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. 2012 Jul;3(7-8):512-20.
doi: 10.1177/1947601912466556.

Cannabidiol Inhibits Growth and Induces Programmed Cell Death in Kaposi Sarcoma-Associated Herpesvirus-Infected Endothelium

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Free PMC article

Cannabidiol Inhibits Growth and Induces Programmed Cell Death in Kaposi Sarcoma-Associated Herpesvirus-Infected Endothelium

Yehoshua Maor et al. Genes Cancer. .
Free PMC article

Abstract

Kaposi sarcoma is the most common neoplasm caused by Kaposi sarcoma-associated herpesvirus (KSHV). It is prevalent among the elderly in the Mediterranean, inhabitants of sub-Saharan Africa, and immunocompromised individuals such as organ transplant recipients and AIDS patients. Current treatments for Kaposi sarcoma can inhibit tumor growth but are not able to eliminate KSHV from the host. When the host's immune system weakens, KSHV begins to replicate again, and active tumor growth ensues. New therapeutic approaches are needed. Cannabidiol (CBD), a plant-derived cannabinoid, exhibits promising antitumor effects without inducing psychoactive side effects. CBD is emerging as a novel therapeutic for various disorders, including cancer. In this study, we investigated the effects of CBD both on the infection of endothelial cells (ECs) by KSHV and on the growth and apoptosis of KSHV-infected ECs, an in vitro model for the transformation of normal endothelium to Kaposi sarcoma. While CBD did not affect the efficiency with which KSHV infected ECs, it reduced proliferation and induced apoptosis in those infected by the virus. CBD inhibited the expression of KSHV viral G protein-coupled receptor (vGPCR), its agonist, the chemokine growth-regulated protein α (GRO-α), vascular endothelial growth factor receptor 3 (VEGFR-3), and the VEGFR-3 ligand, vascular endothelial growth factor C (VEGF-C). This suggests a potential mechanism by which CBD exerts its effects on KSHV-infected endothelium and supports the further examination of CBD as a novel targeted agent for the treatment of Kaposi sarcoma.

Keywords: Kaposi sarcoma; Kaposi sarcoma–associated herpesvirus; cannabidiol; vascular endothelial growth factor receptor 3; viral G protein–coupled receptor.

Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
CBD does not modulate the infection of HMVECs by KSHV. Percentage of KSHV-infected HMVECs by fluorescence. HMVECs were pretreated with various concentrations of CBD or a vehicle control, followed by infection with GFP-KSHV virions or a mock infection. Cells were then washed with 1X PBS and cultured in normal medium. After 48 hours, green fluorescent cells (indicative of GFP-KSHV infection) were counted under a fluorescent microscope. Infection was calculated as a percentage of total cells. Data represent the mean ± SD.
Figure 2.
Figure 2.
CBD preferentially inhibits the proliferation of KSHV-infected HMVECs. (A) Percentage of proliferating cells as measured by an MTS-based assay. Mock-infected HMVECs (HMVEC) and KSHV-infected HMVECs (KSHV/HMVEC) were incubated for 48 hours with various concentrations of CBD or a vehicle control. The proliferation index of untreated HMVECs was established as 100%, and all other cells and conditions were calculated as a percentage of this control. Data indicate the mean ± SD. **P < 0.01 and ***P < 0.001 indicate the difference in proliferation of KSHV/HMVEC versus HMVEC at each CBD concentration. (B) Regression analysis of the effect of CBD on the proliferation of KSHV-infected HMVECs.
Figure 3.
Figure 3.
CBD preferentially induces apoptosis in KSHV-infected HMVECs. (A) Percentage of apoptotic cells as assayed by TUNEL. KSHV-infected HMVECs (KSHV/HMVEC) or mock-infected HMVECs (HMVEC) were treated with various concentrations of CBD or a vehicle control for 24 hours. Data were calculated as apoptotic cells/total cells. Data represent the mean ± SD. ***P < 0.001 indicates the difference in apoptosis levels of KSHV/HMVEC versus HMVEC at each CBD concentration. (B) Regression analysis of the effect of CBD on the apoptosis of KSHV-infected HMVECs.
Figure 4.
Figure 4.
CBD inhibits the expression of KSHV vGPCR. (A) Representative immunohistochemical analysis of KSHV vGPCR expression in cutaneous Kaposi sarcoma lesions (panels II and III). Kaposi sarcoma lesion stained with an antibody isotype control (panel I). For panels I and II, scale bars = 50 µm. For panel III, scale bar =10 µm. (B) Representative Western blot analysis of KSHV vGPCR levels in whole cell lysates of KSHV/HMVECs after incubation with various concentrations of CBD or a vehicle control for 24 hours. GAPDH was used as a loading control.
Figure 5.
Figure 5.
CBD reduces levels of GRO-α in media conditioned by KSHV-infected HMVECs. (A) GRO-α and (C) IL-8 levels in KSHV/HMVEC-conditioned media as assayed by ELISA. After KSHV/HMVECs were incubated with various CBD concentrations or a vehicle control for 18 hours, GRO-α (A) and IL-8 levels (C) were measured by ELISA. Data represent the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 for CBD treatments versus a control (0 µM CBD). (B) Regression analysis of the effect of CBD on GRO-α expression in KSHV/HMVEC-conditioned media.
Figure 6.
Figure 6.
VEGFR-3 is activated by KSHV vGPCR and inhibited by CBD. (A) VEGFR-3 activation as assayed by VEGFR-3 immunoprecipitation and Western blot analysis. 293/VEGFR-3 cells were transfected with a KSHV vGPCR expression plasmid (+) or an empty vector (–). Total cell lysates were collected 48 hours after transfection and used for VEGFR-3 immunoprecipitation. Tyrosine phosphorylation of VEGFR-3 was detected by Western blot analysis. Total VEGFR-3 was used as a loading control. (B) VEGFR-3 activation as assayed by VEGFR-3 immunoprecipitation and Western blot analysis in HMVECs. HMVEC cells were transfected with a KSHV vGPCR expression plasmid (+) or an empty vector (–) and assayed as in A. (C) VEGFR-3 expression by Western blot analysis in KSHV/HMVECs. KSHV/HMVECs were incubated with various CBD concentrations for 18 hours. Total cell lysates were collected and VEGFR-3 expression measured by Western blot analysis. GAPDH was used as a loading control.
Figure 7.
Figure 7.
CBD inhibits the expression of VEGF-C. (A) VEGF-C and (C) VEGF-D levels in KSHV/HMVEC-conditioned media as assayed by ELISA. After KSHV/HMVECs were incubated with various CBD concentrations or a vehicle control for 18 hours, VEGF-C (A) and VEGF-D levels (C) were measured by ELISA. Data indicate the mean ± SD. *P < 0.05 and ***P < 0.001 for CBD treatments versus a control (0 µM CBD). (B) Regression analysis of the effect of CBD on VEGF-C expression in KSHV/HMVEC-conditioned media.

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