Apoptosis of mouse myeloma cells induced by curcumin via the Notch3-p53 signaling axis

Abstract

Resistance to apoptosis is a characteristic of cancer. Curcumin has become a potential anticancer drug for its pro-apoptotic effects, but the underlying mechanisms remain unclear. Furthermore, the Notch3-p53 signaling axis serves an important role in cell fate. The present study was designed to investigate the antitumor effect of curcumin by the Notch3-p53 axis in mouse myeloma P3X63Ag8 cells. The effects of curcumin on the viability of P3X63Ag8 cells were evaluated using an MTT assay. Quantitative expression of the Notch3-p53 signaling axis-associated genes was measured by reverse transcription-quantitative polymerase chain reaction, and western blot analysis was used to investigate the expression of proteins. Additionally, flow cytometry was used to measure the ratio of apoptosis. The results demonstrated that curcumin could significantly inhibit cell viability. No significant pro-apoptotic effect was observed when the concentration of curcumin was <30 µM. At 30 µM, curcumin-treated cells exhibited an apoptotic phenomenon, and the ratio of late apoptosis increased with the concentration of curcumin, and reached 28.4 and 51.8% in the medium- and high-dose groups, respectively. Curcumin inhibited the expression of Notch3, while the middle- and high-dose groups promoted p53. The expression of Notch3-responsive genes Hes family BHLH transcription factor 1 and Hes-related family transcription factor with YRPW motif 1 were notably promoted. Curcumin treatment significantly downregulated B-cell lymphoma-2 (Bcl-2) at the mRNA and protein levels, but upregulated Bcl-2-associated X. These data indicated that curcumin exhibited antitumor effects in mouse myeloma cells with induction of apoptosis by affecting the Notch3-p53 signaling axis.

Keywords: Notch3; P3X63Ag8 cell; apoptosis; curcumin; p53.

Figure 1.

Effect of curcumin on the proliferation of P3X63Ag8 cells. The cells were treated with different concentrations of curcumin (0, 10, 20, 30, 40, 50, 60, 70 and 80 µM) for 24 h. The results are expressed as the mean ± standard error of the mean of eight independent experiments. **P<0.01 vs. the curcumin-untreated group.

Figure 2.

(A) Hes1, (B) Hey1, (C) p53, (D) Bcl-2 and (E) Bax mRNA levels of curcumin-treated (0, 30, 40 and 50 µM) P3X63Ag8 cells for 24 h by reverse transcription-quantitative polymerase chain reaction. The results are expressed as the mean ± standard error of the mean of eight independent experiments. *P<0.05 and **P<0.01, compared with the curcumin-untreated group. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X; Hes1, Hes family BHLH transcription factor 1; Hey1, Hes-related family transcription factor with YRPW motif 1.

Figure 3.

Western blotting of Notch3, p53, Bcl-2, Bax and β-actin in P3X63Ag8 cells stimulated with 0, 30, 40 and 50 µM curcumin for 24 h. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X.

Figure 4.

The effect of curcumin on the apoptosis of P3X63Ag8 cells. (A) Flow cytometric dot plots of one representative experiment. Results are depicted as logarithmic fluorescence intensity on the x-axis (Annexin V-FITC) vs. y-axis (PI). A total of four quadrants represent necrotic cells (Q1-UL:AV-/PI+), late apoptotic cells (Q1-UR:AV+/PI+), early apoptotic cells (Q1-LR:AV+/PI-) and viable cells (Q1-LL:AV-/PI-). The numbers indicate the percentage of cells in each quadrant and a minimum of 10,000 events were read. (B) The histogram depicts the percentage of early and late apoptosis cells ± standard error of three independent experiments. **P<0.01 vs. the curcumin-untreated group. Cells were treated with 0, 30, 40 and 50 µM curcumin for 24 h. FITC, fluorescein isothiocyanate PI, propidium iodide.