SUPPLEMENTARY MATERIALS
MATERIALS AND METHODS
Chemicals: Common laboratory chemicals were from Sigma-Aldrich® (Sigma-Aldrich Chemistry,
Milan, Italy), unless differently stated. Antibodies used are listed below in methods where
appropriate.
In vitro Experiments
Cell line
We used PE/CA-PJ15 human oral squamous carcinoma cell line (European Collection of Cell
Cultures) cultured in Iscove’s modified Dulbecco’s modified Eagle medium (DMEM) (IMDM)
supplemented with 10% fetal bovin serum (FBS) (Biochrom, Germany), 1% penicillin/streptomycin
(10000 U/ml/10000 μg/ml, Biochrom, Germany) and 1% of L-Glutamine 200 mM (Biochrom,
Germany) at 37°C, in an atmosphere of 95% oxygen and 5% CO2.
Drug administration
Curcumin (high purity, cod: C7727) was dissolved in 0.5% Dimethylsulfoxide (DMSO, cod:
34869) and EtOH and sterile H2O (1:1). All manipulations with Curcumin were performed under
subdued light, the range of administered doses was 0.5, 1.0, 3.37 and 6.75 µM. Cisplatin (CDDP,
Code: P4394) was dissolved in sterile saline at and administered at a dose of 1.56 μM. Cells were
trypsinized and seeded on 13 mm cover slips in 24-well plates or in flask in quadruplicate and
allowed to adhere overnight. PE/CA-PJ15 cells were tested in two experimental conditions.
Experiment 1 was performed to analyze the effect of curcumin at different doses on cancer cells,
thus the samples were: a- PE/CA-PJ15 with no drug administration used as control, b- PE/CA-PJ15
treated with 0.5% DMSO and EtOH and sterile H2O (1:1), as control vehicle condition, c- PE/CAPJ15 treated with curcumin 0.5 µM, d- PE/CA-PJ15 treated with curcumin 1.0 µM, e- PE/CA-PJ15
treated with curcumin 3.37 µM and f- PE/CA-PJ15 treated with curcumin 6.75 µM. Experiment 2
was performed to analyze the combined effect of curcumin and CDDP administration, thus the
samples were: a- PE/CA-PJ15 with no drug administration used as control, b- PE/CA-PJ15 treated
with CDDP (1.56 µM), c- PE/CA-PJ15 treated with CDDP and curcumin 0.5 µM, d- PE/CA-PJ15
treated with CDDP and curcumin 1.0 µM, e- PE/CA-PJ15 treated with CDDP and curcumin 3.37
µM, f-PE/CA-PJ15 treated with CDDP and curcumin 6.75 µM. Each experiment was repeated three
times and we ran 4 replicates for each sample. Not significant differences were observed between
control (not treated) and control vehicle cells (data not shown). Cisplatin, curcumin and DMSO
were purchased from Sigma-Aldrich®.
Cell survival
In order to evaluate the toxic effect of curcumin, cisplatin and the combined drug exposure, 1.0×10 4
cells/glass were fixed with 4% paraformaldehyde for 15 minutes, washed twice in PBS and then
incubated in a solution containing DAPI (1:1000 in PBS 0.1 M) and Triton (TX, 0.1% in PBS 0.1
M) for 10 minutes light-protected and at room temperature. DAPI labelling was used to identify
condensed cell nuclei. The sample were, then, washed in PBS and coverslipped with an antifade
medium (ProLong Gold; Invitrogen). Cell count was performed on image acquired (10×) and
processed
with
the
aid
of
ImageJ
Nucleus
Counter
Plugin
(WCIF
ImageJ,
from
http://www.uhnres.utoronto.ca/facilities/wcif/). Each count was based on 10 fields randomly
selected for each of the experimental condition. Moreover, images were taken at higher
magnification (40x) by a confocal laser scanning microscope (TCS-SP2; Leica Microsystem,
GmbH, Wezlar, Germany). To highlight changes in morphology like swelling, nuclear condensation
and cellular fragmentation, DAPI staining was imaged by two-photon excitation (740 nm, < 140 fs,
90 MHz) performed by an ultrafast tunable mode-locked titanium:sapphire laser.
pSTAT3 and Nrf-2 Immunofluorescence
In order to perform immunofluorescence for the molecular targets, Nrf-2 and pSTAT3, cells were
fixed with 4% paraformaldehyde for 15 minutes at room temperature, permeated with 0.1% TX for
15 minutes prior to being blocked in 0.3% BSA for 20 min. Samples were then incubated with
primary antibodies rabbit anti-phospho-STAT3 (Tyr705) (Cell signaling, Tech, Boston, MA, USA)
and mouse anti-Nrf-2 (Abcam, Cambidge, UK) for 3 h in 0.3% BSA in PBS. At the end of
incubation, all samples were washed twice in PBS and incubated at room temperature for 90
minutes, light-protected, with secondary antibody diluted 1:1000 in PBS. To detect pSTAT3 and
Nrf-2 we used goat anti-rabbit 488 (Alexa Fluor) and donkey anti-mouse 546 (Alexa Fluor)
respectively. Moreover, cell nuclei were counterstained with DAPI (1:1000 in PBS) for 10 min at
room temperature, light-protected. Then, the samples were coverslipped with an antifade medium
(ProLong Gold; Invitrogen). Images of pSTAT3 and Nrf-2 immunolabelled specimens (40×) were
taken by the confocal laser scanning microscope equipped with an argon/argon– krypton laser and a
helium/neon laser for 488 and 519 excitation. DAPI staining was imaged by two-photon excitation
(740 nm, <140 fs, 90 MHz) performed by an ultrafast tunable mode-locked titanium:sapphire laser.
pSTAT3, Nrf-2 and DAPI positive cells were identified by green, red and blue fluorescence
respectively. To quantify Nrf-2 and pSTAT3 fluorescence signal, we performed optical density
analysis in the cytoplasmic and nuclear compartment by using Leica Confocal Software (LCS Lite).
Immunofluorescence was performed 24, 48 and 72h after treatment. However, given that no
significant differences were observed among the three time points, only the results at 24h are
discussed.
TUNEL Assay
Apoptosis was evaluated in PE-CA/PJ15 cultures with the APO-BrdU TUNEL assay kit
(Invitrogen) 48h after treatment, according to the manufacturer’s instructions, as previously
described (Piacentini et al, 2008). Briefly, DNA strand breaks in apoptotic cells were labeled with
BrdU by the use of terminal deoxynucleotide transferase. Apoptotic cells were identified
immunocytochemically by means of anti-BrdU antibody labelling with Alexa Fluor 488 dye, and
cell nuclei were identified by means of propidium iodide/RNase staining.
Western blot
PE/CA-PJ15 cells (2x106) were trypsinized 48 h after drug treatment and then centrifuged at 500×g
for 5 minutes. Protein concentration was measured using a Micro BCA kit (Pierce). An equal
amount of sample (30 μg) was separated by 10% SDS-PAGE and then electrophoretically
transferred onto 0.45 μm nitrocellulose membranes using a solution containing 50 mM Tris/HCl,
380 mM glycine, and 20% methanol 1h at 4°C. Afterwards, a Ponceau S staining was performed to
ensure protein transfer (ICN Biochemicals). Nonspecific binding sites were blocked with 5% dry
milk in Tris-buffered saline (TTBS; 20 mM Tris, 500 mM NaCl, and 0.05% Tween 20). Next, the
membranes were incubated overnight at 4°C with the primary antibody anti- Bax (Immunological
Science, Rome, Italy) diluted (1:400) in 3% BSA and 0.05% NaN3/TTBS. After five rinses in
buffer, membranes were incubated for 1h at room temperature with a horseradish peroxidase
conjugated anti-mouse IgG secondary antibody (Cell signaling, Tech, Boston, MA, USA) that was
diluted 1:2500 in 2.5% dry milk/TTBS. Blots were developed using enhanced chemiluminescence
reagents (ECL; GE Healthcare). Equal protein loading among individual lanes was confirmed by
reprobing the membranes with an anti-α-actin mouse monoclonal antibody at a 1:1000 dilution
(Thermo Fisher Scientific). Protein expression was evaluated and documented by using UVItec
Cambridge Alliance. The experiments were repeated three times, and an inter experiment variability
of <10% was found.
In vivo Experiments
Animals
Male adult Wistar rats (UCSC Laboratories, 200-250 gr) were used. The auditory function of each
animal was tested for the presence of Preyer’s reflex. The experiments were performed on 25
animals, randomized and assigned to 4 experimental groups as follows: 1- control (Ctrl; n= 5); 2Cisplatin (CDDP; n= 5); 3- Cisplatin+Curcumin at a dose of 100 mg/kg (CDDPCur 100; n= 5); 4Cisplatin+Curcumin at a dose of 200 mg/kg (CDDPCur 100; n= 5) and 5- Cisplatin+Curcumin at a
dose of 400 mg/kg (CDDPCur 400; n= 5). All animals were sacrificed under deep anesthesia
(ketamine at dose 70 mg/kg and medetomidine-dormitor at dose of 0.5 mg/kg) at day 5 after
treatment onset. For the whole experimental period, the animals were housed 2 per cage at
controlled temperature (22/23°C) and constant humidity (60±5%), under a 12-hour light/dark cycle,
with food (Mucedola 4RF21, Italy) and water ad libitum. All efforts were made to minimize animal
suffering and to reduce their number, in accordance with the European Community Council
Directive of 24 November 1986 (86/609/EEC). All procedures were performed in compliance with
the Laboratory of Animal Care and Use Committee of the Catholic University, School of Medicine
of Rome and were approved by the Italian Department of Health (Ministero della Salute).
Drug administration
As described previously (Fetoni et al, 2014), curcumin was dissolved in DMSO. The diluted
solution was prepared freshly daily and administered intraperitoneally (i.p.) at three different doses
(100, 200 and 400 mg/Kg b.w.). Curcumin solution was injected 1 hour before cisplatin
administration and once daily for the following 3 days. Cisplatin, diluted in sterile saline (1 mg/ml),
was prepared freshly and protected by light. To facilitate the dissolution of cisplatin, the solution
was heated and stirred for a period of 20 minutes. Under deep anaesthesia, a single cisplatin dose of
16 mg/Kg (Fetoni et al, 2014) was delivered i.p. at a rate of 8 ml/h with an infusion pump (Axon
Instruments, Foster City, CA, USA) over about 30 minutes. The animals were hyper-hydrated with
saline solution (subcutaneous injection, 15 ml daily) to limit cisplatin side effects.
Auditory Function Evaluation
Auditory Brainstem Responses (ABR)
Hearing level induced by treatments (cisplatin and curcumin) was estimated by ABR recordings, in
order to identify the acoustic threshold of each animal for each group. ABRs were measured at low
(6 kHz), mid (12, 16, and 20 kHz), and high (24 and 32 kHz) frequencies. In all animals, ABRs
were assessed bilaterally before treatment (day 0) to assure normal hearing and reassessed at all
time points (3 and 5 days from treatment onset) to evaluate the effect of treatments on hearing. All
animals were mildly anesthetized (ketamine, 35 mg/kg and medetomidine-dormitor, 0.25 mg/kg)
and placed in the anechoic room. As described previously (Fetoni et al, 2013; 2014), 3 stainless
steel recording electrodes were subcutaneously inserted posterior to the tested pinna (active), vertex
(reference), and contralateral pinna (ground). A PC-controlled TDT System 3 (Tucker-Davis
Technologies, Alachua, FL, USA) data acquisition system with real-time digital signal processing
was used for ABR recording and auditory stimulus generation. Tone bursts of pure tones from 6 to
32 kHz (1 ms rise/fall time, 10 ms total duration, 20/s repetition rate) were presented monaurally.
Responses were filtered (0.3-3 kHz), digitized, and averaged (across 500 discrete samples at each
frequency-level combination). Threshold value was defined as the lowest stimulus level that yielded
a repeatable waveform-based onset.
Distortion product otoacoustic emissions (DPOAEs)
To determine the effect of cisplatin ototoxicity on OHC function, DPOAEs were measured
unilaterally using an otoacoustic emission system (Tucker-Davis Technologies, Alachua, FL, USA).
The f2/f1 ratio of the primary tones was set to 1.2. DPOAE input/output functions were measured at
f2 frequencies of 4, 8, 12, 16 and 20 kHz. The f1 intensity (L1) always presented +10 dB above the
f2 intensity (L2). Animals were anaesthetized as described above for ABR procedure and placed on
a heating pad in a sound-attenuating booth. The probe assembly was placed in the animal's external
ear canal. Input/output functions were obtained by increasing L1 intensity from 20 to 70 dB SPL at
f2 frequencies of 4, 8, 12, 16 and 20 kHz (32 sweeps per frequency pair). DPOAEs were recorded
before and 3 and 5 days after the onset of treatment.
Morphological analyses and cell viability
The Rhodamine-Phalloidin (Rh-Ph), a high affinity F-actin probe conjugated to the red-orange
fluorescent dye, tetramethylrhodamine (TRITC), was used to visualize the stereociliary arrays and
cuticular plates of hair cells. Rh-Ph staining was performed at day 5 in 5 cochleae/group animals.
As described previously (Fetoni et al, 2014), surface preparations of the organ of Corti were
incubated with a solution containing 0.5% Triton X-100 and Rh-Ph (1:100 dilution; Molecular
Probes, Invitrogen, Carlsbad, CA, USA) in 0.1 M PBS for 1 hour at room temperature protected
from light. Positive cells were counted in segments of approximately 250 µm in length each along
the basilar membrane. Hair cells were considered missing if both the stereocilia bundles and the
cuticular plates were absent, and OHC loss was calculated as percentage with respect to controls.
All morphologic observations were performed with the aid of the confocal laser scanning system.
Nrf-2/ HO-1 and pStat3 Immunostainings
Immunostainings were performed at day 5 in cochlear cryosections (12 m), in order to assess and
quantify the endogenous antioxidant response to cisplatin-induced damage and to evaluate the effect
of curcumin supplementation. As described previously (Fetoni et al, 2013, 2015), the cochleae
(5/group) were quickly removed, and the samples were fixed with 4% paraformaldehyde in PBS at
4°C and a pH 7.5. Next, the cochleae were decalcified for 15 d in EDTA (10% EDTA, changed
daily), incubated for 48 h in sucrose (30%), embedded in OCT, and cryosectioned at a thickness of
12 μm (Cryostat SLEE). The specimens were incubated with a blocking solution (1% BSA, 0.5 %
Triton x-100 and 10% normal goat serum in PBS 0.1 M) and then the slices were incubated
overnight at 4 °C with a solution containing anti-HO-1 or Nrf-2 or anti-Phospho-STAT3 primary
antibodies diluted 1:100 in PBS (anti-HO-1: Stressgen, Ann Arbor, MI, USA; anti-Nrf-2 and antipSTAT3: as above reported). These antibodies cross-reacted with rat tissue. All specimens were
incubated at room temperature for 2 h in labeled conjugated goat anti-rabbit (HO-1 and pSTAT3)
and donkey anti-mouse (Nrf-2) secondary antibody (Alexa Fluor 488 and 546, IgG, Invitrogen)
diluted 1:400 in 0.1 M PBS and DAPI stained (1:500 in 0.1 M PBS). Confocal Z-stacks in series of
15-20 µm-thick were acquired as images of 1024×1024 pixels, reordered at intervals of 0.5 µm, in
order to evaluate the real extent of the nuclear and/or cytoplasmatic fluorescence of HO-1, Nrf2 and
pSTAT3 in high magnifications of the organ of Corti and SGNs (Fetoni et al, 2015). Images (100×)
were obtained with the confocal laser scanning system equipped with an Ar/ArKr laser (for 488 nm
excitation) and HeNe laser (for 543 nm excitation). DAPI staining was imaged by two photon
excitation (740 nm, <140 fs, 90 MHz) performed with an ultrafast, tunable mode-locked Ti:sapphire
laser.
Control experiments (negative controls not shown) were performed by omitting the primary
antibody during processing of tissue randomly selected across experimental groups. Tissues from
all groups were always processed together during the procedures to limit variability related to
antibody penetration, incubation time, post-sectioning age, and condition of tissue.
Statistical Analysis
Results are presented as means ± mean standard error (SEM), and differences were assessed using
variance analysis (ANOVAs). As regard cell survival and proliferation a two-way ANOVA was
performed (group×time point). To analyze TUNEL-positive cells, one way ANOVA was
performed. ABR and DPOAEs data were evaluated by three-way ANOVA (ABR:
group×frequency×time point; DPOAEs: group×dB×time point). Cochleogram data were analyzed
by two-way ANOVA (group×cochlear turn). Post-hoc comparisons were assessed using Tukey’s
test (Statistica, Statsoft, Tulsa, OK, USA); p< 0.05 was considered significant. The magnitude of
tumour progression was expressed as percentage of the ratio of survived cells (cell relative
viability%= experiment values/control*100%) among times points (p<0.05).