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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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Inhibition of NOHA-induced apoptosis in Bcl2 over-expressing MDA-MB-468 (MDA-MB-468/Bcl2) cells.A, Control MDA-MB-468 or MDA-MB-468/Bcl2 cells were treated either with vehicle or NOHA (1mM) for 48 hrs, stained with propidium iodide and cell cycle analysis was performed. B, 75µg of total protein lysates were electrophoresed on 4-15% SDS/PAGE, transferred to PVDF membrane and analysis of proteolytic cleavage of caspase-3 in various treatments was performed by western blot analysis. C, Caspase-3 enzymatic activity in MDA-MB-468 (left panel) and MDA-MB-468/Bcl2 cells (right panel) treated with NOHA (1mM) for various time-points were analyzed. *, p≤ 0.05; **, p≤0.01.
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pone-0079242-g002: Inhibition of NOHA-induced apoptosis in Bcl2 over-expressing MDA-MB-468 (MDA-MB-468/Bcl2) cells.A, Control MDA-MB-468 or MDA-MB-468/Bcl2 cells were treated either with vehicle or NOHA (1mM) for 48 hrs, stained with propidium iodide and cell cycle analysis was performed. B, 75µg of total protein lysates were electrophoresed on 4-15% SDS/PAGE, transferred to PVDF membrane and analysis of proteolytic cleavage of caspase-3 in various treatments was performed by western blot analysis. C, Caspase-3 enzymatic activity in MDA-MB-468 (left panel) and MDA-MB-468/Bcl2 cells (right panel) treated with NOHA (1mM) for various time-points were analyzed. *, p≤ 0.05; **, p≤0.01.

Mentions: The purpose of this experiment was to test whether the mitochondrion is the main target of NOHA-induced apoptosis in human breast cancer cells as suggested previously [7]. Since Bcl2 is a well-established anti-apoptotic protein that is known to block the release of cytochrome c from mitochondria and antagonize apoptotic process, we sought to test whether over-expression of this protein could block NOHA-induced apoptosis. We found that MDA-MB-468 cells stably expressing full-length human Bcl2 (MDA-MB-468/Bcl2) were able to antagonize the NOHA-induced increase in sub-Go population (an indicator of cell death) which occurred in control MDA-MB-468 cells (Figure 2A). We also found a significant increase in caspase-3 proteolytic cleavage (Figure 2B) and caspase-3 enzyme activities at 32 hrs (5.04±0.62 fold) and 48 hrs (11.07±1.22 fold) in control MDA-MB-468 cells after NOHA treatment as expected. However, these increases in apoptotic parameters in NOHA-treated cells were abolished in MDA-MB-468/Bcl2 cells (Figure 2B-C). Our data therefore, provides additional mechanistic insight of possible involvement of mitochondria-dependent pathways that are responsible for apoptosis induction after inhibition of arginase by NOHA in high arginase expressing MDA-MB-468 cells.


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)

Inhibition of NOHA-induced apoptosis in Bcl2 over-expressing MDA-MB-468 (MDA-MB-468/Bcl2) cells.A, Control MDA-MB-468 or MDA-MB-468/Bcl2 cells were treated either with vehicle or NOHA (1mM) for 48 hrs, stained with propidium iodide and cell cycle analysis was performed. B, 75µg of total protein lysates were electrophoresed on 4-15% SDS/PAGE, transferred to PVDF membrane and analysis of proteolytic cleavage of caspase-3 in various treatments was performed by western blot analysis. C, Caspase-3 enzymatic activity in MDA-MB-468 (left panel) and MDA-MB-468/Bcl2 cells (right panel) treated with NOHA (1mM) for various time-points were analyzed. *, p≤ 0.05; **, p≤0.01.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0079242-g002: Inhibition of NOHA-induced apoptosis in Bcl2 over-expressing MDA-MB-468 (MDA-MB-468/Bcl2) cells.A, Control MDA-MB-468 or MDA-MB-468/Bcl2 cells were treated either with vehicle or NOHA (1mM) for 48 hrs, stained with propidium iodide and cell cycle analysis was performed. B, 75µg of total protein lysates were electrophoresed on 4-15% SDS/PAGE, transferred to PVDF membrane and analysis of proteolytic cleavage of caspase-3 in various treatments was performed by western blot analysis. C, Caspase-3 enzymatic activity in MDA-MB-468 (left panel) and MDA-MB-468/Bcl2 cells (right panel) treated with NOHA (1mM) for various time-points were analyzed. *, p≤ 0.05; **, p≤0.01.
Mentions: The purpose of this experiment was to test whether the mitochondrion is the main target of NOHA-induced apoptosis in human breast cancer cells as suggested previously [7]. Since Bcl2 is a well-established anti-apoptotic protein that is known to block the release of cytochrome c from mitochondria and antagonize apoptotic process, we sought to test whether over-expression of this protein could block NOHA-induced apoptosis. We found that MDA-MB-468 cells stably expressing full-length human Bcl2 (MDA-MB-468/Bcl2) were able to antagonize the NOHA-induced increase in sub-Go population (an indicator of cell death) which occurred in control MDA-MB-468 cells (Figure 2A). We also found a significant increase in caspase-3 proteolytic cleavage (Figure 2B) and caspase-3 enzyme activities at 32 hrs (5.04±0.62 fold) and 48 hrs (11.07±1.22 fold) in control MDA-MB-468 cells after NOHA treatment as expected. However, these increases in apoptotic parameters in NOHA-treated cells were abolished in MDA-MB-468/Bcl2 cells (Figure 2B-C). Our data therefore, provides additional mechanistic insight of possible involvement of mitochondria-dependent pathways that are responsible for apoptosis induction after inhibition of arginase by NOHA in high arginase expressing MDA-MB-468 cells.

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