br In addition to confirm that ER stress was regulated
In addition to confirm that ER stress was regulated by ROS, the HC030031 were treated with the ROS scavenger NAC along with CBD. As shown in Fig. 4E, CBD remarkably increased ER stress-related proteins, and NAC partially reversed this eﬀect of CBD, suggesting that ROS induced by CBD contributes to the increased expression levels of Noxa via ER stress.
3.5. CBD inhibits tumor growth in vivo by upregulating the expression of noxa, thereby inducing apoptosis
To verify the results obtained, we used two PDC cell lines. As shown as Fig. 5A, CBD diminished the viability of PDC cell lines. In addition, the intensity of Caspase 3/7-stained cells was increased by CBD treat-ment in PDC cell spheroids in three-dimensional culture (Fig. 5B and C).
Next, we examined the eﬀect of CBD on the tumorigenicity ability in vivo. The HCT116 Luc+ cells (1 × 107 in 100 μL) were subcutaneously injected into BALB/c nude mice, and the tumor size was measured twice a week. When the tumor size was 150 mm3, the luminescence and tumor size in CBD-treated mice were markedly lower than those in the control mice (Fig. 5D, E and 5F), suggesting that CBD decreases the growth of CRC cells in vivo. To unveil whether apoptosis plays a role in CBD-induced growth inhibition, we performed TUNEL assay and im-munohistochemistry on the tumor tissue. The number of TUNEL-posi-tive cells and the level of Noxa protein were higher in CBD-injected tumors, compared to the control tumors (Fig. 5G and H), indicating that CBD induces apoptosis in vivo by Noxa activation.
Because of the diﬀerent problems associated with the chemother-apeutic drugs currently used for CRC, many studies have concentrated on alternative therapies that are safer and less toxic such as those employing natural products . In this study, we focused on the cy-totoxic eﬀect exerted by the natural product CBD. Previous studies have suggested that the anticancer eﬀect of CBD is associated with its ability to induce apoptosis; in fact, several related signaling pathways have been reported [14,19,25,27]. We explored the mechanisms by which CBD mediated apoptosis in human CRC cell lines and investigated the complex relationship between CBD-induced Noxa activation, apoptosis, and generation of ROS/ER stress.
We found that CBD induced dose-dependent growth inhibition and apoptosis in human CRC cells, but not in normal colorectal cells. Apoptosis induction was confirmed by the cleavage of PARP and cas-pase-3, caspase-8, and caspase-9. Moreover, CBD increased the number of Annexin V/PI double-positive cells and TUNEL-positive cells. CBD also decreased tumor volume and promoted apoptosis in a xenograft model. In the xenograft model using prostate cancer cells, an adverse eﬀect was not observed when 100 mg/kg CBD was injected daily for 5 weeks. Thus, the 20 mg/kg dose used in our experiment did not exert any adverse eﬀect on the mice .
One major finding of this study is that Noxa activation is important for CBD-induced apoptosis in human CRC cells; this has not been re-ported previously, to the best of our knowledge. The expression of Bcl-2 Homology 3 domain-only protein, namely, Noxa, was increased by CBD treatment in a dose- and time-dependent manner. Unfortunately, CBD did not aﬀect other Bcl-2 family member proteins except truncated BID (t-BID), which was activated later timepoint than when Noxa was ac-tivated (data not shown). After CBD treatment, MMP was decreased and ROS was produced in a noticeably short time, suggesting that when CBD-induced apoptosis occurs, CBD induces mitochondrial dysfunction directly rather than through other Bcl-2 family proteins. In addition, CBD induced Noxa activation and apoptosis, which could be blocked by siRNA knockdown of Noxa. Noxa expression is regulated by p53 . However, in our system, CBD treatment did not cause any changes in the Noxa levels and cell death in the p53 knockout CRC cell lines, implying that CBD causes Noxa-induced apoptosis through the p53-independent pathway. Cancer Letters 447 (2019) 12–23
Excessive ROS is considered to be toxic, with the ability to induce oxidative damage to biological macromolecules, initiating the perox-idation of membrane lipids and causing the accumulation of lipid per-oxides and damage of DNA and proteins . Mitochondria are the major cellular organelles producing ROS, and within the mitochon-drion, the electron transport chain is the primary site of ROS generation . CBD elevated the oxygen consumption rate and enhanced mi-tochondrial bioenergetics by regulating the mitochondrial complex Ⅰ and Ⅳ . From this point of view, it seems that the CBD-induced mitochondrial ROS overproduction in CRC cells was caused by the regulatory eﬀects of CBD on mitochondrial complex Ⅰ and Ⅳ. However, further studies are necessary to unveil the mechanisms responsible for excessive mitochondrial ROS generation by CBD-treated CRC cells. Moreover, Noxa caused ROS production, which is likely to further ex-acerbate the apoptosis induced by CBD . Thus, these findings to-gether suggest that CBD induces apoptosis of CRC cells by triggering mitochondrial dysfunction.