Limits...
Proteomic identification of mitochondrial targets of arginase in human breast cancer.

Singh R, Avliyakulov NK, Braga M, Haykinson MJ, Martinez L, Singh V, Parveen M, Chaudhuri G, Pervin S - PLoS ONE (2013)

Bottom Line: Both arginase II (Arg II) and mSHMT expressions were higher in human breast tumor tissues compared to the matched normal and there was a strong correlation between Arg II and mSHMT protein expression.MDA-MB-468 xenografts had significant upregulation of Arg II expression that preceded the induction of mSHMT expression.Small inhibitory RNA (siRNA)-mediated inhibition of Arg II in MDA-MB-468 and HCC-1806 cells led to significant inhibition of both the mSHMT gene and protein expression.

View Article: PubMed Central - PubMed

Affiliation: Internal Medicine, Charles Drew University of Medicine and Science, Los Angeles, California, United States of America ; Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America ; Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America.

ABSTRACT
We have previously reported arginase expression in human breast cancer cells and demonstrated that the inhibition of arginase by N(ω) hydroxy L-arginine (NOHA) in MDA-MB-468 cells induces apoptosis. However, arginase expression and its possible molecular targets in human breast tumor samples and potential clinical implications have not been fully elucidated. Here, we demonstrate arginase expression in human breast tumor samples, and several established breast cancer cell lines, in which NOHA treatment selectively inhibits cell proliferation. The over-expression of Bcl2 in MDA-MB-468 cells abolished NOHA-induced apoptosis, suggesting that the mitochondria may be the main site of NOHA's action. We, therefore, undertook a proteomics approach to identify key mitochondrial targets of arginase in MDA-MB-468 cells. We identified 54 non-mitochondrial and 13 mitochondrial proteins that were differentially expressed in control and NOHA treated groups. Mitochondrial serine hydroxymethyltransferase (mSHMT) was identified as one of the most promising targets of arginase. Both arginase II (Arg II) and mSHMT expressions were higher in human breast tumor tissues compared to the matched normal and there was a strong correlation between Arg II and mSHMT protein expression. MDA-MB-468 xenografts had significant upregulation of Arg II expression that preceded the induction of mSHMT expression. Small inhibitory RNA (siRNA)-mediated inhibition of Arg II in MDA-MB-468 and HCC-1806 cells led to significant inhibition of both the mSHMT gene and protein expression. As mSHMT is a key player in folate metabolism, our data provides a novel link between arginine and folate metabolism in human breast cancer, both of which are critical for tumor cell proliferation.

Show MeSH

Related in: MedlinePlus

A, Western blot analysis of high Arg II expressing breast cancer cells MDA-MB-468, HCC 1806 and HCC 70 treated either with NOHA (1mM) alone or in combination with L-ornithine (0.5mM).B, Real-time quantitative PCR analysis showing selective induction of Arg II and mSHMT mRNA expression in fresh/frozen human breast tumor and matched normal tissues of Arg I, Arg II and mSHMT gene expression. C, Western blot analysis of Arg I, Arg II and mSHMT protein expression in normal and human breast tumor tissues (right panel) and densitometric quantitation analysis of protein expression (left panel). D, Top Panel, Immunohistochemical analysis of paraffin-embedded normal and breast tumor sections; Bottom Panel, Quantitative analysis of Arg II and mSHMT immuno-positive cells using ImagePro software as described in “materials and methods”. E, Time-course of induction of Arg II and mSHMT protein and F, gene expression in xenografts obtained from nude mice after injection of MDA-MB-468 (5x106) cells *, p≤0.05; **, p≤0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3818427&req=5

pone-0079242-g004: A, Western blot analysis of high Arg II expressing breast cancer cells MDA-MB-468, HCC 1806 and HCC 70 treated either with NOHA (1mM) alone or in combination with L-ornithine (0.5mM).B, Real-time quantitative PCR analysis showing selective induction of Arg II and mSHMT mRNA expression in fresh/frozen human breast tumor and matched normal tissues of Arg I, Arg II and mSHMT gene expression. C, Western blot analysis of Arg I, Arg II and mSHMT protein expression in normal and human breast tumor tissues (right panel) and densitometric quantitation analysis of protein expression (left panel). D, Top Panel, Immunohistochemical analysis of paraffin-embedded normal and breast tumor sections; Bottom Panel, Quantitative analysis of Arg II and mSHMT immuno-positive cells using ImagePro software as described in “materials and methods”. E, Time-course of induction of Arg II and mSHMT protein and F, gene expression in xenografts obtained from nude mice after injection of MDA-MB-468 (5x106) cells *, p≤0.05; **, p≤0.01.

Mentions: In order to validate our findings from the DIGE-based proteomics analysis, we analyzed the protein expression of mSHMT in MDA-MB-468 as well as in two other high Arg II expressing HCC1806 and HCC 70 cell lines treated either with NOHA (1mM) alone or in combination with L-ornithine (0.5mM) after 48 hrs. We observed that NOHA treatment led to a significant decrease in mSHMT protein expression compared to controls, but simultaneous treatment of these NOHA-treated cells with L-ornithine effectively blocked this NOHA-induced inhibition of mSHMT in all three cell lines that expressed high levels of Arg II (Figure 4A), but not in MDA-MB-157 cell line that expressed only very low levels of arginase (data not shown).


Proteomic identification of mitochondrial targets of arginase in human breast cancer.

Singh R, Avliyakulov NK, Braga M, Haykinson MJ, Martinez L, Singh V, Parveen M, Chaudhuri G, Pervin S - PLoS ONE (2013)

A, Western blot analysis of high Arg II expressing breast cancer cells MDA-MB-468, HCC 1806 and HCC 70 treated either with NOHA (1mM) alone or in combination with L-ornithine (0.5mM).B, Real-time quantitative PCR analysis showing selective induction of Arg II and mSHMT mRNA expression in fresh/frozen human breast tumor and matched normal tissues of Arg I, Arg II and mSHMT gene expression. C, Western blot analysis of Arg I, Arg II and mSHMT protein expression in normal and human breast tumor tissues (right panel) and densitometric quantitation analysis of protein expression (left panel). D, Top Panel, Immunohistochemical analysis of paraffin-embedded normal and breast tumor sections; Bottom Panel, Quantitative analysis of Arg II and mSHMT immuno-positive cells using ImagePro software as described in “materials and methods”. E, Time-course of induction of Arg II and mSHMT protein and F, gene expression in xenografts obtained from nude mice after injection of MDA-MB-468 (5x106) cells *, p≤0.05; **, p≤0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818427&req=5

pone-0079242-g004: A, Western blot analysis of high Arg II expressing breast cancer cells MDA-MB-468, HCC 1806 and HCC 70 treated either with NOHA (1mM) alone or in combination with L-ornithine (0.5mM).B, Real-time quantitative PCR analysis showing selective induction of Arg II and mSHMT mRNA expression in fresh/frozen human breast tumor and matched normal tissues of Arg I, Arg II and mSHMT gene expression. C, Western blot analysis of Arg I, Arg II and mSHMT protein expression in normal and human breast tumor tissues (right panel) and densitometric quantitation analysis of protein expression (left panel). D, Top Panel, Immunohistochemical analysis of paraffin-embedded normal and breast tumor sections; Bottom Panel, Quantitative analysis of Arg II and mSHMT immuno-positive cells using ImagePro software as described in “materials and methods”. E, Time-course of induction of Arg II and mSHMT protein and F, gene expression in xenografts obtained from nude mice after injection of MDA-MB-468 (5x106) cells *, p≤0.05; **, p≤0.01.
Mentions: In order to validate our findings from the DIGE-based proteomics analysis, we analyzed the protein expression of mSHMT in MDA-MB-468 as well as in two other high Arg II expressing HCC1806 and HCC 70 cell lines treated either with NOHA (1mM) alone or in combination with L-ornithine (0.5mM) after 48 hrs. We observed that NOHA treatment led to a significant decrease in mSHMT protein expression compared to controls, but simultaneous treatment of these NOHA-treated cells with L-ornithine effectively blocked this NOHA-induced inhibition of mSHMT in all three cell lines that expressed high levels of Arg II (Figure 4A), but not in MDA-MB-157 cell line that expressed only very low levels of arginase (data not shown).

Bottom Line: Both arginase II (Arg II) and mSHMT expressions were higher in human breast tumor tissues compared to the matched normal and there was a strong correlation between Arg II and mSHMT protein expression.MDA-MB-468 xenografts had significant upregulation of Arg II expression that preceded the induction of mSHMT expression.Small inhibitory RNA (siRNA)-mediated inhibition of Arg II in MDA-MB-468 and HCC-1806 cells led to significant inhibition of both the mSHMT gene and protein expression.

View Article: PubMed Central - PubMed

Affiliation: Internal Medicine, Charles Drew University of Medicine and Science, Los Angeles, California, United States of America ; Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America ; Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America.

ABSTRACT
We have previously reported arginase expression in human breast cancer cells and demonstrated that the inhibition of arginase by N(ω) hydroxy L-arginine (NOHA) in MDA-MB-468 cells induces apoptosis. However, arginase expression and its possible molecular targets in human breast tumor samples and potential clinical implications have not been fully elucidated. Here, we demonstrate arginase expression in human breast tumor samples, and several established breast cancer cell lines, in which NOHA treatment selectively inhibits cell proliferation. The over-expression of Bcl2 in MDA-MB-468 cells abolished NOHA-induced apoptosis, suggesting that the mitochondria may be the main site of NOHA's action. We, therefore, undertook a proteomics approach to identify key mitochondrial targets of arginase in MDA-MB-468 cells. We identified 54 non-mitochondrial and 13 mitochondrial proteins that were differentially expressed in control and NOHA treated groups. Mitochondrial serine hydroxymethyltransferase (mSHMT) was identified as one of the most promising targets of arginase. Both arginase II (Arg II) and mSHMT expressions were higher in human breast tumor tissues compared to the matched normal and there was a strong correlation between Arg II and mSHMT protein expression. MDA-MB-468 xenografts had significant upregulation of Arg II expression that preceded the induction of mSHMT expression. Small inhibitory RNA (siRNA)-mediated inhibition of Arg II in MDA-MB-468 and HCC-1806 cells led to significant inhibition of both the mSHMT gene and protein expression. As mSHMT is a key player in folate metabolism, our data provides a novel link between arginine and folate metabolism in human breast cancer, both of which are critical for tumor cell proliferation.

Show MeSH
Related in: MedlinePlus