Afzelin inhibits migration of MDA-MB-231 cells by suppressing FAK expression and Rac1 activation

Triple-negative breast cancer (TNBC) has the worst prognosis and the highest rate of metastasis among other types ofbreast cancer. These characteristics are supported by the dysregulation of focal adhesion kinase (FAK) and Rac1 whichare the key players of mesenchymal cell migration on TNBC. Afzelin is a secondary metabolite that is contained ina variety of plants. This study explored the anti-migration effect of afzelin and its interaction with FAK and Rac1 onthe highly invasive TNBC cell line, MDA-MB-231. Cell viability was assessed by 3-(4,5-dimethyl 2-thiazolyl)-2,5-diphenyltetrazolium bromide assay, and cell migration was evaluated using in vitro scratch assay. Rac1 activation wasanalyzed using the colorimetric assay, while vinculin and actin filaments were stained through immunofluorescence. Thequantity of total FAK and phosphorylated FAK tyr397 was detected by Western blotting. Afzelin decreased cell viabilityand inhibited two-dimensional cell migration in a dose-dependent manner. Under confocal laser scanning microscopy,vinculin localization at the cell edge demonstrated a reduction of focal adhesion formation by afzelin. Further explorationshowed that afzelin decreased FAK expression but did not affect FAK phosphorylation at tyr397. In addition, afzelindecreased Rac1-GTPase activation, which is a downstream effector of FAK. Taken together, these results suggest thatafzelin suppresses TNBC cell migration, through inhibition of FAK expression and Rac1-GTPase activation.

Strong renal expression of heat shock protein 70, high mobility group box 1, inducible nitric oxide synthase, and nitrotyrosine in mice model of severe malaria

Abstract INTRODUCTION Renal damage is a consequence of severe malaria, and is generally caused by sequestration of Plasmodium falciparum -infected erythrocytes in the renal microcirculation, which leads to obstruction, hypoxia, and ischemia. This triggers high mobility group box 1 (HMGB1) to send a danger signal through toll-like receptors 2 and 4. This signal up-regulates inducible nitric oxide (iNOS) and nitrotyrosine to re-perfuse the tissue, and also increases heat shock protein 70 (HSP70) expression. As no study has examined the involvement of intracellular secondary molecules in this setting, the present study compared the renal expressions of HSP70, HMGB1, iNOS, and nitrotyrosine between mice suffered from severe malaria and normal mice. METHODS C57BL/6 mice were divided into an infected group (intraperitoneal injection of 10 6 P. berghei ANKA) and a non-infected group. Renal damage was evaluated using hematoxylin eosin staining, and immunohistochemistry was used to evaluate the expressions of HSP70, HMGB1, iNOS, and nitrotyrosine. RESULTS Significant inter-group differences were observed in the renal expressions of HSP70, HMGB1, and iNOS (p=0.000, Mann-Whitney test), as well as nitrotyrosine (p=0.000, independent t test). The expressions of HSP70 and HMGB1 were strongly correlated (p=0.000, R=1.000). No correlations were observed between iNOS and HMGB, HMGB1 and nitrotyrosine, HSP70 and nitrotyrosine, or iNOS and nitrotyrosine. CONCLUSIONS It appears that HMGB1, HSP70, iNOS, and nitrotyrosine play roles in the renal damage that is observed in mice with severe malaria. Only HSP70 expression is strongly correlated with the expression of HMGB1.

Detection of immunogenic proteins from Anopheles sundaicussalivary glands in the human serum

The saliva of mosquitoes has an important role in the transmission of several diseases, including malaria, and contains substances with vasomodulating and immunomodulating effects to counteract the host physiological mechanisms and enhance pathogen transmission. As immunomodulatory components, salivary gland proteins can induce the generation of specific IgG antibodies in the host, which can be used as specific biomarkers of exposure to Anopheles sundaicus . The objective of this study was to identify immunogenic proteins from the salivary glands of Anopheles sundaicus by reaction with sera from individuals living in malaria-endemic areas who are thus exposed to Anopheles mosquitoes.

Expression of inducible nitric oxide synthase, caspase-3 and production of reactive oxygen intermediate on endothelial cells culture (HUVECs) treated with P. falciparum infected erythrocytes and tumour necrosis factor-α.

Cytoadherence of P. falciparum infected erythrocytes on endothelial cells is a key factor in development of severe malaria. This process may associated with the activation of local immune that was enhanced by tumour necrosis factor-α (TNF-α). This study was conducted to see the influence of P.falciparum infected erythrocytes cytoadherence and TNF-α treatment in inducing endothelial cells activation in vitro. inducible nitric oxide synthase (iNOS) and caspase-3 expression, also reactive oxygen intermediate (ROI) production were used as parameters. An Experimental laboratory study had been done to observe endothelial cells activation (HUVECs) after treatment with TNF-α for 20 hours or P. falciparum infected erythrocytes for 1 hour or both of them. Normal endothelial cells culture had been used as a control. Using immunocytochemistry local immune activation of endothelial cells was determined by iNOS and caspase-3 expression. Nitro Blue Tetrazolium reduction-assay was conducted to see the ROI production semi quantitatively. inducible nitric oxide synthase expression only found on endothelial cells culture treated with P. falciparum infected erythrocytes or both P. falciparum infected erythrocytes and TNF-α. Caspase-3 expression found slightly on normal endothelial cells culture. This expression increased significantly on endothelial cells culture treated with both P.falciparum infected erythrocytes and TNF-α (p=0.000). The normal endothelial cells release low level of ROI in the presence of non-specific trigger, PMA. In the presence of P. falciparum infected erythrocytes or TNF-α or both of them, some cells showed medium to high levels of ROI. Cytoadherence of P. falciparum infected erythrocytes and TNF α treatment on endothelial cells can induce activation of local immune marked by increase inducible nitric oxide synthase and release of free radicals that cause cell damage.

Identification of point mutations in Glucose-6-Phosphate Dehydrogenase gene in Timor Island people : A preliminary report.

Glucose 6 phosphate dehydrogenase (G6PD) deficiency is common in malaria endemic region, however no molecular study has been performed on G6PD deficiency in Timor Island, Indonesia a malarial hyperendemic area which Proto Malay is the majority of the people in that island. To observe the frequency and molecular type of mutations in G6PD deficient Proto Malay people, 118 native people were screened using formazan ring test. Mutation in the G6PD gene were determined by MPTP (Multiple PCR using Multiple Tandem Forward Primers and a common Reserve Pimer) method and confirmed by automatic sequencer. This study shows that three males have lower G6PD activity. Using MPTP method, a point mutation could be indicated in the two cases. Sequencing of the amplified products in 2 G6PD patients disclosed mutations of T383C in exon 5 and C 592 T in exon 6 in respective case. Our result documents point mutations in exon 5 and exon 6 in the G6PD gene of two Proto Malay people in Timor. These mutations are common in Asia region.