verapamil-induced autophagy-like process in colon adenocarcinoma colo ... · short communication...
TRANSCRIPT
Short communication
Verapamil-induced autophagy-like process
in colon adenocarcinoma COLO 205 cells;
the ultrastructural studies
Beata Paj¹k1, El¿bieta Kania1, Barbara Gajkowska1, Arkadiusz Orzechowski1,2
1Electron Microscopy Platform, Mossakowski Medical Research Centre, Polish Academy of Sciences,Pawiñskiego 5, PL 02-105, Warszawa, Poland2Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences(SGGW), Nowoursynowska 159, PL 02-776 Warszawa, Poland
Correspondence: Beata Paj¹k, e-mail: [email protected]
Abstract:
Background: Verapamil (Ver) is a well known, worldwide used drug to correct cardiac arrhythmias. The main Ver target is the
L-type calcium channel. Modulation of calcium homeostasis vaulted Ver into use in medical applications.
Methods: To examine COLO 205 cells morphology after Ver treatment, an electron microscopy technique was used.
Results: This study shows ultrastructural evidence that Ver initiates autophagy-like process in human colon adenocarcinoma COLO
205 cells. TEM photographs revealed the presence of differently developed autophagic vacuoles in response to Ver administration.
Furthermore, extensive ultrastructural cell alterations confirmed that cancer cells died via necrosis or apoptosis, as demonstrated by
ruptured plasma membrane or condensed chromatin, respectively.
Conclusions: It is the evidence that apoptosis resistant COLO 205 cells are overruled by autophagy-like process. Autophagy-like
cell death could be a promising venue to delete cancer cells. Ver appears to be a new potentially effective anticancer compound.
Key words:
verapamil, autophagy, transmission electron microscopy, colon cancer, cell death
Introduction
COLO 205 cells represent colon adenocarcinomas,
the most common cancer type in human population.
These cells are characterized by an extreme resistance
to cell death induction via natural death receptor
ligands (TNF-a, FasL, TRAIL), as well as chemo-
therapeutic drugs [10]. It was shown that one of the
mechanisms important for resistance to apoptosis re-
lies on changes in calcium signaling in neoplastic
cells [2]. Encouraged by our previous observations
[11], concerning the proapoptotic effects of ethylene
glycol tetraacetic acid (EGTA) and ethylenediamine-
tetraacetic acid (EDTA) (calcium ions chelators), we
investigated the possible mechanisms of calcium sig-
naling in COLO 205 cells. Among several tested
compounds was verapamil, a well-known L-type cal-
cium channel blocker. L-type calcium channels are
expressed in skeletal and heart muscle tissue. Wang et
al. [16] reported that in HT-29, Caco-2 and T84 colon
cancer cells, as well as colon cancer biopsies, the
Pharmacological Reports, 2012, 64, 991�996 991
Pharmacological Reports2012, 64, 991�996ISSN 1734-1140
Copyright © 2012by Institute of PharmacologyPolish Academy of Sciences
L-type calcium channel is upregulated. According to
Loza et al. [6], increased mRNA of the calcium chan-
nel may correlate with enhanced proliferation induced
by epidermal growth factor (EGF). Apart from L-type
channel blockage, Ver has been reported to exert other
cellular responses, such as inhibition of MDR1/P-gp
activity [15] and increased reactive oxygen species
(ROS) production leading to oxidative stress [17].
Oxidative stress results in both autophagy activation
and mitochondrial dysfunction [17]. Furthermore, mi-
tochondrial ROS can further increase calcium release
from the endoplasmic reticulum (ER), thereby caus-
ing protein misfolding. ROS production and protein
misfolding together activate calcium-dependent ki-
nases such as JNK eventually leading to cell death
[7]. On the other hand, changes in calcium homeosta-
sis may foster ER stress triggering autophagy by the
ER-activated autophagy pathway. The latter could be
mediated by limited unfolded protein response (UPR)
signals involving PERK and/or IRE1 as well as
UPR-independent mechanism such as calcium leak-
age (JNK-AKT/mTOR signaling) [4]. Meister et al.
[8] demonstrated that Ver enhanced IRE1- and
PERK-mediated pathways, further activated the UPR
and might trigger an autophagic, caspase-independent
cell death. According to latest studies published by
Kuo et al. [5], the Ver-induced ER stress is deter-
mined by increase in GRP78 chaperone protein ex-
pression and by upregulation of a heat shock protein
70 kDa family member that prevents the unfolded
protein from further transit and secretion [3].
Autophagy, an intracellular degradation system of
cytoplasmic contents and organelles, is required for
normal turnover of cellular components during starva-
tion in eukaryotic cells and is characterized by dis-
torted mitochondria. The autophagosomes, with two
or more membranes enclosed vesicles, engulf various
cellular constituents that fuse with lysosomes for deg-
radation and recycling [12]. Besides promoting cell
survival, autophagy can trigger caspase-independent
cell death [12]. Furthermore, various observations
show that autophagy contributes to cell death induc-
tion in apoptosis competent cells, while it becomes
the major cell death-inducing pathway in apoptosis
deficient cells [9].
We present the first ultrastructural evidence that in
colon adenocarcinoma COLO 205 cells, Ver treat-
ment induces massive autophagy-like process leading
to necrotic cells death.
Materials and Methods
Cell culture
Human colon adenocarcinoma cell line COLO 205
was purchased from American Type Culture Collec-
tion (ATCC). Cells were maintained in the exponen-
tial phase of growth in growth medium [GM, 100 ml/l
Fetal Bovine Serum (FBS)/Dulbecco’s modified
Eagle medium (DMEM) with Glutamax and antibi-
otic-antimycotic mixture (penicillin G sodium salt
50 IU/ml, streptomycin sulfate 50 µg/ml, gentamycin
sulfate 20 µg/ml, fungizone – amphotericin B 1 µg/ml)].
The cells were grown at 37ºC, in a controlled, humidi-
fied 50 ml/l CO2 atmosphere, on 96-well flat-bottomed
or tissue culture Petri dishes (100 mm diameter, BD
Biosciences Pharmingen, San Jose, CA USA).
Experimental procedure
During propagation, the medium was changed every
other day until cultures reached 100% confluence.
One-day (24 h) prior to the experiment, confluent
cells (cells of the same cell density fully covering the
surface of the dish) were switched to post-mitotic
status to induce quiescence (withdrawal from cell cy-
cle) by replacing GM with 20 g/l BSA/DMEM desig-
nated as a control medium (CTRL). In the above-
mentioned conditions divisions of COLO 205 cell
have been completed. Next, cells were treated with
Ver (Sigma-Aldrich Chemical Co., St. Louis, MO,
USA) [50, 100 or 200 µM] for 24 or 48 h.
Ultrastructural studies
Cells were fixed in 2.5% paraformaldehyde and 2%
glutaraldehyde in 0.1 M sodium cacodylate buffer (pH
7.4) for 2 h at 4ºC. Cells were washed with the same
buffer and post-fixed with 1% OsO4 in 0.1 M sodium
cacodylate buffer for 1 h. Cells were dehydrated in
a graded ethanol series, and embedded in Epon 812.
Ultrathin sections were mounted on copper grids, air-
dried, and stained for 10 min with 4.7% uranyl acetate
and for 2 min with lead citrate. The sections were ex-
amined and photographed with a JEOL JEM 1011
electron microscope (Jeol, Tokyo, Japan).
992 Pharmacological Reports, 2012, 64, 991�996
Results
COLO 205 cells were treated with different concentra-
tions of Ver [50, 100, 200 µM] for 24 h. After treat-
ment, cell morphology was examined using transmis-
sion electron microscopy (TEM). The most spectacu-
lar morphological effects were observed when 100
µM Ver concentration was used; however, the pro-
longed treatment [up to 48 h] with lower Ver doses
also affected cell morphology (data not shown). As
shown on Fig. 1B, 1C, Ver-treated [100 µM, 24 h]
COLO 205 cells ultrastructure was drastically
changed as compared to control cells (Fig. 1A). Con-
trol cells showed electron dense cytoplasm, with nu-
merous well developed organelles, such as mitochon-
dria (M), nuclei (N), endosomes (E). Furthermore, the
microvilli around the cell were present (Fig. 1A). Sur-
prisingly, Ver treatment (Fig. 1B, 1C) induced endo-
plasmic reticulum (ER) enlargement, as well as loss
Pharmacological Reports, 2012, 64, 991�996 993
Verapamil and COLO 205 cell deathBeata Paj¹k et al.
Fig. 1. Verapamil induces an auto-phagy process in COLO 205 cells.Transmission electron microscopyanalysis of ultrathin sections of control(A) COLO 205 cells or cells treatedwith 100 µM verapamil for 24 h (B, C).Verapamil-treated cells show dilatedendoplasmic reticulum (ER). Further,verapamil-treated cells contain numer-ous autophagosomes (AU) and auto-phagolysosomes (AL), as well as mul-tivesicular bodies (MVB). Other cellu-lar organelles, such as nuclei (N), mi-tochondria (M), Golgi apparatus (GA),endosomes (E) and lysosomes (L) aremarked
Fig. 2. Series of transmission electronmicroscopy photographs showingautophagic vacuoles � double mem-brane autophagosomes (AU) (A, B),phagofores (PH) (C, D), autophagoly-sosomes (AL) (B, C) and multilamellarbodies (MLB) (D) in verapamil-treated(100 µM, 24 h) COLO 205 cells. Othercellular organelles, such as nuclei (N)and multivesicular bodies (MVB) aremarked
of cellular microvilli. The reduced number of mito-
chondria (M) is also noticeable in comparison to con-
trol cells. Moreover, the Ver-treated cells revealed the
presence of huge electron-permeable areas without
any cellular organelles. In the residue of the cyto-
plasm the phagofores (PH), autophagosomes (AU)
and autophagolysosomes (AL) were found (Fig. 1B,
1C, Fig. 2). Figure 2 shows the diversity of auto-
phagic hallmarks after Ver treatment. Both, the forma-
tion of phagofores (PH) via the elongation of cyto-
plasmic isolation membrane and the first step of auto-
phagic vacuole formation, are visible on Fig. 2C and
2D. Furthermore, numerous multivesicular bodies
(MVB), as well as double-membrane autophago-
somes (precursors to AL) are visible on Fig. 2B and
2C. The mature AL degraded the vesicle content,
leading to electron-permeable vesicles formation
(Fig. 2). If selective resistance to lysosomal degrada-
tion within the autophagic vacuole occurred, mem-
brane lamella called multilamellar bodies (MLBs)
were formed (Fig. 2D). Extensive macroautophagy
process results in cell death induction. According to
TEM analysis, necrosis is likely to occur as mani-
fested by the loss of cellular membrane continuity. It
was predominantly evident at 100 µM of Ver concen-
tration (24 h) (Fig. 3A, 3B). However, some apoptotic
cells were also observed (Fig 3C, 3D). Apoptotic and
necrotic cells could be easily distinguished, as shown
on Figure 3D. Apoptotic cells (Fig. 3D, left side) are
featured by condensed electron-dense chromatin and
cytoplasm, as well as intact cellular membrane. In
contrast, in necrotic cells (Fig. 3D, right side) chro-
matin was scattered and cell nuclei and cytoplasm
were of normal appearance. The cellular content is
shown to be released to the microenvironment via
damaged plasma membrane.
Discussion
Ver is a frequently prescribed calcium channel block-
er used in the treatment of hypertension, angina pec-
toris and cardiac arrhythmias. There are several re-
ports indicating other cellular targets for Ver actions.
In our studies, Ver treatment of colon adenocarcinoma
COLO 205 cells led to massive macroautophagy. As
far as we know, it is the first ultrastructural evidence
showing direct autophagy-like process induction in
response to Ver treatment in colon cancer. Previously,
Meister et al. [8] reported the synergistic effect of Ver
[70 µM, 16 h] to bortezomib-induced endoplasmic re-
ticulum stress, leading to ROS formation and auto-
phagic-like processes in multiple myeloma JK-6L
cells. In JK-6L cells, Ver alone had no effect; addition
of bortezomib to Ver led to autophagosome forma-
994 Pharmacological Reports, 2012, 64, 991�996
Fig. 3. Verapamil treatment (100 µM,24 h) induces necrotic and apoptoticCOLO 205 cell death. Verapamil treat-ment resulted in necrosis inductionmanifested by loss of plasma mem-brane integrity indicated by arrows(A�D). On the contrary, apoptotic cells(C � upper right, D � left) characterizeintact cellular membrane, but con-densed, electron-dense chromatin andcytoplasm are evident. Cellular organ-elles (nuclei � N, mitochondria � M), aswell as autophagic vacuoles (AL) areindicated
tion. Interestingly, Meister et al. [8] observed the pres-
ence of distorted ER after bortezomib treatment
[10 nM, 16 h], cell morphology similar to that visible
on Figure 1B, 1C and 2C in our experiment. The
authors proved that changes in ER ultrastructure and
its colossal enlargement are the consequences of
UPR. Furthermore, western blot analysis demon-
strated the upregulation of UPR-induced chaperones
(Hsp70, CHOP, sXBP-1) in the presence of Ver. Thus,
with regard to COLO 205 cells, we assume that di-
lated ER after Ver action could be mediated via
analogical signaling pathway. Detailed molecular bi-
ology analysis is ongoing in our lab.
According to Meister et al. [8] study, regardless of
described biological effects, Ver neither reduced cells
viability, nor activated caspases if used separately.
Cytotoxic effects of Ver were reported by Suwalsky et
al. [13], who treated erythrocytes with various Ver
concentrations [1 nM – 500 µM, 1, 6, 24 or 48 h].
However, Ver-dependent cytotoxicity was observed
ultimately at 500 µM Ver concentration for at least 48
h. In our study, TEM analysis clearly demonstrate the
presence of necrotic and apoptotic cells in response to
Ver at 100 µM. MTT assay also confirmed the de-
creased percentage of viable cells (data not shown).
Molecular pathways responsible for Ver-dependent
autophagy-like process are under investigation. Our
assumption point to the activity of the calpains, as
well as UPR pathway. In contrary to Meister et al. [8],
we do not expect the involvement of MDR1/P-gp-
dependent pathway in Ver effect on COLO 205 cells.
MDR-1/P-gp is well known target for Ver, however, it
was demonstrated by Ugocsai et al. [14] that COLO
205 cells do not express MDR1/P-gp protein.
Additionall issue to be addressed is the Ver de-
pendent downregulation of mitochondrial counts in
comparison to untreated control cells (Fig. 1). Meister
et al. [8] established ROS-induced damage to mito-
chondria, demonstrated by fall in transmembrane po-
tential of JK-6L cells in response to Ver treatment. In
COLO 205 cells, Ver also induced morphological
changes in mitochondria, such as dilation (Fig. 1C)
and loss of cristae (Fig. 2A). Interestingly, only few
mitochondria could be found in the cytoplasm of
Ver-treated cells. Bearing in mind results published by
Meister et al. [8], we suppose that damaged mitochon-
dria are degraded via mitophagy process, a specific
form of macroautophagy.
Finally, the main cellular target for Ver – L-type
calcium channel, should also be seriously considered
as a mediator of observed effects. Overexpression of
L-type calcium channel was reported previously in
colon cancer cells [16]. Thus, the importance of cal-
cium ion homeostasis and its cellular trafficking, as
well as its connection with autophagy process, is not
ignored in future studies.
Herein, we present the ultrastructural evidence of
Ver-dependent autophagic like process in COLO 205
cells leading to cell death. We believe that further re-
search is needed to elucidate the exact mechanism
how Ver triggers these effects. It should be stressed
that Ver is already a known, commonly used, well-
tolerated drug. Peak plasma concentrations are
reached between 1 and 2 h after Ver oral administra-
tion. Chronic oral administration of 120 mg of Ver
every 6 h resulted in plasma levels of Ver ranging
from 125 to 400 ng/ml, with higher values occasion-
ally reported [1]. Reported plasma concentration is
significantly lower than used in above-mentioned
short-time experiments. Further analysis with chronic
administration of low doses of Ver (400 ng/ml) will be
conducted to verify the possible application of Ver for
cancer patients. Presented data seem to be a promis-
ing perspective for more efficient anti-cancer therapy.
Acknowledgments:
Support for this work was provided by grant No. NN 401 031 538from the Ministry of Science and Higher Education in Poland.Beata Paj¹k is granted by fellowship from the Ministry of Scienceand Higher Education �For Young Outstanding Scientists� in Poland.
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Received: October 20, 2011; in the revised form: January 27, 2012;accepted: April 2, 2012.
996 Pharmacological Reports, 2012, 64, 991�996