After 48 h, cells were collected and washed with cold PBS

After 48 h, cells were collected and washed with cold PBS. exhibited its anti-tumor potential partly through inactivation of Skp2 in breast cancer. Our findings indicate that rottlerin could be a potential safe agent for the treatment of breast cancer. < 0.05, compared to the control (DMSO treatment). (B) Colony formation analysis was conducted to measure the colony numbers in breast cancer cells treated with rottlerin. (C) Flow cytometry was used to detect the cell apoptosis in breast cancer cells treated with rottlerin. (D) Cell cycle was analyzed by Flow cytometry in rottlerin-treated breast cancer cells. Rottlerin induced cell apoptosis Previous studies have shown that rottlerin triggered apoptosis in breast cancer cells [17, 18]. We next aim to further validate whether cell proliferation inhibition by rottlerin treatment could be due to rottlerin-mediated cell apoptotic death. To this end, PI-FITC-annexin assay was performed to detect the cell apoptosis Chlorpromazine hydrochloride in both MCF-7 and MDA-MB-231 cells treated with 3 M Rottlerin for 48 hours. We Rabbit polyclonal to ZCCHC13 observed that rottlerin significantly induced cell apoptosis in both breast cancer cells (Figure ?(Figure1C).1C). Rottlerin treatment led to apoptotic cells from 5% to 13% in MCF-7 cells, and from 8.5% to 19% in MDA-MB-231 cells (Figure ?(Figure1C).1C). Consistent with other studies [17, 18], rottlerin could stimulate apoptosis in breast cancer cells. Rottlerin induced cell cycle arrest Next, we explored whether cell cycle arrest contributed to cell growth inhibition in breast cancer cells after rottlerin treatment. Cell cycle analysis by PI staining and flow cytometry in MCF-7 and MDA-MB-231 cells treated with different concentrations of rottlerin. Our results demonstrated that rottlerin treatment caused cell cycle arrest at G1 phase. Specifically, 3 M and 5 M rottlerin led to G1 cell population from 44% to 73.5% to 83%, respectively, in MCF-7 cells (Figure ?(Figure1D).1D). Similar result was found in MDA-MB-231 cells (Figure ?(Figure1D).1D). Our observations implied that rottlerin could induce G1 cell cycle arrest. Rottlerin inhibited cell migration and invasion To determine whether rottlerin inhibited cell migratory activity, the wound healing assay was conducted in MCF-7 and MDA-MB-231 cells treated with rottlerin. We found that rottlerin treatment remarkably suppressed cell migration in dose-dependent manner in both breast cancer cells (Figure ?(Figure2A).2A). To further define whether rottlerin has invasion inhibition potential, invasion assay was applied for measuring penetration of breast cancer cells via the matrigel-coated membrane. Our Transwell invasion assay revealed that rottlerin decreased the cell numbers of penetration through matrigel, suggesting that rottlerin could retard the cell invasion in breast cancer cells (Figure ?(Figure2B2B). Open in a separate window Figure 2 Effect of rottlerin on cell migration and invasion(A) Top panel: Wound healing assay was performed to detect the inhibitory effect of rottlerin on MCF-7 cells and MDA-MB-231 cells after rottlerin treatment. Bottom panel: Quantitative results are illustrated for left panel. **< 0.01 vs control (DMSO treatment). (B) Left panel: Transwell chambers assay was conducted to measure the cell invasion in MCF-7 cells and MDA-MB-231 cells after rottlerin treatment. Right panel: Quantitative results are illustrated for left panel. **< 0.01 vs control. Rottlerin decreased Skp2 expression Skp2 has Chlorpromazine hydrochloride been characterized as an oncoprotein in breast cancer. Therefore, inhibition of Skp2 could be a promising approach for treating breast cancer. Next, we explored whether rottlerin could down-regulate Skp2 expression, leading to its anti-tumor activity in breast cancer cells. Real-time PCR and Western blotting were used Chlorpromazine hydrochloride to detect the expression of Skp2 at mRNA and protein levels, respectively, in breast cancer cells treated with rottlerin. The results showed that both Skp2 mRNA and its protein level were significantly decreased in rottlerin-treated cells (Figure 3A, 3B). We also found that the expression of p21, one of Skp2 downstream target, was upregulated in cells after rottlerin treatment Chlorpromazine hydrochloride (Figure 3B and.