Cancer Letters 385 (2017) 75e86

Contents lists available at ScienceDirect

Cancer Letters
journal homepage: www.elsevier.com/locate/canlet

Original Article

Doxorubicin anti-tumor mechanisms include Hsp60 post-translational

modications leading to the Hsp60/p53 complex dissociation and
instauration of replicative senescence
Antonella Marino Gammazza a, b, *, Claudia Campanella a, b, Rosario Barone a, b,
Celeste Caruso Bavisotto a, b, Magdalena Gorska c, Michal Wozniak c, Francesco Carini a,
Francesco Cappello a, b, Antonella D'Anneo d, Marianna Lauricella e, Giovanni Zummo a,
Everly Conway de Macario f, g, Alberto J.L. Macario b, f, g, Valentina Di Felice a, b
a

Department of Experimental Biomedicine and Clinical Neurosciences, University of Palermo, Palermo, Italy
Euro-Mediterranean Institute of Science and Technology, Palermo, Italy
c
Department of Medical Chemistry, Medical University of Gdansk, Gdansk, Poland
d
Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, Laboratory of Biochemistry, University of Palermo, Palermo, Italy
e
Department of Experimental Biomedicine and Clinical Neurosciences, Laboratory of Biochemistry, University of Palermo, Palermo, Italy
f
Department of Microbiology and Immunology, School of Medicine, University of Maryland at Baltimore, Baltimore, MD, USA
g
IMET, Columbus Center, Baltimore, MD, USA
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 29 July 2016
Received in revised form
26 October 2016
Accepted 28 October 2016

The chaperone Hsp60 is pro-carcinogenic in certain tumor types by interfering with apoptosis and with
tumor cell death. In these tumors, it is not yet known whether doxorubicin anti-tumor effects include a
blockage of the pro-carcinogenic action of Hsp60. We found a doxorubicin dose-dependent viability
reduction in a human lung mucoepidermoid cell line that was paralleled by the appearance of cell
senescence markers. Concomitantly, intracellular Hsp60 levels decreased while its acetylation levels
increased. The data suggest that Hsp60 acetylation interferes with the formation of the Hsp60/p53
complex and/or promote its dissociation, both causing an increase in the levels of free p53, which can
then activate the p53-dependent pathway toward cell senescence. On the other hand, acetylated Hsp60
is ubiquitinated and degraded and, thus, the anti-apoptotic effect of the chaperonin is abolished with
subsequent tumor cell death. Our ndings could help in the elucidation of the molecular mechanisms by
which doxorubicin counteracts carcinogenesis and, consequently, it would open new roads for the
development of cancer treatment protocols targeting Hsp60.
2016 Elsevier Ireland Ltd. All rights reserved.

Keywords:
Doxorubicin
Hsp60
Acetylation
Ubiquitination
p53
Replicative senescence

Introduction

Abbreviations: DMSO, dimethylsulfoxide; ELISA, enzyme linked immunosorbent

assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GUSB, betaglucuronidase; HDAC, histone deacetylases; H2AX, H2A histone family member X; HPRT1,
hypoxanthine phosphoribosyltransferase 1; Hsps, heat shock proteins; MTT, 3-(4,5dimethylthiazol-2-yl)-2,3-diphenyltetrazolium bromide; OD, optical density; RS,
replicative senescence; SA-b-gal, senescence-associated b-galactosidase; Sirt3, sirtuin 3; 17AAG, 17-(Allylamino)-17-demethoxygeldanamycin.
* Corresponding author. Department of Biomedicine and Clinical Neurosciences,
Human Anatomy Section, Via del Vespro 129, 90127, Palermo, Italy.
E-mail address: [email protected] (A. Marino Gammazza).
http://dx.doi.org/10.1016/j.canlet.2016.10.045
0304-3835/ 2016 Elsevier Ireland Ltd. All rights reserved.

Replicative senescence (RS) or cellular senescence has been

described as a state reached by normal mammalian broblasts
cultured in vitro after a limited number of divisions [1]. In this state,
senescent cells cannot divide and become un-responsive to growth
signaling and resistant to apoptosis. Senescent cells show a attened and enlarged shape and an increase in senescence-associated
b-galactosidase (SA-b-gal) activity [2]. Cytoskeletal proteins may be
involved in RS, for instance vimentin, since this protein is highly
expressed in senescent broblasts [3].
Since cancer cells proliferate indenitely, a requisite for their
immortalization must be bypassing the physiological program that
leads to RS. Abundant data support the notion that RS is a natural

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barrier against tumorigenesis [4,5]. Stress-induced premature

senescence is a program executed by cells in response to chemotherapy, and RS induced by DNA-damaging anticancer drugs is one
of the key determinants of successful chemotherapy [6].
Heat shock proteins (Hsps) are highly expressed in a variety of
cancer cells and are essential to their survival contributing to tumor
cell propagation, metastasis, and protection against apoptosis [7,8].
Several Hsps function as molecular chaperones for other proteins to
prevent their aggregation after environmental stress, for example.
Molecular chaperones participate in the response to anti-cancer
drugs [9,10] and are intensively studied as therapeutic targets and
as diagnostic and/or prognostic markers in many types of cancer, for
example breast cancer, osteosarcomas [11], ovarian carcinoma [12],
and pancreatic carcinoma [13]. As stated above, the senescence
program seems to represent one of the major breaks on cancer
emergence and it has been demonstrated that high levels of chaperones play an important role in suppressing the senescent program,
keeping the p53 signaling under control and thus allowing cancer
cells to proliferate [14]. In fact, specic down-regulation of Hsp70
leads to rapid senescence of various cancer cell lines [15], and human neuroblastoma cells displayed senescence-like characteristics
after inhibition of Hsp90 with tanespimycin (17AAG) [16]. The
mitochondrial chaperonin Hsp60 also named HSPD1 [17] was found
in multiple subcellular sites and function in the folding and intracellular trafcking of many proteins [18,19]. Hsp60 has been found
elevated in a large number of human carcinomas, which opens novel
perspectives for cancer diagnosis and therapy targeting Hsp60
[20,21]. The chaperonin can activate the immune system [22] and
can have both, pro-survival and pro-death functions, depending on
tissue, cell type, and apoptosis inducers [23].
Senescence and apoptosis are alternative cell fates: in some cases
apoptosis is a response to intense stress while senescence is a
consequence of mild damage [4]. For example, doxorubicin, an
anthracycline antitumor drug widely used in clinical chemotherapy,
induces senescence at low doses and apoptosis at high doses in
breast cancer cells and in neonatal rat cardiomyocytes [24,25,26].
Hsp60 over-expression suppressed doxorubicin-induced apoptosis
in cardiomyocytes [27], and doxorubicin-induced apoptosis in HeLa
cells triggering Hsp60 up-regulation [23]. To the best of our
knowledge no data are available that would show a direct participation of Hsp60 in RS. Here we investigated if sub-apoptotic doses of
doxorubicin have an impact on the tumor-favoring Hsp60 action
and, thereby, produces anti-tumor effects via the induction of RS in a
human lung mucoepidermoid cell line.

Materials and methods

Cell culture and treatment conditions
The human lung mucoepidermoid cell line NCI-H292 was obtained from the
American Type Culture Collection. Cells were routinely propagated and maintained
in Roswell Park Memorial Institute medium (RPMI-1640, Sigma Aldrich, Milan, Italy)
with 10% heat-inactivated Fetal Calf Serum (FCS, Life Technology, Milan, Italy),
supplemented with 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. The cell line was grown as monolayers attached to 25 cm2 culture asks and
cultured at 37
C, 5% CO2 in a humidied incubator. Doxorubicin hydrochloride
(Sigma Aldrich) was prepared as an 8 mM stock solution in sterile water, stored
at 20
C and freshly dissolved immediately before use. Working dilutions were
made in sterile water and were added to the complete cell culture medium at the
appropriate concentrations. Twenty-four hours after seeding, when the cells
reached 70% conuency, the cultures were treated with a range of concentrations of
doxorubicin (range 5e1280 nM) for 5 days and untreated cells (UT) were maintained
as control. After that, the cell culture medium was replaced with fresh drug-free
complete medium and cells were left to grow further for 24 h, before harvesting
them for testing. Cells were routinely photographed before and after treatment to
record morphological changes occurring in the cells, using an inverted light microscope equipped with phase contrast rings (LEICA DM-IRB, Leica Microsystem,
Milan, Italy). Measurements of the area of the cell were done and evaluated using
ImageJ Free software (NIH, Bethesda, MD).

MTT assay
The viability of NCI-H292 cells treated with doxorubicin was measured using 3(4,5-dimethylthiazol-2-yl)-2,3-diphenyltetrazolium bromide (MTT) obtained from
Sigma Aldrich. The assay was performed as described [28]. Briey, 5  103 NCI-H292
cells were plated in 200 ml of complete medium per well in 96-well plates and
treated with a series of doses of doxorubicin for 24, 48, or 72 h, or 5 days. MTT was
dissolved in fresh medium and added to the cell cultures at a nal concentration of
0.5 mg/ml. Following a 2 h incubation period, the converted dye was solubilized in
200 ml of dimethylsulfoxide (DMSO)/well and optical density (OD) was measured
with a plate reader (Titertek Multiskan MCC/340, Flow Laboratories, Allschwil
Switzerland) at 570 nm (630 nm as reference). Cell viability was expressed as the
percentage of the OD value of inhibitor-treated cells compared with untreated
controls, according to the following equation: Viability (OD SAMPLE/OD
CONTROL)  100. Untreated cells were used as control and each experiment was
carried out in triplicate.
Cell cycle analysis
Cells cultured in 25 cm2 asks were trypsinized and washed with phosphate
buffered saline (PBS). The cells suspensions in PBS (1  106 cells/ml) were centrifuged and the supernatant was removed. To quantify DNA content, cells were xed
in absolute ethanol and resuspended in 1 ml of hypotonic buffer containing 0.1%
Triton X-100, 0.1% sodium citrate, and 50 mg/ml propidium iodide (Sigma Aldrich) in
propylene FACS tubes. After centrifugation at 1100  g for 5 min and supernatant
removal, the cells were resuspended again in 250 ml of hypotonic buffer and incubated in the dark at 23
C for 15 min. Finally, 250 ml of RNAse A solution (10 mg/ml
RNAse A in PBS) (Sigma Aldrich) was added to each tube following by an incubation
of 15 min in the dark at 23
C. After this treatment and the addition of 0.5 ml of PBS,
the cells were analyzed by ow cytometry (FACScan; Becton-Dickinson, Milan, Italy).
Percentages of cells in G0/G1, G2/M phases were determined, using APC32 acquiring
software and analyzed by APC32 analysis software on a Coulter EPICS cytometer.
Senescence-associated beta-galactosidase (SA-b-gal) activity assay
SA-b-gal activity was detected using in situ b-gal staining kit (Agilent Technologies, Cernusco sul Naviglio, Milan, Italy) according to the manufacturer's protocols.
Briey, attached cells were xed in a buffer including 2% formaldehyde/0.2%
glutaraldehyde for 10 min at 23
C. After removing the xing solution from the wells,
the cells were washed twice with PBS pH 7.6 and incubated with a freshly prepared
staining solution (pH 6) containing X-gal (5-bromo-4 chloro-3-indolyl- b-galactopyranoside) overnight at 37
C in a humidied incubator. Finally, coverslips
were mounted with Vectamount A (Vector Laboratories, Peterborough, UK). The
percentage of blue-stained cells in the total number of cells was determined by
counting cells with a Leica DM 5000B light microscope.
Real-time quantitative PCR (qRT-PCR)
The qRT-PCR technique was performed as previously described [29]. Briey,
total cellular RNA was isolated from both control and treated cell cultures, using
TRIzolREAGENT (Sigma-Aldrich) and according to the manufacturer's instructions.
RNA (50 ng) was retrotranscribed using the ImProm-II Reverse Transciptase Kit
(Promega Corporation, Milan, Italy) to obtain cDNA, which was amplied using the
StepOnePlus Real-Time PCR System (Thermo Scientic, Milan, Italy). qRT-PCR
analysis was performed using GoTaq qPCR Master Mix (A6001, Promega). The
mRNA levels were normalized to the levels obtained for hypoxanthine phosphoribosyltransferase 1 (HPRT1), for betaglucuronidase (GUSB), and for glyceraldehyde3-phosphate dehydrogenase (GAPDH). The cDNA was amplied using the primers
indicated in Table 1. cDNA was amplied using the Rotor-gene 6000 Real-Time PCR
Machine (Qiagen GmbH, Hilden, Germany). Changes in the transcript level were
calculated using the 2eDDCT method [30].
Western blotting
Treated and untreated (control) NCI-H292 cells were harvested with 0.25%
trypsin supplemented with 1 mM EDTA (LONZA, Basel, Switzerland) and centrifuged
at 1100 g for 5 min at 4
C. Pellets were washed twice in PBS and resuspended in
100 ml ice cold RIPA lysis buffer (0.3 M NaCl, 0.1% SDS, 25 mM HEPES pH 7.5, 1.5 mM
MgCl2, 0.2 mM EDTA, 1% Triton X-100, 0.5 mM DTT, 0.5% sodium deoxycholate)
containing proteases and phosphatase inhibitors (0.1 mg/ml phenylmethyl sulfonyl
uoride, 20 mg/ml aprotinin, 20 mg/ml leupeptin, 10 mg/ml NaF, 1 mM DTT, 1 mM
sodium orthovanadate, 20 mM -glycerol phosphate) to obtain lysates. Cell lysates
were incubated for 30 min on ice then centrifuged at 13,000 g for 20 min at 4
C.
The Bradford assay was used to determine the total protein concentration. Equal
amounts (40 mg) of total cellular proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad, Segrate, Milan, Italy). Equal protein
loading was ascertained by Ponceau-S staining of blotted membranes. After 1 h
incubation at 23
C with a blocking solution, 5% milk in Tris buffered saline pH 7.6
(TBS) with 0.05% Tween 20 (Sigma-Aldrich) (T-TBS), membranes were incubated
with primary antibodies (mouse anti-Hsp60, LK1 clone, Sigma Aldrich; mouse antivimentin, V9 clone, Santa Cruz Biotechnology, Heidelberg, Germany; mouse anti-

A. Marino Gammazza et al. / Cancer Letters 385 (2017) 75e86

77

Table 1
Forward and reverse primers used for qRT-PCR.
Primer

Forward

Reverse

GUSBa
GAPDH
HPRT1
HSPD1 var1

5'-ACCACCCCTACCACCTATATC-3'
5'-GAAACCCATCACCATCTTCC-3'
5'-TGTCATGAAGGAGATGGGAG-3'
5'-GAGTAGAGGCGGAGGGAG-3'

5'-ATCCAGTAGTTCACCAGCCC-3'
5'-TCCACGACATACTCAGCAC-3
5'-ATCCAGCAGGTCAGCAAAG-3'
5'-AGTGAGATGAGGAGCCAGTA-3'

a
GUSB, betaglucuronidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPRT1, hypoxanthine phosphoribosyltransferase 1;
HSPD1, Hsp60.

p53, DO-1 clone, Santa Cruz Biotechnology; rabbit anti-phosphorylated Histone

H2AX (Ser 139; gH2AX), Santa Cruz Biotechnology; mouse anti-p21, clone DCS60,
Cell Signaling Technology; mouse anti-b actin, AC-40 clone, Sigma-Aldrich). Membranes were then incubated with HRP-conjugated secondary antibody (ECL antimouse or anti-rabbit IgG HRP-conjugated whole antibody). The specic binding was
detected using ECL (ECL Western Blotting detection, GE Healthcare Europe GmbH,
Milan, Italy). The analysis of bands intensity was performed using ImageJ software.
Immunoprecipitation
Immunoprecipitation was performed as described previously [31] to detect
protein complexes and post-translational modications. Briey, equal amounts of
proteins (500 mg) from total cell lysates for each doxorubicin treatment were
incubated with the primary anti-Hsp60 mouse monoclonal antibody overnight at
4
C with gentle rotation. Antibody/protein complexes were then immunoprecipitated with antibodies linked to Sepharose A beads (GE Healthcare Europe GmbH).
Nonspecically bound proteins were removed by repeated washing with isotonic
lysis buffer. Immunoprecipitated proteins were resolved by 10% SDS-PAGE using a
primary antibody against acetylated lysine (mouse monoclonal anti-acetylated
lysine, Cell Signaling, Pero, Milan, Italy), anti-ubiquitin primary antibody (mouse
monoclonal anti-Ub, clone P4D1, Santa Cruz Biotechnology) and anti-p53 primary
antibody. Each experiment was performed three times.
Immunouorescence
Immunouorescence was performed as described previously [32]. NCI-H292
cells were placed in 8-well microscope chamber slide at the density of 10,000
cells/well, cultured for 24 h and then treated with doxorubicin. Cells were xed with
ice-cold methanol for 30 min, washed in PBS pH 7.4, and then incubated with
unmasking solution (trisodium citrate 10 mM, 0.05% Tween 20, pH 6) for 10 min at
23
C. After rinsing twice with PBS, the cells were blocked with 3% (w/v) bovine
serum albumin (BSA, Sigma Aldrich) in PBS for 30 min at 23
C and incubated with
the primary antibodies directed against Hsp60 and vimentin overnight at 4
C. The
day after, the cells were washed twice in PBS and were incubated with a uorescent
secondary antibody (mouse IgG antibody conjugated with FITC, Sigma-Aldrich) for
1 h at 23
C in a moist chamber. The nuclei were counterstained with Hoechst 33342
(Sigma-Aldrich) for 15 min at 23
C. Finally, the slides were covered with drops of
PBS and mounted with coverslips. Imaging was immediately performed with a Leica
DM5000 upright uorescence microscope.

Results
Inhibition of growth and morphological changes in lung cancer NCIH292 cells exposed to doxorubicin
To determine the effects of doxorubicin on cell viability, NCIH292 cells were cultured in RPMI supplemented with various
concentrations of doxorubicin (range 5 to 1260 nM) at the beginning of the experiments (time 0) and were thereafter harvested at
24, 48, and 72 h, and at 5 days to determine viability and
morphology. Cell growth was not inhibited after 24, 48, and 72 h
(data not shown) but clear effects were observed after a ve-day
exposure to doxorubicin. A dose-dependent reduction of cell
viability was evident as shown by MTT (Fig. 1). High doses of
doxorubicin (640 and 1280 nM) decreased H292 cell viability
inducing apoptotic cell death, as suggested by ow cytometry
analysis (see later). On the basis of these results, three subapoptotic
doses (20, 40, and 80 nM) were chosen to evaluate doxorubicin
effect after 5 days of treatment. Morphological changes were
already visible starting at 20 nM of doxorubicin. Cells showed a
typical senescent phenotype appearing attened and enlarged. In
fact, the measure of their area increased concomitantly with the
increase of doxorubicin doses (Supplementary Fig. S1, p < 0.001).

Doxorubicin induces replicative senescence in NCI-H292 cells

Flow cytometric examination of the cell cycle distribution of
NCI-H292 cells showed a signicant increase of cells with DNA
content corresponding to G2/M at 80 nM of doxorubicin
compared to the control and 20 nM (p < 0.05), whereas the cells

ELISA
Used culture medium was collected after the treatment with doxorubicin and
ELISA was performed as previously described [33] to measure Hsp60, using a
commercial Hsp60 (human) enzyme immunoassay (EIA) kit (Enzo Life Sciences,
Vinci, Italy), according to the manufacturer's instructions. The Hsp60 protein standard was diluted in standard diluent to generate a standard curve with six points,
ranging from 3.125 to 100 ng/ml. Then, 100 ml of prepared standards and samples
was added in duplicate to wells of the immunoassay plate pre-coated with mouse
monoclonal antibody specic for Hsp60 and incubated at 23
C for 1 h and at 37
C
for 2 h. After diluting the primary and secondary antibodies according to the
manufacturer's instructions, 100 ml of anti-Hsp60 goat polyclonal antibody was
added to each well and incubated at 23
C for 1 h and then at 37
C for 1 h. Subsequently, 100 ml of horseradish peroxidase conjugate anti-goat IgG was added to the
plate and incubated at 23
C for 30 min followed by 100 ml of 3,30 ,5,50 -tetramethylbenzidine substrate for 15 min in the dark. Finally, 100 ml of Stop Solution was
added, and absorbance was measured at 450 nm with a microplate photometric
reader (DV990BV4, GDV, Milan, Italy). Sample concentration was calculated by
interpolating the sample measurement in the standard curve. The sensitivity of the
human Hsp60 EIA kit was determined to be 3.125 ng/ml. Human Hsp60 EIA kit is
specic for Hsp60 and the Hsp60 ELISA has been certied for the detection of human
Hsp60.
Statistical analysis
The analysis was performed using the statistical software package GraphPad
Prism4 (San Diego, CA). The data obtained were compared by the One-way ANOVA
analysis of variance using Bonferroni post-hoc multiple comparisons. The data are
expressed as means SD. The statistical signicance threshold was xed at p < 0.05.

Fig. 1. Doxorubicin affects viability of NCI-H292 cells. The impact of doxorubicin was
evaluated at 5 days of treatment at different concentrations by using MTT colorimetric
assay. The viability data shown in the line chart pertain to day 5. After exposure to
increasing concentrations (0e1280 nM) of doxorubicin, the cells showed a dosedependent reduction of viability (growth 50% index: 480 nM).

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A. Marino Gammazza et al. / Cancer Letters 385 (2017) 75e86

A. Marino Gammazza et al. / Cancer Letters 385 (2017) 75e86

with DNA content corresponding to G0/G1 concurrently

decreased (p < 0.05) (Fig. 2AeB). These results indicate that the
drug induced cell cycle arrest. On the contrary, high doxorubicin
doses (640 and 1280 nM) induced a signicant increase in the
number of cells with apoptotic features (sub G0/G1) corresponding to sub-diploid DNA content compared to all other
conditions (p < 0.001) (Fig. 2AeB). In addition, the morphological analyses performed with uorescence microscopy after
staining with Hoechst showed that high doses of doxorubicin
induced the appearance of highly condensed nuclei, which
represent a typical apoptotic hallmark (Fig. 2C).
In order to determine whether the doxorubicin-induced cell
cycle arrest is associated with RS in NCI-H292 cells, SA-b-gal activity and vimentin (which is known to be associated with cytoskeleton remodeling) were also determined. As shown in Fig. 3A,
the number of b-gal-positive, i.e., senescent, cells was markedly
increased in doxorubicin treated by comparison with untreated
NCI-H292 cultures. Moreover, the percentage of b-gal-positive cells
increased signicantly with the dose of doxorubicin reaching the
highest degree at 80 nM (p < 0.01) (Fig. 3B). Western blot analysis
showed a signicant increase in the level of the protein vimentin
after treatment with 80 nM of doxorubicin compared with UT and
20 nM (p < 0.05; Fig. 4AeB). Fluorescence microscopic examination
revealed that in control cells the vimentin network was subtle,
relatively well organized, mainly composed of thin and short laments uniformly distributed throughout the cytoplasm (Fig. 4C,
upper left panel, UT). After the treatment, the cells appeared attened and enlarged and a considerable heterogeneity in appearance and cytoplasmic location of laments also occurred (Fig. 4C
upper right panel, and both bottom panels). At 20 nM both unchanged cells and cells with increased volume were observed.
These enlarged cells were more abundant at 40 and 80 nM of
doxorubicin. Long laments reached the peripheral regions of cells,
but simultaneously few less-regularly arranged and dispersed bers were observed in the vimentin network.
Effect of doxorubicin on p53, gH2AX, and p21 protein levels
In order to determine the effect of doxorubicin on p53 protein
levels western blotting analysis was performed. As showed in
Fig. 5AeB, doxorubicin at 20, 40 and 80 nM induced a signicant
increase of p53 protein levels to 1.7 fold compared to the untreated
condition (p < 0.01). Since p53 can be activated in response to DNA
damage, we analyzed the level of gH2AX, a histone H2A variant that
becomes rapidly phosphorylated at serine 139 in response to DNA
double-strand breaks and is a sensitive indicator of this DNA
damage. By using a specic antibody directed against phosphorylated H2AX (Fig. 5C), we observed that 40 and 80 nM doxorubicin
promoted a signicant phosphorylation of H2AX at the Ser139
residue (gH2AX) compared to 20 nM and UT, favoring its activation
(Fig. 5D, p < 0.001). Data reported in Fig. 5, panels CeE also
demonstrated that the increase of p53 was accompanied by a signicant up-elevation of p21 at 80 nM doxorubicin compared to all
other conditions (p < 0.001). Moreover, p21 protein levels were
signicantly higher at 40 nM doxorubicin compared to UT and
20 nM (p < 0.001).

79

Fig. 3. SA-b-gal activity in NCI-H292 cells after treatment with doxorubicin. A:

Representative images of senescence-associated b-galactosidase staining of untreated
cells (UT), and of cells treated with 80 nM of doxorubicin for ve days. The cells
exhibited the typical blue staining. Bar 100 mm (same for all panels). B: Histograms
showing the percentage of cells positive for b-galactosidase staining. Results are
representative of three independent experiments. * Different from UT, 20, and 40 nM
p < 0.01; DDifferent from UT, and 20 nM p < 0.001. UT: untreated cells. (For interpretation of the references to color in this gure legend, the reader is referred to the
web version of this article.)

Impact of doxorubicin on hsp60 gene expression and Hsp60 protein

levels
To determine the effect of doxorubicin on hsp60 gene expression
qRT-PCR was applied. As shown in Fig. 6A, treatment with 40 and
80 nM of doxorubicin induced the increase of hsp60 gene expression in NCI-H292 cells compared to 20 nM and non-treated cells
(p < 0.01). Moreover, hsp60 expression levels signicantly
increased at 80 nM doxorubicin compared to 40 nM dose (p < 0.01).
On the other hand, the data obtained by Western blotting showed
that Hsp60 protein levels were decreased down to 1.3 fold of the
basal level in cells treated with 20 nM doxorubicin whereas its
levels decreased to 1.8 and 2.5 at 40 and 80 nM doses respectively
(p < 0.05) (Fig. 6BeC). Moreover, Hsp60 protein levels decreased at
80 nM doxorubicin compared to 20 nM dose (p < 0.01). Immunouorescence conrmed that doxorubicin affected the Hsp60 protein levels and cell distribution (Fig. 6D).

Effect of doxorubicin on p53/Hsp60 complex and Hsp60 posttranslational modications and secretion
We next asked whether Hsp60 physically associated with p53,
thus potentially limiting or abolishing its function. The interaction
between Hsp60 and p53 in NCI-H292 cells was investigated using
immunoprecipitation. A complex Hsp60/p53 was apparent in the
untreated cells. The amount of p53 in the complex with Hsp60
signicantly decreased after 40 and 80 nM doxorubicin compared
to 20 nM and the untreated condition (Fig. 7A, top row of blots (a)
and corresponding histogram, B; p < 0.001).

Fig. 2. Cycle distribution of NCI-H292 cells indicative of replicative senescence at sub-apoptotic doses. A: Representative ow cytometry histograms are shown for untreated (UT)
cells, and for cells treated with 20, 40, 80, 640, and 1280 nM of doxorubicin. Cells were classied, according to DNA content, in the following categories: cells with DNA content
corresponding to sub G0/G1, G0/G1, S, and G2/M. B: Cells with DNA content corresponding to G2/M signicantly increased at 80 nM of doxorubicin compared to untreated cells, and
to cells treated with 20, 640, or 1280 nM. DNA content corresponding to the S phase (S) was obtained as the difference between total DNA content (100) and the sum of DNA content
corresponding to sub G0/G1, G2/M, and G0/G1. Cells with DNA content corresponding to sub G0/G1 signicantly increased at 640 and 1280 nM compared to all other conditions.
*Different from UT, 20, 40, 80, and 640 nM, p < 0.001; Different from UT, 20, 40, and 80 nM, p < 0.001; #Different from 80, 640, and 1280 nM, p < 0.05; yDifferent from 80, 640, and
1280 nM, p < 0.05; Different from 640, and 1280 nM, p < 0.05; DDifferent from UT, 20, 640, and 1280 nM, p < 0,05; $Different from 1280 nM, p < 0.01. C: Representative images
showing the nuclei of the cells after staining with Hoechst. High doses of doxorubicin induced the appearance of highly condensed nuclei. Bar 100 mm. UT: untreated cells.

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A. Marino Gammazza et al. / Cancer Letters 385 (2017) 75e86

Fig. 4. Increase of vimentin levels and cytoskeleton remodeling in NCI-H292 cells after treatment with doxorubicin. A: Blots for vimentin showing its increase in cells treated with
40, or 80 nM of doxorubicin compared to untreated (UT) cells and to cells treated with 20 nM; b-actin was used as internal control. B. Ratio vimentin levels/b-actin levels as a
measure of vimentin increase (mean SD). AU: arbitrary unit. *Different from UT, and 20 nM p < 0.05. C. Representative images showing vimentin increase and remodeling of the
laments. Nuclei were counterstained with Hoechst 33,342. Results are representative of three independent experiments. Bar 100 mm (same for all panels).

In order to determine if doxorubicin induced post-translational

modication of Hsp60, a double-labeled immunoprecipitation of
Hsp60/acetylated lysine and Hsp60/ubiquitin was performed. The
results showed that Hsp60 acetylation signicantly increased at 40
and 80 nM doxorubicin compared to 20 nM and UT (Fig. 7A, second
row of blots from the top (b) and corresponding histogram, C;
p < 0.05), while the double-labeled immunoprecipitation of ubiquitin and the chaperonin, revealed that at 40 nM doxorubicin,
Hsp60 was signicantly ubiquitinated compared to other conditions (Fig. 7A third row of blots from the top (c) and corresponding
histogram, D; p < 0.01).
Lastly, ELISA test was used to evaluate Hsp60 extracellular
release. The results showed that Hsp60 release by treated cells into
the extracellular medium was signicantly decreased at 40 nM
dose as compared to all other conditions (p < 0.01), while a
borderline increase occurred at 80 nM compared to 20 nM (Fig. 7E;
p < 0.05).
Discussion
Cellular senescence is a state of permanent cell cycle arrest that
can be triggered by a variety of factors, including DNA damage,

telomere shortening, and oxidative stress. Senescence limits the life

span and proliferative capacity of cells, therefore the induction of
this cellular process is nowadays an objective of anti-cancer treatment [34,35]. Senescence can offer an attractive therapeutic option
if it can be restored in tumor cells, since many cancers cells retain
the ability to senesce either spontaneously or in response to
external stimuli [36]. However, the mechanisms for reactivating
senescence in tumor cells are not well characterized and, therefore,
the use of this therapeutic approach is underappreciated [37,38].
A promising eld of research is the study of the role of Hsps in
carcinogenesis, and of the possible applications of Hsp detection
and quantication in tissues and biological uids for cancer diagnosis, for assessing prognosis and monitoring disease progression,
as well as for developing antitumor therapeutic strategies targeting
Hsps [20]. Some key components of the senescence process are
regulated by chaperones [19]. These proteins in cancer cells are
believed to be involved in tumor progression and cell proliferation,
differentiation, invasiveness, neoangiogenesis, metastasis, and immune system recognition [39,40] as well as in the development of
chemotherapy resistance [41].
In our study, human-lung mucoepidermoid carcinoma cells
were treated with a range of doses of doxorubicin. After treatment

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81

Fig. 5. Doxorubicin induced an increase in the levels of p53, gH2AX, and p21. A and C: Blots of p53, gH2AX, and p21 proteins in cells treated with 20, 40, or 80 nM, and untreated
cells; b-actin was used as internal control. B, D, and E: Histograms representing the ratios p53/b-actin, gH2AX/b-actin, and p21/b-actin levels, respectively, calculated from the
densitometric measurements of the blots in B and C, showing the increase in the three proteins in cells treated with doxorubicin (mean SD). AU, arbitrary unit; UT: untreated cells.
*
Different from 20, 40, and 80 nM p < 0.01; #Different from UT, and 20 nM p < 0.001; Different from UT, 20, and 40 nM p < 0.001.

with 40 and 80 nM doxorubicin, the cells showed enlarged and

attened morphology, positivity to SA-b-gal activity, and cytoskeleton remodeling associated with an increase in the levels of
vimentin, all typical indicators of RS, as also shown by others in
other cell types [2,3]. Moreover, cell cycle analysis showed that the
cells were in the G2/M phase at 80 nM, which is also in agreement
with previous reports demonstrating that cellular senescence was
associated with cell cycle arrest in the G2/M phase [42,43]. The
mechanism by which different doses of doxorubicin may induce
different stress-response programs is not well understood. The data
obtained here demonstrated that doxorubicin induced the

expression of the tumor suppressor protein p53. This is in agreement with previous studies reporting that the drug can intercalate
DNA, generating reactive oxygen species, leading to a DNA damageresponse mediated by p53 [26,44]. Coincident with these ndings,
our data provided evidence that doxorubicin was able to promote in
lung mucoepidermoid NCI-H292 cells the activation of DNA damage response as suggested by the increase in gH2AX. In addition,
other data have indicated the existence of a causeeeffect relationship between the DNA damage response and cellular senescence [45]. In particular, it has been observed that some proteins
involved in DNA damage response, including gH2AX, can localize at

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A. Marino Gammazza et al. / Cancer Letters 385 (2017) 75e86

Fig. 6. hsp60 gene expression and Hsp60 protein levels in NCI-H292 cells. A: Histograms representing qRT-PCR results that show changes in the transcript levels calculated using the
2eDDCT method, which indicate that doxorubicin affected hsp60 gene expression in NCI-H292 cells: namely, hsp60 gene expression increased signicantly in cells treated with 40 and
80 nM compared to untreated cells and to cells treated with 20 nM. B: Blots showing the decrease of Hsp60 protein levels in cells treated with 20, 40, or 80 nM of doxorubicin
compared to untreated cells; b-actin was used as internal control. C: Histograms representing the ratio Hsp60 level/b-actin level that showHsp60 decrease at 40 and 80 nM
compared to UT and 20 nM (mean SD). AU, arbitrary unit. D: Representative immunouorescence images showing Hsp60 (green) decrease in cells treated with 40 or 80 nM of
doxorubicin. Nuclei (blue) were counterstained with Hoechst 33,342. Bar 100 mm (same for all panels). UT: untreated cells. #Different from UT, and 20 nM p < 0.001; *Different
from UT, 20, and 40 nM p < 0.01; Different from all other conditions p < 0.05; yDifferent from 80 nM p < 0.01. (For interpretation of the references to color in this gure legend, the
reader is referred to the web version of this article.)

uncapped telomeres in the senescence-associated DNA damage

[46]. The response activated by telomeric damage has been
considered a potential inducer of senescence or cell death [47].
Moreover, our results showed an up-regulation of p21 after doxorubicin treatment, a transcriptional target of p53 which is involved
in the induction of senescence [48].
Beyond the classical pathways involved in RS, here we provide
experimental data demonstrating that treatment with doxorubicin
inhibits the growth of a lung-cancer cell line via the induction of RS,
which is accompanied by decreased levels of intracellular Hsp60.
An increase of Hsp60 levels has been found correlated with the
normal-to-dysplasia-to-carcinoma transitions in various anatomical sites [40] and with cell cycle progression [49]. Moreover,
immunopositivity for Hsp60 was proposed as biomarker of bronchial carcinogenesis, considering that the levels of the chaperonin
signicantly correlated with the prognosis of lung adenocarcinoma
[50]. Various data have revealed the need for Hsp60 by some types
of tumor cells for their survival and growth. For example, elevated
levels of this chaperonin in tumor cells have been linked to the
ability to survive apoptotic stimuli, loss of RS, uncontrolled proliferation, and neoplastic transformation [51]. In our experimental
model, decreased levels of Hsp60 were associated with appearance

of senescence features and tumor-cell growth arrest, which coincided with Hsp60 modication such as hyperacetylation and
ubiquitination. Hyperacetylation of Hsp60 can be due to the inhibition of sirtuins in line with observations by others who demonstrated that doxorubicin induced senescence in human dermal
broblasts via down-regulation of both Sirtuin 1 and 2 [52].
Hyperacetylated Hsp60 could be tagged for the proteasome system,
via ubiquitinization, which reached the highest degree a 40 nM,
leading to a decrease of the intracellular levels of the chaperonin
and the establishment of RS. This hypothesis is supported by the
ndings that in our model mRNA levels of Hsp60 where found
elevated in contrast with the protein levels. The data obtained are
consistent with in vitro experiments in which Hsp60 knockdown
and inhibition of its expression by short hairpin RNA plasmids
stopped tumor cell proliferation [53,54].
Hsp60 is preeminently a protein-folding machine but it can also
stimulate anti-apoptotic mechanisms involving sequestration of
Bax-containing complexes thus favoring tumor cell survival [51,55].
However, it has been demonstrated that Hsp60 modied with Olinked N-acetylglucosamine (O-GlcNAcylation) in pancreatic bcells, under hyperglycemic conditions, shows a decreased interaction with Bax leading to cell death [56]. Loss of Hsp60 functions in a

A. Marino Gammazza et al. / Cancer Letters 385 (2017) 75e86

83

Fig. 7. Doxorubicin reduced p53/Hsp60 complex, and induced Hsp60 post-translational modication. A: Representative Western blots (WB) for immunoprecipitation experiments.
Cell extracts from untreated cells and from cells treated with 20, 40, or 80 nM doxorubicin were prepared and protein complexes were immunoprecipitated using anti-Hsp60
antibody. Proteins were resolved by SDS-PAGE and identied using anti-p53 (a), anti-acetyl lysine (b), anti-ubiquitin (c), or anti-Hsp60 (d) antibody. BeD: Histograms representing densitometric measurements of the blots in A, showing: (i) the amount of p53 in the Hsp60/p53 complex signicantly decreased in cells treated with 40 or 80 nM
doxorubicin compared to UT and 20 nM (B); (ii) a dose-dependent increase in the levels of Hsp60 acetylation (C); and (iii) the ubiquitination of Hsp60 in cells treated with 40 nM
doxorubicin (D). Histograms in panel E represent ELISA measurements and show a decrease in the levels of Hsp60 in the culture medium of cells treated with 40 nM of doxorubicin
and a marginal increase of the chaperonin in the culture medium of cells treated with 80 nM. Results are representative of three independent experiments. AU, arbitrary unit; UT:
untreated cells. #Different from UT, and 20 nM p < 0.001; *Different from 40, and 80 nM p < 0.05; Different from all other conditions p < 0.01; Different from 20 nM p < 0.05.

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A. Marino Gammazza et al. / Cancer Letters 385 (2017) 75e86

cancer cell, probably as a consequence of hyperacetylation for

example after doxorubicin treatment, could lead to proteotoxic
stress ultimately resulting in the switch of pro-survival signaling to
cell-proliferation arrest. Our results are consistent with previous
data demonstrating that hyperacetylation of Hsp60 was associated
with anticancer activity of geldanamycin in osteosarcoma derived
cells [32]. Hsp60 is one of the proteins that undergo acetylation in
U2OS OS cells with sirtuin 3 (Sirt3) knockout [57]. Treatment with
Histone Deacetylases (HDAC) inhibitors causes hyperacetylation of
Hsp90, Hsp70, and Hsp40 and impairs their functioning. HDAC
inhibitor-mediated deregulation of chaperone function, in turn,
deregulates protein homeostasis and induces protein misfolding
and proteotoxic stress [58,59].
To play a role in DNA damage response, p53 must interact with
the promoter of target genes, an action that can be negatively
affected by the interaction with protein inhibitors, such as Hsp60. In
our control cells, p53 co-immunoprecipitated with Hsp60 and the
complex could be considered as a prerequisite for malignant progression [51,60]. Doxorubicin treatment also caused the decrease of
p53 in the Hsp60/p53 complex favored probably by the Hsp60
lysine (K) acetylation. From a structural point of view, the Hsp60
molecule is composed of various functional modules (and three
structural domains named apical, intermediate, and equatorial)
with distinct roles. If any of these modules is altered by a mutation,
post-translational modication, or by the binding of a specic
chemical compound, its functions may be seriously disrupted. This
might lead to failure of the entire molecule and loss of its biological
functions. For example, the apical domain contains conserved
segments for substrate binding sites presenting lysine residues
[20]. These ndings suggest that stress signals induced by doxorubicin, promote Hsp60 post-translational modications leading to
the disruption of Hsp60/p53 complex and the activation of RS
probably via p53-p21 pathway (Fig. 8), similarly to what has

previously been proposed [61]. In line with these observations, we

demonstrated that doxorubicin treatment increased the level of
p21, a key factor capable of orchestrating cellular senescence [62].
Moreover, the decrease of the intracellular levels of the chaperonin
may lead to a decrease in its extracellular release. Extracellular
chaperones contribute to the intercommunication between
different cells, tissues, and organs, in normal as well as in pathological conditions [18,63].
Extracellular Hsps have been considered to be multifunctional
messengers involved in intercellular signaling [64]. It has been
established that Hsp60 is actively secreted by human tumor cells
and, thus, have potential as useful cancer biomarkers properties
[29,40]. Hsp60 can be released from cells in free, soluble form,
possibly via Golgi participating in extracellular molecular interactions and cell signaling, acting as a paracrine factor when
released into the medium [3,40]. We observed a decrease in the
release of the chaperonin in the culture medium at 40 nM doxorubicin concomitantly with the intracellular decrease of the protein
levels and post-traslational modication. Hsp60 extracellular level
marginally increased at 80 nM doxorubicin reaching the basal level
probably as a result of cell death. A decrease of extracellular Hsp60
could affect the communication between tumor cells, a phenomenon in which Hsp60 would participate and, thereby, modulate the
anti-tumor immuneresponse [65,66].
An overview of the data supports the notion that Hsp60 can
favor carcinogenesis in the cells we studied. Furthermore, our observations shed light on the molecular mechanism by which
doxorubicin acts as antitumor agent, i.e., likely by lowering the
levels of intracellular Hsp60 and possibly also by impairing its
functioning through acetylation. It thus becomes clear that targeting Hsp60 to diminish its levels (or block its functions) must be
included in anti-tumor treatment strategies directed against cancers with elevated intracellular levels of the chaperonin.

Fig. 8. Working hypothesis. In tumor cells there is normal Hsp60 free and Hsp60 bound to p53 (Hsp60/p53 complex). This allows tumor cell growth. Doxorubicin causes acetylation
of Hsp60: it most likely acetylates the free Hsp60 (not bound to p53), but it may also cause acetylation of Hsp60 bound to p53 in the complex Hsp60/p53. The consequences are: (i)
acetylated free Hsp60 cannot bind p53 and the complex Hsp60/p53 does not form; and (ii) acetylation of Hsp60 in the complex Hsp60/p53 causes dissociation of the complex with
liberation of p53 (that goes on to induce replicative senescence via interaction with p21) and acetylated Hsp60. Acetylated Hsp60 is degraded with its consequent diminution inside
the cell, which allows apoptosis to proceed. gH2AX, phosphorylated histone H2AX, considered a biomarker of DNA double-strand breaks.

A. Marino Gammazza et al. / Cancer Letters 385 (2017) 75e86

Author contributions
A.M.G., C.C., and V.D.F. conceived the study and designed the
experiments; A.M.G. wrote the manuscript; A.M.G., R.B., C.C.B, M.G.,
A.D, and M.L. performed experiments and analyzed data; M.W., F.C.
A.D., M.L., G.Z., E.C. de M., and A.J.L.M. contributed to discussions,
data processing and interpretation, and to manuscript writing;
V.D.F., and F.C. provided funding. All authors reviewed the manuscript and approved the nal version submitted.
Acknowledgments
This work was carried out using instruments provided by the
Euro Mediterranean Institute of Science and Technology (IEMEST,
Italy) and funded by the Italian National Operational Programme
for Research and Competitiveness 2007e2013 grant (Project code:
PONa3_00210, European Regional Development Fund). A.J.L.M. and
E.C.de M. were partially supported by IMET; A.J.L.M. and F.C. were
partially supported by IEMEST. This work was done under the
umbrella of the agreement between IEMEST and the Institute of
Marine and Environmental Technology (IMET; USA) signed in
March 2012 (this is IMET contribution number IMET 16-184).
Conicts of interest
None.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.canlet.2016.10.045.
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