Tauroursodeoxycholate br breast cancer cells br Discussion b
breast cancer cells.
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 Tauroursodeoxycholate 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.
Various stimuli are known to promote CHOP binding to the DR5 promoter and, thus, upregulate DR5 expression (Shiraishi et al., 2005; Chen et al., 2016a). Here, we showed that caudatin treatment induced
overexpression of the endoplasmic reticulum (ER) chaperones PERK, BIP, ATF-4 and CHOP. Therefore, we tested the involvement of CHOP in caudatin-induced DR5 expression. As shown in Fig. 6C, knockdown of CHOP by siRNA markedly suppressed the caudatin-induced expression of DR5, suggesting that CHOP plays a critical role in the expression of DR5 induced by caudatin. Additionally, CHOP is known to regulate ER stress-induced cancer cell death, and depletion of CHOP prevents apoptosis against various anticancer drugs (Yao et al., 2017). Then, we investigated whether CHOP upregulation was involved in caudatin-mediated cell apoptosis. Our results showed that silencing of CHOP did not abolish the caudatin-mediated cleavage of caspase-9 and PAPR, suggesting that caudatin-triggered apoptosis is independent of ER stress.
MAPKs, including ERK1/2, p38 MAPK and JNK, have been im-plicated in TRAIL receptor induction (Trivedi et al., 2014; Pennati et al., 2015). Therefore, we analyzed whether caudatin had any eﬀect on the activation of these MAPKs in human breast cancer cells. The phos-phorylation of ERK, p38 MAPK and JNK was assessed following treat-ment with caudatin. We found that the phosphorylation of p38 MAPK and JNK was significantly increased upon caudatin exposure, but the phosphorylation of ERK remained constant. Next, to investigate the direct involvement of p38 MAPK or JNK in caudatin-induced DR5 ex-pression, we performed cotreatment with p38 MAPK- and JNK-specific inhibitors and observed that it abrogated caudatin-induced DR5 in breast cancer cells, which indicated that caudatin-induced DR5 ex-pression was involved both in p38 MAPK and JNK activation.
Overall, our results provide mechanistic evidence that caudatin treatment of breast cancer cells results in the sensitization of cells to TRAIL-induced apoptosis through DR5 upregulation. Our findings provide novel insight into the anticancer activity of caudatin and warrant further evaluation of the combination of caudatin and TRAIL as a potential therapeutic regimen against human breast cancer.
Conflict of interest
The authors declare that there are no conflicts of interest.
This work was supported by grants from the National Natural Science Foundation of China (No. 81703039); Shandong Provincial Natural Science Foundation, China (No ZR2016HL58; No ZR2018MH026).
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