BER, the main medicinal component of Coptis chinensis, is a natural plant product with many applications. BER is accepted as a promising anticancer agent in different types of tumors through multiple mechanisms (Liu et al., 2019b). Recently, Gao et al. reported that BER enhances the sensitivity of breast cancer cells to different conventional chemotherapeutic drugs (Gao et al., 2019). Pretreatment with BER is effective in promoting the antitumor effects of 5-fluorouracil and cisplatin in laryngeal cancer cells (Palmieri et al., 2018). Importantly, BER has attracted much attention because it has low toxicity in relatively high doses and exhibits significant therapeutic activities without major adverse effects (Kumar et al., 2015). However, the efficacy of BER against ATC has rarely been reported, and the potential molecular mechanism remains unknown. The present study showed that BER induced a decrease in cell viability, apoptosis, and autophagy in CAL-62 and BHT-101 cells. Furthermore, combining DOX with BER generated a synergistic anticancer effect in ATC cells.
Autophagy is commonly considered to play a dual role in tumor suppression and cancer promotion, which may provide novel opportunities for anticancer drug development (Singh et al., 2018). BER, as a potential autophagy modulator, may trigger or inhibit autophagy and exert its anticancer effects (Mohammadinejad et al., 2019). For instance, BER induces autophagic cell death in breast cancer cells while inhibiting autophagy in lung cancer cells, suggesting the cytoprotective role of autophagy (Wang et al., 2016; Meng et al., 2017). Further research is consequently needed to determine whether BER exhibits anticancer effects through modulation of autophagy in ATC cells. As previously reported, the presence of LC3-II has been considered an indicator of autophagosome formation (Mohammadinejad et al., 2019). In the present study, we found that BER triggered autophagy and promoted the conversion of LC3-I to LC3-II. Interestingly, the level of p62 also increased, which is related to the degradation of autophagy. As a stress protein, p62 is also affected by oxidative stress and toxic substances. When these conditions act on cells, p62 also increases, which may be accompanied by an increase in autophagy (Moscat et al., 2016). BER also promoted an increase in the LC3-II green fluorescent puncta number in the cytoplasm. In the presented study, we used 3-methyladenine (3-MA) and bafilomycin A1 (BafA1) to understand the role of BER-induced autophagy. When cells were cotreated with BER and BafA1, the BER-induced LC3-II and p62 accumulation was greater than that of cells treated with BER alone. Thus, these findings indicated that autophagy occurred in BER-treated ATC cancer cells. To further investigate the role of autophagy in BER-induced apoptosis of ATC cells, we used 3-MA, a classical inhibitor of autophagy. The combination of BER with 3-MA significantly attenuated the production of LC3-II and cleaved PARP1 compared to treatment with BER alone, which partly indicated that the BER-regulated autophagy may be a form of cell death and not a protective mechanism. These results suggested that BER represses human ATC cell growth by inducing cytostatic autophagy.
Autophagy and apoptosis are two distinct processes that maintain homeostasis, both of which are associated with the death of tumor cells. The present results indicated that the protein levels of cleaved caspase 3 and cleaved PARP1 were significantly upregulated by BER in a concentration-dependent manner. In addition, the inhibition of ATC cell proliferation was decreased after combined treatment with BER and 3-MA compared to BER treatment alone. Rapamycin (RA), an autophagy inducer that targets mTOR (Pan et al., 2009), significantly reversed the BER-reduced cell viability. Moreover, flow cytometry analysis suggested that RA pretreatment enhanced BER-induced apoptosis, while 3-MA reduced BER-induced cell death in ATC cells. These findings indicated that BER‐induced apoptosis is reduced or increased after the inhibition or promotion of autophagy by pharmaceutical tools of autophagy. The present findings also revealed that BER induces apoptosis and autophagy in ATC cells. Therefore, the relationship between apoptosis and autophagy may be mutually regulated with treatment to achieve anticancer effects. Additionally, both autophagy and apoptosis may be triggered by common upstream signals, resulting in the activation of combined autophagy and apoptosis.
Because the PI3K/AKT signaling pathway plays a fundamental role in thyroid carcinogenesis and progression, it is a potential therapeutic target in ATC treatment (Saji et al., 2010; Xing et al., 2010). Previous studies have demonstrated that inactivation of the PI3K/AKT/mTOR signaling pathway is involved in autophagy induction (Wang et al., 2019; Fan et al., 2016). Thus, the PI3K/AKT/mTOR pathway may be associated with both apoptosis and autophagy, and it may play a crucial role in BER-induced apoptosis and autophagy in ATC cells. Here, western lot analysis indicated that BER significantly blocked the activation of the PI3K and mTOR proteins as evidenced by the decreased phosphorylation of these proteins but increased phosphorylation of AKT. Interestingly, our results were consistent with previous studies (Hyun et al., 2010; Eo et al., 2014) demonstrating that BER treatment activates the AKT signaling pathways in chondrosarcoma and HepG2 cells, resulting in apoptosis. Furthermore, blocking the PI3K/AKT/mTOR pathway by WOR, MK2206, and RA enhanced the BER-induced cell growth inhibition. Therefore, we further confirmed that BER represses ATC cell growth through the PI3K/AKT/mTOR pathway. Importantly, in the present study, we simultaneously investigated the roles of PI3K/AKT/mTOR signaling in BER-induced autophagy and apoptosis, and we found that inhibition of PI3K and mTOR significantly increased BER-induced cytostatic autophagy and apoptosis, leading to significant inhibition of cell proliferation, while SC79 (an AKT activator) elicited the opposite effect. These data indicated that PI3K/AKT/mTOR signaling is involved in BER-induced autophagy and apoptosis.
Because the ability of BER to produce free radicals has been reported in various cancer cell lines (Fang et al., 2021; Kim et al., 2021), we investigated the ability of BER to alter intracellular redox potential in ATC cells. In the present study, BER induced a significant increase in ROS generation, which was clearly inhibited by an ROS scavenger (NAC). In addition, a decreased level of ROS through pretreatment with NAC reversed the BER-induced cell viability decrease, apoptosis, and autophagy. These findings demonstrated that BER induces the generation of ROS, which contributes to cell death via apoptosis and/or autophagy.
DOX has been approved by the American Thyroid Association (ATA) guidelines as a conventional single agent in ATC treatment (Smallridge et al., 2012). However, the antitumor activities of combining DOX with BER in ATC are unknown. Thus, we tested the synergistic anticancer effects of DOX and BER in combination on ATC cancer cell lines. The combination therapy of DOX and BER significantly inhibited ATC cell proliferation and induced cell apoptosis compared to DOX or BER monotherapy. In addition, BER combined with DOX exhibited a synergistic inhibitory effect in ATC cells with CI values less than one, indicating that these two compounds in combination markedly exert synergistic growth inhibitory effects on ATC cells. Furthermore, concomitant treatment with DOX and BER enhanced cell autophagy by enhancing the expression of p62 and LC3-II. These data demonstrated that cotreatment with DOX and BER markedly exerts a synergistic growth inhibitory effect on ATC cells by triggering apoptosis and autophagic cell death. Therefore, cotreatment with DOX and BER significantly inhibits cell growth and may be a promising strategy for ATC chemoprevention.
In conclusion, the present results demonstrated that BER exerts antitumor effects in ATC by inhibiting proliferation, promoting apoptosis, and inducing autophagy through the PI3K/AKT/mTOR signaling pathway. In this process, ROS act as a potential target of BER-mediated induction of autophagy and apoptosis. Furthermore, we demonstrated that cotreatment with DOX and BER exhibits synergistic chemopreventive effects by inducing apoptosis and autophagic cell death. Taken together, these results suggested that cotreatment with DOX and BER may be a potential therapeutic approach for ATC. Our findings may shed light on the underlying mechanisms of the anti-ATC effects of BER (Fig. 8), which may serve as a promising drug for treating ATC.