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Research Article
Exposure to Music Alters Cell Viability and Cell Motility of
Human Nonauditory Cells in Culture
Nathalia R. Lestard and Marcia A. M. Capella
Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro,
21941-902 Rio de Janeiro, RJ, Brazil
Correspondence should be addressed to Marcia A. M. Capella; mcapella@biof.ufrj.br
Received January ; Accepted June
Academic Editor: Annarita Stringaro
Copyright © N. R. Lestard and M. A. M. Capella. is is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Although music is part of virtually all cultures in the world, little is known about how it aects us. Since the beginning of this
century several studies suggested that the response to music, and to sound in general, is complex and might not be exclusively
due to emotion, given that cell types other than auditory hair cells can also directly react to audible sound. e present study was
designed to better understand the direct eects of acoustic vibrations, in the form of music, in human cells in culture. Our results
suggest that the mechanisms of cell growth arrest and/or cell death induced by acoustic vibrations are similar for auditory and
nonauditory cells.
1. Introduction
Despite being an integral part of virtually all cultures in the
world, little is known about how music aects us. A number
of studies suggest that music may be useful in medical care,
alleviating stress and nociception in patients undergoing
surgical procedures, as well as in cancer and burned patients
[–] but the mechanisms by which these eects occur are
still unidentied. It is commonly accepted that the eects of
music are secondary to emotional responses, but Møller and
Pedersen armed that vibrotactile sensations and a feeling
of pressure might also occur in the chest and throat while
hearing sounds [].
Since the beginning of this century several studies sug-
gested that the response to music, and to sound in general, is
complex and might not be exclusively due to emotion, given
that cell types other than auditory hair cells can also directly
react to audible sound. For example, it has been shown
that sound wave stimulation makes signicant changes to
protein structure of tobacco cells, producing an increase in
𝛼helix and a decrease in 𝛽turn []; also, sound stimulation
produced eects on the cell cycle of Chrysanthemum [] and
in the callus growth of Dendranthema morifolium []. More
recently,itwasdemonstratedthatthetonalsoundsofkHz
and kHz promoted the growth of Escherichia coli [, ].
When considering mammalian cells, an increase in cor-
ticosterone serum levels aer environmental noise exposure
and a long-term reduction of proliferating cells in the hip-
pocampal of noise exposed rats were observed, suggesting
that exposure to chronic environmental noise at young ages
produces persistent impairment to nonauditory cells, altering
cell proliferation in the hippocampal formation []. It has
also been shown that a frequency of Hz was able to
alter the growth of human gingival broblasts in culture
[]andrecentlyweshowedthatmusic(andnotonlypure
frequencies) can lead to several eects in human cells in
culture, altering cell cycle, proliferation, viability, and binding
of hormone []. Since music is a summation of several
sound frequencies, and since sound is indeed a mechanical
vibration, which can cause mechanical stress, it seems not
odd that music can cause direct eects in mammalian cells.
e present report was thus designed to better understand
the direct eects of acoustic vibrations in the form of music
in human cells in culture.
Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2016, Article ID 6849473, 7 pages
http://dx.doi.org/10.1155/2016/6849473
Evidence-Based Complementary and Alternative Medicine
2. Material and Methods
2.1. Cells. MCF- and MDA-MB- are human breast cancer
cell lines with characteristics of epithelial cells. e cells were
grown in Dulbecco’s Modied Eagle Medium with penicillin
and streptomycin supplemented with % fetal bovine serum
(all from Invitrogen, Brazil) in disposable plastic bottles
(Techno Plastic Product, Germany), at ∘Cuntilconuence.
For each experiment, cells were platted on mm plastic
Petri dishes (TPP, Germany), at 1×10
5cells/dish. For cell
migration experiments 5×104cells/dish were platted on each
well. e experiments were performed h aer seeding, to
ensure uniform attachment of the cells. Each experiment was
repeated at least times.
2.2. Treatment with Music. e cells were exposed for min
to one of the three compositions: Mozart’s Sonata for Two
Pianos in D major, KV. , rst movement; Beethoven’s
th Symphony, rst movement; Ligeti’s Atmospheres, rst
movement; at ∘Cinanincubatorchamber.ecellswere
exposed to the music using a coaxial speaker, model ar c,
ref. , Watts, from UB Natts Eletroac ´
ustica (S˜
ao Paulo,
Brazil), placed at the roof of the incubator chamber, whose
walls were lined with cork and foam. As controls, the cells
were exposed to silence (speakers were turned o in the
incubator) or to the speakers plugged to energy without
anysoundproduced,toobserveapossibleactionofthe
background noise or the magnetic eld produced by the
speakers. Since Beethoven’s Fih and Ligeti’s Atmospheres
move continually from piano to forte and vice versa, there is
no way to imprint a constant sound pressure. erefore, the
sound pressure levels were maintained between and dB
to all compositions.
2.3. Apoptosis Assay. Aer exposure to music the cells were
incubated for , , and h. At each time, the supernatants
of cells cultures were collected into conical tubes (in order
to collect suspension cells) and the remained attached cells
were washed times with phosphate buered saline (PBS),
harvested with trypsin, and collected into the same con-
ical tube. e tubes were centrifuged min ( ×g), the
supernatant was discarded, and the pellet was resuspended
in 𝜇L of annexin V binding buer (Invitrogen, Brazil)
homogenized and transferred to ow cytometry tubes. e
cells were then stained with . 𝜇L annexin V-FITC and 𝜇L
propidium iodide (PI) ( 𝜇g/mL) for min in the dark.
Aer this time 𝜇L of annexin binding buer was added
and homogenized and the cells were analyzed in a FACScan
ow cytometer (Becton and Dickinson, USA).
2.4. Signaling Pathway to Apoptosis. MCF- cells were cul-
tured and exposed to Ligeti’s Atmospheres as described
above. Aer h of incubation, the cells were collected
and the levels of p, phospho-p, Bad, phospho-Bad,
CleavedCaspase,andCleavedPARPweremeasuredby
ELISA with commercially available kits (PathScan Apoptosis
Multi-Target Sandwich ELISA Kit , CST Inc., USA).
Absorbance was read at nm.
2.5. Cell Migration. Cell migration was characterized using
Tran s well system 𝜇m pore size (Corning, USA). e cells
weregrownuntil%conuence,washedtimeswith
PBS, and incubated with serum-free medium supplemented
with .% BSA. Aer h starvation cells were washed
times with PBS, harvested with trypsin, and collected into
conical tubes. Aer centrifugation for min ( ×g), the
supernatant was discarded and the pellet was resuspended
in serum-free medium at a density of 5×10
5cells/mL.
An aliquot of 𝜇L of the cell suspension was added into
the upper chamber of the Transwell system and 𝜇Lof
medium with % SFB was added into the lower chamber. e
cells were randomly exposed for min to the compositions
and then incubated for hours. e inserts were xed in
% paraformaldehyde for min and stained with % crystal
violet for min. e inserts were then washed and the upper
surface of the membranes was wiped with a cotton swab to
remove nonmigratory cells. Migrated cells were counted in
verandomlyselectedelds.eresultsarepresentedas
mean cell numbers for membrane.
2.6. Statistical Analysis. Each experiment was repeated at
least times. Data are expressed as means ±standard error
of the mean and were analyzed using Student’s t-test or
one-way ANOVA with Dunnett post test for comparison of
the dierences. Values of 𝑝less than . were considered
statistically signicant.
3. Results
In a previous study we observed that the human breast
cancer cell line MCF- responded to music by altering cell
cycle and diminishing its viability []. However, the method
used, Trypan blue assay, only permits visualization of dead
cells, which lost membrane integrity. at method could not
distinguish between necrotic and late apoptotic cells. It was
of interest to know whether cell death was due to apoptosis,
which is a more physiological type of cell death, or necrosis,
which would indicate an abrupt, sudden, death. erefore,
we now used the annexin-PI assay. According to Figure ,
living cells are gated in region R of Figure (a), showing
unlabeled cells (A−Pi−); cells undergoing apoptosis are gated
in region R of Figure (a), which shows cells labeled only
with annexin V (A+Pi−);andlateapoptotic,deadcells,labeled
with both annexin V and PI (A+Pi+), are gated in region R
of Figure (a).
In Figure (c) it is shown that the percentage of live cells
(R) is signicantly diminished h aer acoustic exposure
to Beethoven’s th Symphony or Ligeti’s Atmospheres. e
speaker alone and Mozart’s composition did not reduce sig-
nicantly the cell viability. Moreover, no signicant dierence
was observed h or h aer music exposition (Figures (b)
and (d), resp.).
In Figure it is shown that Ligeti’s composition increased
signicantly the percentage of apoptotic cells (region R,
A+Pi−) h aer acoustic treatment. Beethoven’s and Moz-
art’s compositions, as well as the speaker alone, have a tend-
ency to increase the percentage of apoptotic cells, although
Evidence-Based Complementary and Alternative Medicine
104
103
102
101
100
PI log
Annexin log
100101102103104
(a)
0
20
40
60
80
100
% of cells gated in R1
Control Speaker Beethoven Ligeti Mozart
R1 (A−Pi−)
(b)
0
20
40
60
80
100
% of cells gated in R1
∗∗
Control Speaker Beethoven Ligeti Mozart
R1 (A−Pi−)
(c)
% of cells gated in R1
0
20
40
60
80
100
Control Speaker Beethoven Ligeti Mozart
R1 (A−Pi−)
(d)
F : Apoptosis induced by music in MCF- cells. e cells were exposed to each composition as explained in Section and incubated
for , , or h. Apoptotic cells were analyzed by ow cytometry with propidium iodide (PI) and annexin V-FITC staining. (a) Dot-plot
of control cells, showing the regions used in Figures and . (b) Percentage of cells gated in region R (live cells) h aer exposure to the
compositions. (c) Percentage of cells gated in region R h aer exposure to the compositions. (d) Percentage of cells gated in R h aer
exposure to the compositions. Data are presented as means ±SE of four independent experiments. ∗𝑝< 0.05 (signicant when compared
with control).
0
5
10
15
20
Control Speaker Beethoven Ligeti Mozart
∗
A+
% of cells
Pi−
A+Pi−
(a)
0
5
10
15
20
25
Control Speaker Beethoven Ligeti Mozart
∗
∗
∗
% of cells
A+Pi+
A+Pi+
(b)
F : Apoptosis induced by music in MCF- cells. e cells were exposed to each composition and incubated for h. (a) % of cells gated
in region R of Figure (a) (positive for annexin V only). (b) % of cells gated in region R of Figure (a) (positive for annexin V and PI). Data
are presented as means ±SE of four independent experiments. ∗𝑝< 0.05 (signicant when compared with control).
Evidence-Based Complementary and Alternative Medicine
3
2.5
2
1.5
1
0.5
0
Speaker
Ligeti
Phospho-Bad
Phospho-p53
Bad
p53
Cleaved Caspase 3
Cleaved PARP
(a)
2.0
1.5
1.0
0.5
0.0
Ratio Ligeti/speaker
Phospho-Bad
Phospho-p53
Bad
p53
Cleaved Caspase 3
Cleaved PARP
(b)
F : Treatment of MCF- cells with Ligeti’s Atmospheres induced phosphorylation of p at Ser, as well as cleavage of Caspase as
detected by PathScanApoptosis Multi-Target Sandwich ELISA Kit . e cells were exposed to the composition or to the speaker alone
and incubated for h and the lysates were assayed as explained in Section . e absorbance readings at nm are shown as a -dimensional
representation in (a), while the ratio between Ligeti’s Atmospheres and speaker is shown in (b).
the dierences were not statistically signicant in relation to
the control. In addition, the three compositions increased
signicantly the percentage of dead cells (late apoptotic cells),
gated in region R (A+Pi+).
Since these results suggest that cells are dying by apopto-
sis, we tried to identify the possible apoptosis pathway. To do
this, we used Ligeti’s Atmospheres, since it was the compo-
sitionthatledtothemajorincreaseinapoptoticcells(both
A+Pi−and A+Pi+regions).ePathScanApoptosisMulti-
Target Sandwich ELISA Kit was used, which detects
endogenous levels of p protein, phospho-p protein, Bad,
phospho-Bad, Cleaved Caspase , and Cleaved PARP, key
molecules in signaling pathways controlling survival and
apoptosis. In Figure a representative result from one ex-
periment (Figure (a)) and the ratio between cells exposed
to Ligeti’s Atmospheres and cells exposed to speaker (Fig-
ure (b)) are shown. It can be seen that only phosphorylated
p and Cleaved Caspase were increased in cells exposed to
Ligeti’s Atmospheres, in relation to the speaker alone.
Because until now only one cell line was studied, we tested
whether the eects observed so far could be observed in
other cell lines: MDCK, a canine renal cell line with charac-
teristics of distal nephron []; K and K-Lucena, two
human erythroleukemia cell lines, the latest being multidrug
resistant []; and MDA-MB-, a human breast cancer cell
line previously used by our group that do not respond to
estrogens []. e rst three cell lines did not alter their
viability aer exposure to any compositions used (data not
shown). However, the breast cancer cells MDA-MB- have
also undergone apoptosis aer exposure to music, but their
response was dierent from that observed for MCF- cells, as
canbeseeninFigure.
In this gure it is shown that the three compositions, as
well as the white noise of the speaker, reduced the percentage
of viable cells (Figure (a)). Correspondingly, they increased
the number of cells undergoing both early and late apoptosis.
Sincethiscelllineispotentiallymetastatic,wetried
to evaluate whether music could alter this potential. For
this,aerexposuretomusicorspeakeralonethecells
were submitted to the cell migration assay, as described in
Section . In Figure it is shown that both Beethoven’s th
Symphony and Mozart’s sonata signicantly diminished the
migration of MDA-MB- cells.
4. Discussion
In a previous study we showed that music could act directly
on MCF-, a human breast cancer cell line, altering cell
cycle, proliferation, and viability. In the present report we
extend our studies, trying to understand such eects and
evaluating whether they could be obser ved in other cell types.
Interestingly, although we tested four cell lines, we noticed
that only the breast cancer cell line MBA-MD- reacted
to music and that such reaction was dierent from that
previously observed for MCF- cells. Possible explanations
for this fact could be that the human erythroleukemia cell
lines, being from blood origin, retain characteristics that help
them to cope with mechanical stress. e same argument
may be plausible for MDCK cells: being from distal nephron
origin, they are familiar with mechanical stress due to tubular
ow. erefore, it is possible that the absence of response of
these cell lines was related to their origins. is hypothesis
takes into account that in vitro direct eects of music in
nonauditory cells are related to mechanical stress, which is
reasonable, since music is, aer all, a mechanical vibration,
which can cause mechanical stress. However, at present it is
not possible to say whether such stress occurs externally (in
the culture medium), inside cells themselves or both.
We observed that the two breast cancer cell lines reacted
to music in dierent ways. While Mozart’s composition did
not alter cell viability of MCF- cells, it led MDA-MD-
to apoptosis. Moreover, the speaker alone was able to
signicantly reduce MDA-MD- viability, while it had
no eect on MCF- cells. Moreover, the compositions of
Beethoven and Mozart inhibited MDA-MB- migration,
without altering cell viability. ese results suggest that the
Evidence-Based Complementary and Alternative Medicine
0
20
40
60
80
100
Control Speaker Beethoven Ligeti Mozart
∗∗ ∗∗
% of cells
A−Pi−
A−Pi−
(a)
0
5
10
15
Control Speaker Beethoven Ligeti Mozart
∗
∗
∗
% of cells
A+Pi−
A+Pi−
(b)
0
2
4
6
8
Control Speaker Beethoven Ligeti Mozart
∗
% of cells
A+Pi+
A+Pi+
(c)
F : Apoptosis induced by music in MDA-MB- cells. e cells were exposed to each composition and incubated for h. (a) % of
cells gated in region R of Figure (a) (negative for both annexin V and PI). (b) % of cells gated in region R of Figure (a) (positive for annexin
V only). (c) % of cells gated in region R of Figure (a) (positive for annexin V and PI). Data are presented as means ±SE of four independent
experiments. ∗𝑝< 0.05 (signicant when compared with control).
0
Control Speaker Beethoven Ligeti Mozart
100
200
300
400
Number of cells migrated
∗∗
F : Exposition to music reduced the migration of MDA-MB- cells in vitro. Cells were exposed to each one of the three compositions,
as explained in Section , and the cell migration was observed. ∗𝑝< 0.05 (signicant when compared with control).
cellular response to music, and perhaps to sound in general,
is dependent not only on the nature of the sound, but also
on intrinsic characteristics of the cellular type. Since this
is the second report about the direct eects of music in
nonauditory cells in culture, more studies are needed to
achieve comprehension of these phenomena.
In the present report we also tried to observe the pathway
of apoptosis elicited by Ligeti’s music in MCF- cells. By
using an ELISA kit, we tested the key apoptotic molecules p
protein, phospho-p protein, Bad, phospho-Bad, Cleaved
Caspase , and Cleaved PARP and observed that only p and
Caspase were increased.
Evidence-Based Complementary and Alternative Medicine
e p protein is associated with DNA repair, growth
arrest, and apoptosis []. It is known that, aer a DNA
damage, p upregulates Bad transcription and that dephos-
phorylated Bad can heterodimerize with p and translocate
to mitochondria []. Moreover, PARP- is also activated
aer DNA damage and it has been shown that the interplay
between PARP- and p is dependent on the type of damage
induced to DNA []. Since there was no alteration on both
PARP- and Bad status aer exposure of MCF- cells to music,
it seems that the apoptosis induced by Ligeti’s Atmospheres
isnotduetoDNAdamage,whichisveryreasonable,given
that we do not expect that music will lead to DNA damage.
We have previously shown that Ligeti’s Atmospheres induced
MCF- cells growth arrest in S-phase and also an increase in
sub-G phase, which is in accordance to our present results.
erefore, taken together, our results suggest that exposure
of MCF- cells to music can induce cell growth arrest and/or
apoptosis. Interestingly, it has been shown that intense noise
leads to hair cells apoptosis by a pathway dependent on p
and Caspase [, ]. erefore, our results suggest that the
mechanisms of cell death or growth arrest induced by sound
(including music) are similar for auditory and nonauditory
cells.
Finally, in the present study we evaluated cell fate within
days of experimentation. Longer observations for follow-
upwouldbringothervaluabledataandcouldbeinteresting
to understand the properties observed by music intervention
to people with disease. We expect to do this in the future.
Competing Interests
e authors declare that there are no competing interests
regarding the publication of this paper.
Acknowledgments
is work was supported by grants from CNPq and Fundac¸˜
ao
do Cancer.
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