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A MALDI-MS-based quantitative analytical method for endogenous estrone in human breast cancer cells.

Kim KJ, Kim HJ, Park HG, Hwang CH, Sung C, Jang KS, Park SH, Kim BG, Lee YK, Yang YH, Jeong JH, Kim YG - Sci Rep (2016)

Bottom Line: The one-step modification of endogenous estrone provided good linearity (R(2) > 0.99) and significantly increased the sensitivity of the platform (limit of quantitation: 11 fmol).Finally, by applying the MALDI-MS-based quantitative method to endogenous estrones, we successfully monitored changes in the metabolic expression level of estrones (17.7 fmol/10(6) letrozole-treated cells) in MCF-7 cells resulting from treatment with an aromatase inhibitor.Taken together, these results suggest that this MALDI-MS-based quantitative approach may be a general method for the targeted metabolomics of ketone-containing metabolites, which can reflect clinical conditions and pathogenic mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Soongsil University, Seoul 156-743, Korea.

ABSTRACT
The level of endogenous estrone, one of the three major naturally occurring estrogens, has a significant correlation with the incidence of post-menopausal breast cancer. However, it is challenging to quantitatively monitor it owing to its low abundance. Here, we develop a robust and highly sensitive mass-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based quantitative platform to identify the absolute quantities of endogenous estrones in a variety of clinical specimens. The one-step modification of endogenous estrone provided good linearity (R(2) > 0.99) and significantly increased the sensitivity of the platform (limit of quantitation: 11 fmol). In addition, we could identify the absolute amount of endogenous estrones in cells of the breast cancer cell line MCF-7 (34 fmol/10(6) cells) by using a deuterated estrone as an internal standard. Finally, by applying the MALDI-MS-based quantitative method to endogenous estrones, we successfully monitored changes in the metabolic expression level of estrones (17.7 fmol/10(6) letrozole-treated cells) in MCF-7 cells resulting from treatment with an aromatase inhibitor. Taken together, these results suggest that this MALDI-MS-based quantitative approach may be a general method for the targeted metabolomics of ketone-containing metabolites, which can reflect clinical conditions and pathogenic mechanisms.

No MeSH data available.


Related in: MedlinePlus

Bar graphs showing the decrease in endogenous estrone level in MCF-7 cells in response to the letrozole treatment.The intensities of GP-E1 from natural and letrozole-treated MCF-7 cells (each 106 cells) correspond to 34 fmol and 17.7 fmol of estrone, respectively (***P value < 0.001; P values were derived from the two-tailed Student’s t test, n = 3; different MCF-7 cells/letrozole-treated MCF-7 cells. Error bars show SEM.).
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f5: Bar graphs showing the decrease in endogenous estrone level in MCF-7 cells in response to the letrozole treatment.The intensities of GP-E1 from natural and letrozole-treated MCF-7 cells (each 106 cells) correspond to 34 fmol and 17.7 fmol of estrone, respectively (***P value < 0.001; P values were derived from the two-tailed Student’s t test, n = 3; different MCF-7 cells/letrozole-treated MCF-7 cells. Error bars show SEM.).

Mentions: Our MALDI-MS-based method was used to quantify endogenous estrone in cultured MCF-7 cells, a breast cancer cell line. After the lysis of the cells, d4-estrones were spiked for absolute quantitation of the endogenous estrones. The extracted estrones were labeled with GP and subsequently analyzed using our MALDI-MS-based method. Based on the peak intensity of d4-estrone (44 fmol), the amount of endogenous estrone was determined to be 34 fmol in 106 MCF-7 cells (Figs 5, S4 and S5), which is comparable to a previous report (29 fmol estrone) and is physiologically realistic3940. In addition, we would like to monitor changes in the levels of endogenous estrones. Letrozole has been effectively used as a medical therapy for hormonally responsive breast cancers because it can inhibit aromatase activity and thereby regulate endogenous estrone levels. The inhibitor was added to the cell culture, and then the cells were collected to quantify the endogenous estrone. As we expected, letrozole significantly decreased the absolute amount of estrone in the MCF-7 cells (Figs 5 and S5). An MTT assay was used to investigate the cytotoxic effects of letrozole on MCF-7 cells. As shown in Fig. S6A, no significant changes in the viability of cells were observed following treatment with less than 20 μM of letrozole. Treatment with 20 μM of letrozole for 48 h also does not affect the monolayered morphology of the cells (Fig. S6). Therefore, this treatment condition (i.e., 20 μM of letrozole for 48 h) was used in following investigations. This result demonstrates that the MALDI-MS-based estrone quantitation method may be applicable for diagnosing and monitoring hormone-related diseases, such as breast cancers.


A MALDI-MS-based quantitative analytical method for endogenous estrone in human breast cancer cells.

Kim KJ, Kim HJ, Park HG, Hwang CH, Sung C, Jang KS, Park SH, Kim BG, Lee YK, Yang YH, Jeong JH, Kim YG - Sci Rep (2016)

Bar graphs showing the decrease in endogenous estrone level in MCF-7 cells in response to the letrozole treatment.The intensities of GP-E1 from natural and letrozole-treated MCF-7 cells (each 106 cells) correspond to 34 fmol and 17.7 fmol of estrone, respectively (***P value < 0.001; P values were derived from the two-tailed Student’s t test, n = 3; different MCF-7 cells/letrozole-treated MCF-7 cells. Error bars show SEM.).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836303&req=5

f5: Bar graphs showing the decrease in endogenous estrone level in MCF-7 cells in response to the letrozole treatment.The intensities of GP-E1 from natural and letrozole-treated MCF-7 cells (each 106 cells) correspond to 34 fmol and 17.7 fmol of estrone, respectively (***P value < 0.001; P values were derived from the two-tailed Student’s t test, n = 3; different MCF-7 cells/letrozole-treated MCF-7 cells. Error bars show SEM.).
Mentions: Our MALDI-MS-based method was used to quantify endogenous estrone in cultured MCF-7 cells, a breast cancer cell line. After the lysis of the cells, d4-estrones were spiked for absolute quantitation of the endogenous estrones. The extracted estrones were labeled with GP and subsequently analyzed using our MALDI-MS-based method. Based on the peak intensity of d4-estrone (44 fmol), the amount of endogenous estrone was determined to be 34 fmol in 106 MCF-7 cells (Figs 5, S4 and S5), which is comparable to a previous report (29 fmol estrone) and is physiologically realistic3940. In addition, we would like to monitor changes in the levels of endogenous estrones. Letrozole has been effectively used as a medical therapy for hormonally responsive breast cancers because it can inhibit aromatase activity and thereby regulate endogenous estrone levels. The inhibitor was added to the cell culture, and then the cells were collected to quantify the endogenous estrone. As we expected, letrozole significantly decreased the absolute amount of estrone in the MCF-7 cells (Figs 5 and S5). An MTT assay was used to investigate the cytotoxic effects of letrozole on MCF-7 cells. As shown in Fig. S6A, no significant changes in the viability of cells were observed following treatment with less than 20 μM of letrozole. Treatment with 20 μM of letrozole for 48 h also does not affect the monolayered morphology of the cells (Fig. S6). Therefore, this treatment condition (i.e., 20 μM of letrozole for 48 h) was used in following investigations. This result demonstrates that the MALDI-MS-based estrone quantitation method may be applicable for diagnosing and monitoring hormone-related diseases, such as breast cancers.

Bottom Line: The one-step modification of endogenous estrone provided good linearity (R(2) > 0.99) and significantly increased the sensitivity of the platform (limit of quantitation: 11 fmol).Finally, by applying the MALDI-MS-based quantitative method to endogenous estrones, we successfully monitored changes in the metabolic expression level of estrones (17.7 fmol/10(6) letrozole-treated cells) in MCF-7 cells resulting from treatment with an aromatase inhibitor.Taken together, these results suggest that this MALDI-MS-based quantitative approach may be a general method for the targeted metabolomics of ketone-containing metabolites, which can reflect clinical conditions and pathogenic mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Soongsil University, Seoul 156-743, Korea.

ABSTRACT
The level of endogenous estrone, one of the three major naturally occurring estrogens, has a significant correlation with the incidence of post-menopausal breast cancer. However, it is challenging to quantitatively monitor it owing to its low abundance. Here, we develop a robust and highly sensitive mass-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based quantitative platform to identify the absolute quantities of endogenous estrones in a variety of clinical specimens. The one-step modification of endogenous estrone provided good linearity (R(2) > 0.99) and significantly increased the sensitivity of the platform (limit of quantitation: 11 fmol). In addition, we could identify the absolute amount of endogenous estrones in cells of the breast cancer cell line MCF-7 (34 fmol/10(6) cells) by using a deuterated estrone as an internal standard. Finally, by applying the MALDI-MS-based quantitative method to endogenous estrones, we successfully monitored changes in the metabolic expression level of estrones (17.7 fmol/10(6) letrozole-treated cells) in MCF-7 cells resulting from treatment with an aromatase inhibitor. Taken together, these results suggest that this MALDI-MS-based quantitative approach may be a general method for the targeted metabolomics of ketone-containing metabolites, which can reflect clinical conditions and pathogenic mechanisms.

No MeSH data available.


Related in: MedlinePlus