Cannabidiol hydroxyquinone

Toxicol Lett . 2010 May 19;195(1):68-74.

Cannabidiol hydroxyquinone-induced apoptosis of splenocytes is mediated predominantly by thiol depletion[Pubmed: 20184945]

Cannabidiol, the major nonpsychotropic phytocannabinoid, has been recently demonstrated to induce apoptosis in primary lymphocytes via an oxidative stress-dependent mechanism. Cannabidiol can be converted by microsomal enzymes to the hydroxyquinone metabolite HU-331 that forms adducts with glutathione. The present study tested the hypothesis that HU-331 could cause apoptosis via the depletion of thiols in splenocytes. Our results showed that HU-331 treatment significantly enhanced splenocyte apoptosis in a time- and concentration-dependent manner. Concordantly, a gradual diminishment in the cellular thiols and glutathione was detected in HU-331-treated splenocytes. The apoptosis and thiol diminishment induced by HU-331 were abrogated in the presence of thiol antioxidants, including N-acetyl-(L)-cysteine and N-(2-mercaptopropionyl) glycine, whereas the non-thiol antioxidants catalase and pyruvate were ineffective. In comparison, both thiol and non-thiol antioxidants were capable of attenuating H(2)O(2)-induced thiol diminishment and reactive oxygen species generation in splenocytes. Collectively, these results suggest that HU-331 might be an active metabolite of cannabidiol potentially contributing to the induction of apoptosis in splenocytes, and that the apoptosis is primarily mediated by the loss of cellular thiols.

Chem Res Toxicol . 1998 Oct;11(10):1209-1216.

Characterization of cytochrome P450 3A inactivation by cannabidiol: possible involvement of cannabidiol-hydroxyquinone as a P450 inactivator[Pubmed: 9778318]

Cannabidiol (CBD) is a major constituent of marijuana and a potent inhibitor of P450-mediated hepatic drug metabolism. Mouse P450 3A11 metabolism of [14C]CBD resulted in the formation of radiolabeled P450, which after digestion with lysyl endopeptidase C (Lys-C) and HPLC resolution of peptides, revealed one major broadly eluting peak of radioactivity. Electrophoresis/autoradiography of this peak identified several peptide bands, one of which was predominantly radiolabeled and had an apparent molecular mass of approximately 6 kDa. Amino-terminal sequence determination of this band revealed the presence of two peptides whose sequences identified them as Ala344-Lys379 and Gly426-Lys454. To characterize the reactive species that may be generated during P450 3A11-catalyzed CBD metabolism, reduced glutathione (GSH) was used as a trapping agent for possible electrophilic metabolites. Incubation of P450 3A11 in the presence of cofactors NADPH, CBD, and [3H]GSH resulted in the formation of a radiolabeled product which was absent in incubations lacking any of the cofactors. The UV absorption spectra of this compound indicated absorbances at approximately 220, 275, and 350 nm, and mass spectral analysis revealed prominent ions at m/z 634, 599, 505, 402, and 359, ions consistent with that of a GSH adduct of CBD-hydroxyquinone. A synthetic CBD-hydroxyquinone-GSH adduct was also prepared and had UV absorption and mass spectra nearly identical to that of the P450-mediated CBD-GSH adduct. CBD-hydroxyquinone formation may be the penultimate oxidative step involved in CBD-mediated modification and inactivation of P450 3A11.

Chem Res Toxicol . 2018 Feb 19;31(2):137-144.

HU-331 and Oxidized Cannabidiol Act as Inhibitors of Human Topoisomerase IIα and β[Pubmed: 29272108]

Topoisomerase II is a critical enzyme in replication, transcription, and the regulation of chromatin topology. Several anticancer agents target topoisomerases in order to disrupt cell growth. Cannabidiol is a major non-euphoriant, pharmacologically active component of cannabis. Previously, we examined the cannabidiol derivative HU-331 in order to characterize the mechanism of the compound against topoisomerase IIα. In this current work, we explore whether cannabidiol (CBD) impacts topoisomerase II activity, and we additionally examine the activity of these compounds against topoisomerase IIβ. CBD does not appear to strongly inhibit DNA relaxation and is not a poison of topoisomerase II DNA cleavage. However, oxidation of CBD allows this compound to inhibit DNA relaxation by topoisomerase IIα and β without poisoning DNA cleavage. Additionally, we found that oxidized CBD, similar to HU-331, inhibits ATP hydrolysis and can result in inactivation of topoisomerase IIα and β. We also determined that oxidized CBD and HU-331 are both able to stabilize the N-terminal clamp of topoisomerase II. Taken together, we conclude that while CBD does not have significant activity against topoisomerase II, both oxidized CBD and HU-331 are active against both isoforms of topoisomerase II. We hypothesize that oxidized CBD and HU-331 act against the enzyme through interaction with the N-terminal ATPase domain. According to the model we propose, topoisomerase II inactivation may result from a decrease in the ability of the enzyme to bind to DNA when the compound is bound to the N-terminus.

Expert Opin Investig Drugs . 2007 Sep;16(9):1405-13.

HU-331: a cannabinoid quinone, with uncommon cytotoxic properties and low toxicity[Pubmed: 17714026]

The oxidation of cannabis constituents has given rise to their corresponding quinones, which have been identified as cytotoxic agents. Out of these molecules the quinone of cannabidiol--the most abundant non-psychoactive cannabinoid in Cannabis sativa--has shown the highest cytotoxicity. This compound was named HU-331 and it exerts antiangiogenic properties, induces apoptosis to endothelial cells and inhibits topoisomerase II in nanomolar concentrations. Unlike other quinones, it is not cardiotoxic and does not induce the formation of free radicals. A comparative in vivo study in mice has shown HU-331 to be less toxic and more effective than the commonly used doxorubicin. This review summarises the properties of HU-331 and compares it with doxorubicin and other topoisomerase II inhibitors.

Life Sci . 2008 Nov 21;83(21-22):717-724.

Generation of reactive oxygen species during mouse hepatic microsomal metabolism of cannabidiol and cannabidiol hydroxy-quinone[Pubmed: 18929579]

We investigated whether cannabidiol (CBD) and cannabidiol hydroxy-quinone (CBDHQ) generate reactive oxygen species (ROS) during metabolism with mouse hepatic microsomes. CBD and CBDHQ (91.5 microM) significantly suppressed lipid peroxidation in the mouse hepatic microsomes. CBDHQ also significantly decreased NADH-cytochrome b5 reductase (fp1) activity by 25% of the control activity in the hepatic microsomes, and tended to increase NADPH-cytochrome c (P450) reductase (fp2) activity. CBDHQ also significantly inhibited superoxide dismutase and catalase activities in mouse hepatic 105,000 xg supernatant. Moreover, CBDHQ significantly increased glutathione reductase activity and significantly inhibited NAD(P)H-quinone reductase activity. CBD exhibited similar effects on these enzymes, except that cannabinoid significantly inhibited glutathione reductase activity in mouse hepatic 105,000 xg supernatant. These results suggest that CBDHQ is easily converted to the semiquinone form rather than the hydroquinone form. It was also suggested that CBDHQ and CBD were capable of generating ROS as superoxide anion radicals during their metabolism with mouse hepatic microsomes or with purified fp2 by electron spin resonance spin trapping methods with 5,5-dimethyl-1-pyrroline-N-oxide. The present results suggest that CBDHQ formed during hepatic microsomal metabolism of CBD is capable of generating ROS and inducing cell toxicity.

Chem Res Toxicol . 2014 Dec 15;27(12):2044-2051

HU-331 is a catalytic inhibitor of topoisomerase IIα[Pubmed: 25409338]

Topoisomerases are essential enzymes that are involved in DNA metabolism. Topoisomerase II generates transient DNA strand breaks that are stabilized by anticancer drugs, such as doxorubicin, causing an accumulation of DNA damage. However, doxorubicin causes cardiac toxicity and, like etoposide and other topoisomerase II-targeted agents, can induce DNA damage, resulting in secondary cancers. The cannabinoid quinone HU-331 has been identified as a potential anticancer drug that demonstrates more potency in cancer cells with less off-target toxicity than that of doxorubicin. Reports indicate that HU-331 does not promote cell death via apoptosis, cell cycle arrest, caspase activation, or DNA strand breaks. However, the precise mechanism of action is poorly understood. We employed biochemical assays to study the mechanism of action of HU-331 against purified topoisomerase IIα. These assays examined DNA binding, cleavage, ligation, relaxation, and ATPase activities of topoisomerase IIα. Our results demonstrate that HU-331 inhibits topoisomerase IIα-mediated DNA relaxation at micromolar levels. We find that HU-331 does not induce DNA strand breaks in vitro. When added prior to the DNA substrate, HU-331 blocks DNA cleavage and relaxation activities of topoisomerase IIα in a redox-sensitive manner. The action of HU-331 can be blocked, but not reversed, by the presence of dithiothreitol. Our results also show that HU-331 inhibits the ATPase activity of topoisomerase IIα using a noncompetitive mechanism. Preliminary binding studies also indicate that HU-331 decreases the ability of topoisomerase IIα to bind DNA. In summary, HU-331 inhibits relaxation activity without poisoning DNA cleavage. This action is sensitive to reducing agents and appears to involve noncompetitive inhibition of the ATPase activity and possibly inhibition of DNA binding. These studies provide a promising foundation for the exploration of HU-331 as a catalytic inhibitor of topoisomerase IIα.

Mol Pharmacol . 2006 Jul;70(1):51-59.

A cannabinoid quinone inhibits angiogenesis by targeting vascular endothelial cells[Pubmed: 16571653]

Recent findings on the inhibition of angiogenesis and vascular endothelial cell proliferation by anthracycline antibiotics, which contain a quinone moiety, make this type of compound a very promising lead in cancer research/therapy. We have reported that a new cannabinoid anticancer quinone, Cannabidiol hydroxyquinone (HU-331), is highly effective against tumor xenografts in nude mice. For evaluation of the antiangiogenic action of cannabinoid quinones, collagen-embedded rat aortic ring assay was used. The ability of cannabinoids to cause endothelial cell apoptosis was assayed by TUNEL staining and flow cytometry analysis. To examine the genes and pathways targeted by HU-331 in vascular endothelial cells, human cDNA microarrays and polymerase chain reaction were used. Immunostaining with anti-CD31 of tumors grown in nude mice served to indicate inhibition of tumor angiogenesis. HU-331 was found to be strongly antiangiogenic, significantly inhibiting angiogenesis at concentrations as low as 300 nM. HU-331 inhibited angiogenesis by directly inducing apoptosis of vascular endothelial cells without changing the expression of pro- and antiangiogenic cytokines and their receptors. A significant decrease in the total area occupied by vessels in HU-331-treated tumors was also observed. These data lead us to consider HU-331 to have high potential as a new antiangiogenic and anticancer drug.

J Pharmacol Exp Ther . 2007 Aug;322(2):646-653.

A cannabinoid anticancer quinone, HU-331, is more potent and less cardiotoxic than doxorubicin: a comparative in vivo study[Pubmed: 17478614]

Several quinones have been found to be effective in the treatment of some forms of cancer; however, their cumulative heart toxicity limits their use. The cannabinoid quinone HU-331 [3S,4R-p-benzoquinone-3-hydroxy-2-p-mentha-(1,8)-dien-3-yl-5-pentyl] is highly effective against tumor xenografts in nude mice. We report now a comparison of the anticancer activity of HU-331 and its cardiotoxicity with those of doxorubicin in vivo. General toxicity was assayed in Sabra, nude and SCID-NOD mice. The anticancer activity in vivo was assessed by measurement of the tumors with an external caliper in HT-29 and Raji tumor-bearing mice and by weighing the excised tumors. Left ventricular function was evaluated with transthoracic echocardiography. Myelotoxicity was evaluated by blood cell count. Cardiac troponin T (cTnT) plasma levels were determined by immunoassay. HU-331 was found to be much less cardiotoxic than doxorubicin. The control and the HU-331-treated groups gained weight, whereas the doxorubicin-treated group lost weight during the study. In HT-29 colon carcinoma, the tumor weight in the HU-331-treated group was 54% smaller than in the control group and 30% smaller than in the doxorubicin-treated group. In Raji lymphoma, the tumor weight in the HU-331-treated group was 65% smaller than in the control group and 33% smaller than in the doxorubicin-treated group. In contrast to doxorubicin, HU-331 did not generate reactive oxygen species in mice hearts (measured by protein carbonylation levels and malondialdehyde levels). In vivo, HU-331 was more active and less toxic than doxorubicin and thus it has a high potential for development as a new anticancer drug.

J Exp Clin Cancer Res . 2013 Apr 30;32(1):24.

Cyclohexa-2,5-diene-1,4-dione-based antiproliferative agents: design, synthesis, and cytotoxic evaluation[Pubmed: 23631805]

Background: Tumors are diseases characterized by uncontrolled cell growth and, in spite of the progress of medicine over the years, continue to represent a major threat to the health, requiring new therapies. Several synthetic compounds, such as those derived from natural sources, have been identified as anticancer drugs; among these compounds quinone represent the second largest class of anticancer agents in use. Several studies have shown that these act on tumor cells through several mechanisms. An important objective of this work is to develop quinoidscompounds showing antitumor activity, but with fewer side effects. The parachinone cannabinol HU-331, is a small molecule that with its core 4-hydroxy-1,4-benzoquinone, exhibits a potent and selective cytotoxic activity on different tumor cell lines. A series of derivatives 3-hydroxy-1,4-benzochinoni were thus developed through HU-331 chemical modifications. The purpose of the work is to test the ability of the compounds to induce proliferative inhibition and study the mechanisms of cell death. Methods: The antitumor activities were evaluated in vitro by examining their cytotoxic effects against different human cancer cell lines. All cell lines tested were plated in 96-multiwell and treated with HU-100-V at different concentrations and cell viability was evaluated byMTT assay. Subsequently via flow cytometry (FACS) it was possible to assess apoptosis by the system of double labeling with PI and Annexin-V, and the effect of the compounds on ROS formation by measuring the dichlorofluorescein fluorescence. Results: The substitution by n-hexyl chain considerably enhanced the bioactivity of the compounds. In details, 2-hexyl-5-hydroxycyclohexa-2,5-diene-1,4-dione (V), 2,5-Dimethoxy-3-hexyl-2,5-cyclohexadiene-1,4-dione (XII) and 2-hydroxy-5-methoxy-3-hexyl-cyclohexa-2,5-diene-1,4-dione (XIII) showed most prominent cytotoxicity against almost human tumour cell lines. Compound V was further subjected to downstream apoptotic analysis, demostrating a time-dependent pro-apoptotic activity on human melanoma M14 cell line mediated by caspases activation and poly-(ADP-ribose)-polymerase (PARP) protein cleavage. Conclusions: These findings indicate that 2-hexyl-5-idrossicicloesa-2,5-diene-1,4-dione can be a promising compound for the design of a new class of antineoplastic derivatives.Carmen Petronzi, Michela Festa, Antonella Peduto and Maria Castellano: equally contributed equally to this work.