Antitumor Activity of Plant Cannabinoids with Emphasis on the Effect of Cannabidiol on Human Breast Carcinoma, Journal of Pharmacology and Experimental Therapeutics, 2006, Alessia Ligresti, Aniello Schiano Moriello, Katarzyna Starowicz, Isabel Matias, Simona Pisanti, Luciano De Petrocellis, Chiara Laezza, Giuseppe Portella, Maurizio Bifulco and Vincenzo Di Marzo
L’articolo pubblicato nel 2006 dimostra come il cannabidiolo esibisca un effetto antitumorale in numerose cellule cancerogene nettamente superiore rispetto al THC ed agli altri cannabinoidi, senza, tra l’altro avere alcun tipo di effetto sulle cellule non tumorali. Lo studio indica che, sia in vitro che in vivo, il cannabidiolo o gli estratti di cannabis arricchiti con cannabinoidi naturali rappresentino una potente strategia antineoplatistica non psicoattiva, in particolar modo per dei tumori al seno molto maligni.
“Δ9-Tetrahydrocannabinol (THC) exhibits antitumor effects on various cancer cell types, but its use in chemotherapy is limited by its psychotropic activity. We investigated the antitumor activities of other plant cannabinoids, i.e., cannabidiol, cannabigerol, cannabichromene, cannabidiol acid and THC acid, and assessed whether there is any advantage in using Cannabis extracts (enriched in either cannabidiol or THC) over pure cannabinoids. Results obtained in a panel of tumor cell lines clearly indicate that, of the five natural compounds tested, cannabidiol is the most potent inhibitor of cancer cell growth (IC50 between 6.0 and 10.6 μM), with significantly lower potency in noncancer cells. The cannabidiol-rich extract was equipotent to cannabidiol, whereas cannabigerol and cannabichromene followed in the rank of potency. Both cannabidiol and the cannabidiol-rich extract inhibited the growth of xenograft tumors obtained by s.c. injection into athymic mice of human MDA-MB-231 breast carcinoma or rat v-K-ras-transformed thyroid epithelial cells and reduced lung metastases deriving from intrapaw injection of MDA-MB-231 cells.
Our data support the further testing of cannabidiol and cannabidiol-rich extracts for the potential treatment of cancer.
The therapeutic properties of the hemp plant, Cannabis sativa, have been known since antiquity, but the recreational use of its euphoric and other psychoactive effects has restricted for a long time research on its possible pharmaceutical application. The isolation of Δ9-tetrahydrocannabinol (THC), the main psychoactive component of Cannabis (Gaoni and Mechoulam, 1964), opened the way to further investigations. After the discovery of the two specific molecular targets for THC, CB1, and CB2 (for review, see Pertwee, 1997), it became clear that most of the effects of marijuana in the brain and peripheral tissues were due to activation of these two G-protein-coupled cannabinoid receptors. However, evidence is also accumulating that some pharmacological effects of marijuana are due to Cannabis components different from THC. Indeed, C. sativa contains at least 400 chemical components, of which 66 have been identified to belong to the class of the cannabinoids (Pertwee, 1997).
To date, cannabinoids have been successfully used in the treatment of nausea and vomiting (for review, see Robson, 2005), two common side effects that accompany chemotherapy in cancer patients. Nevertheless, the use of cannabinoids in oncology might be somehow underestimated since increasing evidence exist that plant, synthetic, and endogenous cannabinoids (endocannabinoids) are able to exert a growth-inhibitory action on various cancer cell types. However, the precise pathways through which these molecules produce an antitumor effect has not been yet fully characterized, also because their mechanism of action appears to be dependent on the type of tumor cell under study. It has been reported that cannabinoids can act through different cellular mechanisms, e.g., by inducing apoptosis, cell-cycle arrest, or cell growth inhibition, but also by targeting angiogenesis and cell migration (for review, see Bifulco and Di Marzo, 2002; Guzman, 2003; Kogan, 2005). Furthermore, the antitumoral effects of plant, synthetic and endocannabinoids can be mediated by activation of either CB1 (Melck et al., 2000; Bifulco et al., 2001; Ligresti et al., 2003; Mimeault et al., 2003) or CB2 receptors or both (Sanchez et al., 2001; Casanova et al., 2003; McKallip et al., 2005), and, at least in the case of the endocannabinoid anandamide, by transient receptor potential vanilloid type-1 (TRPV1) receptors (Maccarrone et al., 2000; Jacobsson et al., 2001; Contassot et al., 2004) as well as by noncannabinoid, nonvanilloid receptors (Ruiz et al., 1999). Additionally, cannabidiol has been suggested to inhibit glioma cell growth in vitro and in vivo independently from cannabinoid and vanilloid receptors (Massi et al., 2004; Vaccani et al., 2005).
The main limitation of the possible future use of THC in oncology might be represented by adverse effects principally at the level of the central nervous system, consisting mostly of perceptual abnormalities, occasionally hallucinations, dysphoria, abnormal thinking, depersonalization, and somnolence (Walsh et al., 2003). However, most non-THC plant cannabinoids seem to be devoid of direct psychotropic proprieties. In particular, it has been ascertained that cannabidiol is nonpsychotropic (for review, see Mechoulam et al., 2002; Pertwee, 2004) and may even mitigate THC psychoactivity by blocking its conversion to the more psychoactive 11-hydroxy-THC (Bornheim and Grillo, 1998; Russo and Guy, 2006). At least cannabidiol, cannabigerol, and cannabichromene lack psychotropic activity and indicate that for a promising medical profile in cancer therapy, research should focus on these compounds, which instead have been poorly studied with regard to their potential antitumor effects.
For in vitro studies, the cannabinoids under investigation were screened for their ability to reduce cell proliferation on a collection of tumoral cell lines. Cannabidiol always exhibited the highest potency with IC50 values ranging between 6.0 ± 3.0 and 10.6 ± 1.8 μM (Table 1). Cannabidiol acid was the least potent compound. Among the other plant cannabinoids, cannabigerol was almost always the second most potent compound, followed by cannabichromene (Table 1). The effect of the two Cannabis extracts (enriched in cannabidiol or THC) was next investigated, and in some circumstances, the cannabidiol-rich extract appeared slightly more potent than pure cannabidiol (Table 1). Finally, to investigate the selectivity of cannabidiol effect in tumoral versus nontumoral cells, various concentrations (from 1–100 μM) of cannabidiol on different stabilized nontumor cell lines such as HaCat (human keratinocyte), 3T3-F442A (rat preadipocytes), and RAW 264.7 (mouse monocytemacrophages) were also tested. Cannabidiol, at a dose similar to its IC50 values in the various tumoral cell lines, did not affect the vitality of nontumor cell lines (Fig. 2E). Only at a concentration of 25 μM, which exerts nearly 100% inhibition of cancer cell growth, cannabidiol exhibited a cytotoxic effect in these nontumoral cell lines (Fig. 2E). Lastly, it was examined the selectivity of cannabidiol versus a primary cell line derived from mammary glands (human mammary epithelial cells) since several experiments on the mechanism of action of cannabidiol were performed using a human breast carcinoma cell line (MBA-MD-231 cells). Cannabidiol affected significantly the vitality of this cell line only at a 25 μM concentration (Fig. 2F).
For the in vivo studies, the efficacy of cannabidiol and its enriched extract at reducing tumor size and volume was evaluated. Mice treated with either pure cannabidiol or the cannabidiol-rich extract exhibited significantly smaller tumors in comparison with control mice. A strong and statistically significant antitumor effect was observed with both treatments and with both in vivo xenograft tumor models used (Fig. 3, A and B). The effect of cannabidiol and cannabidiolrich compounds on the formation of lung metastatic nodules of MBA-MD-231 cells injected into the paw was also investigated. Both cannabidiol and cannabidiol-rich exhibited a strong and significant reduction of metastatic lung infiltration (Fig. 3C). With the intention to evaluate if the inhibitory effect on cell growth of cannabidiol was associated with apoptotic events or blockade of mitogenesis, the percentage of G1 population cells was estimated by flow cytometry. In MCF-7 cells, a hormone-sensitive cell line, cannabidiol, exerted antiproliferative effect by causing a cell cycle block at the G1/S phase transition (Fig. 4A; Table 2). A similar result was observed in another hormone-sensitive cell line KiMol cells, where, however, the antiproliferative effect of cannabidiol was also accompanied by a proapoptotic action (Fig. 4C; Table 2). Finally, in C6 glioma and MDA-MB-231 cells (two nonhormone-sensitive cell lines), cannabidiol provoked a pure proapoptotic effect (Fig. 4D; Table 2). The proapoptotic effect of cannabidiol on MDA-MB-231 cells was also established by evaluating the involvement of caspase-3. The proapoptotic effect of cannabidiol was confirmed in this cell line but not in DU-145 cells.
part from cannabidiol, only cannabigerol and cannabidiol acid activated TRPV1 receptors, with a significantly lower potency than cannabidiol, whereas cannabichromene, THC, and THC acid were almost inactive (Fig. 5). The cannabidiol-rich extract was as efficacious and potent as cannabidiol, whereas the THC-rich extract was more efficacious and potent than THC, possibly due to the presence of other TRPV1-active cannabinoids, including cannabidiol and cannabigerol (Fig. 5).
To assess whether plant cannabinoids, which are very weak agonists of CB1 and CB2 receptors, activate these receptors indirectly, i.e., by elevating endocannabinoid levels, we studied their effects on anandamide cellular uptake and enzymatic hydrolysis (Bifulco et al., 2004). Although most of the compounds tested did inhibit anandamide metabolism (Table 4), particularly at the level of cellular uptake, their rank of potency (cannabichromene = cannabigerol > cannabidiol = THC) did not reflect their potency at inhibiting cancer cell proliferation.
MDA-MB-231 cells were selected also to investigate the implications of cannabidiol effects on oxidative stress phenomena. Already at 0.1 μM concentration, α-tocopherol significantly prevented, although in a partial manner, the antiproliferative effects of cannabidiol on these cells (Fig. 9A); also, vitamin C and astaxantine, at 25 μM concentration, were able to counteract the inhibitory effect of cannabidiol by ∼30% (data not shown).
The aim of this study was to identify natural cannabinoids with antitumor activities at least similar to those of THC and devoid of the potential central effects of this compound. Given that the efficacy of cannabinoids as antitumoral agents appears to be strictly correlated to the cell type under investigation, we screened a panel of plant cannabinoids in a wide range of tumoral cell lines distinct in origin and typology. We found that, surprisingly, cannabidiol acted as a more potent inhibitor of cancer cell growth than THC and that cannabigerol and cannabichromene usually followed cannabidiol in the rank of potency. The cell growth-inhibitory effect of cannabidiol depended on its chemical structure since the addition of a carboxylic acid group (as in cannabidiol acid) dramatically reduced its activity. We also found that the cannabidiol-rich Cannabis extract was as potent as pure cannabidiol in most cases or even more potent in some cell lines. These results suggest the use in cancer therapy for cannabidiol, a compound lacking the psychotropic effects typical of THC. Indeed, the efficacy of cannabidiol and of the cannabidiol-rich extract were confirmed in vivo in two different models of xenograft tumors obtained by inoculation in athymic mice of either v-K-ras-transformed thyroid epithelial cells or of the highly invasive MDA-MB-231 breast cancer cells. Furthermore, cannabidiol and the cannabidiolrich extract also inhibited the formation of lung metastases subsequent to inoculation of MDA-MB-231 cells, in agreement with the inhibitory actions on cancer cell migration.
The weak effects observed here with THC might be regarded as surprising. In fact, THC was reported to induce apoptosis in both C6 glioma and human prostate PC-3 cells (Sanchez et al., 1998; Ruiz et al., 1999; Sarfaraz et al., 2005), although it may even enhance breast cancer growth and metastasis (McKallip et al., 2005). The low potency found here for this compound, at least in glioma and prostate cancer, could be explained by the different experimental conditions used and supports the notion that the efficacy of cannabinoids is strongly dependent on the cell type utilized.
In conclusion, our data indicate that cannabidiol, and possibly Cannabis extracts enriched in this natural cannabinoid, represent a promising nonpsychoactive antineoplastic strategy. In particular, for a highly malignant human breast carcinoma cell line, we have shown here that cannabidiol and a cannabidiol-rich extract counteract cell growth both in vivo and in vitro as well as tumor metastasis in vivo. Cannabidiol exerts its effects on these cells through a combination of mechanisms that include either direct or indirect activation of CB2 and TRPV1 receptors and induction of oxidative stress, all contributing to induce apoptosis. Additional investigations are required to understand the mechanism of the growth-inhibitory action of cannabidiol in the other cancer cell lines studied here”.
Testo estratto da Antitumor Activity of Plant Cannabinoids with Emphasis on the Effect of Cannabidiol on Human Breast Carcinoma, Journal of Pharmacology and Experimental Therapeutics, 2006, Alessia Ligresti, Aniello Schiano Moriello, Katarzyna Starowicz, Isabel Matias, Simona Pisanti, Luciano De Petrocellis, Chiara Laezza, Giuseppe Portella, Maurizio Bifulco and Vincenzo Di Marzo