Proliferation and survival molecules implicated in the inhibition of BRAF pathway in thyroid cancer cells harbouring different genetic mutations

<p>Abstract</p> <p>Background</p> <p>Thyroid carcinomas show a high prevalence of mutations in the oncogene BRAF which are inversely associated with RAS or RET/PTC oncogenic activation. The possibility of using inhibitors on the BRAF pathway as became an interesting therapeutic approach. In thyroid cancer cells the target molecules, implicated on the cellular effects, mediated by inhibition of BRAF are not well established. In order to fill this lack of knowledge we studied the proliferation and survival pathways and associated molecules induced by BRAF inhibition in thyroid carcinoma cell lines harbouring distinct genetic backgrounds.</p> <p>Methods</p> <p>Suppression of BRAF pathway in thyroid cancer cell lines (8505C, TPC1 and C643) was achieved using RNA interference (RNAi) for BRAF and the kinase inhibitor, sorafenib. Proliferation analysis was performed by BrdU incorporation and apoptosis was accessed by TUNEL assay. Levels of protein expression were analysed by western-blot.</p> <p>Results</p> <p>Both BRAF RNAi and sorafenib inhibited proliferation in all the cell lines independently of the genetic background, mostly in cells with BRAF^V600Emutation. In BRAF^V600Emutated cells inhibition of BRAF pathway lead to a decrease in ERK1/2 phosphorylation and cyclin D1 levels and an increase in p27^Kip1. Specific inhibition of BRAF by RNAi in cells with BRAF^V600Emutation had no effect on apoptosis. In the case of sorafenib treatment, cells harbouring BRAF^V600Emutation showed increase levels of apoptosis due to a balance of the anti-apoptotic proteins Mcl-1 and Bcl-2.</p> <p>Conclusion</p> <p>Our results in thyroid cancer cells, namely those harbouring BRAF^V600Emutation showed that BRAF signalling pathway provides important proliferation signals. We have shown that in thyroid cancer cells sorafenib induces apoptosis by affecting Mcl-1 and Bcl-2 in BRAF^V600Emutated cells which was independent of BRAF. These results suggest that sorafenib may prove useful in the treatment of thyroid carcinomas, particularly those refractory to conventional treatment and harbouring BRAF mutations.</p

Cytoplasmic Skp2 Expression Is Increased in Human Melanoma and Correlated with Patient Survival

BACKGROUND: S-phase kinase protein 2 (Skp2), an F-box protein, targets cell cycle regulators via ubiquitin-mediated degradation. Skp2 is frequently overexpressed in a variety of cancers and associated with patient survival. In melanoma, however, the prognostic significance of subcellular Skp2 expression remains controversial. METHODS: To investigate the role of Skp2 in melanoma development, we constructed tissue microarrays and examined Skp2 expression in melanocytic lesions at different stages, including 30 normal nevi, 61 dysplastic nevi, 290 primary melanomas and 146 metastatic melanomas. The TMA was assessed for cytoplasmic and nuclear Skp2 expression by immunohistochemistry. The Kaplan-Meier method was used to evaluate the patient survival. The univariate and multivariate Cox regression models were performed to estimate the hazard ratios (HR) at five-year follow-up. RESULTS: Cytoplasmic but not nuclear Skp2 expression was gradually increased from normal nevi, dysplastic nevi, primary melanomas to metastatic melanomas. Cytoplasmic Skp2 expression correlated with AJCC stages (I vs II-IV, P<0.001), tumor thickness (≤2.00 vs >2.00 mm, P<0.001) and ulceration (P = 0.005). Increased cytoplasmic Skp2 expression was associated with a poor five-year disease-specific survival of patients with primary melanoma (P = 0.018) but not metastatic melanoma (P>0.05). CONCLUSION: This study demonstrates that cytoplasmic Skp2 plays an important role in melanoma pathogenesis and its expression correlates with patient survival. Our data indicate that cytoplasmic Skp2 may serve as a potential biomarker for melanoma progression and a therapeutic target for this disease

RNA-Binding Protein Musashi1 Modulates Glioma Cell Growth through the Post-Transcriptional Regulation of Notch and PI3 Kinase/Akt Signaling Pathways

Musashi1 (MSI1) is an RNA-binding protein that plays critical roles in nervous-system development and stem-cell self-renewal. Here, we examined its role in the progression of glioma. Short hairpin RNA (shRNA)-based MSI1-knock down (KD) in glioblastoma and medulloblastoma cells resulted in a significantly lower number of self renewing colony on day 30 (a 65% reduction), compared with non-silencing shRNA-treated control cells, indicative of an inhibitory effect of MSI1-KD on tumor cell growth and survival. Immunocytochemical staining of the MSI1-KD glioblastoma cells indicated that they ectopically expressed metaphase markers. In addition, a 2.2-fold increase in the number of MSI1-KD cells in the G2/M phase was observed. Thus, MSI1-KD caused the prolongation of mitosis and reduced the cell survival, although the expression of activated Caspase-3 was unaltered. We further showed that MSI1-KD glioblastoma cells xenografted into the brains of NOD/SCID mice formed tumors that were 96.6% smaller, as measured by a bioluminescence imaging system (BLI), than non-KD cells, and the host survival was longer (49.3±6.1 days vs. 33.6±3.6 days; P<0.01). These findings and other cell biological analyses suggested that the reduction of MSI1 in glioma cells prolonged the cell cycle by inducing the accumulation of Cyclin B1. Furthermore, MSI1-KD reduced the activities of the Notch and PI3 kinase-Akt signaling pathways, through the up-regulation of Numb and PTEN, respectively. Exposure of glioma cells to chemical inhibitors of these pathways reduced the number of spheres and living cells, as did MSI1-KD. These results suggest that MSI1 increases the growth and/or survival of certain types of glioma cells by promoting the activation of both Notch and PI3 kinase/Akt signaling

Evaluating Gene Drive Approaches for Public Benefit

Gene drive approaches—those which bias inheritance of a genetic element in a population of sexually reproducing organisms—have the potential to provide important public benefits. The spread of selected genetic elements in wild populations of organisms may help address certain challenges, such as transmission of vector-borne human and animal diseases and biodiversity loss due to invasive animals. Adapting various naturally occurring gene drive mechanisms to these aims is a long-standing research area, and recent advances in genetics have made engineering gene drive systems significantly more technically feasible. Gene drive approaches would act through changes in natural environments, thus robust methods to evaluate potential research and use are important. Despite the fact that gene drive approaches build on existing paradigms, such as genetic modification of organisms and conventional biological control, there are material challenges to their evaluation. One challenge is the inherent complexity of ecosystems, which makes precise prediction of changes to the environment difficult. For gene drive approaches that are expected to spread spatially and/or persist temporally, responding to this difficulty with the typical stepwise increases in the scale of studies may not be straightforward after studies begin in the natural environment. A related challenge is that study or use of a gene drive approach may have implications for communities beyond the location of introduction, depending on the spatial spread and persistence of the approach and the population biology of the target organism. This poses a particular governance challenge when spread across national borders is plausible. Finally, community engagement is an important element of responsible research and governance, but effective community engagement for gene drive approaches requires addressing complexity and uncertainty and supporting representative participation in decision making. These challenges are not confronted in a void. Existing frameworks, processes, and institutions provide a basis for effective evaluation of gene drive approaches for public benefit. Although engineered gene drive approaches are relatively new, the necessities of making decisions despite uncertainty and governing actions with potential implications for shared environments are well established. There are methodologies to identify potential harms and assess risks when there is limited experience to draw upon, and these methodologies have been applied in similar contexts. There are also laws, policies, treaties, agreements, and institutions in place across many jurisdictions that support national and international decision making regarding genetically modified organisms and the potential applications of gene drive approaches, such as public health and biodiversity conservation. Community engagement is an established component of many decision-making processes, and related experience and conceptual frameworks can inform engagement by researchers. The existence of frameworks, processes, and institutions provides an important foundation for evaluating gene drive approaches, but it is not sufficient by itself. They must be rigorously applied, which requires resources for risk assessment, research, and community engagement and diligent implementation by governance institutions. The continued evolution of the frameworks, processes, and institutions is important to adapt to the growing understanding of gene drive approaches. With appropriate resources and diligence, it will be possible to responsibly evaluate and make decisions on gene drive approaches for public benefit

A review of zoonotic infection risks associated with the wild meat trade in Malaysia.

The overhunting of wildlife for food and commercial gain presents a major threat to biodiversity in tropical forests and poses health risks to humans from contact with wild animals. Using a recent survey of wildlife offered at wild meat markets in Malaysia as a basis, we review the literature to determine the potential zoonotic infection risks from hunting, butchering and consuming the species offered. We also determine which taxa potentially host the highest number of pathogens and discuss the significant disease risks from traded wildlife, considering how cultural practices influence zoonotic transmission. We identify 51 zoonotic pathogens (16 viruses, 19 bacteria and 16 parasites) potentially hosted by wildlife and describe the human health risks. The Suidae and the Cervidae families potentially host the highest number of pathogens. We conclude that there are substantial gaps in our knowledge of zoonotic pathogens and recommend performing microbial food safety risk assessments to assess the hazards of wild meat consumption. Overall, there may be considerable zoonotic risks to people involved in the hunting, butchering or consumption of wild meat in Southeast Asia, and these should be considered in public health strategies

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Not AvailableA total of 12 genotypes of moth bean were evaluated for desiccation tolerance at germination and seedling stage. Differential response was observed at 8% and 15% PEG on germination and seedling level respectively where Varieties RMO-435, RMM-12-Single, RMM12-Poly And CZM-105 Were foundtolerant. Certain protein were found to be upregulated with treatment in all the samples while some showed upregulation only in tolerant types through SDS PAGE analysis. Twenty four primer combinations of anchored poly T (3) and random primers (8) were used to amplify the transcripts and amplicons were resolved on agarose gel with ethidium bromide. This modified DDRT-PCR assay amplified a total of 95 bands with an average of 4.04 per primer combination. 43 gene fragments were found to be up-regulated whereas 34 were downregulated in response to drought as compared to the control. A total of 23 unigenes (4 contigs and 19 singletons), were derived by cluster assembly and sequence alignment of 29 ESTs Out of 29 ESTs, 4 EST (13.79%) was found to be novel to moth bean. Significant BLASTX similarity (<1E06) in the NCBI non-redundant (nr) database was detected for 25 ESTs (86.20%). Gene ontology unctional classification terms (BLASTX results and GO terms), were retrieved for all sequences and 23 sequences were annotated with 8 enzyme commission (EC) codes and were mapped to 11 different KGG pathways Our study lists mainly the transcripts that are differentially expressed under PEG induced desiccation stress, where 9 chaperones including HSP 70, HSP 101 and Dnaj like proteins were nfound.Not Availabl