Propranolol induced G0/G1/S phase arrest and apoptosis in melanoma cells via AKT/MAPK pathway

Abstract

Both preclinical and epidemiology studies associate β-adrenoceptors-blockers (β-blockers) with activity against melanoma. However, the underlying mechanism is still unclear, especially in acral melanoma. In this study, we explored the effect of propranolol, a non-selective β-blocker, on the A375 melanoma cell line, two primary acral melanoma cell lines (P-3, P-6) and mice xenografts. Cell viability assay demonstrated that 50μM-400μM of propranolol inhibited viability in a concentration and time dependent manner with an IC50 ranging from 65.33μM to 148.60μM for 24h -72h treatment, but propranolol (less than 200μM) had no effect on HaCaT cell line. Western blots showed 100μM propranolol significantly reduced the expression of Bcl-2 while increasing the expressions of Bax, cytochrome c, cleaved capase-9 and cleaved caspase-3, and down-regulated the levels of p-AKT, p-BRAF, p-MEK1/2 and p-ERK1/2 in melanoma cells, after a 24h incubation. The in vivo data confirmed the isolation results. Mice received daily ip. administration of propranolol at the dose of 2 mg/kg for 3 weeks and the control group was treated with the same volume of saline. The mean tumor volume at day 21 in A375 xenografts was 82.33 ± 3.75mm3vs. 2044.67 ± 54.57mm3 for the propranolol-treated mice and the control group, respectively, and 31.66 ± 4.67 mm3vs. 1074.67 ± 32.17 mm3 for the P-3 xenografts. Propranolol also reduced Ki67, inhibited phosphorylation of AKT, BRAF, MEK1/2 and ERK1/2 in xenografts. These are the first data to demonstrate that propranolol might inhibit melanoma by activating the intrinsic apoptosis pathway and inactivating the MAPK and AKT pathways.

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Oncotarget1
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www.impactjournals.com/oncotarget/ Oncotarget, Advance Publications 2016
Propranolol induced G0/G1/S phase arrest and apoptosis in
melanoma cells via AKT/MAPK pathway
Chengfang Zhou1, Xiang Chen3, Weiqi Zeng3, Cong Peng3, Gang Huang4, Xian’an
Li4, Zhengxiao Ouyang4, Yi Luo4, Xuezheng Xu4, Biaobo Xu1, Weili Wang1, Ruohui
He1, Xu Zhang3, Liyang Zhang5, Jie Liu1, Todd C. Knepper2, Yijing He1,2,3, Howard
L. McLeod1,2
1Department of Clinical Pharmacology, XiangYa Hospital, Central South University, Institute of Clinical Pharmacology, Central
South University, Hunan Key Laboratory of Pharmacogenetics, Changsha, China
2Moftt Cancer Center, DeBartolo Family Personalized Medicine Institute, Tampa, FL, USA
3Department of Dermatology, XiangYa Hospital, Central South University, Changsha, China
4Department of Orthopedics, Hunan Tumor Hospital, Changsha, China
5Department of Neurosurgery, Xiang-Ya Hospital, Central South University, Changsha, China
Correspondence to: Howard L. McLeod, email: Howard.McLeod@moftt.org
Yijing He, email: yijing.he@moftt.org
Keywords: melanoma, propranolol, apoptosis, AKT pathway, MAPK pathway
Received: February 15, 2016 Accepted: August 12, 2016 Published: August 25, 2016
ABSTRACT
Both preclinical and epidemiology studies associate β-adrenoceptors-blockers
(β-blockers) with activity against melanoma. However, the underlying mechanism
is still unclear, especially in acral melanoma. In this study, we explored the effect
of propranolol, a non-selective β-blocker, on the A375 melanoma cell line, two
primary acral melanoma cell lines (P-3, P-6) and mice xenografts. Cell viability assay
demonstrated that 50μM-400μM of propranolol inhibited viability in a concentration
and time dependent manner with an IC50 ranging from 65.33μM to 148.60μM for 24h
-72h treatment, but propranolol (less than 200μM) had no effect on HaCaT cell line.
Western blots showed 100μM propranolol signicantly reduced the expression of Bcl-2
while increasing the expressions of Bax, cytochrome c, cleaved capase-9 and cleaved
caspase-3, and down-regulated the levels of p-AKT, p-BRAF, p-MEK1/2 and p-ERK1/2
in melanoma cells, after a 24h incubation. The in vivo data conrmed the isolation
results. Mice received daily ip. administration of propranolol at the dose of 2 mg/kg
for 3 weeks and the control group was treated with the same volume of saline. The
mean tumor volume at day 21 in A375 xenografts was 82.33 ± 3.75mm3vs. 2044.67
± 54.57mm
3
for the propranolol-treated mice and the control group, respectively,
and 31.66 ± 4.67 mm3 vs. 1074.67 ± 32.17 mm3 for the P-3 xenografts. Propranolol
also reduced Ki67, inhibited phosphorylation of AKT, BRAF, MEK1/2 and ERK1/2 in
xenografts. These are the rst data to demonstrate that propranolol might inhibit
melanoma by activating the intrinsic apoptosis pathway and inactivating the MAPK
and AKT pathways.
INTRODUCTION
Melanoma, a cancer most commonly affecting
the skin, is derived from pigment-containing cells
known as melanocytes [1]. In China, the most common
subtype is acral lentiginous melanoma, which accounts
for approximately 40% of cutaneous melanoma
[2, 3]. Surgery followed by systemic chemotherapy
is recommended by National Comprehensive Cancer
Network (NCCN) for early stage melanoma patients.
For unresectable melanoma, BRAF/MEK inhibitors and
immune check points inhibitors are the rst line treatment
[4]. BRAF inhibitors, vemurafenib and dabrafenib, have
been approved for use in melanoma patients who are

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BRAF V600E/V600K mutation carriers, these agents have
been shown to signicantly improve survival in BRAF-
mutated melanoma patients [5, 6]. The MEK inhibitors
trametinib can also improve survival in melanoma when
used in combination with BRAF inhibitors [7]. However,
the duration of response for single-agent vemurafenib
varied between 2 to 18 months [8]. Most patients
developed an acquired resistance to vemurafenib after 6
months of treatment [9]. Lack of durable response and the
development of acquired drug resistance are limitations to
the use of BRAF inhibitors, particularly as single-agents.
Mitogen-activated protein kinase (MAPK) and
AKT pathways play an important role in BRAF inhibitor
antitumor activity. Thep42/p44 MAPK and AKT are
highly activated during the growth stage of melanoma [10,
11]. Ras ultimately activates several signaling pathways
that are related to cell proliferation and anti-apoptotic
signalling cascades, including the Raf/MEK/extracellular
signal-regulated kinase (ERK) and phosphatidylinositol
3-kinase (PI3K)/ ATP-dependent tyrosine kinases (AKT)/
Phosphatase and PTEN pathways and NF-κB [12, 13].
β-adrenoceptors are also expressed in melanoma cells [14,
15]. Because of the important role of β-adrenergic receptor
in regulating AKT/MAPK pathway, we hypothesized
that blockage of β-adrenergic receptor may inhibit AKT/
MAPK pathway and lead to the death of melanoma cells.
Propranolol is a non-selective β-blocker widely
used for the treatment of hypertension, which has also
been demonstrated safety and efcacy for the treatment
of large hemangiomas in infants [16, 17]. Propranolol
has anti-proliferative, anti-migratory, anti-angiogenic and
cytotoxic properties in a wide variety of cancers [18–26].
In addition, a retrospective study revealed that melanoma
patients received β-blockers for hypertension achieved
longer survival than patients did not [27]. Recently,
Wrobelet al. reported that propranolol, not metoprolol,
inhibited proliferation of melanoma cell lines in vitro and
shrank the tumor size in vivo[15]. However, the underlying
mechanism of how β-blockers inhibit melanoma remains
unknown, especially with the acral melanoma subtype.
This study ascertained if propranolol could inhibit
proliferation and induce apoptosis in cutaneous/acral
melanoma and explored the potential mechanism of this
effect.
RESULTS
Propranolol cytotoxicity in cutaneous/acral
melanoma
The direct effect of propranolol on melanoma
proliferation was assessed using A375 cell, two acral
melanoma cell lines (P-3 and P-6 cell lines), and HaCaT,
a normal skin keratinocytes cell line. Cell viability was
measured by AlamarBlue® Cell Viability Assay. After
treatment for 24h, 48h and 72h with 25μM, 50μM,
100μM, 200μM and 400μM, propranolol respectively, cell
proliferation was signicantly reduced from 0.911 to 0.056
(Figure 1A, 1B, 1C, P<0.0001) in A375, 0.906 to 0.074
(Figure 1A, 1B, 1C, P<0.0001) in P-3, and 0.912 to 0.096
(Figure 1A, 1B, 1C, P<0.0001) in P-6, respectively. In
this assay, propranolol signicantly reduced cell viability
of the three melanoma cell lines (the range of IC50 in
A375: 65.33μM to 98.17, P-3: 116.86μM to 148.60μM,
P-6: 88.24μM to 118.23μM, Figure 1D, P < 0.05) in a
concentration and time dependent manner. Interestingly,
compared with melanoma cell lines, propranolol slightly
inhibited proliferation of HaCaT cell (the range of IC50
in HaCaT: 88.24μM to 466.11μM, Figure 1D) at the same
concentration (less than 200μM).
Propranolol induced apoptosis and inhibited cell
cycle in melanoma cell lines
After 100μM propranolol treatment for 24h, the
population of cells in G0/G1 greatly increased from
57.8%, 56.8% and 62.5% to 68.1%, 73.1% and 73.5%
in A375, P-3 and P-6 respectively (Figure 2A, Aa-
Ac, P< 0.0001), the distribution of S phase obviously
reduced from 27.9% to 9.0% in A375, 26.8% to 12.5%
in P-3 and 27.1% to 13.5% in P-6 (Figure 2A, Aa-Ac,
P< 0.0001), the number of G2M phase also signicantly
increased from 8.2% to 22.1% in A375, 7.6% to 13.1%
in P-6 (Figure 2A, Aa-Ac, P< 0.001), but no signicant
change in P-3 (Figure 2Ab). This redistribution of cells
within the cell cycle may be, at least in part, accounted for
propranolol-mediated apoptosis. Hoechst stain showed
the apoptotic cell number, dening with chromatin
condensation, nuclear shrinkage and formation of
apoptotic bodies, signicantly increased from 9.9% to
95.4% in A375 (Figure 2B, 2Ba, P< 0.0001), 6.6% to
88.3% in P-3 (Figure 2B, 2Bb, P< 0.0001), and 6.4% to
48.5% in P-6 (Figure 2B, 2Bc, P< 0.0001). These ndings
suggested that the propranolol might inhibit melanoma
cell lines by arresting cell progression at G0/G1 and S
phase and then inducing apoptosis.
Propranolol induced apoptosis by activating an
intrinsic apoptosis pathway
An inuence of propranolol on apoptosis may
contribute to the mechanistic effects. Melanoma cell lines
were exposed to 100μM propranolol for 24h, Bax was
signicantly increased in A375 (Figure 3A, Aa, P <0.001),
P-3 (Figure 3A, Ab, P <0.01) and P-6 (Figure 3A, Ac,
P <0.01) cell lines, while Bcl-2 was decreased in the
three melanoma cell lines (Figure 3B, Ba-Bc, P <0.01).
Propranolol also caused releasing of cytochrome c in
three cell lines (Figure 3C, Ca-Cc, P <0.01). Capase-9
and caspase-3, initiator caspases of the intrinsic apoptotic
pathway and one of the downstream target proteins of
cytochrome c, were activated by propranolol. An obvious

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increase of the cleavage of caspase-9 and caspase-3
was observed in A375 (Figure 3D, Da, Figure 3E, Ea, P
<0.001), P-3 (Figure 3D, Db, Figure 3E, Eb, P <0.01) and
P-6 (Figure 3D, Dc, Figure 3E, Ec, P <0.01) cell lines.
These data strongly suggested that propranolol induced
apoptosis of melanoma cells by activating intrinsic
apoptosis pathway.
Propranolol inhibited proliferation by inhibiting
MAPK pathway
Since AKT and MAPK pathways play a key role in
tumor proliferation and apoptosis, we hypothesized that
propranolol might produce its strong function through
down-regulating activities in the two pathways. As shown
in Figure 4, p-BRAF (Figure 4A, Aa-Ac, P < 0.001),
p-MEK1/2 (Figure 4B, Ba-Bc, P < 0.001), p-Erk1/2
(Figure 4C, Ca-Cc, P < 0.001) and p-AKT (Figure 4D,
Da-Dc, P < 0.001) were greatly reduced while total
expressions of them were slightly decreased in propranolol
100μM group after a 24-hour incubation, suggesting that
the anti-proliferation might be mediated by simultaneously
targeting both ERK and AKT phosphorylation.
Propranolol inhibited melanoma growth in vivo
To evaluate whether the strong action observed
against melanoma in vitro could be transferred to
xenografts, the three human melanoma cells were
engrafted in BALB/C nude mice, while P-6 xenografts
were failed to develop to a solid tumor, we compared
the effect of propranolol on tumor growth. Previous
studies showed propranolol was effective in suppressing
melanoma tumor growth in vivo at dosages of 2-10 mg/
kg/day [15, 24]. In this study, mice received a daily
ip. injection of propranolol at the dose of 2 or 10mg/
kg for 3 weeks, the control group was treated with the
same volume of saline, but 80% animals were weight
loss and hemafecal after the higher dosage propranolol
administration (Data not shown), interestingly, the lower
showed better effect. As shown in Figure 5, the mean
tumor sizes of propranolol-treated mice were smaller
than the PBS group on day 21 in A375 xenografts (82.33
±3.75mm3 versus 2044.67 ± 54.57mm3, unpaired t-test,
P<0.0001, Figure 5A, 5C) and P-3 xenografts (31.66
±4.67 mm3 versus 1074.67 ± 32.17 mm3, unpaired t-test
P<0.0001, Figure 5B, 5D). Notably, no mice in the
Figure 1: The effect of propranolol on cell survival in melanoma cell lines and normal skin cell line. A-C. Cell viability was
determined following treatment with increasing propranolol concentration (25, 50, 100, 200, 400μM) for 24h, 48h and 72h by AlamarBlue®
Cell Viability Assay, relative growth was calculated as the ratio of treated to untreated cells at each dose for each replicate. D. IC50 of
propranolol after a 24h, 48h and 72h incubation in the four cell lines, respectively. ****P<0.0001. All experiments were repeated for at least
three times independently.

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Figure 2: Propranolol induced cell cycle arrest and apoptosis in melanoma cells. A. A375, P-3 and P-6 cells were exposed
to100μM propranolol for 24h and then stained by propidium iodide (PI) to determine cell cycle assay by ow cytometry. B. Hoechst staining
was performed on the three cell lines after 100μM propranolol treatment. Aa-Ac, Data from (A) in histogram. Ba-Bc, Quantication of
Hoechst staining. ***P<0.001, ****P<0.0001. All experiments were repeated for at least three times independently.

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propranolol group lost body weight (more than 10%) both
in A375 and P-3 xenografts (Figure 5E, 5F), indicating the
absence of gross toxicity for the treatment.
Propranolol inhibited proliferation and induced
apoptosis in melanoma xenografts
To determine whether the inducing apoptosis by
propranolol observed in vitro also functioned in vivo, we
performed IHC analyses on tumor sections from all the
experimental groups. H&E staining assay was applied to
investigate the role of propranolol on cell morphology (Figure
6A). When compared with PBS treated group, the number of
nuclei with karyorhexis and karyolysis was greatly increased in
propranolol treated xenografts, implying that propranolol could
promote cell necrosis in tumors (Figure 6A, Aa, Ab, P <0.01).
We also assessed the proliferation level indicated by cell
marker Ki-67 in tumor sections. The Ki67 index signicantly
decreased in both A375 (47.67% ± 3.84 % versus 16.00% ±
2.65%, N=6, P= 0.0025, Figure 6B, Ba) and P-3 (41.67% ±
2.60% versus 21.33% ± 2.60%, N=6, P=0.0052, Figure 6B,
Bb) xenografts. To demonstrate the role of propranolol on cell
death, tunnel terminal deoxynucleotidyl transferase dUTP nick
end labeling (TUNEL) assay was performed. In A375 derived
tumors, TUNEL positive cell was signicantly (P < 0.0001)
higher of propranolol group (92.67% ± 1.86%) than PBS
group (3.67% ± 0.88%, Figure 6C, Ca). In P-3 derived tumors,
higher proportion of TUNEL positive cells was observed in the
propranolol treated group than in the control group (89.0% ±
2.65% vs 6.33% ± 1.76%, P<0.0001, N=6, Figure 6D, Da).
Figure 3: Propranolol activated mitochondria-mediated apoptosis pathway in melanoma. A-E. The expressions of Bax,
Bcl-2, cytochrome c, caspase-9 and caspase-3 following exposure to propranolol 100μM for 24h in A375, P-3 and P-6 cell lines. Aa-Ec.
Quantication of A-D. Results are presented as mean±SEM, *P<0.05, ***P<0.001, ****P<0.0001. All experiments were repeated for at least
three times independently.

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Propranolol inhibited AKT pathway and MAPK
pathway in vivo
To determine whether the down-regulation of
MAPK and AKT signaling by propranolol observed in
vitro also functioned in vivo, immunohistochemistry
analysis was applied to determine the phosphorylation of
the AKT and MAPK pathways in tumors. As observed in
Figure 7, the data showed that the expression of p-AKT
was signicantly (P<0.0001) reduced in the propranolol
treated group compared with PBS group both in A375
(Figure 7A, Aa) and P-3 xenografts (Figure 7A, Ab). The
Figure 4: Propranolol inhibited MAPK pathway. A-D. BRAF, MEK1/2, ERK1/2 and AKT phosphorylation following treatment
with propranolol 100μM for 24h in A375, P-3 and P-6 cell lines. Aa-Df, Quantication of A-D. Results are presented as mean±SEM,
*P<0.05, **P<0.01,***P<0.001, ****P<0.0001. All experiments were repeated for at least three times independently.

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expression of p-BRAF and p-MEK1/2 were moderately
lowered by propranolol in A375 (Figure 7B, 7C, Ba, Ca)
and P-3 xenografts (Figure 7B, 7C, Bb, Cb). Furthermore,
the phosphorylated ERK1/2 was reduced in A375 (Figure
7D, Da) and P-3 (Figure 7D, Db) derived tumors in the
propranolol treated group. These data revealed that
propranolol could inhibit the growth of melanoma by
inhibiting the phosphorylation of AKT and MAPK
pathway in vivo.
DISCUSSION
Accumulative studies have reported that
β-blockers have anti-tumor effects by inhibiting
proliferation, inducing apoptosis and inhibiting
metastasis of many types of tumor [24–26]. Calvani M
and colleagues found β3-AR activation in melanoma
accessory cells drives stromal reactivity by inducing
pro-inammatory cytokines secretion and de novo
angiogenesis, sustaining tumor growth and melanoma
aggressiveness, which validated selective β3-AR
antagonists as potential promising anti-metastatic agents
[28]. Another study demonstrated the combination
of propranolol with 5-FU or paclitaxel resulted in
more profound and sustained anti-tumor effects and
signicantly increased the survival benets in MDA-
MB-231 cell xenografts [20]. In 2015, Wrobelet al.
reported that propranolol could inhibit proliferation in
Figure 5: Propranolol inhibits tumor development in xenografts. A, B. Tumors excised from xenografts of A375 and P-3 model
mice, untreated mice (PBS group) and mice treated with propranolol (propranolol group). C, D. The growth curves of tumor measured by
average volume of 6 tumors in each group. E, F. Body weights of A375 and P-3 model mice were measured before and after the beginning
of the treatment by three days interval.

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Figure 6: The effect of propranolol on tumor histology in vivo. A. Hematoxylin- -eosin staining (HE) for cell morphology in
propranolol and PBS group. B. Ki67 was assessed by immunohistochemistry assay. C-D. Cell death was measured by TUNEL assay in
A375 and P-3 xenografts from untreated (left panels) and propranolol treated mice (right panels). Ba,-Bb, Quantication of Ki67 staining
in A375 (n=30 estimations on 6 tumors, both in propranolol and PBS groups) and P-3 (n=30 estimations on 6 tumors, both in propranolol
treated and PBS groups) xenografts mice. Ca, Da, Quantication of TUNEL staining in A375 (n=36 estimations on 6 tumors, both in
propranolol and PBS groups) and P-3 (n=36 estimations on 6 tumors, both in propranolol and PBS groups) xenografts mice. Results are
presented as mean±SEM, **P<0.01, ****P<0.0001. All experiments were repeated for at least three times independently.

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melanoma cell lines without knowing its underlying
mechanism [15]. This study demonstrated that
propranolol inhibited melanoma growth by regulating
the AKT pathway and MAPK pathways and inducing
apoptosis. It is also the rst evidence showing that
activation of apoptosis by propranolol could down-
regulate the activity of AKT and MAPK pathways both
in cutaneous and acral melanoma.
Figure 7: The effect of propranolol on Akt and MAPK pathway in vivo. A-D. p-Akt, p-BARF, p-MEK, and p-ERK were
assessed by immunohistochemistry assay in A375 (n=30 estimations on 6 tumors, both in propranolol and PBS groups) and P-3 (n=40
estimations on 6 tumors, both in propranolol and PBS groups) derived xenografts. Aa-Db. Quantication of p-AKT, p-BRAF, p-MEK1/2
and p-ERK1/2 staining in A375 (n=30 estimations on 6 tumors, both in propranolol and PBS groups) and P-3 (n=30 estimations on 6
tumors, both in propranolol treated and PBS groups) xenografts mice, respectively. Results are presented as mean±SEM, ***P<0.001,
****P<0.0001. All experiments were repeated for at least three times independently.

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Propranolol inhibited proliferation in the three
melanoma cell lines. Wrobel and his colleague also
addressed that propranolol signicantly reduced melanoma
cell viability with 100μM for 24h [15]. Similar result was
reported by Albiñana who found that propranolol reduced
viability and induced apoptosis in hemangioblastoma
cells from von Hippel-Lindau patients [29]. Consistent
with the in vitro studies, propranolol shrunk tumor size
and inhibited the expression of Ki67 in xenograft models.
In neuroblastoma and breast cancer, propranolol was
also observed to reduce tumor volume in xenografts
models [20, 24]. Propranolol inhibition of proliferation
of cutaneous and acral melanoma occurred in a time
and concentration dependent manner. This observation
is consistent with the hemangioblastoma study, which
found 50 and 100μM propranolol signicantly reduced
cell viability with longer incubation time (24h-96h)
[29]. Liao and his colleague also demonstrated that
propranolol reduced proliferation of gastric cancer cells
in a concentration-dependent manner (25μM-300μM)
[23]. Pharmacokinetic data suggested that the peak serum
concentrations of propranolol ranged from 200-400 ng/
ml, which is equivalent to 0.77-1.5μM in vivo[30]. But
the IC50 value of propranolol was more than 60 μM after
a 24-hour incubation in three melanoma lines. Large
discrepancy of drug concentration was observed between
in vivo and in vitro models. This may because propranolol
could exert the anti-tumor effect via multiple pathways in
vivo compared with in vitro models, such as propranolol,
could inuence angiogenesis [31, 32] and attenuate
immune-suppressionin tumor [33], thus may require less
drug concentrations to achieve similar effect. On the other
hand, propranolol showed no effect on normal skin cell
line at the same concentration. This is consistent with a
recent study, which demonstrated that propranolol did not
show any cytotoxicity in normal skin cells and normal
melanocytes [15].
The mechanisms of anti-tumor activity of β-blockers
have been explored by other investigators in other cancer.
Lin X et al. found that the non-selective β-AR agonist
isoproterenol signicantly increased the activation of
ERK/MAPK signal pathway in pancreatic cancer cell [25].
Huang’s study showed that norepinephrine also stimulated
pancreatic cancer cell proliferation, migration and invasion
via β-AR dependent activation of P38/MAPK pathway
[34]. Propranolol and other β-blockers reduced the activity
of MAPK in pancreatic cancer and Hemangioma [35, 36].
Meanwhile, inhibition of MAPK is a widely used strategy
for melanoma treatment [37]. Vemurafenib, a BRAFV600E
inhibitor, has achieved high response rate (more than 50%)
at the initial stage of vemurafenib treatment in advanced
melanoma patients [38–40]. However, more than 60%
patients developed acquired drug resistance (ADR) after
6 months [41–43]. One possible reason of vemurafenib
induced ADR was believed to be the reactivation of
MAPK pathway in melanoma [44]. This study further
advanced our knowledge on the therapeutic mechanism
of propranolol in melanoma. More signicantly, our data
also provide a scientic basis on a strategy of using the
propranolol inhibitory effects on the MAPK pathway
to overcoming the vemurafenib-induced resistance in
melanoma treatment.
In summary, this data implied that propranolol could
inhibit melanoma in vitro and in vivo. The study highlights
for the rst time that propranolol may exert the anti-tumor
effect in cutaneous and acral melanoma by suppressing
AKT and MAPK pathways. Clinical trials are needed to
verify the treatment effect of propranolol in melanoma
patients as an adjuvant regimen.
MATERIALS AND METHODS
Cell lines and reagents
The A375 cell line, derived from chronic sun-
induced damage (CSD) cutaneous melanoma [45], was
obtained from American type culture collection (ATCC).
All cell lines were cultured in DMEM medium (Gibco,
Life Technologies, China) supplemented with 10% FBS
(Gibco, Life Technologies Australia) at 37°C and 5%
CO2 in tissue culture incubator. The A375 cell line was
regularly veried to be mycoplasmae –free and no cross
cell contaminationusing ABI 3100 type genetic analyzer
(Applied Biosystems, USA). (Supplementary Figure S1).
Human melanoma primary cultures
Patient 3 cell line (P-3) and patient 6 cell line
(P-6) were derived from surgical resection samples
of acral melanoma from two patients (Supplementary
Figure S2). Written informed consent was obtained from
the patients. Melanoma specic antigens were detected
via western blotting for CD31, CD63, CD166 and CD146
conrmed that the cells were melanocytes (Supplementary
Figure S3, Supplementary Figure S6). Tumor tissues were
mechanically dissociated with a small tissue chopper prior
to sequential enzymatic digestion in 2 mg/ml Collagenase
(Sigma-Aldrich, Schnelldorf, Germany) and 1 mg/ml
Dispase I (Gibco/Invitrogen, Carlsbad, CA) in DMEM for
30 min at 37°C. Cells were ltered (100 μm cell strainer)
to obtain a single cell suspension and re-supplemented by
phosphate-buffered saline, thereafter centrifuged at 1000
r.p.m. for 5 minutes. Pellet was re-suspended in DMEM
with 10% FBS and then cultured in 75 cm
3
plastic asks
(Themo Fisher Scientic, China).
Adenylyl cyclase activity
Adenylyl cyclase activity was measured by a
protein binding assay [46]. Cell pellets were thawed and
homogenised in a glass douncehomogeniser containing
ice-cold homogenization buffer (0.3 M sucrose, 25

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mMTris, pH 7.4). A 40 μL sample of homogenate
was then added to 30μL premix buffer (nal assay
concentration 50 mMTris (pH 7.5), 5 mM Mg2+, 1 mM
ATP (Sigma-Aldrich, Schnelldorf, Germany), 1μM GTP
(G9002-25MG,Sigma-Aldrich, Schnelldorf, Germany),
250μM 4- (3-Butoxy- 4-methoxybenzyl) imidazolidin-
2-one (RO20-1724, Abcam, USA), 20 mMcreatine
phosphate (000000010621714001, Roche) and 130 U/mL
creatine phosphokinase (C7886-500UN, Sigma-Aldrich,
Schnelldorf, Germany)). The tubes were incubated at 37
°C for 10 min and the reaction terminated by the addition
of 20 μL of 100% trichloroacetic acid and the tubes placed
on ice for 10 min. Precipitated protein was pelleted by
centrifugation at 2,900 × g for 20 min at 4 °C, the resulting
supernatant was used to determine cAmp level by cAMP
Direct Immunoassay Kit (Catalog#K371-100, BioVison,
USA) according to the instruction. The adenylyl cyclase
activity expressed as pmol cyclic AMP min
-1
mg
-1
protein.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from the three melanoma
and HaCaT cell lines by using Trizol reagent (Invitrogen,
Carlsbad, CA) according to the manufacturer’s
instructions. qRT-PCR was performed using the
SYBR® Green Realtime PCR Master Mix assay kit
(Toyobo, Osaka, Japan) according to the manufacturer’s
instructions. The primers of target genes were as follows:
β1AR: 5’-ATCGAGACCCTGTGTGTCATT-3’ (forward)
and 5’-GTAGAAGGAGACTACGGACGAG-3’ (reverse),
β2AR: 5’-TTGCTGGCACCCAATAGAAGC-3’ (forward)
and 5’-CAGACGCTCGAACTTGGCA-3’ (reverse),
β3AR: 5’-GACCAACGTGTTCGTGACTTC-3’ (forward)
and 5’-GCACAGGGTTTCGATGCTG-3’ (reverse),
and GAPDH: 5’-GAGTCAACGGATTTGGTCGT-3’
(forward) and 5’-TTGATTTTGGAGGGATCTCG-3’
(reverse). The results were analyzed using the 2-ΔΔCt
method as the following formula: ΔΔCt = ΔCtβ2/β3AR-
ΔCtβ1AR, ΔCt = Ct target gene -CtGAPDH.
Cell viability assays
The half-maximal inhibitory concentration (IC50)
value was determined by AlamarBlue
®
assay (Invitrogen,
Burlington, ON, Canada). A375 cells were plated in
96-well plates at a density of 2×103 and treated with
25μM-400μM propranolol (propranolol hydrochloride,
P0884, Sigma-Aldrich, USA) for 24h, 48h and 72h.
Cells were incubated with 10% of AlamarBlue overnight.
Fluorescence of each plate was measured using a
spectrophotometer at excitation 530 nm and emission 590
nm (Spectra MAX Gemini EM, Molecular Devices).
Cell cycle analysis by ow cytometry
A375,P-3 and P-6 cell lines were cultured at a
density of 1 × 106 cells in 100 mm2 culture dishes (Corning,
China) and were treated with 100 spectrophotometer 24h.
Cells were then harvested, washed, xed overnight in 70
% ethanol at −20°C, after washed with PBS, digested
with RNase A, stained with propidium iodide (50 with
propidiumilyzed by ow cytometry using BD Accuri
TM
C6
(Becton, Dickinson and Company, USA).
Western blot analysis
Western blot analysis was performed on cell extracts
of A375,P-3 and P-6 cell lines treated with 100μM
propranolol for 24h. Immunoblots were performed from
whole cell lysate prepared using RIPA Buffer (Cell
Signaling Technology) supplemented with dithiothreitol
(DTT), phenylmethylsulphonyl uoride (Sigma), and fresh
protease and phosphatase inhibitors (Sigma). Cell lysates
were quantied for protein content using a bicinchoninic
acid (BCA) protein assay kit (Thermo). Protein samples
were resolved on NuPAGE 12% Bis-Tris gels with MOPS
buffer or 3–8% Tris acetate gels with Tris acetate buffer
(Life Technologies) and then transferred to 0.45-mm
nitrocellulose membrane (Bio-Rad). After saturation in
Tris-buffered saline supplemented with 5% BSA, the
membranes were incubated with antibodies (diluted at
1:2,000) overnight at 4°C. Antibodies specic for the
following proteins were purchased from Cell Signaling
Technology: AKT (rabbit, 9272), B-Raf (rabbit, 9433),
MEK1/2 (L38C12) (mouse, 4694), ERK1/2 (rabbit, 9102),
phospho-ser473-AKT (rabbit, 9271), phosphor-ser445-
BRAF (rabbit, 2696), phosphor-ser221-MEK (rabbit,
2338), phospho-ERK1/2-Thr202/Tyr204 (rabbit, 4370),
cytochrome c-D18C7 (rabbit, 11940), caspase-9 (mouse,
9508) and caspase-3 (rabbit, 9662). The antibodies specic
for Bax (rabbit, sc-6236) and Bcl-2 (rabbit, sc-492), were
purchased from Santa Cruz Biotechnology. The antibodies
for BAD (rabbit, ab32445), p-BAD (rabbit, ab28824),
CD31 (mouse, ab9498), CD147 (rabbit, ab108317),
CD146 (mouse, ab24577), CD63 (mouse, ab8219) and
CD166 were purchased from Abcam. The antibody
specic for GAPDH (mouse, clone 6C5, MAB374) was
purchased from Millipore. Quantication of the bands was
done with Image J.
Hoechst staining
Cells treated with propranolol were xed, washed
twice with PBS and stained with Hoechst 33258 staining
solution according to the manufacturer’s instructions
(Beyotime, Jiangsu, China). Stained nuclei were observed
under a uorescence microscope (Leica-microsystems-
DM5000B+DFC1300,Leitz Wild Group, Germany).
Xenografts of human melanoma
The six weeks old NOD/SCID male mice (Hunan
Silaike experimental Animals Inc, China) was injected
subcutaneously with 106living melanoma cells that were

Oncotarget12
www.impactjournals.com/oncotarget
maintained in DMEM with 2% FBS and 15% Matrigel
(BD Bioscience, Franklin Lakes, NJ) in the left ank. A
solid tumor develops in A375 and P-3 xenografts within
two weeks, while P-6 xenografts were failed to develop.
In vivo propranolol treatment
The NOD/SCID mice with xenografts were
randomly divided into three groups after the tumor volume
reached 0.3 cm3. The propranolol group (n=6) received
a daily ip. injectionof propranolol (Sigma-Aldrich) at the
dose of 2 or 10mg/kgfor 3 weeks and the control group
(n=6) was treated with the same amount of saline. Tumor
volume was measured by length (l), width (w), and height
(h) twice a week with an external caliper and tumor size
was calculated by the modied ellipsoidal formula (Tumor
volume =π/6× l × w × h). The mice were sacriced at the
end of 3 weekstreatment. The xenografts were removed,
weighted and then snap-frozen in liquid nitrogen. Parafn-
embedded tumor blocks were prepared for further analysis
at the same time.
Immunohistochemistry and TUNEL assay
Deparafnized tissue sections were treated with
Antigen Retrieval Solution (made from citrate buffer, pH
6.0, concentrated 103, T0050 Diapath). Tissue sections
were then incubated with Peroxidase Blocking Solution
(S2023, Dako) for 15 min and Protein Block (X0909,
Dako) for 20 min. Primary antibody specic for phosphor-
ser221-MEK (rabbit, 2338, CST), phospho-ERK1/2-
Thr202/Tyr204 (rabbit, 4370, CST) and phospho-ser473-
AKT (rabbit, 9271, CST) and p-RAF-B (goat, sc-28006,
Santa Cruz Biotechnology) were applied, and the slides
were incubated overnight at 4°C. Signals were visualized
using rabbit HRP-conjugated secondary antibody (K4003,
Dako) and a haematoxylin (MHS32, Sigma) counterstain.
TUNEL assay was done using Cell-LightTMEdUTP
TUNEL In situ Detection Kit (Ruibo, China).
Statistical procedures
Drug sensitivity of propranolol and cell cycle
population analysis were done using an analysis of
variance on repeated measures with the Graphpad Prism
software (GraphPad Software, Inc., version 6.0).
ACKNOWLEDGMENTS
This work was supported by the National Natural
Science Foundation of China (Grant No. 81070902) and
by the Young Scientists Fund of the National Natural
Science Foundation of China (Grant No. 30600774
and No.81403022). Project supported by the China
Postdoctoral Science Foundation (Grant No. 201104515
and No. 2012M510138). The authors thank Prof.
William Tse from Division of Blood and Bone Marrow
Transplantation James Graham Brown Cancer Center
Department of Medicine University of Louisville School
of Medicine for editorial assistance on the text and study
suggestions.
CONFLICTS OF INTEREST
The authors state no conict of interest.
HIGHLIGHTS
1. Propranolol could inhibit the cell proliferation and
induce apoptosis of melanoma via activating the intrinsic
apoptosis pathway and inactivating the MAPK and AKT
pathways in vitro and in vivo.
2. Acral melanoma cells deprived from patients
were showed to response to propranolol treatment in vivo
and in vitro.
REFERENCES
1.
Melanoma Treatment–for health professionals (PDQ®).
National Cancer Institute, June 26, 2015.
2. Chi, Z., S. Li, X. Sheng, L. Si, C. Cui, M. Han, and J. Guo,
Clinical presentation, histology, and prognoses of malignant
melanoma in ethnic Chinese: a study of 522 consecutive
cases. BMC Cancer, 2011. 11: p. 85.
3.
Chang, J.W., Acral melanoma: a unique disease in Asia.
JAMA Dermatol, 2013. 149: p. 1272-3.
4.
NCCN Guidelines Version 1.2016 Melanoma. National
Comprehensive Ccancer Network, 2015.
5.
Sosman, J.A., K.B. Kim, L. Schuchter, R. Gonzalez,
A.C. Pavlick, J.S. Weber, G.A. McArthur, T.E. Hutson,
S.J. Moschos, K.T. Flaherty, P. Hersey, R. Kefford, D.
Lawrence, et al., Survival in BRAF V600-mutant advanced
melanoma treated with vemurafenib. N Engl J Med, 2012.
366: p. 707-14.
6.
Hauschild, A., J.J. Grob, L.V. Demidov, T. Jouary, R.
Gutzmer, M. Millward, P. Rutkowski, C.U. Blank,
W.H. Miller, Jr., E. Kaempgen, S. Martin-Algarra, B.
Karaszewska, C. Mauch, et al., Dabrafenib in BRAF-
mutated metastatic melanoma: a multicentre, open-label,
phase 3 randomised controlled trial. Lancet, 2012. 380:
p. 358-65.
7.
Long, G.V., D. Stroyakovskiy, H. Gogas, E. Levchenko,
F. de Braud, J. Larkin, C. Garbe, T. Jouary, A. Hauschild,
J.J. Grob, V. Chiarion-Sileni, C. Lebbe, M. Mandala, et al.,
Dabrafenib and trametinib versus dabrafenib and placebo
for Val600 BRAF-mutant melanoma: a multicentre, double-
blind, phase 3 randomised controlled trial. Lancet, 2015.
386: p. 444-51.
8.
Poulikakos, P.I. and N. Rosen, Mutant BRAF melanomas--
dependence and resistance. Cancer Cell, 2011. 19: p. 11-5.

Oncotarget13
www.impactjournals.com/oncotarget
9.
Flaherty, K.T., I. Puzanov, K.B. Kim, A. Ribas, G.A.
McArthur, J.A. Sosman, P.J. O’Dwyer, R.J. Lee, J.F.
Grippo, K. Nolop, and P.B. Chapman, Inhibition of mutated,
activated BRAF in metastatic melanoma. N Engl J Med,
2010. 363: p. 809-19.
10.
Govindarajan, B., J.E. Sligh, B.J. Vincent, M. Li, J.A.
Canter, B.J. Nickoloff, R.J. Rodenburg, J.A. Smeitink,
L. Oberley, Y. Zhang, J. Slingerland, R.S. Arnold, J.D.
Lambeth, et al., Overexpression of Akt converts radial
growth melanoma to vertical growth melanoma. J Clin
Invest, 2007. 117: p. 719-29.
11.
Cohen, C., A. Zavala-Pompa, J.H. Sequeira, M. Shoji, D.G.
Sexton, G. Cotsonis, F. Cerimele, B. Govindarajan, N.
Macaron, and J.L. Arbiser, Mitogen-actived protein kinase
activation is an early event in melanoma progression. Clin
Cancer Res, 2002. 8: p. 3728-33.
12.
McCubrey, J.A., L.S. Steelman, W.H. Chappell, S.L.
Abrams, E.W. Wong, F. Chang, B. Lehmann, D.M. Terrian,
M. Milella, A. Tafuri, F. Stivala, M. Libra, J. Basecke,
et al., Roles of the Raf/MEK/ERK pathway in cell growth,
malignant transformation and drug resistance. Biochim
Biophys Acta, 2007. 1773: p. 1263-84.
13.
Agell, N., O. Bachs, N. Rocamora, and P. Villalonga,
Modulation of the Ras/Raf/MEK/ERK pathway by Ca(2+),
and calmodulin. Cell Signal, 2002. 14: p. 649-54.
14.
Yang, E.V., S.J. Kim, E.L. Donovan, M. Chen, A.C.
Gross, J.I. Webster Marketon, S.H. Barsky, and R.
Glaser, 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: p. 267-75.
15.
Wrobel, L.J. and F.A. Le Gal, Inhibition of human
melanoma growth by a non-cardioselective beta-blocker. J
Invest Dermatol, 2015. 135: p. 525-31.
16.
Leaute-Labreze, C., E. Dumas de la Roque, T. Hubiche,
F. Boralevi, J.B. Thambo, and A. Taieb, Propranolol for
severe hemangiomas of infancy. N Engl J Med, 2008. 358:
p. 2649-51.
17.
Leaute-Labreze, C., P. Hoeger, J. Mazereeuw-Hautier, L.
Guibaud, E. Baselga, G. Posiunas, R.J. Phillips, H. Caceres,
J.C. Lopez Gutierrez, R. Ballona, S.F. Friedlander, J.
Powell, D. Perek, et al., A randomized, controlled trial of
oral propranolol in infantile hemangioma. N Engl J Med,
2015. 372: p. 735-46.
18.
Yang, E.V., A.K. Sood, M. Chen, Y. Li, T.D. Eubank,
C.B. Marsh, S. Jewell, N.A. Flavahan, C. Morrison,
P.E. Yeh, S. Lemeshow, and R. Glaser, 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: p. 10357-64.
19.
Wang, H., D.R. Gomez, and Z. Liao, beta-Blockers and
metastasis in non-small-cell lung cancer. Expert Rev
Anticancer Ther, 2013. 13: p. 641-3.
20.
Pasquier, E., J. Ciccolini, M. Carre, S. Giacometti, R.
Fanciullino, C. Pouchy, M.P. Montero, C. Serdjebi, M.
Kavallaris, and N. Andre, Propranolol potentiates the anti-
angiogenic effects and anti-tumor efcacy of chemotherapy
agents: implication in breast cancer treatment. Oncotarget,
2011. 2: p. 797-809.
21. Coelho, M., M. Moz, G. Correia, A. Teixeira, R. Medeiros,
and L. Ribeiro, Antiproliferative effects of beta-blockers
on human colorectal cancer cells. Oncol Rep, 2015. 33:
p. 2513-20.
22.
Landen, C.N., Jr., Y.G. Lin, G.N. Armaiz Pena, P.D. Das,
J.M. Arevalo, A.A. Kamat, L.Y. Han, N.B. Jennings, W.A.
Spannuth, P.H. Thaker, S.K. Lutgendorf, C.A. Savary, A.M.
Sanguino, et al., Neuroendocrine modulation of signal
transducer and activator of transcription-3 in ovarian
cancer. Cancer Res, 2007. 67: p. 10389-96.
23.
Liao, X., X. Che, W. Zhao, D. Zhang, T. Bi, and G. Wang,
The beta-adrenoceptor antagonist, propranolol, induces
human gastric cancer cell apoptosis and cell cycle arrest
via inhibiting nuclear factor kappaB signaling. Oncol Rep,
2010. 24: p. 1669-76.
24.
Wolter, J.K., N.E. Wolter, A. Blanch, T. Partridge, L. Cheng,
D.A. Morgenstern, M. Podkowa, D.R. Kaplan, and M.S.
Irwin, Anti-tumor activity of the beta-adrenergic receptor
antagonist propranolol in neuroblastoma. Oncotarget,
2014. 5: p. 161-72.
25.
Lin, X., K. Luo, Z. Lv, and J. Huang, Beta-adrenoceptor
action on pancreatic cancer cell proliferation and tumor
growth in mice. Hepatogastroenterology, 2012. 59: p. 584-8.
26.
Powe, D.G., M.J. Voss, K.S. Zanker, H.O. Habashy, A.R.
Green, I.O. Ellis, and F. Entschladen, Beta-blocker drug
therapy reduces secondary cancer formation in breast
cancer and improves cancer specic survival. Oncotarget,
2010. 1: p. 628-38.
27.
De Giorgi, V., M. Grazzini, S. Gandini, S. Benemei, T.
Lotti, N. Marchionni, and P. Geppetti, Treatment with beta-
blockers and reduced disease progression in patients with
thick melanoma. Arch Intern Med, 2011. 171: p. 779-81.
28.
Calvani, M., F. Pelon, G. Comito, M.L. Taddei, S. Moretti,
S. Innocenti, R. Nassini, G. Gerlini, L. Borgognoni,
F. Bambi, E. Giannoni, L. Filippi, and P. Chiarugi,
Norepinephrine promotes tumor microenvironment
reactivity through beta3-adrenoreceptors during melanoma
progression. Oncotarget, 2015. 6: p. 4615-32.
29.
Albinana, V., K. Villar Gomez de Las Heras, G. Serrano-
Heras, T. Segura, A.B. Perona-Moratalla, M. Mota-Perez,
J.M. de Campos, and L.M. Botella, Propranolol reduces
viability and induces apoptosis in hemangioblastoma cells
from von Hippel-Lindau patients. Orphanet J Rare Dis,
2015. 10: p. 118.
30. Dey, M., J. Brisson, G. Davis, R. Enever, K. Pray, B. Zaim,
and D. Dvornik, Relationship between plasma propranolol
concentration and dose in young, healthy volunteers.
Biopharm Drug Dispos, 1986. 7: p. 103-11.

Oncotarget14
www.impactjournals.com/oncotarget
31.
Xie, W., H. Xie, F. Liu, W. Li, J. Dan, Y. Mei, L. Dan, X.
Xiao, J. Li, and X. Chen, Propranolol induces apoptosis
of human umbilical vein endothelial cells through
downregulation of CD147. Br J Dermatol, 2013. 168:
p. 739-48.
32.
Pasquier, E., J. Street, C. Pouchy, M. Carre, A.J.
Gifford, J. Murray, M.D. Norris, T. Trahair, N. Andre,
and M. Kavallaris, beta-blockers increase response to
chemotherapy via direct antitumour and anti-angiogenic
mechanisms in neuroblastoma. Br J Cancer, 2013. 108: p.
2485-94.
33.
Shakhar, G. and S. Ben-Eliyahu, In vivo beta-adrenergic
stimulation suppresses natural killer activity and
compromises resistance to tumor metastasis in rats. J
Immunol, 1998. 160: p. 3251-8.
34.
Huang, X.Y., H.C. Wang, Z. Yuan, J. Huang, and Q.
Zheng, Norepinephrine stimulates pancreatic cancer cell
proliferation, migration and invasion via beta-adrenergic
receptor-dependent activation of P38/MAPK pathway.
Hepatogastroenterology, 2012. 59: p. 889-93.
35.
Munabi, N.C., R.W. England, A.K. Edwards, A.A. Kitajewski,
Q.K. Tan, A. Weinstein, J.E. Kung, M. Wilcox, J.K.
Kitajewski, C.J. Shawber, and J.K. Wu, Propranolol Targets
Hemangioma Stem Cells via cAMP and Mitogen-Activated
Protein Kinase Regulation. Stem Cells Transl Med, 2015.
36.
Zhang, D., Q. Ma, Z. Wang, M. Zhang, K. Guo, F. Wang,
and E. Wu, beta2-adrenoceptor blockage induces G1/S
phase arrest and apoptosis in pancreatic cancer cells via
Ras/Akt/NFkappaB pathway. Mol Cancer, 2011. 10: p. 146.
37.
Burotto, M., V.L. Chiou, J.M. Lee, and E.C. Kohn, The
MAPK pathway across different malignancies: a new
perspective. Cancer, 2014. 120: p. 3446-56.
38.
Lott, J.P., Vemurafenib in melanoma with BRAF V600E
mutation. N Engl J Med, 2011. 365: p. 1449-50; author
reply 1450.
39.
Dalle, S., N. Poulalhon, and L. Thomas, Vemurafenib in
melanoma with BRAF V600E mutation. N Engl J Med,
2011. 365: p. 1448-9; author reply 1450.
40.
Chapman, P.B., A. Hauschild, C. Robert, J.B. Haanen, P.
Ascierto, J. Larkin, R. Dummer, C. Garbe, A. Testori, M.
Maio, D. Hogg, P. Lorigan, C. Lebbe, et al., Improved
survival with vemurafenib in melanoma with BRAF V600E
mutation. N Engl J Med, 2011. 364: p. 2507-16.
41.
Toy, E.L., F. Vekeman, M.C. Lewis, A.K. Oglesby, and M.S.
Duh, Costs, resource utilization, and treatment patterns
for patients with metastatic melanoma in a commercially
insured setting. Curr Med Res Opin, 2015. 31: p. 1561-72.
42.
Zhang, C., W. Spevak, Y. Zhang, E.A. Burton, Y. Ma, G.
Habets, J. Zhang, J. Lin, T. Ewing, B. Matusow, G. Tsang,
A. Marimuthu, H. Cho, et al., RAF inhibitors that evade
paradoxical MAPK pathway activation. Nature, 2015. 526:
p. 583-6.
43. Brady, D.C., M.S. Crowe, M.L. Turski, G.A. Hobbs, X. Yao,
A. Chaikuad, S. Knapp, K. Xiao, S.L. Campbell, D.J. Thiele,
and C.M. Counter, Copper is required for oncogenic BRAF
signalling and tumorigenesis. Nature, 2014. 509: p. 492-6.
44.
Johannessen, C.M., J.S. Boehm, S.Y. Kim, S.R. Thomas,
L. Wardwell, L.A. Johnson, C.M. Emery, N. Stransky, A.P.
Cogdill, J. Barretina, G. Caponigro, H. Hieronymus, R.R.
Murray, et al., COT drives resistance to RAF inhibition
through MAP kinase pathway reactivation. Nature, 2010.
468: p. 968-72.
45.
Giard, D.J., S.A. Aaronson, G.J. Todaro, P. Arnstein, J.H.
Kersey, H. Dosik, and W.P. Parks, In vitro cultivation of human
tumors: establishment of cell lines derived from a series of
solid tumors. J Natl Cancer Inst, 1973. 51: p. 1417-23.
46.
Brown, B.L., J.D. Albano, R.P. Ekins, and A.M. Sgherzi,
A simple and sensitive saturation assay method for the
measurement of adenosine 3’:5’-cyclic monophosphate.
Biochem J, 1971. 121: p. 561-2.