br Noxa activation is responsible for
3.2. Noxa activation is responsible for CBD-induced apoptosis
Since Noxa plays important roles in the regulation of apoptosis , we confirmed the levels of Noxa mRNA and protein. Immunoblotting analysis showed that CBD increased the mRNA and protein levels of Noxa in a time-dependent manner (Fig. 2A, B and 2C). In addition, the fluorescence intensity of Noxa was significantly higher in CBD-treated cells, compared to the control HC-030031 (Fig. 2D).
To unveil the role of Noxa activation in CBD-induced apoptosis, CRC cells were transfected with Noxa-specific siRNA. Compared to the cells treated with control siRNA, Noxa knockdown attenuated CBD-induced apoptotic cell death in both cell lines, as shown by the numbers of Annexin V/PI double-stained cells as well as levels of PARP, caspase-3, -8 and -9 cleavage (Fig. 2E, F, and 2G).
Since Noxa is regulated by p53 , we examined whether CBD-induced Noxa activation is associated with p53. Microarray and wes-tern blot results showed that p53 was increased by CBD. However, when p53 was knocked out, p53 was not changed, but Noxa and apoptosis were increased (Suppl. Fig. 1A and 1B). Taken together, these findings demonstrated that Noxa plays an important role in apoptotic cell death by CBD independently of p53 in CRC cells. Cancer Letters 447 (2019) 12–23
3.3. CBD induces ROS generation and endoplasmic reticulum (ER) stress
To elucidate the mechanisms underlying CBD-induced apoptosis, microarray analysis was performed. Gene set enrichment analysis showed that ROS and unfolded protein response (UPR) were increased by CBD treatment (Fig. 3A). Therefore, we examined the potential in-volvement of ROS production in CBD-induced apoptosis. The eﬀect of CBD on the generation of superoxide was investigated by monitoring the intensity of DCF-DA. CBD treatment enhanced intracellular super-oxide production, and treatment with NAC, an antioxidant, eﬀectively blocked the levels of intracellular superoxide (Fig. 3B and Suppl. Fig. 2A). Because ROS is mainly produced in the mitochondria , we measured mitochondrial ROS production to determine whether the CBD-induced ROS generation was derived in the mitochondria. As shown in Fig. 3C, Suppl. Fig. 2B and 2C, CBD increased mitochondrial superoxide levels from an early time and NAC inhibited this eﬀect of CBD. CBD also induced mitochondrial dysfunction by TMRE fluores-cence, an indicator of MMP (Suppl. Fig. 2D). Moreover, antioxidant enzymes including SODs and Catalase were decreased by CBD treat-ment (Fig. 3D), implying that CBD causes mitochondrial dysfunction and excessive ROS generation especially mitochondrial superoxide.
ROS and ER stress are closely related . Previous studies showed that ROS induces ER stress in CRC cells [1,34]. DLD-1 and HCT116 cells were treated with 6 μM CBD, and the levels of ER stress-related proteins were examined by western blot. CBD dramatically elevated ER trans-membrane receptors (IRE1α and PERK), ER chaperone protein (Bip, GRP94), and transcription factor CHOP in a dose- and time-dependent manner (Fig. 3E and F). Together, CBD triggers mitochondrial dys-function-induced ROS overproduction and excessive ER stress through IRE1α and PERK.
3.4. CBD-induced noxa regulation leads to apoptosis by causing excessive ROS and ER stress
To further explore the role of ROS generation in CBD-induced CRC cell death further, NAC-mediated blockade of ROS was performed. The results showed that the NAC suppressed CBD-induced apoptosis as re-vealed by the level of cleaved PARP (Fig. 4A) and the number of An-nexin V/PI double-stained cells (Fig. 4B), suggesting that CBD triggers ROS overproduction and subsequent apoptosis in human CRC cells. Because Noxa causes mitochondrial dysfunction, which is mainly in-duced by ROS [22,37], we investigated the possible link between ROS overproduction and Noxa activation in CBD-treated CRC cells. We found that CBD-induced increase in Noxa levels was remarkably atte-nuated by NAC pre-treatment in both CRC cell lines (Fig. 4A). Together, these findings indicated that ROS overproduction is linked to the Noxa activation, and that CBD-induced apoptotic cell death is responsible for the ROS-mediated Noxa activation.
To confirm that the relationship between ER stress and CBD-induced apoptosis, CRC cells were first transfected with CHOP siRNA. Then, the number of Annexin V/PI double-stained cells and the levels of Cleaved PARP were measured by western blotting and flow cytometry, respec-tively. CHOP knockdown significantly reversed the increases in both the Cleaved PARP level and Annexin V/PI double-stained cell count (Fig. 4D). Furthermore, CBD activated the Noxa protein and increased the extent of staining levels of Noxa, which could be further blocked by CHOP siRNA (Fig. 4C and F). ChIP assay was performed to confirm that CHOP directly regulates Noxa. As shown in Suppl. Fig. 3A and 3B, CHOP did not bind to the Noxa promoter. Since ATF3 and ATF4 bind to Noxa and CHOP promoter to regulate the apoptosis , we examined whether CBD aﬀects the activity of binding to the Noxa and CHOP promoter regions. As shown in Suppl. Fig. 3C and 3D, CBD increased the binding of ATF3 and ATF4 to the promoters of Noxa and CHOP. These data together imply that excessive ER stress contributes to the CBD-dependent Noxa activation and apoptotic cell death by ATF3 and ATF4.