br ROS is well known ER
ROS is well-known ER stress inducer . ER stress triggers the activation of UPR, which functions as an adaptive response and further leads to apoptosis . UPR is distinguished by three ER transmem-brane receptor proteins, namely, IRE1α, PERK, and ATF6 . In resting cells, all three receptors are associated with the ER resident chaperone Bip, which maintains them in an inactive state. On accu-mulation of unfolded proteins in the ER, the three receptors dissociate from Bip, leading to their activation. On activation, the three receptors induce signal transduction, which reduces the accumulation of mis-folded proteins by increasing the expression of ER chaperones [21,29]. Consistent with this, we found that the expression of ER stress-related genes was reduced in CBD-treated CRC cells, and that it is regulated by ROS. NAC, a ROS scavenger, eﬀectively blocked CBD-induced genera-tion of ROS and ER stress and attenuated CBD-induced apoptotic cell death. Moreover, ROS and ER stress are associated with Noxa activa-tion. Interestingly, CHOP indirectly regulates Noxa activation. We speculated that activating transcription factor 3 (ATF3) and ATF4 are involved in CBD-induced CHOP and Noxa activation. ER stress activates ATF3 and ATF4 and increases their complex formation to activate Noxa and CHOP . Our results showed that ATF3 and ATF4 bind directly to ATF/cAMP responsive element-binding sites in the promoter regions of Noxa and CHOP, inducing CBD-induced Noxa and CHOP activation. Thus, CBD-induced Noxa and CHOP are expected to be mediated by upregulation of ATF3 and ATF4 binding to their promoter regions.
To summarize, CBD induces apoptotic cell death in CRC cells. This increase in apoptosis can be attributed to its ability to trigger the generation of excessive mitochondrial ROS and ER stress and sub-sequent activation of Noxa (Fig. 6). Thus, our results provide important evidence for the use of CBD as an alternative therapeutic agent for CRC.
Conflicts of interest
No potential conflicts of interest to declare.
Soyeon Jeong and Hye Kyeong Yun participated in study design and conception, execution of the experiments, data analysis and inter-pretation, and manuscript writing; Yoon A Jeong participated in mice tumor tissue processing and immunofluorescence of tumor tissue; Min Jee Jo participated in the primary culture of patient derived CRC Tigecycline and in vivo experiments; Sang Hee Kang participated in the study con-ception and analysis of microarray data; Jung Lim Kim and Dae Yeong Kim participated in the collection and assembly of data and in vivo experiments; Sun Il Lee, Seong Hae Park, Bo Ram Kim and Yoo Jin Na participated in interpretation of data and designing of figures; Han Do Kim and Dae Hyun Kim provided Cannabidiol; Dae-Hee Lee and Sang Cheul Oh participated in the conception and design, financial support, interpretation of study, and supervised all of the experiments. All
Fig. 6. Scheme of apoptosis pathway induced by CBD.
authors approved the submission of the final manuscript.
This work was supported by the National Research Foundation of Korea grant funded by the Korea government (MSIP) (NRF-2017R1D1A1B03030703) and supported by the Business for Cooperative R & D between Industry, Academy, and Research Institute funded Korea Small and Medium Business Administration in 20 (C0566291) and a Korea University Grant.
Appendix A. Supplementary data
 M. Guzman, Cannabinoids: potential anticancer agents, Nat. Rev. Canc. 3 (2003) 745–755.  N. Hatsugai, V. Perez Koldenkova, H. Imamura, H. Noji, T. Nagai, Changes in cy-tosolic ATP levels and intracellular morphology during bacteria-induced hy-persensitive cell death as revealed by real-time fluorescence microscopy imaging, Plant Cell Physiol. 53 (2012) 1768–1775.  K. Iﬄand, F. Grotenhermen, An update on safety and side eﬀects of cannabidiol: a review of clinical data and relevant animal studies, Cannabis Cannabinoid Res. 2
S. Iida, Lower expression of activating transcription factors 3 and 4 correlates with shorter progression-free survival in multiple myeloma patients receiving borte-zomib plus dexamethasone therapy, Blood cancer J 5 (2015) e373.  E. Oda, R. Ohki, H. Murasawa, J. Nemoto, T. Shibue, T. Yamashita, T. Tokino,
B. Hinz, COX-2 and PPAR-gamma confer cannabidiol-induced apoptosis of human lung cancer cells, Mol. Canc. Therapeut. 12 (2013) 69–82.  S.K. Saha, A.R. Khuda-Bukhsh, Molecular approaches towards development of purified natural products and their structurally known derivatives as eﬃcient anti-cancer drugs: current trends, Eur. J. Clin. Pharmacol. 714 (2013) 239–248.