*3.4. B Induces Caspase-Dependent Apoptosis and Autophagy in Caco2 Cells*

Activation of the caspase dependent apoptosis by **B** in Caco2 cells was further confirmed by Western blot analysis in the presence or absence of Z-VAD-FMK. The level of the pro apoptotic protein BAX was significantly increased upon treatment with **B** or EL000327 at cytotoxic concentrations for 12 h. However, a significant change in the levels of BAX were not observed in cells that had been pretreated with Z-VAD-FMK. In contrast, the expression level of the anti-apoptotic protein Bcl-xL was decreased in the Caco2 cells after treatment with **B** or EL000327 for 24 h, but no change in the presence of Z-VAD-FMK (Figure 4a,b). Clear cleavage of the main apoptotic markers PARP and caspase-3 was detected after treatment with **B** (20, 60 μg/mL) or EL000327 (60 μg/mL) for 48 h in the absence of Z-VAD-FMK (Figure 4c). The effect of **B** and EL000327 on the activation of the JNK/c-jun and Akt signaling pathways was assessed, as these pathways eventually lead to the induction of apoptosis as well as autophagy in cells. Phosphorylation of JNK and c-jun was markedly induced upon treatment with **B** at IC50 concentrations for 48 h and decreased the phosphorylation of Akt. However, no significant changes in protein levels were detected in cells treated with EL000327 (Figure 4d). Regulation of the autophagy related proteins Beclin 1, p62, and LC3BI/II was also examined in the presence or absence of Z-VAD-FMK. Upregulation of Beclin 1 and LC3BI/II and downregulation of the p62 protein levels were observed in Caco2 cells upon treatment with cytotoxic concentrations of **B** or EL000327, indicating the inhibition of autophagosome degradation in these cells. However, consistent regulation patterns were not observed for these autophagy markers in the presence of Z-VAD-FMK (Figure 4e,f).

In order to confirm the major cell death pathway induced by **B** and crude extract EL000327, cell viability was assessed after pretreatment with apoptotic and autophagic inhibitors. Treatment with Z-VAD-FMK significantly increased the cell viability in Caco2 cells in response to treatment with **B** or EL000327. In contrast, the application of 3MA (3-methylladenine), a blocker of autophagosome formation, and CQ (chloroquine), an inhibitor of lysosomal acidification and autophagosome degradation, resulted in a dose dependent decrease in cell viability, indicating that the inhibition of autophagy enhanced the cytotoxicity of **B** toward Caco2 cells (Figure 4g). Of the autophagy blockers, CQ had a more potent effect on the induction of cell death than 3MA. Application of CQ markedly increased the levels of cleaved PARP and caspase-3 and decreased the level of Bcl-xL in the cells treated with **B** for 48 h (Figures 4h and S5b). These data suggest that **B** induced caspase dependent apoptosis mainly via the activation of the JNK/c-jun pathway in Caco2 cells. Furthermore, the observed induction of autophagy by **B** may activate the protective mechanism in cells.

**Figure 4.** Inhibition of **B** induced autophagy increases apoptosis in Caco2 cells. (**a**) Western blot analysis of the pro-apoptotic protein BAX and the anti-apoptotic protein Bcl-xL treated by **B** (20 or 60 μg/mL) or EL000327 (60 μg/mL) for 12 or 24 h in the presence or absence of Z-VAD-FMK (10 μM).

(**b**) Quantification of BAX and Bcl-XL protein expressions. (**c**) Western blot of apoptotic proteins; PARP, Caspase-3treated by **B** (20 or 60 μg/mL) or EL000327 (60 μg/mL) for 48 h in the presence or absence of Z-VAD-FMK. (**d**) Expressions of the apoptotic signaling pathway related proteins p-JNK, JNK, p-c-jun, c-jun, p-AKT, and AKT in Caco2 cells treated with **B** (20 μg/mL) or EL000327 (60 μg/mL) for 48 h, as analyzed by Western blotting. (**e**) Western blot of autophagy related proteins; Beclin 1 (12 h), p62 (24 h), and LC3BI/II (48 h) in Caco2 cells pre-incubated with or without Z-VAD-FMK, and treated with **B** (20, 60 μg/mL) or EL000327 (60 μg/mL). (**f**) Quantification of Beclin 1 and p62 protein expressions. (**g**) The relative percentage cell viability of Caco2 cells treated with **B** (20, 60 μg/mL) or EL000327 (60 μg/mL) for 48 h, with or without Z-VAD-FMK (10 μM) and autophagy inhibitors 3 MA (1 mM) and CQ (10 μM). (**h**) Expression levels of PARP, caspase-3, and Bcl-xL determined by Western blot analysis after treatment with **B** (20, μg/mL) or EL000327 (60 μg/mL) for 48 h in the presence or absence of CQ (10 μM). Data represent the mean ± S.D. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001; NS: no significant difference (*p* > 0.05), compared with the Z-VAD-FMK, 3MA, and CQ treated groups or the DMSO-treated control.

#### *3.5. B Activates JNK/c-Jun Signaling Pathway via Triggering ROS Generation*

Intracellular ROS play a vital role in activating cellular apoptotic and autophagic functions. To assess the effect of **B** on ROS generation, cells were treated with **B** or EL000327 at toxic concentrations for 12 h in the presence or absence of the antioxidant N-acetyl cysteine (NAC), and ROS were detected by staining cells with DCFH-DA, followed by fluorescence microscopy or flow cytometry. While exposure of Caco2 cells to **B** dramatically increased the green fluorescent signals indicative of ROS generation, pretreatment with NAC significantly reduced the fluorescent signals, suggesting a block in ROS generation (Figure 5a). The number of fluorescently labeled cells decreased upon treatment with NAC, as detected by flow cytometry. No significant change in fluorescence emission was detected in the EL000327 treated cells after 12 h compared to the control cells (Figure 5b,c). Thus, pretreatment with NAC decreased the expression of apoptotic and autophagy markers and rescued cells from **B**-induced apoptosis and autophagy. Furthermore, NAC reversed the phosphorylation of JNK and c-jun in cells treated with IC50 concentrations of **B** (Figure 5d,e). Collectively, these results indicate that **B** activates the ROS/JNK signaling pathway in Caco2 cells.

**Figure 5.** *Cont*.

**Figure 5. B** induces ROS generation and activates JNK signaling in Caco2 cells. (**a**) Intracellular ROS generation was detected by fluorescence microscopy using DCFH-DA (10 μM) in Caco2 cells treated with **B** (20, 60 μg/mL) or EL000327 (60 μg/mL) for 12 h with or without the ROS inhibitor NAC (5 mM). (**b**) Flow cytometric analysis of the fluorescence intensity of Caco2 cells preincubated with DCFH-DA (10 μM) and treated with **B** (20, 60 μg/mL) or EL000327 (60 μg/mL) for 12 h with or without NAC (5 mM). (**c**) Quantification of the mean fluorescence intensity of Caco2 cells preincubated with DCFH-DA (10 μM) and treated with the indicated concentrations of **B** for 12 h in the presence or absence of NAC. (**d**) Western blot analysis of PARP, caspase-3, Bcl-xL, p62, LC3BI/II, p-JNK, JNK, p-c-jun, and c-jun protein expression after treatment with **B** (20, μg/mL) or EL000327 (60 μg/mL) for 24 or 48 h, with or without NAC (5 mM). (**e**) Quantification of Bcl-XL and p62 protein expressions in the presence or absence of NAC. Data represent the mean ± S.D. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001; NS: no significant difference (*p* > 0.05) compared with the NAC-treated group or the DMSO-treated control.
