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      17-allyamino-17-demethoxygeldanamycin treatment results in a magnetic resonance spectroscopy-detectable elevation in choline-containing metabolites associated with increased expression of choline transporter SLC44A1 and phospholipase A2

      research-article
      1 , 1 , 1 ,
      Breast Cancer Research : BCR
      BioMed Central

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          Abstract

          Introduction

          17-allyamino-17-demethoxygeldanamycin (17-AAG), a small molecule inhibitor of Hsp90, is currently in clinical trials in breast cancer. However, 17-AAG treatment often results in inhibition of tumor growth rather than shrinkage, making detection of response a challenge. Magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) are noninvasive imaging methods than can be used to monitor metabolic biomarkers of drug-target modulation. This study set out to examine the MRS-detectable metabolic consequences of Hsp90 inhibition in a breast cancer model.

          Methods

          MCF-7 breast cancer cells were investigated, and MRS studies were performed both on live cells and on cell extracts. 31P and 1H MRS were used to determine total cellular metabolite concentrations and 13C MRS was used to probe the metabolism of [1,2- 13C]-choline. To explain the MRS metabolic findings, microarray and RT-PCR were used to analyze gene expression, and in vitro activity assays were performed to determine changes in enzymatic activity following 17-AAG treatment.

          Results

          Treatment of MCF-7 cells with 17-AAG for 48 hours caused a significant increase in intracellular levels of choline (to 266 ± 18% of control, P = 0.05) and phosphocholine (PC; to 181 ± 10% of control, P = 0.001) associated with an increase in expression of choline transporter SLC44A1 and an elevation in the de novo synthesis of PC. We also detected an increase in intracellular levels of glycerophosphocholine (GPC; to 176 ± 38% of control, P = 0.03) associated with an increase in PLA2 expression and activity.

          Conclusions

          This study determined that in the MCF-7 breast cancer model inhibition of Hsp90 by 17-AAG results in a significant MRS-detectable increase in choline, PC and GPC, which is likely due to an increase in choline transport into the cell and phospholipase activation. 1H MRSI can be used in the clinical setting to detect levels of total choline-containing metabolite (t-Cho, composed of intracellular choline, PC and GPC). As Hsp90 inhibitors enter routine clinical use, t-Cho could thus provide an easily detectable, noninvasive metabolic biomarker of Hsp90 inhibition in breast cancer patients.

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          Most cited references56

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          Malignant transformation alters membrane choline phospholipid metabolism of human mammary epithelial cells.

          Transduction of mitogenic signals in cells can be mediated by molecules derived from the synthesis and breakdown of the major membrane phospholipid, phosphotidylcholine. Studies were performed on human mammary epithelial cells in culture to understand the impact of malignant transformation and progression on membrane phospholipid metabolism. In the model system used here, phosphocholine levels and total choline-containing phospholipid metabolite levels increased with progression from normal to immortalized to oncogene-transformed to tumor-derived cells. These changes occurred independently of cell doubling time. A "glycerophosphocholine to phosphocholine switch" was apparent with immortalization. This alteration in phenotype of increased phosphocholine relative to glycerophosphocholine was observed in oncogene-transformed and for all human breast tumor cell lines analyzed. The results demonstrate that progression of human mammary epithelial cells from normal to malignant phenotype is associated with altered membrane choline phospholipid metabolism.
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            Hsp90 and co-chaperones twist the functions of diverse client proteins.

            Hsp90 molecular chaperones are required for the stability and activity of a diverse range of client proteins that have critical roles in signal transduction, cellular trafficking, chromatin remodeling, cell growth, differentiation, and reproduction. Mammalian cells contain three types of Hsp90s: cytosolic Hsp90, mitochondrial Trap-1, and Grp94 of the endoplasmic reticulum. Each of the Hsp90s, as well as the bacterial homolog, HtpG, hydrolyze ATP and undergo similar conformational changes. Unlike the other forms of Hsp90, cytosolic Hsp90 function is dependent on a battery of co-chaperone proteins that regulate the ATPase activity of Hsp90 or direct Hsp90 to interact with specific client proteins. This review will summarize what is known about Hsp90's ability to mediate the folding and activation of diverse client proteins that contribute to human diseases, such as cancer and fungal and viral infections. (c) 2009 Wiley Periodicals, Inc.
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              Alterations of choline phospholipid metabolism in ovarian tumor progression.

              Recent characterization of abnormal phosphatidylcholine metabolism in tumor cells by nuclear magnetic resonance (NMR) has identified novel fingerprints of tumor progression that are potentially useful as clinical diagnostic indicators. In the present study, we analyzed the concentrations of phosphatidylcholine metabolites, activities of phosphocholine-producing enzymes, and uptake of [methyl-14C]choline in human epithelial ovarian carcinoma cell lines (EOC) compared with normal or immortalized ovary epithelial cells (EONT). Quantification of phosphatidylcholine metabolites contributing to the 1H NMR total choline resonance (3.20-3.24 ppm) revealed intracellular [phosphocholine] and [total choline] of 2.3 +/- 0.9 and 5.2 +/- 2.4 nmol/10(6) cells, respectively, with a glycerophosphocholine/phosphocholine ratio of 0.95 +/- 0.93 in EONT cells; average [phosphocholine] was 3- to 8-fold higher in EOC cells (P < 0.0001), becoming the predominant phosphatidylcholine metabolite, whereas average glycerophosphocholine/phosphocholine values decreased significantly to < or =0.2. Two-dimensional (phosphocholine/total choline, [total choline]) and (glycerophosphocholine/total choline, [total choline]) maps allowed separate clustering of EOC from EONT cells (P < 0.0001, 95% confidence limits). Rates of choline kinase activity in EOC cells were 12- to 24-fold higher (P < 0.03) than those in EONT cells (basal rate, 0.5 +/- 0.1 nmol/10(6) cells/h), accounting for a consistently elevated (5- to 15-fold) [methyl-14C]choline uptake after 1-hour incubation (P < 0.0001). The overall activity of phosphatidylcholine-specific phospholipase C and phospholipase D was also higher ( approximately 5-fold) in EOC cells, suggesting that both biosynthetic and catabolic pathways of the phosphatidylcholine cycle likely contribute to phosphocholine accumulation. Evidence of abnormal phosphatidylcholine metabolism might have implications in EOC biology and might provide an avenue to the development of noninvasive clinical tools for EOC diagnosis and treatment follow-up.
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                Author and article information

                Journal
                Breast Cancer Res
                Breast Cancer Research : BCR
                BioMed Central
                1465-5411
                1465-542X
                2010
                14 October 2010
                : 12
                : 5
                : R84
                Affiliations
                [1 ]Department of Radiology and Biomedical Imaging, University of California, San Francisco, 1700 4th Street, San Francisco, CA 94158, USA
                Article
                bcr2729
                10.1186/bcr2729
                3096977
                20946630
                687419c7-2142-4e9e-9c5e-eded063963a6
                Copyright ©2010 Brandes et al.; licensee BioMed Central Ltd

                This is an open access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

                History
                : 23 July 2010
                : 29 September 2010
                : 14 October 2010
                Categories
                Research Article

                Oncology & Radiotherapy
                Oncology & Radiotherapy

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