Figure 1.
Effect of KPT185 on cell viability in NSCLC cells.
Six NSCLC cell lines were treated with KPT-185 for 48 h or 72 h. Cell viability was suppressed by KPT-185 in a dose- and time-dependent manner (A∼F). KPT-185 inhibited cell viability in H1975 (G) and HCC827 (H) cells when compared to the effect of EGFR-TKI gefitinib treatment. (N = 3).
Figure 2.
Effect of KPT-185 on the regulation of NSCLC cell cycle.
Cells were arrested at the G1 phase of the cell cycle in KPT-185 sensitive cells. (N = 3).
Figure 3.
Effect of KPT-185 on apoptosis in NSCLC cells.
The six NSCLC cell lines were treated with KPT-185 and then tumor cell apoptosis was assessed by flow cytometry assay. The data indicated a dose-dependent induction of cell apoptosis after 48 h of treatment (A). KPT-185 induced apoptosis in H1975 (B) and HCC827 (C) cells when compared with the EGFR-TKI gefitinib treatment. A representative data of apoptosis assay in H1975 cells was shown (D). (N = 3).
Figure 4.
Effect of KPT-185 on the regulation of protein expression in NSCLC cells.
The six NSCLC cell lines were treated with KPT-185 for 48 h. The expression of CRM1 protein was down-regulated (A & E), while the CRM1 mRNA levels were up-regulated (B, N = 3). In the presence of the proteasome inhibitor bortezomib, the reduction of CRM1 protein following KPT-185 treatment was almost blocked in H1975, HCC827, and H1650GR cells (C). EGFR expression was downregulated following KPT-185 treatment. The NF-κB and IκB-α proteins levels were not significantly affected. KPT-185 upregulated wild type p53 in A549 cells, downregulated mutant p53 in H1975 cell, and there was no effect in HCC827 and H2228 cell. In addition, p53 was not detected in H1650 and H1650GR cells (D&E, 400×).
Figure 5.
Effect of KPT-185 on the regulation of apoptosis related protein expression in NSCLC cells.
The six NSCLC cell lines were treated with KPT-185 for 48 h. Caspases-8, -9 and -3 and PARP were activated or cleaved, but survivin was down-regulated (A). In the presence of the pan-caspase inhibitor Z-VAD-FMK, KPT-185-induced apoptosis was completely blocked in H1975 cells (B, N = 3).
Figure 6.
Effects of KPT-276 on mouse H1975 cell xenograft growth inhibition.
NSCLC H1975 cells were transplanted into NOD-SCID mice. Mice with an established NSCLC cell xenograft were orally administered with vehicle, gefitinib, or KPT-276. Tumor cell xenograft growth was assessed for 30 days. Tumor growth curves showed a statistically significant suppression of H1975 cell growth in vivo when compared to vehicle or gefitinib treatment (P<0.05; A). Mice administered oral KPT-276 (100 mg/kg, three times a week for three weeks) tolerated the treatment well (B). The protein level of CRM1was detected in xenograft tumors by immunohistochemistry (C, 400×).