In Vitro and In Vivo Prostate Cancer Metastasis and Chemoresistance Can Be Modulated by Expression of either CD44 or CD147

CD44 and CD147 are associated with cancer metastasis and progression. Our purpose in the study was to investigate the effects of down-regulation of CD44 or CD147 on the metastatic ability of prostate cancer (CaP) cells, their docetaxel (DTX) responsiveness and potential mechanisms involved in vitro and in vivo. CD44 and CD147 were knocked down (KD) in PC-3M-luc CaP cells using short hairpin RNA (shRNA). Expression of CD44, CD147, MRP2 (multi-drug resistance protein-2) and MCT4 (monocarboxylate tranporter-4) was evaluated using immunofluorescence and Western blotting. The DTX dose-response and proliferation was measured by MTT and colony assays, respectively. The invasive potential was assessed using a matrigel chamber assay. Signal transduction proteins in PI3K/Akt and MAPK/Erk pathways were assessed by Western blotting. An in vivo subcutaneous (s.c.) xenograft model was established to assess CaP tumorigenecity, lymph node metastases and DTX response. Our results indicated that KD of CD44 or CD147 decreased MCT4 and MRP2 expression, reduced CaP proliferation and invasive potential and enhanced DTX sensitivity; and KD of CD44 or CD147 down-regulated p-Akt and p-Erk, the main signal modulators associated with cell growth and survival. In vivo, CD44 or CD147-KD PC-3M-luc xenografts displayed suppressed tumor growth with increased DTX responsiveness compared to control xenografts. Both CD44 and CD147 enhance metastatic capacity and chemoresistance of CaP cells, potentially mediated by activation of the PI3K and MAPK pathways. Selective targeting of CD44/CD147 alone or combined with DTX may limit CaP metastasis and increase chemosensitivity, with promise for future CaP treatment.     

Ten years of pathway analysis: current approaches and outstanding challenges

Pathway analysis has become the first choice for gaining insight into the underlying biology of differentially expressed genes and proteins, as it reduces complexity and has increased explanatory power. We discuss the evolution of knowledge base-driven pathway analysis over its first decade, distinctly divided into three generations. We also discuss the limitations that are specific to each generation, and how they are addressed by successive generations of methods. We identify a number of annotation challenges that must be addressed to enable development of the next generation of pathway analysis methods. Furthermore, we identify a number of methodological challenges that the next generation of methods must tackle to take advantage of the technological advances in genomics and proteomics in order to improve specificity, sensitivity, and relevance of pathway analysis.     

The role of nitric oxide in cancer

Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes, including va-sodilatation, neurotransmission and macrophage-mediated immunity. The family of nitric oxide synthases (NOS) comprises inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). Interestingly, various studies have shown that all three isoforms can be involved in promoting or inhibiting the etiology of cancer. NOS activity has been detected in tumour cells of various histogenetic origins and has been associated with tumour grade, proliferation rate and expression of important signaling components associated with cancer development such as the oestrogen receptor. It appears that high levels of NOS expression (for example, generated by activated macrophages) may be cytostatic or cytotoxic for tumor cells, whereas low level activity can have the opposite effect and promote tumour growth. Paradoxically therefore, NO (and related reactive nitrogen species) may have both genotoxic and angiogenic pro     

Rapid ecosystem service assessment of the impact of Koshi Tappu Wildlife Reserve on wetland benefits to local communities

Wetlands are important for biodiversity and are critical for human livelihoods, providing ecosystem services such as clean water, food and global climate regulation. Many wetlands are threatened by land-use conversion, but creating protected areas to conserve them can benefit both biodiversity and people. However, protected areas can also have socio-economic costs for local communities. At Koshi Tappu Wildlife Reserve in Nepal, there has been historical conflict over the creation of the reserve. In light of a recent proposal to expand the protected area, we explored the use of a rapid ecosystem service assessment tool (TESSA) to assess the impact of the reserve on some of the key ecosystem services the site provides. Based on the ecosystem services assessed we estimated that the economic value of KTWR as a protected area is $350,000 y^?1 ($20 ha^?1y^?1) less than the value of the wetland in an unprotected state. However, this difference is relatively small and is affected by the limitations of the approach and sensitivity of the values to market prices and the assumptions made, so we cannot draw clear conclusions on the overall impact of the reserve in relation to local livelihoods. However, we found TESSA to be a useful tool for engaging with the stakeholder community and for highlighting the potential impacts that land use decisions can have on key ecosystem services. In the context of informing the potential expansion of the reserve, it is clear that further intensive socio-economic assessment of the potential costs and benefits is necessary.     

A review of methods for interpretation of glycopeptide tandem mass spectral data

Despite the publication of several software tools for analysis of glycopeptide tandem mass spectra, there remains a lack of consensus regarding the most effective and appropriate methods. In part, this reflects problems with applying standard methods for proteomics database searching and false discovery rate calculation. While the analysis of small post-translational modifications (PTMs) may be regarded as an extension of proteomics database searching, glycosylation requires specialized approaches. This is because glycans are large and heterogeneous by nature, causing glycopeptides to exist as multiple glycosylated variants. Thus, the mass of the peptide cannot be calculated directly from that of the intact glycopeptide. In addition, the chemical nature of the glycan strongly influences product ion patterns observed for glycopeptides. As a result, glycopeptidomics requires specialized bioinformatics methods. We summarize the recent progress towards a consensus for effective glycopeptide tandem mass spectrometric analysis.     

Entropy and barrier-controlled fluctuations determine conformational viscoelasticity of single biomolecules

Biological macromolecules have complex and nontrivial energy landscapes, endowing them with a unique conformational adaptability and diversity in function. Hence, understanding the processes of elasticity and dissipation at the nanoscale is important to molecular biology and emerging fields such as nanotechnology. Here we analyze single molecule fluctuations in an atomic force microscope, using a generic model of biopolymer viscoelasticity that includes local "internal" conformational dissipation. Comparing two biopolymers, dextran and cellulose (polysaccharides with and without local bistable transitions), demonstrates that signatures of simple conformational change are minima in both the elastic and internal friction constants around a characteristic force. A novel analysis of dynamics on a bistable energy landscape provides a simple explanation: an elasticity driven by the entropy, and friction by a barrier-controlled hopping time of populations between states, which is surprisingly distinct to the well-known relaxation time. This nonequilibrium microscopic analysis thus provides a means of quantifying new dynamical features of the energy landscape of the glucopyranose ring, revealing an unexpected underlying roughness and information on the shape of the barrier of the chair-boat transition in dextran. The results presented herein provide a basis toward probing the viscoelasticity of macromolecular conformational transitions on more complex energy landscapes, such as during protein folding.     

mGluR long‐term depression regulates GluA2 association with COPII vesicles and exit from the endoplasmic reticulum

mGluR long‐term depression (mGluR‐LTD) is a form of synaptic plasticity induced at excitatory synapses by metabotropic glutamate receptors (mGluRs). mGluR‐LTD reduces synaptic strength and is relevant to learning and memory, autism, and sensitization to cocaine; however, the mechanism is not known. Here we show that activation of Group I mGluRs in medium spiny neurons induces trafficking of GluA2 from the endoplasmic reticulum (ER) to the synapse by enhancing GluA2 binding to essential COPII vesicle proteins, Sec23 and Sec13. GluA2 exit from the ER further depends on IP3 and Ryanodine receptor‐controlled Ca²⁺ release as well as active translation. Synaptic insertion of GluA2 is coupled to removal of high‐conducting Ca²⁺‐permeable AMPA receptors from synapses, resulting in synaptic depression. This work demonstrates a novel mechanism in which mGluR signals release AMPA receptors rapidly from the ER and couple ER release to GluA2 synaptic insertion and GluA1 removal.     

Role of Calmodulin-Calmodulin Kinase II,cAMP/Protein Kinase A and ERK 1/2 on Aeromonas hydrophila-Induced Apoptosis of Head Kidney Macrophages

The role of calcium (Ca²⁺) and its dependent protease calpain in Aeromonas hydrophila-induced head kidney macrophage (HKM) apoptosis has been reported. Here, we report the pro-apoptotic involvement of calmodulin (CaM) and calmodulin kinase II gamma (CaMKIIg) in the process. We observed significant increase in CaM levels in A. hydrophila-infected HKM and the inhibitory role of BAPTA/AM, EGTA, nifedipine and verapamil suggested CaM elevation to be Ca²⁺-dependent. Our studies with CaM-specific siRNA and the CaM inhibitor calmidazolium chloride demonstrated CaM to be pro-apoptotic that initiated the downstream expression of CaMKIIg. Using the CaMKIIg-targeted siRNA, specific inhibitor KN-93 and its inactive structural analogue KN-92 we report CaM-CaMKIIg signalling to be critical for apoptosis of A. hydrophila-infected HKM. Inhibitor studies further suggested the role of calpain-2 in CaMKIIg expression. CaMK Kinase (CaMKK), the other CaM dependent kinase exhibited no role in A. hydrophila-induced HKM apoptosis. We report increased production of intracellular cAMP in infected HKM and our results with KN-93 or KN-92 implicate the role of CaMKIIg in cAMP production. Using siRNA to PKACA, the catalytic subunit of PKA, anti-PKACA antibody and H-89, the specific inhibitor for PKA we prove the pro-apoptotic involvement of cAMP/PKA pathway in the pathogenicity of A. hydrophila. Our inhibitor studies coupled with siRNA approach further implicated the role of cAMP/PKA in activation of extracellular signal-regulated kinase 1 and 2 (ERK 1/2). We conclude that the alteration in intracellular Ca²⁺ levels initiated by A. hydrophila activates CaM and calpain-2; both pathways converge on CaMKIIg which in turn induces cAMP/PKA mediated ERK 1/2 phosphorylation leading to caspase-3 mediated apoptosis of infected HKM.     

Caprylate-Conjugated Cisplatin for the Development of Novel Liposomal Formulation

Cisplatin, first (platinum) compound to be evolved as an anticancer agent, has found its important place in cancer chemotherapy. However, the dose-dependent toxicities of cisplatin, namely nephrotoxicity, ototoxicity, peripheral neuropathy, and gastrointestinal toxicity hinder its widespread use. Liposomes can reduce the toxicity of cisplatin and provide a better therapeutic action, but the low lipid solubility of cisplatin hinders its high entrapment in such lipid carrier. In the present investigation, positively charged reactive aquated species of cisplatin were complexed with negatively charged caprylate ligands, resulting in enhanced interaction of cisplatin with lipid bilayer of liposomes and increase in its encapsulation in liposomal carrier. Prepared cisplatin liposomes were found to have a vesicular size of 107.9 ± 6.2 nm and zeta potential of −3.99 ± 3.45 mV. The optimized liposomal formulation had an encapsulation efficiency of 96.03 ± 1.24% with unprecedented drug loading (0.21 mg cisplatin / mg of lipids). The in vitro release studies exhibited a pH-dependent release of cisplatin from liposomes with highest release (67.55 ± 3.65%) at pH 5.5 indicating that a maximum release would occur inside cancer cells at endolysosomal pH. The prepared liposomes were found to be stable in the serum and showed a low hemolytic potential. In vitro cytotoxicity of cisplatin liposomes on A549 lung cancer cell line was comparable to that of cisplatin solution. The developed formulation also had a significantly higher median lethal dose (LD₅₀) of 23.79 mg/kg than that of the cisplatin solution (12 mg/kg). A promising liposomal formulation of cisplatin has been proposed that can overcome the disadvantages associated with conventional cisplatin therapy and provide a higher safety profile.Key Words: cisplatin, complexation, cytotoxicity, LD₅₀, liposome