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: bepaj@wp.pl

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.

References:

1. Borges NC, Mendes GD, Barrientos-Astigarraga RE,

Galvinas P, Oliveira CH, de Nucci G: Varapamil quantifica-

tion in human plasma by liquid chromatography coupled to

tandem mass spectrometry. An application for bioequiva-

lence study. J Chromatogr B, 2005, 827, 165–172.

2. Capiod T, Shuba Y, Skryma R, Prevarskaya N: Calcium

signalling and cancer growth. Subcell Biochem, 2007,

45, 405–427.

3. Chen X, Shen J, Prywes R: The luminal domain of ATF6

senses endoplasmic reticulum (ER) stress and causes

translocation of ATF6 from ER to the Golgi. J Biol

Chem, 2002, 277, 13045–13053.

4. Ding WX, Yin XM: Sorting, recognition and activation

of the misfolded protein degradation pathways through

macroautophagy and the proteasome. Autophagy, 2008,

16, 141–150.

5. Kuo TC, Chiang PC, Yu CC, Nakagawa-Goto K, Bastow

KF, Lee KH: A unique P-glycoprotein interacting agent

displays anticancer activity against hepatocellular carci-

Pharmacological Reports, 2012, 64, 991�996 995

Verapamil and COLO 205 cell deathBeata Paj¹k et al.

noma through inhibition of GRP78 and mTOR pathways.

Biochem Pharmacol, 2011, 81, 1136–1144.

6. Loza J, Carpio L, Lawless G, Marzec N, Dziak RD:

Role of extracellular calcium influx in EGF-induced

osteoblastic cell proliferation. Bone, 1995, 16, Suppl 4,

314S–347S.

7. Malhotra JD, Kaufman RJ: Endoplasmic reticulum stress

and oxidative stress: a vicious cycle or a double-edged

sword? Antioxid Redox Signal, 2007, 9, 2277–2293.

8. Meister S, Frey B, Lang VR, Gaipl US, Schett G,

Schlotzer-Schrehardt U, Voll RE: Calcium channel

blocker verapamil enhances endoplasmic reticulum

stress and cell death induced by proteasome inhibition in

myeloma cells. Neoplasia, 2010, 12, 550–561.

9. Notte A, Leclere L, Michiels C: Autophagy as a madia-

tor of chemotherapy-induced cell death in cancer. Bio-

chem Pharmacol, 2011, 82, 427–434.

10. Paj¹k B, Orzechowski A: Complex forms of immune in-

sufficiency to some cytokines (TNF-alpha, interferons)

in colorectal cancer as exemplified by the COLO 205

cell line. Mechanisms of resistance with special refer-

ence to signaling proteins and cytokines (Polish). Pos-

tepy Hig Med Dosw, 2004, 58, 428–457.

11. Pajak B, Orzechowski A: Ethylenediaminetetraacetic

acid affects subcellular expression of clusterin protein in

human colon adenocarcinoma COLO 205 cell line.

Anti-Cancer Drugs, 2007, 18, 55–63.

12. Sun Y, Peng ZL: Programmed cell death and cancer.

Postgrad Med J, 2009, 85, 134–140.

13. Suwalsky M, Munoz M, Mennickent S, Sotomayor CP,

Bolognin S, Zatta P: Structural effects of verpamil on

cell membranes and molecular models. J Chil Chem Soc,

2010, 55, 1–4.

14. Ugocsai K, Varga A, Molnar P, Artus S, Monar J: Effects

of selected flavonoids and carotenoids on drug accumu-

lation and apoptosis induction in multidrug-resistant co-

lon cancer cells expressing MDR1/LRP. In Vivo, 2005,

19, 433–438.

15. Wagner-Souza K, Diamond HR, Ornellas MH, Gomes

BE, Almeida-Oliveira A, Abdelhay E, Bouzas LF et al.:

Rhodamine 123 efflux in human subpopulations of he-

matopoietic stem cells: comparison between bone mar-

row, umbilical cord blood and mobilized peripheral

blood CD34+ cells. Int J Mol Med, 2008, 22, 237–242.

16. Wang XT, Nagaba Y, Cross HS, Wrba F, Zhang L,

Guggino SE: The mRNA of L-type calcium channel ele-

vated in colon cancer: protein distribution in normal and

cancerous colon. Am J Pathol, 2000, 157, 1549–1562.

17. Zhang K, Kaufman RJ: From endoplasmic-reticulum

stress to the inflammatory response. Nature, 2008, 454,

455–462.

Received: October 20, 2011; in the revised form: January 27, 2012;accepted: April 2, 2012.

996 Pharmacological Reports, 2012, 64, 991�996