Korean J Physiol Pharmacol.  2013 Aug;17(4):291-297. 10.4196/kjpp.2013.17.4.291.

Mutant p53-Notch1 Signaling Axis Is Involved in Curcumin-Induced Apoptosis of Breast Cancer Cells

• Bae YH
• Ryu JH
• Park HJ
• Kim KR
• Wee HJ
• Lee OH
• Jang HO
• Bae MK
• Kim KW
• Bae SK

• KMID: 2285463
• DOI: http://doi.org/10.4196/kjpp.2013.17.4.291

Abstract

Notch1 has been reported to be highly expressed in triple-negative and other subtypes of breast cancer. Mutant p53 (R280K) is overexpressed in MDA-MB-231 triple-negative human breast cancer cells. The present study aimed to determine whether the mutant p53 can be a potent transcriptional activator of the Notch1 in MDA-MB-231 cells, and explore the role of this mutant p53-Notch1 axis in curcumin-induced apoptosis. We found that curcumin treatment resulted in an induction of apoptosis in MDA-MB-231 cells, together with downregulation of Notch1 and its downstream target, Hes1. This reduction in Notch1 expression was determined to be due to the decreased activity of endogenous mutant p53. We confirmed the suppressive effect of curcumin on Notch1 transcription by performing a Notch1 promoter-driven reporter assay and identified a putative p53-binding site in the Notch1 promoter by EMSA and chromatin immunoprecipitation analysis. Overexpression of mutant p53 increased Notch1 promoter activity, whereas knockdown of mutant p53 by small interfering RNA suppressed Notch1 expression, leading to the induction of cellular apoptosis. Moreover, curcumin-induced apoptosis was further enhanced by the knockdown of Notch1 or mutant p53, but it was decreased by the overexpression of active Notch1. Taken together, our results demonstrate, for the first time, that Notch1 is a transcriptional target of mutant p53 in breast cancer cells and suggest that the targeting of mutant p53 and/or Notch1 may be combined with a chemotherapeutic strategy to improve the response of breast cancer cells to curcumin.

Keyword

Apoptosis; Breast cancer cells; Curcumin; Mutant p53; Notch1

MeSH Terms

Apoptosis
Axis, Cervical Vertebra
Breast
Breast Neoplasms
Chromatin Immunoprecipitation
Curcumin
Down-Regulation
Humans
RNA, Small Interfering
Curcumin
RNA, Small Interfering

Figure

• 

  Fig. 1 Localization and identification of a 53-binding site in human Notch1 promoter. MDA-MB-231 cells were treated with different concentrations of curcumin (0~20 µM) for 24 h. (A) The mRNA levels of human Notch1, Notch2, Notch3, Notch4, and β-actin were detected by RT-PCR analysis. (B) The protein levels of Notch1, c-Notch1, Hes1, and β-actin were confirmed by Western blot analysis using the specific antibodies. (C) A schematic representation showing the deletion constructs of the human Notch1 promoter. (D) MDA-MB-231 cells were transfected with the indicated luciferase reporter vectors and subsequently treated with curcumin (20 µM) for 24 h. Cell extracts were prepared and analyzed for luciferase activity. **p<0.001 vs control, n=3. (E) The p53-binding activity in nuclear extracts was measured by EMSA. MDA-MB-231 cells were incubated with curcumin (20 µM) for 24 h. Nuclear extracts from MDA-MB-231 cells were incubated with biotinlabeled P(p53) oligonucleotide as a Notch1 promoter-specific p53 probe. In competition assay, 20x and 50x excess unlabeled probe were added to the reaction mixture. ns: nonspecific band. (F) MDA-MB-231 cells were treated with or without curcumin (20 µM) for 24 h. ChIP analysis was performed with control IgG or anti-p53 antibodies. IP, immunoprecipitation.

• 

  Fig. 2 Effects of p53 silencing on Notch1 expression. MDA-MB-231 cells were transfected with control siRNA or with p53 siRNA for 48 h. (A) The mRNA levels of human p53, Notch1, Notch2, Notch3, Notch4, Hes1, and β-actin were detected by RT-PCR analysis; representative RT-PCR. (B) The protein levels of human p53, total Notch1 (t-Notch1), cleaved Notch1 (c-Notch1), Hes1, and β-actin were detected by Western blot analysis; representative Western blot. (C) MDA-MB-231 cells were cotransfected with p(484)luc vector together with the indicated siRNAs, followed 48 h later by luciferase activity assay. *p<0.01 vs control siRNA, n=3. (D) MDA-MB-231 cells were cotransfected with p(484)luc together with a mammalian expression vector encoding wild-type p53 (p53WT) or mutant p53 (p53 Mut), or with control vector (pCMV-neo). After 48 h of transfection, cell extracts were prepared and analyzed for luciferase activity. **p<0.001; *p<0.01 vs control vector, n=3.

• 

  Fig. 3 Effects of down-regulation of mutant p53-Notch1 axis on curcumin-induced apoptosis. MDA-MB-231 cells were transfected with control siRNA or Notch1 siRNA. After 24 h of transfection, cells were treated with curcumin (20 µM) and incubated for 24 h. (A) Apoptosis was detected by TUNEL assay. DAPI stains nuclear DNA. Scale bar: 50 µm. (B) Values are expressed as the percentage of TUNEL-positive cells divided by total DAPI-positive cells counted. **p<0.001 vs control siRNA without curcumin, ##p<0.001 vs control siRNA with curcumin, n=3.

• 

  Fig. 4 Effects of overexpression of active Notch1 on curcumininduced apoptosis. MDA-MB-231 cells were transfected with an empty vector control or a mammalian expression vector encoding Myc-tagged NICD. After 24 h of transfection, cells were treated with curcumin (20 µM) and incubated for 48 h. (A) Apoptosis was detected by the TUNEL assay. DAPI stains nuclear DNA. Scale bar: 50 µm. (B) Values are expressed as the percentage of TUNELpositive cells divided by total DAPI-positive cells counted. *p<0.01; **p<0.001 vs control without curcumin, #p<0.01; ##p< 0.001 vs control with curcumin n=3. (C) Western blot analysis of Myc, NICD, PARP, Caspase-3, and Bcl-2 protein levels in NICD-transfected cells in the presence or absence of curcumin (20 µM).

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