Journal: PLoS ONE

Article Title: NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway

doi: 10.1371/journal.pone.0011994

Figure Lengend Snippet: ( A ) H1229 cells treated with empty vector and IC 20 of cisplatin (3.0 µM), NPRL2, or NPRL2+ with IC 20 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 IC 20 dose of cisplatin or NPRL2, P-Chk1 increased in a time-dependent manner. In contrast, in those treated with NPRL2 and IC 20 dose of cisplatin, P-Chk1 was dramatically enhanced. Although P-Chk2 was not detected in H1299 cells treated with empty vector and IC 20 dose of cisplatin, it was clearly increased in a time-dependent manner in those treated with NPRL2 or NPRL2+ IC 20 dose of cisplatin. ( B ) P-Chk2 expression was immunohistochemically analyzed in an orthotopic model of H322 pleural dissemination, as described in the 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 + IC 20 dose of cisplatin, NPRL2 or NPRL2+ IC 20 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.

Article Snippet: Anti-phospho-Chk1 (Ser345), anti-phospho-Chk2 (Thr68), anti-phospho-Chk1 (Ser345), anti-NBS1, anti-phospho-NBS1 (Ser343), anti-SMC1, anti-phospho-Cdc25C (Ser216), anti-phospho-Cdc2 (Tyr15), anti-caspase-3, and anti-caspase-9 were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Plasmid Preparation, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway

doi: 10.1371/journal.pone.0011994

Figure Lengend Snippet: NPRL2+ cisplatin ( cis -diamminedichloroplatinum (II) [CDDP]) treatment effector caspase, caspase-3, is activated and PARP is cleaved. Therefore, our result that the combination treatment of NPRL2 and CDDP activates a caspase cascade and hyperphosphorylates H2AX suggests that this combination treatment can strongly enhance the apoptotic pathway. Adding NPRL2 treatment to CDDP significantly enhanced Chk2 and Chk1 kinase activity, and NPRL2+ CDDP treatment remarkably degraded the interaction of Cdc2 and cyclin B1, leading to the inactivation of the Cdc2/cyclin B1 complex and arrest in G2/M. The cells arrested at the G2/M phase ultimately promote apoptosis with characteristic nuclear fragmentation. This combination treatment strongly inactivates Cdc25A and Cdc25C, and phospho-Cdc2 (Tyr-15), an inactivated type of Cdc2, is clearly increased in NPRL2+ CDDP treatment.

Article Snippet: Anti-phospho-Chk1 (Ser345), anti-phospho-Chk2 (Thr68), anti-phospho-Chk1 (Ser345), anti-NBS1, anti-phospho-NBS1 (Ser343), anti-SMC1, anti-phospho-Cdc25C (Ser216), anti-phospho-Cdc2 (Tyr15), anti-caspase-3, and anti-caspase-9 were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Activity Assay