Combination of NPRL2 and cisplatin increases the activation of both Chk1 and Chk2.

(A) H1229 cells treated with empty vector and IC20 of cisplatin (3.0 µM), NPRL2, or NPRL2+ with IC20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment and analyzed for expression of Chk1, P-Chk1 (Ser345), Chk2, and P-Chk2 (Thr68). In cells treated with empty vector and IC20 dose of cisplatin or NPRL2, P-Chk1 increased in a time-dependent manner. In contrast, in those treated with NPRL2 and IC20 dose of cisplatin, P-Chk1 was dramatically enhanced. Although P-Chk2 was not detected in H1299 cells treated with empty vector and IC20 dose of cisplatin, it was clearly increased in a time-dependent manner in those treated with NPRL2 or NPRL2+ IC20 dose of cisplatin. (B) P-Chk2 expression was immunohistochemically analyzed in an orthotopic model of H322 pleural dissemination, as described in the Materials and Methods section. P-Chk2 expression was slightly detected in pleural tumor cells from mice treated with NPRL2 nanoparticles, but not in those treated with LacZ or LacZ + cisplatin. In contrast, treatment by NPRL2 nanoparticles and cisplatin induced high phospho-Chk2 expression in tumor cells. Magnification: ×400. (C) The kinase activity of Chk1 and Chk2 in H1299 cells treated with empty vector + IC20 dose of cisplatin, NPRL2 or NPRL2+ IC20 dose of cisplatin was analyzed with use of a K-LISA Checkpoint Activity Kit, an enzyme-linked immunosorbent assay (ELISA)-based activity assay. Chk1 kinase activity was greater in cells treated with NPRL2+ cisplatin than in cells treated with empty vector or empty vector + cisplatin (P<0.003). Chk2 kinase activity was more strongly enhanced in cells treated with NPRL2 or NPRL2+ cisplatin than in cells treated with empty vector or empty vector + cisplatin (P<0.0001). Bars, SDs of the mean in four individual experiments.