Tang X, Feng Y, Ye K. 1 Fyn is over-expressed in NK malignant cells(A) qRT-PCR of mRNA in PBMCs from 10 HDs and from 8 patients with CLPD (UPN: 1C8). Statistical significance was determined using an unpaired test and < 0.001. (B) WB analysis of Fyn in 3 HDs, 3 CLPD patients (UPN: 6C7) and two NK cell lines, KHYG1 and NK92. Quantification of Fyn protein levels was normalized with -actin bands. 4c pyrazolo[3,4-< 0.01) to reach 50% reduction of viability (EC50) (Table ?(Table1).1). Interestingly, 4c compound had negligible effect in PBMCs and in purified NK cells from HDs (Figure ?(Figure2A).2A). Same results were obtained in primary NK cells from Aminocaproic acid (Amicar) HDs treated with 4c compound and cultured without KCTD18 antibody IL2 (Supplementary Figure S1). Open in a separate window Figure 2 4c compound reduced cell viability inducing apoptosis and cell cycle arrest in NK leukemic cells(A) Viability of KHYG1, NK92, 3 PBMCs from HDs (HDs) and NK primary cells sorted from 3 HDs (HD-NK cells) was evaluated by MTS assay after treatment with 4c compound at different concentration (2C10 M) for 24C72 hours. Results are expressed as percent of cell viability normalized to DMSO-treated control cells. The bar-graphs represent mean with S.D. from three independent experiments. (B) Apoptosis and (C) cell cycle analysis were evaluated by flow cytometer in NK cell Aminocaproic acid (Amicar) line Aminocaproic acid (Amicar) after 4c or DMSO vehicle treatment at 4 M for 24 hours. Dot plots and cell cycle histograms show a single representative experiment, the bar-graphs represent mean with S.D. from three independent experiments. < 0.001. Table 1 EC50 obtained in two cell lines and PBMCs and in purified NK cells from HD samples after 4c compound treatment (value is < 0.01 for both cell lines vs HDs) (n.c.: not calculable) < 0.001) and cell cycle arrest in G2/M phase in treated KHYG1 respect to their control (Figure 2BC2C). Fyn phosphorylation is reduced after 4c compound treatment and it decreased Akt and P70 S6 kinase activation To verify Fyn inhibition we performed its immunoprecipitation in KHYG1 cell line treated with 4c compound or with DMSO vehicle control and we detected its phosphorylation. We observed that Fyn phosphorylation significantly decreased after treatment (< 0.01; Figure ?Figure3A3A). Open in a separate window Figure 3 Inhibition on Fyn phosphorylation by 4c compound decreased Akt and P70 phosphorylationWB analysis of (A) phospho-Fyn and (B) total and phosphorylated Akt and P70 S6 kinase in NK cell line treated with 4c compound compared to control. Protein levels were normalized on -actin signals. Bar-graphs represent mean with S.D. from three independent experiments. < 0.05, **< 0.01. We also explored, by WB, the activation of two protein involved in Fyn pathway, Akt and P70 S6 kinase. Our data showed that there was a decrease of phosphorylation of Akt and P70 S6 kinase after treatment with 4c compound (< 0.05; Figure ?Figure3B3B). Gene expression and protein profile of treated NK leukemic cells showed the activation of apoptotic pathways We performed gene expression profile (GEP) analysis of KHYG1 cell line after 4 M drug incubation at 24 hours compared to control. A total of 697 genes (297 up-regulated vs 400 down-regulated genes) were differentially expressed. Ingenuity Pathway Analysis (IPA) gene ontology analysis demonstrated that most of genes were involved in cellular growth, death, development and cell cycle (Table ?(Table2).2). Moreover, analysis carried out with DAVID let us to cluster all genes in the same functions indicated by IPA. In fact, the first up-regulated cluster genes [e.g. ((and in KHYG1 treated vs control. Bar-graphs represent mean with S.D. from three independent experiments. (D) Bar-graphs of protein levels resulted from apoptotic protein array and (E) WB analysis of caspase 3 in KHYG1 treated vs control. < 0.05, **< 0.01, ***< 0.001. We validated GEP data by qRT-PCR confirming the down-regulation of anti-apoptotic gene, (< 0.05), and the up-regulation of two pro-apoptotic genes, CFLAR (< 0.05) and (< 0.001), in KHYG1 treated vs Aminocaproic acid (Amicar) control (Figure ?(Figure4C4C). Subsequently, to corroborate apoptosis and GEP data, we also performed an array analysis.