br Upregulation of DR contributes to the caudatin mediated
Upregulation of DR5 contributes to the caudatin-mediated enhancement of TRAIL-induced apoptosis
Because TRAIL-induced apoptosis is mediated by the DR4 and DR5 receptors, we investigated whether caudatin modulates the expression of DR4 and DR5 in breast cancer cells. Breast cancer PX-478 were treated with various concentrations of caudatin for 24 h, and western blotting was then used to detect the expression of DR4 and DR5. We found that caudatin increased the level of DR5 in a dose-dependent manner in both MDA-MB-231 and MCF-7 cells (Fig. 5A). However, there was no sig-nificant change in DR4 expression upon caudatin treatment.
To determine whether caudatin treatment enhances TRAIL-induced apoptosis via upregulation of DR5, we used siRNA specific to DR5 to examine whether suppression of DR5 could abrogate the sensitizing eﬀects of caudatin on the TRAIL-induced cleavage of caspases. As shown in Fig. 5B, the eﬀect of caudatin on the TRAIL-induced cleavage of caspase-8, -9 and PARP was eﬀectively abolished in cells transfected with DR5 siRNA, suggesting that induction of DR5 is critical for the sensitization of breast cancer cells to the eﬀect of caudatin on TRAIL-induced apoptosis.
The ER stress pathway is activated in caudatin-treated human breast cancer cells
One mechanism for inducing DR5 expression is through activation of the ER stress signaling pathway (Na et al., 2017). Therefore, we in-vestigated the eﬀect of caudatin on the activation of the ER stress marker CHOP and signaling proteins downstream of ER stress. As shown in Fig. 6A, exposure to caudatin markedly increased the protein expression of CHOP in MDA-MB-231 and MCF-7 cells. Moreover, the levels of PERK, BIP and ATF4 were significantly enhanced in response to caudatin treatment.
Since apoptosis occurs subsequent to ER stress, we hypothesized that it could be a consequence of caudatin-triggered ER stress. We transiently transfected breast cancer cells with CHOP siRNA in the presence or absence of caudatin to confirm whether ER stress induced by caudatin led to the cleavage of caspase-9 and PARP. Western blotting results showed that CHOP knockdown did not aﬀect the caudatin-in-duced cleavage of caspase-9 and PARP (Fig. 6B), suggesting that cau-datin-triggered apoptosis was unrelated to ER stress in MDA-MB-231 and MCF-7 cells.
To elucidate the role of CHOP in the caudatin-induced upregulation of DR5, we silenced the expression of CHOP by using siRNA and de-tected the change in caudatin-induced DR5 levels. Western blotting results indicated that CHOP knockdown significantly suppressed cau-datin-induced DR5 upregulation in both MDA-MB-231 and MCF-7 cells
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 cells 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.