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Siah1 proteins enhance radiosensitivity of human breast cancer cells.

He HT, Fokas E, You A, Engenhart-Cabillic R, An HX - BMC Cancer (2010)

Bottom Line: In addition, Siah1 and Siah1L significantly reduced cell clonogenic survival and proliferation.Siah1 or Siah1L significantly reduced invasion ability of SKBR3 and suppressed Tcf/Lef factor activity.Importantly, Siah1 siRNA demonstrated opposite effects in MCF-7 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Radiotherapy and Radiation Oncology, Philipps-University Marburg, Baldingerstr, D-35043 Marburg, Germany.

ABSTRACT

Background: Siah proteins play an important role in cancer progression. We evaluated the effect of Siah1, its splice variants Siah1L and the Siah1 mutant with the RING finger deleted (Siah1DeltaR) on radiosensitization of human breast cancer cells.

Methods: The status of Siah1 and Siah1L was analysed in five breast cancer cell lines. To establish stable cells, SKBR3 cells were transfected with Siah1, Siah-1L and Siah1DeltaR. Siah1 function was suppressed by siRNA in MCF-7 cells. The impact of Siah1 overexpression and silencing on apoptosis, proliferation, survival, invasion ability and DNA repair was assessed in SKBR3 and MCF-7 cells, also in regards to radiation.

Results: Siah1 and Siah1L mRNA expression was absent in four of five breast cancer cells lines analysed. Overexpression of Siah1 and Siah1L enhanced radiation-induced apoptosis in stable transfected SKBR3 cells, while Siah1DeltaR failed to show this effect. In addition, Siah1 and Siah1L significantly reduced cell clonogenic survival and proliferation. Siah1L sensitization enhancement ratio values were over 1.5 and 4.0 for clonogenic survival and proliferation, respectively, pointing to a highly cooperative and potentially synergistic fashion with radiation. Siah1 or Siah1L significantly reduced invasion ability of SKBR3 and suppressed Tcf/Lef factor activity. Importantly, Siah1 siRNA demonstrated opposite effects in MCF-7 cells. Siah1 and Siah1L overexpression resulted in inhibition of DNA repair as inferred by increased levels of DNA double-strand breaks in irradiated SKBR3 cells.

Conclusion: Our results reveal for the first time how overexpression of Siah1L and Siah1 can determine radiosensitivity of breast cancer cells. These findings suggest that development of drugs augmenting Siah1 and Siah1L activity could be a novel approach in improving tumor cell kill.

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Knockdown of Siah1 expression in MCF7 cells. A: Western blot showed that siRNA as well as siRNA-2 and siRNA-3 for Siah1 resulted in suppressed protein expression in MCF-7 cells. B: Siah2 expression is decreased in SKBR3 and MCF-7 cells. α-Tubulin was used as an internal control.
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Figure 2: Knockdown of Siah1 expression in MCF7 cells. A: Western blot showed that siRNA as well as siRNA-2 and siRNA-3 for Siah1 resulted in suppressed protein expression in MCF-7 cells. B: Siah2 expression is decreased in SKBR3 and MCF-7 cells. α-Tubulin was used as an internal control.

Mentions: The expression of Siah1 and its splice variant Siah1L in breast cancer cell lines was determined using RT-PCR with specific primers recognizing the common sequences of both siah1 and siah1L. In line with previous report [10,27], MCF7 breast cancer cells exhibited strong expression of Siah1 and Siah1L, whereas BT-20, MDA-MB-231, SKBR3 and ZR75-1 breast cancer lines lacked any expression of Siah1 and Siah1L analysed (Figure 1A). The SKBR3 cells without detectable endogenous expression of Siah1 and Sial1L were chosen to establish stable gene expression. Stable clones were selected for G418 resistance and screened for relative equivalent levels of exogenous expression of Siah constructs detected by using immunoblotting (Figure 1B). Additionally, we performed siRNA for Siah1 in MCF-7 to further investigate its role in radiosensitization. Western blot confirmed blockade of Siah1 in MCF-7. Off-target effects can occur during siRNA [28] and therefore two additional Siah1 siRNAs (siRNA-2 and siRNA-3) were tested as well. They also confirmed blockade of Siah1 protein in MCF-7 cells. Control siRNA was used as well (Figure 2A). Siah2 protein expression was absent in both SKBR-3 and MCF-7 cells (Figure 2B), as previously demonstrated [29].


Siah1 proteins enhance radiosensitivity of human breast cancer cells.

He HT, Fokas E, You A, Engenhart-Cabillic R, An HX - BMC Cancer (2010)

Knockdown of Siah1 expression in MCF7 cells. A: Western blot showed that siRNA as well as siRNA-2 and siRNA-3 for Siah1 resulted in suppressed protein expression in MCF-7 cells. B: Siah2 expression is decreased in SKBR3 and MCF-7 cells. α-Tubulin was used as an internal control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2921397&req=5

Figure 2: Knockdown of Siah1 expression in MCF7 cells. A: Western blot showed that siRNA as well as siRNA-2 and siRNA-3 for Siah1 resulted in suppressed protein expression in MCF-7 cells. B: Siah2 expression is decreased in SKBR3 and MCF-7 cells. α-Tubulin was used as an internal control.
Mentions: The expression of Siah1 and its splice variant Siah1L in breast cancer cell lines was determined using RT-PCR with specific primers recognizing the common sequences of both siah1 and siah1L. In line with previous report [10,27], MCF7 breast cancer cells exhibited strong expression of Siah1 and Siah1L, whereas BT-20, MDA-MB-231, SKBR3 and ZR75-1 breast cancer lines lacked any expression of Siah1 and Siah1L analysed (Figure 1A). The SKBR3 cells without detectable endogenous expression of Siah1 and Sial1L were chosen to establish stable gene expression. Stable clones were selected for G418 resistance and screened for relative equivalent levels of exogenous expression of Siah constructs detected by using immunoblotting (Figure 1B). Additionally, we performed siRNA for Siah1 in MCF-7 to further investigate its role in radiosensitization. Western blot confirmed blockade of Siah1 in MCF-7. Off-target effects can occur during siRNA [28] and therefore two additional Siah1 siRNAs (siRNA-2 and siRNA-3) were tested as well. They also confirmed blockade of Siah1 protein in MCF-7 cells. Control siRNA was used as well (Figure 2A). Siah2 protein expression was absent in both SKBR-3 and MCF-7 cells (Figure 2B), as previously demonstrated [29].

Bottom Line: In addition, Siah1 and Siah1L significantly reduced cell clonogenic survival and proliferation.Siah1 or Siah1L significantly reduced invasion ability of SKBR3 and suppressed Tcf/Lef factor activity.Importantly, Siah1 siRNA demonstrated opposite effects in MCF-7 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Radiotherapy and Radiation Oncology, Philipps-University Marburg, Baldingerstr, D-35043 Marburg, Germany.

ABSTRACT

Background: Siah proteins play an important role in cancer progression. We evaluated the effect of Siah1, its splice variants Siah1L and the Siah1 mutant with the RING finger deleted (Siah1DeltaR) on radiosensitization of human breast cancer cells.

Methods: The status of Siah1 and Siah1L was analysed in five breast cancer cell lines. To establish stable cells, SKBR3 cells were transfected with Siah1, Siah-1L and Siah1DeltaR. Siah1 function was suppressed by siRNA in MCF-7 cells. The impact of Siah1 overexpression and silencing on apoptosis, proliferation, survival, invasion ability and DNA repair was assessed in SKBR3 and MCF-7 cells, also in regards to radiation.

Results: Siah1 and Siah1L mRNA expression was absent in four of five breast cancer cells lines analysed. Overexpression of Siah1 and Siah1L enhanced radiation-induced apoptosis in stable transfected SKBR3 cells, while Siah1DeltaR failed to show this effect. In addition, Siah1 and Siah1L significantly reduced cell clonogenic survival and proliferation. Siah1L sensitization enhancement ratio values were over 1.5 and 4.0 for clonogenic survival and proliferation, respectively, pointing to a highly cooperative and potentially synergistic fashion with radiation. Siah1 or Siah1L significantly reduced invasion ability of SKBR3 and suppressed Tcf/Lef factor activity. Importantly, Siah1 siRNA demonstrated opposite effects in MCF-7 cells. Siah1 and Siah1L overexpression resulted in inhibition of DNA repair as inferred by increased levels of DNA double-strand breaks in irradiated SKBR3 cells.

Conclusion: Our results reveal for the first time how overexpression of Siah1L and Siah1 can determine radiosensitivity of breast cancer cells. These findings suggest that development of drugs augmenting Siah1 and Siah1L activity could be a novel approach in improving tumor cell kill.

Show MeSH
Related in: MedlinePlus