Induction in melanoma cells (Beck et al.,

Induction of prolonged ER stress or suppression of ER stress adaptation mechanisms were proposed as alternative strategies to overcome PLX4032 resistance in melanoma cells (Beck et al., 2013; Cerezo et al., 2016). We used immunoblotting to check the expression of PERK, ATF6 and IRE1? in A375-R cells upon CUMA treatment as representative markers of ER stress response. CUMA treatment (20 µM) resulted in the initiation of ER stress, concluded shown by the time-dependent increase in the expression of IRE1?, but with no significant changes in the expression of PERK or ATF6 (Figure 5A). The cleavage form of apoptotic hallmark PARP was increased upon the compound treatment (Figure 5A).
Increasing evidence shows that prolonged ER stress cause not only apoptosis but also another type of programmed cell death, autophagic-cell death (Salazar et al., 2009; Cerezo et al., 2016). Our quantitative real-time polymerase chain reaction (qPCR) data showed that expression of autophagy-related genes including ATG13, ATG12, LC3B, LAMP2 were increased in A375-R cells treated with 20 µM CUMA after 24 h (Figure 5B). To test the effect of CUMA in the induction of autophagic-cell death, we used two different approaches: fluorescence microscopy to visualize the accumulation of LC3B puncta; and immunoblotting to measure the conversion of LC3B-I to LC3B-II as those are indicators of autophagosomes formation (Klionsky et al., 2016; Murugan and Amaravadi, 2016). As expected, CUMA promoted the accumulation of autophagosomes as observed by the increased LC3B puncta and enhanced LC3B fluorescence compared to the vehicle-treated cells (Figure 5C). Accumulation of autophagosomes may result from induction (activation of autophagy) or blockage (inhibition of autophagy) of autophagy flux. As presented in Figure 5D, the level of LC3B-II induced by CUMA can be further enhanced when co-incubated with autophagy inhibitor Bafilomycin A1 (Baf A1), suggesting that CUMA increased the autophagic flux, rather than blockage of its degradation (Figure 5D). The activation of autophagy was observed in vivo as well, as indicated by the increased LC3 staining in the tumors of mice treated with CUMA, PLX4032 and CUMA in combination, but to a much lesser degree in PLX4032 treated mice (Figure 5E).
To further investigate the role of CUMA-induced ER stress in cell death we co-treated A375-R cells with CUMA and a chemical chaperone 4-phenylbutiric acid (4-PBA) which attenuates ER stress by promoting protein folding and protein stabilization (Zhang et al., 2013). Thapsigargin (Tg) was used as a positive control for ER stress (Healy et al., 2009) and autophagy induction. As expected, 4-PBA reduced the expression of IRE1? both basally and in response to CUMA (Figure 5F). Interestingly, CUMA-induced cleavage of PARP was moderately reversed when co-treated with 4-PBA, implying that CUMA-induced ER stress is partially responsible for the A375-R apoptosis (Figure 5F). Furthermore, conversion of LC3B-I to LC3B-II was also reduced suggesting that autophagy may be the downstream effect of CUMA-induced ER stress (Figure 5F). Emerging evidence shows that autophagy plays a dual role in tumor cell death, cytoprotective, to restore the cellular homeostasis, or cell damaging to promote cancer cell death (Scarlatti et al., 2009; Tomic et al., 2011; Liu and Debnath, 2016, Giglio et al., 2015)). To investigate the role autophagy plays in CUMA-induced cell death, we used two autophagy inhibitors, 3-methyladenine (3-MA), which blocks autophagosome formation and chloroquine (CQ), which blocks autophagosome-lysosome fusion (Klionsky et al., 2016). However, when A375-R cells were first pretreated for one hour with either 3-MA (4 mM) or CQ (40 µM) and then additionally treated with CUMA for 24 h, there was no significant alteration in A375-R cell viability compared to CUMA-only treated cells (Figure S4B). The role of autophagy in CUMA-induced cell death and interplay with ER stress requires further evaluation.