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Midostaurin preferentially attenuates proliferation of triple-negative breast cancer cell lines through inhibition of Aurora kinase family.

Kawai M, Nakashima A, Kamada S, Kikkawa U - J. Biomed. Sci. (2015)

Bottom Line: It is, thus, required to develop an effective therapeutic reagent to treat TNBC.Following studies indicated that midostaurin attenuates the phosphorylation reaction mediated by Aurora kinase in the cells and directly inhibits this protein kinase in vitro, and that this reagent induces apoptosis accompanying accumulation of 4N and 8N DNA cells in TNBC cells.The precise study of midostaurin on cell growth will contribute to the development of the drug for the treatment of TNBC.

View Article: PubMed Central - PubMed

Affiliation: Biosignal Research Center, Kobe University, Kobe, 657-8501, Japan. 090s381s@stu.kobe-u.ac.jp.

ABSTRACT

Background: Breast cancer is classified into three subtypes by the expression of biomarker receptors such as hormone receptors and human epidermal growth factor receptor 2. Triple-negative breast cancer (TNBC) expresses none of these receptors and has an aggressive phenotype with a poor prognosis, which is insensitive to the drugs that target the hormone receptors and human epidermal growth factor receptor 2. It is, thus, required to develop an effective therapeutic reagent to treat TNBC.

Results: The study using a panel of 19 breast cancer cell lines revealed that midostaurin, a multi-target protein kinase inhibitor, suppresses preferentially the growth of TNBC cells comparing with non-TNBC cells. Clustering analysis of the drug activity data for the panel of cancer cell lines predicted that midostaurin shares the target with Aurora kinase inhibitors. Following studies indicated that midostaurin attenuates the phosphorylation reaction mediated by Aurora kinase in the cells and directly inhibits this protein kinase in vitro, and that this reagent induces apoptosis accompanying accumulation of 4N and 8N DNA cells in TNBC cells.

Conclusion: Midostaurin suppresses the proliferation of TNBC cells among the breast cancer cell lines presumably through the inhibition of the Aurora kinase family. The precise study of midostaurin on cell growth will contribute to the development of the drug for the treatment of TNBC.

No MeSH data available.


Related in: MedlinePlus

Growth inhibition of breast cancer cell lines by midostaurin. The 19 breast cancer cell lines were treated with 1 μM midostaurin for 72 h, and cell viability was measured. a Cell viability shown as a ratio relative to the control sample without treatment. Bars are 1 s.d. of quintuple experiments. Breast cancer subtypes are indicated as follows: gray, TNBC; light gray, HER2; white, ER+. b Box plot showing relative viability according to breast cancer subtypes. TNBC vs. non-TNBC, p = 0.00075 by Welch’s t-test
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Fig1: Growth inhibition of breast cancer cell lines by midostaurin. The 19 breast cancer cell lines were treated with 1 μM midostaurin for 72 h, and cell viability was measured. a Cell viability shown as a ratio relative to the control sample without treatment. Bars are 1 s.d. of quintuple experiments. Breast cancer subtypes are indicated as follows: gray, TNBC; light gray, HER2; white, ER+. b Box plot showing relative viability according to breast cancer subtypes. TNBC vs. non-TNBC, p = 0.00075 by Welch’s t-test

Mentions: A panel of 19 cell lines, representing three subtypes of human breast cancer, 3 of ER+, 7 of HER2, and 9 of TNBC, were treated with different concentrations of midostaurin, and cell viability was measured (Additional file 2). The effect of midostaurin differed among the cell lines, and thus the viability was compared at 1 μM (Fig. 1a), because the plasma concentrations of the drug in clinical trial for AML have been reported to be a few μM [9]. The TNBC cells except for one line were more sensitive to midostaurin than non-TNBC subtypes such as ER+ and HER2 cells (Fig. 1a): the mean viability values of TNBC and non-TNBC cell lines were 0.53 and 0.91, respectively. The difference between TNBC and non-TNBC subtypes was shown by box plot and was statistically significant (Fig. 1b). The effect of midostaurin on cell death was examined by measuring the cleavage of PARP, as a marker of apoptosis (Fig. 2). In consistent with the result of cell viability, midostaurin brought the cleavage of PARP in TNBC cell lines, BT-20 and MDA-MB-468, but the fragment was not detected in non-TNBC cell lines, BT-474 and HCC1419. These results indicate that midostaurin induces apoptosis preferentially in TNBC cells. Midostaurin was initially generated as a PKC inhibitor [6], and the expression level of the PKC isoforms was evaluated in the breast cancer cell lines by Western blot analysis. PKC isoforms were detected in the breast cancer cell lines such as PKC-α and PKC-βII of the conventional PKC group as well as PKC-δ and PKC-ε of the novel PKC group (Additional file 3). Midostaurin suppressed the PKC-mediated protein phosphorylation as judged by Western blot analysis using the p-Serine PKC substrates antibody in MDA-MB-468 cell line (Additional file 4). The correlation of the expression level of the PKC isoforms with the TNBC cell lines was, however, not observed. On the other hand, it is well known that TNBC cancer cells frequently express EGF receptor although other two subtypes do not [28]. Therefore, the effect of midostaurin was examined on the phosphorylation of EGF receptor and its downstream EGF signaling mechanisms including Akt and Erk kinases. While the treatment of midostaurin at 1 μM induced apoptosis by 24 h as judged by the cleavage of PARP, no significant suppression of the phosphorylation of EGFR (p-EGFR Tyr1068), GSK-3β (p-GSK-3β Ser9), and Erk (p-Erk Thr202/Thr204) was observed during the period (Additional file 4). In addition, lapatinib, a potent inhibitor of the EGF receptor kinase, did not suppress viability of MDA-MB-468 cells, as described previously [29], or enhance the effect of midostaurin (data not shown). Namely, these observations indicate that midostaurin does not target EGF receptor in the TNBC cells.Fig. 1


Midostaurin preferentially attenuates proliferation of triple-negative breast cancer cell lines through inhibition of Aurora kinase family.

Kawai M, Nakashima A, Kamada S, Kikkawa U - J. Biomed. Sci. (2015)

Growth inhibition of breast cancer cell lines by midostaurin. The 19 breast cancer cell lines were treated with 1 μM midostaurin for 72 h, and cell viability was measured. a Cell viability shown as a ratio relative to the control sample without treatment. Bars are 1 s.d. of quintuple experiments. Breast cancer subtypes are indicated as follows: gray, TNBC; light gray, HER2; white, ER+. b Box plot showing relative viability according to breast cancer subtypes. TNBC vs. non-TNBC, p = 0.00075 by Welch’s t-test
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4491224&req=5

Fig1: Growth inhibition of breast cancer cell lines by midostaurin. The 19 breast cancer cell lines were treated with 1 μM midostaurin for 72 h, and cell viability was measured. a Cell viability shown as a ratio relative to the control sample without treatment. Bars are 1 s.d. of quintuple experiments. Breast cancer subtypes are indicated as follows: gray, TNBC; light gray, HER2; white, ER+. b Box plot showing relative viability according to breast cancer subtypes. TNBC vs. non-TNBC, p = 0.00075 by Welch’s t-test
Mentions: A panel of 19 cell lines, representing three subtypes of human breast cancer, 3 of ER+, 7 of HER2, and 9 of TNBC, were treated with different concentrations of midostaurin, and cell viability was measured (Additional file 2). The effect of midostaurin differed among the cell lines, and thus the viability was compared at 1 μM (Fig. 1a), because the plasma concentrations of the drug in clinical trial for AML have been reported to be a few μM [9]. The TNBC cells except for one line were more sensitive to midostaurin than non-TNBC subtypes such as ER+ and HER2 cells (Fig. 1a): the mean viability values of TNBC and non-TNBC cell lines were 0.53 and 0.91, respectively. The difference between TNBC and non-TNBC subtypes was shown by box plot and was statistically significant (Fig. 1b). The effect of midostaurin on cell death was examined by measuring the cleavage of PARP, as a marker of apoptosis (Fig. 2). In consistent with the result of cell viability, midostaurin brought the cleavage of PARP in TNBC cell lines, BT-20 and MDA-MB-468, but the fragment was not detected in non-TNBC cell lines, BT-474 and HCC1419. These results indicate that midostaurin induces apoptosis preferentially in TNBC cells. Midostaurin was initially generated as a PKC inhibitor [6], and the expression level of the PKC isoforms was evaluated in the breast cancer cell lines by Western blot analysis. PKC isoforms were detected in the breast cancer cell lines such as PKC-α and PKC-βII of the conventional PKC group as well as PKC-δ and PKC-ε of the novel PKC group (Additional file 3). Midostaurin suppressed the PKC-mediated protein phosphorylation as judged by Western blot analysis using the p-Serine PKC substrates antibody in MDA-MB-468 cell line (Additional file 4). The correlation of the expression level of the PKC isoforms with the TNBC cell lines was, however, not observed. On the other hand, it is well known that TNBC cancer cells frequently express EGF receptor although other two subtypes do not [28]. Therefore, the effect of midostaurin was examined on the phosphorylation of EGF receptor and its downstream EGF signaling mechanisms including Akt and Erk kinases. While the treatment of midostaurin at 1 μM induced apoptosis by 24 h as judged by the cleavage of PARP, no significant suppression of the phosphorylation of EGFR (p-EGFR Tyr1068), GSK-3β (p-GSK-3β Ser9), and Erk (p-Erk Thr202/Thr204) was observed during the period (Additional file 4). In addition, lapatinib, a potent inhibitor of the EGF receptor kinase, did not suppress viability of MDA-MB-468 cells, as described previously [29], or enhance the effect of midostaurin (data not shown). Namely, these observations indicate that midostaurin does not target EGF receptor in the TNBC cells.Fig. 1

Bottom Line: It is, thus, required to develop an effective therapeutic reagent to treat TNBC.Following studies indicated that midostaurin attenuates the phosphorylation reaction mediated by Aurora kinase in the cells and directly inhibits this protein kinase in vitro, and that this reagent induces apoptosis accompanying accumulation of 4N and 8N DNA cells in TNBC cells.The precise study of midostaurin on cell growth will contribute to the development of the drug for the treatment of TNBC.

View Article: PubMed Central - PubMed

Affiliation: Biosignal Research Center, Kobe University, Kobe, 657-8501, Japan. 090s381s@stu.kobe-u.ac.jp.

ABSTRACT

Background: Breast cancer is classified into three subtypes by the expression of biomarker receptors such as hormone receptors and human epidermal growth factor receptor 2. Triple-negative breast cancer (TNBC) expresses none of these receptors and has an aggressive phenotype with a poor prognosis, which is insensitive to the drugs that target the hormone receptors and human epidermal growth factor receptor 2. It is, thus, required to develop an effective therapeutic reagent to treat TNBC.

Results: The study using a panel of 19 breast cancer cell lines revealed that midostaurin, a multi-target protein kinase inhibitor, suppresses preferentially the growth of TNBC cells comparing with non-TNBC cells. Clustering analysis of the drug activity data for the panel of cancer cell lines predicted that midostaurin shares the target with Aurora kinase inhibitors. Following studies indicated that midostaurin attenuates the phosphorylation reaction mediated by Aurora kinase in the cells and directly inhibits this protein kinase in vitro, and that this reagent induces apoptosis accompanying accumulation of 4N and 8N DNA cells in TNBC cells.

Conclusion: Midostaurin suppresses the proliferation of TNBC cells among the breast cancer cell lines presumably through the inhibition of the Aurora kinase family. The precise study of midostaurin on cell growth will contribute to the development of the drug for the treatment of TNBC.

No MeSH data available.


Related in: MedlinePlus