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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.

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Analysis of Arg I and Arg II in human breast tumor specimen and established breast cancer cells.A, 50 µg of breast tumor lysates (1-36) obtained from CHTN and NDRI were analyzed by Western blot analysis using anti-Arg I or anti-Arg II antibodies. B, 50 µg of cell lysates obtained from established breast cancer cells were analyzed by Western blot analysis using anti-Arg I or anti-Arg II antibodies. C, Selective Inhibition of cell proliferation by NOHA (1mM) in high Arg II expressing breast cancer MDA-MB-468, HCC1806, HCC 70 and MDA-MB-157 cells and blockade of this effect by exogenous L-ornithine (L-Orn) (500µM). *, p≤0.05; **,p≤0.01.
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pone-0079242-g001: Analysis of Arg I and Arg II in human breast tumor specimen and established breast cancer cells.A, 50 µg of breast tumor lysates (1-36) obtained from CHTN and NDRI were analyzed by Western blot analysis using anti-Arg I or anti-Arg II antibodies. B, 50 µg of cell lysates obtained from established breast cancer cells were analyzed by Western blot analysis using anti-Arg I or anti-Arg II antibodies. C, Selective Inhibition of cell proliferation by NOHA (1mM) in high Arg II expressing breast cancer MDA-MB-468, HCC1806, HCC 70 and MDA-MB-157 cells and blockade of this effect by exogenous L-ornithine (L-Orn) (500µM). *, p≤0.05; **,p≤0.01.

Mentions: We have previously demonstrated high levels of arginase expression in some selected human breast cancer cell lines, which are highly sensitive to NOHA treatment, an arginase inhibitor. In our present study, we analyze the expression of both Arg I and Arg II in human breast tumor samples obtained from various breast tumor patients as well as in some additional breast cancer cells that were not investigated before. We observed that 29 out of 36 human breast tumors expressed Arg II but only 18 out of 36 tumors expressed Arg I. The overall expression levels of Arg II was significantly higher compared to the Arg I protein expression levels (Figure 1A). Analysis of 9 human breast cancer cell lines demonstrated very high levels of Arg II expression in at least 6 cells lines (Figure 1B), including MDA-MB-468 which was previously reported to express high levels of Arg II [2]. Our current data validates our previous reports that Arg II is the predominant isoform expressed in breast cancer cells, and is also abundantly present in human breast tumor tissues obtained from both estrogen receptor positive (ER+) as well as from triple negative (TN) tumors. While Arg II was expressed in 78% of the samples, Arg I was expressed in only 47% of these samples at a lower level.


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)

Analysis of Arg I and Arg II in human breast tumor specimen and established breast cancer cells.A, 50 µg of breast tumor lysates (1-36) obtained from CHTN and NDRI were analyzed by Western blot analysis using anti-Arg I or anti-Arg II antibodies. B, 50 µg of cell lysates obtained from established breast cancer cells were analyzed by Western blot analysis using anti-Arg I or anti-Arg II antibodies. C, Selective Inhibition of cell proliferation by NOHA (1mM) in high Arg II expressing breast cancer MDA-MB-468, HCC1806, HCC 70 and MDA-MB-157 cells and blockade of this effect by exogenous L-ornithine (L-Orn) (500µM). *, p≤0.05; **,p≤0.01.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0079242-g001: Analysis of Arg I and Arg II in human breast tumor specimen and established breast cancer cells.A, 50 µg of breast tumor lysates (1-36) obtained from CHTN and NDRI were analyzed by Western blot analysis using anti-Arg I or anti-Arg II antibodies. B, 50 µg of cell lysates obtained from established breast cancer cells were analyzed by Western blot analysis using anti-Arg I or anti-Arg II antibodies. C, Selective Inhibition of cell proliferation by NOHA (1mM) in high Arg II expressing breast cancer MDA-MB-468, HCC1806, HCC 70 and MDA-MB-157 cells and blockade of this effect by exogenous L-ornithine (L-Orn) (500µM). *, p≤0.05; **,p≤0.01.
Mentions: We have previously demonstrated high levels of arginase expression in some selected human breast cancer cell lines, which are highly sensitive to NOHA treatment, an arginase inhibitor. In our present study, we analyze the expression of both Arg I and Arg II in human breast tumor samples obtained from various breast tumor patients as well as in some additional breast cancer cells that were not investigated before. We observed that 29 out of 36 human breast tumors expressed Arg II but only 18 out of 36 tumors expressed Arg I. The overall expression levels of Arg II was significantly higher compared to the Arg I protein expression levels (Figure 1A). Analysis of 9 human breast cancer cell lines demonstrated very high levels of Arg II expression in at least 6 cells lines (Figure 1B), including MDA-MB-468 which was previously reported to express high levels of Arg II [2]. Our current data validates our previous reports that Arg II is the predominant isoform expressed in breast cancer cells, and is also abundantly present in human breast tumor tissues obtained from both estrogen receptor positive (ER+) as well as from triple negative (TN) tumors. While Arg II was expressed in 78% of the samples, Arg I was expressed in only 47% of these samples at a lower level.

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