Sanguinarine overcomes P-glycoprotein-mediated multidrug-resistance via induction of apoptosis and oncosis in CEM-VLB 1000 cells

ARTICLE IN PRESS

Experimental and Toxicologic Pathology 58 (2006) 21–30

EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp

Sanguinarine overcomes P-glycoprotein-mediated multidrug-resistance via induction of apoptosis and oncosis in CEM-VLB 1000 cells Priya Weerasinghea,b,, Sarathi Hallocka,b, Shou-Ching Tanga,c, Benjamin Trumpb, Andrejs Liepinsa a

Faculty of Medicine, Memorial University of Newfoundland, 300 Prince Phillip Drive, St. John’s, Newfoundland, Canada, A1B 3V6 b AMC Cancer Research Center, 1600 Pierce Street, Denver, CO 80214, USA c Sylvester Comprehensive Cancer Center, University of Miami, 1475 NW 12th Ave. (D8-4), Suite 3400, Miami, FL 33136, USA Received 11 November 2005; accepted 27 January 2006

Abstract Permeability-glycoprotein (Pgp) positive cells are known to be encoded by the multidrug-resistance gene (MDR1), and characterized by a reduced ability to accumulate drugs. The vinblastin-resistant, Pgp positive CEM-VLB 1000 and its wild type (Pgp-negative and vinblastin-sensitive) counterpart CEM-T4 human leukemia cells, when treated with the alkaloid sanguinarine, were both found to undergo apoptosis at concentrations of 1.5 mg/ml and oncosis/blister cell death (BCD) at concentrations of 12.5 mg/ml. The aim of this study was to assess the ability of sanguinarine to overcome Pgp-mediated multidrug-resistance (MDR), and also to characterize the cell death processes of apoptosis and oncosis (or bimodal cell death) induced by sanguinarine in MDR cells. The cell death processes of apoptosis and oncosis in CEM-VLB 1000 and CEM-T4 cell lines were found to be qualitatively similar when assessed by light microscopy, terminal deoxynucleotidyl transferase (TdT) end-labeling, annexin-V-binding, trypan blue exclusion and western blot analysis. Western blotting revealed an increase in the Bax/Bcl-2 ratio and activation of caspase-3 in apoptosis but not oncosis in both cell lines. The Pgp-positive CEM-VLB 1000 cells and their wild type CEM-T4 cells were both equally sensitive to sanguinarine. Thus, sanguinarine may overcome the phenomenon of Pgp-mediated MDR by inducing apoptosis through increasing the Bax/Bcl-2 ratio and activating caspase-3, and oncosis, which involved neither. r 2006 Elsevier GmbH. All rights reserved. Keywords: Oncosis; Apoptosis; Bimodal cell death; Necrosis; Sanguinarine; Multidrug-resistance; P-glycoprotein; Lymphoma; Anticancer-agent

Introduction

Corresponding author. Baylor College of Medicine, Department of Medicine, BCM 286, N 1319, 1 Baylor Plaza, Houston, TX 77030, USA. Tel.: +832 878 6237; fax: +713 796 9552. E-mail address: [email protected] (P. Weerasinghe).

0940-2993/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2006.01.008

Sanguinarine (bloodroot) belongs to the benzophenanthridine alkaloid group, which are biosynthesized from phenylalanine in plants of the papaveraceae family (Dostal and Pota´cek, 1990; Imanek, 1985). This alkaloid has been reported to display a wide spectrum of

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biological effects, such as antifungal, antimicrobial, analgesic and moreover, anti-cancer as well as chemopreventative properties (Dostal and Pota´cek, 1990; Imanek, 1985; Vavreckova and Ulrichova´, 1994; Ahmad et al., 2000; Adhami et al., 2003). Its principal use today is in over-the-counter products including toothpaste, mouthwash, cough and cold remedies, and homeopathic preparations (Babich et al., 1996). Sanguinarine has also been reported to be a protein kinase C (PKC) and NFkB (nuclear transcription factor kB) inhibitor (Gopalakrishna et al., 1995; Chaturvedi et al., 1997), a DNA intercalator (binding to guanosine-cytosine rich regions) (Babich et al., 1996; Sen and Maiti, 1994), a topoisomerase I and II inhibitor (Hsiang et al., 1989; Zhang et al., 1990), an inhibitor of tubulin assembly (Wolff and Knipling, 1993), an inhibitor of Na+/K+ ATPase (Seifen et al., 1979; Straub and Carver, 1975) and a generator of DNA adducts (Stiborova et al., 2002). Recently, it was reported that sanguinarine inhibited Mitogen-activated protein kinase phosphatase-1 (MKP1), a dual specificity phosphatase that is overexpressed in many human tumors and can protect cells from apoptosis caused by DNA-damaging agents or cellular stress (Vogt et al., 2005). Sanguinarine is also thought to suppress angiogenesis via the inhibition of vascular endothelial growth factor (VEGF) (Eun and Koh, 2004). Resistance of malignant tumors to chemotherapeutic agents remains a major cause of failure in cancer therapy. A multigene family designated MDR (multidrug-resistant) (Endicot and Ling, 1989; Kang et al., 2000) has been identified, the members of which encode a family of membrane glycoproteins termed P-glycoproteins (P-gp) (Nooter and Herweijer, 1991; Borst, 1991; Juranka et al., 1989; Endicot and Ling, 1989). Pgp appears to function as an energy-dependent transport pump capable of effluxing cytotoxic agents and thereby decreasing their intracellular concentration (Hamada and Tsuruo, 1988). The MDR gene, overexpressed in multidrug-resistant cell lines, is associated with poor prognosis for therapy (Ling, 1997; Bosch and Croop, 1996). In addition to protecting cells from chemotherapy-induced apoptosis, P-gp also protects cells from other caspase-dependent death stimuli such as CD95L, TNF and UV irradiation (Igney and Krammer, 2002). The present study shows that the multi-drug resistant, P-glycoprotein (Pgp)-positive CEM-VLB 1000 human lymphoblastic cells are sensitive to sanguinarine; treatment with sanguinarine resulted in cells undergoing apoptosis at lower doses, and oncosis at higher doses. The functionality of the P-gp was maintained in CEMVLB 1000 cells by passaging them in the presence of vinblastin. Our previous work has also found sanguinarine to be an efficient chemotherapeutic agent. In this regard, we have found sanguinarine to overcome the multidrug resistant phenomenon in several cell lines.

For example, we have shown that cisplatin-resistant human papillomavirus (HPV) type 16-immortalized endocervical cells are sensitive to sanguinarine (Ding et al., 2002). Also, Sanguinarine was found to efficiently induce cell death in p53 null k562 cells (Weerasinghe et al., 2001a), which in several previous reports have been found to be rather resistant to the induction of apoptosis (Kobayashi et al., 1998). Thus, sanguinarine may prove to be a valuable chemotherapeutic agent for most cancers which carry p53 mutations. These findings emphasize the need to further study the cell death pathways of apoptosis and oncosis induced by sanguinarine. Several recent studies have implicated that sanguinarine may also be an effective anti-genotoxic and chemopreventive agent (Biswas and KhudaBukhsh, 2002; Adhami et al., 2003). The objective of the present study was to analyze the affects of sanguinarine-treatment in the multidrugresistant CEM-VLB 1000 human leukemia cells and in their wild type CEM-T4 cells. We found that treatment with sanguinarine at concentrations of 1.5 mg/ml for 2 h resulted in the induction of maximum apoptosis and treatment with concentrations of 12.5 mg/ml for 2 h resulted in maximum oncosis/blister cell death (BCD) (i.e. formation of a large blister on the cell surface) in Pgp-positive CEM-VLB 1000 cells and in their wild type Pgp-negative CEM-T4 cells. The induction of apoptosis and oncosis by sanguinarine was termed ‘‘bimodal cell death’’ (Weerasinghe et al., 2001b). Interestingly, at the intermediate concentration of 6.25 mg/ml, both cell lines displayed the morphology of diffuse cell swelling, which may represent a variant of oncosis. Compared to apoptosis, oncosis is by far the older term; however, it is less understood from the standpoint of molecular genetics. Thus, this report is also an attempt to better understand the possible molecular mechanisms by which sanguinarine may induce oncosis/BCD, in comparison to apoptosis, in a Pgp-positive, multidrug-resistant cell line.

Materials and methods The CEM-VLB 1000 and CEM-T4 cell lines were kindly provided by Dr. Victor Ling (University of British Columbia, Canada). These cells were cultured in RPMI 1640 medium supplemented with L. glutamate and 10% fetal calf serum. In addition, the multidrugresistant CEM-VLB 1000 cells were grown in the presence of vinblastin in the media. They were maintained in a 37 1C/5% CO2 fully humidified incubator and passaged 4–5 times weekly. Annexin-V-fluos staining kit for the detection and quantification of apoptosis and differentiation from necrosis at the single cell level was purchased from

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Boehringer Mannheim Corp (Indianapolis, IN). The kit was stored and handled according to manufacturer’s instructions. The drug sanguinarine ([1,3]benzodioxolo[5,6-c]-1,3dioxolo[4,5-I]phenanthridinium, 13-methyl-, chloride[9CI]) was purchased from Sigma Chemicals (St. Louis, MO) and maintained as a dry powder at room temperature. It was dissolved in deionized water at 1 mg/ml and used as a stock solution. Sanguinarine was subsequently diluted with RPMI 1640 in order to make a working solution of 25 mg/ml.

Drug treatment The following experimental format was used in order to prepare the cells for subsequent experiments. Logarithmically growing CEM-VLB 1000 and CEMT4 cells were placed in 60 mm petri dishes at approximately 3
106 cells/ml. Thereafter, serially diluted sanguinarine concentrations of 0.19, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5 and 25 mg/ml were added to the petri dishes and incubated for 2 h in a 37 1C/5% CO2 incubator. Cells that underwent apoptosis and oncosis/ BCD (at concentrations of 1.5 and 12.5 mg/ml respectively) were selected for use in subsequent experiments.

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preferentially repaired by terminal deoxynucleotidyl transferase (TdT) (Gavrieli et al., 1992). The TdT-mediated nick end-labeling assay has been developed to label these strand-breaks with streptovidinhorseradish peroxidase conjugated nucleotides followed by the addition of a substrate (TACS Blue LabelTM or TBL). After sanguinarine treatment, cells were removed from individual wells, washed in phosphate buffered saline (PBS) and fixed in 10% neutral buffered formaldehyde for 10 min. Thereafter, cells were resuspended in 80% ethanol at 1
106 cells per ml. Fiftythousand cells were placed onto an electrostatically treated glass slide and air-dried at room temperature. Cells were then permeabilized with proteinase K (Gavrieli et al., 1992) and incubated for 60 min at 37 1C in the presence and absence of exogenous TdT and streptavidin-horseradish peroxidase conjugated dNTP (deoxynucleotide triphosphate), followed by the substrate TBL according to the manufacturer’s instructions (Sigma TACSTM in situ Apoptosis Detection Kit, St. Louis, MO). Thereafter, cells were examined and photographed under phase microscopy and counted to determine the percentage of cells with DNA nicking to total cells.

Light microscopy Fluorescein-conjugated annexin-V binding assay Cells treated with sanguinarine were studied and quantified by light microscopy. The key morphological criterion for the detection of apoptosis was the formation of apoptotic bodies and the key criterion for oncosis/BCD was the formation of cell surface blisters.

Trypan blue assay The charged cationic dye trypan blue is excluded from putative live cells with intact plasma membranes (O’Brien et al., 1997; Majno and Joris, 1995). Changes in membrane permeability in CEM-VLB 1000 and CEM-T4 cells exposed to sanguinarine treatment, and in untreated controls were evaluated in triplicate, at 2
104 cells per well after 2 h of incubation at 37 1C using flat bottom 96 well plates. After 10 min of incubation with trypan blue (0.2%), the number of trypan blue excluding cells was counted using a hemocytometer. The exclusion of 0.2% trypan blue was used as an indicator of the number of non-necrotic cells.

Terminal deoxynucleotidyl transferase (TdT) endlabeling assay Specific 30 -hydroxyl ends of DNA fragments generated by endonuclease-mediated apoptosis are

After treatment with sanguinarine concentrations that induce apoptosis and BCD, CEM-VLB 1000 and CEMT4 cells (1
106 cells) were washed with PBS and incubated with annexin-V fluorescein isothiocyanate (FITC) conjugate and propidium iodide (PI) utilizing an annexin-V-fluos staining kit (Boehringer Mannheim Corp, Indianapolis, IN). After labeling, cells were resuspended in binding buffer and analyzed using flow cytometry. FITC-fluorescence was measured at 530–545 nm and fluorescence of DNA-PI complexes at 575–606 nm. Cell debris was excluded from analysis by appropriate forward light scatter threshold setting (Leist et al., 1997).

Expression of Pgp in cell surface CEM-VLB 1000 and CEM-T4 Cells were incubated for 30 min with MDR1 antibody (0.5 mg for 106 cells) or non-specific murine IgG2a. After washing with PBS twice, cells were incubated with FITC conjugated F(ab0 )2 fragment of sheep anti-mouse immunoglobulin (50 ml of 1:200 dilution). After 30 min, cells were washed with PBS twice and held at 4 1C in the dark until analysis by flowcytometry (Kunikane et al., 1997).

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SDS-PAGE and western blotting analysis

Statistical analysis

Following sanguinarine treatment (as described above), the expression of Bcl-2, Bax, Bak, Bad, Bik/ Nbk, Bcl-XS, Bid, Bcl-XL, p53, and CPP-32 proteins were determined by western blotting analysis using previously described methods with minor modifications (Poxton, 1990; Towbin et al., 1992). Briefly, whole cell pellets (1
107 cell/dish) were washed twice in PBS, resuspended in 50 ml PBS and lysed by adding lysis buffer solution. The homogenates were quantified using DC protein assay. Equal amounts of proteins (20 mg/ well) were boiled for 5 min, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (5% stacker and 12% resolving) and electroblotted to nitrocellulose. After blocking with PBS tween 20 (0.05%) and with 5% non-fat dried milk for 1 h at 22 1C, the blots were incubated in fresh blocking solution with an appropriate dilution of primary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). For the study of CPP-32, primary antibody was used at 1:250. Blots were washed 3 times for 5 min in TBS-T and then incubated with 1:2500 dilution of horseradish peroxidase conjugated secondary antibody (Santa Cruz) for 1 h at 22 1C. Blots were again washed 3 times for 5 min in TBS-T and then developed by chemiluminescence (Amersham, Arlington Heights, IL).

The results are presented as means7SE. Statistical analyses were performed using the student’s t-test. Differences were considered significant when po0:05.

Results The affects of sanguinarine-treatment in CEM-VLB 1000 (and its wild type CEM-T4 cells) were studied at different doses and time points. In Fig. 1, we have summarized the percentages of apoptosis, oncosis/ blister cell death and ruptured cells/necrosis, when A 100 apo

90

onco/bed necro

% of Cell Death

80 70 60 50 40 30 20 10 0

0

0.19 0.36 0.78

1.5

3.1

6.2

12.5

25

Sanguinarine Concentration (micro g/ml) B

90 apo

80

onco/bed necro

% of Cell Death

70 60 50 40 30 20 10 0 0

0.19 0.36 0.78 1.5 3.1 6.2 12.5 Sanguinarine Concentration (micro g/ml)

25

C 120 apo onco/bed necro

100 % of Cell Death

Fig. 1. Dose–response studies showed a similar pattern of morphological changes for both CEM-VLB 1000 and its wild type counterpart CEM-T4 cells. Shown below are 3 dose–response curves for sanguinarine in CEM-VLB 1000 cells (data for CEM-T4 not shown) at different time points: (A) 2 h, (B) 12 h and (C) 24 h. Quantitative morphological analysis was done in CEM-VLB 1000 (and CEM-T4) cells treated with gradually increasing concentrations (0.19, 0.36, 0.78, 1.5, 3.1, 6.2, 12.5 and 25.0 mg/ml) of sanguinarine at 2 h of drugexposure (A). As shown in (A), maximum apoptosis (apo) (i.e. in 485% of cells) was observed at 1.5 mg/ml, and maximum oncosis/blister cell death (onco/bcd) (in 490% of cells) at 12.5 mg/ml, respectively. Ruptured cells not extruding the dye – trypan blue were taken as necrosis (necro), and this peaked when exposed to a sanguinarine concentration of 25 mg/ml. Quantitative morphology at 12 h of sanguinarine-exposure (B) showed maximum apoptosis (480% of cells) at 0.78 mg/ml, maximum oncosis/BCD (440% of cells) at 6.25 mg/ml, and ruptured cells were seen from 6.2 mg/ml. Additionally, for each cell population exposed to sanguinarine for 12 h, the morphologies of cell death observed were more heterogenous when compared with 2 h of drug-exposure; i.e. at 12 h of exposure to each drug concentration, mixed populations of apoptosis, oncosis/BCD, and ruptured cells were observed. Quantitative morphology at 24 h of drug-exposure (C) showed maximum apoptosis (450% of cells) at 0.36 mg/ml, maximum oncosis/ BCD (440% of cells) at 3.1 mg/ml, and ruptured cells were seen from 0.78 mg/ml. At 25 mg/ml all cells were found to be ruptured/necrotic. Thus, from the above dose–response study with time, a clear shift to the left is evident.

80 60 40 20 0 0

0.19 0.36 0.78

1.5

3.1

6.2

12.5

Sanguinarine Concentration (micro g/ml)

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The TdT end-labeling method and the annexin-Vassay were used to assess both apoptosis and oncosis. TdT end-labeling method showed DNA nicking in about 70% of apoptotic cells in both cell lines, and an absence of DNA nicking during oncosis (Fig. 4). The annexin-V-assay was used to detect the cell surface % of Cells Excluding Trypan Blue

CEM-VLB 1000 cells were treated with ascending concentrations of sanguinarine for 2, 12 and 24 h, respectively. Maximum apoptosis was seen with cells exposed to sanguinarine at concentrations of 1.5 mg/ml for 2 h; and maximum oncosis/blister cell death at 12.5 mg/ml for 2 h. A salient feature of this study was that the dose–response curves were found to shift to the left as the duration of exposure of cells to the drug gradually increased. Both cell lines showed the formation of apoptotic bodies during apoptosis, and the formation of a cell surface blister during oncosis (Fig. 2). The percentages of trypan blue excluding cells, denoting cell viability (O’Brien et al., 1997; Majno and Joris, 1995), were measured at 2 and 12 h of sanguinarine treatment. Treatment with sanguinarine at 1.5 and 12.5 mg/ml for 2 h showed about 90% of cells excluding trypan blue at both concentrations (Fig. 3). However, after 12 h of sanguinarine-exposure at concentrations of 1.5 and 12.5 mg/ml, the percentage of trypan blue excluding cells were 490% and about 62%, respectively. After exposure to sanguinarine for 24 h, the percentage of cells excluding trypan blue at 1.5 and 12.5 mg/ml were 63% and 7%, respectively (results not shown).

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100 90 80 70 60 50 40 30 20 10 0

VLB 0

T4 VLB T4 VLB T4 1.5 1.5 12.5 12.5 0 Sanguinarine Concentration (µg / ml)

Fig. 3. Quantitative trypan blue exclusion in sanguinarinetreated CEM-VLB and CEM-T4 cells for 2 h show all cell samples of both cell lines excluding trypan blue. Ech data point represents the mean7SE for three experiments carried out in duplicate.

Fig. 2. Light micrographs (original magnification—
282) of CEM-VLB 1000 and CEM-T4 cells treated with sanguinarine. Control, apoptosis and oncosis/BCD of CEM-VLB 1000 cells are shown in (A), (B) and (C), while control, apoptosis and oncosis/ BCD of CEM-T4 cells are represented by (D), (E) and (F), respectively. Both cell lines treated with sanguinarine at 1.5 and 12.5 mg/ ml respectively for 2 h showed the morphology of apoptosis (arrows) and oncosis/BCD (arrows).

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100 90 80 70 60 50 40 30 20 10 0

p value > 0.005 * *

VLB T4 VLB T4 VLB T4 0 0 1.5 1.5 12.5 12.5 Sanguinarine Concentration (micro g / ml)

Fig. 4. TdT end-labeling assay in CEM-VLB 1000 and CEMT4 cells treated with sanguinarine was used to detect DNA nicking. As shown in the graph, sanguinarine-induced apoptotic cells of both cell lines were positive for DNA nicking (7275.8 for CEM-VLB 1000 and 75.575.6 for CEMT4), whereas oncosis/BCD of both cell lines (1876.1 for CEM-VLB 1000 and 22.578.3 for CEM-T4) as well as untreated controls were negative. Each data point represents the mean7SE for 3 experiments carried out in duplicate (p value o0.005).

membrane phosphatidyl serine (PS) flip, usually associated with apoptosis (Leist et al., 1997). Results showed about 55% of annexin-V positive cells in both cell lines after 2 h of sanguinarine exposure at apoptosis-inducing concentrations (i.e. 1.5 mg/ml). However, the percentage of annexin-V positive cells during oncosis was only about 20% in both cell lines (Fig. 5). Detection of cell-surface P-gp expression using immunofluorescence-flow cytometry with FITC fluorescence was done to confirm the P-gp-positive status of the CEM-VLB 1000 cells, and the lack thereof in wild type CEM-T4 cells. Over 99% of CEM-VLB 1000 cells displayed FITC fluorescence when treated with the MDR1 antibody. In contrast, MDR1 antibodytreated CEM-T4 cells showed no FITC fluorescence. CEM-T4 and CEM-VLB 1000 cells treated with the isotype IgG (negative controls) did not show fluorescence (Fig. 6). Western blotting analysis was done to study the protein expression during sanguinarine-induced apoptosis and oncosis/BCD in both cell lines. These studies were limited to analyzing the expression of the Bcl-2 family gene products. The expression of the 21 kDa proapoptotic Bax protein was found to be elevated in sanguinarine-induced apoptosis of both cell lines as compared to untreated control cells. In contrast, sanguinarine-induced oncosis/BCD did not show any significant change in Bax protein expression. No change was observed in the expression of the 26 kDa antiapoptotic Bcl-2 protein in apoptosis nor oncosis/BCD in either cell line. The 32 kDa endogenous caspase 3 was inherent in both cell lines. However, it’s 20 kDa

* p value < 0.05

70 % of Annexin V Positive Cells

% TUNEL positive cells

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* *

60 50 40 30 20 10 0

VLB 0

T4 VLB T4 VLB T4 1.5 1.5 12.5 12.5 0 Sanguinarine Concentration (micro g / ml)

Fig. 5. Annexin-V-binding assay in CEM-VLB 1000 and CEM-T4 cells treated with sanguinarine. The cell surface membrane phosphatidyl serine flip in sanguinarine induced apoptosis (1.5 mg/ml) and Oncosis/BCD (12.5 mg/ml) in both cell lines were measured using annexin-V and FITC staining with subsequent quantification of annexin-V positive cells by FACS analysis. Each data point represents the mean7SE from three independent experiments. As shown, apoptosis of both cell lines had annexin-V binding. The difference in annexin-V binding between sanguinarine-treated CEM-VLB 1000/CEM-T4 cells at apoptosis-inducing concentrations and controls of both cell lines was statistically significant (P value o0.05). All other cell samples (control and oncosis/BCD) failed to show the PS flip.

Fig. 6. Cell surface P-gp expression in CEM-VLB 1000 and CEM-T4 cells. Peaks ‘‘b’’ and ‘‘d’’ show P-gp-positive CEMVLB 1000 cells and P-gp-negative CEM-T4 cells, respectively. As shown in the histogram over 95% of CEM-VLB 1000 cells were P-gp positive. Both ‘‘a’’ and ‘‘c’’ peaks show P-gpnegative controls. The X-axis shows the intensity of FITC fluorescence and Y-axis shows cell counts.

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0

0

1.5

1.5

12.5 12.5

VLB T4

VLB

T4

VLB

Sanguinarine (µM)

T4

A

Bax

B

Bel 2

C

Caspase 3

D

β Actin

Fig. 7. Western blots for Bax, Bcl-2, CPP-32 and b actin protein expression is shown in (A), (B), (C) and (D), respectively. Lanes 1, 3 and 5 of (A) show Bax protein expression in control, apoptosis and oncosis/BCD of CEMVLB 1000 cells, while lanes 2, 4 and 6 show control, apoptosis and oncosis/BCD of CEM-T4 cells. The 21 kDa Bax band is elevated in apoptosis of both cell lines. However, the elevation of the Bax band in oncosis/BCD was not significant. Lanes 1, 3 and 5 of (B) show Bcl-2 protein expression in control, apoptosis and oncosis/BCD of CEM-VLB 1000 cells, while lanes 2, 4 and 6 show control, apoptosis and oncosis/BCD of CEM-T4 cells. Results show no change in the 26 kDa Bcl-2 levels in sanguinarine-treated cells. Lanes 1, 3 and 5 of (C) show CPP-32 protein expression and of its 20 Kd breakdown product in control, apoptosis and oncosis/BCD of CEM-VLB 1000 cells, whereas lanes 2, 4 and 6 show control, apoptosis and oncosis/BCD of CEM-T4 cells. Results show the 20 kDa breakdown product only in apoptosis of both cell lines. (D) shows the expression of b actin house-keeping protein in each sample.

breakdown products, denoting activation, was only observed in apoptosis of both cell lines but not in oncosis/BCD (Fig. 7). The expression of pro-apoptotic Bak, Bad, Bik/Nbk, Bcl-XS, Bid, p53 and anti-apoptotic Bcl-XL proteins was not significantly affected by sanguinarine in either cell line at low level or high level (individual data not shown).

Discussion Results of the present study show that sanguinarine overcomes the P-gp mediated MDR phenomenon. P-gppositive CEM-VLB 1000 and P-gp-negative CEM-T4 cell lines underwent similar patterns of apoptosis and oncosis/BCD, both qualitatively and quantitatively, during sanguinarine treatment. When exposed to sanguinarine at concentrations of 1.5 and 12.5 mg/ml for 2 h, both cell lines showed the morphology of apoptosis in over 85% of cells and oncosis/BCD in over

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90% of cells, respectively. Also, in contrast to oncosis/ BCD, apoptosis induced by sanguinarine showed DNA nicking and PS flipping. However, both apoptosis and oncosis were found to have signs of cell viability by exclusion of trypan blue. The Bcl-2 family of genes serves as both positive and negative regulators of cell death (Antonsson and Martinou, 2000). The intracellular level and the ratio of Bcl-2 and Bcl-2-related proteins dictate if a cell should engage in the apoptotic program (Reed, 1997). Sanguinarine-induced apoptosis showed a moderate upregulation of the Bax protein in both cell lines, while Bcl-2 levels remained unchanged. Our previous studies have shown that high levels of endogenous Bcl-2 protect JM1 pre-B lymphoblastic cells from undergoing apoptosis and oncosis, when treated with sanguinarine (Weerasinghe et al., 2001b, c). These findings illustrate the importance of the Bax/Bcl-2 ratio (Weerasinghe et al., 2001c; Adhami et al., 2003; Korsmeyer, 1999) for the induction of apoptosis. Furthermore, this ratio has been shown to correlate with the chemotherapeutic effect (Nakata et al., 1998; Yin et al., 1994). On the other hand, protein expression analysis of oncosis/BCD revealed no significant changes in Bcl-2 family protein expression as compared to untreated control cells and did not appear to show the increased expression of Bax seen in apoptosis. Other members of the Bcl-2 family, including Bak, Bad, Bik, Bcl-XS, Bcl-XL and Bid, as well as the tumor suppressor p53 appear not to play any significant role in either apoptosis or oncosis. Apoptosis is known to be executed by a family of cysteine proteases called caspases (Nun˜ez et al., 1998). Considerable attention has been devoted to studying the activation of the protease cascade in drug-induced and other forms of apoptosis, as well as on the ability of Bcl2 and related proteins to inhibit this process (Wang et al., 1997). It is known that the activation of caspase-3 leads to proteolytic activation of downstream effector caspases and the cleavage of a number of vital proteins, resulting in the orderly demise of cells (Nun˜ez et al., 1998; Salvesen, 1999). Results of the present study concurred with the above findings, in that, sanguinarineinduced apoptosis activated caspase 3; however, oncosis failed to show caspase 3 activation. Our previous studies have also shown that sanguinarine treatment of Bcl-2 low-expressing K562 cells resulted in the activation/cleavage of caspase-3 into 20 kd CPP-32 breakdown products, whereas the Bcl-2 over-expressing JM1 cells showed no breakdown products of CPP-32 (Weerasinghe et al., 2001c). Therefore, the presence of elevated Bcl-2 abrogated sanguinarinetriggered caspase 3 activation, which is in agreement with several reports in the literature (Kojima et al., 1996; Irabo et al., 1996). Our results also appear to be consistent with several other reports, which suggest that in the case of some, but perhaps not all cytotoxic

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stimuli, Bcl-2 functions upstream of a critical cysteine protease involved in the degradation phase of apoptosis (Wang et al., 1997). As opposed to Bcl-2, our results confirm that increased levels of Bax promote caspase 3 activity in sanguinarine-induced apoptosis. In contrast, our results also indicate that the activation of caspase 3 may not be a necessary element in the execution of oncosis. Recent literature has shown evidence of alternate nonapoptotic forms of cell death pathways – an emerging interest in toxicogenomics. These pathways are thought to share some, but not all, the characteristics of classical apoptotic pathways (Igney and Krammer, 2002). For example, it has been reported that there are molecularly less-well defined cell-death pathways that do not require caspase activation (Borner and Monney, 1999; Sperandio et al., 2000). Several other reports indicate that apoptosis may share common molecular pathways with other types of cell death at an early stage (Shirai, 1999). Our findings show that, at least in the case of sanguinarine, tumor cell death may also be induced by oncosis, which is caspase 3independent. The cell death process of oncosis is, more often than not, confused with necrosis (Majno and Joris, 1995, 2004). Necrosis represents not a form of cell death but changes secondary to cell death by any mechanism (Majno and Joris, 1995; Levin et al., 1999). It is known that early necrotic cells lose plasma membrane integrity, thus permitting the entry of trypan blue and propidium iodide into the cell (O’Brien et al., 1997). The exclusion of trypan blue and propidium iodide by oncosis in cells indicate that oncosis is not representative of early necrosis. The morphology of sanguinarine-induced blister formation resembles that of ‘‘oncosis’’ as previously described by Majno and Joris (1995) and Phelps et al. (1989). Majno and Joris (1995) describe oncosis as a form of accidental cell death accompanied by cellular swelling, organelle swelling, blebbing and increased membrane permeability caused by the failure of the ionic pumps of the plasma membrane. Trump et al. (1997) and Trump and Berezesky (1992) associate oncosis (blister formation) with increases in concentrations of cytosolic calcium and rearrangement of cytoskeletal proteins. It has also been shown that oncosis, like apoptosis, can be triggered by the activation of cell surface receptors (Ma et al., 2001). Our findings have shown that treatment with sanguinarine at doses that induce apoptosis and oncosis in CEM-VLB 1000 and CEM-T4 lymphoblastic cells, cause minimal or no damage to normal human T lymphocytes (data not shown). However, Debiton et al. (2003) has reported that sanguinarine inhibits the growth of normal human fibroblasts at the same concentrations that inhibit the growth in a panel of human solid cancer cell lines via the induction of

apoptosis. Contrary to these findings and in corrroboration of our findings, others (Adhami et al., 2003; Ahmad et al., 2000) have shown that sanguinarine treatment of squamous cell carcinoma resulted in the inhibition of growth in cancerous cells via the induction of apoptosis, but did not result in apoptosis of normal human epidermal keratinocytes at similar doses. In a related study it was shown that animals treated with a mixture of sanguinarine and chelerythrine at 5 mg/kg showed no hematological, biochemical, histological nor clinical changes from that of untreated controls (Kosina et al., 2004). Thus, the anticancer potential of sanguinarine might be dose as well as cell line dependent. Several reports in the literature indicate that sanguinarine is effective against many cancers, including skin cancer, prostate cancer and uveal melanoma (KemenyBeke et al., 2005; Adhami et al., 2003, 2004). Moreover, these reports also indicate that sanguinarine induces cancer cell death via the induction of bimodal cell death (Kemeny-Beke et al., 2005; Ahmad et al., 2000). In this report, we have shown that sanguinarine may overcome the phenomenon of Pgp-mediated MDR in the CEMVLB 1000 cell line by inducing bimodal cell death. Interestingly, sanguinarine has also been shown to overcome drug resistance mediated by Glutathione Stransferase P1-1 (GSTP1-1), a phase II drug metabolism enzyme implicated in carcinogenesis and development of resistance to anti-cancer drugs (Duvoix et al., 2004). Sanguinarine may therefore be explored as a potential anti-cancer drug in reversing drug-resistance in the chemotherapy of common malignancies.

Acknowledgments This work was supported by a Medical Research Council of Canada Grant (MT-13178).

References Adhami VM, Aziz MH, Mukhtar H, et al. Activation of prodeath Bcl-2 family proteins and mitochondrial apoptosis pathway by sanguinarine in immortalized human HaCaT keratinocytes. Clin Cancer Res 2003;9:3176–82. Adhami VM, Aziz MH, Reagan-Shaw SR, et al. Sanguinarine causes cell cycle blockade and apoptosis of human prostate carcinoma cells via modulation of cyclin kinase inhibitorcyclin-cyclin-dependent kinase machinery. Mol Cancer Ther 2004;3:933–40. Ahmad N, Gupta S, Husain MM, et al. Differential antiproliferative and apoptotic response of sanguinarine for cancer cells vs normal cells. Clin Cancer Res 2000;6:1524–8. Antonsson B, Martinou J. The Bcl-2 protein family. Exp Cell Res 2000;256:50–7. Babich H, Zuckerbraun HL, Barber IB, et al. Cytotoxicity of sanguinarine chloride to cultured human cells from oral tissue. Pharmacol Toxicol 1996;78:397–403.

ARTICLE IN PRESS P. Weerasinghe et al. / Experimental and Toxicologic Pathology 58 (2006) 21–30

Biswas SJ, Khuda-Bukhsh AR. Effect of a homeopathic drug, chelidonium, in amelioration of p-DAB induced hepatocarcinogenesis in mice. BMC Complement Altern Med 2002;2:4. Borner C, Monney L. Apoptosis without caspases: an inefficient molecular guillotine? Cell Death Differ 1999; 6:497–507. Borst P. Genetic mechanisms of multidrug resistance – a review. Acta Oncol 1991;30:87–105. Bosch I, Croop J. P-glycoprotein multidrug resistance and cancer. Biochim Biophys Acta 1996;1288:F37–54. Chaturvedi MM, Kumar A, Darnay BG, et al. Sanguinarine (pseudochelerythrine) is a potent inhibitor of NF-kB activation, I-kB phosphorylation and degradation. J Biol Chem 1997;272:30129–34. Debiton E, Madelmont J, Legault J, et al. Sanguinarineinduced apoptosis is associated with an early and severe cellular glutathione depletion. Cancer Chemother Pharmacol 2003;51:474–82. Ding Z, Tang SC, Weerasinghe P, et al. The alkaloid sanguinarine is effective against multidrug resistance in human cervical cells via bimodal cell death. Biochem Pharmacol 2002;63:1415–21. Dostal J, Pota´cek M. Quaternary Benzo[c]phenanthridine alkaloids. Coll Czech Chem Commun 1990;55: 2840–73. Duvoix A, Delhalle S, Blasius R, et al. Effect of chemopreventive agents on glutathione S-transferase P1-1 gene expression mechanisms via activating protein 1 and nuclear factor kappaB inhibition. Biochem Pharmacol 2004;68: 1101–11. Endicot JA, Ling V. The biochemistry of P-glycoprotein mediated multidrug resistance. Annu Rev Biochem 1989;58:137–71. Eun JP, Koh GY. Suppression of angiogenesis by the plant alkaloid, sanguinarine. Biochem Biophys Res Commun 2004;317:618–24. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation. J Cell Biol 1992;119: 493–501. Gopalakrishna R, Chen ZH, Gundimeda U. Modifications of cystein-rich regions in protein kinase C induced by oxidant tumor promoters and enzyne specific inhibitors. Methods Enzymol 1995;252:132–46. Hamada H, Tsuruo T. Characterization of the ATPase activity of the Mr 170,000-180,000 membrane glycoprotein (Pglycoprotein) associated with multidrug resistance in K562/ ADM cells. Cancer Res 1988;48:4926–32. Hsiang YH, Liu LF, Wall ME, et al. DNA topoisomerase 1mediated DNA cleavage and cytoxocity of camptothesin analogues. Cancer Res 1989;49:4385–9. Igney FH, Krammer PH. Death and Anti-Death: Tumour resistance to apoptosis. Nat Rev Cancer 2002;2:277–88. Imanek V. The alkaloids. San Diego, CA: Academic Press; 1985. Irabo AM, Huang Y, Fang G, et al. Overexpression of Bcl-2 or Bcl-xL inhibits ara-c-induced CPP32/Yama protease activity and apoptosis of human acute myelogenous leukemia HL-60 cells. Cancer Res 1996;56:4743–8.

29

Juranka PF, Zastawny RL, Ling V. P-glycoprotein: multidrug resistance and a superfamily of membrane associated transport proteins. FASEB J 1989;3:2583–92. Kang C, Ahn B, Jeong C, et al. Down-regulation of JNK/ SAPK activity is associated with the cross-resistance to Pglycoprotein – unrelated drugs in multidrug resistant FM3A/M cells overexpressing P-glycoprotein. Exp Cell Res 2000;256:300–7. Kemeny-Beke A, Aradi J, Damjanovich J, et al. Apoptotic response of uveal melanoma cells upon treatment with chelidonine, sanguinarine and chelerythrine. Cancer Lett 2005. Kobayashi T, Ruan S, Clodi K, et al. : Overexpression of Bax gene sensitizes K562 erythroleukemia cells to apoptosis induced by selective chemotherapeutic agents. Oncogene 1998;16:1587–91. Kojima H, Talanian RV, et al. : Activation of the CPP 32 protease in apoptosis induced by 1-3-D arabinofuranosylcytosine and other DNA damaging agents. Blood 1996;88:1936–43. Korsmeyer SJ. Bcl2 gene family and the regulation of programmed cell death. Cancer Res 1999;59(Suppl): 1693S–700S. Kosina P, Walterova D, Ulrichova J, et al. Sanguinarine and chelerythrine: assessment of safety on pigs in ninety days feeding experiment. Food Chem Toxicol 2004; 42:85–91. Kunikane H, Zalupski MM, Ramachandran C, et al. Flowcytometric analysis of P-glycoprotein expression and drug efflux in human soft tissue and bone sarcomas. Cytometry 1997;30:197–203. Leist M, Single B, Castoldi AF, et al. Intracellular ATP concentration: a switch deciding between apoptosis and necrosis. J Exp Med 1997;185:1481–6. Levin S, Bucci TJ, Cohen SM, et al. The nomenclature of cell death: recommendations of an ad hoc committee of the society of toxicologic pathologists. Toxicol Pathol 1999;27:484–90. Ling V. Multidrug resistance: molecular mechanisms and clinical relevance. Cancer Chemother Pharmacol 1997;40(Suppl):S3–8. Ma F, Zhang C, Prasad KV, et al. Molecular cloning of porimin, a novel cell surface receptor mediating oncotic cell death. Proc Natl Acad Sci USA 2001;98: 9778–83. Majno G, Joris I. Apoptosis, oncosis and necrosis. An overview of cell death. Am J Pathol 1995;146:3–15. Majno G, Joris I. Cell injury and cell death. In: Cells tissues and disease-principles of general pathology. 2nd ed. New York: Oxford University Press; 2004. p. 186–245. Nakata B, Muguruma K, Hirakawa K, et al. Predictive value of Bcl-2 and Bax protein expression for chemotherapeutic effect in gastric cancer. Oncology 1998;55:543–7. Nooter K, Herweijer H. Multidrug resistance (MDR) genes in human cancer. Br J Cancer 1991;63:663–9. Nun˜ez G, Benedict MA, Hu Y, et al. Caspases: the proteases of the apoptotic pathway. Oncogene 1998;17:3237–45. O’Brien MC, Healy Jr SF, Raney SR, et al. Discrimination of late apoptotic/necrotic cells (Type III) by flow cytometry in solid tumors. Cytometry 1997;28:81–9.

ARTICLE IN PRESS 30

P. Weerasinghe et al. / Experimental and Toxicologic Pathology 58 (2006) 21–30

Phelps PC, Smith MW, Trump BF. Cytosolic ionized calcium and bleb formation after acute cell injury of cultured rabbit renal tubule cells. Lab Invest 1989;60:630–42. Poxton IR. Immunoblotting technics. Curr Opin Immunol 1990;2:905–9. Reed JC. Double identity for the proteins of the Bcl-2 family. Nature 1997;387:773–6. Salvesen GS. Programmed cell death and the caspases. APMIS 1999;107:73–9. Seifen ER, Adems RJ, Reimer RK. Sanguinarine: a positive inotropic alkaloid which inhibits cardiac Na+ K+ ATPase. Eur J Pharmacol 1979;60:377–9. Sen A, Maiti M. Interaction of sanguinarine iminium and alkalolamine form with calf thymus DNA. Biochem Pharmacol 1994;48:2097–3012. Shirai T. Commentary: Oncosis and apoptosis: two faces of necrosis in a new proposal to clear up confusion regarding cell death. Toxicol Pathol 1999;27:1495–6. Sperandio S, De Belle I, Bredesen DE. An alternative, nonapoptotic form of programmed cell death. Proc Natl Acad Sci USA 2000;97:14376–81. Stiborova M, Simanek V, Frei E, et al. DNA adduct formation from quaternary benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine as revealed by the 32P-postlabeling technique. Chem Biol Interact 2002;140:231–42. Straub KD, Carver P. Sanguinarine, inhibitor of Na–K dependent ATPase. Biochem Biophys Res Commun 1975;62:913–7. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications 1979. Biotechnology 1992;24:145–9. Trump BF, Berezesky IK. The role of cytosolic Ca++ in cell injury, necrosis and apoptosis. Curr Opin Cell Biol 1992;4:227–32.

Trump BF, Berezesky IK, Chang SH, et al. The pathways of cell death: Oncosis, Apoptosis and Necrosis. Toxicol Pathol 1997;25:82–8. Vavreckova C, Ulrichova´ J. Biological activity of Quaternary Benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine. Chem Listy 1994;88:238–48. Vogt A, Tamewitz A, Skoko J, et al. The benzo[c]phenanthridine alkaloid, sanguinarine, is a selective, cell-active inhibitor of mitogen-activated protein kinase phosphatase1. J Biol Chem 2005;280:19078–86. Wang S, Vrana JA, Bartimole TM, et al. Agents that downregulate or inhibit protein kinase C circumvent resistance to 1-b-D-Arabinofuranosylcytosine-induced apoptosis in human leukemia cells that overexpress Bcl-2. Mol Pharmacol 1997;52:1000–9. Weerasinghe P, Hallock S, Liepins A. Bax, Bcl-2 and NF-kB expression in sanguinarine induced bimodal cell death. Exp Mol Pathol 2001a;71:89–98. Weerasinghe P, Hallock S, Tang S, et al. : Sanguinarine induces bimodal cell death in K562 but not in high Bcl-2 expressing JM1 cells. Path Res Pract 2001b;197: 717–26. Weerasinghe P, Hallock S, Tang S, et al. Role of Bcl-2 family proteins and caspase-3 in sanguinarine-induced bimodal cell death. Cell Biol Toxicol 2001c;17:371–81. Wolff J, Knipling L. Antimicrotubule properties of benzophenanthridine alkaloids. Biochemistry 1993;32: 13334–9. Yin XM, Oltvai ZN, Korsmeyer SJ. BH1 and BH2 domains of bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 1994;369: 272–3. Zhang H, D’Arpa P, Liu LF. A model for tumor cell killing by topoisomerase poisons. Cancer Cells 1990;2:23–7.