cannabidiol and cannabidiol-hydroxyquinone

A cannabinoid anticancer para-quinone, HU-331, which was synthesized by our group five decades ago, was shown to have very high efficacy against human cancer cell lines in-vitro and against in-vivo grafts of human tumors in nude mice. The main mechanism was topoisomerase IIα catalytic inhibition. Later, several groups synthesized related compounds. In the present presentation, we review the publications on compounds synthesized on the basis of HU-331, summarize their published activities and mechanisms of action and report the synthesis and action of novel quinones, thus expanding the structure-activity relationship in these series.

Topics: Animals; Cannabidiol; DNA Topoisomerases, Type II; Humans; Mice; Mice, Nude; Neoplasm Proteins; Neoplasms, Experimental; Poly-ADP-Ribose Binding Proteins; Quinones; Topoisomerase II Inhibitors

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.

Topics: Angiogenesis Inhibitors; Animals; Cannabidiol; Cannabinoids; Cell Death; Cytotoxins; Humans; Quinones; Tumor Cells, Cultured

The reported characteristics of cannabidiol (CBD) have encouraged significant growth in commercial CBD products. There is limited information on the stability of CBD and some researchers have noted significant reductions of CBD in products. In this study, the chemical profiles of plant-based and chemically synthesized CBD in a prototype e-liquid formulation were assessed during 4 weeks of storage under varying conditions. Samples were analysed on days 1, 8, 15, 22, and 29 by untargeted analysis using ultra-high performance liquid chromatography-trapped ion mobility-time-of-flight mass spectrometry (UHPLC-TIMS-TOF-MS). On day 1, analysis of plant-based and synthetic CBD formulations showed small differences in their composition, with plant-based CBD e-liquid containing trace levels of a higher number of phytocannabinoid-related impurities. Storage for 4 weeks under stress (40 °C, 75% relative humidity, dark) and ambient (25 °C, 60% relative humidity, daylight) conditions led to increases in the number and abundance of cannabinoid-related degradation products, including cannabielsoin (CBE) and CBD-hydroxyquinone (HU-331), which are products of the oxidation of CBD, and other unidentified cannabinoid-related compounds. The unidentified cannabinoid-related compounds were probed by accurate mass measurement and MS

Topics: Cannabidiol; Cannabinoids; Chromatography, High Pressure Liquid

Topoisomerase II (Topo2) inhibitors in combination with cisplatin represent a common treatment modality used for glioma patients. The main mechanism of their action involves induction of DNA double-strand breaks (DSBs). DSBs are repaired via the homology-dependent DNA repair (HRR) and non-homologous end-joining (NHEJ). Inhibition of the NHEJ or HRR pathway sensitizes cancer cells to the treatment. In this work, we investigated the effect of three Topo2 inhibitors-etoposide, NK314, or HU-331 in combination with cisplatin in the U-87 human glioblastoma cell line. Etoposide as well as NK314 inhibited Topo2 activity by stabilizing Topo2-DNA cleavable complexes whereas HU-331 inhibited the ATPase activity of Topo2 using a noncompetitive mechanism. To increase the effectiveness of the treatment, we combined cisplatin and Topo2 inhibitor treatment with DSB repair inhibitors (DRIs). The cells were sensitized with NHEJ inhibitor, NU7441, or the novel HRR inhibitor, YU238259, prior to drug treatment. All of the investigated Topo2 inhibitors in combination with cisplatin efficiently killed the U-87 cells. The most cytotoxic effect was observed for the cisplatin + HU331 treatment scheme and this effect was significantly increased when a DRI pretreatment was used; however, we did not observed DSBs. Therefore, the molecular mechanism of cytotoxicity caused by the cisplatin + HU331 treatment scheme is yet to be evaluated. We observed a concentration-dependent change in DSB levels and accumulation at the G2/M checkpoint and S-phase in glioma cells incubated with NK314/cisplatin and etoposide/cisplatin. In conclusion, in combination with cisplatin, HU331 is the most potent Topo2 inhibitor of human glioblastoma cells.

Topics: Apoptosis; Benzamides; Brain Neoplasms; Cannabidiol; Cell Cycle; Cell Line, Tumor; Chromones; Cisplatin; DNA Breaks, Double-Stranded; DNA Repair; Etoposide; Glioblastoma; Humans; Morpholines; Phenanthrenes; Sulfonamides; Topoisomerase II Inhibitors

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.

Topics: Cannabidiol; DNA; DNA Cleavage; DNA Topoisomerases, Type II; Humans; Models, Molecular; Molecular Structure; Oxidation-Reduction; Plasmids; Poly-ADP-Ribose Binding Proteins; Topoisomerase II Inhibitors

Trinucleotide repeats are involved in various neurodegenerative diseases and are highly unstable both in dividing or non-dividing cells. In Huntington disease (HD), the age of onset of symptoms is inversely correlated to the number of CAG repeats within exon 1 of the HTT gene. HD shows paternal anticipation as CAG repeats are increased during spermatogenesis. CAG expansion were indeed found to be generated during the chromatin remodeling in spermatids where most histones are evicted and replaced by protamines. This process involves striking change in DNA topology since free supercoils must be eliminated. Using an in vitro CAG repeat reporter assay and a highly active nuclear extracts from spermatids, we demonstrate that free negative supercoils result in CAG TNR expansion at a stabilized hairpin. We also suggest a possible role for protamines in promoting localized torsional stress and consequently TNR expansion. The transient increase in torsional stress during spermiogenesis may therefore provide an ideal context for the generation of such secondary DNA structures leading to the paternal anticipation of trinucleotidic diseases.

Topics: Animals; Cannabidiol; Chromatin Assembly and Disassembly; DNA Topoisomerases, Type II; DNA, Superhelical; Exons; Histones; Huntingtin Protein; Huntington Disease; Male; Mice; Protamines; Reproducibility of Results; Spermatids; Spermatogenesis; Topoisomerase II Inhibitors; Torsion, Mechanical; Trinucleotide Repeats

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.

Topics: Antigens, Neoplasm; Cannabidiol; Catalysis; DNA; DNA Topoisomerases, Type II; DNA-Binding Proteins; Topoisomerase II Inhibitors

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.. 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.. 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.. 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.

Topics: Antineoplastic Agents; Apoptosis; Benzoquinones; Cannabidiol; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Design; Humans; Inhibitory Concentration 50; Reactive Oxygen Species; Signal Transduction; Topoisomerase Inhibitors

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.

Topics: Animals; Apoptosis; Cannabidiol; Caspase 8; Lymphocytes; Male; Mice; Mice, Inbred BALB C; Spleen; Sulfhydryl Compounds

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.

Topics: Aerobiosis; Animals; Cannabidiol; Catalase; Cytochrome-B(5) Reductase; Electron Spin Resonance Spectroscopy; Glutathione Peroxidase; Glutathione Reductase; In Vitro Techniques; Lipid Peroxidation; Male; Mice; Microsomes, Liver; NADPH-Ferrihemoprotein Reductase; Reactive Oxygen Species; Superoxide Dismutase

Anthracyclines, a large group of quinonoid compounds, are used to treat some forms of cancer. Although highly effective in cancer therapy, the mechanism of action of these compounds is not specific; they act on cancer and other cells by numerous mechanisms. A new anticancer quinone (HU-331) was synthesized from cannabidiol. It shows significant high efficacy against human cancer cell lines in vitro and against in vivo tumor grafts in nude mice. In this study, we investigated its mode of action and present evidence on its unique mechanism. HU-331 does not cause cancer cell cycle arrest, cell apoptosis, or caspase activation. HU-331-caused cell death of human cancer cell lines is not mediated by reactive oxygen intermediates/species, as exposure to HU-331 failed to elicit the generation of reactive oxygen species. HU-331 inhibits DNA topoisomerase II even at nanomolar concentrations but has only a slight nonsignificant effect on DNA topoisomerase I action. The cannabinoid quinone HU-331 is a highly specific inhibitor of topoisomerase II, compared with most known anticancer quinones. It might represent a new potent anticancer drug.

Topics: Antigens, Neoplasm; Antineoplastic Agents; Apoptosis; Cannabidiol; Cannabinoid Receptor Antagonists; Caspase 3; Cell Cycle; DNA Breaks; DNA Topoisomerases, Type II; DNA-Binding Proteins; DNA, Neoplasm; HT29 Cells; Humans; Jurkat Cells; NAD(P)H Dehydrogenase (Quinone); Nucleic Acid Conformation; Reactive Oxygen Species; Receptors, Cannabinoid; Topoisomerase II Inhibitors

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.

Topics: Animals; Antineoplastic Agents; Blood Cell Count; Body Weight; Cannabidiol; Cell Line, Tumor; Doxorubicin; Echocardiography; Heart; HT29 Cells; Humans; Lipid Peroxidation; Male; Malondialdehyde; Mice; Mice, Inbred NOD; Mice, Inbred Strains; Mice, Nude; Mice, SCID; Molecular Structure; Myocardium; Neoplasms; Protein Carbonylation; Troponin T; Xenograft Model Antitumor Assays

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.

Topics: Animals; Aorta; Apoptosis; Benzoquinones; Cannabidiol; Cannabinoids; Cattle; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Endothelial Cells; Gene Expression; HT29 Cells; Humans; Immunohistochemistry; In Vitro Techniques; Mice; Mice, Nude; Neovascularization, Pathologic; Neovascularization, Physiologic; Platelet Endothelial Cell Adhesion Molecule-1; Rats; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Cannabidiol hydroxy-quinone (CBDHQ) was identified as an air oxidation product of cannabidiol (CBD). The in vitro incubation of mouse hepatic microsomes with CBDHQ resulted in a decrease of cytochrome P-450 content. CBDHQ inhibited the hepatic microsomal drug-metabolizing enzymes of mice. This inhibitory effect was stronger than that of CBD. CBDHQ (150 microM) inhibited aniline hydroxylase, p-nitroanisole O-demethylase and aminopyrine N-demethylase in the microsomes by 70, 52 and 77%, respectively, whereas the same concentration of CBD caused the inhibition by 39, 30 and 26%, respectively. CBDHQ (91.5 microM) significantly decreased total heme content by 21% and free SH groups by 11% in the microsomes. The results indicate that CBDHQ, which is an oxidation product of CBD, inhibits the hepatic microsomal drug-metabolizing enzymes through the decrease of cytochrome P-450 content.

Topics: Animals; Cannabidiol; Gas Chromatography-Mass Spectrometry; In Vitro Techniques; Male; Mice; Mice, Inbred Strains; Microsomes, Liver; Oxidation-Reduction