SP cells exhibit molecular markers of stem like cells Recent reports Regorafenib CAS suggest that epithelial cells acquire can cer stem cell properties upon induction of epithelial to mesenchymal transition. To evaluate whether SP cells show features of EMT, SP and MP cells from A549, H1650 and H1975 were examined for the levels of EMT markers like E cadherin, Vimentin and Fibronectin. As shown in Figure 2A, ABCG2 expression was significantly higher in the SP fraction in all the three cell lines. The levels of E cadherin was lower in H1650 SP cells as compared to MP cells, however, it was un detectable in A549 and unchanged in H1975 cells. Fibro nectin was detected at higher levels in A549 and H1975 SP cells, but undetectable in H1650 cells. Inhibitors,Modulators,Libraries Vimentin level was higher in A549 SP cells, but low in H1975 and H1650 SP cells.

Although the levels vary in a cell type dependent manner, these results suggest that, SP cells express proteins indicative of EMT without any external stimuli to the cells. The molecular basis for the differential expression of the EMT markers was then examined. Transcription factors like Twist, Slug and Snail Inhibitors,Modulators,Libraries have been demonstrated Inhibitors,Modulators,Libraries to be capable of coordin ating the EMT program during embryonic development and in cancers. Therefore, we next assessed the expression of these transcription factors in SP and MP cells. Real time PCR analysis revealed that Twist, Slug and Snail transcription factors Inhibitors,Modulators,Libraries are expressed at higher levels in SP cells in all the three NSCLC cell lines. The expression of Oct4, Sox2 and Nanog transcrip tion factors was next examined in SP cells.

Real time PCR analysis showed elevated levels of ABCG2, Oct4, Sox2, and Nanog in the SP fraction in all the three cell lines.Further, SP cells from H1650 cells growing as spheres showed expression of ABCG2, Oct4, Sox2 and Nanog proteins by fluores cence microscopy, indicating the undiffer entiated growth of self renewing SP cells within the spheres. EGFR tyrosine kinase Inhibitors,Modulators,Libraries inhibitors downregulate self renewal and SP phenotype Experiments were conducted to explore the molecular mechanisms involved in the self renewal of SP cells. Since aberrant EGFR signaling is implicated with the initiation and progression of lung cancer, we first assessed SP fre quency and expression of ABCG2 in the presence of an antagonistic antibody against EGFR.

Cells were mixed with 10 ug ml anti EGFR antibody or an isotype find more information control and plated in 2% FBS containing media for 5 days. Block ing EGF receptors resulted in a significant decrease in SP frequency in both A549 and H1650 cells, along with decreased EGFR phosphorylation as well as ABCG2 expression in both the cell lines. Con firming these results, depletion of EGFR expression by a siRNA resulted in decreased SP frequency and ABCG2 ex pression in A549, H1650 and H1975 cells.