INVESTIGATION OF THE BETA 2 ADRENERGIC RECEPTOR (Β2-AR) PATHWAY IN CANINE HEMANGIOSARCOMA BY ROBERTA PORTELA THESIS Submitted in partial fulfillment of the requirements for the degree of Master of Science in VMS – Veterinary Clinical Medicine in the Graduate College of the University of Illinois at Urbana – Champaign, 2014 Urbana, Illinois Master’s Committee: Assistant Professor Jackie M. Wypij, Chair Associate Professor Timothy M. Fan Assistant Professor Stephane Lezmi
74
Embed
INVESTIGATION OF THE BETA 2 ADRENERGIC RECEPTOR (Β2-AR ...
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
INVESTIGATION OF THE BETA 2 ADRENERGIC RECEPTOR (Β2-AR) PATHWAY IN CANINE HEMANGIOSARCOMA
BY
ROBERTA PORTELA
THESIS
Submitted in partial fulfillment of the requirements
for the degree of Master of Science in VMS – Veterinary Clinical Medicine in the Graduate College of the
University of Illinois at Urbana – Champaign, 2014
Urbana, Illinois
Master’s Committee: Assistant Professor Jackie M. Wypij, Chair
Associate Professor Timothy M. Fan Assistant Professor Stephane Lezmi
ii
ABSTRACT
Canine hemangiosarcoma is a highly metastatic cancer arising from vascular endothelial cells. It
is one of the most aggressive canine cancers and most dogs die from this disease within a few
months of the diagnosis. Despite advancements in veterinary oncology, there has been minimal
improvement in the overall survival time even with standard treatment, which includes surgery
and chemotherapy. Propranolol, an oral drug originally developed for the treatment of
cardiovascular diseases, has been successfully used for the treatment of infantile hemangioma
which is a benign neoplasia of vascular endothelial cells. Propranolol blocks adrenergic
receptors, which would otherwise bind to catecholamines responsible for the “stress signal”
leading to many physiologic changes. Stress has been implicated in many models of
carcinogenesis and tumor progression. Given the relationship between stress and cancer, as well
as similarities between canine hemangiosarcoma and infantile hemangioma, we sought to
investigate the presence of the beta 2 adrenergic receptor and the effects of propranolol in canine
hemangiosarcoma. We demonstrated the presence of the beta 2 adrenergic receptor via
immunohistochemistry in all 18 tissue samples of spontaneous canine splenic hemangiosarcoma
and in canine hemangiosarcoma cell lines Fitz and DEN, cell line receptor expression was also
confirmed with Western blot. Src, a possible intermediary downstream protein involved in
adrenergic signaling was also investigated and both Fitz and DEN exhibited the presence of the
Src protein in Western blot. Activation of this pathway would involve phosphorylation of Src
upon catecholamine binding to the receptor, which was investigated through Western blot. Fitz
and DEN exhibited basal phosphorylation of Src and after treatment with norepinephrine, and
Fitz exhibited greater phosphorylation (23% increase compared to basal control) after 60-minute
exposure to the agonist. Fitz cells were pretreated with a biologically achievable dose of
iii
propranolol followed by the agonist, and a modest decrease in phosphorylation was observed
(11% decrease compared to basal level). Further investigation into the biological effects of
propranolol in Fitz and DEN revealed a decrease in VEGF secretion, increase in proliferation
and decrease in cell migration. Reduction in VEGF secretion was evaluated via ELISA and it
was present at propranolol doses greater than 10 µM for DEN and Fitz, achieving a maximum
reduction of 21% in DEN and 44% in Fitz compared to untreated cells. Cell proliferation was
measured through MTS assay, which revealed an increase in cell proliferation only in Fitz cells
treated with 0.1 µM of NE (25% increase) and cells treated with 0.1 µM of propranolol (30%
increase). Cell migration was evaluated with a scratch assay and was decreased only when cells
were treated with propranolol at a high dose (100 µM). Taken together, the findings of this study
show that beta 2 adrenergic receptors are expressed by canine hemangiosarcoma, Src may be
involved in the downstream signaling from the receptor and blockade of the receptor leads to
mild to moderate effects in cell angiogenesis, proliferation and migration.
iv
For my family,
Luiz, Thais, Rodrigo and Victoria Portela
for never letting me feel alone
and for always believing in me
v
ACKNOWLEDGEMENTS
Thank you to my advisor, Dr. Jackie Wypij, for all her help in this project.
My sincere thank you to Dr. Timothy Fan, for the enormous patience, without whom I
could not have accomplished this.
Thank you to Dr. Stephane Lezmi for the support not only with immunohistochemistry
but also with ideas and encouragement that inspired me to keep going.
A huge thank-you to my friend Evelyn Caporali for her incredible help and support when
I needed it most. Thank you so much.
Thanks to Kevin LeBoedec for taking the time to help me with statistics.
Special thank you to Holly Pondenis, for all the technical knowledge shared and
assistance with experiments. Her expertise is invaluable.
Thank you so much to my resident-mates, Sharon Shor, Zach Neumann and Alycen
Lundberg; technicians Jenny Byrd, Rebecca Kamerer and Tara Bailey, and Dr. Laura Garrett
from the Oncology Service for their words of encouragement throughout the years.
My gratitude to James R. Harkness, Wayne D. and Josephine H Spangler Endowment
fund for the financial assistance in this project.
Lastly, thank you to all my friends everywhere in the world, who at some point have
given me instructions, directions, shelter, a shoulder to cry on, and specially have pushed me to
move forward into success. Their kindness will never be forgotten.
Given that Src and phosphorylated Src (Tyr416) are intermediaries involved in signal
transduction from the β2-AR to the nucleus, we sought to determine if the β2-AR agonist (NE)
would lead to phosphorylation of the protein, and if this process could be blocked by the
antagonist (P). To determine the effects of NE and P on Src and p-Src, we performed WB on
cHSA cells. In DEN and Fitz cells treated with different concentrations of NE and P (up to 10
µM), there was no qualitative difference in the expression of total Src (Fig. 4.3b). Both DEN and
Fitz demonstrated significant basal phosphorylation of Src, suspected to be caused by the FBS
used in culture. Overnight serum-starvation in DEN and Fitz cell lines did not result in an
apparent difference in p-Src protein expression with P (low dose 0.1 µM) and increased time-
exposure to NE (Fig. 4.3c). To optimize phosphorylation, Fitz cells were serum-starved for 24
hours, followed by increasing time exposure to NE. Increased protein expression of p-Src was
identified, with phosphorylation more pronounced starting at 15 minutes and persisting through
60 minutes (Fig. 4.3d). Once the positive effects of the agonist NE on phosphorylation were
demonstrated, we proceeded to evaluate if pre-exposure to low-dose propranolol (0.1 µM) was
40
sufficient to block the receptor to the effects of the agonist. In Fitz cells, NE caused a modest
phosphorylation of Src and treatment with low-dose propranolol for 24 hours prior to exposure
led to a modest decrease in phosphorylation. These effects were visible in the WB and therefore
we proceeded to quantify these results by normalizing the adjusted volume of the p-Src band to
the matching β-actin band, to ensure that the increased intensity of the band observed was not
due to increased protein loading (Fig. 4.3e). To determine if propranolol could rescue NE
agonism, DEN and Fitz cells were serum-starved, pre-treated with NE (0-10 µM), then
propranolol (0-100 µM). In Fitz cells, low doses of propranolol reduce p-Src compared to
untreated cells, which is lost at the highest dose in unstimulated cells. In NE-stimulated Fitz
cells, propranolol reduces p-Src compared to basal levels at all doses. Conversely, in DEN cells,
propranolol increases p-Src in untreated and NE stimulated cells (Fig. 4.3f)
4.4. Propranolol affects VEGF secretion of cHSA in vitro
After demonstrating the presence of the receptor as well as a possible intermediate
protein involved in signal transduction, we proceeded to investigate the effects of NE and P on
cell function activities of cHSA cells. Since propranolol’s ability to inhibit tumor angiogenesis
has been largely implicated as a primary anti-neoplastic effect, we sought to evaluate the effects
of NE and P on the ability of cHSA cells to secrete VEGF, a key pro-angiogenic cytokine. In the
first experiment, we used NE at varying doses (0-10 µM) to assess optimal agonism. Normalized
to cell count, basal secretion of VEGF was 1098.83 pg/mL and 464.42 pg/mL for DEN and Fitz
cells, respectively. All doses of NE induced VEGF secretion compared to untreated cells for both
cell lines. Optimal NE agonism was obtained at 1 µM NE, with a significant increase in VEGF
secretion compared to control. At optimal NE agonism, there was an increase in VEGF of 29%
41
for DEN cells (p<0.05 for all treatments compared to control), and 2.6-fold increase for Fitz cells
(p<0.001 for all treatments compared to control) (Fig. 4.4a). Cells were then incubated with the
selected NE dose of 1 µM combined with varying doses of P starting at a biologically achievable
dose (0.1 µM) up to 100 µM. No significant differences were noted at low doses, however there
was a reduction in VEGF secretion in DEN at P ≥ 10 µM (p<0.01) with a maximum decrease in
VEGF of 21% at 100 µM. Similar results were seen with Fitz, with reduced VEGF secretion at P
≥ 10 µM (p<0.001) with maximum 44% reduction at 100 µM; a dose dependence was noted in
the Fitz cell line (p<0.05) (Fig 4.4b). Similar but less profound results were seen without NE
stimulation (results not shown).
4.5. Propranolol affects cell proliferation of cHSA in vitro
The metabolic activity of Fitz and DEN, an indirect measurement of cell viability and
proliferation, was evaluated upon exposure to different doses of NE and P. While there was no
significant difference noted after 24h incubation, when treated for 48h Fitz exhibited significant
difference in proliferation between untreated vs 0.1 µM of NE (25% increase) (p < 0.05) and
untreated vs 0.1 µM of P (30% increase) (p < 0.01). DEN did not exhibit significant difference
between groups (p > 0.05) (Fig. 4.5)
4.6. Propranolol affects cell migration of cHSA in vitro
The ability of cancer cells to move within the microenviroment towards the nearby
vasculature is important in the process of metastasis and therefore we elected to evaluate if P had
42
any effect on cell mobility by evaluating the cells ability to close a gap created between the cells.
Increasing concentrations of P were used (0-100 µM) and an artificially created gap was
measured before and 24 hours after treatment. Data is represented as a percentage of the original
gap (time 0), with being 100% a completely closed gap. The gap remained more noticeably open
at 10 and 100 µM, being statistically significant at 100 µM when compared to untreated control
cells (p<0.05) (Fig. 4.6).
43
CHAPTER 5
DISCUSSION AND CONCLUSIONS
In veterinary medicine, the emotional component of stress may not play an important role
as it does in humans, however the presence of cancer can lead to microenvironmental stressors
such as hypoxia and inflammation which may lead to catecholamine release [222, 223].
Therefore, stress can play a part in canine tumor progression. In this study we were able to
demonstrate the presence of β2-AR in all spontaneous splenic cHSA as well as cell lines
demonstrating the clinical relevance and consistency of expression, and suggesting that this
pathway may be activated by stress-related catecholamines. The Fitz cell line appeared to have
stronger protein expression than the DEN cell line. The expression of β2-AR in spontaneous
tumor samples was heterogeneous in intensity and localization within the cells. The variability of
IHC staining could be explained by a variable expression of the receptor within the neoplastic
cells, as well as integrity and detectability of the antigen depending on the time the tissue has
been stored and the fixative used to initially preserve it as described in a study looking at EGFR
[224, 225]. Since the samples tested were from different cases obtained over several years, the
intensity of the staining obtained could be a reflection of the particular sample rather than
variability within the tumors. However, since tumors undergo frequent mutations, some may
express more of a particular antigen or receptor compared to others and this variability may
account for the difference between cytoplasmic versus nuclear staining. Regarding cellular
localization, upon stimulation β2-AR undergoes internalization, intracellular trafficking and
recycling and thus variability in cytoplasmic localization may reflect variation in stimulation and
internalization [226]. Nuclear membrane localization of β1- and β3-AR as well as downstream
44
signaling partners, but not β2-AR, has been reported in normal cardiac myocytes [227]. Our IHC
results are in concordance with similar studies in human cancer. In a study of β2-AR IHC
expression in hepatocellular carcinoma, 60% of tumors had moderate/strong staining intensity,
31% weak staining intensity and 9% negative staining with nuclear and cytoplasmic distribution
[228]. Another study showed strong β2-AR staining in 41% of angiosarcoma samples that were
analyzed and a 77% total expression indicating heterogeneity in receptor expression, similar to
the results obtained in this project [34]. Further evaluation of localization of β2-AR and signaling
partners could include Western blotting of fractionated cell lysates, confocal microscopy with
immunofluorescence and other methods.
The non-receptor tyrosine kinase Src has been reported as one of the many downstream
proteins involved in β2-AR activation and therefore we elected to investigate if this pathway is
indeed activated and blocked by β2-AR agonism/antagonism [19]. After initially demonstrating
the presence of Src within the cell, we proceeded to verify if this protein is phosphorylated by
NE. The initial challenge was to overcome the strong basal phosphorylation of this protein
within the cHSA, which did not allow for a difference in visualization between untreated and
treated cells. Part of this phosphorylation was probably due to the presence of FBS, which
contains growth factors that can stimulate the Src pathway. After 24h serum-starvation there was
still p-Src activity within the untreated cells, however it was diminished enough that an
appreciable difference was seen compared to NE-treated cells. Norepinephrine agonism of Src
phosphorylation was confirmed, supporting a role for catecholamine-induced tumor stimulation.
The subsequent experiment was performed with pre-treatment of cells with P, showing a
decrease in phosphorylation at low doses of P. With NE agonism prior to P treatment (i.e.
propranolol “rescue”), which may most closely resemble the clinical scenario, P successfully
45
decreased Src phosphorylation in NE pretreated cells, while phosphorylation was variable in
DEN cells. This may be a factor of β2-AR expression, which was qualitatively higher in Fitz
cells. The preliminary results of this experiment warrants further investigation of this pathway,
however due to multiple pathways involved in β2-AR signaling it is possible that alternative
pathways are stimulated or inhibited with propranolol leading to the effects seen. These effects
were related to proliferation, VEGF secretion and cell migration. With respect to VEGF
secretion, cHSA cells secrete basal levels of VEGF with NE causing increased VEGF secretion,
more evident in the Fitz cell line. This supports our hypothesis that NE would increase VEGF
secretion as demonstrated in other studies [229]. The increase in VEGF may be a result of Src
phosphorylation as hypothesized, and the more profound reduction in VEGF in the Fitz cell line
may be related to the increased β2-AR expression as well as the more consistent phosphorylation
effects of P. Taken together, this data supports a role for the β2-AR and the potential role of
propranolol in inhibiting VEGF-mediated HSA angiogenesis.
The effects of NE and P on cell proliferation were also investigated. The initial
hypothesis was that NE would lead to increased proliferation and P would decrease proliferation
[230]. Interestingly, we found that both NE and P caused an increase in proliferation in Fitz cells
at 0.1 µM. There is one report in the literature of P inhibiting proliferation of pulmonary artery
smooth muscle cells at a 10 µM but not at 0.1 µM. The mechanism for this effect on proliferation
remained unexplained [231]. One possible explanation may be that this β-blocker could act as an
agonist to a different receptor or pathway involved in cell proliferation. Additional studies such
as time-dependent effects may elucidate this further. In one rodent study, propranolol caused a
transient increase in markers of cell proliferation (PCNA, mitotic index) that were abrogated
with longer term treatment [232]. The variation observed between the different cell lines in this
46
project was also observed in another study, where beta blockade effectively reduced proliferation
rate among multiple cell lines however some lines were more resistant to these effects than
others [214].
Our last finding was that P at a high dose (100 µM) had an effect on cell migration. This
effect has been described previously in human infantile hemangioma endothelial cells and it also
decreased phosphorylation of cofilin, as a possible mechanism involved (since cofilin is an actin-
severing protein and phosphorylation is an inhibitory event) [136, 233]. The dose necessary for a
significant effect is not biologically achievable, however a time-dependent effect at lower doses
was not investigated in this study and is an avenue for further investigation. Decrease in cell
migration was detected at a 50 µM dose in one study, which was careful to evaluate the effects of
this dose on net cell proliferation to eliminate inhibition of proliferation as a contributing factor
[214]. It is possible that our dose of 100 µM is affecting cell proliferation, which is indirectly
leading to part of the migration effects seen on the scratch assay.
In relating this study to the clinical scenario of canine hemangiosarcoma, the finding of
β2-AR expression in both spontaneous tumors and two cell lines, strong basal phosphorylation of
Src, as well as consistent agonism of downstream signaling by NE supports a role of this
pathway in tumor progression. Variability in expression may suggest a variable response to
pathway blockade. Effects on VEGF secretion and cell migration were most significant at the
highest doses of propranolol, which are not biologically achievable with single-dose treatment at
standard doses. The high doses of propranolol needed to impair tumor cell function is consistent
with that seen in infantile hemangioma – most of these studies show in vitro effect at 100-300
µM, which is not clinically achievable. However, in infantile hemangioma, continuous treatment
is used clinically, which may account for the discordancy between in vitro and in vivo effect. The
47
finding of positive effects on Src phosphorylation at low doses in cHSA may also support a
potential for low-dose propranolol time-dependent effects on VEGF secretion or cell migration,
although this was beyond the scope of this study. Continuous low dose treatment with
propranolol would be reasonable in an adjunctive setting in cHSA due to its low cost and side
effect profile.
Although this study indicated the presence of the β2-ARs in cHSA and that propranolol
exerts some biological effects in cHSA cell lines, it is important to acknowledge some
limitations of this project. There is no direct evidence that Src is the intermediary pathway
connecting β2-AR agonism and cell proliferation, VEGF secretion or cell migration since there
are many other possible pathways involved [19]. We attempted to elicit phosphorylation of Src
by exposing the cells to NE over time in order to detect the optimal moment at which most of the
protein is phosphorylated and thus more easily detectable. We were only able to detect a small
percentage of phosphorylation and even smaller attenuation by propranolol in one experiment.
The necessary amount of phosphorylated Src protein required for downstream activation of
subsequent proteins (such as STAT3 [234]) is unknown and could be as low as 1% and as high
as 100%, therefore the amount of necessary blockade of phosphorylation for biological effect
could also be variable. The use of an agonist with a stronger affinity for β2-AR, such as
isoproterenol [235], could lead to greater phosphorylation of Src and allow for more objective
visualization of the effects of propranolol. The cells were treated with 0.1 µM of propranolol for
one hour, which is a biologically achievable dose; however it does not represent the exposure
time to which an animal is subjected to, being a drug of continuous use. The same critique can be
applied to the other assays in which the cells were treated for either 24 or 48 hours, revealing
only statistically significant results at higher doses, which are not biologically achievable. Most
48
studies looking at the in vitro effects of norepinephrine or propranolol used high doses of
agonist/antagonist, probably because it was necessary to achieve significant findings. Drug
absorption and delivery to the tumor can be variable in each patient depending on tumor blood
flow and microenviroment, which is one limitation of translating in vitro findings to in vivo
effects. Additionally, as noted in one study where propranolol did not completely abolish tumor
cell proliferation in vivo as noted by positive PCNA staining in propranolol-treated tumors,
single-agent propranolol may not be as effective in tumor control however synergistic effects
may be observed with chemotherapeutics warranting further investigation into combination
therapy [214].
In conclusion, our study showed that cHSA express β2-ARs, Src kinase is one pathway
activated by this receptor and agonism/antagonism of this receptor exerts anti-angiogenic effects
in VEGF secretion and minimal effect on cell proliferation. Propranolol at high doses affects cell
migration. Further studies looking at other possible intermediate pathways as well as other
mechanisms involved in cancer development (matrix metalloproteinases or apoptosis) are areas
for further investigation.
49
CHAPTER 6
FIGURES
Figure 2.1. G Protein-coupled receptor activation (Belmonte S.L and Blaxall B.C., Circulation Research 109 (3), 309-319, July 2011).
Figure 2.2. G Protein-coupled receptor downstream signaling. (Cole S.W. and Sood A.K., Clin Cancer Res 18 (5), 1201-1206, March 2012).
50
Figure 4.1. Expression of β2-AR in spontaneous cHSA. Immunohistochemistry was performed on formalin-fixed paraffin-embedded sections containing tissue from splenic cHSA. All samples stained positive for the β2-AR antibody and the degree of staining was variable. Examples of strong β2-AR stain intensity (top row) and mild β2-AR stain intensity (bottom row) are shown. The diagnosis of splenic cHSA was confirmed after review of H&E staining and CD31. Negative control was performed using an isotypic control antibody (1000x).
Figure 4.2a. Expression of β2-AR in cHSA cell lines. Immunohistochemistry was performed on formalin-fixed paraffin-embedded sections of agar-embedded cell pellets containing cHSA cells Fitz and DEN. Human mammary carcinoma cell line MDA-MB-231 was used as a positive control. Negative control was the absence of the primary antibody (10x).
51
Figure 4.2b. Expression of β2-AR protein in cHSA cell lines. Western blot was performed using whole cell lysates of Fitz and DEN. Cell lines HeLa and MDA-MB-231 were used as positive control.
Figure 4.3a. Expression of Src and p-Src in cHSA cell lines. Western blot using an antibody for Src and p-Src was performed using whole cell lysates of Fitz, DEN and MDCK (control). For better visualization of p-Src the cells were serum-starved and stimulated with 3 mM of H2O2 for 5 minutes. All cells demonstrated the presence of basal Src protein as well as phosphorylated Src protein.
Figure 4.3b. Expression of Src in cHSA cell lines exposed to different concentrations of NE and P. Fitz and DEN cells were treated with different concentrations of NE and P for 24 hours as indicated below. Western blot revealed bands of similar size across all treatments, indicating that the presence of total Src protein is independent of the stimulus.
52
Figure 4.3c. Expression of p-Src in cHSA cell lines during increasing exposure to NE and P. Fitz and DEN cells were serum-starved overnight and treated with 10 µM of NE and 0.1 µM of P for different lengths of time as indicated below. Despite 12hr serum-starvation there was still marked baseline phosphorylation of Src, which did not allow for qualitative distinction between control and treated groups.
Figure 4.3d. Expression of p-Src in Fitz cell line during increasing exposure to NE. Fitz was serum-starved for 24 hr and subsequently increasingly exposed to 10 µM of NE. There was a qualitative difference between untreated control and 15-60 minute exposure.
53
Figure 4.3e. Expression of p-Src in Fitz cell line during increasing exposure to NE after pretreatment with P. Fitz was serum-starved for 24 hr and cells received either no pre-treatment or 0.1µM of propranolol for 24 hours. Both groups were subsequently increasingly exposed to 10 µM of NE. There was a modest increase in phosphorylation of Src over time (23% at 60’) and there was modestly decreased phosphorylation when the cells had been pretreated with propranolol (11% at 60’). This difference was quantified as the ratio of chemiluminescence emitted by p-Src and β-actin for each band.
Figure 4.3f. Expression of p-Src in DEN and Fitz cell line pretreated with NE and rescued with propranolol. DEN and Fitz were serum-starved for 24 hr and cells received either no pre-treatment or 10µM of NE for 45 minutes. Cells were subsequently exposed to 0-100µM of P. In non-pretreated Fitz cells, low doses of propranolol reduce p-Src, which is lost at the highest dose. With NE agonism in Fitz cells, propranolol reduces p-Src compared to basal levels at all doses. In DEN cells, propranolol increases p-Src in untreated and NE stimulated cells.
54
Figure 4.4a. VEGF secretion by cHSA cell lines after treatment with NE. The graph represent an average of VEGF secretion by Fitz and DEN quantified by ELISA and normalized to cell count. Cells were treated with increasing doses of NE (0-10 µM) for 24 hours. In both cell lines, compared to untreated cells, there is a significant difference in VEGF secretion (DEN p<0.05, Fitz p<0.001). Error bars represent SD.
Figure 4.4b. VEGF secretion by cHSA cell lines after treatment with NE and P. The graphs represent an average of VEGF secretion by Fitz and DEN quantified by ELISA and normalized to cell count. All cells were treated with 1 µM NE (agonist) and P at 0-100µM for 24 hours. At propranolol doses of ≥10 µM, there was a significant difference of VEGF secretion by both cell lines compared to untreated cells (DEN p<0.01, Fitz p<0.001). For Fitz, there was a significant difference between 10 and 100 µM. p<0.05. Error bars represent SD.
55
Figure 4.5. Proliferation activity of cHSA cell lines after treatment with NE and P. Fitz and DEN were treated with different concentrations of NE and P for 24 hours (top two graphs) and subsequently analyzed by MTS assay. There was no statistical difference between the groups in both cell lines (p > 0.5). When cells were treated for 48 hours (bottom 2 graphs), Fitz exhibited significant difference between untreated vs 0.1 µM of NE (p < 0.05) and untreated vs 0.1 µM of P (p < 0.01). Error bars represent SD.
56
Figure 4.6. Migration of cHSA cells when exposed to increasing doses of propranolol. A scratch assay was performed with Fitz and DEN exposed to increasing concentrations of propranolol for 24 hours, and an average measurement of the residual gap in each treatment group was compared to an untreated control. There was a significant difference between the untreated group and 100 µM (p < 0.05). Error bars represent SD.
57
Figure 4.6 (cont). Migration of cHSA cells when exposed to increasing doses of propranolol. A scratch assay was performed with Fitz and DEN exposed to increasing concentrations of propranolol for 24 hours, and an average measurement of the residual gap in each treatment group was compared to an untreated control. There was a significant difference between the untreated group and 100 µM (p < 0.05). Error bars represent SD.
58
REFERENCES
1. Priester, W.A., Hepatic angiosarcomas in dogs: an excessive frequency as compared with man. J Natl Cancer Inst, 1976. 57(2): p. 451-‐4.
2. Priester, W.A. and F.W. McKay, The occurrence of tumors in domestic animals. Natl Cancer Inst Monogr, 1980(54): p. 1-‐210.
3. Spangler, W.L. and M.R. Culbertson, Prevalence, type, and importance of splenic diseases in dogs: 1,480 cases (1985-‐1989). J Am Vet Med Assoc, 1992. 200(6): p. 829-‐34.
4. Thamm, D.H., Hemangiosarcoma, in Small Animal Clinical Oncology, S.J. Withrow, Vail D.M., Page, R.L., Editor. 2013, Elsevier: St. Louis, MO. p. 679-‐688.
5. Leaute-‐Labreze, C., et al., Propranolol for severe hemangiomas of infancy. N Engl J Med, 2008. 358(24): p. 2649-‐51.
6. Leboulanger, N., et al., Propranolol in the therapeutic strategy of infantile laryngotracheal hemangioma: A preliminary retrospective study of French experience. Int J Pediatr Otorhinolaryngol, 2010. 74(11): p. 1254-‐7.
7. Kurzyna, A., et al., [Propranolol for treatment of subglottic hemangioma]. Otolaryngol Pol, 2010. 64(6): p. 388-‐91.
8. Jadhav, V.M. and S.N. Tolat, Dramatic response of propranolol in hemangioma: report of two cases. Indian J Dermatol Venereol Leprol, 2010. 76(6): p. 691-‐4.
9. Fay, A., et al., Propranolol for isolated orbital infantile hemangioma. Arch Ophthalmol, 2010. 128(2): p. 256-‐8.
10. Baetz, J., et al., [Infantile hemangioma. Successful treatment with propranolol]. Hautarzt, 2010. 61(4): p. 290-‐2.
11. Denoyelle, F., et al., Role of Propranolol in the therapeutic strategy of infantile laryngotracheal hemangioma. Int J Pediatr Otorhinolaryngol, 2009. 73(8): p. 1168-‐72.
12. Barron, T.I., et al., Beta blockers and breast cancer mortality: a population-‐ based study. J Clin Oncol, 2011. 29(19): p. 2635-‐44.
13. Powe, D.G., et al., Beta-‐blocker drug therapy reduces secondary cancer formation in breast cancer and improves cancer specific survival. Oncotarget, 2010. 1(7): p. 628-‐38.
14. Buhler, H.U., et al., Plasma adrenaline, noradrenaline and dopamine in man and different animal species. J Physiol, 1978. 276: p. 311-‐20.
15. http://www.oxforddictionaries.com/us/definition/american_english/stress). . 16. Reiche, E.M., S.O. Nunes, and H.K. Morimoto, Stress, depression, the immune system, and
cancer. Lancet Oncol, 2004. 5(10): p. 617-‐25. 17. Chrousos, G.P., The hypothalamic-‐pituitary-‐adrenal axis and immune-‐mediated inflammation. N
Engl J Med, 1995. 332(20): p. 1351-‐62. 18. Dimsdale, J.M., M, Plasma catecholamines in stress and exercise. JAMA, 1980. 243(2): p. 340-‐
342. 19. Cole, S.W. and A.K. Sood, Molecular pathways: beta-‐adrenergic signaling in cancer. Clin Cancer
Res, 2012. 18(5): p. 1201-‐6. 20. Daly, C.J. and J.C. McGrath, Previously unsuspected widespread cellular and tissue distribution of
beta-‐adrenoceptors and its relevance to drug action. Trends Pharmacol Sci, 2011. 32(4): p. 219-‐26.
21. Carroll, B.T., et al., Screening for depression and anxiety in cancer patients using the Hospital Anxiety and Depression Scale. Gen Hosp Psychiatry, 1993. 15(2): p. 69-‐74.
22. Sellick, S.M. and A.D. Edwardson, Screening new cancer patients for psychological distress using the hospital anxiety and depression scale. Psychooncology, 2007. 16(6): p. 534-‐42.
23. Mantovani, A., et al., Cancer-‐related inflammation. Nature, 2008. 454(7203): p. 436-‐44.
59
24. Candido, J. and T. Hagemann, Cancer-‐related inflammation. J Clin Immunol, 2013. 33 Suppl 1: p. S79-‐84.
25. Pollock, R.E. and J.A. Roth, Cancer-‐induced immunosuppression: implications for therapy? Semin Surg Oncol, 1989. 5(6): p. 414-‐9.
26. Jenkins, F.J., B. Van Houten, and D.H. Bovbjerg, Effects on DNA Damage and/or Repair Processes as Biological Mechanisms Linking Psychological Stress to Cancer Risk. J Appl Biobehav Res, 2014. 19(1): p. 3-‐23.
27. Obeid, E.I. and S.D. Conzen, The role of adrenergic signaling in breast cancer biology. Cancer Biomark, 2013. 13(3): p. 161-‐9.
28. Yang, E.V. and T.D. Eubank, The impact of adrenergic signaling in skin cancer progression: possible repurposing of beta-‐blockers for treatment of skin cancer. Cancer Biomark, 2013. 13(3): p. 155-‐60.
29. Bridle, P.A., et al., Basal levels of plasma epinephrine and norepinephrine in the dog. Hypertension, 1983. 5(6 Pt 3): p. V128-‐33.
30. Sloan, E.K., et al., The sympathetic nervous system induces a metastatic switch in primary breast cancer. Cancer Res, 2010. 70(18): p. 7042-‐52.
31. Taylor, M.R., Pharmacogenetics of the human beta-‐adrenergic receptors. Pharmacogenomics J, 2007. 7(1): p. 29-‐37.
32. Audet, M. and M. Bouvier, Insights into signaling from the beta2-‐adrenergic receptor structure. Nat Chem Biol, 2008. 4(7): p. 397-‐403.
33. Rosenbaum, D.M., S.G. Rasmussen, and B.K. Kobilka, The structure and function of G-‐protein-‐coupled receptors. Nature, 2009. 459(7245): p. 356-‐63.
34. Chisholm, K.M., et al., beta-‐Adrenergic receptor expression in vascular tumors. Mod Pathol, 2012. 25(11): p. 1446-‐51.
35. Lipka, E., et al., In vivo non-‐linear intestinal permeability of celiprolol and propranolol in conscious dogs: evidence for intestinal secretion. Eur J Pharm Sci, 1998. 6(1): p. 75-‐81.
36. Vauquelin, G.a.v.M., B. , G-‐protein coupled receptors. G Protein-‐Coupled Receptors: Molecular Pharmacology from Academic Concept to Pharmaceutical Research, 2007: p. 77-‐230.
37. Luttrell, L.M., et al., Beta-‐arrestin-‐dependent formation of beta2 adrenergic receptor-‐Src protein kinase complexes. Science, 1999. 283(5402): p. 655-‐61.
38. Armaiz-‐Pena, G.N., et al., Src activation by beta-‐adrenoreceptors is a key switch for tumour metastasis. Nat Commun, 2013. 4: p. 1403.
39. Guarino, M., Src signaling in cancer invasion. J Cell Physiol, 2010. 223(1): p. 14-‐26. 40. Summy, J.M. and G.E. Gallick, Src family kinases in tumor progression and metastasis. Cancer
Metastasis Rev, 2003. 22(4): p. 337-‐58. 41. Shor, A.C., et al., Dasatinib inhibits migration and invasion in diverse human sarcoma cell lines
and induces apoptosis in bone sarcoma cells dependent on SRC kinase for survival. Cancer Res, 2007. 67(6): p. 2800-‐8.
42. Fredriksson, J.M., et al., Norepinephrine induces vascular endothelial growth factor gene expression in brown adipocytes through a beta -‐adrenoreceptor/cAMP/protein kinase A pathway involving Src but independently of Erk1/2. J Biol Chem, 2000. 275(18): p. 13802-‐11.
43. Fredriksson, J.M. and J. Nedergaard, Norepinephrine specifically stimulates ribonucleotide reductase subunit R2 gene expression in proliferating brown adipocytes: mediation via a cAMP/PKA pathway involving Src and Erk1/2 kinases. Exp Cell Res, 2002. 274(2): p. 207-‐15.
44. Dickerson, E.B., et al., Imatinib and Dasatinib Inhibit Hemangiosarcoma and Implicate PDGFR-‐beta and Src in Tumor Growth. Transl Oncol, 2013. 6(2): p. 158-‐68.
45. Powe, D.G. and F. Entschladen, Targeted therapies: Using beta-‐blockers to inhibit breast cancer progression. Nat Rev Clin Oncol, 2011. 8(9): p. 511-‐2.
60
46. Guise, T.A., et al., Evidence for a causal role of parathyroid hormone-‐related protein in the pathogenesis of human breast cancer-‐mediated osteolysis. J Clin Invest, 1996. 98(7): p. 1544-‐9.
47. Campbell, J.P., et al., Stimulation of host bone marrow stromal cells by sympathetic nerves promotes breast cancer bone metastasis in mice. PLoS Biol, 2012. 10(7): p. e1001363.
48. Katayama, Y., et al., Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow. Cell, 2006. 124(2): p. 407-‐21.
49. Calvi, L.M., Osteoblastic activation in the hematopoietic stem cell niche. Ann N Y Acad Sci, 2006. 1068: p. 477-‐88.
50. Elefteriou, F., Neuronal signaling and the regulation of bone remodeling. Cell Mol Life Sci, 2005. 62(19-‐20): p. 2339-‐49.
51. Melhem-‐Bertrandt, A., et al., Beta-‐blocker use is associated with improved relapse-‐free survival in patients with triple-‐negative breast cancer. J Clin Oncol, 2011. 29(19): p. 2645-‐52.
52. Haffty, B.G., et al., Locoregional relapse and distant metastasis in conservatively managed triple negative early-‐stage breast cancer. J Clin Oncol, 2006. 24(36): p. 5652-‐7.
53. Lin, N.U., et al., Sites of distant recurrence and clinical outcomes in patients with metastatic triple-‐negative breast cancer: high incidence of central nervous system metastases. Cancer, 2008. 113(10): p. 2638-‐45.
54. Powe, D.G., et al., Alpha-‐ and beta-‐adrenergic receptor (AR) protein expression is associated with poor clinical outcome in breast cancer: an immunohistochemical study. Breast Cancer Res Treat, 2011. 130(2): p. 457-‐63.
55. Jansen, L., et al., Beta blocker use and colorectal cancer risk: population-‐based case-‐control study. Cancer, 2012. 118(16): p. 3911-‐9.
56. Friedman, G.D., N. Udaltsova, and L.A. Habel, Norepinephrine antagonists and cancer risk. Int J Cancer, 2011. 128(3): p. 737-‐8; author reply 739.
57. Shah, S.M., et al., Does beta-‐adrenoceptor blocker therapy improve cancer survival? Findings from a population-‐based retrospective cohort study. Br J Clin Pharmacol, 2011. 72(1): p. 157-‐61.
58. Fitzgerald, P.J., Beta blockers, norepinephrine, and cancer: an epidemiological viewpoint. Clin Epidemiol, 2012. 4: p. 151-‐6.
59. Bangalore, S., et al., Antihypertensive drugs and risk of cancer: network meta-‐analyses and trial sequential analyses of 324,168 participants from randomised trials. Lancet Oncol, 2011. 12(1): p. 65-‐82.
60. Huang, X.Y., et al., Norepinephrine stimulates pancreatic cancer cell proliferation, migration and invasion via beta-‐adrenergic receptor-‐dependent activation of P38/MAPK pathway. Hepatogastroenterology, 2012. 59(115): p. 889-‐93.
61. Guo, K., et al., Norepinephrine-‐induced invasion by pancreatic cancer cells is inhibited by propranolol. Oncol Rep, 2009. 22(4): p. 825-‐30.
62. Zhang, D., et al., beta2-‐adrenergic antagonists suppress pancreatic cancer cell invasion by inhibiting CREB, NFkappaB and AP-‐1. Cancer Biol Ther, 2010. 10(1): p. 19-‐29.
63. Al-‐Wadei, H.A., M.H. Al-‐Wadei, and H.M. Schuller, Prevention of pancreatic cancer by the beta-‐blocker propranolol. Anticancer Drugs, 2009. 20(6): p. 477-‐82.
64. Lin, X., et al., Beta-‐adrenoceptor action on pancreatic cancer cell proliferation and tumor growth in mice. Hepatogastroenterology, 2012. 59(114): p. 584-‐8.
65. Wisler, J.W., et al., A unique mechanism of beta-‐blocker action: carvedilol stimulates beta-‐arrestin signaling. Proc Natl Acad Sci U S A, 2007. 104(42): p. 16657-‐62.
66. Thaker, P.H., et al., Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma. Nat Med, 2006. 12(8): p. 939-‐44.
67. Lutgendorf, S.K., et al., Stress-‐related mediators stimulate vascular endothelial growth factor secretion by two ovarian cancer cell lines. Clin Cancer Res, 2003. 9(12): p. 4514-‐21.
61
68. Jemal, A., et al., Cancer statistics, 2004. CA Cancer J Clin, 2004. 54(1): p. 8-‐29. 69. Hlatky, L., P. Hahnfeldt, and J. Folkman, Clinical application of antiangiogenic therapy:
microvessel density, what it does and doesn't tell us. J Natl Cancer Inst, 2002. 94(12): p. 883-‐93. 70. Lee, J.W., et al., The effect of surgical wound on ovarian carcinoma growth in an animal model.
Anticancer Res, 2013. 33(8): p. 3177-‐84. 71. Lee, J.W., et al., Surgical stress promotes tumor growth in ovarian carcinoma. Clin Cancer Res,
2009. 15(8): p. 2695-‐702. 72. Allendorf, J.D., et al., Increased tumor establishment and growth after open vs laparoscopic
bowel resection in mice. Surg Endosc, 1998. 12(8): p. 1035-‐8. 73. Abramovitch, R., et al., Stimulation of tumour growth by wound-‐derived growth factors. Br J
Cancer, 1999. 79(9-‐10): p. 1392-‐8. 74. Belizon, A., et al., Major abdominal surgery increases plasma levels of vascular endothelial
growth factor: open more so than minimally invasive methods. Ann Surg, 2006. 244(5): p. 792-‐8. 75. Ramos-‐Jimenez, J., et al., Histamine augments beta2-‐adrenoceptor-‐induced cyclic AMP
accumulation in human prostate cancer cells DU-‐145 independently of known histamine receptors. Biochem Pharmacol, 2007. 73(6): p. 814-‐23.
76. Zhang, P., et al., beta-‐arrestin2 mediates beta-‐2 adrenergic receptor signaling inducing prostate cancer cell progression. Oncology reports, 2011. 26(6): p. 1471-‐7.
77. Palm, D., et al., The norepinephrine-‐driven metastasis development of PC-‐3 human prostate cancer cells in BALB/c nude mice is inhibited by beta-‐blockers. Int J Cancer, 2006. 118(11): p. 2744-‐9.
78. Hassan, S., et al., Behavioral stress accelerates prostate cancer development in mice. J Clin Invest, 2013. 123(2): p. 874-‐86.
79. Ramberg, H., et al., Hormonal regulation of beta2-‐adrenergic receptor level in prostate cancer. Prostate, 2008. 68(10): p. 1133-‐42.
80. Prowatke, I., et al., Expression analysis of imbalanced genes in prostate carcinoma using tissue microarrays. Br J Cancer, 2007. 96(1): p. 82-‐8.
81. Pippig, S., et al., Overexpression of beta-‐arrestin and beta-‐adrenergic receptor kinase augment desensitization of beta 2-‐adrenergic receptors. J Biol Chem, 1993. 268(5): p. 3201-‐8.
82. Grytli, H.H., et al., Use of beta-‐blockers is associated with prostate cancer-‐specific survival in prostate cancer patients on androgen deprivation therapy. Prostate, 2013. 73(3): p. 250-‐60.
83. Lin, Q., et al., Effect of chronic restraint stress on human colorectal carcinoma growth in mice. PLoS One, 2013. 8(4): p. e61435.
84. Hicks, B.M., et al., beta-‐Blocker usage and colorectal cancer mortality: a nested case-‐control study in the UK Clinical Practice Research Datalink cohort. Ann Oncol, 2013. 24(12): p. 3100-‐6.
85. Moretti, S., et al., beta-‐adrenoceptors are upregulated in human melanoma and their activation releases pro-‐tumorigenic cytokines and metalloproteases in melanoma cell lines. Lab Invest, 2013. 93(3): p. 279-‐90.
86. Guo, Y., et al., Interleukin-‐6 signaling pathway in targeted therapy for cancer. Cancer Treat Rev, 2012. 38(7): p. 904-‐10.
87. Waugh, D.J. and C. Wilson, The interleukin-‐8 pathway in cancer. Clin Cancer Res, 2008. 14(21): p. 6735-‐41.
88. Yang, E.V., et al., Norepinephrine upregulates VEGF, IL-‐8, and IL-‐6 expression in human melanoma tumor cell lines: implications for stress-‐related enhancement of tumor progression. Brain Behav Immun, 2009. 23(2): p. 267-‐75.
89. De Giorgi, V., et al., beta-‐adrenergic-‐blocking drugs and melanoma: current state of the art. Expert Rev Anticancer Ther, 2012. 12(11): p. 1461-‐7.
62
90. De Giorgi, V., et al., Effect of beta-‐blockers and other antihypertensive drugs on the risk of melanoma recurrence and death. Mayo Clin Proc, 2013. 88(11): p. 1196-‐203.
91. Kozanoglu, I., et al., New indication for therapeutic potential of an old well-‐known drug (propranolol) for multiple myeloma. J Cancer Res Clin Oncol, 2013. 139(2): p. 327-‐35.
92. Yang, E.V., et al., VEGF is differentially regulated in multiple myeloma-‐derived cell lines by norepinephrine. Brain Behav Immun, 2008. 22(3): p. 318-‐23.
93. Shand, D.G., Pharmacokinetics of propranolol: a review. Postgrad Med J, 1976. 52 Suppl 4: p. 22-‐25.
94. Mills, P.C., G.A. Siebert, and M.S. Roberts, A model to study intestinal and hepatic metabolism of propranolol in the dog. J Vet Pharmacol Ther, 2004. 27(1): p. 45-‐8.
95. Akin, A., et al., The efficacy of amiodarone-‐propranolol combination for the management of childhood arrhythmias. Pacing Clin Electrophysiol, 2013. 36(6): p. 727-‐31.
96. Hedera, P., F. Cibulcik, and T.L. Davis, Pharmacotherapy of Essential Tremor. J Cent Nerv Syst Dis, 2013. 5: p. 43-‐55.
97. Schneider, S.A. and G. Deuschl, The treatment of tremor. Neurotherapeutics, 2014. 11(1): p. 128-‐38.
98. Hepp, Z., L.M. Bloudek, and S.F. Varon, Systematic review of migraine prophylaxis adherence and persistence. J Manag Care Pharm, 2014. 20(1): p. 22-‐33.
99. Shamliyan, T.A., et al., Preventive pharmacologic treatments for episodic migraine in adults. J Gen Intern Med, 2013. 28(9): p. 1225-‐37.
100. Hruska, B., P.K. Cullen, and D.L. Delahanty, Pharmacological modulation of acute trauma memories to prevent PTSD: Considerations from a developmental perspective. Neurobiol Learn Mem, 2014.
101. Fitzgerald, P.J., J.R. Seemann, and S. Maren, Can fear extinction be enhanced? A review of pharmacological and behavioral findings. Brain Res Bull, 2013.
102. Tawa, J. and S. Murphy, Psychopharmacological treatment for military posttraumatic stress disorder: an integrative review. J Am Assoc Nurse Pract, 2013. 25(8): p. 419-‐23.
103. de Kleine, R.A., B.O. Rothbaum, and A. van Minnen, Pharmacological enhancement of exposure-‐based treatment in PTSD: a qualitative review. Eur J Psychotraumatol, 2013. 4.
104. Searcy, C.P., et al., Pharmacological prevention of combat-‐related PTSD: a literature review. Mil Med, 2012. 177(6): p. 649-‐54.
105. Black, J.W., et al., A NEW ADRENERGIC BETARECEPTOR ANTAGONIST. Lancet, 1964. 1(7342): p. 1080-‐1.
106. Basu, B., et al., A Case of Akathisia induced by Escitalopram: Case Report & Review of Literature. Curr Drug Saf, 2014. 9(1): p. 56-‐9.
107. Fitzgerald, P.J., Is elevated norepinephrine an etiological factor in some cases of schizophrenia? Psychiatry Res, 2014. 215(3): p. 497-‐504.
108. Hogan, S.R., J. Mandrell, and D. Eilers, Adrenergic urticaria: Review of the literature and proposed mechanism. J Am Acad Dermatol, 2013.
109. Finnerty, C.C. and D.N. Herndon, Is propranolol of benefit in pediatric burn patients? Adv Surg, 2013. 47: p. 177-‐97.
110. Herndon, D.N., et al., Long-‐term propranolol use in severely burned pediatric patients: a randomized controlled study. Ann Surg, 2012. 256(3): p. 402-‐11.
111. Olah, G., et al., Increased poly(ADP-‐ribosyl)ation in skeletal muscle tissue of pediatric patients with severe burn injury: prevention by propranolol treatment. Shock, 2011. 36(1): p. 18-‐23.
112. Rojas, Y., et al., Burns: an update on current pharmacotherapy. Expert Opin Pharmacother, 2012. 13(17): p. 2485-‐94.
63
113. Nandhra, H.S., C.L. Murphy, and A. Sule, Novel pharmacological agents targeting memory and cognition in the treatment of anxiety disorders. Hum Psychopharmacol, 2013.
114. Altamura, A.C., et al., Understanding the pharmacokinetics of anxiolytic drugs. Expert Opin Drug Metab Toxicol, 2013. 9(4): p. 423-‐40.
115. Lindgren, M.E., et al., Beta-‐blockers may reduce intrusive thoughts in newly diagnosed cancer patients. Psychooncology, 2013. 22(8): p. 1889-‐94.
116. Lawley, L.P., E. Siegfried, and J.L. Todd, Propranolol treatment for hemangioma of infancy: risks and recommendations. Pediatr Dermatol, 2009. 26(5): p. 610-‐4.
117. Pavlakovic, H., et al., Hyperkalemia complicating propranolol treatment of an infantile hemangioma. Pediatrics, 2010. 126(6): p. e1589-‐93.
118. Abbott, J., et al., Diarrhea associated with propranolol treatment for hemangioma of infancy (HOI). Pediatr Dermatol, 2010. 27(5): p. 558.
119. Giron-‐Vallejo, O., et al., Dental caries as a side effect of infantile hemangioma treatment with propranolol solution. Pediatr Dermatol, 2010. 27(6): p. 672-‐3.
120. Bonifazi, E., et al., Severe hypoglycemia during successful treatment of diffuse hemangiomatosis with propranolol. Pediatr Dermatol, 2010. 27(2): p. 195-‐6.
121. McBride, J.T., M.C. McBride, and P.H. Viles, Hypoglycemia associated with propranolol. Pediatrics, 1973. 51(6): p. 1085-‐7.
122. Holland, K.E., et al., Hypoglycemia in children taking propranolol for the treatment of infantile hemangioma. Arch Dermatol, 2010. 146(7): p. 775-‐8.
123. Kwon, E.K., et al., Retrospective review of adverse effects from propranolol in infants. JAMA Dermatol, 2013. 149(4): p. 484-‐5.
124. Volmer, P.A., Human Drugs of Abuse, in Kirk's Current Veterinary Therapy XIV, J.D.B.D.C. Twedt, Editor. 2009, Saunders Co.: Philadelphia. p. 144-‐145.
125. Wright, K.N., Assessment and Treatment of Supraventricular Tachyarrhythmias, in Kirk's Current Veterinary Therapy XIV, J.D.B.D.C. Twedt, Editor. 2009, Saunders Co: Philadelphia. p. 722-‐727.
126. Schober, K.E., Myocarditis, in Kirk's Current Veterinary Therapy XIV, J.D.B.D.C. Twedt, Editor. 2009, Saunders Co: Philadelphia. p. 804-‐808.
127. Kates, R.E., B.W. Keene, and R.L. Hamlin, Pharmacokinetics of propranolol in the dog. Journal of Veterinary Pharmacology and Therapeutics, 1979. 2(1): p. 21-‐26.
128. Trepanier, L.A., Medical Treatment of Feline Hyperthyroidism, in Kirk's Current Veterinary Therapy XIV, J.D.B.D.C. Twedt, Editor. 2009, Saunders Co: Philadelphia. p. 175-‐179.
129. Westropp, I.F.L.J.L., Urinary Incontinence and Micturition Disorders: Pharmacologic Management, in Kirk's Current Veterinary Therapy XIV, J.D.B.D.C. Twedt, Editor. 2009, Saunders Co.: Philadelphia. p. 955.
130. Dana G. Allen, J.K.P., Dale Smith, Handbook of Veterinary Drugs. 1993. 131. Feng, X.M., et al., Preparation and evaluation of a novel delayed-‐onset sustained-‐release system
of propranolol hydrochloride. J Pharm Pharmacol, 2008. 60(7): p. 817-‐22. 132. Papich, M.G., Table of Common Drugs: Approximate Dosages, in Kirk's Current Veterinary
Therapy XIV, J.D.B.D.C. Twedt, Editor. 2009, Saunders Co: Philadelphia. p. 1329. 133. Plumb, Plumb's Veterinary Drug Handbook. Vol. 6th. 2008. 134. Fonseca Junior, N.L., et al., [Therapeutical effectiveness of interferon alpha in a child with
craniofacial giant hemangioma: case report]. Arq Bras Oftalmol, 2008. 71(3): p. 423-‐6. 135. Enjolras, O., et al., [Vincristine treatment for function-‐ and life-‐threatening infantile
hemangioma]. Arch Pediatr, 2004. 11(2): p. 99-‐107. 136. Stiles, J., et al., Propranolol treatment of infantile hemangioma endothelial cells: A molecular
analysis. Exp Ther Med, 2012. 4(4): p. 594-‐604.
64
137. Drolet, B.A., et al., Initiation and use of propranolol for infantile hemangioma: report of a consensus conference. Pediatrics, 2013. 131(1): p. 128-‐40.
138. Shah, S. and I.J. Frieden, Treatment of infantile hemangiomas with beta-‐blockers: a review. Skin Therapy Lett, 2013. 18(6): p. 5-‐7.
139. Frieden, I.J., et al., Infantile hemangiomas: current knowledge, future directions. Proceedings of a research workshop on infantile hemangiomas, April 7-‐9, 2005, Bethesda, Maryland, USA. Pediatr Dermatol, 2005. 22(5): p. 383-‐406.
140. Haggstrom, A.N., et al., Measuring the severity of infantile hemangiomas: instrument development and reliability. Arch Dermatol, 2012. 148(2): p. 197-‐202.
141. Boye, E. and B.R. Olsen, Signaling mechanisms in infantile hemangioma. Curr Opin Hematol, 2009. 16(3): p. 202-‐8.
142. Grimmer, J.F., et al., Familial clustering of hemangiomas. Arch Otolaryngol Head Neck Surg, 2011. 137(8): p. 757-‐60.
143. Khan, Z.A., et al., Multipotential stem cells recapitulate human infantile hemangioma in immunodeficient mice. J Clin Invest, 2008. 118(7): p. 2592-‐9.
144. Guimaraes, S. and D. Moura, Vascular adrenoceptors: an update. Pharmacol Rev, 2001. 53(2): p. 319-‐56.
145. Wolter, N.E., et al., Propranolol as a novel adjunctive treatment for head and neck squamous cell carcinoma. J Otolaryngol Head Neck Surg, 2012. 41(5): p. 334-‐44.
146. Neufeld, G., et al., Vascular endothelial growth factor (VEGF) and its receptors. Faseb j, 1999. 13(1): p. 9-‐22.
147. Ji, Y., et al., Effects of propranolol on the proliferation and apoptosis of hemangioma-‐derived endothelial cells. J Pediatr Surg, 2012. 47(12): p. 2216-‐23.
148. Zhao, Z.F., et al., [The change of serum vascular endothelial growth factor and matrix metalloproteinases-‐9 in proliferative hemangioma treated with propranolol]. Zhonghua Zheng Xing Wai Ke Za Zhi, 2011. 27(5): p. 359-‐61.
149. Chim, H., et al., Propranolol induces regression of hemangioma cells through HIF-‐1alpha-‐mediated inhibition of VEGF-‐A. Ann Surg, 2012. 256(1): p. 146-‐56.
150. Schieven, G.L., The biology of p38 kinase: a central role in inflammation. Curr Top Med Chem, 2005. 5(10): p. 921-‐8.
151. Bamburg, J.R., Proteins of the ADF/cofilin family: essential regulators of actin dynamics. Annu Rev Cell Dev Biol, 1999. 15: p. 185-‐230.
152. Street, C.A. and B.A. Bryan, Rho kinase proteins-‐-‐pleiotropic modulators of cell survival and apoptosis. Anticancer Res, 2011. 31(11): p. 3645-‐57.
153. Kelly, B.D., et al., Cell type-‐specific regulation of angiogenic growth factor gene expression and induction of angiogenesis in nonischemic tissue by a constitutively active form of hypoxia-‐inducible factor 1. Circ Res, 2003. 93(11): p. 1074-‐81.
154. Kleinman, M.E., et al., Hypoxia-‐induced mediators of stem/progenitor cell trafficking are increased in children with hemangioma. Arterioscler Thromb Vasc Biol, 2007. 27(12): p. 2664-‐70.
155. Greenberger, S., et al., Targeting NF-‐kappaB in infantile hemangioma-‐derived stem cells reduces VEGF-‐A expression. Angiogenesis, 2010. 13(4): p. 327-‐35.
156. Bagazgoitia, L., A. Hernandez-‐Martin, and A. Torrelo, Recurrence of infantile hemangiomas treated with propranolol. Pediatr Dermatol, 2011. 28(6): p. 658-‐62.
157. Chang, L., et al., Recurrence of infantile hemangioma after termination of propranolol treatment. Ann Plast Surg, 2014. 72(2): p. 173-‐5.
158. Shehata, N., et al., Late rebound of infantile hemangioma after cessation of oral propranolol. Pediatr Dermatol, 2013. 30(5): p. 587-‐91.
65
159. Wong, A., et al., Propranolol accelerates adipogenesis in hemangioma stem cells and causes apoptosis of hemangioma endothelial cells. Plast Reconstr Surg, 2012. 130(5): p. 1012-‐21.
160. Zhang, L., et al., Propranolol inhibits angiogenesis via down-‐regulating the expression of vascular endothelial growth factor in hemangioma derived stem cell. Int J Clin Exp Pathol, 2013. 7(1): p. 48-‐55.
161. Zou, H.X., et al., Propranolol inhibits endothelial progenitor cell homing: a possible treatment mechanism of infantile hemangioma. Cardiovasc Pathol, 2013. 22(3): p. 203-‐10.
162. Rostami, M., et al., Tumors in domestic animals examined during a ten-‐year period (1980 to 1989) at Miyazaki University. J Vet Med Sci, 1994. 56(2): p. 403-‐5.
163. Day, M.J., V.M. Lucke, and H. Pearson, A review of pathological diagnoses made from 87 canine splenic biopsies. J Small Anim Pract, 1995. 36(10): p. 426-‐33.
164. Spangler, W.L. and P.H. Kass, Pathologic factors affecting postsplenectomy survival in dogs. J Vet Intern Med, 1997. 11(3): p. 166-‐71.
165. Schultheiss, P.C., A retrospective study of visceral and nonvisceral hemangiosarcoma and hemangiomas in domestic animals. J Vet Diagn Invest, 2004. 16(6): p. 522-‐6.
166. Oksanen, A., Haemangiosarcoma in dogs. J Comp Pathol, 1978. 88(4): p. 585-‐95. 167. Arp, L.H. and R.L. Grier, Disseminated cutaneous hemangiosarcoma in a young dog. J Am Vet
Med Assoc, 1984. 185(6): p. 671-‐3. 168. Brown, N.O., A.K. Patnaik, and E.G. MacEwen, Canine hemangiosarcoma: retrospective analysis
of 104 cases. J Am Vet Med Assoc, 1985. 186(1): p. 56-‐8. 169. Srebernik, N. and E.C. Appleby, Breed prevalence and sites of haemangioma and
haemangiosarcoma in dogs. Vet Rec, 1991. 129(18): p. 408-‐9. 170. Moe, L., et al., Canine neoplasia-‐-‐population-‐based incidence of vascular tumours. APMIS Suppl,
2008(125): p. 63-‐8. 171. Hargis, A.M., et al., A retrospective clinicopathologic study of 212 dogs with cutaneous
hemangiomas and hemangiosarcomas. Vet Pathol, 1992. 29(4): p. 316-‐28. 172. Ward, H., et al., Cutaneous hemangiosarcoma in 25 dogs: a retrospective study. J Vet Intern
Med, 1994. 8(5): p. 345-‐8. 173. Ware, W.A. and D.L. Hopper, Cardiac tumors in dogs: 1982-‐1995. J Vet Intern Med, 1999. 13(2):
p. 95-‐103. 174. Aupperle, H., et al., Primary and secondary heart tumours in dogs and cats. J Comp Pathol, 2007.
136(1): p. 18-‐26. 175. Goritz, M., et al., Canine splenic haemangiosarcoma: influence of metastases, chemotherapy and
growth pattern on post-‐splenectomy survival and expression of angiogenic factors. J Comp Pathol, 2013. 149(1): p. 30-‐9.
176. Sabattini, S. and G. Bettini, An immunohistochemical analysis of canine haemangioma and haemangiosarcoma. J Comp Pathol, 2009. 140(2-‐3): p. 158-‐68.
177. Yonemaru, K., et al., Expression of vascular endothelial growth factor, basic fibroblast growth factor, and their receptors (flt-‐1, flk-‐1, and flg-‐1) in canine vascular tumors. Vet Pathol, 2006. 43(6): p. 971-‐80.
178. Clifford, C.A., et al., Plasma vascular endothelial growth factor concentrations in healthy dogs and dogs with hemangiosarcoma. J Vet Intern Med, 2001. 15(2): p. 131-‐5.
179. Clifford, C.A., et al., Vascular endothelial growth factor concentrations in body cavity effusions in dogs. J Vet Intern Med, 2002. 16(2): p. 164-‐8.
180. Kato, Y., et al., Gene expressions of canine angiopoietin-‐1 and -‐2 in normal tissues and spontaneous tumours. Res Vet Sci, 2006. 81(2): p. 280-‐6.
181. Asa, S.A., et al., Expression of platelet-‐derived growth factor and its receptors in spontaneous canine hemangiosarcoma and cutaneous hemangioma. Histol Histopathol, 2012. 27(5): p. 601-‐7.
66
182. Mayr, B., et al., Tumour suppressor gene p53 mutation in a case of haemangiosarcoma of a dog. Acta Vet Hung, 2002. 50(2): p. 157-‐60.
183. Yonemaru, K., et al., The significance of p53 and retinoblastoma pathways in canine hemangiosarcoma. J Vet Med Sci, 2007. 69(3): p. 271-‐8.
184. Tamburini, B.A., et al., Gene expression profiling identifies inflammation and angiogenesis as distinguishing features of canine hemangiosarcoma. BMC Cancer, 2010. 10: p. 619.
185. Dickerson, E.B., et al., Mutations of phosphatase and tensin homolog deleted from chromosome 10 in canine hemangiosarcoma. Vet Pathol, 2005. 42(5): p. 618-‐32.
186. Murakami, M., et al., Expression of the anti-‐apoptotic factors Bcl-‐2 and survivin in canine vascular tumours. J Comp Pathol, 2008. 139(1): p. 1-‐7.
187. Bertazzolo, W., et al., Canine angiosarcoma: cytologic, histologic, and immunohistochemical correlations. Vet Clin Pathol, 2005. 34(1): p. 28-‐34.
188. Gamlem, H. and K. Nordstoga, Canine vascular neoplasia-‐-‐histologic classification and inmunohistochemical analysis of 221 tumours and tumour-‐like lesions. APMIS Suppl, 2008(125): p. 19-‐40.
189. Jakab, C., et al., Claudin-‐5 protein is a new differential marker for histopathological differential diagnosis of canine hemangiosarcoma. Histol Histopathol, 2009. 24(7): p. 801-‐13.
190. Thamm, D.H., Miscellaneous tumors (hemangiosarcoma), in Withrow and MacEwen's Small Animal Clinical Oncology, S.J.W.D.M.V.R.L. Page, Editor. 2013, Saunders Co. p. 679-‐688.
191. Hammer, A.S., et al., Efficacy and toxicity of VAC chemotherapy (vincristine, doxorubicin, and cyclophosphamide) in dogs with hemangiosarcoma. J Vet Intern Med, 1991. 5(3): p. 160-‐6.
192. Alvarez, F.J., et al., VAC protocol for treatment of dogs with stage III hemangiosarcoma. J Am Anim Hosp Assoc, 2013. 49(6): p. 370-‐7.
193. Sorenmo, K., et al., Canine hemangiosarcoma treated with standard chemotherapy and minocycline. J Vet Intern Med, 2000. 14(4): p. 395-‐8.
194. Sorenmo, K.U., et al., Efficacy and toxicity of a dose-‐intensified doxorubicin protocol in canine hemangiosarcoma. J Vet Intern Med, 2004. 18(2): p. 209-‐13.
195. Ogilvie, G.K., et al., Surgery and doxorubicin in dogs with hemangiosarcoma. J Vet Intern Med, 1996. 10(6): p. 379-‐84.
196. Ogilvie, G.K., et al., Phase II evaluation of doxorubicin for treatment of various canine neoplasms. J Am Vet Med Assoc, 1989. 195(11): p. 1580-‐3.
197. Wiley, J.L., et al., Efficacy of doxorubicin-‐based chemotherapy for non-‐resectable canine subcutaneous haemangiosarcoma. Vet Comp Oncol, 2010. 8(3): p. 221-‐33.
198. Payne, S.E., et al., Treatment of vascular and soft-‐tissue sarcomas in dogs using an alternating protocol of ifosfamide and doxorubicin. Vet Comp Oncol, 2003. 1(4): p. 171-‐9.
199. Rassnick, K.M., et al., Evaluation of ifosfamide for treatment of various canine neoplasms. J Vet Intern Med, 2000. 14(3): p. 271-‐6.
200. Kim, S.E., et al., Epirubicin in the adjuvant treatment of splenic hemangiosarcoma in dogs: 59 cases (1997-‐2004). J Am Vet Med Assoc, 2007. 231(10): p. 1550-‐7.
201. Sorenmo, K., et al., Clinical and pharmacokinetic characteristics of intracavitary administration of pegylated liposomal encapsulated doxorubicin in dogs with splenic hemangiosarcoma. J Vet Intern Med, 2007. 21(6): p. 1347-‐54.
202. Vail, D.M., et al., Liposome-‐encapsulated muramyl tripeptide phosphatidylethanolamine adjuvant immunotherapy for splenic hemangiosarcoma in the dog: a randomized multi-‐institutional clinical trial. Clin Cancer Res, 1995. 1(10): p. 1165-‐70.
203. Hillers, K.R., et al., Effects of palliative radiation therapy on nonsplenic hemangiosarcoma in dogs. J Am Anim Hosp Assoc, 2007. 43(4): p. 187-‐92.
67
204. Lana, S., et al., Continuous low-‐dose oral chemotherapy for adjuvant therapy of splenic hemangiosarcoma in dogs. J Vet Intern Med, 2007. 21(4): p. 764-‐9.
205. Kahn, S.A., et al., Doxorubicin and deracoxib adjuvant therapy for canine splenic hemangiosarcoma: a pilot study. Can Vet J, 2013. 54(3): p. 237-‐42.
206. Gardner, H.L.L., C.A; Portela, R.F.; Nguyen, S.; Rosenberg, M.P.; Klein, M.K.; Clifford, C.; Thamm, D.H.; Vail, D.M; Bergman, P.J.; Crawford-‐Jakubiak, M.; Henry, C.; Locke, J.; Garrett, L.D.; Cronin, K.L., Maintenance therapy with toceranib following doxorubicin-‐based chemotherapy for canine splenic hemangiosarcoma. Manuscript submitted for publication., 2014.
207. Prymak, C., et al., Epidemiologic, clinical, pathologic, and prognostic characteristics of splenic hemangiosarcoma and splenic hematoma in dogs: 217 cases (1985). J Am Vet Med Assoc, 1988. 193(6): p. 706-‐12.
208. Wood, C.A., et al., Prognosis for dogs with stage I or II splenic hemangiosarcoma treated by splenectomy alone: 32 cases (1991-‐1993). J Am Anim Hosp Assoc, 1998. 34(5): p. 417-‐21.
209. Dunning, D., et al., Analysis of prognostic indicators for dogs with pericardial effusion: 46 cases (1985-‐1996). J Am Vet Med Assoc, 1998. 212(8): p. 1276-‐80.
210. Aronsohn, M., Cardiac hemangiosarcoma in the dog: a review of 38 cases. J Am Vet Med Assoc, 1985. 187(9): p. 922-‐6.
211. Szivek, A., et al., Clinical outcome in 94 cases of dermal haemangiosarcoma in dogs treated with surgical excision: 1993-‐2007*. Vet Comp Oncol, 2012. 10(1): p. 65-‐73.
212. Burton, J.H., B.E. Powers, and B.J. Biller, Clinical outcome in 20 cases of lingual hemangiosarcoma in dogs: 1996-‐2011. Vet Comp Oncol, 2012.
213. Shiu, K.B., et al., Predictors of outcome in dogs with subcutaneous or intramuscular hemangiosarcoma. J Am Vet Med Assoc, 2011. 238(4): p. 472-‐9.
214. Stiles, J.M., et al., Targeting of beta adrenergic receptors results in therapeutic efficacy against models of hemangioendothelioma and angiosarcoma. PLoS One, 2013. 8(3): p. e60021.
215. Rada, T., L. Okruhlicova, and J. Slezak, Immunohistochemical localization of beta-‐adrenergic receptors in the heart. Bratisl Lek Listy, 1991. 92(3-‐4): p. 138-‐41.
216. Vroon, A., et al., Taxol normalizes the impaired agonist-‐induced beta2-‐adrenoceptor internalization in splenocytes from GRK2+/-‐ mice. Eur J Pharmacol, 2007. 560(1): p. 9-‐16.
217. Neubauer, B., et al., Renin expression in large renal vessels during fetal development depends on functional beta1/beta2-‐adrenergic receptors. Am J Physiol Renal Physiol, 2011. 301(1): p. F71-‐7.
218. Ferrer, L., et al., Immunohistochemical detection of CD31 antigen in normal and neoplastic canine endothelial cells. J Comp Pathol, 1995. 112(4): p. 319-‐26.
219. Slotkin, T.A., et al., Beta-‐adrenoceptor signaling and its control of cell replication in MDA-‐MB-‐231 human breast cancer cells. Breast Cancer Res Treat, 2000. 60(2): p. 153-‐66.
220. Tallman, J.F., C.C. Smith, and R.C. Henneberry, Induction of functional beta-‐adrenergic receptors in HeLa cells. Proc Natl Acad Sci U S A, 1977. 74(3): p. 873-‐7.
221. Liang, C.C., A.Y. Park, and J.L. Guan, In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc, 2007. 2(2): p. 329-‐33.
222. Avni, R., B. Cohen, and M. Neeman, Hypoxic stress and cancer: imaging the axis of evil in tumor metastasis. NMR Biomed, 2011. 24(6): p. 569-‐81.
223. Coussens, L.M. and Z. Werb, Inflammation and cancer. Nature, 2002. 420(6917): p. 860-‐7. 224. Ramos-‐Vara, J.A., Technical aspects of immunohistochemistry. Vet Pathol, 2005. 42(4): p. 405-‐
26. 225. Atkins, D., et al., Immunohistochemical detection of EGFR in paraffin-‐embedded tumor tissues:
variation in staining intensity due to choice of fixative and storage time of tissue sections. J Histochem Cytochem, 2004. 52(7): p. 893-‐901.
68
226. Xiao, K. and S.K. Shenoy, Beta2-‐adrenergic receptor lysosomal trafficking is regulated by ubiquitination of lysyl residues in two distinct receptor domains. J Biol Chem, 2011. 286(14): p. 12785-‐95.
227. Boivin, B., et al., Functional beta-‐adrenergic receptor signalling on nuclear membranes in adult rat and mouse ventricular cardiomyocytes. Cardiovasc Res, 2006. 71(1): p. 69-‐78.
228. Chen, D., et al., The beta2-‐adrenergic receptor is a potential prognostic biomarker for human hepatocellular carcinoma after curative resection. Ann Surg Oncol, 2012. 19(11): p. 3556-‐65.
229. Yang, E.V., et al., Norepinephrine up-‐regulates the expression of vascular endothelial growth factor, matrix metalloproteinase (MMP)-‐2, and MMP-‐9 in nasopharyngeal carcinoma tumor cells. Cancer Res, 2006. 66(21): p. 10357-‐64.
230. Hajighasemi F. , M.A., Propranolol effect on proliferation and vascular endothelial growth factor secretion in human immunocompetent cells. Journal of Clinical Immunology and Immunopathology Research, 2010. 2(2): p. 22-‐27.
231. Fujio, H., et al., Carvedilol inhibits proliferation of cultured pulmonary artery smooth muscle cells of patients with idiopathic pulmonary arterial hypertension. J Cardiovasc Pharmacol, 2006. 47(2): p. 250-‐5.
232. Cardani, R., et al., Influence of beta-‐adrenergic antagonists on cell proliferation rates in the kidney of untreated and diethylnitrosamine-‐treated male F344 rats. Chem Biol Interact, 1999. 118(3): p. 217-‐31.
233. Arber, S., et al., Regulation of actin dynamics through phosphorylation of cofilin by LIM-‐kinase. Nature, 1998. 393(6687): p. 805-‐9.
234. Schreiner, S.J., A.P. Schiavone, and T.E. Smithgall, Activation of STAT3 by the Src family kinase Hck requires a functional SH3 domain. J Biol Chem, 2002. 277(47): p. 45680-‐7.
235. Lefkowitz, R.J., E. Haber, and D. O'Hara, Identification of the cardiac beta-‐adrenergic receptor protein: solubilization and purification by affinity chromatography. Proc Natl Acad Sci U S A, 1972. 69(10): p. 2828-‐32.