Apoptosis assay Apoptosis was evaluated using Annexin V-FITC/PI apoptosis detection kit purchased from BIO-BOX Biotech (Nanjing, China) following the manufacturer’s instructions. Briefly, 2×106cells were harvested and washed twice with pre-cold PBS and then resuspended in 500 μl binding buffer. 5 μl of annexin V-FITC and 5 μl of Propidium Iodide (PI) were added to each sample and then incubated at room temperature in dark for 10 minutes. Analysis was performed by FACScan flow cytometer (Becton Dickinson, San Jose, CA). Results Parthenolide effectively inhibits the growth of human lung cancer cells through induction of apoptosis and cell cycle arrest It has

been reported that parthenolide has antitumor effects on various cancer cells. Hence, we examined the inhibition effect of PTL on AZD9291 order human NSCLC cells by treating the cells with various concentrations for 48 h and then

conducting SRB and MTT assay. As is shown, PTL had a dose-dependent growth inhibition effect on NSCLC cells Calu-1, H1792, A549, H1299, H157, and H460 (Figure 1A, B). To characterize the mechanism by which PTL induces growth inhibition in human NSCLC cells, we first determined the effect of PTL on induction of NCT-501 in vivo apoptosis by western blot analysis. The data showed that PTL could induce cleavage of apoptotic proteins such as CASP8, CASP9, CASP3 and PARP1 both in concentration- and time-dependent manner in tested lung cancer cells, indicating that apoptosis was trigged after PTL AR-13324 concentration exposure (Figure 1C, D). In addition to induction of apoptosis, PTL also induced G0/ G1 cell cycle arrest in a concentration- dependent manner in A549 cells and G2/M cell cycle arrest in H1792 cells (Additional file 1: Figure S1). The difference in cell cycle arrest induced in these two cell lines may be due to the p53 status [37, 38]. Collectively, these results show that PTL inhibits the growth of human lung cancer cells through induction of apoptosis and/or tuclazepam cell-cycle arrest. Figure 1 Parthenolide inhibits cell growth (A, B) and induces apoptosis in a concentration-dependent (C) and a time-dependent manner (D).

The indicated cell lines were seeded in 96-well plates and treated with the given concentration of PTL for 48 hrs. Cell survival was estimated using SRB assay (A) and MTT assay (B). Points: mean of four replicate determinations; bars: S.D. The indicated cells were treated with indicated concentrations of PTL for 24 hrs (C) or treated with 20 μmol/L PTL for various lengths of time and harvested for Western blot analysis (D). CF: cleaved form. Parthenolide triggers extrinsic apoptosis by up-regulation of TNFRSF10B expression In order to understand the molecular mechanism of PTL-induced apoptosis in NSCLC cell lines, several apoptosis-related proteins were examined. Data showed that TNFRSF10B was up-regulated after exposure to PTL (Figure 2A, B).