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Identification of proteins responsible for adriamycin resistance in breast cancer cells using proteomics analysis.

Wang Z, Liang S, Lian X, Liu L, Zhao S, Xuan Q, Guo L, Liu H, Yang Y, Dong T, Liu Y, Liu Z, Zhang Q - Sci Rep (2015)

Bottom Line: Basing on the human protein-protein interactions (PPI), we have retrieved the associated functional interaction networks for the DEPs and analyzed the biological functions.The identified proteins in biological networks served to resistant drug and to select critical candidates for validation analyses by western blot.The glucose-6-phosphate dehydrogenase (G6PD), gamma-glutamyl cyclotransferase (GGCT), isocitrate dehydrogenase 1 (NADP+,soluble)(IDH1), isocitrate dehydrogenase 2 (NADP+,mitochondrial) (IDH2) and glutathione S-transferase pi 1(GSTP1), five of the critical components of GSH pathway, contribute to chemoresistance.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Medical Oncology, The Third Affiliated Hospital of Harbin Medical University, Haping Road 150 of Nangang District, Harbin 150081, Heilongjiang Province, China [2] Department of Medical Oncology, The Fourth Affiliated Hospital of Harbin Medical University, Harbin, China.

ABSTRACT
Chemoresistance is a poor prognostic factor in breast cancer and is a major obstacle to the successful treatment of patients receiving chemotherapy. However, the precise mechanism of resistance remains unclear. In this study, a pair of breast cancer cell lines, MCF-7 and its adriamycin-resistant counterpart MCF-7/ADR was used to examine resistance-dependent cellular responses and to identify potential therapeutic targets. We applied nanoflow liquid chromatography (nLC) and tandem mass tags (TmT) quantitative mass spectrometry to distinguish the differentially expressed proteins (DEPs) between the two cell lines. Bioinformatics analyses were used to identify functionally active proteins and networks. 80 DEPs were identified with either up- or down-regulation. Basing on the human protein-protein interactions (PPI), we have retrieved the associated functional interaction networks for the DEPs and analyzed the biological functions. Six different signaling pathways and most of the DEPs strongly linked to chemoresistance, invasion, metastasis development, proliferation, and apoptosis. The identified proteins in biological networks served to resistant drug and to select critical candidates for validation analyses by western blot. The glucose-6-phosphate dehydrogenase (G6PD), gamma-glutamyl cyclotransferase (GGCT), isocitrate dehydrogenase 1 (NADP+,soluble)(IDH1), isocitrate dehydrogenase 2 (NADP+,mitochondrial) (IDH2) and glutathione S-transferase pi 1(GSTP1), five of the critical components of GSH pathway, contribute to chemoresistance.

No MeSH data available.


Related in: MedlinePlus

Enzyme-catalyzed reactions in the γ-glutamyl cycle.1: γ-glutamyl cysteine synthetase; 2: glutathione synthetase; 3: γ-glutamyl transpeptidase; 4: GGCT; 5: 5-oxoprolinase.
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f6: Enzyme-catalyzed reactions in the γ-glutamyl cycle.1: γ-glutamyl cysteine synthetase; 2: glutathione synthetase; 3: γ-glutamyl transpeptidase; 4: GGCT; 5: 5-oxoprolinase.

Mentions: The GSH metabolism pathway contributes to the detoxification and elimination of a wide range of xenobiotic compounds, underscoring the role of redox regulation of MDR mediated by drug efflux pumps323334. In several previous studies GGCT, a critical component of the GSH pathway, has been implicated as a cancer marker with a potential role in cell proliferation. Despite the differential expression of GGCT in tumor tissues353637, little is known about the function of GGCT in cancer resistant cells. The γ-glutamyl cycle is a pathway that encompasses the synthesis and degradation of GSH and is thought to contribute to the uptake of amino acids across cellular membranes38. GGCT is a pivotal enzyme that contributes to the γ-glutamyl cycle regulating GSH metabolism through catalyzing the formation of 5-oxoproline (pyroglutamic acid) from γ-glutamyl dipeptides3439. Aaron J Oakley et al. reported39 that the inhibition of GGCT in cases of GSH synthetase deficiency blocks the degradation of γ-glutamylcysteine and allows it to accumulate to a level where it may partially substitute for GSH in redox and detoxification reactions (Fig. 6). There is a significant turnover of GSH and γ-glutamyl-amino acid dipeptides via GGCT and the γ-glutamyl cycle under normal metabolic conditions (Fig. 2 and Fig. 6). The position of GGCT in the γ-glutamyl cycle suggests that it could play a significant role in regulating the synthesis of GSH by limiting the availability of γ-glutamylcysteine. Aaron J Oakley et al.39 also discuss the feedback inhibition of γ-glutamyl cysteine synthetase by GSH. Based on this process, we further hypothesize that GSH expression is modulated by GGCT dependent negative feedback. In our analysis GGCT was found down-regulated in MCF-7/ADR cells (Table 2), while GSH synthetase was not found statistically regulated in the mixed samples. Moreover, the expression of GGCT was also independently shown by observations from western blot analyses, the expression of GGCT was down-regulated in MCF-7/ADR cells compared with MCF-7 cells which supporting proteomic results (Fig. 5). These results suggest that the decreased activity of GGCT is necessary for MCF-7/ADR cells to maintain a high level of GSH, which in turn exports adriamycin out of the cell. Our data provide a novel mechanism for the acquisition of adriamycin resistance.


Identification of proteins responsible for adriamycin resistance in breast cancer cells using proteomics analysis.

Wang Z, Liang S, Lian X, Liu L, Zhao S, Xuan Q, Guo L, Liu H, Yang Y, Dong T, Liu Y, Liu Z, Zhang Q - Sci Rep (2015)

Enzyme-catalyzed reactions in the γ-glutamyl cycle.1: γ-glutamyl cysteine synthetase; 2: glutathione synthetase; 3: γ-glutamyl transpeptidase; 4: GGCT; 5: 5-oxoprolinase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4377623&req=5

f6: Enzyme-catalyzed reactions in the γ-glutamyl cycle.1: γ-glutamyl cysteine synthetase; 2: glutathione synthetase; 3: γ-glutamyl transpeptidase; 4: GGCT; 5: 5-oxoprolinase.
Mentions: The GSH metabolism pathway contributes to the detoxification and elimination of a wide range of xenobiotic compounds, underscoring the role of redox regulation of MDR mediated by drug efflux pumps323334. In several previous studies GGCT, a critical component of the GSH pathway, has been implicated as a cancer marker with a potential role in cell proliferation. Despite the differential expression of GGCT in tumor tissues353637, little is known about the function of GGCT in cancer resistant cells. The γ-glutamyl cycle is a pathway that encompasses the synthesis and degradation of GSH and is thought to contribute to the uptake of amino acids across cellular membranes38. GGCT is a pivotal enzyme that contributes to the γ-glutamyl cycle regulating GSH metabolism through catalyzing the formation of 5-oxoproline (pyroglutamic acid) from γ-glutamyl dipeptides3439. Aaron J Oakley et al. reported39 that the inhibition of GGCT in cases of GSH synthetase deficiency blocks the degradation of γ-glutamylcysteine and allows it to accumulate to a level where it may partially substitute for GSH in redox and detoxification reactions (Fig. 6). There is a significant turnover of GSH and γ-glutamyl-amino acid dipeptides via GGCT and the γ-glutamyl cycle under normal metabolic conditions (Fig. 2 and Fig. 6). The position of GGCT in the γ-glutamyl cycle suggests that it could play a significant role in regulating the synthesis of GSH by limiting the availability of γ-glutamylcysteine. Aaron J Oakley et al.39 also discuss the feedback inhibition of γ-glutamyl cysteine synthetase by GSH. Based on this process, we further hypothesize that GSH expression is modulated by GGCT dependent negative feedback. In our analysis GGCT was found down-regulated in MCF-7/ADR cells (Table 2), while GSH synthetase was not found statistically regulated in the mixed samples. Moreover, the expression of GGCT was also independently shown by observations from western blot analyses, the expression of GGCT was down-regulated in MCF-7/ADR cells compared with MCF-7 cells which supporting proteomic results (Fig. 5). These results suggest that the decreased activity of GGCT is necessary for MCF-7/ADR cells to maintain a high level of GSH, which in turn exports adriamycin out of the cell. Our data provide a novel mechanism for the acquisition of adriamycin resistance.

Bottom Line: Basing on the human protein-protein interactions (PPI), we have retrieved the associated functional interaction networks for the DEPs and analyzed the biological functions.The identified proteins in biological networks served to resistant drug and to select critical candidates for validation analyses by western blot.The glucose-6-phosphate dehydrogenase (G6PD), gamma-glutamyl cyclotransferase (GGCT), isocitrate dehydrogenase 1 (NADP+,soluble)(IDH1), isocitrate dehydrogenase 2 (NADP+,mitochondrial) (IDH2) and glutathione S-transferase pi 1(GSTP1), five of the critical components of GSH pathway, contribute to chemoresistance.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Medical Oncology, The Third Affiliated Hospital of Harbin Medical University, Haping Road 150 of Nangang District, Harbin 150081, Heilongjiang Province, China [2] Department of Medical Oncology, The Fourth Affiliated Hospital of Harbin Medical University, Harbin, China.

ABSTRACT
Chemoresistance is a poor prognostic factor in breast cancer and is a major obstacle to the successful treatment of patients receiving chemotherapy. However, the precise mechanism of resistance remains unclear. In this study, a pair of breast cancer cell lines, MCF-7 and its adriamycin-resistant counterpart MCF-7/ADR was used to examine resistance-dependent cellular responses and to identify potential therapeutic targets. We applied nanoflow liquid chromatography (nLC) and tandem mass tags (TmT) quantitative mass spectrometry to distinguish the differentially expressed proteins (DEPs) between the two cell lines. Bioinformatics analyses were used to identify functionally active proteins and networks. 80 DEPs were identified with either up- or down-regulation. Basing on the human protein-protein interactions (PPI), we have retrieved the associated functional interaction networks for the DEPs and analyzed the biological functions. Six different signaling pathways and most of the DEPs strongly linked to chemoresistance, invasion, metastasis development, proliferation, and apoptosis. The identified proteins in biological networks served to resistant drug and to select critical candidates for validation analyses by western blot. The glucose-6-phosphate dehydrogenase (G6PD), gamma-glutamyl cyclotransferase (GGCT), isocitrate dehydrogenase 1 (NADP+,soluble)(IDH1), isocitrate dehydrogenase 2 (NADP+,mitochondrial) (IDH2) and glutathione S-transferase pi 1(GSTP1), five of the critical components of GSH pathway, contribute to chemoresistance.

No MeSH data available.


Related in: MedlinePlus