Nitrofen suppresses cell proliferation and promotes mitochondria-mediated apoptosis in type II pneumocytes.
OBJECTIVE
To characterize the molecular mechanisms of nitrofen-induced pulmonary hypoplasia.


METHODS
After administration of nitrofen to cultured type II A549 pneumocytes, cell proliferation and DNA synthesis were investigated by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetry, colony formation assay, flow cytometry and [3H]-thymidine incorporation assay. Apoptosis was measured by terminal transferase-mediated dUTP nick-end-labeling, acridine orange-ethidium bromide staining and flow cytometry. Expression of proliferating cell nuclear antigen (PCNA) and apoptosis-related genes was assayed by immunofluorescence, RT-PCR and Western blot.


RESULTS
Nitrofen inhibited the cell proliferation of A549 cells in a dose- and time-dependent manner, accompanied by downregulation of PCNA. As a result, the DNA synthesis of nitrofentreated A549 cells decreased, while cell cycle was arrested at G0/G1 phase. Moreover, nitrofen induced apoptosis of A549 cells, which was not abolished by Z-Val-Ala- Asp(OCH3)- fluoromethylketone. In addition, nitrofen decreased the expression of Bcl-x( L), but not of Bcl-2, Bax, and Bak, resulting in a loss of mitochondrial membrane potential and the nuclear translocation of apoptosis-inducing factor (AIF). Meanwhile, nitrofen strongly activated the p38 mitogen-activated protein kinase (p38-MAPK). Pretreatment of cells with SB203580 (5 micromol/L) blocked nitrofen-induced phosphorylation of p38-MAPK and abolished nitrofen-induced AIF translocation and apoptosis in A549 cells.


CONCLUSIONS
Nitrofen suppresses the proliferation of cultured type II pneumocytes accompanied by the downregulation of PCNA, and induces mitochondria-mediated apoptosis involving the activation of p38-MAPK.
Tong
Qiang-Song
QS
Department of Surgery, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. qstong@mails.tjmu.edu.cn
Zheng
Li-Duan
LD
Tang
Shao-Tao
ST
Jiang
Guo-Song
GS
Ruan
Qing-Lan
QL
Zeng
Fu-Qing
FQ
Dong
Ji-Hua
JH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D020169:Caspases / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured / Q000187:drug effects / Q000502:physiology; D006540:Herbicides / Q000494:pharmacology*; D006801:Humans; D008168:Lung / Q000166:cytology / Q000187:drug effects; D020935:MAP Kinase Signaling System / Q000502:physiology; D008928:Mitochondria / Q000378:metabolism*; D010647:Phenyl Ethers / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D006540:Herbicides; D010647:Phenyl Ethers; D048051:p38 Mitogen-Activated Protein Kinases; D020169:Caspases; C007350:nitrofen