The indicated cells were treated with indicated concentrations of PTL for 24 hrs (A) or treated with 20 μmol/L PTL for various lengths of time and harvested for Western blot Selleck TH-302 analysis (B). A549 (C, D) and H1299 (C, D) cells were seeded in 6-well plates and on the second day transfected with control or ATF4 (C) or DDIT3 (D) siRNA. A549 cells were treated with 20 μmol/L PTL while H1299 cells with 10 μmol/L for 24 hours after 48 hrs of transfection and harvested for Western blot analysis. Figure 6 Parthenolide up-regulates endoplasmic reticulum hallmarks ERN1, HSPA5 and p-EIF2A in a dose-dependent (A) and a time-dependent (B) manner. The indicated cells were treated with indicated concentrations of
PTL for 24 hrs (A) or treated with 20 μmol/L PTL for
various lengths of time and harvested for Western blot analysis (B). Parthenolide selectively eradicates lung cancer stem-like cells Weinberg et al. has demonstrated that selleck screening library knocking down of CDH1/E-cadherin with shRNA could make the cells have stem-like properties . We had demonstrated that A549/shCDH1 cells in which CDH1/E-cadherin expression is inhibited had stronger capacity of proliferation, migration and invasiveness . Furthermore, we found that the Temsirolimus mouse expression of SOX2 and POU5F1 which were considered to be the makers of stem cells were up-regulated in A549/shCDH1 cells (Additional file 1: Figure S2) [41, 42]. So in order to determine why PTL could selectively eradicate cancer stem-like cells, A549/shCDH1 cell line was used to mimic cancer stem cells and the A549/shCtrl cell line served as control. SRB assay showed PTL was more effective in inhibiting the growth of A549/shCDH1 cells than that of A549/shCtrl cells (Figure 7A). Western blot data showed that PTL could induce stronger cleavage of pro-caspases and PARP1 in A549/shCDH1 cell line (Figure 7B), which means that
PTL could trigger stronger apoptosis in A549/shCDH1 cells compared with control cells. Furthermore, apoptosis-related proteins were detected in A549/shCtrl and A549/shCDH1 cells side by side. Both long form and short form of CFLAR levels were down-regulated even more clearly in A549/shCDH1 PAK6 cells than that in control cells after PTL treatment. We also found that MCL1 was reduced more dramatically in A549/shCDH1 cells, while PMAIP1 was up-regulated on contrary after PTL treatment compared with the control cells (Figure 7C). Taken together, we conclude that both extrinsic apoptosis and intrinsic apoptosis induced by PTL are enhanced in A549/shCDH1 cells. The levels of p-EIF2A, ATF4 and DDIT3 were also examined. Data showed that their expression was further up-regulated in A549/shCDH1 cells after PTL treatment compared with A549/shCtrl cells (Figure 7C). DDIT3 was knocked down in the two cell lines simultaneously, and PMAIP1 was down-regulated and apoptosis was receded (Figure 7D).