Production of hydrogen peroxide and redox cycling can explain how sanguinarine and chelerythrine induce rapid apoptosis

Archives of Biochemistry and Biophysics 477 (2008) 43–52

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Production of hydrogen peroxide and redox cycling can explain how sanguinarine and chelerythrine induce rapid apoptosis Smita S. Matkar a, Lisa A. Wrischnik b, Utha Hellmann-Blumberg a,* a b

Department of Chemistry, University of the Pacific, 3601 Pacific Avenue, Stockton, CA 95211, USA Department of Biological Sciences, University of the Pacific, 3601 Pacific Avenue, Stockton, CA 95211, USA

a r t i c l e

i n f o

Article history: Received 10 February 2008 and in revised form 28 April 2008 Available online 4 June 2008 Keywords: Sanguinarine Chelerythrine Reactive oxygen species Hydrogen peroxide Redox cycling Rapid apoptosis Potential anticancer agent

a b s t r a c t Sanguinarine and chelerythrine are naturally occurring benzophenanthridines with multiple biological activities. Sanguinarine is believed to be a potential anticancer agent but its mechanism of action has not been fully elucidated. We previously found that it causes oxidative DNA damage and very rapid apoptosis that is not mediated by p53-dependent DNA damage signaling. Here we show that sanguinarine and chelerythrine cause the production of large amounts of reactive oxygen species (ROS), in particular hydrogen peroxide, which may deplete cellular antioxidants and provide a signal for rapid execution of apoptosis. Several oxidoreductases contribute to cell death induced by sanguinarine and chelerythrine which appear to be reduced upon entering the cell. We propose a model in which the generation of lethal amounts of hydrogen peroxide is explained by enzyme-catalyzed redox cycling between the reduced and oxidized forms of the phenanthridines and discuss the implications of such a mechanism for potential pharmaceutical applications. Ó 2008 Elsevier Inc. All rights reserved.

The benzophenanthridine alkaloids sanguinarine (13-methyl [1,3]benzodioxolo [5,6-c]-1,3-dioxolo [4,5-i] phenanthridinium or pseudochelerythrine) and chelerythrine (1,3-dimethoxy-12methyl [1,3]benzodioxolo [5,6-c] phenanthridinium) have many biological activities. They are found in bloodroot (Sanguinaria canadensis), Macleaya species and a number of poppy species, where they appear to function as a defense against fungi [1–4]. While chelerythrine has been used as an inhibitor of protein kinase C (PKC)1 for many years [5], sanguinarine, which was briefly used as an antimicrobial agent, has been investigated for use as an anticancer agent [6–9]. The cytotoxicity of sanguinarine has been correlated with induction of apoptosis and other types of cell death [6,7], activation of pro-death Bcl-2 family proteins [10], down-regulation of cyclins and cyclin-dependent kinases [11], and other alterations in the abundance, cellular localization or activity of proteins involved in cellular signaling. None of these studies led to a comprehensive mechanism of action. Reactive oxygen species (ROS) such as superoxide anions (O2  ) or hydrogen peroxide (H2O2) have also been observed in sanguinarine-treated cells [9,12,13]. However, it is neither clear to which extent ROS contrib-

* Corresponding author. Fax: +1 209 946 2607. E-mail address: ublumberg@pacific.edu (U. Hellmann-Blumberg). 1 Abbreviations used: PKC, protein kinase C; CHO, Chinese hamster ovary; ROS, reactive oxygen species; NAC, N-acetylcysteine; DTT, dithiothreitol; GSH, glutathione; DPI, diphenylene iodonium; NADH, nicotinamide adenine dinucleotide reduced form; TLC, thin layer chromatography. 0003-9861/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2008.05.019

ute to the antiproliferative effects of sanguinarine nor how they are generated. ROS are important for many biological processes and have recently been implicated in the action of many antibiotics [14]. Our previous observation that sanguinarine causes oxidative DNA damage and rapid, p53-independent apoptosis [15] led to the hypothesis that sanguinarine increases the production of H2O2, which is more stable than other ROS and can damage DNA through Fenton-type reactions. Since H2O2 can also cause apoptosis we investigate here whether H2O2 production can explain the rapid cell death seen in cells treated with sanguinarine or chelerythrine. The two major systems for generating ROS in mammalian cells are the mitochondria and the Nox-family NAD(P)H oxidases which are linked [16–18]. Minor sources include xanthine oxidase. The plasma membrane-associated enzymes of the Nox-family are involved in regulation of the cellular redox status and reduce extracellular compounds that can potentially cause oxidative damage to cells [19]. Some Nox-family enzymes are associated with internal membranes [20,21]. Nox-family enzymes and another type of plasma membrane-associated NAD(P)H oxidase, the small cell-surface NAD(P)H oxidases or ECTO-NOX proteins [22], exhibit increased activity in cancer cells [19,22–24]. Many ROS-producing enzymes are inhibited by the flavoprotein inhibitor diphenylene iodonium (DPI), including the Nox-family NAD(P)H oxidases [24–26], nitric oxide synthase [27], xanthine oxidase [28] and some of the enzymes involved in mitochondrial electron transfer [29]. We studied the effects of DPI, the xanthine

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oxidase inhibitor allopurinol, and the mitochondrial electron transfer inhibitors rotenone (inhibits complex I) and antimycin A (inhibits complex III) on rapid apoptosis induced by sanguinarine and chelerythrine. These studies and other experiments described here led to a model in which sanguinarine and chelerythrine cycle between an oxidized and a reduced form and cause the production of very high levels of H2O2 which are likely responsible for rapid cell death. Materials and methods Chemicals Sanguinarine hydrochloride (P98% pure) and chelerythrine hydrochloride (P97% pure and completely free of biologically active impurities such as sanguinarine) were purchased from Alexis Biochemicals through Axxora; catalase, glutathione, antimycin and dihydrodichlorofluorescein (dichlorofluorescin) acetate are from Sigma; dihydroethidium is from Invitrogen, monoclonal antibodies for actin, catalase and phosphorylated histone H2AX (c-H2AX) were purchased from Chemicon International (now Millipore) and the antibody for the 89 KDa poly(ADP-ribose) polymerase (PARP) fragment from Cell Signaling Technology. The reverse-phase thin layer chromatography (RP-TLC) plates were from Whatman. N-acetylcysteine, diphenylene iodonium chloride and rotenone were from VWR. Cell culture The cell lines MCF-7 and MDA-MB231 were provided by Dr. G. Wurz, the AA-8 cell line by Dr. R. Tebbs, and the cell lines HCT116, SW480 and OVCAR-3 were purchased from ATCC. All cell lines were grown in DMEM-F12 medium supplemented with 10% fetal bovine serum (Hyclone Laboratories), 50 U/ml penicillin and 50 lg/ ml streptomycin at 37 °C in 95% air and 5% CO2. Viability studies Treatment consisted of adding appropriate volumes of 1 mM sanguinarine or chelerythrine in DMSO to cells while controls were treated with DMSO only. Approximately 10,000 cells per well were seeded and allowed to attach for 24 h. They were treated at 30–50% confluence in 200 ll of medium for 24 h after which the medium was replaced with 100 ll medium containing the MTS assay reagents (Cell titer 96Ò aqueous non-radioactive cell proliferation assay from Promega). Viable cells were quantified by measuring absorbance at 490 nm using a Macintosh plate reader. At least 18 measurements (6 wells from three independent experiments) were averaged for each data point. Western blots For the detection of phosphorylated H2AX (c-H2AX), the protocol from MacPhail et al. [30] was used with minor modifications. Approximately 105 cells were seeded and allowed to attach for 24 h after which they were treated with sanguinarine and/or other compounds for 4 h. The cells were collected in PBS, centrifuged at 500g for 5 min and lysed in 100 ll ice-cold lysis buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 0.1% SDS; 1% Triton X-100; 5 mM EDTA, pH 8.0) containing protease inhibitors. Ten microliter of protein solution were fractionated on 15% SDS–PAGE gels and transferred to nitrocellulose membranes at 75 V. After blocking for at least 1 h, membranes were incubated overnight at 4 °C with monoclonal mouse anti-cH2AX antibody (diluted 1:1000 in 5% nonfat dry milk/PBST) and washed three times. Blots were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (diluted 1:2500 in PBST) and washed. Bands were visualized on X-ray film using ECL detection (Amersham/GE Healthcare). The monoclonal mouse anti-actin antibody (control) was detected simultaneously. Similar protocols were used for the detection of catalase and cleaved PARP. In order to compare relative levels of catalase expression, the catalase and actin bands were scanned and processed with Un-Scan-It software (Silk Scientific, Inc.) which yielded catalase/actin ratios. Fluorescence microscopy Cells were grown overnight in 4-chamber cover glass dishes (Nalgene Nunc) and observed with Chroma filters for three different, non-overlapping excitation and emission ranges (DAPI, FITC and TRITC). For the detection of sanguinarine and chelerythrine in live cells, fluorescence was observed with DAPI and FITC filters. For the detection of intracellular ROS, cells grown overnight in 4-chamber cover glass dishes were pretreated with either 3 lM sanguinarine, 7.5 lM chelerythrine or 200 lM H2O2 (control) for 1.5 h, then treated with either dihydroethidium or dihydrodichlorofluorescein acetate for 30 min in the dark, after which the medium

was removed, cells were washed with PBS and fluorescence was observed in fresh medium using a Leica fluorescence microscope with the TRITC filter in the case of ethidium and FITC filter in the case of fluorescein. In vitro reaction of the phenanthridines with NADH Equal volumes of 1 mM sanguinarine or chelerythrine and 1 mM freshly prepared aqueous NADH solution were mixed, incubated for 5 min at room temperature, and loaded onto a 2% agarose gel with wells located roughly in the middle after addition of 5% glycerol. Electrophoresis was carried out in TAE buffer for approximately 1 h. Alternatively, the reaction mixture containing phenanthridine and NADH or a mixture of 1 mM sanguinarine or chelerythrine and excess sodium borohydride in ethanol were spotted onto a reverse-phase TLC plate and developed with a mobile phase consisting of 90% ethanol and 10% 0.25 M sodium phosphate, pH 7.4. The reaction products were visualized under UV light. Detection of DNA damage DNA single strand breaks were detected by alkaline single cell electrophoresis (comet) assay [31]. Briefly, cells were seeded at 105 cells per plate and allowed to attach for 24 h after which they were treated with sanguinarine or chelerythrine (with or without additional compounds), harvested, suspended at 106 cells/ml and stored on ice. Frosted microscope slides were coated with 1.5% regular agarose. Ten microliter of cell suspension were mixed with 120 ll of low-melting agarose and added to the slides. The slides were placed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris–HCl, pH 9.5. 1% Triton X-100 and 10% DMSO) at 4 °C for at least 1 h and equilibrated in electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH >13) for 20 min. Electrophoresis was carried out at 25 V for 20 min. The slides were stained with ethidium bromide (20 lg/ml) and comets were detected at 525 nm using a Leica fluorescence microscope. Analysis of comet tails and tail moment was carried out using the ‘‘Comet Score” software provided by TriTek Corp. Statistical analysis All experiments were repeated at least three times under the same conditions. Numerical values were averaged and the P-values are indicated in the legends for Fig. 4 and Supplemental Figure 2.

Results The effects of sanguinarine and chelerythrine on the viability of different cell lines The antiproliferative effects of sanguinarine and chelerythrine were studied in several mammalian cell lines including AA-8, a non-cancerous Chinese hamster ovary (CHO) cell line and cell lines derived from several human solid tumors (Table 1). All cells were treated at 30–50% confluency since we found that fully confluent cells are not affected by these compounds (data not shown). Viability assays performed after 24 h of continuous exposure show that the growth of all cell lines is inhibited by sanguinarine and chelerythrine in a non-linear fashion with sanguinarine being more potent (Table 1 and Fig. 2). Little inhibition is seen until a threshold concentration is reached. Furthermore, in most cell lines treatment with 5 lM sanguinarine or 10 lM chelerythrine induces apoptosis within 2–4 h as evident from morphological changes such as membrane blebbing. At 3 lM sanguinarine or 7.5 lM chelerythrine apoptosis occurs somewhat later. The failure of MCF-7 cells to show apoptotic features including blebbing, DNA laddering or H2AX phosphorylation in response to phenanthridine treatment (data not shown) could be due to a defect in apoptosis execution.

Table 1 IC50 values for 24-h treatment with phenanthridine IC50 values for

Cell type

Sanguinarine (lM)

Chelerythrine (lM)

MCF-7 MDA-MB231 HCT116 SW480 OVCAR-3 AA-8

Breast cancer Breast cancer Colon cancer Colon cancer Ovarian cancer CHO

4.4 1.6 1.8 1.6 1.8 1.4

12.3 6.7 6.6 6.6 6.6 6.7

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S.S. Matkar et al. / Archives of Biochemistry and Biophysics 477 (2008) 43–52

OR1

OH

OR2

CH3 N

OR1 OR2

CH3

OH

-

N

O

O

O

O

2e-

2e-

H+ OR1 CH3

OR 1

OR2

OR 2

CH 3

N

N

+

H

O

O

O

O

OR1

OR 1 CH 3

CH3

OR 2

OR2 N

N

O

O

O

O

Fig. 1. Different forms of sanguinarine (R1, R2 = –CH2–) and chelerythrine (R1 = R2 = –CH3): Upon UV exposure the colored iminium form (A) shows orange (sanguinarine) or yellow (chelerythrine) fluorescence, which appears green with a FITC filter. Raising the pH in vitro by adding NaOH generates the pseudobase (B, blue fluorescence). Reduction with NaBH4 produces the dihydro form (C, light blue fluorescence) whereas reduction with NADH produces a negatively charged species. The proposed anion (dark blue fluorescence) and two resonance structures are shown (D–F).

Since they lack functional caspase-3 [32] they may undergo a different type of cell death at higher phenanthridine concentrations. Treatment conditions were established by monitoring the appearance of the 89 KDa fragment of poly(ADP-ribose) polymerase (PARP), a sensitive marker for early apoptosis [33], in order to determine when cell death occurs. Fig. 3 shows that 5 lM sanguinarine or 10 lM chelerythrine cause PARP cleavage within 2 h whereas 3 lM sanguinarine or 7.5 lM chelerythrine require at least 3 h of treatment. Thus, we studied phenanthridine-induced apoptosis after 2–4 h of treatment. Oxidative processes and H2O2 are involved in rapid cell death caused by sanguinarine and chelerythrine The importance of oxidation for the antiproliferative effects of sanguinarine and chelerythrine was supported by the observation that 10 mM NAC restore viability almost completely in all cell lines (Fig. 2 and data not shown). In addition, NAC prevents the appearance of apoptotic features. Apoptotic DNA cleavage leads to phosphorylation of histone H2AX to c-H2AX in cells treated with sanguinarine [15]. All cell lines assayed (except MCF-7) show

strong c-H2AX bands after treatment with 3 lM sanguinarine or 7.5 lM chelerythrine for 4 h (Fig. 7A–C, lane 7). Fig. 7 further shows that co-treatment with 5 mM dithiothreitol (DTT), 10 mM glutathione (GSH) or 10 mM NAC and either sanguinarine or chelerythrine prevents the formation of c-H2AX. Since we had previously ruled out the possibility that NAC reacts directly with phenanthridines (data not shown) the observation that DTT, GSH and NAC prevent the rapid apoptotic cell death supports the hypothesis that ROS play an important role in cell death caused by these compounds. We investigated catalase because it specifically destroys H2O2. While catalase does reduce the growth-inhibitory effects of the phenanthridines (Fig. 2), the enzyme appears to be less effective than NAC, GSH or DTT. This could indicate that other reactive species such as O2  help mediate cell death induced by sanguinarine or chelerythrine. However, the addition of superoxide dismutase did not provide protection or show any significant effect on cell viability (data not shown). It has been reported that sanguinarine does not generate enough O2  to induce apoptosis [9].Thus, we focused on H2O2. Possible explanations for the partial rescue by catalase include limited stability of the enzyme preparation used or a

S.S. Matkar et al. / Archives of Biochemistry and Biophysics 477 (2008) 43–52

HCT116

120 100 80 60 40 20 0

MDA-MB231

% Cell Viability

% Cell Viability

46

0

1 2 3 Conc (micromolar)

4

0

HCT116

80 60 40 20 0

1 2 3 Conc (micromolar)

4

MDA-MB231

120

120 100

% Cell Viability

% Cell Viability

120 100 80 60 40 20 0

100 80 60 40 20 0

0

5 10 Conc (micromolar)

15

0

5 10 Conc (micromolar)

15

Fig. 2. Viability studies of HCT116 and MDA-MB231 cells treated with sanguinarine (A and B) and chelerythrine (C and D): Diamonds () show treatment with the phenanthridine alone, j indicate co-treatment with catalase and N indicate pretreatment with 10 mM NAC.

San (5µM) + + Che(10µM) - Time 1hr 2hr

+ - - + + + 3hr 1hr 2hr 3hr PARP Actin

San(3µM) + Che(7.5µM)Time 2hr

+ + - - + + + 3hr 4hr 2hr 3hr 4hr PARP

Actin Fig. 3. Time required for the onset of apoptosis as determined by the appearance of the 89 kDa fragment of PARP. (A) lanes 1–3: 5 lM sanguinarine for 1, 2 and 3 h; lanes 4–6: 10 lM chelerythrine for 1, 2 and 3 h; (B) lanes 1–3: 3 lM sanguinarine for 2, 3, and 4 h; lanes 4–6: 7.5 lM chelerythrine for 2, 3, and 4 h; lane 7: untreated control.

rate of H2O2 transport that is not high enough for all of the H2O2 produced in the cells to react with the catalase added to the outside of the cells. Differences in H2O2 transport efficiency [34] across the plasma membrane could explain why the degree of catalase protection varies between cell lines. We also found some variability in protection depending on the catalase preparation that was used. More evidence for a critical role of H2O2 in the cellular response to sanguinarine and chelerythrine was obtained by assaying catalase levels in different cell lines. Fig. 4 shows that most cells lines have similar catalase levels. However, MCF-7 cells contain much more catalase. This result provides another explanation why MCF-7 cells are less sensitive to sanguinarine and chelerythrine. Data from the literature support an inverse correlation between catalase expression and sensitivity to phenanthridines. For the prostate cancer cell lines DU-145 and LNCaP, IC50 values of 2.3 and 6.3 lM were reported for sanguinarine while the IC50 values for chelerythrine were 6.0 and 22 lM, respectively [35]. The more

Fig. 4. Relative amount of catalase protein in different cell lines as determined by Western blotting. The average of at least three experiments was calculated and the ratios of catalase over actin protein were plotted. The ratio for for MCF-7 cells was set to 100%. (*P < .005)

sensitive DU-145 cell line has a catalase activity of 20 KU/ mg protein compared to 60 KU/mg for LNCaP [36]. Similarly, many strains of bacteria are inhibited by 50 lM sanguinarine [37] but Heliobacter pylori with their extremely high catalase activity [38] require 150 lM sanguinarine or 300 lM chelerythrine [39]. Fluorescence microscopy of cells treated with sanguinarine or chelerythrine shows increased ROS levels We wanted to investigate ROS levels in cells treated with sanguinarine or chelerythrine but we were concerned that the strong auto-fluorescence of the phenanthridines [40–42] might interfere with ROS detection. Hydrogen peroxide is commonly detected using fluorescein derivatives [43] which are oxidized to products emitting in the yellow-green (FITC) range while superoxide production is studied with dihydroethidium, which yields products with orange fluorescence upon oxidation [44]. The potential overlap of fluorescent signals was evaluated by adding up to 5 lM sanguinarine or up to 10 lM chelerythrine to different cell lines and observing them directly by fluorescence microscopy (in the absence of dyes). Within 2 min, we saw blue fluorescence throughout each cell, whereas the expected fluorescence in the FITC-range appeared several minutes later and subsequently increased in inten-

S.S. Matkar et al. / Archives of Biochemistry and Biophysics 477 (2008) 43–52

sity. Emission continued in both ranges but showed distinct cellular distributions with the FITC emission being more localized to internal membranes (Supplemental Figure 1). While the blue fluorescence does not interfere with the detection of ROS, the fluorescence in the FITC-range was found to cause errors during the quantitative evaluation of fluorescein derivatives by flow cytometry. However, since the phenanthridines have a different cellular distribution than fluorescein, it is possible to use fluorescence microscopy to visually evaluate ROS production. We observed a large increase in H2O2 levels and a substantial increase in O2  levels when cells were treated with sanguinarine or chelerythrine and the appropriate reduced dye. Fig. 5 shows HCT116 cells treated with 3 lM sanguinarine (B and E) or 7.5 lM chelerythrine (C and F) and either dihydroethidium (A–C) or dihydrodichlorofluorescein (D–F). The intense fluorescence of the fluorescein generated by H2O2 could be seen throughout the cells while the distribution of the dihydrohydroethidium oxidized by O2  was more limited. Similar results were obtained when other cell lines were treated with 5 lM sanguinarine or 10 lM chelerythrine. Reduction of sanguinarine and chelerythrine in vitro The observation that the phenanthridines fluorescence changes to blue when they enter cells indicates that they undergo a chemical reaction. It has been reported that the pseudobase (Fig. 1), a derivative with blue fluorescence, crosses the cell membrane [41,45]. However, the pKa of sanguinarine is around pH 8 [41,42,45] and the pKa of chelerythrine around pH 9 [41]. Since many tumor cells have an intracellular pH of 7.1–7.3 [46] and an acidic extracellular environment, not much pseudobase would be formed under physiological conditions. Moreover, we found that the pseudobase generated in vitro by addition of NaOH to sanguinarine or chelerythrine is quickly converted to the iminium form at pH 7–8. Thus, the bluefluorescing form observed in cells treated with sanguinarine or chelerythrine is more likely a reduced form such as the ‘‘dihydro” form (Fig. 1). Dihydro-phenanthridines should enter cells as easily as the related compound dihydroethidium.

47

Initially, our hypothesis that the phenanthridines are reduced upon entering the cell did not seem compatible with the increased production of O2  and H2O2. Thus, we wondered whether ROS are generated by intracellular reoxidation of dihydrophenanthridines. We also found that the phenanthridines are easily reduced. While investigating enzyme-catalyzed reductions in vitro, we made the unexpected observation that sanguinarine and chelerythrine are reduced by NADH in the absence of any enzyme. When mixed in equimolar concentrations, NADH converts the iminium salts (orange-yellow with yellow fluorescence) to colorless compounds with blue fluorescence within minutes. When an excess of NADH is used, the reaction occurs instantly in the case of sanguinarine while chelerythrine reacts somewhat slower. In order to visualize the products better we separated them from excess NADH by gel electrophoresis. To our great surprise, the main products moved in the same direction as NADH indicating that this reduced form is negatively charged (Fig. 6A). We suspected that the anions become dihydrophenanthridines upon protonation but observed that increasing the proton concentration beyond pH 6.0 leads to rapid production of the iminium form (data not shown). An uncharged dihydrophenanthridine might be less stable than either an iminium cation or an anion because the electrons can not be delocalized over the entire structure. Reduction of sanguinarine with excess sodium borohydride (NaBH4) yields dihydrosanguinarine, an uncharged molecule with blue fluorescence [4]. Because it is difficult to visualize this reduced form on agarose gels, we compared the products of the reduction with NaBH4 and NADH after separation by reverse-phase thin layer chromatography (TLC). Fig. 6B shows that NADH produces mostly the dark blue-fluorescing anion, which travels almost to the top of the TLC plate, and a small amount of less hydrophilic dihydro form, which travels less than half-way. In contrast, reduction with NaBH4 yields only the latter. After 2 h, the dihydro form is completely oxidized to the iminium form whereas only a small fraction of the anion has been converted (the dark blue appears slightly purple, Fig. 6C). This indicates that the anion is more stable than the dihydro form in the presence of air. The ease of the reduction and the relative stability of the (anionic) reduced form support

Fig. 5. Fluorescence microscopy shows the intracellular production of ROS in HCT116 cells treated with no phenantridine (A and D), 3 lM sanguinarine (B and E) or 7.5 lM chelerythrine (C and F) plus dihydroethidium (A–C) or dihydrodichlorofluorescein acetate (D–F).

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- Electrode

1

2

3

4

5

+NaBH4

+NADH

Chelerythrine Iminium

+NaBH4

+NADH Anion

Sanguarine Iminium

Start

Dihydro/ Iminium Anion NADH

Start + Electrode Fig. 6. (A) Agarose gel showing that the reaction of phenanthridines with NADH in vitro produces a negatively charged species with dark blue fluorescence. Lane 1: sanguinarine (magenta iminium cation). Lane 2: sanguinarine + NADH (1:2) giving the reduced anion (dark blue) and excess NADH (turquoise). Lane 3: chelerythrine (orange iminium form). Lane 4 chelerythrine + NADH (1:2) giving the reduced anion (dark blue), excess NADH (turquoise), traces of unreacted iminium cation and perhaps traces of neutral dihydroform in the well (‘‘Start”). Lane 5: NADH. (B) and (C): Reverse phase thin layer chromatogram showing the products of the reduction of sanguinarine with NaBH4 and NADH. (B) Immediately after TLC only the reduced forms are seen, (C) After being exposed to air for 2 h, the blue dihydro form is converted to the orange iminium form while the anion band near the top of the NADH lane has not changed much. Fluorescence was visualized with a Fotodyne 3–300 UV transilluminator.

the hypothesis that cells can accumulate reduced sanguinarine or chelerythrine as long as enough NADH is available for the reduction. Rapidly proliferating cells have high levels of NAD(P)H because they require reduced nucleotides for DNA synthesis. This could explain why the antiproliferative effects of sanguinarine and chelerythrine are only observed in rapidly proliferating cells. Inhibitors of oxidoreductases prevent apoptosis induced by sanguinarine and chelerythrine While sanguinarine and chelerythrine are readily reduced to what appears to be a relatively stable anion we suspect that reoxidation under physiological conditions requires catalysis. Thus, we investigated whether redox enzymes are required in order for sanguinarine and chelerythrine to induce apoptosis using the flavoprotein inhibitor DPI, the specific mitochondrial electron transport inhibitors rotenone and antimycin A and the xanthine oxidase inhibitor allopurinol. Their effects were assayed by monitoring H2AX phosphorylation and cleavage of PARP (Fig. 7) after 4 h of treatment with a phenanthridine and an inhibitor. The 4-h time point was chosen in order to obtain a strong apoptotic signal from the phenanthridines alone (lane 1 in Fig. 7) while minimizing the possibility that one of enzyme inhibitors alone might have a significant effect on cell death. Despite reports that DPI can have a pro-apoptotic effect [47,48] we found that, under our experimental conditions, 10 lM DPI completely prevented the apoptotic effects of sanguinarine or chelerythrine (Fig. 7A, B and D). We concluded that some flavoproteins must be required in order for phenanthridines to induce rapid apoptosis. Our data also indicate that 20 lM rotenone prevents cell death induced by the phenanthridines at least temporarily. Further studies indicated that cell death occurs later in the presence of rotenone (data not shown). Similar results were obtained in the presence of 20 lM antimycin A, indicating that functioning electron transfer in mitochondria accelerates cell death induced by sanguinarine and chelerythrine

but is not absolutely required. Xanthine oxidase does not appear to be involved since 20 lM allopurinol had very little effect. Production of ROS can explain DNA damage caused by sanguinarine We previously noted that oxidative DNA damage in sanguinarine-treated cells was not associated with apoptosis and occurred prior to the onset of cell death [15]. Since early DNA damage could be the result of reactive oxygen species we investigated whether DTT, GSH, NAC, catalase, and DPI were able to prevent DNA single strand breaks. Supplemental Figure 2 shows that treating cells with these reagents in addition to sanguinarine leads to a significant reduction in DNA single strand breaks detected by alkaline comet assay. DNA damage prior to apoptosis was not detected in cells treated with chelerythrine, which shows a different intracellular distribution and lower reactivity towards NADH. Discussion The antiproliferative effects of sanguinarine and other phenanthridines, and the perceived safety of these agents, make them attractive candidates for anticancer therapy. However, a comprehensive mechanism of action has not been proposed. Sanguinarine is not extensively metabolized by mammals. A single reduced metabolite known as ‘‘dihydrosanguinarine” was detected by mass spectrometry in rat liver and plasma [49] but has not been implicated in any biological effects. Dihydrosanguinarine also occurs in plants, where its blue fluorescence has been observed [4]. The blue fluorescence we observed in human cancer and CHO cells treated with sanguinarine or chelerythrine is consistent with the presence of dihydrophenanthridines in mammalian cells. We found that sanguinarine and chelerythrine are easily reduced by NADH and yield two products with blue fluorescence. The major product is an anion which has not been described before and which is more stable than the uncharged

S.S. Matkar et al. / Archives of Biochemistry and Biophysics 477 (2008) 43–52

dihydro form. It might be generated by a nucleophilic attack of an electron pair on the carbon next to the electron-deficient nitrogen. Subsequent delocalization of the negative charge (Fig. 1) provides an explanation for the relative stability of the anion

San + NAC DPI DTT GSH Rotenone -

+ + -

+ + -

+ + -

+ + -

+ +

+ Actin

γ H2AX Che + NAC DPI DTT GSH Rotenone -

+ + -

+ + -

+ + -

+ + -

+ -

+ +

Actin

γ H2AX San + Che Antimycin Allopurinol -

+ + -

+ +

-

+ -

+

Actin

H2AX

γ H2AX San + Che DPI Rotenone -

+ + -

+ +

+ -

+ + -

+ +

PARP Actin

San + Che Antimycin Allopurinol -

+ + -

+ +

+ -

+ + -

+ +

PARP Actin

49

at neutral or alkaline pH. In fact, both the anion and the (oxidized) iminium form are highly conjugated and much more stable than the neutral dihydro form. We suspect that the anion has not been reported before because the method used to detect dihydrosanguinarine can not distinguish the two reduced forms when both are protonated during analysis. The initial reduction of sanguinarine or chelerythrine as they enter mammalian cells might be catalyzed by the trans-plasma membrane electron transport [50] and ECTO-NOX proteins which reduce a variety of substrates such as the cell-impermeable tetrazolium salt WST-1 [19,22]. A sanguinarine reductase was recently found in plants [4] while dihydrophenanthridine oxidases, which reduce O2 to H2O2 while oxidizing dihydrosanguinarine to sanguinarine, were isolated from poppies years ago [2,51]. It has been proposed that sanguinarine occurs in the oxidized form only on the outside of plant cells and as dihydrosanguinarine on the inside [4]. The toxic extracellular form serves as a defense against fungi whereas the non-toxic intracellular form is used for biosynthesis of other plant compounds [4]. In contrast, mammals do not seem to metabolize the reduced form(s) further. Instead, we saw evidence of reoxidation in several intracellular locations. This indicates that the reduced phenanthridines (and perhaps the oxidized forms as well) interact with different mammalian enzymes. Oxidation might be catalyzed by Nox-family NAD(P)H oxidases associated with internal membranes and organelles, by enzymes involved in mitochondrial electron transfer or by both. Our observation that rotenone and antimycin A, inhibitors of mitochondrial electron transfer, block phenanthridine-induced apoptosis temporarily indicates that participation of the mitochondrial electron transfer facilitates redox cycling but is not absolutely required. Mitochondrial electron transfer could provide electrons for reduction as well as protons for the formation of neutral dihydrophenanthridines. Although the neutral dihydrophenanthridines are more easily oxidized than the anions, the reaction with oxygen under physiological conditions probably requires enzymatic catalysis. A scenario in which multiple enzymes at various locations in the cell, on the cell surface and in mitochondria facilitate redox reactions involving phenanthridines is illustrated in Fig. 8. The production of H2O2 at different locations provides another explanation for the observation that addition of extracellular catalase restores viability only partially. Several lines of evidence indicate that H2O2 is responsible for the rapid apoptosis in cells treated with sanguinarine or chelerythrine. Although it is difficult to measure H2O2 levels accurately, one estimate places them on the order of 100 nM in normal cells [52]. Small increases in H2O2 concentration (10 nM–1 lM) are thought to increase cellular proliferation [53], whereas treatment of cells with approximately 50 lM H2O2 induces apoptosis [54–56]. Concentrations of 500 lM or more lead to cell death by necrosis [54,57]. The exact amount of H2O2 required for induction of cell

3 Fig. 7. Effect of antioxidants and inhibitors of electron transfer on apoptosis induced by sanguinarine and chelerythrine. In (A)–(C) DNA apoptotic double strand breaks were detected by H2AX phosphorylation. Cells in A (lanes 1–6) and C (lanes 1–3) were treated with 3 lM sanguinarine, cells in B (lanes 1–6) and C (lanes 4–6) were treated with 7.5 lM chelerythrine for 4 h. In addition, lanes A2 and B2 were treated with 10 mM NAC, lanes A3 and B3 with 10 lM DPI, lanes A4 and B4 with 1 mM DTT, lanes A5 and B5 with 10 mM GSH, lanes A6 and B6 with 20 lM rotenone, lanes C2 and C5 with 20 lM antimycin and lanes C3 and C6 with 20 lM allopurinol. Untreated controls are in lane 7 (A–C). The effect of the inhibitors on apoptosis was also shown by PARP cleavage (D, E). Lanes 1 – 3 were treated with 3 lM sanguinarine, lanes 4–6 were treated with 7.5 lM chelerythrine and lane 7 contains untreated control. In addition, lanes D2 and D5 were treated with 10 lM DPI, lanes D3 and D6 with 20 lM rotenone, lanes E2 and E5 with 20 lM antimycin and lanes E3 and E6 with 20 lM allopurinol.

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Pox

NAD+ 1.

H2O2 O2

NADH

NADH

NAD+

3.

Pox

PH2

Pred I.

2.

NADH

NAD+

H2O2 O *2

O2 H2O2

O2

Pox

PH2 III.

Fig. 8. Model illustrating how several different enzymes could be involved in the production of large amounts of H2O2 and redox cycling of the phenanthridines (Pox, Pred and PH2) and the generation of ROS: 1. cell surface NAD(P)H oxidase or ECTO-NOX protein, 2. and 3. intracellular NAD(P)H oxidases, I. and III. mitochondrial complexes.

death depends on cell type and density [52]. We noticed that sanguinarine and chelerythrine behave like H2O2 in several experiments, for example in the comet assays. Like H2O2, sanguinarine and chelerythrine can induce different types of cell death depending on the concentration but their concentrations are approximately 50–100 times lower. Thus, we suspect that sanguinarine and chelerythrine generate large amounts of H2O2 by undergoing repeated cycles of reduction and oxidation. In addition to the biological effects of large amounts of H2O2 produced during redox cycling, the repeated reduction of each molecule of sanguinarine or chelerythrine likely depletes reduced coenzymes, which further accelerates apoptosis in cells treated with these compounds. The production of very large amounts of H2O2 explains also why phenanthridine-induced apoptosis does not require time-consuming signaling and transcriptional activation such as p53-mediated DNA damage response. Since ROS are downstream signals in the p53-mediated mitochondrial apoptosis pathway [58–61], the production of H2O2 would be expected to short-circuit this pathway and explain our previous observation that apoptosis initiated by sanguinarine is independent of p53 [15]. Induction of p53-independent cell death could be used as an argument for promoting the use of sanguinarine and chelerythrine for the treatment of cancer since loss of p53 function reduces the efficacy of many anticancer agents. It has also been suggested that sanguinarine is particularly valuable for the treatment of cancer because it acts specifically on cancer cells [12,13]. This suggestion was based on a study involving a single cancer cell line (A431) and a single non-cancerous cell line (NHEK) [6]. However, we studied several cell lines including the non-cancerous CHO cell line AA-8 and found that sanguinarine and chelerythrine induce apoptosis regardless of cell type as long as the cells are proliferating, have all the factors required for apoptosis execution and do not express excessive amounts of catalase. The resistance to phenanthridineinduced cell death in slowly dividing and near-confluent cells could be due to diminished levels of reduced coenzymes or lower activity of crucial oxidoreductases. There is evidence that some redox enzymes, perhaps the enzymes which interact with sanguinarine and chelerythrine, are particularly active in fast-growing cells [19,53,62]. Since rapidly dividing cells are sensitive to the antipro-

liferative effect of sanguinarine and chelerythrine, these compounds likely cause the same side effects as other anticancer agents which target rapidly proliferating cells. In addition, cells with a high density of mitochondria may be particularly sensitive to the effects of sanguinarine and chelerythrine. In the case of anthracyclines, which can also undergo redox cycling, the interaction with enzymes involved in mitochondrial electron transfer has been linked to their cardiotoxicity [63]. Thus, the interaction of sanguinarine and chelerythrine with mitochondrial enzymes may also cause cardiac side effects. However, the chemistry involved in the redox cycling of phenanthridines is different from the chemistry of anthracyclines and requires further investigation. Acknowledgments This research was supported by internal grants from the University of the Pacific. We acknowledge Ms. Christy Young for her work on the chemical synthesis and chromatography of dihydrosanguinarine. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.abb.2008.05.019. References [1] S. Blechert, W. Brodschelm, S. Hoelder, L. Kammerer, T.M. Kutchan, M.J. Mueller, Z.-Q. Xia, M.H. Zenk, The octadecanoic pathway: signal molecules for the regulation of scondary pathways, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 4099–4105. [2] H. Arakawa, W.G. Clark, M. Psenak, C.J. Coscia, Purification and characterization of dihydrobenzophenanthridine oxidase from elicited Sanguinaria canadensis cell cultures, Arch. Biochem. Biophys. 299 (1992) 1–7. [3] S.E. Newman, M.J. Roll, A naturally occuring compound for controlling powdery mildew of greenhouse roses, Hort. Science 34 (1999) 686–689. [4] D. Weiss, A. Baumert, M. Vogel, W. Roos, Sanguinarine reductase, a key enzyme of benzophenanthridine detoxification, Plant, Cell and Environment 29 (2006) 291–302. [5] J.M. Herbert, J.M. Augereau, J. Gleye, J.P. Maffrand, Chelerythrine is a potent and specific inhibitor of protein kinase C, Biochem. Biophys. Res. Comm. 172 (1990) 993–999.

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