The differential susceptibilities of MCF-7 and MDA-MB-231 cells to the cytotoxic effects of curcumin are associated with the PI3K/Akt-SKP2-Cip/Kips pathway

Abstract

Background: The mechanism underlying the differential cytotoxicity of curcumin in various cancer types, however, remains largely unclear. The aims of this study is to examine the concentration- and time-related effects of curcumin on two different breast cancer cells, MCF-7 and MDA-MB-231, and investigated the functional changes induced by curcumin treatment, as well as their relationship to the PI3K/Akt-SKP2-Cip/Kips pathway.

Methods: First, WST-1 and clonogenic assay were performed to determine the cytotoxicity of curcumin in MCF-7 and MDA-MB-231 cells. Then, the expression of CDK interacting protein/Kinase inhibitory protein (Cip/Kips) members (p27, p21 and p57) and S-phase kinase-associated protein-2 (SKP2) was investigated by QRT PCR and Western Blotting. Curcumin's effect on PI3K (phosphatidylinositol 3-kinase) /Akt and its substrates Foxo1 and Foxo3a were then studied by Western Blotting. Small interfering RNAs (siRNAs) targeting SKP2 was used to explore the relationship between SKP2 and Cip/Kips members. Finally, WST-1 assay was tested to explore the concomitant treatment with curcumin and the inhibition of PKB or SKP2 signaling on curcumin sensitivity in MCF-7 and MDA-MB-231 cells.

Results: We demonstrated MCF-7 and MDA-MB-231 cells exhibited differential responses to curcumin by WST-1 and clonogenic assay (MDA-MB-231 cells was sensitive, and MCF-7 cells was resistant), which were found to be related to the differential curcumin-mediated regulation of SKP2-Cip/Kips (p21 and p27 but not p57) signaling. The differential cellular responses were further linked to the converse effects of curcumin on PI3K/Akt and its substrates Foxo1 and Foxo3a. Importantly, PI3K inhibitor wortmannin could counteract both curcumin-induced phosphorylation of Akt and up-regulation of SKP2 in MCF-7 cells. Subsequent WST-1 assay demonstrated concomitant treatment with curcumin and wortmannin or SKP2 siRNA not only further augmented curcumin sensitivity in MDA-MB-231 cells but also overcame curcumin resistance in MCF-7 cells.

Conclusions: Our study established PI3K/Akt-SKP2-Cip/Kips signaling pathway is involved in the mechanism of action of curcumin and revealed that the discrepant modulation of this pathway by curcumin is responsible for the differential susceptibilities of these two cell types to curcumin.

Keywords: Breast cancer; Cip/Kips; Curcumin sensitivity; FOXO; PI3K; SKP2.

Figure 1

Curcumin’s responsiveness of MCF-7 and MDA-MB-231 cells. (A) WST-1 assay of MCF-7 and MDA-MB-231 cells after curcumin treatment. The indicated breast cancer cells were treated with indicated concentrations of curcumin for 12 or 24 h as described in the text. Data represents the fluorescent changes (n = 3). The experiments were performed three times. (B) Clonogenic analysis of MCF-7 and MDA-MB-231 cells after curcumin treatment. The indicated breast cancer cells were treated with 40 μM curcumin for 3 or 6 h, trypsinized, and plated in duplicates at low density (2,000 per well in 6 well plate). After 7–12 days, formed colonies were stained with crystal violet. Clones in a given area were counted for each condition. Represented is the percentage of viable clones after curcumin treatment respective to untreated cells. Columns, mean of three independent determinations; bars, SD.

Figure 2

Analysis of Cip/Kips expression in response to curcumin in MCF-7 and MDA-MB-231 cells. (A) Analysis of p21 (1:1000), p27 (1:1000) and p57 (1:500) changes induced by indicated concentration of curcumin for 24 h in MCF-7 and MDA-MB-231 cells was took by western blotting. Also shown is a blot for β-actin as a loading control. The representative data of at least two independent experiments are shown. (B) Analysis of p21 (1:1000), p27 (1:1000) and p57 (1:500) changes induced by 40 μM curcumin for indicated time in MCF-7 and MDA-MB-231 cells was took by western blotting. Also shown is a blot for β-actin as a loading control. The representative data of at least two independent experiments are shown. Quantitative analysis of Cip/Kips protein expression using Quantity One software (Bio-Rad, USA). Graphs show quantification of Cip/Kips protein expression after normalizing the data to respective control group.

Figure 3

Analysis of SKP2 at both protein and transcriptional level in response to curcumin. (A) Analysis of (1:1000) changes induced by indicated curcumin for 24 h in MCF-7 and MDA-MB-231 cells was taken by western blotting. Also shown is a blot for GAPDH as a loading control. The representative data of at least two independent experiments are shown. (B) Analysis of SKP2 (1:1000) changes induced by 40 μM curcumin for indicated time in MCF-7 and MDA-MB-231 cells was took by western blotting. Also shown is a blot for GAPDH as a loading control. The representative data of at least two independent experiments are shown. Quantitative analysis of SKP2 protein expression using Quantity One software (Bio-Rad, USA). Graphs show quantification of SKP2 protein expression after normalizing the data to respective control group. (C) MCF-7 or MDA-MB-231 cells were treated with indicated concentration of curcumin for 24 h. The bar graph indicates the fold change of SKP2 mRNA level in curcumin treated cells compared with non-treated cells. One-way ANOVA was studied, followed by Student’s t test. Each bar represents the mean ± SD of three independent experiments. *,P <0.05; **,P <0.01, compared with control. RT-Q-PCR, quantitative RT-PCR.

Figure 4

Knockdown of SKP2 expression in MCF-7 and MDA-MB-231 cells induced up-regulation of p27 and p21. MCF-7 or MDA-MB-231 cells were transfected with SKP2-SiRNA or negative SiRNA (control-SiRNA) for 24 h or 48 h, (NEG means control group). Down-regulation of SKP2 expression was evaluated by western blotting. A protein level of p21 and p27 in all cells was examined by western blotting. Also shown is a blot for GAPDH as a loading control. The representative data of at least two independent experiments are shown.

Figure 5

Curcumin modulated different PI3K/Akt pathway in MCF-7 and MDA-MB-231 cells. (A) Analysis of p-Akt (1:5000), Akt (1:1000) changes induced by indicated curcumin for 24 h or 40 μM curcumin for indicated time in MCF-7 and MDA-MB-231 cells was took by western blotting. (B) MCF-7 cells were treated with 1 μM concentration of wortmannin for 1 h prior to treatment with curcumin (40 μM) for 6 h. Expression of p-Akt (1:5000) and SKP2 (1:1000) in indicated condition was examined by western blotting. Also shown is a blot for GAPDH as a loading control. (C) Analysis of p-Foxo1 (1:1000) and p-Foxo3a (1:1000) changes induced by 40 μM curcumin for indicated time in MCF-7 and MDA-MB-231 cells was taken by western blotting. Also shown is blot for β-actin as a loading control. The representative data of at least two independent experiments are shown.

Figure 6

Cell viability assay for treatment of curcumin with PKB inhibitor or SKP2 SiRNA. (A) Wortmannin alters curcumin response in MCF-7 and MDA-MB-231 cells. MCF-7 and MDA-MB-231 cells were treated with 1 μM concentration of wortmannin for 1 h prior to treatment with curcumin (40 μM) for 12 h and WST-1 assay was performed as described in the “Materials and methods”. Each bar represents the mean ± SD of three independent experiments. *,P <0.05; **,P <0.01, compared with control (DMSO). (B) Silencing of SKP2 increases cellular sensitivity to curcumin in MCF-7 and MDA-MB-231 cells. Cells were seeded at a density of 5000 cells/well in 96-well plate and cultured overnight. Then the medium was replaced by 1%(V/V) FBS, 20nM SKP2-SiRNA and control-SiRNA was added to indicated wells and cells were cultured for 48 h. SKP2-SiRNA transfection cells and control-SiRNA transfection cells were obtained. Cells were treated with 40 μM curcumin for 12 h and WST-1 assay was performed to detect viability of cells. One-way ANOVA was studied, followed by Student’s t test. Each bar represents the mean ± SD of three independent experiments. *,P <0.05; **,P <0.01, compared with control (DMSO).