The generation of ROS in addition to eliciting oxidative str
The generation of ROS, in addition to eliciting oxidative stress, is an important proapoptotic signal (Ott et al., 2007). Apoptosis is clearly associated with the production of ROS, since ROS generation occurs in the mitochondrial respiratory chain and disturbs mitochondrial homeostasis, which plays a pivotal role in regulating apoptosis and proapoptotic signals (Ryter et al., 2007). In the present study, we observed increased ROS generation, as well as an increase in [Ca2+]i levels, which is considered an important trigger for neuronal cell death (Lee et al., 2012a, Lee et al., 2012b, Cao et al., 2013). Elevated intracellular Ca2+ could either directly activate caspases in neuronal (R)-(-)-Niguldipine hydrochloride kinase or increase Ca2+ flux into the mitochondria, which would subsequently trigger the release of cytochrome c from the mitochondria (Lee et al., 2015). In this study, excessive ROS generation together with increased [Ca2+]i could trigger apoptosis in TDCIPP-treated SH-SY5Y cell cells. A previous study has shown that exposure to neurotoxicants such as chlorpyrifos could cause ROS generation, leading to apoptosis in cultured SH-SY5Y cells (Ki et al., 2013). Thus, generation of excess ROS and increase in [Ca2+]i are important factors triggering apoptosis in neuronal cells. The endoplasmic reticulum (ER) is involved in many apoptotic pathways, and ER stress plays a key role in apoptosis (Rao et al., 2004, Ron and Walter, 2007). Several studies have shown that ROS can cause oxidative damage to the ER, leading to ER stress (Zhang et al., 2014, Park et al., 2014). Hence, we further investigated the role of ER signaling in apoptosis. Excessively oxidization in the ER can cause the accumulation of misfolded proteins and trigger the unfolded protein response (UPR) (Ron and Walter, 2007). In the current study, we observed increased expression of the ER stress markers GRP78, p-eIF2α, and ATF4. eIF2a-ATF4 signaling has a pro-apoptotic function; CHOP is expressed at low levels, but it can be dramatically induced by ER stress at the transcriptional level via ATF4, ATF6, and XBP-1 pathways in response to ER stress-mediated apoptosis (Huang et al., 2011, Oyadomari and Mori, 2004). The initiation of ER stress-related apoptotic pathways increases CHOP gene expression, triggering ER stress-specific apoptotic cascades (Su et al., 2013). In the present study, we showed that the expression of the proapoptotic factor CHOP is increased in SH-SY5Y cells following TDCIPP treatment. Thus, ER stress induced by TDCIPP is sufficient to activate the proapoptotic pathways of the UPR. Our results thus demonstrated that TDCIPP induces ER stress through the PERK/eIF2a pathway in SH-SY5Y cells. To confirm the role of ER stress in TDCIPP-induced apoptotic cell death in SH-SY5Y cell, PBA, an inhibitor of ER stress was applied (Boussabbeh et al., 2015, Su et al., 2013), and effectively abolished TDCIPP-induced ER stress, as shown by decreased GRP78 and CHOP expression. Consequently, reduced cell death and a decrease in apoptotic ratio were observed in PBA-pretreated cells. Prolonged activation of CHOP promotes apoptosis through down-regulation of the anti-apoptotic protein Bcl-2 and up-regulation of the proapoptotic protein Bax in Bax-mediated mitochondrial dysfunction and apoptosis (Tabas and Ron, 2011). We showed that Bax plays an important role in the induction of apoptosis, since pretreatment with PBA could reduce ER stress and the Bax/Bcl-2 ratio, and reduced cell death. Therefore, TDCIPP-induced apoptotic cell death in SH-SY5Y cells likely involved the ER stress pathway. Furthermore, we showed that scavenging of ROS by the antioxidant NAC inhibits the ER stress response and effectively protects cells from TDCIPP-induced apoptosis. Elimination of ROS generation was accompanied by reduced cell death, a decreased apoptotic ratio, and the loss of ΔΨm. This further demonstrated the role of ROS in TDCIPP-induced cell apoptosis. Thus, exposure to TDCIPP causes the generation of excess ROS and an increase in [Ca2+]i, thereby triggering ER stress and mitochondrial damage, resulting in apoptotic death in the SH-SY5Y cells (Fig. 6).