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Rapid Quantification of Oxidized and Reduced Forms of Glutathione Using Ortho -phthalaldehyde in Cultured Mammalian Cells In Vitro
Published on: June 28, 2024
Tumor-suppressive maspin regulates cell response to oxidative stress by direct interaction with glutathione
Shuping Yin1, Xiaohua Li, Yonghong Meng
1Department of Pathology, Center of Molecular Medicine and Genetics, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Abstract:
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.
Insights
Maspin, a serine protease inhibitor, interacts with glutathione S-transferase (GST) to reduce reactive oxygen species (ROS) and inhibit tumor progression. This interaction is key to maspin
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Maspin, a serine protease inhibitor, is known to suppress tumor progression but its molecular targets and mechanisms remain unclear.
- Identifying maspin's molecular targets is crucial for understanding its anti-cancer functions, particularly in prostate cancer bone metastasis.
Purpose of the Study:
- To identify the molecular targets of maspin using a yeast two-hybrid screen.
- To elucidate the functional significance of the maspin-glutathione S-transferase (GST) interaction in cancer cells.
Main Methods:
- Yeast two-hybrid screening using full-length maspin cDNA bait against prostate tumor and HeLa cDNA prey libraries.
- Confirmation of protein interactions using purified proteins, cell lines (MDA-MB-435, DU145), and human prostate tissues.
- Assays for GST activity, reactive oxygen species (ROS) generation, and vascular endothelial growth factor A (VEGF-A) expression.
Main Results:
- Maspin was found to interact with heat shock protein 90, GST, and heat shock protein 70.
- The interaction between maspin and GST was confirmed, with a specific point mutation (Mas(R340A)) decreasing affinity.
- Maspin expression correlated with decreased ROS levels and inhibited oxidative stress-induced VEGF-A expression, mediated by GST.
Conclusions:
- Maspin directly interacts with GST, forming a complex that plays a significant role in regulating cellular oxidative stress.
- This maspin-GST interaction inhibits ROS generation and vascular endothelial growth factor A induction, suggesting a novel mechanism for maspin's tumor-suppressive activity.
- The findings provide new insights into maspin's molecular mechanisms and its potential as a therapeutic target in cancer treatment.
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