time of Cy5-S6 in A549 tumor was observed and a clear targeted cancer imaging result was presented. On this basis, to further promote the application to imaging other carcinomas, LS2 and ZY8, which are two aptamers selected by our group against Bel-7404 and SMMC-7721 liver carcinoma cells respectively, were tested in a similar way, both in vitro and in vivo. Results showed that these aptamers were even effective in differentiating liver carcinomas of different subtypes in the same body. Conclusions/Significance: This work might greatly advance the application of whole cell-SELEX aptamers to carcinomas-related in vivo researches. [ABSTRACT FROM AUTHOR]

T-STAR (testis-signal transduction and activation of RNA) is an RNA binding protein, containing an SH3-binding domain and thus potentially playing a role in integration of cell signaling and RNA metabolism. The specific function of T-STAR is unknown and its implication in cancer is poorly characterized. Expression of T-STAR has been reported in human testis, muscle and brain tissues, and is associated with a growth-inhibitory role in immortalized fibroblasts. The aim of this paper was to investigate the functional role of T-STAR through (i) survival analysis of patients with primary invasive breast cancer and (ii) experimental evaluation of the effect of T-STAR on breast cancer cell growth. T-STAR protein expression was analysed by immunohistochemistry (IHC) in tissue microarrays with tumors from 289 patients with primary invasive breast cancer, and correlations to clinicopathological characteristics, recurrence-free and overall survival (RFS and OS) and established tumor markers such as HER2 and ER status were evaluated. In addition, the function of T-STAR was investigated using siRNA-mediated knock-down and overexpression of the gene in six breast cancer cell lines. Of the tumors analysed, 86% showed nuclear T-STAR expression, which was significantly associated with an improved RFS and strongly associated with positive HER2 status and negative hormone receptor status. Furthermore, experimental data showed that overexpression of T-STAR decreased cellular growth while knock-down increased it, as shown both by thymidine incorporation and metabolic activity. In summary, we demonstrate that T-STAR protein expression correlates with an improved RFS in primary breast cancer. This is supported by functional data, indicating that T-STAR regulation is of importance both for breast cancer biology and clinical outcome but future studies are needed to determine a potential role in patient stratification. [ABSTRACT FROM AUTHOR]

First reported as a vasoactive peptide in the cardiovascular system, intermedin (IMD), also known as adrenomedullin 2 (ADM2), is a hormone with multiple potent roles, including its antioxidant action on the pulmonary, central nervous, cardiovascular and renal systems. Though IMD may play certain roles in trophoblast cell invasion, early embryonic development and cumulus cell-oocyte interaction, the role of IMD in the male reproductive system has yet to be investigated. This paper reports our findings on the gene expression of IMD, its receptor components and its protein localization in the testes. In a rat model, bacterial lippolysaccharide (LPS) induced atypical orchitis, and LPS treatment upregulated the expression of IMD and one of its receptor component proteins, i.e. receptor activity modifying protein 2 (RAMP2). IMD decreased both plasma and testicular levels of reactive oxygen species (ROS) production, attenuated the increase in the gene expression of the proinflammatory cytokines tumor necrosis factor alpha (TNFα), interleukin 6 (IL6) and interleukin 1 beta (IL1β), rescued spermatogenesis, and prevented the decrease in plasma testosterone levels caused by LPS. The restorative effect of IMD on steroidogenesis was also observed in hydrogen peroxide-treated rat primary Leydig cells culture. Our results indicate IMD plays an important protective role in spermatogenesis and steroidogenesis, suggesting therapeutic potential for IMD in pathological conditions such as orchitis. [ABSTRACT FROM AUTHOR]

T-STAR (testis-signal transduction and activation of RNA) is an RNA binding protein, containing an SH3-binding domain and thus potentially playing a role in integration of cell signaling and RNA metabolism. The specific function of T-STAR is unknown and its implication in cancer is poorly characterized. Expression of T-STAR has been reported in human testis, muscle and brain tissues, and is associated with a growth-inhibitory role in immortalized fibroblasts. The aim of this paper was to investigate the functional role of T-STAR through (i) survival analysis of patients with primary invasive breast cancer and (ii) experimental evaluation of the effect of T-STAR on breast cancer cell growth. T-STAR protein expression was analysed by immunohistochemistry (IHC) in tissue microarrays with tumors from 289 patients with primary invasive breast cancer, and correlations to clinicopathological characteristics, recurrence-free and overall survival (RFS and OS) and established tumor markers such as HER2 and ER status were evaluated. In addition, the function of T-STAR was investigated using siRNA-mediated knock-down and overexpression of the gene in six breast cancer cell lines. Of the tumors analysed, 86% showed nuclear T-STAR expression, which was significantly associated with an improved RFS and strongly associated with positive HER2 status and negative hormone receptor status. Furthermore, experimental data showed that overexpression of T-STAR decreased cellular growth while knock-down increased it, as shown both by thymidine incorporation and metabolic activity. In summary, we demonstrate that T-STAR protein expression correlates with an improved RFS in primary breast cancer. This is supported by functional data, indicating that T-STAR regulation is of importance both for breast cancer biology and clinical outcome but future studies are needed to determine a potential role in patient stratification. [ABSTRACT FROM AUTHOR]

An enhanced anti-apoptotic capacity of tumor cells plays an important role in the process of breakpoint cluster region/Abelson tyrosine kinase gene (BCR/ABL)-independent imatinib resistance. We have previously demonstrated that brain expressed X-linked 1 (BEX1) was silenced in secondary imatinib-resistant K562 cells and that re-expression of BEX1 can restore imatinib sensitivity resulting in the induction of apoptosis. However, the mechanism by which BEX1 executes its pro-apoptotic function remains unknown. We identified B-cell lymphoma 2 (BCL-2) as a BEX1-interacting protein using a yeast two-hybrid screen. The interaction between BEX1 and BCL-2 was subsequently confirmed by co-immunoprecipitation assays. Like BCL-2, BEX1 was localized to the mitochondria. The region between 33K and 64Q on BEX1 is important for its localization to the mitochondria and its ability to interact with BCL-2. Additionally, we found that this region is essential for BEX1-regulated imatinib-induced apoptosis. Furthermore, we demonstrated that the interaction between BCL-2 and BEX1 promotes imatinib-induced apoptosis by suppressing the formation of anti-apoptotic BCL-2/BCL-2-associated X protein (BAX) heterodimers. Our results revealed an interaction between BEX1 and BCL-2 and a novel mechanism of imatinib resistance mediated by the BEX1/BCL-2 pathway. [ABSTRACT FROM AUTHOR]

YAP is a key component of the Hippo signaling pathway and plays a critical role in the development and progression of multiple cancer types, including ovarian cancer. However, the effects of YAP on ovarian cancer development in vivo and its downstream effectors remain uncertain. In this study we found that strong YAP expression was associated with poor ovarian cancer patient survival. Specifically, we showed for the first time that high YAP expression levels were positively correlated with TEAD4 gene expression, and their co-expression was a prognostic marker for poor ovarian cancer survival. Hyperactivation of YAP by mutating its five inhibitory phosphorylation sites (YAP-5SA) increased ovarian cancer cell proliferation, resistance to chemotherapeutic drugs, cell migration, and anchorage-independent growth. In contrast, expression of a dominant negative YAP mutant reversed these phenotypes in ovarian cancer cells both in vitro and in vivo. Our results suggested that YAP caused these effects by promoting an epithelial-to-mesenchymal transition. Thus, YAP promotes ovarian cancer cell growth and tumorigenesis both in vitro and in vivo. Further, high YAP and TEAD4 expression is a prognostic marker for ovarian cancer progression and a potential target for ovarian cancer treatment. [ABSTRACT FROM AUTHOR]

: Telomere length is considered to be a risk factor in adults due to its proved association with cancer incidence and mortality. Since newborn present a wide interindividual variation in mean telomere length, it is relevant to demonstrate if these differences in length can act also as an early risk indicator. To answer this question, we have measured the mean telomere length of 74 samples of cord blood from newborns and studied its association with the basal genetic damage, measured as the frequency of binucleated cells carrying micronuclei. In addition, we have challenged the cells of a subgroup of individuals (N = 35) against mitomycin-C (MMC) to establish their sensitivity to induced genomic instability. Results indicate that newborn with shorter telomeres present significantly higher levels of genetic damage when compared to those with longer telomeres. In addition, the cellular response to MMC was also significantly higher among those samples from subjects with shorter telomeres. Independently of the causal mechanisms involved, our results show for the first time that telomere length at delivery influence both the basal and induced genetic damage of the individual. Impact: Individuals born with shorter telomeres may be at increased risk, especially for those biological processes triggered by genomic instability as is the case of cancer and other age-related diseases. [ABSTRACT FROM AUTHOR]

Tumour response to therapy is assessed primarily in the clinic by monitoring reductions in tumour size. However, this approach lacks sensitivity since in many cases several weeks may elapse before there is evidence of tumour shrinkage. There is therefore a need to develop non-invasive imaging techniques for monitoring tumour treatment response in the clinic. Here, we assessed the pre-clinical utility of 18F-ICMT-11 positron emission tomography - a method for detecting caspase 3/7 activation - in non-small cell lung cancer (NSCLC). 18F-ICMT-11 uptake was compared to molecular biochemical measures of cell death in PC9 and A549 NSCLC cells following treatment with carboplatin in vitro and in vivo. Carboplatin-induced apoptosis in the ERCC1 low/mutant EGFR PC9 cells was characterised by time and dose-related increased caspase-3/7 activation, poly-ADP-ribose polymerase cleavage and Annexin V staining. 18F-ICMT-11 uptake was consequently increased up to 14-fold at 200 µM carboplatin compared to vehicle treated cells (P<0.01). In contrast, necrosis was the predominant death mechanism in ERCC1 high/wt EGFR A549 cells and no change in 18F-ICMT-11 uptake was detected. In vivo, histological analysis of PC9 tumour xenografts indicated high pre-therapy necrosis. A 4.6-fold increase in cleaved caspase-3/7 was measured in non-necrotic regions of PC9 tumours at 48h post carboplatin therapy. Average PET-derived tumour 18F-ICMT-11 uptake was insensitive to changes in apoptosis in the presence of substantial pre-existing necrosis. PET-based voxel intensity sorting however, identified intra-tumoural regions of high 18F-ICMT-11 uptake, enabling accurate assessment of apoptosis and therefore therapy response. In A549 tumours that lacked high pre-therapy necrosis, carboplatin induced growth inhibition that was only minimally associated with apoptosis and thus not detectable by 18F-ICMT-11 PET. [ABSTRACT FROM AUTHOR]

Background: Esophageal squamous cell carcinoma (ESCC) is a highly aggressive cancer whose underlying molecular mechanisms are poorly understood. The natural antisense transcript (NAT) WRAP53 regulates p53 expression and WRAP53 protein is a component of telomerase. NATs play key roles in carcinogenesis, and although WRAP53 is known to increase cancer cell survival, its role in ESCC clinicopathology is unknown. The aim of this study was to investigate WRAP53 expression in ESCC and to correlate it with clinicopathological characteristics. Methods: WRAP53 mRNA and protein expression was measured by quantitative PCR (qRT-PCR) and western blotting, respectively, in 4 ESSC cells lines and in 45 paired ESCC and non-neoplastic esophageal mucosa tissues. To correlate WRAP53 protein expression with clinicopathological characteristics, immunohistochemistry (IHC) was performed on 134 ESCC and 85 non-neoplastic esophageal mucosa tissues. Results: Expression of WRAP53 was detected in all ESCC cell lines and was upregulated in the ESCC tissues compared with the corresponding non-neoplastic tissues (P<0.01). More cells expressed WRAP53 protein in the ESCC tissues than in the non-neoplastic tissues (P<0.01). Overexpression of WRAP53 was significantly correlated with tumor infiltration depth (P = 0.000), clinical stage (P = 0.001), and lymph node metastasis (P = 0.025). Wrap53 expression was not correlated with age, gender, or tumor differentiation. Conclusion: This report indicates increased expression of WRAP53 in ESCC and that WRAP53 overexpression is correlated with tumor progression. WRAP53 may play a significant role in ESCC; accordingly, WRAP53 could be a useful biomarker for ESCC. [ABSTRACT FROM AUTHOR]

We aimed to identify a prostate cancer DNA hypermethylation microarray signature (denoted as PHYMA) that differentiates prostate cancer from benign prostate hyperplasia (BPH), high from low-grade and lethal from non-lethal cancers. This is a non-randomized retrospective study in 111 local Asian men (87 prostate cancers and 24 BPH) treated from 1995 to 2009 in our institution. Archival prostate epithelia were laser-capture microdissected and genomic DNA extracted and bisulfite-converted. Samples were profiled using Illumina GoldenGate Methylation microarray, with raw data processed by GenomeStudio. A classification model was generated using support vector machine, consisting of a 55-probe DNA methylation signature of 46 genes. The model was independently validated on an internal testing dataset which yielded cancer detection sensitivity and specificity of 95.3% and 100% respectively, with overall accuracy of 96.4%. Second validation on another independent western cohort yielded 89.8% sensitivity and 66.7% specificity, with overall accuracy of 88.7%. A PHYMA score was developed for each sample based on the state of methylation in the PHYMA signature. Increasing PHYMA score was significantly associated with higher Gleason score and Gleason primary grade. Men with higher PHYMA scores have poorer survival on univariate (p = 0.0038, HR = 3.89) and multivariate analyses when controlled for (i) clinical stage (p = 0.055, HR = 2.57), and (ii) clinical stage and Gleason score (p = 0.043, HR = 2.61). We further performed bisulfite genomic sequencing on 2 relatively unknown genes to demonstrate robustness of the assay results. PHYMA is thus a signature with high sensitivity and specificity for discriminating tumors from BPH, and has a potential role in early detection and in predicting survival. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) regulate diverse cellular functions by triggering signal transduction events, such as Src and mitogen-activated protein (MAP) kinases. Here, we report the role of caveolin-1 and Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP-2) in H2O2-induced signaling pathway in brain astrocytes. H2O2-mediated oxidative stress induced phosphorylation of caveolin-1 and association between p-caveolin-1 and SHP-2. SHP-2 specifically bound to wild-type caveolin-1 similarly to c-Src tyrosine kinase (CSK), but not to phosphorylation-deficient mutant of caveolin-1 (Y14A), and interfered with complex formation between caveolin-1 and CSK. In the presence of CSK siRNA, binding between caveolin-1 and SHP-2 was enhanced by H2O2 treatment, which led to reduced Src phosphorylation at tyrosine (Tyr) 530 and enhanced Src phosphorylation at Tyr 419. In contrast, siRNA targeting of SHP-2 facilitated H2O2-mediated interaction between caveolin-1 and CSK and enhanced Src phosphorylation at Tyr 530, leading to subsequent decrease in Src downstream signaling, such as focal adhesion kinase (FAK) and extracellular signal-related kinase (ERK). Our results collectively indicate that SHP-2 alters Src kinase activity by interfering with the complex formation between CSK and phosphotyrosine caveolin-1 in the presence of H2O2, thus functions as a positive regulator in Src signaling under oxidative stress in brain astrocytes. [ABSTRACT FROM AUTHOR]

Aim: Histone deacetylase inhibitors (HDACIs)-based therapies have stimulated interest via their anti-tumor activities, including apoptosis induction, cell cycle arrest, cell differentiation, and autophagy. However, the mechanisms of HDACI-associated anti-tumor activity are not yet clearly defined. The aim of this study was to explore the key events of Trichostatin A (TSA), a classic HDACI agent, against breast cancer cells. Methods: The MCF-7, MDA-MB-231 and MCF-10A cell lines were evaluated with colony-forming and cell viability assays. Apoptosis and cell cycle distribution were detected by flow cytometry. Mitochondrial function was measured with biochemical assays, flow cytometry and transmission electron microscopy. Results: TSA inhibited breast cancer cell viability and proliferation, without affecting MCF-10A cell. TSA-induced breast cancer cell apoptosis was initiated by G2-M arrest and depended on mitochondrial reactive oxygen species (ROS) produced subsequent to reduced mitochondrial respiratory chain activity. The enhanced mitochondrial ROS production and apoptosis in cancer cells were markedly attenuated by antioxidants, such as N-acetyl cysteine (NAC), reduced glutathione (GSH) and Vitamin C. Conclusion: The present study demonstrated that TSA-induced cell death by arresting cell cycle in G2-M phase and was dependent on production of mitochondria-derived ROS, which was derived from impaired mitochondrial respiratory chain. [ABSTRACT FROM AUTHOR]

Pulsed electromagnetic field (PEMF) has been successfully applied to accelerate fracture repair since 1979. Recent studies suggest that PEMF might be used as a nonoperative treatment for the early stages of osteonecrosis. However, PEMF treatment requires a minimum of ten hours per day for the duration of the treatment. In this study, we modified the protocol of the single-pulsed electromagnetic field (SPEMF) that only requires a 3-minute daily treatment. In the in vitro study, cell proliferation and osteogenic differentiation was evaluated in the hBMSCs. In the in vivo study, new bone formation and revascularization were evaluated in the necrotic bone graft. Results from the in vitro study showed no significant cytotoxic effects on the hBMSCs after 5 days of SPEMF treatment (1 Tesla, 30 pulses per day). hBMSC proliferation was enhanced in the SPEMF-treated groups after 2 and 4 days of treatment. The osteogenic differentiation of hBMSCs was significantly increased in the SPEMF-treated groups after 3–7 days of treatment. Mineralization also increased after 10, 15, 20, and 25 days of treatment in SPEMF-treated groups compared to the control group. The 7-day short-course treatment achieved similar effects on proliferation and osteogenesis as the 25-day treatment. Results from the in vivo study also demonstrated that both the 7-day and 25-day treatments of SPEMF increased callus formation around the necrotic bone and also increased new vessel formation and osteocyte numbers in the grafted necrotic bone at the 2nd and 4th weeks after surgery. In conclusion, the newly developed SPEMF accelerates osteogenic differentiation of cultured hBMSCs and enhances bone repair, neo-vascularization, and cell growth in necrotic bone in mice. The potential clinical advantage of the SPEMF is the short daily application and the shorter treatment course. We suggest that SPEMF may be used to treat fractures and the early stages of osteonecrosis. [ABSTRACT FROM AUTHOR]

The lack of knowledge about molecular pathology of uterine sarcomas with a representation of 3–7% of all malignant uterine tumors prevents the establishment of effective therapy protocols. Here, we explored advanced therapeutic options to the previously discovered antitumorigenic effects of the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) by combined treatment with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L). In addition, we investigated the uterine sarcoma cell lines, MES-SA and ESS-1, regarding the underlying molecular mechanisms of SAHA and TRAIL-induced apoptosis and their resistance towards TRAIL. Compared to single SAHA or TRAIL treatment, the combination of SAHA with TRAIL led to complete cell death of both tumor cell lines after 24 to 48 hours. In contrast to single SAHA treatment, apoptosis occurred faster and was more pronounced in ESS-1 cells than in MES-SA cells. Induction of SAHA- and TRAIL-induced apoptosis was accompanied by upregulation of the intrinsic apoptotic pathway via reduction of mitochondrial membrane potential, caspase-3, -6, and -7 activation, and PARP cleavage, but was also found to be partially caspase-independent. Apoptosis resistance was caused by reduced expression of caspase-8 and DR 4/TRAIL-R1 in ESS-1 and MES-SA cells, respectively, due to epigenetic silencing by DNA hypermethylation of gene promoter sequences. Treatment with the demethylating agent 5-Aza-2'-deoxycytidine or gene transfer therefore restored gene expression and increased the sensitivity of both cell lines against TRAIL-induced apoptosis. Our data provide evidence that deregulation of epigenetic silencing by histone acetylation and DNA hypermethylation might play a fundamental role in the origin of uterine sarcomas. Therefore, tumor growth might be efficiently overcome by a cytotoxic combinatorial treatment of HDAC inhibitors with TRAIL. [ABSTRACT FROM AUTHOR]

Chordomas are rare malignant tumors that originate from the notochord remnants and occur in the skull base, spine and sacrum. Due to a very limited understanding of the molecular pathogenesis of chordoma, there are no adjuvant and molecular therapies besides surgical resection and radiation therapy. microRNAs (miRNAs) are small noncoding regulatory RNA molecules with critical roles in cancer. The role of miRNAs in chordomas is mostly unknown. We uncover microRNA-608 (miR-608) and microRNA-34a (miR-34a) as novel tumor suppressive microRNAs that regulate malignancy in chordoma. We find that miR-608 and miR-34a expressions are downregulated in human chordoma cell lines and primary cells at least partially via alteration of their genes’ copy numbers. We identify the commonly deregulated oncogenes EGFR and Bcl-xL as direct targets of miR-608 and the receptor tyrosine kinase MET as direct target of miR-34a. We show that EGFR and MET activations promote chordoma cell proliferation and invasion and that pharmacological inhibition of EGFR and MET inhibits chordoma cell proliferation and survival. We demonstrate that restoration of miR-608 and miR-34a inhibits cell proliferation and invasion and induces apoptosis in chordoma cells. We find that miR-34a inversely correlates with MET expression and miR-608 inversely correlates with EGFR expression in chordoma cells. These findings demonstrate for the first time that miR-608 and miR-34a regulate chordoma malignancy by regulating EGFR, MET and Bcl-xL. [ABSTRACT FROM AUTHOR]

KIF14 (kinesin family member 14) is a mitotic kinesin and an important oncogene in several cancers. Tumor KIF14 expression levels are independently predictive of poor outcome, and in cancer cells KIF14 can modulate metastatic behavior by maintaining appropriate levels of cell adhesion and migration proteins at the cell membrane. Thus KIF14 is an exciting potential therapeutic target. Understanding KIF14's regulation in cancer cells is crucial to the development of effective and selective therapies to block its tumorigenic function(s). We previously determined that close to 30% of serous ovarian cancers (OvCa tumors) exhibit low-level genomic gain, indicating one mechanism of KIF14 overexpression in tumors. We now report on transcriptional and epigenetic regulation of KIF14. Through promoter deletion analyses, we identified one cis-regulatory region containing binding sites for Sp1, HSF1 and YY1. siRNA-mediated knockdown of these transcription factors demonstrated endogenous regulation of KIF14 overexpression by Sp1 and YY1, but not HSF1. ChIP experiments confirmed an enrichment of both Sp1 and YY1 binding to the endogenous KIF14 promoter in OvCa cell lines with high KIF14 expression. A strong correlation was seen in primary serous OvCa tumors between Sp1, YY1 and KIF14 expression, further evidence that these transcription factors are important players in KIF14 overexpression. Hypomethylation patterns were observed in primary serous OvCa tumors, suggesting a minor role for promoter methylation in the control of KIF14 gene expression. miRNA expression analysis determined that miR-93, miR-144 and miR-382 had significantly lower levels of expression in primary serous OvCa tumors than normal tissues; treatment of an OvCa cell line with miRNA mimics and inhibitors specifically modulated KIF14 mRNA levels, pointing to potential novel mechanisms of KIF14 overexpression in primary tumors. Our findings reveal multiple mechanisms of KIF14 upregulation in cancer cells, offering new targets for therapeutic interventions to reduce KIF14 in tumors, aiming at improved prognosis. [ABSTRACT FROM AUTHOR]

The transmembrane and secreted protein delta-like 1 homolog (DLK1) belongs to the EGF-like family. It is widely accepted that DLK1 plays important roles in regulating cell differentiation, such as adipogenesis and osteogenesis. Aberrant expression of DLK1 has been found in various types of human cancers, including lung cancer. A previous study in this lab has revealed that DLK1 is associated with tumor invasion, although the mechanism is still unknown. To explore the potential effects that DLK1 might have on invasion, DLK1 was overexpressed or knocked down in the human lung cancer cell lines. The protein's influences on cell invasion were subsequently evaluated. A transwell assay showed that DLK1 overexpression significantly promoted cancer cell invasion. Western blotting and gelatin zymography analysis indicated that DLK1 could affect both matrix metalloproteinase-9 (MMP9) expression and its extracellular activity. An analysis of NOTCH1 and HES1 gene expression and Notch intracellular domain (NICD) nuclear translocation during DLK1 stimulation or depletion demonstrated that DLK1 could activate Notch signaling in lung cancer cells. Additionally, the elevated expression of MMP9 induced by DLK1 stimulation could be significantly decreased by inhibiting Notch signaling using γ-secretase inhibitor (GSI). The data presented in this study suggest that DLK1 can promote the invasion of lung cancer cells by upregulating MMP9 expression, which depends on Notch signaling. [ABSTRACT FROM AUTHOR]

Heat-shock proteins (HSPs) are molecular chaperones that protect proteins from damage. HSP27 expression is associated with cancer transformation and invasion. Ginkgo biloba extract (EGb761), the most widely sold herbal supplement, has antiangiogenic effects and induces tumor apoptosis. Data regarding the effect of EGb761 on HSP expression is limited, particularly in cancer. HSP27 expression in paired tumors and normal lung tissues of 64 patients with non-small cell lung cancer (NSCLC) were detected by real-time PCR, western blotting, and immunohistochemistry. NSCLC cell lines (A549/H441) were used to examine the migratory abilities in vitro. NSCLC tissue showed higher HSP27 expression than normal lung tissue. Kaplan–Meier survival analysis showed that NSCLC patients with low HSP27 expression ratio (<1) had significantly longer survival time than those with a high expression ratio (>1) (p = 0.04). EGb761 inhibited HSP27 expression and migratory ability of A549/H441 cells, which is the same as HSP27-siRNA transfection effect. Moreover, EGb761 treatment activated the AKT and p38 pathways and did not affect the expression of PI3K, ERK, and JNK pathways. HSP27 is a poor prognostic indicator of NSCLC. EGb761 can decrease the migration ability of A549/H441 by inhibiting HSP27 expression most likely through AKT and p38 MAPK pathways activation. [ABSTRACT FROM AUTHOR]

Studies have indicated that the aggregation of activated platelets with cancer cells facilitates tumor metastasis; the adhesion molecule P-selectin may be an important mediator of this process, but the detailed mechanism is unclear. In the current study, we established a B16F10 (B16) cell metastatic model in P-selectin knockout (P-sel−/−) mice to determine the effect of P-selectin-mediated platelet adhesion on metastasis. Compared with C57 mice, P-sel−/− mice developed fewer metastatic foci, and cell proliferation within the metastatic tumors was inhibited by P-selectin deficiency. The platelet refusion assay demonstrated that mice with P-sel−/− platelets developed fewer lung metastatic foci (P<0.01) with a lower microvascular density (MVD) than mice with wild-type platelets. A co-culture model of platelets and B16 cells was utilized to determine the difference in VEGF concentration in the supernatants. The results demonstrated that the supernatant from the P-sel−/− platelet/B16 co-culture had a lower concentration of VEGF. Therefore, our findings indicated that P-selectin deficiency inhibited the metastasis of B16 cells and that wild-type platelet refusion reversed this inhibition. The P-selectin-mediated interaction between platelets and B16 cells promoted angiogenesis by up-regulating VEGF. [ABSTRACT FROM AUTHOR]

Accumulating evidence indicates that numerous microRNAs are involved in the tumorigenesis and progression of gastric cancer, while the clinical significance of microRNA-214 in gastric cancer is poorly understood and the exact role of microRNA-214 in gastric cancer remains obscure. In the present study, expression levels of microRNA-214 in 80 gastric carcinoma tissues, 18 nontumourous gastric tissues, and 4 types of gastric cancer cell lines were quantified by reverse transcription followed by real-time quantitative polymerase chain reaction (RT-qPCR), and the relationship between microRNA-214 expression and cliniopathological characteristics including prognosis was explored. To investigate the potential role of microRNA-214 in gastric cancer cell biological behaviour, we performed cell proliferation, apoptosis, migration and invasion assays in four gastric cancer cell lines and an immortalized gastric cell line in vitro. Our results showed that microRNA-214 was dramatically downregulated in gastric cancer tissues and gastric cancer cell lines, compared with nontumourous gastric tissues. Stepwise downregulation of microRNA-214 expression was observed among nontumourous gastric mucosa, nonmetastasis gastric cancer tissues, and metastasis gastric cancer tissues. The expression of microRNA-214 was significantly inversely correlated with lymph node metastasis and tumour size but had no correlation with the patient's prognosis. Ectopic expression of microRNA-214 could inhibit cell migration and invasion ability in SGC7901 and MKN45 gastric cancer cells. And knockdown of microRNA-214 significantly facilitated cell proliferation, migration and invasion in a cell-specific manner in MKN28, BGC823 and GES-1 cells. Colony stimulating factor 1 (CSF1) was identified as a target gene of microRNA-214. In summary, our data demonstrated that microRNA-214 is a promising novel biomarker for lymph node metastasis in patients with gastric cancer. And we identified that downregulation of microRNA-214 may regulate the proliferation, invasion and migration of gastric cancer cells by directly targeting CSF1. [ABSTRACT FROM AUTHOR]

Background: Based on the potential of Schiff base compounds to act as sources for the development of cancer chemotherapeutic agents, this in vivo study was performed to investigate the inhibitory properties of the synthetic Schiff base compound Cu(BrHAP)2 on colonic aberrant crypt foci (ACF). Methodology: This study involved five groups of male rats. The negative control group was injected with normal saline once a week for 2 weeks and fed 10% Tween 20 for 10 weeks, the cancer control group was subcutaneously injected with 15 mg/kg azoxymethane once per week for two consecutive weeks, the positive control group was injected with 15 mg/kg azoxymethane once per week for two consecutive weeks and 35 mg/kg 5-fluorouracil (injected intra-peritoneally) for 4 weeks, and the experimental groups were first injected with 15 mg/kg azoxymethane once per week for two consecutive weeks and then fed 2.5 or 5 mg/kg of the Schiff base compound once a day for 10 weeks. Application of the Schiff base compound suppressed total colonic ACF formation by up to 72% to 74% (P<0.05) when compared with the cancer control group. Analysis of colorectal specimens revealed that treatments with the Schiff base compound decreased the mean crypt scores in azoxymethane-treated rats. Significant elevations of superoxide dismutase, glutathione peroxidase and catalase activities and a reduction in the level of malondialdehyde were also observed. Histologically, all treatment groups exhibited significant decreases in dysplasia compared to the cancer control group (P<0.05). Immunohistochemical staining demonstrated down-regulation of the PCNA protein. Comparative western blot analysis revealed that COX-2 and Bcl2 were up-regulated and Bax was down-regulated compared with the AOM control group. Conclusion: The current study demonstrated that the Cu(BrHAP)2 compound has promising chemoprotective activities that are evidenced by significant decreases in the numbers of ACFs in azoxymethane-induced colon cancer. [ABSTRACT FROM AUTHOR]

We previously reported that calycosin, a natural phytoestrogen structurally similar to estrogen, successfully triggered apoptosis of estrogen receptor (ER)-positive breast cancer cell line, MCF-7. To better understand the antitumor activities of calycosin against breast cancer, besides MCF-7 cells, another ER-positive cell line T-47D was analyzed here, with ER-negative cell lines (MDA-231, MDA-435) as control. Notably, calycosin led to inhibited cell proliferation and apoptosis only in ER-positive cells, particularly in MCF-7 cells, whereas no such effect was observed in ER-negative cells. Then we investigated whether regulation of ERβ, a subtype of ER, contributed to calycosin-induced apoptosis in breast cancer cells. The results showed that incubation of calycosin resulted in enhanced expression ERβ in MCF-7 and T-47D cells, rather than MDA-231 and MDA-435 cells. Moreover, with the upregulation of ERβ, successive changes in downstream signaling pathways were found, including inactivation of insulin-like growth factor 1 receptor (IGF-1R), then stimulation of p38 MAPK and suppression of the serine/threonine kinase (Akt), and finally poly(ADP-ribose) polymerase 1 (PARP-1) cleavage. However, the other two members of the mitogen-activated protein kinase (MAPK) family, extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK), were not consequently regulated by downregulated IGF-1R, indicating ERK 1/2 and JNK pathways were not necessary to allow proliferation inhibition by calycosin. Taken together, our results indicate that calycosin tends to inhibit growth and induce apoptosis in ER-positive breast cancer cells, which is mediated by ERβ-induced inhibition of IGF-1R, along with the selective regulation of MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductase M1 (RRM1) is required for mammalian deoxyribonucleotide (dNTP) metabolism. It is the primary target of the antimetabolite drug gemcitabine, which is among the most efficacious and most widely used cancer therapeutics. Gemcitabine directly binds to RRM1 and irreversibly inactivates ribonucleotide reductase. Intra-tumoral RRM1 levels are predictive of gemcitabine’s therapeutic efficacy. The mechanisms that regulate intracellular RRM1 levels are largely unknown. Here, we identified the E3 ubiquitin-protein ligases RNF2 and Bmi1 to associate with RRM1 with subsequent poly-ubiquitination at either position 48 or 63 of ubiquitin. The lysine residues 224 and 548 of RRM1 were identified as major ubiquitination sites. We show that ubiquitinated RRM1 undergoes proteasome-mediated degradation and that targeted post-transcriptional silencing of RNF2 and Bmi1 results in increased RRM1 levels and resistance to gemcitabine. Immunohistochemical analyses of 187 early-stage lung cancer tumor specimens revealed a statistically significant co-expression of RRM1 and Bmi1. We were unable to identify suitable reagents for in situ quantification of RNF2. Our findings suggest that Bmi1 and possibly RNF2 may be attractive biomarkers of gemcitabine resistance in the context of RRM1 expression. They also provide novel information for the rational design of gemcitabine-proteasome inhibitor combination therapies, which so far have been unsuccessful if given to patients without taking the molecular context into account. [ABSTRACT FROM AUTHOR]

Background: Association between the single nucleotide polymorphism rs3218536 (known as Arg188His) located in the X-ray repair cross complementing group 2 (XRCC2) gene and cancer susceptibility has been widely investigated. However, results thus far have remained controversial. A meta-analysis was performed to identify the impact of this polymorphism on cancer susceptibility. Methods: PubMed and Embase databases were searched systematically until September 7, 2013 to obtain all the records evaluating the association between the XRCC2 Arg188His polymorphism and the risk of all types of cancers. We used the odds ratio (OR) as measure of effect, and pooled the data in a Mantel-Haenszel weighed random-effects meta-analysis to provide a summary estimate of the impact of this polymorphism on breast cancer, ovarian cancer and other cancers. All the analyses were carried out in STATA 12.0. Results: With 30868 cases and 38656 controls, a total of 45 case-control studies from 26 publications were eventually included in our meta-analysis. No significant association was observed between the XRCC2 Arg188His polymorphism and breast cancer susceptibility (dominant model: OR = 0.94, 95%CI = 0.86–1.04, P = 0.232). However, a significant impact of this polymorphism was detected on decreased ovarian cancer risk (dominant model: OR = 0.83, 95%CI = 0.73–0.95, P = 0.007). In addition, we found this polymorphism was associated with increased upper aerodigestive tract (UADT) cancer susceptibility (dominant model: OR = 1.51, 95%CI = 1.04–2.20, P = 0.032). Conclusion: The Arg188His polymorphism might play different roles in carcinogenesis of various cancer types. Current evidence did not suggest that this polymorphism was directly associated with breast cancer susceptibility. However, this polymorphism might contribute to decreased gynecological cancer risk and increased UADT cancer risk. More preclinical and epidemiological studies were still imperative for further evaluation. [ABSTRACT FROM AUTHOR]

Previously three oncogene transgenic zebrafish lines with inducible expression of xmrk, kras or Myc in the liver have been generated and these transgenic lines develop oncogene-addicted liver tumors upon chemical induction. In the current study, comparative transcriptomic approaches were used to examine the correlation of the three induced transgenic liver cancers with human liver cancers. RNA profiles from the three zebrafish tumors indicated relatively small overlaps of significantly deregulated genes and biological pathways. Nevertheless, the three transgenic tumor signatures all showed significant correlation with advanced or very advanced human hepatocellular carcinoma (HCC). Interestingly, molecular signature from each oncogene-induced zebrafish liver tumor correlated with only a small subset of human HCC samples (24–29%) and there were conserved up-regulated pathways between the zebrafish and correlated human HCC subgroup. The three zebrafish liver cancer models together represented nearly half (47.2%) of human HCCs while some human HCCs showed significant correlation with more than one signature defined from the three oncogene-addicted zebrafish tumors. In contrast, commonly deregulated genes (21 up and 16 down) in the three zebrafish tumor models generally showed accordant deregulation in the majority of human HCCs, suggesting that these genes might be more consistently deregulated in a broad range of human HCCs with different molecular mechanisms and thus serve as common diagnosis markers and therapeutic targets. Thus, these transgenic zebrafish models with well-defined oncogene-induced tumors are valuable tools for molecular classification of human HCCs and for understanding of molecular drivers in hepatocarcinogenesis in each human HCC subgroup. [ABSTRACT FROM AUTHOR]

UL16 binding proteins (ULBPs) are a family of cell surface proteins that are present in transformed and stressed cells and ligands for NKG2D. Soluble NKG2D ligands have been found in sera from cancer patients with their protein concentrations correlated with poor cancer prognosis. Here we show, for the first time, that human tumor cells lost their surface expression of ULBP2, but not ULBP1 and ULBP3, during NK cell-mediated cytolysis. In contrast to spontaneous shedding of NKG2D ligands, NK cytolysis-mediated shedding of ULBP2 was linked to target cell apoptosis, although both resulted from metalloproteinase cleavages. Inhibition of ULBP2 shedding by a metalloproteinase inhibitor BB-94 lead to reduced NK cell-mediated cytotoxicity and cytokine production. These results illustrate a regulation of NK cell effector functions through cytolysis-induced NKG2D ligand shedding. Consequently, compounds inhibiting NKG2D ligand shedding may offer therapeutic means to reduce excessive pathogenic NK cell activities. [ABSTRACT FROM AUTHOR]

Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells) and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382). Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells. [ABSTRACT FROM AUTHOR]

Purpose: OLC1 was recently identified to be a potential oncogene. However, the role of OLC1 in human esophageal cell carcinoma (ESCC) is unknown. The aim of this study was therefore to evaluate the expression of OLC1 in human ESCC from normal, premalignant, and malignant lesions, and to clarify the mechanisms by which OLC1 contributes to the progression of ESCC. Experimental Design: Two hundred and fourteen paired ESCC specimens, and an independent set from 28 ESCC patients, were used to analyze the correlation between OLC1 expression and the pathological characteristics of tumors using immunohistochemistry. Stable OLC1-overexpressing and OLC1-interfering esophageal cancer cells were established and a series of experimental methods were used to investigate the biological functions and mechanisms of action of OLC1. Results: We showed that OLC1 was overexpressed in 145 of 214 (67.8%) of human ESCC specimens, compared with in only 59 of 214 (27.57%) paired adjacent normal tissues (P<0.001). OLC1 overexpression occurred at a rate of 35% (10/28) at the stage of mild/moderate dysplasia, but was significantly upregulated to 66% (22/33) at the stages of severe dysplasia and in situ carcinoma, while 71% positive staining (22/28) was observed in invasive carcinoma tissues compared with normal tissues (P<0.05). We also provided evidence that OLC1 abnormalities significantly altered the cell proliferation and apoptosis induced by cytotoxic agents. OLC1 overexpression suppressed apoptosis, and was associated with attenuated caspase-3 activation and increased Bcl-2 stability. Conclusion: Our study provides strong evidence suggesting OLC1 abnormalities may contribute to the development of human ESCC and have some important clinical significance. [ABSTRACT FROM AUTHOR]

Premature infants exposed to hyperoxia suffer acute and long-term pulmonary consequences. Nevertheless, neonates survive hyperoxia better than adults. The factors contributing to neonatal hyperoxic tolerance are not fully elucidated. In contrast to adults, heme oxygenase (HO)-1, an endoplasmic reticulum (ER)-anchored protein, is abundant in the neonatal lung but is not inducible in response to hyperoxia. The latter may be important, because very high levels of HO-1 overexpression are associated with significant oxygen cytotoxicity in vitro. Also, in contrast to adults, HO-1 localizes to the nucleus in neonatal mice exposed to hyperoxia. To understand the mechanisms by which HO-1 expression levels and subcellular localization contribute to hyperoxic tolerance in neonates, lung-specific transgenic mice expressing high or low levels of full-length HO-1 (cytoplasmic, HO-1-FL(H) or HO-1-FL(L)) or C-terminally truncated HO-1 (nuclear, Nuc-HO-1-TR) were generated. In HO-1-FL(L), the lungs had a normal alveolar appearance and lesser oxidative damage after hyperoxic exposure. In contrast, in HO-1-FL(H), alveolar wall thickness with type II cell hyperproliferation was observed as well worsened pulmonary function and evidence of abnormal lung cell hyperproliferation in recovery from hyperoxia. In Nuc-HO-1-TR, the lungs had increased DNA oxidative damage, increased poly (ADP-ribose) polymerase (PARP) protein expression, and reduced poly (ADP-ribose) (PAR) hydrolysis as well as reduced pulmonary function in recovery from hyperoxia. These data indicate that low cytoplasmic HO-1 levels protect against hyperoxia-induced lung injury by attenuating oxidative stress, whereas high cytoplasmic HO-1 levels worsen lung injury by increasing proliferation and decreasing apoptosis of alveolar type II cells. Enhanced lung nuclear HO-1 levels impaired recovery from hyperoxic lung injury by disabling PAR-dependent regulation of DNA repair. Lastly both high cytoplasmic and nuclear expression of HO-1 predisposed to long-term abnormal lung cellular proliferation. To maximize HO-1 cytoprotective effects, therapeutic strategies must account for the specific effects of its subcellular localization and expression levels. [ABSTRACT FROM AUTHOR]

Background: Active cancer-associated fibroblasts (CAFs) or myofibroblasts play important roles not only in the development and progression of breast carcinomas, but also in their prognosis and treatment. Therefore, targeting these cells through suppressing their supportive procarcinogenic paracrine effects is mandatory for improving the current therapies that are mainly targeting tumor cells. To this end, we investigated the effect of the natural and pharmacologically safe molecule, caffeine, on CAF cells and their various procarcinogenic effects. Methodology/Principal Findings: We have shown here that caffeine up-regulates the tumor suppressor proteins p16, p21, p53 and Cav-1, and reduces the expression/secretion of various cytokines (IL-6, TGF-β, SDF-1 and MMP-2), and down-regulates α-SMA. Furthermore, caffeine suppressed the migratory/invasiveness abilities of CAF cells through PTEN-dependent Akt/Erk1/2 inactivation. Moreover, caffeine reduced the paracrine pro-invasion/−migration effects of CAF cells on breast cancer cells. These results indicate that caffeine can inactivate breast stromal myofibroblasts. This has been confirmed by showing that caffeine also suppresses the paracrine pro-angiogenic effect of CAF cells through down-regulating HIF-1αand its downstream effector VEGF-A. Interestingly, these effects were sustained in absence of caffeine. Conclusion/Significance: The present findings provide a proof of principle that breast cancer myofibroblasts can be inactivated, and thereby caffeine may provide a safe and effective prevention against breast tumor growth/recurrence through inhibition of the procarcinogenic effects of active stromal fibroblasts. [ABSTRACT FROM AUTHOR]

Liver cancer is one of leading causes of cancer-related deaths. A deeper mechanistic understanding of liver cancer could lead to the development of more effective therapeutic strategies. In our previous work, we screened 646 miRNAs and identified 11 that regulate liver cancer cell migration. The current study shows that miR-525-3p is frequently up-regulated in liver cancer tissues, and enhanced expression of miR-525-3p can promote liver cancer cell migration and invasion. Zinc finger protein 395 (ZNF395) is the direct functional target gene for miR-525-3p, and it is frequently down-regulated in liver cancer tissues. High expression of ZNF395 can significantly inhibit while knockdown of ZNF395 expression can markedly enhance the migration and invasion of liver cancer cells, suggesting that ZNF395 suppresses metastasis in liver cancer. Down-regulation of ZNF395 can mediate miR-525-3p induced liver cancer cell migration and invasion. In conclusion, miR-525-3p promotes liver cancer cell migration and invasion by directly targeting ZNF395, and the fact that miR-525-3p and ZNF395 both play important roles in liver cancer progression makes them potential therapeutic targets. [ABSTRACT FROM AUTHOR]

There is growing interest in development of natural products as anti-cancer and chemopreventive agents. Many triterpenoids have been proved as potential agents for chemoprevention and therapy of breast cancer. Ginsenosides from ginseng, which mostly belong to dammarane-type triterpenoids, have gained great attention for their anti-breast cancer activity with diverse mechanisms. However, studies of other kinds of triterpenoid saponins on breast cancer are limited. Previously, we purified and identified a novel oleanane-type triterpene saponin named D Rhamnose β-hederin (DRβ-H) from Clematis ganpiniana, a Chinese traditional anti-tumor herb. In the present study, DRβ-H showed strong inhibitory activity on the growth of various breast cancer cells and induced apoptosis in these cells. DRβ-H inhibited PI3K/AKT and activated ERK signaling pathway. PI3K inhibitor LY294002 synergistically enhanced DRβ-H-induced apoptosis whereas MEK inhibitor U0126 reduced the apoptosis rate. Moreover, DRβ-H regulated the ratio of pro-apoptotic and anti-apoptotic Bcl-2 family proteins. Furthermore, DRβ-H induced depolarization of mitochondrial membrane potential which released Apaf-1 and Cytochrome C from the inter membrane space into the cytosol, where they promoted caspase-9 and caspase-3 activation. This is the first report on the pro-apoptotic effects of DRβ-H, a novel oleanane-type triterpenoid saponin, on breast cancer cells and its comprehensive apoptosis pathways. It implied that oleanane-type triterpenoid saponin DRβ-H could be a promising candidate for chemotherapy of breast cancer. [ABSTRACT FROM AUTHOR]

Osteosarcoma is the most common primary malignant bone tumour characterized by osteoid production and/or osteolytic lesions of bone. A lack of response to chemotherapeutic treatments shows the importance of exploring new therapeutic methods. Imatinib mesylate (Gleevec, Novartis Pharma), a tyrosine kinase inhibitor, was originally developed for the treatment of chronic myeloid leukemia. Several studies revealed that imatinib mesylate inhibits osteoclast differentiation through the M-CSFR pathway and activates osteoblast differentiation through PDGFR pathway, two key cells involved in the vicious cycle controlling the tumour development. The present study investigated the in vitro effects of imatinib mesylate on the proliferation, apoptosis, cell cycle, and migration ability of five osteosarcoma cell lines (human: MG-63, HOS; rat: OSRGA; mice: MOS-J, POS-1). Imatinib mesylate was also assessed as a curative and preventive treatment in two syngenic osteosarcoma models: MOS-J (mixed osteoblastic/osteolytic osteosarcoma) and POS-1 (undifferentiated osteosarcoma). Imatinib mesylate exhibited a dose-dependent anti-proliferative effect in all cell lines studied. The drug induced a G0/G1 cell cycle arrest in most cell lines, except for POS-1 and HOS cells that were blocked in the S phase. In addition, imatinib mesylate induced cell death and strongly inhibited osteosarcoma cell migration. In the MOS-J osteosarcoma model, oral administration of imatinib mesylate significantly inhibited the tumour development in both preventive and curative approaches. A phospho-receptor tyrosine kinase array kit revealed that PDGFRα, among 7 other receptors (PDFGFRβ, Axl, RYK, EGFR, EphA2 and 10, IGF1R), appears as one of the main molecular targets for imatinib mesylate. In the light of the present study and the literature, it would be particularly interesting to revisit therapeutic evaluation of imatinib mesylate in osteosarcoma according to the tyrosine-kinase receptor status of patients. [ABSTRACT FROM AUTHOR]

Background: Accumulating evidence supports the concept that melanoma is highly heterogeneous and sustained by a small subpopulation of melanoma stem-like cells. Those cells are considered as responsible for tumor resistance to therapies. Moreover, melanoma cells are characterized by their high phenotypic plasticity. Consequently, both melanoma stem-like cells and their more differentiated progeny must be eradicated to achieve durable cure. By reevaluating compounds in heterogeneous melanoma populations, it might be possible to select compounds with activity not only against fast-cycling cells but also against cancer stem-like cells. Natural compounds were the focus of the present study. Methods: We analyzed 120 compounds from The Natural Products Set II to identify compounds active against melanoma populations grown in an anchorage-independent manner and enriched with cells exerting self-renewing capacity. Cell viability, cell cycle arrest, apoptosis, gene expression, clonogenic survival and label-retention were analyzed. Findings: Several compounds efficiently eradicated cells with clonogenic capacity and nanaomycin A, streptonigrin and toyocamycin were effective at 0.1 µM. Other anti-clonogenic but not highly cytotoxic compounds such as bryostatin 1, siomycin A, illudin M, michellamine B and pentoxifylline markedly reduced the frequency of ABCB5 (ATP-binding cassette, sub-family B, member 5)-positive cells. On the contrary, treatment with maytansine and colchicine selected for cells expressing this transporter. Maytansine, streptonigrin, toyocamycin and colchicine, even if highly cytotoxic, left a small subpopulation of slow-dividing cells unaffected. Compounds selected in the present study differentially altered the expression of melanocyte/melanoma specific microphthalmia-associated transcription factor (MITF) and proto-oncogene c-MYC. Conclusion: Selected anti-clonogenic compounds might be further investigated as potential adjuvants targeting melanoma stem-like cells in the combined anti-melanoma therapy, whereas selected cytotoxic but not anti-clonogenic compounds, which increased the frequency of ABCB5-positive cells and remained slow-cycling cells unaffected, might be considered as a tool to enrich cultures with cells exhibiting melanoma stem cell characteristics. [ABSTRACT FROM AUTHOR]

We have recently reported that the homologous Rho kinases, ROCK1 and ROCK2, play different roles in regulating stress-induced stress fiber disassembly and cell detachment, and the ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has remarkable anti-apoptotic, anti-detachment and pro-survival effects against doxorubicin, a chemotherapeutic drug. This study investigated the roles of ROCK isoforms in doxorubicin-induced reactive oxygen species (ROS) generation which is believed to be the major mechanism underlying its cytotoxicity to normal cells, and especially to cardiomyocytes. Different antioxidants have been shown to provide a protective role reported in numerous experimental studies, but clinical trials of antioxidant therapy showed insufficient benefit against the cardiac side effect. We found that both ROCK1−/− and ROCK2−/− MEFs exhibited reduced ROS production in response to doxorubicin treatment. Interestingly, only ROCK1 deficiency, but not ROCK2 deficiency, significantly enhanced the protective effects of antioxidants against doxorubicin-induced cytotoxicity. First, ROCK1 deficiency and N-acetylcysteine (an anti-oxidant) treatment synergistically reduced ROS levels, caspase activation and cell detachment. In addition, the reduction of ROS generation in ROCK1−/− MEFs in response to doxorubicin treatment was in part through inhibiting NADPH oxidase activity. Furthermore, ROCK1 deficiency enhanced the inhibitory effects of diphenyleneiodonium (an inhibitor of NADPH oxidase) on ROS generation and caspase 3 activation induced by doxorubicin. Finally, ROCK1 deficiency had greater protective effects than antioxidant treatment, especially on reducing actin cytoskeleton remodeling. ROCK1 deficiency not only reduced actomyosin contraction but also preserved central stress fiber stability, whereas antioxidant treatment only reduced actomyosin contraction without preserving central stress fibers. These results reveal a novel strategy to enhance the protective effect of antioxidant therapy by targeting the ROCK1 pathway to stabilize the actin cytoskeleton and boost the inhibitory effects on ROS production, apoptosis and cell detachment. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is the most frequent and aggressive primary adult brain tumor with poor prognosis. Epidermal growth factor receptor variant III (EGFRvIII) is the most common and highly oncogenic EGFR mutant in GBM. With the aim to generate specific molecular probes able to target EGFRvIII with high affinity, we selected four DNA aptamers (U2, U8, U19 and U31) specifically bound to U87-EGFRvIII cells that over expressed EGFRvIII with Kd values in the nanomole range by a cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX) process. U87MG cells were introduced as control cells for counter selection. We further affirmed U2 and U8 identified EGFRvIII on the surface of target cells specifically. Then we radiolabeled U2 with 188Re to serve as a molecular imaging probe and observed 188Re -labeled U2 significantly targeted EGFRvIII over-expressing glioblastoma exnografts in mice. In conclusion, aptamers obtained from whole cell-SELEX strategy have great potential as molecular imaging probes that are probably beneficial to GBM diagnoses. [ABSTRACT FROM AUTHOR]