Biochemical and Biophysical Research Communications xxx (2016) 1e7

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

Fasting boosts sensitivity of human skin melanoma to cisplatin-

induced cell death
Fernanda Antunes a, Marco Corazzari b, c, *, Gustavo Pereira a, Gian Maria Fimia b, d,
Mauro Piacentini b, c, **, Soraya Smaili a
a
Department of Pharmacology, Federal University of Sa ~o Paulo, Brazil
b
Department of Biology, University of Rome “Tor Vergata”, Italy
c
National Institute for Infectious Diseases IRCCS “Lazzaro Spallanzani”, Italy
d
Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento, Lecce, 73100, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Melanoma is one of leading cause of tumor death worldwide. Anti-cancer strategy includes combination
Received 23 September 2016 of different chemo-therapeutic agents as well as radiation; however these treatments have limited ef-
Accepted 28 September 2016 ficacy and induce significant toxic effects on healthy cells. One of most promising novel therapeutic
Available online xxx
approach to cancer therapy is the combination of anti-cancer drugs with calorie restriction. Here we
investigated the effect Cisplatin (CDDP), one of the most potent chemotherapeutic agent used to treat
Keywords:
tumors, in association with fasting in wild type and mutated BRAFV600E melanoma cell lines. Here we
Melanoma
show that nutrient deprivation can consistently enhance the sensitivity of tumor cells to cell death in-
CDDP
Starvation
duction by CDDP, also of those malignancies particularly resistant to any treatment, such as oncogenic
BRAF BRAF melanomas. Mechanistic studies revealed that the combined therapy induced cell death is char-
Apoptosis acterized by ROS accumulation and ATF4 in the absence of ER-stress. In addition, we show that auto-
phagy is not involved in the enhanced sensitivity of melanoma cells to combined CDDP/EBSS-induced
apoptosis. While, the exposure to 2-DG further enhanced the apoptotic rate observed in SK Mel 28 cells
upon treatment with both CDDP and EBSS.
© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction pathways, ER stress and autophagy dysregulation, and resistance to

The incidence of melanoma has been increasing significantly
over the years. Although is the less frequent type of skin cancer, it is
the most lethal due to the high incidence of metastasis and lack of
effective treatments [1,2]. Although UV-radiation exposure repre-
sents the main cause of melanomagenesis it is now evident the
association also with several gene mutations [3,4]. Among these,
the RAS/RAF/MEK pathway seems to be the most affected, with
BRAFV600E present in approximately 70% of all melanomas [5,6]. The
mutation leads to constitutively active enzyme which determines
an uncontrolled cell proliferation, inactivation of cell death
* Corresponding author. Department of Biology, University of Rome “Tor Ver-
gata”, Via della Ricerca Scientifica 1, 00133, Rome, Italy.
** Corresponding author. Department of Biology, University of Rome “Tor Ver-
gata”, Via della Ricerca Scientifica 1, 00133, Rome, Italy.
E-mail addresses: marco.corazzari@uniroma2.it (M. Corazzari), mauro.
piacentini@uniroma2.it (M. Piacentini).
any treatment [7]. This mutation was also correlated with alter-
ations in the metabolic profile of melanoma cancer cells, favoring
the glycolytic metabolism and reducing the rate of mitochondrial
catabolic pathways [8]. This is in accordance with the “Warburg
effect”, one of the hallmarks of cancer cells that have an alteration
of metabolic pathways compared to normal cells. One of the most
important change is the up-regulation of glycolysis as a primer
source of energy and building blocks even in the presence of
abundant oxygen [9,10]. However, the metabolic profile is high
heterogeneous in melanoma cells, even between those harboring a
BRAFV600E mutation, which render difficult the use of a metabolism
targeted therapy [11].
It has been recently demonstrated that caloric intake modula-
tion can influence both the development and treatment outcome of
many cancers, in part by reprogramming the metabolism [12e14].
One of these protocols consists in severe nutrients restriction cycles
(fasting) combined with chemotherapeutic treatment, which in-
creases the susceptibility of cancer cells to cell death [12]. Dietary

http://dx.doi.org/10.1016/j.bbrc.2016.09.149
0006-291X/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: F. Antunes, et al., Fasting boosts sensitivity of human skin melanoma to cisplatin-induced cell death,
Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.09.149
2 F. Antunes et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7
interventions also up-regulates the AMPK signalling, an important
pathway with different targets for cancer treatment [15]. This
pathway is also involved in autophagy regulation [16], a possible
mechanism involved in the action of fasting since during the stress
induced by nutritional deprivation autophagy is up-regulated as a
survival mechanism [17]. Though, it is known that aggressive tu-
mors that harbor Ras mutations are energetically compromised
under starvation conditions since this mutation limits a higher
induction of the already increased basal autophagy. In this way
there is not enough energy supply, leading tumor cells to cell death
[18,19].
Cisplatin (CDDP) is one of the most potent chemotherapeutic
agent used to treat a wide range of malignancy as lung cancers,
head and neck tumors, ovarian carcinoma, among others [20].
Before the development of targeted therapies, cisplatin was one of
the first choice to treat melanoma, however with a poor response
rate [1]. CDDP resistance may occur at several levels as the cellular
extrusion of the drug [21], mechanism of DNA repair activation [22]
and autophagy induction [23]. Nevertheless, identifying new uses
for existing drugs might be an effective therapeutic approach to
overcome drug resistance in diverse malignancy and sustain pa-
tient's survival [29]. Therefore, we aimed to evaluate the opportu-
nity of using fasting cycles combined with cisplatin treatment as a
novel valuable clinical approach to treat human skin melanoma
independently of its mutational status.
2. Materials and methods
2.1. Materials
Cisplatin (Sigma Aldrich), Bafilomycin A1 (Sigma Aldrich), Z-
VAD-fmk (Santa Cruz Biotechnology), Necrostatin-1 (Sigma
Aldrich), N-acetyl-cysteine (Sigma Aldrich), 2-deoxy-glucose
(Sigma Aldrich), EBSS (Sigma Aldrich).
2.2. Cell culture
CHL-1 and SK Mel 28 metastatic melanoma cells were cultured
in DMEM high glucose (Sigma-Aldrich) supplemented with 10% FBS
(Life Technologies), 2 mM L-glutamine, and 1% penicillin/strepto-
mycin solution, at 37
C under 5% CO2.
2.3. Apoptosis evaluation
Briefly, 1,5  105 CHL-1 or SK Mel 28 cells were treatment as
indicated, for 24 h. Then, cells were fixed with methanol-acetone,
pelleted and resuspended in RNAse (50 mg/ml in PBS) and incu-
bated at 37
C for 15 min followed by propidium iodide (25 mg/ml in
PBS) staining. The cell cycle distribution was evaluated by flow
cytometry. 10.000 events were acquired using a FACS Calibur cy-
tometer (Becton-Dickinson, Mountain View,CA, USA) and the sub-
G1 phase analyzed using FlowJo software [24].
2.4. ROS and DJm assessment
Briefly, 1,5  105 CHL-1 or SK Mel 28 cells were treatment as
indicated and cells harvested at indicate time points. Then, cells
were pelleted and resuspended in H2-DCFDA (10 mM in PBS) or in
TMRM (50 nM in PBS), to detect ROS or to evaluate the DJm,
respectively. Cells were incubated at 37
C for 15 min in the dark,
and 10.000 events were acquired by using a FACS Calibur cytometer
(Becton-Dickinson, Mountain View,CA, USA). Data analysis was
performed using FlowJo software.
Please cite this article in press as: F. Antunes, et al., Fasting boosts sensitivity of human skin melanoma to cisplatin-induced cell death,
Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.09.149
2.5. Real time PCR analysis
Total RNA was extracted by using Trizol reagent (Invitrogen) as
recommended by the supplier. cDNA synthesis was performed
using a reverse transcription kit (Promega) according to the man-
ufacturer's recommendations. Quantitative PCR reactions were
performed by using a Rotor-Gene 6000 (Corbett Research Ltd)
thermocycler. Maxima SYBR Green/ROX qPCR Master Mix (Thermo
Scientific) was used to produce fluorescently labeled PCR products.
Primer sets for all amplicons were designed using Primer-Express
1.0 software (Roche).
ATF4 forward: 50 -GTGGCCAAGCACTTCAAACC-30
ATF4 reverse: 50 -CCCGGAGAAGGCATCCTC-30
ATF6 forward: 50 -TTTGCTGTCTCAGCCTACTGTGGT-30
ATF6 reverse: 50 -TCCATTCACTGGGCTATTCGCTGA-30
Xbp-I forward: 50 -GAATGAAGTGAGGCCAGTG-30
Xbp-I reverse: 50 -GAGTCAATACCGCCAGAATC-30
L34 forward: 50 -GTCCCGAACCCCTGGTAATAGA-30
L34 reverse: 50 -GGCCCTGCTGACATGTTTCTT-30
L34 mRNA level was used as an internal control and results were
expressed as previously described [24].
2.6. Western blotting analysis
Cells were lysed in Cell Lytic buffer (Sigma-Aldrich) supple-
mented with protease and phosphatase inhibitors (protease in-
hibitor cocktail plus 5 mM sodium fluoride, 0.5 mM sodium
orthovanadate, 1 mMsodium molybdate, 50 mM 2-
chloroacetamide, 2 mM 1,10-phenanthroline monohydrate, and
0,5 mM PMSF; Sigma-Aldrich). Lysates were incubated at 4
C for
30 min in ice. After centrifugation at 4
C for 10 min at 13,000 rpm
to remove insoluble debris, protein concentration was determined
using a Bradford assay (Biorad). 20 mg of total proteins were
resolved by using NuPAGEBis-Tris gels (Life Technologies) and
electroblotted onto nitrocellulose (Protran, Schleicher&Schuell) or
PVDF (Millipore) membranes. Blots were incubated with primary
antibodies resuspended in 5% non-fat dry milk in PBS plus 0.1%
Tween-20 overnight at 4
C. Detection was achieved using a
horseradish peroxidase-conjugate secondary antibody (1:5000 in
5% non-fat dry milk in PBS plus 0.1% Tween-20, Jackson Immuno
Research Laboratories), visualised with ECL (GE Healthcare) and
images were recorded by using a Chemidoc apparatus (BioRad).
Primary antibodies were: anti-PARP cleaved (1:1000, Cell Signal-
ling), anti-p62 (1:2000 MBL), anti-LC3B (1:1000, Cell Signalling),
anti-Ambra1 (1:1000, Cell Signalling), anti-Beclin-1 (1: 500, Santa
Cruz Biotechnology); anti-Atg5 (1:500, Santa Cruz Biotechnology);
anti-Actin (1:5000, Santa Cruz Biotechnology) and anti-GAPDH
(1:20000, Calbiochem).
2.7. Statistics
All values are represented as the mean ± SD. Significance was
evaluated by ANOVA one-way followed by Tukey test for multiple
comparisons among control and treatments. ANOVA two-way fol-
lowed by Bonferroni post-test was used for group analysis. Differ-
ences were considered significant with p  0.05. At least three
independent experiments were conducted to warrant that the re-
sults were representative.
3. Results and discussion
3.1. Starvation enhances melanoma cells sensitivities to cisplatin-
induced cell death
Previous results show severe nutrient restriction (starvation)
 F. Antunes et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7 3

associated to chemo-therapeutic drugs regimen results in
enhanced cell death induction in many cancer, ameliorating clinical
outcome of many therapy resistant malignancies [12e14]. Due to
the notorious resistance of human melanoma to clinical treatment
and the absence of a definitive therapy, we decided to explore this
opportunity in human skin melanoma. To this aim, we used a wild
type (CHL-1) and a BRAFV600E cell line (SK Mel 28) as a model. Both
cell lines were treated with CDDP (50 mM) or cultured in EBSS
(nutrient deprivation) alone or in combination for 24 h, and cell
death induction was evaluated by measuring the sub-G1 distribu-
tion of cells, by flow cytometric analysis of propidium iodide-
stained (PI) samples. Data reported in Fig. 1A indicate CHL-1 cells
are sensitive to both CDDP or EBSS treatment, while SK Mel 28
resulting highly resistant as expected, due to the presence of
oncogenic BRAF [25]. However, combined exposure to both CDDP
and EBSS resulted in consistently enhanced cell death induction in
both wild-type and oncogenic BRAF melanoma cells, compared to
individual treatments (70% and 35% respectively). To better char-
acterize the synergy of combined treatment, CDDPþEBSS, on cell
death induction we performed a time-course experiment in which
both cell lines were untreated or treated for 6 or 8 h with CDDP,
EBSS or CDDPþEBSS, and cell death induction was evaluated by
western blotting analysis of cleaved PARP, a well-characterized
apoptotic marker. As showed in Fig. 1B, combined treatment of
CHL-1 with CDDPþEBSS resulted in earlier cell death induction,
compared to individual treatments. Moreover, no PARP cleavage
was observed in SK Mel 28 cells, indicating that cell death induction
could be delayed by constitutive active pro-survival pathways
characterizing the oncogenic BRAF melanoma cells [7,25].
Since the observed cleavage of PARP and positive staining with
PI are indicative of an involvement of the apoptotic pathway, we
decided to unveil the molecular pathway activated by the com-
bined CDDP/EBSS treatment in both cell lines. Therefore, both cell
lines were untreated or treated with CDDPþEBSS for 24 h in
presence or absence of the pan-caspase inhibitor Z-VAD-FMK
(ZVAD, 50 mM), or the necroptosis inhibitor Necrostatin-1 (NEC,
50 mM), or with the ROS (reactive oxygen species) scavenger NAC
(N-acetyl-cysteine, 10 mM), and cell death induction was evaluated
by flow cytometric analysis of PI-stained samples. Interestingly, the
inhibition of caspases resulted in almost complete abrogation of
CDDPþEBSS-induced cell death, confirming apoptosis as the main
pathway activated by combined treatment in both cell lines
(Fig. 1C). However, NEC was also able to partially inhibit the cell
death induction in both cell line, indicating the involvement of
other cell death pathways (Fig. 1C). Importantly, NAC displayed a
significant cell death inhibition similar to ZVAD administration,
thus indicating a key role of ROS in the apoptotic pathway stimu-
lated by the combined administration of CDDP and EBSS (Fig. 1C), in
both cell lines.
Collectively these data indicate that nutrient deprivation can

Fig. 1. Starvation potentiates CDDP-induced apoptosis in melanoma cells. (A) Flow cytometric analysis of cell death (sub-G1 phase) in CHL-1 and SK Mel 28 cells treated 24 h
with EBSS (STV ¼ nutrient shortage), CDDP (50 mM) or CDDPþSTV, and stained with propidium iodide. (B) Representative immunoblots of western blotting analysis of cleaved PARP
levels in CHL-1 and SK Mel 28 cells treated as indicated. Actin was used as loading control. (C) Quantitative analyzes of cell death (sub-G1 phase) in CHL-1 and SK Mel 28 cells
treated for 24 h with STV (EBSS), CDDP (50 mM), CDDP, Z-VAD-fmk (50 mM), Necrostatin-1 (NEC, 50 mM), N-acetyl-cysteine (NAC, 10 mM), as indicated. (mean ± SD; n ¼ 3)
*p < 0.0001 compared with control cells. #p < 0.0001 compared to cisplatin.  p < 0.0001 compared to CDDP plus STV. One-way ANOVA, pos-test Tukey.

Please cite this article in press as: F. Antunes, et al., Fasting boosts sensitivity of human skin melanoma to cisplatin-induced cell death,
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4 F. Antunes et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7
consistently enhance the sensitivity of tumor cells to cell death
induction by chemotherapeutic drugs, also of those malignancies
particularly resistant to any treatment, such as oncogenic BRAF
melanomas.
3.2. Role of mitochondria and ER stress in nutrient deprivation-
enhanced melanoma cell death induction
Results reported in Fig. 1C clearly indicate the involvement of
ROS in the apoptotic process stimulated by combined CDDP and
EBSS exposure of melanoma cells. To confirm this data, the pro-
duction of ROS was evaluated in both melanoma cell lines 2 h after
CDDP, EBSS or CDDPþEBSS administration, by analyzing the fluo-
rescence of the ROS specific probe DCFDA (20 ,70 -dichlorofluorescein
diacetate) by flow cytometry. Data reported in Fig. 2A confirmed a
significant increase in ROS production in cells exposed to combined
CDDP and EBSS. However, it is important to note the major
contribution of nutrient deprivation (STV ¼ EBSS) to ROS produc-
tion in both cell lines treated with CDDPþEBSS (compare 2nd and
4th histograms of each panel in Fig. 2A, respectively).
ROS production during apoptosis induction/execution is often
the result of imbalanced mitochondrial homeostasis. Therefore, to
evaluate the status of this organelle, both CHL-1 and SK Mel 28 cells
were untreated or treated with CDDP, EBSS or CDDPþEBSS for 2, 4
or 8 h, and the mitochondrial membrane potential (MMP) was
monitored by flow cytometric analysis of TMRM-stained cells. Our
analysis, reported in Fig. 2B, revealed an early (2 h) pronounced
alteration of MMP in EBSS or CDDPþEBBS treated oncogenic BRAF
cells (SK Mel 28, Fig. 2B right panel), possibly accounting for the
early ROS production observed in this cell line (Fig. 2A, right panel).
However, a minor effect was observed in CHL-1 cells at any time
point, with a major effect observed after 8 h of treatment. These
data parallel the different amount of ROS produced by the two cell
lines and confirm nutrient deprivation (EBSS) as the main cause of
cellular stress.
Nutrient shortage typically results in protein synthesis attenu-
ation through eIF2a (eukaryotic initiation of translation) phos-
phorylation, resulting in the inhibition of cap-dependent
translation while favoring the translation of IRES-containing
mRNAs, such as ATF4, and thus resulting in downstream up-
regulation of this factor. The analysis of ATF4 mRNA levels in both
melanoma cell lines upon CDDP or EBSS treatment indeed revealed
an enhanced expression of this factor under nutrient deprivation
(STV ¼ EBSS) alone or in combination with CDDP (Fig. 2C). Since
CDDP alone resulted in a slight up-regulation of ATF4, that may also
indicate the induction of an ER stress response, we asked if this
process was involved in the apoptotic pathway stimulated by
combined treatment of melanoma cells with CDDP and EBSS. To
answer this question we evaluated the expression of two other ER
stress marker, ATF6 and XBP1, in the same experimental conditions.
Results reported in Fig. 2C (right panels) evidenced no significant
modulation of ATF6 while a slight up-regulation of XBP1 in SK Mel
28 in response to CDDP. Collectively these data indicate a minor or
no involvement of ER stress in our model.
3.3. Autophagy does not contribute to nutrient deprivation
enhanced cell death sensitivity of melanoma cells to CDDP
Autophagy is a catabolic process promptly activated by cells
under several stress conditions such as hypoxia, infection, nutrient
deprivation and in presence of cytotoxic compounds, such as CDDP
[26]. Although it represents primarily a pro-survival process, pro-
longed or deregulated autophagy may result or contribute to cell
death induction/execution [27]. Therefore, we asked whether
autophagy was involved in the enhanced cell sensitivity to CDDP-
Please cite this article in press as: F. Antunes, et al., Fasting boosts sensitivity of human skin melanoma to cisplatin-induced cell death,
Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.09.149
induced cell death in melanoma cells under nutrient deprivation.
To this aim, we evaluated the autophagic flux in cells subjected to
nutrient deprivation, CDDP treatment or a combination of the two
stimuli, evaluating the expression of the two well-known auto-
phagic markers p62 and LC3, in presence or absence of bafilomycin
A1 (BAF, 5 nM). Therefore, both CHL-1 and SK Mel 28 cells were
subjected to starvation or exposed to CDDP for 2, 4 and 6 h and p62
protein levels or LC3 conversion (LC3-II) were monitored by
western blotting analysis. Data reported in Fig. 3A and B show CHL-
1 are sensitive to both CDDP or EBSS administration, while SK Mel
28 were sensitive to CDDP-induced while resistant to STV-induced
autophagy. The latter is in line with previous results showing
intrinsic basal autophagy deregulation in oncogenic BRAF mela-
noma cells [7]. Interestingly, combined treatment of both mela-
noma cell lines with CDDP and EBSS showed no significant
modulation of either p62 protein levels nor LC3-II accumulation,
thus indicating that autophagy is not involved in enhanced sensi-
tivity of melanoma cells to combined CDDP/EBSS-induced
apoptosis (Fig. 3C). This conclusion are supported by results re-
ported in Fig. 3D showing no accumulation of key autophagic
proteins Ambra1, Beclin-1 or Atg5, in the same experimental
conditions.
3.4. Glucose metabolism is involved in melanoma cells survival
It is well-known that cancer cells are characterized by an altered
metabolism, favoring glycolysis even in aerobic conditions, an
event knows as “Warburg effect” [8]. Since cancer cells are addicted
to use glucose as primary source of energy and building blocks
supplier, we asked whether nutrient deprivation (also resulting in
glucose shortage) might be responsible for cancer cell deregulation
of metabolism resulting in enhanced sensitivity to pro-apoptotic
stimuli. To this end, we exposed our cell lines to CDDP in absence
or presence of 2-deoxy-glucose (2-DG, 10 mM), an irreversible in-
hibitor of glycolysis, supplemented to normal cell culture (high
glucose) or EBSS medium, and evaluated the apoptotic rates after
24 h. Our data indicate that inhibition of glycolysis enhanced cell
sensitivity to pro-apoptotic stimuli (CDDP þ 2-DG) similarly to
general nutrient deprivation (CDDP þ EBSS) in BRAF wild-type cells
(CHL-1, Fig. 4, left panel). On the other hand, oncogenic BRAF cells
seems to be more sensitive to combined EBSS þ 2-DG compared to
CDDP þ 2-DG treatment, possibly indicating that the glucose
metabolism is fundamental for the survival or these cancer cells.
Importantly, the exposure to 2-DG further enhanced the apoptotic
rate observed in SK Mel 28 cells upon treatment with both CDDP
and EBSS, indicating that a ‘complete’ nutrient deprivation might
consistently boost pro-apoptotic induction in the notoriously
resistant oncogenic BRAF melanoma cells (Fig. 4, right panel,
compare the two right-most histograms).
Several studies in animal models have suggested that calorie
restriction is able to significantly increase the life span and decrease
the risk of cancer development [28]. The data presented in this
study confirm this hypothesis indicating that nutrient deprivation
increases the efficacy of CDDP treatment in melanoma irre-
spectively of the BRAF status. Particularly, interesting is the effect of
the combined treatment with 2-DG that is able to induce similar
cell death levels both wild type and mutated BRAF cells. However,
further in vivo experiments are required to better define the
regulation/deregulation of glucose metabolism in wild-type and
oncogenic BRAF melanoma cells, and its role in their survival under
stress conditions [29e30].
Conflict of interests
None.
 F. Antunes et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7 5

Fig. 2. Involvement of mitochondria and ER stress on CDDP/STV induced cell death of melanoma cells. (A) Quantitative analyzes of DCFDA fluorescence by flow cytometry in
CHL-1 and SK Mel 28 cells treated for 2 h with STV, CDDP (50 mM) and CDDP plus STV (B) Measuring of DJm alteration with TMRM by flow cytometry in CHL-1 and SK Mel 28 cells
treated for 2e4 and 8 h with STV, CDDP (50 mM) and CDDP plus STV. CCCP (50 mM) was added as a positive control to loss of DJm. (C) Gene expression analyzes by qPCR of ATF4,
ATF6 and XBP-1 in CHL-1 and SK Mel 28 cells treated for 6e8 h with STV, CDDP (50 mM) and CDDP plus STV. (mean ± SD; n ¼ 3) *p < 0.005 compared with control cells. One-way
ANOVA, pos-test Tukey.

Please cite this article in press as: F. Antunes, et al., Fasting boosts sensitivity of human skin melanoma to cisplatin-induced cell death,
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6 F. Antunes et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7

Fig. 3. Analysis of autophagy induced by EBSS or CDDP in melanoma cells. p62 and LC3-II western blotting analysis of CHL-1 and SK Mel 28 cells untreated or treated 2, 4 or 6 h
with EBSS (STV) (A) or CDDP (50 mM) (B), (BAF, 5 nM). (C) p62 and LC3-II western blotting analysis of CHL-1 and SK Mel 28 cells treatment for 6 h with STV (EBSS), CDDP (50 mM),
CDDP plus STV, in presence or absence of bafilomycin A1. (D) Western blotting analysis of the autophagic proteins Ambra1, Beclin-1 and Atg5 in CHL-1 and SK Mel 28 cells treated
for 6e8 h with STV, CDDP (50 mM) or a combination. GAPDH was used as loading control.

Fig. 4. Glucose metabolism is involved in melanoma cells survival. Quantitative analysis of apoptosis (sub-G1 phase) in CHL-1 and SK Mel 28 cells treated with STV, or CDDP
(50 mM) as indicated, in presence or absence of 2-deoxy-glucose (2-DG e 10 mM) for 24 h. Cells were stained with propidium iodide and analyzed by flow cytometry (mean ± SD;
n ¼ 3) *p < 0.0001 compared with control cells. One-way ANOVA, pos-test Tukey. #p < 0.0001 compared to cisplatin. Two-way ANOVA, post-test Bonferroni.

Please cite this article in press as: F. Antunes, et al., Fasting boosts sensitivity of human skin melanoma to cisplatin-induced cell death,
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 F. Antunes et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7
Acknowledgments
This work was supported in part by grants from the Telethon
Foundation (GEP12072), AIRC (IG2015 n. 17404 to GMF and IG2014
n. 15244 to MP), the Italian Ministry of University and Research
(FIRB Accordi di Programma 2011), the Italian Ministry of Health
(Ricerca Corrente and Ricerca Finalizzata), Fondazione Fibrosi Cis-
tica (Progetto FFC#8/2015).
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.bbrc.2016.09.149.
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