Cytotoxic Effects of Cannabinoids on Human HT-29 Colorectal Adenocarcinoma Cells : Different Mechanisms of THC, CBD, and CB83
In this study, we investigated the effects of exposition to IC50 dose for 24 h of a new synthetic cannabinoid (CB83) and of phytocannabinoids Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) on HT-29 colorectal carcinoma cells. Cell viability and proliferative activity evaluated using the MTT, lactate dehydrogenase (LDH), and CyQUANT assays showed that cell viability was significantly affected when CB83, THC, and CBD were administered to cells. The results obtained showed that the reduced glutathione/oxidized glutathione ratio was significantly reduced in the cells exposed to CBD and significantly increased in the cells treated with the CB83 when compared to the controls. CBD treatment causes a significant increase in malondialdehyde content. The catalase activity was significantly reduced in HT-29 cells after incubation with CB83, THC, and CBD. The activities of glutathione reductase and glutathione peroxidase were significantly increased in cells exposed to THC and significantly decreased in those treated with CBD. The ascorbic acid content was significantly reduced in cells exposed to CB83, THC, and CBD. The ultrastructural investigation by TEM highlighted a significantly increased percentage of cells apoptotic and necrotic after CB83 exposition. The Annexin V-Propidium Iodide assay showed a significantly increased percentage of cells apoptotic after CB83 exposition and necrotic cells after CBD and THC exposition. Our results proved that only CBD induced oxidative stress in HT-29 colorectal carcinoma cells via CB receptor-independent mechanisms and that CB83 caused a mainly CB2 receptor-mediated antiproliferative effect comparable to 5-Fuorouracil, which is still the mainstay drug in protocols for colorectal cancer.
Keywords : HT-29 cells; apoptosis; cannabidiol; oxidative stress; synthetic cannabinoid; Δ9-tetrahydrocannabinol.
Cannabinoids obtained from Cannabis sativa and their derivatives produce many biological eects, mainly through interactions with specific receptors such as CB1 and CB2, which have been cloned and characterized [1,2]. Moreover, the orphan G protein coupled receptor 55 (GPR55), the transient receptor potential cation channel subfamily V member 1 (TRPV1), and peroxisome proliferator-activated receptors (PPARs) have been reported as possible receptors for endogenous cannabinoids [3,4]. Given the many eects of cannabinoids and the evidence demonstrated by preclinical studies, it is possible to assume a potential use of these substances in the medical field. To date, cannabinoids have been used in the treatment of nausea and vomiting in cancer patients undergoing chemotherapy, but the use of cannabinoids in oncology is likely to be limited, although there is evidence showing that cannabinoids are able to inhibit cell growth in dierent cancer cell lines  and to exert antitumor effects in experimental animal models .
Through cannabinoid receptor and nonreceptor signaling pathways, cannabinoids show specific cytotoxicity against tumor cells while protecting healthy tissue from apoptosis. Bogdanovi´c et al.  investigated the proapoptotic and antiproliferative eects of cannabinoids and associated signaling pathways in dierent cancer cell lines, and it has been demonstrated that natural and synthetic cannabinoids cause a CB1 and/or CB2 receptor-dependent decrease in the proliferation of breast and
intestinal cancer cells [5,8]. Cannabinoids impair tumor progressions at various levels. Their main
effect is the induction of cancer cell death by apoptosis and the inhibition of cancer cell proliferation.
At least one of those actions has been demonstrated in almost all cancer cell types tested . Cannabinoid treatments aect directly the viability of a great variety of cancer cells via the induction of apoptosis or cell cycle arrest [10,11]. The psychotropic cannabinoid, the D9-tetrahydrocannabinol (THC, Figure 1), induces apoptosis in a variety of transformed and nontransformed cells, including those of immune origin [10,12]. It was observed that THC treatment induces significant levels of apoptosis in leukemias and lymphocytes in culture, as well as in the murine thymus and spleen [12–14], showing that THC may impair T-cell functions through the induction of apoptosis. Moreover, cannabidiol (CBD, Figure 1), a nonpsychotropic cannabinoid, has also been reported to induce apoptosis in several transformed or immortalized cells, including K-ras-thyroid epithelial, C6 glioma, malondhyaldhehyde (MDA)-MB-231 breast carcinoma, HL-60, and Jurkat leukemia cells [5,15,16].
In addition, many evidences suggest that cannabinoids damage tumor angiogenesis and block invasion and metastasis [6,17]. The role of reactive oxygen species (ROS) in regulating apoptosis is supported by many evidences , and the production of ROS during apoptosis has been described in various models of apoptotic cell death . Cancer cells seem to possess higher levels of endogenous ROS compared to normal cells, but events that increase ROS levels above a certain threshold seem to be incompatible with the cellular survival. Thus, compounds that increase the ROS level or that impair the cellular antioxidant system will shift the redox balance, inducing cancer cell cytotoxicity .
Our previous study in the cannabinoid field led to the development of a new class of synthetic cannabinoid ligands [21–25] chemically characterized by a substituted resorcinol nucleus linked to fatty acid amides. In fact, their structure merges the crucial pharmacophoric requirements for the cannabinoid receptor binding of both THC and anandamide (AEA, Figure 1), the main endogenous cannabinoid, such as a rigid aromatic backbone bearing an alkyl tail and a flexible saturated chain
with an amidic head. Among these derivatives, CB83 , Figure 1, belonging to the 5-(10,10 dimethylheptyl)resorcinol class, was selected for its balanced potency (Ki CB1 = 310 nM and Ki CB2 = 30 nM) and selectivity.