mAtNOS1 induces apoptosis of human mammary adenocarcinoma cells
Introduction
AtNOS1 is an Arabidopsis thaliana gene that regulates mitochondrial nitric oxide (NO) in plants (Guo et al., 2003). The role of AtNOS1 in mitochondrial NO and plant's functions have been tested using AtNOS1 mutants (Crawford, 2006). It has been shown that about 80% of NOS activity was eliminated in AtNOS1 mutants (Guo et al., 2003, He et al., 2004, Zeidler et al., 2004), indicating that AtNOS1 accounts for most NOS activity in the plant. It has been shown that AtNOS1 is critical for various plant's functions including germination, root and shoot growth, seed fertility (Guo et al., 2003), and control of flower timing (He et al., 2004). AtNOS1 mutants are more susceptible to bacterial pathogen and fail to respond to lipopolysaccharide treatment (Zeidler et al., 2004), suggesting a crucial role for AtNOS1 in plant defense responses. Mammalian ortholog of AtNOS1, mAtNOS1, has been reported in fibroblast cell line COS1 (Zemojtel et al., 2006). Using cells transfected with V5-His-tagged mAtNOS1, it has been shown that mitochondria of transfected cells cross-react with anti-V5 antibody, suggesting that mAtNOS1 is localized at the mitochondria of those cells (Zemojtel et al., 2006). mAtNOS1 that is the mammalian ortholog of AtNOS1 has been shown to involve in the development of neural, haematopoeitic and bone systems in mouse embryos (Zemojtel et al., 2006). However, exact functions of mAtNOS1 are not fully understood. The present study tested functions of mAtNOS1 for mammary adenocarcinoma MCF-7 cells. We generated MCF-7 cells overexpressing and underexpressing mAtNOS1 and show that mAtNOS1 plays a pivotal role in regulating mitochondrial NO and apoptosis of those cells. The molecular mechanisms underlying mAtNOS1 functions and importance of mitochondrial Ca2+ ([Ca2+]m) will be discussed.
Section snippets
Materials
Human mammary adenocarcinoma cells (MCF-7) and cDNA were purchased from ATCC (Manassas, VA, USA). Small interfering RNA (siRNA) was designed and synthesized by Sigma-Proligo (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), l-glutamine, penicillin-streptomycin, Rhodamine 2AM (Rhod2), Hoechst 33342, propidium iodide (PI), mitotracker red (MTR), mitotracker green (MTG), Alexa Fluor 488, pcDNA 3.1/ myc His(-)A, primers, lipofectamine 2000, plasmid
mAtNOS1 expression level
First, we analyzed the expression level of mAtNOS1 in MCF-7 cells. Fig. 1 shows higher mAtNOS1 expression in overexpressed and lower expression in underexpressed cells, compared to the control. Barograms represent Mean densitometric analysis of PCR products.
Mitochondrial NO and calcium
We measured mitochondrial NO and [Ca2+]m. Fig. 2A, B shows higher mitochondrial NO in overexpressed and lower mitochondrial NO in underexpressed cells, as compared to the controls. Co-localization of DAF and mitotracker red signal is
Discussion
The present investigation was conducted to study functions of mAtNOS1 in human cancer cells. First, we generated mAtNOS1 over- and underexpressing cells. Our results show higher mAtNOS1 expression in overexpressing cells and lower expression in underexpressing MCF-7 cells, compared to the controls. AtNOS1 that is plant ortholog of mAtNOS1 regulates plant's mitochondrial NO (Guo and Crawford, 2005) that is involved in various functions including oxidative stress and apoptosis (Crawford and Guo,
Acknowledgements
Financial support was provided by the National Institute on Aging (award AG023264-02) and American Heart Association (award 0565221B).
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