Apoptotic response of uveal melanoma cells upon treatment

with chelidonine, sanguinarine and chelerythrine

Adam Kemeny-Bekea, Janos Aradib, Judit Damjanovicha, Zoltan Beckc,

Andrea Facskoa, Andras Bertaa, Andrea Bodnard,*

aDepartment of Ophthalmology, Research Center for Molecular Medicine, Medical and Health Science Center,

University of Debrecen, Nagyerdei krt. 98, H-4012 Debrecen, HungarybDepartment of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, Medical and Health Science Center,

University of Debrecen, Nagyerdei krt. 98, H-4012 Debrecen, HungarycDepartment of Microbiology, Research Center for Molecular Medicine, Medical and Health Science Center,

University of Debrecen, Nagyerdei krt. 98, H-4012 Debrecen, HungarydCell Biophysics Research Group of the Hungarian Academy of Sciences, Research Center for Molecular Medicine,

Medical and Health Science Center, University of Debrecen, Nagyerdei krt. 98, H-4012 Debrecen, Hungary

Received 12 March 2005; received in revised form 8 May 2005; accepted 22 May 2005

Abstract

The benzophenanthridine alkaloids sanguinarine, chelerythrine and chelidonine were reported previously to provoke cell

death in a variety of tumor cells suggesting their potential application as anticancer agents. Here we tested their effects on a

primary human uveal melanoma cell line, OCM-1. Flow cytometric analysis of annexin V binding/PI exclusion and DNA

fragmentation disclosed that all these alkaloids could induce apoptosis in OCM-1 cells. Moreover, necrotic cell death was also

observed upon alkaloid treatment. As it was also evidenced by light microscopic inspection of cellular morphology, chelidonine

primarily caused apoptosis, while sanguinarine and chelerythrine were effective via a so-termed bimodal cell death (apoptosis

and primary necrosis). The relative efficiencies of the two modes depended on the applied dose. This study is the first

implication for the possible use of these alkaloids in the therapy of uveal melanomas, for which no really efficient therapeutic

regimen is available so far.

q 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Uveal melanoma; Benzophenanthridine alkaloids; Cell death; Annexin V; DNA fragmentation; Flow cytometry

1. Introduction

Uveal melanomas (UM) are the most common

primary intraocular tumors in adults with a high

0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights re

doi:10.1016/j.canlet.2005.05.037

* Corresponding author. Tel./fax: C36 52 412 623.

E-mail address: bodnar@jaguar.dote.hu (A. Bodnar).

mortality rate due to frequent liver metastases [1].

Since UM was found to be highly resistant to

chemotherapeutic drugs used to date [2], at present

no really efficient chemotherapy regimen is available

for either intraocular or metastatic uveal melanomas.

Development of successful immune therapies is

hampered by the fact, that the eye, at least to a certain

Cancer Letters 237 (2006) 67–75

www.elsevier.com/locate/canlet

served.

A. Kemeny-Beke et al. / Cancer Letters 237 (2006) 67–7568

extent, is an immunologically privileged site, where

both the adaptive and the innate immune responses are

suppressed [3]. In addition UM cells exert lympho-

cyte-inhibitory actions endowed by their ocular

microenvironment [4]. Consequently, there is an

urgent call for the development of new approaches

to treat uveal melanomas.

Identification of melanoma-associated antigens on

fresh and cultured UM cells indicated that in select

circumstances these tumors can also be immunogenic

[5,6], which makes them an attractive target for

dendritic cell (DC)-based immune therapies. Indeed,

it was shown recently that DCs pulsed with apoptotic

UM cells were able to stimulate proliferative and

cytolitic T cell responses [7]. It is suggested that DCs

produced in this way may either augment the

efficiency of apoptosis-inducing anticancer drugs

in vivo or can be used to generate effector T cells

in vitro for adoptive transfer therapy [7]. Therefore,

screening for new reagents with a high degree of

apoptotic potential against UM cells is not only

important for chemotherapeutic trials but it also has a

key significance in the rational design of DC-based

antitumor strategies.

In the last few years clinical trials using plant-

derived drugs for the prevention and/or treatment of

tumors became increasingly widespread in cancer

therapy [8,9]. Benzophenanthridine alkaloids cheler-

ythrine and sanguinarine (pseudochelerythrine) of

Chelidonium majus, L. (and other Papaveraceae

plants) were shown to mediate a broad variety of

biological activities, among others anti-microbial and

anti-inflammatory effects [10–12]. Most notably both

of them were reported to exert cell growth-inhibitory

effect via the induction of apoptosis in numerous

cancer cells [13–17], suggesting their potential

application as proapoptotic drugs in cancer therapy.

Furthermore, they were effective against certain

tumors that are otherwise resistant to standard

therapies [18,19]. Although up to now only a few

reports are available on the biological actions of

chelidonine, the major alkaloid component of Cheli-

donium majus, recent data indicate that this benzo-

phenanthridine alkaloid could also induce apoptosis in

some transformed or malignant cell lines [20].

Since it has not been tested so far on UM cells,

herein we investigated the effect of sanguinarine,

chelerythrine and chelidonine on a primary, human

uveal melanoma cell line, OCM-1. Apoptotic poten-

tial of benzophenanthridine alkaloids was determined

by flow cytometry, using two assays probing different

characteristics of apoptotic cells. Combined analysis

of PI exclusion/annexin V binding (i.e. probing

plasma membrane integrity and phosphatidylserine

exposure) and DNA fragmentation revealed that albeit

in different extent, all alkaloids induced apoptosis in

OCM-1 cells. Moreover, alkaloid treatment also

resulted in necrotic cell death. As it was supported

by light microscopic inspection of cellular mor-

phology, chelidonine predominantly induced apopto-

sis, whereas sanguinarine and chelerythrine were

effective against OCM-1 cells via bimodal cell death

(i.e. apoptosis and primary necrosis). Whereas the

efficiency of necrotic cell death monotonously

increased throughout the applied concentration

range, the amount of apoptotic cells exhibited a

‘biphasic’ behavior for these alkaloids. Our data

implicate the possible application of benzophenan-

thridine alkaloids investigated in this study as

anticancer reagents in the chemotherapy or DC-

based immune therapy of uveal melanomas.

2. Materials and methods

2.1. Alkaloids

Benzophenanthridine alkaloids were purchased

from Sigma Chemical Co. (St Louis, OR, USA).

Chelidonine was dissolved in dimethylsulfoxide

(DMSO), while stock solutions of chelerythrine

chloride and sanguinarine chloride were prepared in

DMSO/water (1:2).

2.2. Cell culture

OCM-1 human primary uveal melanoma cell line

(kindly provided by Dr H. M. H. Hurks, Department

of Ophthalmology, Leiden University Medical Cen-

ter, Leiden, The Netherlands) was cultured in RPMI

1640 medium supplemented with 10% FCS,

L-glutamine and gentamycin at 37 8C, in a humidified

5% CO2 atmosphere. Cells were subcultured twice or

three times a week using the standard trypsinization

procedure.

A. Kemeny-Beke et al. / Cancer Letters 237 (2006) 67–75 69

2.3. Treatment with benzophenanthridine alkaloids

If otherwise not indicated cells were seeded in

24-well tissue culture plates (w1!105 cells/well in

500 ml medium). Cells grown to w80–90% con-

fluence were treated with varying concentrations of

the alkaloids (1, 4 and 8 mg/ml in 500 ml medium/

well) and cultured for 4, 24 or 48 h afterwards. Taking

into account the similar molecular weight of the tested

alkaloids, the applied doses meant nearly identical

molar concentrations (2.6–2.7, 10.4–10.8 and 20.8–

21.8 mM, respectively). Before use aliquots of stock

solutions were serially diluted with the appropriate

solvents so that the final concentration of DMSO was

the same in the related samples. Control cells were

treated with the same amount of DMSO and were kept

at the same experimental conditions. At the indicated

time points, cells were trypsinized, washed with PBS

and prepared for the DNA fragmentation or the

annexin V/PI assays as described below. Due to

alkaloid treatment a fraction of the cells detached

from the surface of the culture plates and floated in the

medium. In order to avoid the loss of floating cells,

they were collected before trypsinization and there-

after were pooled with the trypsinized ones.

2.4. DNA fragmentation assay

DNA content of cells was determined by flow

cytometry as described [21,22]. Briefly, cells were

centrifuged at 200!g and the pellet was gently

suspended in 0.5 ml hypotonic fluorochrome solution

(50 mg/ml PI in 0.1% sodium citrate plus 0.1% Triton

X-100) and stored overnight at 4 8C before flow

cytometry. The samples were applied in a FACScan

flow cytometer (Becton Dickinson, Franklin Lakes,

NJ) using the FL-2 channel to detect the PI

fluorescence. Data were analyzed by the softwares

WinMDI 2.8 (Copyrightq 1993–1998, Joseph Trot-

ter) or FLEX [23]. Apoptotic cells were recognized by

the diminished amount of their DNA, i.e. the presence

of the characteristic sub-G1 peak on the DNA-content

(PI-intensity) frequency histogram.

2.5. Analysis of annexin V-FITC/PI staining of cells

Apoptotic cells were differentiated from viable or

necrotic ones by combined application of annexin

V-FITC and PI, using the Annexin V-FITC Apoptosis

Detection Kit (Sigma, St Louis, OR). Briefly, cells

were centrifuged and the cell pellet was suspended in

the binding buffer (10 mM HEPES/NaOH, 0.14 M

NaCl, 2.5 mM CaCl2, pH 7.5) at a concentration of

approximately 1!106 cells/ml. Samples were incu-

bated with 0.5 mg/ml annexin V-FITC and 2 mg/ml PI

for exactly 10 min at room temperature and then were

measured on a FACScan flow cytometer. Annexin

V-FITC and PI fluorescence was detected in the FL-1

(green) and FL-2 (red) channels, respectively, after

correction to the spectral overlap between the two

channels. Data were analyzed by the softwares

WinMDI 2.8 or FLEX (see above). Apoptotic and

necrotic cells were distinguished on the basis of

annexin V-FITC reactivity and PI exclusion. Live,

non-apoptotic cells were not stained with any of the

reagents. Apoptotic cells exhibited intense green

(FITC) and low or intermediate red (PI) fluorescence

(early and late stages of apoptosis, respectively).

Permeability of late apoptotic cells for PI is the

consequence of the compromised integrity of their

plasma membrane. Necrotic cells were stainable with

both reagents and exhibited strong green and red

fluorescence [24,25].

2.6. Analysis of cell morphology

In order to study the effect of alkaloids on cell

morphology, cells were seeded in 8-chamber slides.

Cells grown to w80–90% confluence were treated

with the alkaloids, incubated for 4 h and then

examined under light microscopy using a Zeiss

LSM 510 confocal laser scanning microscope.

2.7. MTT assay

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-

nyltetrazolium bromide) assay was performed as

described earlier [26] and specified by the manual of

American Type Culture Collection (ATCC). Briefly,

cells seeded in 48-well plates (w0.6!105 cells/well

in 200 ml medium) were cultured for 15 h and then

treated with the indicated doses of alkaloids or the

solvent alone. After 24-h incubation the cultures

were mixed with 20 ml of MTT solution and incubated

for further 3 h, then treated with 200 ml acidic

iso-propanol solution, suspended and 200 ml of this

A. Kemeny-Beke et al. / Cancer Letters 237 (2006) 67–7570

homogenous solution was transferred into 96-well

plates and measured with ELISA reader.

Fig. 1. Effect of chelidonine, sanguinarine and chelerythrine on the

growth/survival of OCM-1 uveal melanoma cells. Viability of cells

exposed to increasing concentrations of the alkaloids for 24 h was

determined by the MTT assay as detailed in Section 2. The data are

expressed as the percentage of viable cells and display the mean (G

SEM) values of a representative experiment where each treatment

was performed in 3 wells.

3. Results and discussion

3.1. Differential effects of chelidonine versus

sanguinarine and chelerythrine on the

growth/survival of OCM-1 cells

Benzophenanthridine alkaloids used in this study

were previously demonstrated to inhibit the growth of

various cancer cells [11,13,15–17,20]. Therefore, by

using the colorimetric MTT assay we tested whether

they also impart similar effect in OCM-1 uveal

melanoma cells. As it is shown in Fig. 1, 24-h

treatment with sanguinarine and chelerythrine

resulted in a potent and dose-dependent decrease in

the viability of OCM-1 cells. At the same time

chelidonine affected cell viability only slightly and in

the applied concentration range its effect did not

exhibit significant dose-dependence. Differential anti-

proliferative response for chelidonine versus sangui-

narine and chelerythrine was also reported for other

cell types [11].

3.2. Morphological changes upon alkaloid treatment

of OCM-1 cells

Although it proved to be only a weak inhibitor of

cell growth (Fig. 1), light microscopic inspection of

cellular morphology revealed that even a 4-h

treatment with chelidonine could induce apoptosis

in OCM-1 cells. Compared to the vehicle-treated

control sample containing adherent cells (Fig. 2A), a

fraction of chelidonine-treated cells detached from

the surface of the culture plate and many of them

also manifested plasma membrane blebbing, a

characteristic feature of apoptosis (Fig. 2D). Light

microscopy also demonstrated dose-dependent

effect of sanguinarine and chelerythrine on cell

death morphology. Cells treated with high doses

(8 mg/ml) of sanguinarine or chelerythrine mainly

exhibited cell swelling, a marker of early stages of

necrosis (Fig. 2B and C). Lowering the concentration

the amount of necrotic cells decreased and we could

also observe cells with apoptotic morphology. At the

lowest concentration (w0.5 mg/ml) most of the cells

had normal morphology similar to that of the control

cells (images not shown). These observations hinted

at the existence of two alternative mechanisms

(apoptosis and necrosis) by which sanguinarine and

chelerythrine kills uveal melanoma cells.

3.3. Quantitative analysis of apoptosis induced

by benzophenanthridine alkaloids in OCM-1 cells

Morphological changes detected by light

microscopy indicated that benzophenanthridine alka-

loids used in this study could induce apoptosis in

OCM-1 uveal melanoma cells. In order to quantify the

apoptotic potential of these alkaloids against OCM-1

cells, two methods exploring different attributes of

apoptotic cells were applied.

3.3.1. DNA fragmentation analysis

One of the hallmarks of apoptosis is the appearance

of fragmented, low molecular weight DNA, which can

be detected by flow cytometry. Hence we first

evaluated the induction of apoptosis via the capability

of the alkaloids to cause DNA degradation in OCM-1

cells. Whereas neither chelidonine nor sanguinarine

caused significant DNA degradation after 4 h, the

fraction of cells with fragmented DNA was enhanced

considerably upon 24-h incubation with these alka-

loids (Figs. 3A and B). Chelerythrine was capable to

Fig. 2. Effect of benzophenanthridine alkaloids on the morphology of OCM-1 uveal melanoma cells as assessed by light microscopy. Cells

treated with vehicle (A) or 8 mg/ml sanguinarine (B), chelerythrine (C) or chelidonine (D) were cultured for 4 hrs and then examined on a Zeiss

LSM 510 confocal laser scanning microscope. (Image size: 70!70 mm).

A. Kemeny-Beke et al. / Cancer Letters 237 (2006) 67–75 71

induce significant fragmentation even after 4 h

(Fig. 3C) and the amount of apoptotic cells increased

further upon longer treatment. In the case of

chelidonine the evoked response did not show notable

dose-dependence within the applied concentration

range (Fig. 3A). At the same time the effect of

sanguinarine and chelerythrine exhibited a ‘biphasic’

pattern (Figs. 3B and C).

3.3.2. Analysis of annexin V-FITC/PI staining

Simultaneously with the DNA fragmentation assay

we also prepared samples which were stained with

annexin V-FITC and PI and then were observed by

flow cytometry. The ability of cells to bind annexin V,

i.e. the exposure of phosphatidylserine on the outer

surface of the plasma membrane, is another specific

marker for apoptosis. Moreover, contrary to the DNA

degradation test, dual staining of cells with fluor-

ophore-tagged annexin V and a plasma membrane

integrity probe (e.g. PI) reveals not only apoptotic

cells, but necrotic and live ones could also be

distinguished. It should be noted, that the absolute

amount of apoptotic cells determined by the two

assays are not necessarily equal to each other. Beside

the fact, that distinct cellular responses are detected,

this could also be caused by the different sample

preparation and sample processing (measurement,

analysis) procedures.

Figs. 4A–C show the fraction of apoptotic cells after

4- and 24-h incubations with the benzophenanthridine

Fig. 3. DNA fragmentation induced by benzophenanthridine alkaloids in OCM-1 uveal melanoma cells as assessed by flow cytometry (see

Section 2). Cells were treated with vehicle or specified doses of chelidonine (A), sanguinarine (B) or chelerythrine (C) and cultured for 4 or 24 h

afterwards (white and black bars, respectively). Data are expressed as percentages of sub-G1 cells and represent the means (GSD) of at least

three independent experiments.

A. Kemeny-Beke et al. / Cancer Letters 237 (2006) 67–7572

alkaloids. Similar to the DNA degradation analysis, no

significant apoptosis was observed after 4-h culturing

of cells with chelidonine (Fig. 4A). Prolonging the

incubation time increased dramatically the amount of

apoptotic cells, which also exhibited some dose-

dependence not detected by the DNA fragmentation

assay (Figs. 3A and 4A). While it was hardly

Fig. 4. Induction of apoptotic (A–C) and necrotic (D–F) cell death by benzo

FITC binding assay (for the details see Section 2). Cells were cultured for 4

of chelidonine (A, D), sanguinarine (B, E) or chelerythrine (C, F). Th

experiments.

detectable by the other method (Fig. 3B), annexin

V-FITC/PI staining of OCM-1 cells provided clear

evidence, that within the tested concentration range

sanguinarine could induce remarkable apoptosis even

after 4 h (Fig. 4B). This is in a good accordance with a

recent finding indicating that sanguinarine stimulates a

rapid apoptotic response (within a few hours) via an

phenanthridine alkaloids as revealed by the PI exclusion/annexin V-

or 24 h (white and black bars, respectively) with the indicated doses

e data represent the means (GSD) of at least three independent

A. Kemeny-Beke et al. / Cancer Letters 237 (2006) 67–75 73

early and severe glutathione depletion of cells [27].

‘Biphasic’ pattern of sanguinarine-mediated apoptotic

response was also evidenced by these experiments

(Fig. 4B). Although the amount of apoptotic cells

exhibited opposite time-dependence as compared to

the DNA fragmentation test, otherwise the results of

the two types of experiments were similar for

chelerythrine (Figs. 3C and 4C).

Despite the observed differences the two assays

unequivocally demonstrated apoptotic potential of

benzophenanthridine alkaloids against OCM-1 uveal

melanoma cells and revealed differential dose-

dependence of the evoked responses.

3.4. Induction of necrotic cell death in OCM-1 cells

by benzophenanthridine alkaloids

Analysis of annexin V-FITC/PI staining disclosed,

that benzophenanthridine alkaloids of this study also

induced a dose-dependent necrosis of OCM-1 cells

(Figs. 4D–F). This effect was the less pronounced for

chelidonine (Fig. 4D), while sanguinarine and

chelerythrine were more efficient (Figs. 4E and F).

Even a 4-h treatment with sanguinarine or cheler-

ythrine could induce considerable necrosis, whereas

chelidonine raised the fraction of necrotic cells only

slightly (it did not exceed 20–25% even after a 48-h

treatment (the basal level was w10%)). It appears that

these remarkable differences in the extent of necrotic

response induced by chelidonine versus sanguinarine

or chelerythrine are account for the differential

antiproliferative response of OCM-1 cells for these

alkaloids (Fig. 1).

Intriguing conclusions could be drawn if we

compare the amount of necrotic and apoptotic cells

for a given alkaloid (Fig. 4). In the case of

chelidonine the fraction of apoptotic cells signifi-

cantly exceeded that of the necrotic ones, implying

that under the applied experimental conditions

apoptosis is the predominant form of chelidonine-

induced cell death. At the same time it appears that

for sanguinarine and chelerythrine the two modes of

cell death (apoptosis and necrosis) are competing

with each other and the net effect (the actual ratio of

apoptotic and necrotic cells) depends on the concen-

tration, the incubation time and the condition (e.g.

confluence) of cells. As a rule of thumb at higher

concentration necrosis is the dominant form of cell

death, whereas for lower doses the efficiency of

apoptosis could exceed that of necrosis. These results

are in a good accordance with morphological data

obtained by light microscopy (Fig. 2) and explain the

‘biphasic’ pattern of the apoptotic response observed

for sanguinarine and chelerythrine (Figs. 3B, C

and 4B, C). The above findings were also corrobo-

rated by analyzing light scattering properties (i.e. size

and morphology) of cells by flow cytometry. Changes

in the forward and the right angle light scatter signals

supported that upon chelidonine treatment cells

mainly undergo apoptosis, while sanguinarine and

chelerythrine induces bimodal cell death (data not

shown). Sanguinarine-induced bimodal cell death

was reported earlier in several other cell types

[18,28], suggesting that this is a general phenomenon.

Similarities between sanguinarine- and chelerythrine-

mediated cellular responses presumably arose from

the structural homology of these alkaloids [29].

4. Conclusion

Taken together, our data demonstrated that albeit

in different extent, benzophenanthridine alkaloids

investigated in this study could induce apoptotic as

well as necrotic cell death in uveal melanoma cells.

Whereas chelidonine was predominantly effective via

apoptosis, sanguinarine and chelerythrine induced a

so-termed bimodal cell death (both apoptosis and

necrosis). These observations turn our attention to the

possible use of these benzophenanthridine alkaloids in

the treatment of uveal melanomas. Due to their

apoptotic potential these alkaloids are not only good

candidates for chemotherapeutic regimens, but may

also contribute to the development of successful

immune therapies of uveal melanomas. In addition to

the promising use of apoptotic uveal melanoma cells

in dendritic cell-based immune therapies [7], their

application in combination with the expression of

costimulatory molecules could provide a novel

adjuvant therapy for these tumors [30]. To our

knowledge this is the first study showing the cancer

therapeutic potential of chelidonine, sanguinarine and

chelerythrine against uveal melanoma cells. However,

further studies are required to unravel the exact

mechanism(s) of the responses evoked by these

alkaloids as well as to verify their effectiveness in

A. Kemeny-Beke et al. / Cancer Letters 237 (2006) 67–7574

a model system. The possible side effects should be

also investigated.

Acknowledgements

We thank Dr Sandor Damjanovich for his valuable

criticism and discussion. The skillful technical

assistance of Rita Szabo and Szilvia Banhalmine

Szilagyi in the preparation of cells and samples is

gratefully acknowledged. This work was supported by

research grants OTKA F046497 (A.B.), OTKA

T038163 (J.A.), OTKA T038348 (A.F.), ETT

603/2003 (A.B.), Mec-5/2002 (J.D.) and a Bolyai

Janos Research Fellowship (to A.B.).

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