br Results br Caudatin inhibits the viability of human breas
Caudatin inhibits the viability of human breast cancer cells
Both MDA-MB-231 and MCF-7 cells were treated with various concentrations of caudatin for 24 h, and the in vitro eﬀects of caudatin were then evaluated by the CCK-8 assay. As shown in Fig. 1A, caudatin treatment resulted in a concentration-dependent decrease in cell via-bility.
Caudatin induces PX478 arrest in human breast cancer cells
To determine whether the growth arrest of breast cancer cells caused by caudatin is related to cell cycle arrest, cells were treated with caudatin for 24 h, followed by cell cycle determination via flow cyto-metry. As shown in Fig. 2A, caudatin treatment induced an increase in the G1-phase cell population and a decrease in the S- or G2/M-phase cell population in both MDA-MB-231 and MCF-7 cells, indicating that caudatin was able to arrest the cell cycle in G1 phase.
To further investigate the mechanism by which caudatin blocks cell
Fig. 3. The apoptotic eﬀect of caudatin on the human breast cancer cells. (A) MDA-MB-231 and MCF-7 cells were treated with diﬀerent concentrations of caudatin for 24 h. Apoptosis was analyzed by TUNEL staining according to the manufacturer's instructions. (B) Caudatin treatment induced the cleavage of caspase-8, -9 and PARP. After cells were treated with caudatin for 24 h, total cell lysates were harvested and analyzed by western blotting analysis.
cycle progression, we analyzed the expression of several cell cycle regulators upon caudatin treatment. Significant accumulation of p21 and p27 in response to caudatin treatments in breast cancer cells was observed (Fig. 2B). However, the level of p53 was upregulated in caudatin-treated MCF-7 cells, whereas it remained unchanged in MDA-MB-231 cells. The expression levels of cyclinB1, p-Cdc2 and Cdk4 were downregulated upon treatment of breast cancer cells with caudatin.
Caudatin triggers cell apoptosis in human breast cancer cells
Our previous studies have shown that caudatin can induce apoptosis in hepatocellular cancer cells (Fei et al., 2012a); hence, we investigated
whether apoptosis occurred in caudatin-treated breast cancer cells. TUNEL assay results indicated that higher concentrations of caudatin could trigger cell apoptosis (Fig. 3A). Western blotting results showed increased cleavage of caspase-8, caspase-9 and PARP, the hallmark feature of apoptosis, in both MDA-MB-231 and MCF-7 cells (Fig. 3B), suggesting that caudatin can induce cell apoptosis in human breast cancer cells.
Caudatin sensitizes human breast cancer cells to TRAIL-induced cell death
The identification of small-molecule compounds that potently sen-sitize cancer cells to TRAIL-induced apoptosis is an eﬀective way to
Fig. 4. Eﬀect of caudatin plus TRAIL treatment on apoptosis of human breast cancer cells. (A) MDA-MB-231 and MCF-7 cells were treated with 80 ng/ml TRAIL in the presence or absence of 10 μg/ml caudatin for 24 h, and cell apoptosis was assessed by TUNEL staining. (B) Cells were treated with TRAIL (80 ng/ml) in the absence or the presence of caudatin (10 μg/ml) or triptolide (5 nM) for 24 h. The cleavage of caspase-8, caspase-9 and PARP were analyzed by western blotting analysis.
overcome acquired TRAIL resistance (Zoller et al., 2016). Hence, we examined the cytotoxic and apoptotic eﬀects of caudatin in combina-tion with TRAIL in breast cancer cells. Both MDA-MB-231 and MCF-7 cells were treated with caudatin or TRAIL or subjected to concomitant treatment with TRAIL and caudatin for 24 h. TUNEL staining was per-formed to detect cell apoptosis. We found a significant increase in
apoptotic cells in both cancer cell lines upon exposure to caudatin combined with TRAIL (Fig. 4A). We also measured caspase-8, -9 and PARP cleavage to confirm that caudatin increased TRAIL-triggered apoptosis in breast cancer cells. As shown in Fig. 4B, compared with the agent-alone group, the cleavage of caspase-8, -9 and PARP was more obvious in both of the cancer cell lines when treated with caudatin plus
Fig. 5. Caudatin treatment sensitizes human breast cancer cells to TRAIL-induced apoptosis by upregulating DR5. (A) Cells were treated with caudatin at the indicated concentrations for 24 h. DR4 and DR5 protein levels in whole cell lysates were determined by western blotting analysis. β-actin was used as a loading control. (B) MDA-MB-231 and MCF-7 cells were transfected with DR5 siRNA or control siRNA and then exposed to caudatin for 24 h. Western blotting analysis was used to detect the protein expression.
TRAIL. Collectively, these data indicate that treatment of breast cancer cells with the combination of caudatin and TRAIL sensitizes breast cancer cells to TRAIL-induced cell apoptosis.