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Open Access Full Text Article R apid C ommu n icatio n

Suppression of vascular endothelial growth

factor expression by cannabinoids in a canine
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osteosarcoma cell line

This article was published in the following Dove Press journal:
Veterinary Medicine: Research and Reports
4 July 2013
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Andreza S Figueiredo 1 Abstract: Vascular endothelial growth factor (VEGF) is a key regulator in both physiologic
Hiram J García-Crescioni 1 and pathologic angiogenesis, and cannabinoids decrease VEGF release in human and murine
Sandra C Bulla 1 cancer cells. The aim of this study was to assess the in vitro effects of a synthetic ­cannabinoid,
Matthew K Ross 2 ­WIN-55,212-2, on the expression of the proangiogenic factor VEGF-A in the canine ­osteosarcoma
For personal use only.

Chelsea McIntosh 1 cell line 8. After analysis of gene expression by quantitative real-time polymerase chain reaction,
the compound decreased VEGF-A expression by 35% ± 10% (P , 0.0001) as compared with
Kari Lunsford 3
the control. This synthetic cannabinoid shows promise as a potential inhibitor of angiogenesis,
Camilo Bulla 1
and further studies are warranted to investigate its in vivo effects and to explore the potential
1
Department of Pathobiology and of this and related compounds as adjuvant cancer therapy in the dog.
Population Medicine, 2Department
of Basic Sciences, 3Department Keywords: dog, cancer, angiogenesis, cannabinoids
of Clinical Sciences and Animal
Health Center, College of Veterinary
Medicine, Mississippi State University, Introduction
Mississippi State, MS, USA Vascular endothelial growth factor (VEGF) is a proangiogenic signaling protein that
induces proliferation and migration of vascular endothelial cells. The VEGF family
comprises several glycoproteins, including VEGF-A, which is a major regulator of
normal and abnormal angiogenesis.1 It plays a key role in tumor angiogenesis and has
been found to be overexpressed in malignant tumors.2 Neoplastic tumors are dependent
on angiogenesis because the formation of new blood vessels is required for delivery
of the necessary nutrients and oxygen. In the absence of angiogenesis, tumors cannot
grow larger than approximately 2 mm in diameter. Cells in rapidly growing masses
are triggered by hypoxia to increase cellular VEGF expression in order to induce new
vessel growth.3
Endocannabinoids, phytocannabinoids, and synthetic cannabinoids bind to
­cannabinoid 1 (CB1) and 2 (CB2) receptors,4 and can inhibit tumor growth in several
cancers, including mouse glioma,5 and human pancreatic,6 the uterine,7 and prostatic
cancer.8 CB1 and CB2 receptors are found in many tissues in mammals,4 including the
bone cells of human9 and mouse10 species. WIN-55,212-2 is a synthetic ­cannabinoid
that binds to both CB1 and CB2 receptors and had been demonstrated to impede
­angiogenesis, induce apoptosis in mouse cancer cells,9 and inhibit VEGF expression
Correspondence: Camilo Bulla
Department of Pathobiology and in both human and mouse cancer cells.11,12
Population Medicine, College of Cancer biology and tumor behavior have been shown to be similar in humans and
Veterinary Medicine, Mississippi
State University, 240 Wise Center Drive, dogs for several common cancers,13 and the dog is emerging as a valuable naturally
Mississippi State, MS 39762-6100, USA occurring cancer model.14 Examples of these similarities are the canine and human
Tel +1 662 325 1189
Fax +1 662 325 4548
leukemias and lymphomas, that have a similar clinical presentation, tumor biology,
Email [email protected] and response to therapy, and have been demonstrated to share evolutionary conserved

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chromosomal aberrations and conserved mutations within had the same dimethylsulfoxide content of 0.1% (v/v). Three
key oncogenes.15 Mammary malignancies share the same independent experiments were performed.
pattern of deregulated genes.16 Specifically, studies conducted After total RNA extraction (RNeasy Mini Kit, Qiagen,
with canine osteosarcoma were the first to yield results appli- Valencia, CA, USA), RNA quality and concentration was
cable to humans.17 The similarities between osteosarcoma in evaluated by spectrophotometry. Real-time quantitative
both species include a large patient size, 75% or more cases polymerase chain reaction was performed by a one-step
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affecting the appendicular skeleton, metaphyseal location, reaction with a primer pair and labeled probe specific for
less than 10% of patients having documented metastasis canine VEGF-A (TaqMan Gene Expression Assay, Invitro-
at presentation, over 90% of tumors showing high-grade gen, Carlsbad, CA, USA).
­histology, 75% of tumors showing aneuploidy, the metastatic A specific canine GAPDH primer pair and labeled probe
rate being 80% or more with amputation alone, the lung being (Eurofins MWG Operon, Huntsville, AL, USA) was designed
the most common site of metastasis, and improved survival for normalization purposes. It was demonstrated to be a suit-
with adjuvant chemotherapy,18 as well as deregulation of able gene for normalization under the experimental conditions
key cellular proteins, like STAT3.19 Recently, the study of after following procedures described elsewhere.24 GAPDH
molecular profiles derived from canine osteosarcoma helped was considered to be stably expressed during the experiment
to group complex human osteosarcoma into biologically and on the basis of a −1.068-fold change because of treatment and
clinically relevant molecular subtypes.20 checked by the Student’s t-test with a P value of 0.48.
Despite aggressive treatment including surgery and chemo- The comparative quantification method was used for rela-
For personal use only.

therapy, little improvement in survival time has been achieved tive quantification25 and all samples were run in triplicate. The
in either humans or dogs.21,22 Added to the great importance of amplification efficiencies were analyzed by 10-fold dilution
osteosarcoma in both humans and dogs described above is the of a series of pooled RNA samples by 96.3% and 99.9% for
fact that VEGF levels are predictive of pulmonary metastasis VEGF-A and GAPDH, respectively.
and a poor prognosis in human osteosarcoma,23 leading to the Data are presented as the mean ± standard deviation. The
hypothesis that impairment of the proangiogenic pathway with statistical analysis was done by analysis of variance followed
cannabinoids can improve the prognosis of the disease. by the Student’s t-test26 using SAS version 9.2 (SAS Institute
To the best of our knowledge, there are no published Inc, Cary, NC, USA). A value of P , 0.05 was considered
studies evaluating VEGF levels in canine osteosarcoma or to be statistically significant.
the effects of cannabinoids in canine cancer or on canine
tumor cells. Thus, the aim of this study was to assess the Results
impact of treatment with WIN-55,212-2 on VEGF-A expres- As shown in Figure 1, OSA-8 cells incubated with ­WIN-55,212-2
sion in the canine osteosarcoma 8 (OSA-8) cell line.20 To for 48 hours showed a decrease in VEGF-A expression by
the authors’ knowledge, this is the first study using a canine 35% ± 10% on average (P , 0.0001) compared with the
model to evaluate the possible use of cannabinoids to impair control (dimethyl sulfoxide), which was 100% ± 23%.
angiogenesis in osteosarcoma.
Discussion
Materials and methods In this paper, we detail the use of a highly sensitive and spe-
The OSA-8 cells were maintained in Dulbecco’s Modified cific quantitative real-time polymerase chain reaction method
Eagle’s Medium (Gibco, Grand Island, NY, USA) supple- for initial assessment of VEGF-A mRNA levels in a canine
mented with 10% fetal bovine serum (Gibco), 0.2% Primocin tumor cell line following treatment with a cannabinoid. We
(InvivoGen, San Diego, CA, USA), and 1% HEPES (BioRe- have demonstrated that WIN-55,212-2 decreased VEGF-A
agent, Sigma-Aldrich, St Louis, MO, USA). Twenty-four hours expression in the cultured OSA-8 cells by 35%. This find-
before the experiments, 8 × 106 cells were seeded per well. ing is in accordance with other reports of the antiangiogenic
A stock solution of WIN-55,212-2 (Cayman Chemical Com- effect of WIN-55,212-2, with a greater than 25% decrease
pany, Ann Arbor, MI, USA) was prepared in dimethylsulfoxide in VEGF release from human and rat glioma cells,12 a 40%
(Thermo Fisher Scientific Inc, Waltham, MA, USA). The decrease from human prostate cancer cells,8 and marked
solution was added to OSA-8 cells to a final concentration of inhibition of VEGF expression in human skin carcinoma.11
1 µM in supplemented Dulbecco’s Modified Eagle’s Medium In addition to the antiangiogenic effects of cannabinoids
and incubated for 48 hours (n = 8). Control ­incubations (n = 6) observed here and previously, these compounds have been

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 Dovepress Antiangiogenic effects of cannabinoids in an osteosarcoma cell line

140% and human osteosarcoma,18 make the canine OSA-8 cell line
an attractive in vitro model for assessing the antiangiogenic
120% effects of cannabinoid treatment, and future work will benefit
both comparative angiogenesis research and canine patients
VEGF mRNA levels

100%
with cancer.
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80% * Conclusion
In conclusion, the cannabinoid agonist, WIN-55,212-2,
60%
100% ± was shown to reduce the expression of VEGF-A in a canine
23% osteosarcoma cell line. On the basis of the results reported
40%
65% ± here, we suggest that cannabinoid receptor agonists may have
10%
20% the potential for use as clinical angiogenesis inhibitors. Our
results suggest a first step toward the use of cannabinoids
0% as potential adjuvants to chemotherapeutics in the treatment
Control (n = 6) WIN-55,212-2 of canine cancers.
(n = 8)

Figure 1 Comparison between vascular endothelial growth factor (VEGF)

messenger RNA (mRNA) levels in control OSA-8 cells cultured for 48 hours with
Acknowledgment
dimethylsulfoxide (0.1%, v/v) or WIN-55,212-2 (1 µM). We thank Jaime F Modiano for kindly providing the canine
For personal use only.

Notes: Data are reported as the mean ± standard deviation of three independent
osteosarcoma cell line. This study was supported by the
experiments. *Statistically significant at P , 0.05.
Merial Veterinary Scholars Program and the Mississippi
State University, College of Veterinary Medicine.
used successfully in patients with human cancer for the
treatment of symptoms resulting from both cancer and anti- Disclosure
neoplastic therapies, including nausea and vomiting, weight The authors report no conflicts of interest in this work.
loss, lack of appetite, and pain. Although these benefits have
been known for years, it is only recently that the clinical anti- References
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