Oxidative stress in NSC-741909-induced apoptosis of cancer cells.
BACKGROUND
NSC-741909 is a novel anticancer agent that can effectively suppress the growth of several cell lines derived from lung, colon, breast, ovarian, and kidney cancers. We recently showed that NSC-741909-induced antitumor activity is associated with sustained Jun N-terminal kinase (JNK) activation, resulting from suppression of JNK dephosphorylation associated with decreased protein levels of MAPK phosphatase-1. However, the mechanisms of NSC-741909-induced antitumor activity remain unclear. Because JNK is frequently activated by oxidative stress in cells, we hypothesized that reactive oxygen species (ROS) may be involved in the suppression of JNK dephosphorylation and the cytotoxicity of NSC-741909.


METHODS
The generation of ROS was measured by using the cell-permeable nonfluorescent compound H2DCF-DA and flow cytometry analysis. Cell viability was determined by sulforhodamine B assay. Western blot analysis, immunofluorescent staining and flow cytometry assays were used to determine apoptosis and molecular changes induced by NSC-741909.


RESULTS
Treatment with NSC-741909 induced robust ROS generation and marked MAPK phosphatase-1 and -7 clustering in NSC-741909-sensitive, but not resistant cell lines, in a dose- and time-dependent manner. The generation of ROS was detectable as early as 30 min and ROS levels were as high as 6- to 8-fold above basal levels after treatment. Moreover, the NSC-741909-induced ROS generation could be blocked by pretreatment with antioxidants, such as nordihydroguaiaretic acid, aesculetin, baicalein, and caffeic acid, which in turn, inhibited the NSC-741909-induced JNK activation and apoptosis.


CONCLUSIONS
Our results demonstrate that the increased ROS production was associated with NSC-741909-induced antitumor activity and that ROS generation and subsequent JNK activation is one of the primary mechanisms of NSC-741909-mediated antitumor cell activity.
Wei
Xiaoli
X
Department of Biochemical Pharmacology, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China.
Guo
Wei
W
Wu
Shuhong
S
Wang
Li
L
Huang
Peng
P
Liu
Jinsong
J
Fang
Bingliang
B
D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D053181:Caspase 8 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D016000:Cluster Analysis; D004354:Drug Screening Assays, Antitumor; D054637:Dual-Specificity Phosphatases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008175:Lung Neoplasms / Q000201:enzymology / Q000473:pathology*; D009637:Masoprocol / Q000494:pharmacology; D054639:Mitogen-Activated Protein Kinase Phosphatases / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
D000975:Antioxidants; D007211:Indoles; C544349:NSC-741909; D016755:Proto-Oncogene Proteins c-jun; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C532252:oncrasin-1; D009637:Masoprocol; D011065:Poly(ADP-ribose) Polymerases; D048031:JNK Mitogen-Activated Protein Kinases; D054639:Mitogen-Activated Protein Kinase Phosphatases; C439065:DUSP16 protein, human; D054637:Dual-Specificity Phosphatases; D053181:Caspase 8
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