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  • br Fig E ect of

    2022-08-18


    Fig. 6. Effect of caudatin on the expression of ER stresses proteins in human breast cancer cells. (A) Western blotting analyses of markers of ER stress and unfolded protein response. Cells were treated with different concentrations of caudatin for 24 h. β-actin was used as a loading control. (B) MDA-MB-231 and MCF-7 Chloramphenicol were transfected with control siRNA or CHOP siRNA and then exposed to 50 μg/ml caudatin for 24 h. Whole-cell extracts were prepared and analyzed by western blotting.
    (C) Breast cancer cells were transfected with control siRNA or CHOP siRNA and then exposed to 10 μg/ml caudatin for 24 h. DR5 expression was analyzed by western blotting analysis.
    (Fig. 6C). These results confirmed the caudatin-induced activation of the CHOP-DR5 signaling cascade.
    Caudatin-induced DR5 upregulation requires p38 MAPK and JNK but not ERK
    MAPK activation is also known to regulate TRAIL receptor induction (Lepage et al., 2011). Hence, we investigated the effects of caudatin exposure on the activation of ERK1/2, JNK and p38 MAPK in breast cancer cells. As shown in Fig. 7A, caudatin caused phosphorylation of 
    p38 MAPK and JNK in both MDA-MB-231 and MCF-7 cells, while the phosphorylation of ERK1/2 was unchanged.
    As the activation of p38 MAPK and JNK was observed upon cau-datin treatment, we further explored whether it was directly linked to DR5 upregulation. We used pharmacological inhibitors of p38 MAPK (SB203580) and JNK (SP600125) and examined caudatin-induced DR5 expression. Our results showed that caudatin-induced DR5 expression was suppressed in the presence of either a p38 MAPK or JNK pathway-specific inhibitor (Fig. 7B), suggesting that p38 MAPK and JNK play a critical role in mediating the effects of caudatin on DR5 upregulation in
    Fig. 7. Effect of caudatin on the MAPK activation in human breast cancer cells. (A) MDA-MB-231 and MCF-7 cells were treated with different concentrations of caudatin for 24 h; equal amounts of total proteins were subjected to SDS-PAGE. Expressions of ERK, p-ERK, JNK, p-JNK, p38, p-p38 and β-actin were detected by western blotting analysis. (B) The effect of cotreatment with inhibitors of p38 MAPK (SB203580) or JNK (SP600125) on the caudatin-induced DR5 expression.
    breast cancer cells.
    Discussion
    In the present study, we first evaluated the effects of caudatin on cell growth in breast cancer cells. We found that caudatin inhibited cell viability in a dose-dependent manner. Although caudatin mediated G1 phase arrest in both MDA-MB-231 and MCF-7 cells, some differences in the cell cycle phase distribution were observed. In MDA-MB-231 cells, caudatin-induced G1 phase arrest was accompanied by reduced cell numbers in G2/M phase. However, treatment of MCF-7 cells with caudatin caused a progressive decrease in the S-phase cell population, which has resulted from the upregulation of p53 in MCF-7 cells after caudatin treatment.
    As a promising and effective anticancer therapy agent, TRAIL has been shown to induce apoptosis in breast carcinoma (Naik et al., 2015). Unfortunately, TRAIL will probably not be viable as a single agent, since the majority of tumor cells are TRAIL-resistant. Therefore, com-bination therapy is essential for the use of TRAIL against refractory tumors (Lin et al., 2011). Cell surface DR5 expression has been broadly found in TRAIL-sensitive tumor cell lines and in primary tumors, and upregulation of DR5 may be an effective way to increase the sensitivity of TRAIL-induced tumor cell apoptosis (Yagita et al., 2004; Shishodia et al., 2018). In this study, we found that the levels of DR5 were strongly elevated by caudatin treatment. Using siRNA transfection, we demonstrated for the first time that DR5 upregulation is a critical event in the enhancement of TRAIL-induced apoptosis, since gene silencing of DR5 attenuated the effect of caudatin on the TRAIL-induced cleavage of caspase-8, -9 and PARP.