RESUMEN
Introduction: Laser radiation is a promising strategy against various malignancies. Recent studies have shown that the application of low-power laser therapy (LPLT) at different doses and exposure times could modulate the growth dynamic of tumor cells. Based on the type of laser, LPLT could potentially trigger cell proliferation, differentiation, and apoptosis in different cell lines. Methods: In this study, MTT assay was used to monitor the effect of low and high laser intensities on the viability of normal and cancer lymphocytes. The protein levels of Ki-67 (a proliferation marker) and Caspase-3 (an apoptosis factor) were measured in human peripheral mononuclear cells (PBMCs) and the B-lymphoblastic cell line (Nalm-6) using flow cytometry after being-exposed to 630-nm LPLT at low (2, 4, 6, and 10 J/cm2 ) and high (15, 30, 60, and 120 J/cm2) energy densities in a continuous mode for 48 and 72 hours. Results: By using higher energy densities, 60 and 120 J/cm2 , a significant decrease was shown in the viability of Nalm-6 cells, which reached 6.6 and 10.1% after 48 hours compared to the control cells (P<0.05). Notably, Cell exposure to doses 30, 60, and 120 J/cm2 yielded 7.5, 12.9, and 21.6 cell viability reduction after 72 hours. The collected data showed that the high-intensity parameters of LPLT (15 to 120 J/cm2) promoted significant apoptotic changes in the exposed cells coincided with the activation of Caspase-3 compared to the none-treated control cells (P<0.05). The data further showed the stimulation of the Ki-67 factor both in primary PBMCs and the lymphoblastic cell line treated with LPLT at energy densities of 4 and 6 J/cm2 (P<0.05), indicating enhanced cell proliferation. Similar to Nalm-6 cells, primary PBMCs showed apoptosis after 48 hours of being exposed to doses 60, and 120 J/cm2 , indicated by increased Caspase-3 levels (P<0.05). As expected, the Nalm-6 cells were resistant to cytotoxic effects of laser irradiation in the first 48 hours (P>0.05) compared to normal PBMCs. The exposure of Nalm-6 cells to low-intensity laser intensities increased a proliferation rate compared to the PBMCs treated with the same doses. Conclusion: We showed the potency of LPLT in the induction of apoptosis and proliferation in human primary PBMCs and Nalm-6 cells in a dose and time-dependent manner after 72 hours.
RESUMEN
Purpose: Generation of antibodies which potentially discriminate between malignant and healthy cells is an important prerequisite for early diagnosis and treatment of gastric cancer (GC). Comparative analysis of cell surface protein landscape will provide an experimental basis for biomarker discovery, which is essential for targeted molecular therapies. This study aimed to isolate phage-displayed antibody fragments recognizing cell surface proteins, which were differently expressed between two closely related GC cell lines, namely AGS and MKN-45. Methods: We selected and screened a semisynthetic phage-scFv library on AGS, MKN-45, and NIH-3T3 cell lines by utilizing a tailored selection scheme that was designed to isolate phagescFvs that not only recognize the differentiated AGS cells but also distinguish them from NIH3T3 fibroblasts and the poorly differentiated MKN-45 cells. Results: After four rounds of subtractive whole cell panning, 14 unique clones were identified by ELISA screening and nucleotide sequencing. For further characterization, we focused on four phage-scFvs with strong signals in screening, and their specificity was confirmed by cell-based ELISA. Furthermore, the selected phage-scFvs were able to specifically stain AGS cells with 38.74% (H1), 11.04% (D11), 76.93% (G11), and 69.03% (D1) in flow cytometry analysis which supported the ability of these phage scFvs in distinguishing AGS from MKN-45 and NIH-3T3 cells. Conclusion: Combined with other proteomic techniques, these phage-scFvs can be applied to membrane proteome analysis and, subsequently, identification of novel tumor-related antigens mediating proliferation and differentiation of cells. Furthermore, such antibody fragments can be exploited for diagnostic purposes as well as targeted drug delivery of GC.
RESUMEN
IL-10, an anti-inflammatory cytokine, has been shown to exhibit stimulatory functions including CD14 up-regulation on human monocytic cells. CD14-mediated signaling following LPS stimulation of monocytic cells results in the synthesis of proinflammatory cytokines. Our results show that LPS-induced CD14 expression on monocytic cells may be mediated by endogenously produced IL-10. To investigate the molecular mechanism by which IL-10 enhances CD14 expression, both human monocytes and the promyelocytic HL-60 cells were used as model systems. IL-10 induced the phosphorylation of PI3K and p42/44 ERK MAPK. By using specific inhibitors for PI3K (LY294002) and ERK MAPKs (PD98059), we demonstrate that LY294002 either alone or in conjunction with PD98059 inhibited IL-10-induced phosphorylation of STAT-1 and consequently CD14 expression. However, IL-10-induced STAT-3 phosphorylation remained unaffected under these conditions. Finally, STAT-1 interfering RNA inhibited IL-10-induced CD14 expression. Taken together, these results suggest that IL-10-induced CD14 up-regulation in human monocytic cells may be mediated by STAT-1 activation through the activation of PI3K either alone or in concert with the ERK MAPK.