ABSTRACT
Title
Antiangiogenic properties of the non psychoactive cannabinoid compound Cannabidiol: in vitro and in vivo studies
Authors
M. Solinas
Doctorate in Neurobiology, Doctorate School in Biological and Medical Sciences
Dept. of Structural and Functional Biology, University of Insubria, Busto Arsizio (VA), Italy
Doctorate in Neurobiology, Doctorate School in Biological and Medical Sciences
Dept. of Structural and Functional Biology, University of Insubria, Busto Arsizio (VA), Italy
Abstract
Malignant gliomas are the most common and aggressive primary tumors of the CNS, characterized by resistance to the conventional chemotherapy, invasion and extensive angiogenesis. In fact, among solid tumors, gliomas are characterized by the highest grade of vascular proliferation, and new blood vessel formation correlates with their degree of malignancy, aggressiveness, clinical recurrence and decreased survival. Recently, we have reported that the non-psychoactive cannabinoid compound cannabidiol (CBD) is highly effective at inhibiting in vitro and in vivo gliomas growth and at down-regulating some pro-angiogenic signal produced by human glioma cells. Thus, in the present work we aim at studying CBD ability to modulate tumor angiogenic process, to support its possible use to reduce new blood vessel formation and tumor growth itself.
At first, we evaluated CBD anti-proliferative effect on human endothelial HUVE cells treated with increasing doses of the drug for 24 h. MTT assay showed that CBD was able to inhibit in a dose-related manner cell proliferation with an IC50 of 9.9 μM. FACS analysis demonstrated that the antiproliferative effect exibited by CBD does not involve induction of apoptosis. Besides, we found that in vitro CBD induced a significant inhibition of HUVE cells motility both in a classical Boyden chamber test and in a wound-healing assay. The efficacy of CBD in these two tests was different with an EC50 of 1 μM in the Boyden chamber and 9 μM in the scratch assay. We next evaluated the effects of CBD on different proteins implied in angiogenic process and released by endothelial cells by the use of both an angiogenic Array Kit and an enzyme-linked immunosorbent assay (ELISA) designed to measure VEGF levels. CBD significantly down-regulated the expression profile of several proteins specifically involved in basal membrane degradation and neovascularisation within a range from 30 to 50% inhibition, depending on the considered factor. Finally we evaluated in vitro angiogenesis, testing the ability of endothelial cells to invade a Matrigel layer, mimicking the extracellular matrix, and create capillary-like structures. As a consequence of CBD treatment, the capillary-like tube structures formation was far less organized than control. In vitro angiogenensis was also evaluated in HUVEC spheroids embedded into collagen gels. In-gel angiogenesis was quantified by measuring the cumulative length of all of the capillary-like sprouts originating from the individual spheroids: CBD significantly reduces new vessel formation already at the concentration of 1 μM.
Based on these in vitro results, we further characterized CBD effects in ex vivo angiogenesis assay, using Matrigel sponge model of angiogenesis. Matrigel sponges containing increasing doses of the drug were inoculated subcutaneously into the flank of C57Bl6 mice. Four days after injection, striking inhibition of angiogenesis by CBD was observed both microscopically and by spectrophotometric analysis of haemoglobin content, with respect to the control.
In the whole, the present study reveals that CBD inhibits angiogenesis by multiple mechanisms and its dual effect both on glioma and on endothelial cells reinforces the hypothesis that CBD could represent an effective tool in glioma therapy.
Acknowledgements: Funded by GW Pharmaceuticals
At first, we evaluated CBD anti-proliferative effect on human endothelial HUVE cells treated with increasing doses of the drug for 24 h. MTT assay showed that CBD was able to inhibit in a dose-related manner cell proliferation with an IC50 of 9.9 μM. FACS analysis demonstrated that the antiproliferative effect exibited by CBD does not involve induction of apoptosis. Besides, we found that in vitro CBD induced a significant inhibition of HUVE cells motility both in a classical Boyden chamber test and in a wound-healing assay. The efficacy of CBD in these two tests was different with an EC50 of 1 μM in the Boyden chamber and 9 μM in the scratch assay. We next evaluated the effects of CBD on different proteins implied in angiogenic process and released by endothelial cells by the use of both an angiogenic Array Kit and an enzyme-linked immunosorbent assay (ELISA) designed to measure VEGF levels. CBD significantly down-regulated the expression profile of several proteins specifically involved in basal membrane degradation and neovascularisation within a range from 30 to 50% inhibition, depending on the considered factor. Finally we evaluated in vitro angiogenesis, testing the ability of endothelial cells to invade a Matrigel layer, mimicking the extracellular matrix, and create capillary-like structures. As a consequence of CBD treatment, the capillary-like tube structures formation was far less organized than control. In vitro angiogenensis was also evaluated in HUVEC spheroids embedded into collagen gels. In-gel angiogenesis was quantified by measuring the cumulative length of all of the capillary-like sprouts originating from the individual spheroids: CBD significantly reduces new vessel formation already at the concentration of 1 μM.
Based on these in vitro results, we further characterized CBD effects in ex vivo angiogenesis assay, using Matrigel sponge model of angiogenesis. Matrigel sponges containing increasing doses of the drug were inoculated subcutaneously into the flank of C57Bl6 mice. Four days after injection, striking inhibition of angiogenesis by CBD was observed both microscopically and by spectrophotometric analysis of haemoglobin content, with respect to the control.
In the whole, the present study reveals that CBD inhibits angiogenesis by multiple mechanisms and its dual effect both on glioma and on endothelial cells reinforces the hypothesis that CBD could represent an effective tool in glioma therapy.
Acknowledgements: Funded by GW Pharmaceuticals