Constitutive Wnt signalling is characterized by excessive levels of β-catenin protein and is a frequent occurrence in cancer. APC and Axin are key components of the β-catenin destruction complex that acts to promote β-catenin degradation. The levels of Axin are in turn controlled by tankyrases, members of the PARP-family of poly-ADP-ribosylation enzymes. In colorectal cancer cells, which typically harbor APC mutations, inhibition of tankyrase activity promotes Axin stabilization and attenuates Wnt signalling. Here, we examined the effect of inhibiting tankyrases in breast cancer cells with normal APC. We show that application of the small molecule tankyrase inhibitor, XAV939 or siRNA-mediated abrogation of tankyrase expression increases Axin1 and Axin2 protein levels and attenuates Wnt-induced transcriptional responses in several breast cancer lines. In MDA-MB-231 cells, inhibiton of tankyrase activity also attenuate Wnt3a induced cell migration. Moreover, in both MDA-MB-231 and colorectal cancer cells, XAV939 inhibits cell growth under conditions of serum-deprivation. However, the presence of serum prevents this growth inhibitory effect, although inhibition of Wnt-induced transcriptional and migratory responses was maintained. These results indicate that stabilization of Axin by inhibition of tankyrases alone, may not be an effective means to block tumor cell growth and that combinatorial therapeutic approaches should be considered.
Chinese tarantula, Chilobrachys jingzhao is one of the most venomous spiders in southern China and its venom is a mixture of various compounds with diversified biological activities. The proteome of C. jingzhao venom was analyzed by proteomic techniques. Proteins with molecular weight of over 10 kDa, indicated by gel-filtration and SDS-PAGE, were analyzed using 2-DE and MALDI-TOF/TOF and LC/ESI-Q-TOF MS. More than 90 proteins were detected, with 47 confirmed by sequence similarity search using mass spectrum driven basic local alignment search tool (MS BLAST). On the other hand, peptides with MW lower than 10 kDa were separated by HPLC and identified by MALDI-TOF MS and Edman degradation sequencing. About 120 peptides were detected, 60 of which were fully or partially sequenced. Our results indicate that peptides with MW lower than 10 kDa are the major components in the crude venom of C. jingzhao. Like those of other tarantulas, these peptides are very likely to act on various ion channels. These results pave a way for further detailed structure-function correlation analysis of the individual toxins present in the venom of C. jingzhao.
Using differential display, we isolated DDC-4, a secreted frizzled-related protein (sFRP), which is induced in the physiological apoptosis of hormonally regulated, reproductive tissues such as mammary gland, prostate, corpus luteum and uterus. The role of this gene in apoptosis was studied in animals overexpressing ectopic DDC-4/sFRP-4. Transgenic mice bearing the DDC-4/sFRP-4 cDNA under the control of the MMTV-LTR promoter showed lactational insufficiency and many apoptotic cells in the alveoli between day 19 of pregnancy and day 4 of lactation as demonstrated by TUNEL reaction and the presence of activated caspase-3. We performed a PKB/Akt kinase assay and studied several of its substrates using phosphorylation-specific antibodies to show reduced phosphorylation in PKB/Akt itself, as well as in glycogen synthetase kinase-3b (GSK-3b), BAD, and Forkhead. Taken together, our results show a role for DDC-4/sFRP-4 in abrogating an epithelial cell survival pathway at the onset of mammary gland involution.
Black widow spider is one of the most poisonous spiders in the world. Up to now, there have been few systematic analyses of the spider venom components, and the mechanism of action of the venom has not been completely understood. In this work, we employed combinative proteomic strategy to analyze the venom collected from living adult spider Latrodectus tredecimguttatus by electrical stimulation. The experiments demonstrated that the venom is primarily composed of high molecular weight proteins and has high abundance proteins around 100 kDa. The content of peptides and proteins with low molecular weight is low. A total of 75 nonredundant venom proteins with distinct function were unambiguously identified. Besides the known black widow spider venom proteins including latrotoxins, a variety of hydrolases and other proteins with special activity were found in the venom, such as proteinase, phospholipase, phosphatase, nuclease, fucolectin, venom allergen antigen 5-like protein and trypsin inhibitor, and so on. Their possible biological actions and relationship with latrodectism were discussed. The results help to understand the complexity and action mechanism of L. tredecimguttatus venom.
The L. tredecimguttatus venom was collected by electrical stimulation and systematically analyzed. Gel electrophoresis and RP-HPLC showed that the venom consisted primarily of proteins with molecular weights above 10 kDa, most of which were high-molecular-mass acidic proteins, with fewer proteins and peptides below 10 kDa. The most abundant proteins in the venom were concentrated at around 100 kDa, which included latrotoxins- the principal toxic components of the venom. Injection of the venom in mice and cockroaches P. americana gave rise to obvious poisoned symptoms, with LD50 values of 0.16 mg/kg and 1.87 microg/g, respectively. Electrophysiological experiments showed that the venom could block the neuromuscular transmission in isolated mouse phrenic nerve-hemidiaphragm and rat vas deferens preparations. The low-molecular-weight fraction (<10 kDa) of the venom had no effect on the transmission. Enzymatic analysis indicated that the venom possess activities of several kinds of hydrolases including hyaluronidase and proteases. These results demonstrated that L. tredecimguttatus venom was basically a large-protein-constituted venom and is one of the most poisonous spider venoms known in the world. The mammalian toxicity of the venom was based on its larger proteins rather than on smaller proteins and peptides, and its hydrolase activities might be involved in the latrodectism. The use of electrical stimulation method to collect the venom has the advantages of avoiding contamination and repeated use of the valuable L. tredecimguttatus venom resources.
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