Tumor-suppressive maspin regulates cell response to oxidative stress by direct interaction with glutathione S-transferase.
Maspin, a novel serine protease inhibitor, suppresses tumor progression in several cancer models, including an in vivo model for prostate cancer bone metastasis. However, the molecular mechanism of maspin remains illusive, primarily because its molecular targets are unknown. To this end, we used a full-length maspin cDNA bait to screen against both a primary prostate tumor cDNA prey library and a HeLa cDNA prey library by the yeast two-hybrid method. We found that heat shock protein 90, glutathione S-transferase (GST), and heat shock protein 70 interacted with maspin with the highest frequencies. We confirmed the maspin/GST interaction using purified proteins, human epithelial cell lines, and human prostate tissues. A maspin variant that has a point mutation of Arg(340) to Ala (Mas(R340A)) showed a significantly decreased affinity for GST. Although purified maspin had no effect on the activity of purified GST in vitro, intracellular interaction between endogenous maspin and GST correlated with an elevated total GST activity in both MDA-MB-435- and DU145-derived stably transfected cells. Consistently, tumor cells treated with purified wild type maspin, but not Mas(R340A), enhanced cellular GST activity. Maspin expression in cancer cell lines also correlated with decreased basal levels of reactive oxygen species (ROS). Furthermore, H(2)O(2) treatment not only induced GST expression but also increased intracellular maspin/GST interaction, which was inversely correlated with the level of ROS generation. Conversely, maspin knockdown by small interfering RNA increased the basal, as well as H(2)O(2)-induced, ROS generation. Furthermore, the maspin effect on ROS generation was completely abolished by a GST inhibitor, indicating an essential role of GST in maspin-mediated cellular response to oxidative stress. Consistently, oxidative stress-induced vascular endothelial growth factor A expression was significantly inhibited in maspin-expressing cells. Together, our data suggest a new mechanism by which maspin, through its direct interaction with GST, may inhibit oxidative stress-induced ROS generation and vascular endothelial growth factor A induction, thus preventing further adverse effects on tumor genetics and stromal reactivity.
Yin
Shuping
S
Department of Pathology, Center of Molecular Medicine and Genetics, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Li
Xiaohua
X
Meng
Yonghong
Y
Finley
Russell L
RL
Sakr
Wael
W
Yang
Heng
H
Reddy
Neelima
N
Sheng
Shijie
S
D000409:Alanine / Q000737:chemistry; D001120:Arginine / Q000737:chemistry; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000473:pathology; D045744:Cell Line, Tumor; D002469:Cell Separation; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D016147:Genes, Tumor Suppressor / Q000502:physiology*; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D047468:Immunoprecipitation; D008297:Male; D008856:Microscopy, Fluorescence; D009362:Neoplasm Metastasis; D018384:Oxidative Stress; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011471:Prostatic Neoplasms / Q000473:pathology; D011485:Protein Binding; D017382:Reactive Oxygen Species; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015843:Serpins / Q000737:chemistry / Q000502:physiology*; D014162:Transfection; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D017382:Reactive Oxygen Species; C085344:SERPIN-B5; D015843:Serpins; D001120:Arginine; D006861:Hydrogen Peroxide; D005982:Glutathione Transferase; D005978:Glutathione; D000409:Alanine