• Journal of Microbiology and Biotechnology
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• v.28 no.10
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• pp.1736-1748
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• 2018

Brucella abortus can survive and replicate within host macrophages, and great efforts have been made to demonstrate the genes involved in pathogenicity, such as internalization, in Brucella research. Here, intracellular responses were compared between THP-1 macrophage cells stimulated with B. abortus wild-type and four mutants (C1, C10, C27, and C32) using microarray to demonstrate the role of genes related to intracellular survival and replication. These mutants were generated by deleting genes encoding BAB_RS13225 (4-hydrobenzoate 3-monooxygenase, PHBH), BAB_RS00455 (heme exporter protein cytochrome C, CcmC), BAB_RS03675 (exopolyphosphatase, PPX), and BAB_RS13225 (peptidase M24). The results showed that mutants C1 and C10 induced significant suppression of survival levels and cytokine expression relative to wild-type in the THP-1 macrophage cells. These findings suggest that the BAB_RS13225 and BAB_RS00455 genes play important roles in survival within human macrophages. Conversely, mutants C27 and C32 induced significantly higher survival level than wild-type in the cells inhibiting cellular signal transduction. It is assumed that the BAB_RS03675 and BAB_RS13225 genes play a role in cellular resistance to B. abortus. Therefore, the disrupted genes are involved in B. abortus intracellular growth, and especially in its survival, and they could be effective targets for understanding the intracellular bacterium, B. abortus.

• Parasites, Hosts and Diseases
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• v.54 no.1
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• pp.31-38
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• 2016

Specific gene expressions of host cells by spontaneous STAT6 phosphorylation are major strategy for the survival of intracellular Toxoplasma gondii against parasiticidal events through STAT1 phosphorylation by infection provoked $IFN-{\gamma}$. We determined the effects of small molecules of tyrosine kinase inhibitors (TKIs) on the growth of T. gondii and on the relationship with STAT1 and STAT6 phosphorylation in ARPE-19 cells. We counted the number of T. gondii RH tachyzoites per parasitophorous vacuolar membrane (PVM) after treatment with TKIs at 12-hr intervals for 72 hr. The change of STAT6 phosphorylation was assessed via western blot and immunofluorescence assay. Among the tested TKIs, Afatinib (pan ErbB/EGFR inhibitor, $5{\mu}M$) inhibited 98.0% of the growth of T. gondii, which was comparable to pyrimethamine ($5{\mu}M$) at 96.9% and followed by Erlotinib (ErbB1/EGFR inhibitor, $20{\mu}M$) at 33.8% and Sunitinib (PDGFR or c-Kit inhibitor, $10{\mu}M$) at 21.3%. In the early stage of the infection (2, 4, and 8 hr after T. gondii challenge), Afatinib inhibited the phosphorylation of STAT6 in western blot and immunofluorescence assay. Both JAK1 and JAK3, the upper hierarchical kinases of cytokine signaling, were strongly phosphorylated at 2 hr and then disappeared entirely after 4 hr. Some TKIs, especially the EGFR inhibitors, might play an important role in the inhibition of intracellular replication of T. gondii through the inhibition of the direct phosphorylation of STAT6 by T. gondii.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.2
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• pp.581-590
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• 2016

Resistance to anoikis, a cell-detachment induced apoptosis, is one of the malignant phenotypes which support tumor metastasis. Molecular mechanisms underlying the establishment of this phenotype require further investigation. This study aimed at exploring protein expression profiles associated with anoikis resistance of a metastatic breast cancer cell. Cell survival of suspension cultures of non-metastatic MCF-7 and metastatic MDA-MB-231 cells were compared with their adherent cultures. Trypan blue exclusion assays demonstrated a significantly higher percentage of viable cells in MDA-MB-231 than MCF-7 cell cultures, consistent with analysis of annexin V-7-AAD stained cells indicating that MDA-MB-231 possess anti-apoptotic ability 1.7 fold higher than MCF-7 cells. GeLC-MS/MS analysis of protein lysates of MDA-MB-231 and MCF-7 cells grown under both culture conditions identified 925 proteins which are differentially expressed, 54 of which were expressed only in suspended and adherent MDA-MB-231 but not in MCF-7 cells. These proteins have been implicated in various cellular processes, including DNA replication and repair, transcription, translation, protein modification, cytoskeleton, transport and cell signaling. Analysis based on the STITCH database predicted the interaction of phospholipases, PLC and PLD, and 14-3-3 beta/alpha, YWHAB, with the intrinsic and extrinsic apoptotic signaling network, suggesting putative roles in controlling anti-anoikis ability. MDA-MB-231 cells grown in the presence of inhibitors of phospholipase C, U73122, and phospholipase D, FIPI, demonstrated reduced ability to survive in suspension culture, indicating functional roles of PLC and PLD in the process of anti-anoikis. Our study identified intracellular mediators potentially associated with establishment of anoikis resistance of metastatic cells. These proteins require further clarification as prognostic and therapeutic targets for advanced breast cancer.