Molecular cytotoxic mechanisms of anticancer hydroxychalcones

Chemico-Biological Interactions 148 (2004) 57–67

Molecular cytotoxic mechanisms of anticancer hydroxychalcones Omid Sabzevari a,b , Giuseppe Galati c , Majid Y. Moridani a , Arno Siraki a , Peter J. O’Brien a,c,∗ a

b

Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, ON, Canada M5S 2S2 Department of Toxicology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box: 14155/6451, Tehran, Iran c Department of Pharmacology, University of Toronto, Toronto, ON, Canada M5S 2S2 Received 25 January 2004; received in revised form 18 April 2004; accepted 20 April 2004

Abstract Chalcones are being considered as anticancer agents as they are natural compounds that are particularly cytotoxic towards K562 leukemia or melanoma cells. In this study, we have investigated phloretin, isoliquiritigenin, and 10 other hydroxylated chalcones for their cytotoxic mechanisms towards isolated rat hepatocytes. All hydroxychalcones partly depleted hepatocyte GSH and oxidized GSH to GSSG. These chalcones also caused a collapse of mitochondrial membrane potential and increased oxygen uptake. Furthermore, glycolytic or citric acid cycle substrates prevented cytotoxicity and mitochondrial membrane potential collapse. The highest pKa chalcones were the most effective at collapsing the mitochondrial membrane potential which suggests that the cytotoxic activity of hydroxychalcones are likely because of their ability to uncouple mitochondria. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Chalcone; Mitochondria; Toxicity; Hepatocytes; Glutathione; Anticancer

1. Introduction Abbreviations: 2 -HC, 2 -hydroxychalcone; 2-HC, 2-hydroxychalcone; 4 -HC, 4 -hydroxychalcone; 4-HC, 4-hydroxychalcone; 2 ,2-DHC, 2 ,2-dihydroxychalcone; 2 ,3-DHC, 2 ,3-dihydroxychalcone; 2 ,4-DHC, 2 ,4-dihydroxychalcone; 2 ,4 -DHC, 2 ,4 -dihydroxychalcone; 2 ,5 -DHC, 2 ,5 -dihydroxychalcone; 2 ,3 ,4 THC, 2 ,3 ,4 -trihydroxychalcone; Isoliquiritigenin, 2 ,4 ,4-trihydroxychalcone; NQO1, NAD(P)H: quinone reductase; Phloretin, 2 ,4 ,6 ,4-tetrahydroxydihydrochalcone; DTNB, 5,5 -dithiobis-(2-nitrobenzoic acid); GSH, glutathione (reduced); GSSG, glutathione (oxidized); GST, glutathione S-transferase ∗ Corresponding author. Tel.: +1-416-9782716; fax: +1-416-9788511. E-mail address: [email protected] (P.J. O’Brien).

Polyphenolics form a major part of the dietary antioxidant capacity of fruits and vegetables and have been identified as chemopreventive or anticancer agents by many researchers using several animal cancer models and cell cultures. Hydroxychalcones are abundantly distributed throughout the plant kingdom and are compounds with two aromatic rings (benzene or phenol) and an unsaturated side chain, i.e., phenyl ketone analogs of cinnamic acid. In plants, flavonoids, and isoflavonoids are synthesized from hydroxychalcones which are synthesized from p-coumaroyl-CoA

0009-2797/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2004.04.004

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O. Sabzevari et al. / Chemico-Biological Interactions 148 (2004) 57–67

and malonyl-CoA by enzyme catalyzed condensation and cyclization reactions [1–3]. Isoliquiritigenin (2 ,4 ,4-trihydroxychalcone, a licorice chalcone), is currently in use as a phosphodiesterase III inhibitor for the treatment of cardiovascular diseases [4]. Chalcones are also potent as anti-inflammatory agents [5–7]. Plant hydroxychalcones have recently provoked attention because of their effectiveness as antitumor agents (antimitotic, cell growth inhibitor) towards cultured tumor cells [8–14]. Isoliquiritigenin or the dihydrochalcone phloretin (a major apple chalcone) induced apoptosis in B16 melanoma cells which was suggested to result from the inhibition of glucose transmembrane transport, the inhibition of lipoxygenase activity, promotion of Bax protein expression or activation of caspases [12,14,15]. Isoliquiritigenin-induced apoptosis in human gastric cancer cells was proposed to occur through a calcium dependent mitochondrial toxicity pathway [16]. Chalcones are also effective in vivo in preventing tumor cell proliferation in rats [9,10]. In vivo, chalcones (e.g., isoliquiritigenin) are also potent anti-tumor-promoting agents [17–19]. Chalcones are also effective in vivo as chemopreventive agents in several rat carcinogenesis models [17,20,21]. Induction of phase II enzymes and increasing glutathione levels are major chemopreventive strategies for preventing chemical carcinogenesis. Various chalcones incubated with cultured cells readily induced NAD(P)H: quinone reductase (NQO1) [22,23] as well as glutathione S-transferase (GST) [24] and would be expected to alleviate oxidative stress and detoxify mutagenic xenobiotics. The most effective chalcones for inducing NQO1 and GST were 2,2 -dihydroxychalcone and 2 ,3 ,4 -trihydroxychalcone [22,24]. Recently, we reviewed the tumor cell apoptosis mechanisms induced by dietary flavonoids and other polyphenolics, and shown that the chalcone phloretin was among the most cytotoxic agents [25]. However, for chalcones to be effective anticancer agents, they should not be toxic to normal cells. In the following, the molecular cytotoxic mechanisms of phloretin, isoliquiritigenin, and 10 other chalcones (Fig. 1) towards isolated rat hepatocytes was investigated using “accelerated cytotoxic mechanism screening” (ACMS) techniques, utilizing high doses and short

incubation times [25–27]. Our findings suggest that the cytotoxicity of chalcones to hepatocytes and possibly tumor cells is likely due to their ability to form prooxidant phenoxyl radicals and uncouple mitochondria. Furthermore, quantitative structure toxicity relationships (QSTRs) were derived in order to identify significant physicochemical parameters of chalcones that correlated with cytotoxicity. Such parameters could aid in the development of anticancer drugs. 2. Materials and methods 2.1. Materials 2 -Hydroxychalcone (2 -HC) was obtained from Extrasynthese (France). 4 -Hydroxychalcone (4 -HC), 2-hydroxychalcone (2-HC), 4-hydroxychalcone (4HC), 2 ,2-dihydroxychalcone (2 ,2-DHC), 2 ,3-dihydroxychalcone (2 ,3-DHC), 2 ,4-dihydroxychalcone (2 ,4-DHC), 2 ,4 -dihydroxychalcone (2 ,4 -DHC), 2 ,5 -dihydroxychalcone (2 ,5-DHC), 2 ,4 ,4-trihydroxychalcone (isoliquiritigenin), and 2 ,3 ,4 -trihydroxy chalcone (2 ,3 ,4 -THC) were purchased from Indofine Chemical Company Inc. (NJ, USA). 2 ,4 ,6 ,4Tetrahydroxydihydrochalcone (phloretin) was obtained from Toronto Research Chemicals (Canada). Collagenase (from Clostridium histolyticum), bovine serum albumin (BSA), and Hepes were purchased from Boehringer–Mannheim (Montreal, QU, Canada). Trypan blue, Tris–HCl, sodium azide, βNADPH, DTNB (5,5 -dithio-bis-(2-nitrobenzoic acid)), iodoacetic acid, 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), trichloroacetic acid, dicumarol, dimethyl sulfoxide, fluoro-2,4-dinitrobenzene, digitonin, rhodamine 123, heparin, sodium pentobarbital, β-d(−)-fructose, d-sorbitol, pyruvate, and xylitol were obtained from Sigma (St. Louis, MO, USA). 1-Bromoheptane was purchased from Aldrich Chemical Company (Milwaukee, WI, USA). HPLC-grade solvents were obtained from Caledon (Georgetown, ON, Canada). All chemicals were of the highest available commercial grade. 2.2. Animals Male Sprague–Dawley rats (280–300 g), fed a standard chow diet and given water ad libitum, were used in all experiments.

O. Sabzevari et al. / Chemico-Biological Interactions 148 (2004) 57–67 5 6

5' 4' 3'

6'

1 2

A 2'

B

59

4 3 OH

1'

OH O

O

2'-Hydroxychalcone (2'-HC)

2-Hydroxychalcone (2-HC) OH

HO

O

O

4'-Hydroxychalcone (4'-HC)

4-Hydroxychalcone (4-HC)

OH OH OH

OH O

O

2',2-Dihydroxychalcone (2',2-DHC)

2',3-Dihydroxychalcone (2',3-DHC)

OH HO

OH O

OH O

2',4-Dihydroxychalcone (2',4-DHC)

2',4'-Dihydroxychalcone (2',4'-DHC)

OH HO HO OH O

OH

2',5'-Dihydroxychalcone (2',5'-DHC)

O

2',3',4'-Trihydroxychalcone (2',3',4'-THC)

OH HO

OH HO

OH

O

2',4',4-Trihydroxychalcone (Isoliquiritigenin)

OH

OH O

2',4',6',4-Tetrahydroxydihydrochalcone (Phloretin)

Fig. 1. Chemical structures of the hydroxychalcones used in this study.

2.3. Isolation and incubation of hepatocytes Freshly isolated hepatocytes were prepared by collagenase perfusion of the liver of rats as described by Moldeus et al. [28]. Approximately 85–90% of the hepatocytes excluded trypan blue. The cells were sus-

pended at a density of 106 cells/ml in round bottomed flasks rotating in a water bath maintained at 37 ◦ C in Krebs–Henseleit buffer (pH 7.4), supplemented with 12.5 mM Hepes under an atmosphere of 10% O2 : 85% N2 : 5% CO2 . Stock solutions of chemicals were made in DMSO and aliquots were added to the hepa-

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O. Sabzevari et al. / Chemico-Biological Interactions 148 (2004) 57–67

tocyte suspensions following a 30 min pre-incubation period. 2.4. Cell viability The viability of isolated hepatocytes and the LD50 (2 h) were assessed from the integrity of the plasma membrane as determined by the trypan blue (0.2% (w/v)) exclusion test in aliquots taken at different time points during the 3 h incubation period [28]. 2.5. HPLC analysis of hepatocyte GSH/GSSG The total amount of GSH and GSSG in hepatocytes and isolated microsomes were measured by HPLC analysis of deproteinized samples (25% metaphosphoric acid) after derivatization with iodoacetic acid and fluoro-2,4-dinitrobenzene [29] using a Waters HPLC system (Model 510 pumps, WISP 710B auto injector, and model 410 UV-Vis detector) equipped with a Waters ␮Bondapak® NH2 (10 ␮m) 3.9 mm × 300 mm column. GSH and GSSG were used as external standards. 2.6. GSH depleted hepatocytes Bromoheptane rapidly depletes hepatocyte GSH by >95%, whereas, inhibiting hepatocyte GSH synthesis with buthionine sulfoxide depletes GSH by 50–62%. GSH-depleted hepatocytes were therefore prepared by pre-incubating the hepatocytes (10 ml, 106 cells/ml) with 200 ␮M 1-bromoheptane (a GSH S-transferase substrate) for 30 min as described previously [30].

cence spectrophotometer set at 490 nm excitation and 520 nm emission wavelengths. The capacity of mitochondria to take up the rhodamine 123 was calculated as the difference between control and treated cells. The control hepatocytes had a mitochondrial membrane potential equal to 502 ± 22 fluorescent units for 106 hepatocytes per ml. 2.8. Oxygen uptake Hepatocyte respiration was measured using a Clark type oxygen electrode (Yellow Springs Instrument Co., Inst. Model 53) in a 2 ml chamber maintained at room temperature. Following a 1 h pre-incubation period of hepatocyte suspension (at a density of 106 cells/ml in round bottom flasks rotating in a water bath maintained at 37 ◦ C), a 2 ml aliquot was transferred to the chamber and the oxygen uptake was recorded before and after addition of chalcones [32]. 2.9. QSTR analysis and statistical parameters The QSTR analysis was carried out using the log LD50 (i.e., the chalcone concentration required to cause 50% cell death in 2 h) and the number of hydroxy groups on the A-ring and/or the B-ring. The statistical parameters used in the studies describing the QSTR equations are: n, the number of data points upon which the equation is based; r2 , the square value for the correlation coefficient; s, the standard deviation; and P, the significance coefficient.

3. Results 2.7. Mitochondrial membrane potential assay The uptake of the cationic fluorescent dye, rhodamine 123, was used for the estimation of mitochondrial membrane potential [31]. Aliquots of the cell suspension (0.5 ml) were separated from the incubation medium by centrifugation at 1000 g for 1 min. The cell pellet was then resuspended in 2 ml of fresh incubation medium containing 1.5 ␮M rhodamine 123 and incubated at 37 ◦ C in a thermostatic bath for 10 min with gentle shaking. Hepatocytes were then separated by centrifugation and the amount of rhodamine 123 remaining in the incubation medium was measured fluorimeterically using a Shimadzu RF5000U fluores-

We have investigated phloretin, isoliquiritigenin, and 10 other chalcones (Fig. 1) for their cytotoxic mechanisms towards isolated rat hepatocytes. The LD50 (2 h), i.e., the concentration of chalcones required to cause 50% cytotoxicity after a 2 h incubation at 37 ◦ C was obtained (Table 1). Cytotoxicity was induced by 100–400 ␮M chalcone concentrations. The cytotoxic effectiveness of chalcones in order of increasing LD50 (2 h) were as follows: 2 ,2-dihydroxychalcone (2 ,2-DHC) ≥ 2 ,4-dihydroxychalcone (2 ,4DHC) > 2 ,3-dihydroxychalcone (2 ,3-DHC) > 2 hydroxychalcone (2 -HC) > 2 ,5 -dihydroxychalcone (2 ,5 -DHC) > 2-hydroxychalcone (2-HC) > 2 ,4 -di-

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61

Table 1 Hepatocyte LD50 (2 h) for dietary hydroxychalcones Hydroxychalcone

Cytotoxicity

Number of OH groups

LD50 (␮M) (2 h) 2 -Hydroxychalcone 2-Hydroxychalcone 4 -Hydroxychalcone 4-Hydroxychalcone 2 ,2-Dihydroxychalcone 2 ,3-Dihydroxychalcone 2 ,4-Dihydroxychalcone 2 ,4 -Dihydroxychalcone 2 ,5 -Dihydroxychalcone 2 ,3 ,4 -Trihydroxychalcone Isoliquiritigenin Phloretin

115 125 200 300 100 110 100 140 120 180 215 400

± ± ± ± ± ± ± ± ± ± ± ±

13 15 22 29 11 12 11 16 14 22 27 35

Calculated log LD50

log LD50

A-ring

B-ring

Total (A + B)

Eq. (1)

Eq. (2)

Eq. (3)

2.06 2.10 2.30 2.48 2.00 2.04 2.00 2.15 2.08 2.26 2.33 2.60

1 0 1 0 1 1 1 2 2 3 2 3

0 1 0 1 1 1 1 0 0 0 1 1

1 1 1 1 2 2 2 2 2 3 3 4

2.02 1.82 2.01 1.82 2.02 2.02 2.02 2.21 2.21 2.41 2.21 2.41

1.90 1.90 1.90 1.90 2.10 2.10 2.10 2.10 2.10 2.31 2.31 2.51

1.92 1.84 1.92 1.84 2.06 2.06 2.06 2.14 2.14 2.36 2.28 2.50

Mean ± S.E.M. for three separate experiments are given.

hydroxychalcone (2 ,4 -DHC) > 2 ,3 ,4 -trihydroxychalcone (2 ,3 ,4 -THC) > 4 -hydroxychalcone (4-HC) > isoliquiritigenin > 4-hydroxychalcone (4-HC) > phloretin (Table 1). All chalcones, at LD50 (2 h) concentrations, depleted hepatocyte GSH either by GSH oxidation to GSSG or likely by conjugate formation with GSH as GSH was depleted without GSH oxidation (Table 2). 2 ,3 ,4 -THC was the most potent GSH de-

pleting chalcone while phloretin and isoliquiritigenin had the least effect on hepatocyte GSH (Table 2). 2 -HC, 2-HC, 2 ,2-DHC, 2 ,3-DHC, 2 ,4-DHC, and 2 ,4 -DHC caused mainly GSH oxidation to GSSG, however, GSH depletion by 2 ,3 ,4 -THC did not involve significant GSH oxidation (Table 2). GSH depletion with some GSH oxidation also occurred with 2 ,5 -DHC. The cytotoxicity of all studied chalcones was greater in GSH depleted hepatocytes particularly

Table 2 Modulation of GSH by hydroxychalcones in hepatocytes Hydroxychalconea

GSH Control 2 -Hydroxychalcone 2-Hydroxychalcone 4 -Hydroxychalcone 4-Hydroxychalcone 2 ,2-Dihydroxychalcone 2 ,3-Dihydroxychalcone 2 ,4-Dihydroxychalcone 2 ,4 -Dihydroxychalcone 2 ,5 -Dihydroxychalcone 2 ,3 ,4 -Trihydroxychalcone Isoliquiritigenin Phloretin

GSH-depleted hepatocytesb LD50 (␮M) (2 h)

Normal hepatocytes (nmol/106 cells at 1 h)

52 13 11 22 20 18 18 25 24 6 4 40 36

± ± ± ± ± ± ± ± ± ± ± ± ±

GSSG 5 1 1 2 2 3 2 3 2 1 1 4 4

10 22 22 16 15 24 21 19 18 18 13 12 12

± ± ± ± ± ± ± ± ± ± ± ± ±

2 3 3 2 2 3 2 2 2 2 2 1 2

Mean ± S.E.M. for three separate experiments are given. a Hydroxychalcone concentration used was the LD 50 (2 h) concentration shown in Table 1. b GSH-depleted hepatocytes contained <5 nmol GSH per 106 hepatocytes.

– 75 85 145 200 60 70 70 80 75 75 195 220

± ± ± ± ± ± ± ± ± ± ± ±

8 9 17 26 7 8 7 9 9 8 21 25

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Table 3 Hepatocyte mitochondrial membrane potential collapse initiated by hydroxychalcones

Table 4 Effect of glycolytic substrates on phloretin induced cytotoxicity and mitochondrial membrane potential

Hydroxychalconea

Mitochondrial membrane potential (%) ψ (1 h)

Cytotoxicity (%) (1 h)

Addition

Mitochondrial membrane potential (%) ψ (2 h)

Cytotoxicity (%) (2 h)

Control 2 -Hydroxychalcone 2-Hydroxychalcone 4 -Hydroxychalcone 4-Hydroxychalcone 2 ,2-Dihydroxychalcone 2 ,3-Dihydroxychalcone 2 ,4-Dihydroxychalcone 2 ,4 -Dihydroxychalcone 2 ,5 -Dihydroxychalcone 2 ,3 ,4 -Trihydroxychalcone Isoliquiritigenin Phloretin Dicumarol (20 ␮M)

100 23 23 26 28 22 27 33 30 32 43 40 38 84

19 20 22 24 24 25 24 24 25 25 20 26 25 21

Control Phloretin (400 ␮M) +Sorbitola (10 mM) +Xylitola (10 mM) +Fructosea (10 mM) +Pyruvatea (10 mM)

100 26 98 96 89 86

23 52 31 26 29 32

± ± ± ± ± ± ± ± ± ± ± ± ±

4 3 4 5 2 4 5 5 6 7 7 5 9

± ± ± ± ± ± ± ± ± ± ± ± ± ±

2 2 3 3 3 4 4 3 4 3 2 5 3 2

Mean ± S.E.M. for three separate experiments are given. a Hydroxychalcone concentration used was the LD (2 h) con50 centration shown in Table 1.

with phloretin and 2 ,3 ,4 -THC, whereas, isoliquiritigenin and 2 ,4-DHC showed a smaller increase in cytotoxic effectiveness (Table 2). As shown in Table 3, all chalcones caused a collapse of mitochondrial membrane potential before cytotoxicity ensued. This occurred with low concentrations of 2 ,2-DHC, 2 -HC, and 2 ,3-DHC, whereas, higher concentrations of isoliquiritigenin, 2 ,3 ,4 -THC and phloretin, were required to collapse the mitochondrial membrane potential. Table 4 shows that the collapse of the mitochondrial membrane potential and cytotoxicity induced by phloretin, were significantly prevented by the glycolytic or citric acid cycle substrates sorbitol, xylitol, fructose, and pyruvate. All of the chalcones studied, at their LD50 concentrations, caused an increase in hepatocyte respiration indicating an uncoupling of mitochondrial oxidative phosphorylation. A dose–response study of isoliquiritigenin showed that hepatocyte respiration was induced over a concentration range 10–215 ␮M (results not shown). The inhibition of hepatocyte NQO1 with dicumarol markedly increased 2 ,3-DHC, 2 ,4 -DHC, 2 ,5 -DHC, isoliquiritigenin, and 2 ,3 ,4 -THC-induced cytotoxicity, but not the phenols 2 -HC, 2-HC, 4 -HC, 2 ,2-DHC, and 2 ,4-DHC, suggesting that cytotoxic-

± ± ± ± ±

3 9∗ 9∗ 8∗ 7∗

± ± ± ± ± ±

3 6 3∗ 3∗ 3∗ 3∗

a Sorbitol, xylitol, fructose, and pyruvate were pre-incubated for 30 min prior to the addition of phloretin. Mean ± S.E.M. for three separate experiments are given. ∗ Significant difference from phloretin treatment (ANOVA, P < 0.001). Mean ± S.E.M. for three separate experiments are given.

ity induced by quinone metabolite forming chalcones were detoxified by NQO1 (Fig. 2).

4. Discussion We have investigated the cytotoxic mechanism of phloretin, isoliquiritigenin, and 10 other hydroxylated chalcones towards isolated rat hepatocytes using ACMS techniques [25–27]. The cytotoxic effectiveness of chalcones in order of decreasing toxicity was as follows: 2 ,2-DHC ≥ 2 ,4-DHC > 2 ,3-DHC > 2 -HC > 2 ,5 -DHC > 2-HC > 2 ,4 -DHC > 2 ,3 ,4 -THC > 4-HC > isoliquiritigenin > 4 -HC > phloretin. This suggests that chalcones with phenols on both A- and B-rings were more toxic than chalcones that contained a benzene B-ring and a polyphenol (i.e., hydroquinone, resorcinol, and pyrogallol) A-ring. Phloretin may be the least cytotoxic because it has the most hydroxy groups and also lacks a double bond. All chalcones depleted hepatocyte GSH either by GSH oxidation to GSSG or by conjugate formation with GSH. The chalcone 2 ,3 ,4 -THC was the most potent GSH depleting chalcone while phloretin and isoliquiritigenin had the least effect on hepatocyte GSH. The chalcones 2 -HC, 2-HC, 2 ,2-DHC, 2 ,3-DHC, 2 ,4-DHC, and 2 ,4 -DHC caused mainly GSH oxidation to GSSG most likely as a result of oxidation to phenoxyl radicals. The cytotoxicity of 70 phenols towards L1210 tumor cells correlated with

O. Sabzevari et al. / Chemico-Biological Interactions 148 (2004) 57–67

63

Fig. 2. Effect of NQO1 inhibition on hydroxychalcone-induced hepatocyte toxicity. Isolated rat hepatocytes (106 cells/ml) were incubated in round-bottomed flasks rotating in a water bath maintained at 37 ◦ C in Krebs–Henseleit buffer (pH 7.4) with low toxic concentrations of hydroxychalcones. Cytotoxicity was determined as the percentage of cells that take up trypan blue after 3 h. NQO1 inactivation was achieved by pre-incubation of hepatocytes (10 ml, 106 cells/ml) with 20 ␮M dicumarol for 30 min. ∗ Significant difference from normal hepatocytes (ANOVA, P < 0.001).

their bond dissociation energy which indicated that cytotoxicity is related to the phenols hydrophobicity and ease of oxidation to phenoxyl radicals [33]. We have previously published data showing that GSH is readily oxidized to GSSG by the phenoxyl radicals formed when flavonoids or chalcones undergo a peroxidase-catalyzed oxidation [34]. In addition, the chalcones have been shown to inhibit glutathione reductase in the following order of IC50 effectiveness: 4 -HC (47 ␮M) > butein, phloretin (71 ␮M) > 2 -HC (82 ␮M) > 2-HC (123 ␮M) [35,36]. However, GSH depletion without significant GSSG formation by other chalcones were likely due to conjugate formation with GSH. The chalcones 4 -HC, 4-HC, 2 ,5 -DHC, 2 ,3 ,4 -THC, isoliquiritigenin, and phloretin may have formed GSH conjugates by a Michael addition to the α,β-unsaturated carbonyl moiety as occurred for curcumin [37]. The order of cytotoxic effectiveness we found in isolated rat hepatocytes, i.e., 2,2 -DHC > 2 -HC > 2-HC > 4 -HC > 4-HC was identical to the order of poten-

cies of these hydroxychalcones on inducing NQO1 activity in Hepa 1c1c7 murine hepatoma cells found by Dinkova-Kostova et al. [22]. This order was also similar to the order of chalcone effectiveness in depleting hepatoctye GSH and forming GSSG. The similarity between the two results suggests similar chalcone molecular cytotoxic mechanisms towards isolated rat hepatocytes and tumor cells. Recently, we published a similar cytotoxic effectiveness of flavonoids towards isolated rat hepatocytes and HeLa tumor cells [38]. Hepatocyte GSH depletion induced by chalcones preceded cytotoxicity, and GSH-depleted hepatocytes were much more susceptible to chalcones particularly 2 ,3 ,4 -THC (2.4-fold decrease of LD50 ). The increase in chalcone toxicity in GSH-depleted hepatocytes were as follows: 2 ,3 ,4 -THC > phloretin > 2 ,4 -DHC > 2 ,2-DHC ≥ 2 ,5 -DHC > 2 ,3-DHC > 2-HC > 2 -HC > 4-HC > 4 -HC > 2 ,4-DHC > isoliquiritigenin. This suggests that GSH plays an important role in chalcone detoxification, e.g., by forming GSH conjugates with quinone metabolites.

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Furthermore, NQO1-inactivated hepatocytes were also much more susceptible to isoliquiritigenin, 2 ,3-DHC, 2 ,4 -DHC, 2 ,5 -DHC, and 2 ,3 ,4 -THC than control hepatocytes suggesting that these chalcones underwent metabolism to a reactive quinone which could be detoxified by NQO1. However, other phenolic chalcones were not more susceptible suggesting that any quinone metabolites formed were not sufficient to induce cytotoxicity. These phenolic chalcones were also more effective at collapsing the mitochondrial membrane potential making it unlikely that the dicumarol used to inactivate NQO1 also acted as a mitochondrial uncoupler. Furthermore, dicumarol at the concentration used (20 ␮M) only slightly decreased the hepatocyte mitochondrial membrane potential (Table 3). The microsomal catalyzed GSH depletion by chalcones in order of decreasing effectiveness were as follows: 2 ,3 ,4 -THC > 2 ,5 -DHC > 2-HC > 2 ,4 -DHC > 2 -HC > 2 ,3-DHC > 4 -HC > 4-HC > phloretin. The accelerating effect of chalcone ortho-hydroxy groups on the reactivity of the α,β-unsaturated carbonyl with GSH may result from the lowering of the pKa of the SH group through intermolecular inductive hydrogen bonding with the neighboring phenolic hydroxy groups. This explanation was suggested for the sulfhydryl reactivity of benzylidenealkanones [22]. As is evident from the cytotoxicity and GSH depletion studies, a QSTR pattern could be established between chalcones and their LD50 -induced cytotoxicity in isolated hepatocytes: i.e., the cytotoxicity decreases by an increase in the number of hydroxy groups on the chalcone A-ring. Using cytotoxicity data presented in Table 1, we have derived three QSTRs for the chalcones studied here: log LD50 (␮M) = 0.198(±0.050)A + 1.818(±0.097) (1) n = 9, r 2 = 0.691, s = 0.118, P < 0.005; outliers: 2-HC, 4-HC, 4 -HC; “A” is the number of hydroxy groups on the chalcone A-ring log LD50 (␮M) = 0.204(±0.039)(A + B) + 1.693(±0.097) r2

(2)

n = 9, = 0.795, s = 0.096, P < 0.001; outliers: 2-HC, 4-HC, 4 -HC; “A+B” is the total number of hydroxy groups on both of the chalcone rings combined

log LD50 (␮M) = 0.221(±0.041)A + 0.145(±0.065)B + 1.696(±0.095)

(3)

n = 9, r 2 = 0.830, s = 0.094, P < 0.002 (“A” parameter and intercept), P < 0.07 (“B” parameter); outliers: 2-HC, 4-HC, 4 -HC; “A” and “B” are the numbers of hydroxy groups on the chalcone A-ring and B-ring, respectively. The calculated LD50 (␮M) values for chalcones derived from Eqs. (1)–(3) were close to the actual cytotoxicity measurements (Table 1). The three different equations included similar parameters, i.e., the presence of hydroxyl groups on A- and/or B-rings, which validated the importance of hydroxy groups in chalcone cytotoxicity. Furthermore, the regression of the equations gradually improved, resulting in the derivation of a more statistically significant QSTR. 2 -HC, 4 -HC, 2 ,2-DHC, 2 ,3-DHC, and 2 ,4-DHC all have one hydroxy group on their A-ring. The order of cytotoxicity for this sub-group of chalcones was as follows: 2 ,2-DHC > 2 ,4-DHC > 2 ,3-DHC > 2 -HC > 4 -HC. Although, 2 ,2-DHC, 2 ,4-DHC, and 2 ,3-DHC have one hydroxy group more than 2 -HC and 4 -HC, they were slightly more toxic likely because their extra hydroxy group on the chalcone B-ring can be potentially metabolized to a phenoxyl radical which can readily oxidize a glutathione molecule. Alternatively, these compounds may undergo ring hydroxylation to form a catechol, which can then be further metabolized to a quinone, which form GSH conjugates. The order of cytotoxicity for the second sub-group of chalcones which have two hydroxy groups on their A-ring was 2 ,5 -DHC > 2 ,4 -DHC > 2 ,4 ,4-THC (isoliquiritigenin). 2 ,5 -DHC is the only chalcone from this sub group which does not require a ring hydroxylation or ring epoxidation prior to bioactivation to form a reactive intermediate species (i.e., a hydroquinone) and explains why it is the most toxic chalcone. On the other hand, isoliquiritigenin (2 ,4 ,4-THC) may be a less effective prooxidant than 2 ,4 -DHC because it has an extra hydroxy group on its B-ring which makes the molecule less prooxidant. The third sub-group of chalcones has three hydroxy groups on their A-ring and their cytotoxic effectiveness was 2 ,3 ,4 -THC > 2 ,4 ,6 ,4-dihydrochalcone (phloretin). As is evident from the chemical struc-

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tures of these two chalcones, 2 ,3 ,4 -THC can readily form a quinone reactive intermediate species whereas, phloretin not only cannot form this reactive intermediate species but also is less prooxidant because of an extra hydroxy group on the B-ring. In addition, the three hydroxy groups present in the phloretin chemical structure are arranged on the A-ring in such a fashion that they cannot form a quinone reactive species upon their oxidation or metabolism to a phenoxyl radical. It should also be noted that an increase in the number of hydroxy groups on an aromatic ring decreases the pKa value of the first hydroxy group for a polyphenolic compound: e.g., pKa (pyrogallol) < pKa (catechol) < pKa (phenol). Based on this criterion, it seems that chalcones with lower pKa values are less toxic to hepatocytes because they have more hydroxy groups on their A-ring than those with higher pKa values. However, it should be also borne in mind that the arrangement of hydroxy groups on the A-ring or B-ring plays a crucial role in determining the mechanism of chalcone cytotoxicity towards isolated rat hepatocytes. For instance, if the hydroxy groups are in the ortho position, the chalcone can be potentially metabolized to a quinone reactive intermediate species, which can react with cell nucleophiles and deplete GSH. It was also found that the chalcones which have a fewer number of hydroxy groups on their aromatic rings, e.g., 2-HC, 2 -HC, 4-HC, and 2 ,2-DHC, have a higher pKa and collapse the mitochondrial membrane potential more readily. They are also more likely to oxidize GSH to GSSG. By contrast chalcones with a greater number of hydroxy groups on their aromatic rings, have relatively lower pKa values and are less effective at collapsing the mitochondrial membrane potential. They are also more likely to form GSH conjugates. Uncoupling properties of some chalcones and dihydrochalcones including 2 -HC on oxidative phosphorylation in mung bean hypocotyl and potato tuber mitochondria have been shown to be associated with the presence of hydrogen or hydroxyl groups in the 2 -position and hydrogen or hydroxyl groups in the 4 -position [39]. Butein, a possible catecholic metabolite of isoliquiritigenin, was also found to be a more potent mitochondrial succinoxidase inhibitor than other flavonoids [40].

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Outliers found in this study included 2-HC, 4-HC, and 4 -HC which were less toxic than predicted by the equations. The calculated LD50 values for the outliers were greater or equal to two-fold different than their experimental LD50 values. Initially, the correlations included the outliers which allowed their identification, since, the inclusion to these points resulted in a poor regression. As the outliers were removed, the regression improved significantly (r2 increased by ≥0.5). The chalcones 2-HC and 4-HC, both not containing any hydroxy groups in the A-ring, were also less toxic than predicted. This suggests that at least one hydroxy group on the A-ring is necessary for increased toxicity involvement in the cytotoxic mechanism as demonstrated with other phenolics [26,33]. This and the GSH oxidation may possibly indicate phenoxyl radical formation. Furthermore, both 2 -HC and 4 -HC were more toxic than 2-HC and 4-HC, indicating that a chalcone with one hydroxy group on the A-ring would be more toxic than a chalcone with only one hydroxy group on the B-ring. 4 -HC was also less toxic than predicted by the equations, as it was predicted to be as toxic as 2 -HC. The 2 -hydroxy position was found to contribute to toxicity more than the 4 -hydroxy position. With the exception of these few outliers, Eqs. (1)–(3) are good predictors for chalcone toxicity. In summary, the cytotoxic mechanism of all of the hydroxychalcones was partly due to mitochondrial uncoupling as is evident from an observed increase in oxygen consumption together with a collapse in mitochondrial membrane potential. Furthermore, at least for phloretin, glycolytic substrates prevented the cytotoxicity and collapse in mitochondrial membrane potential. The cytotoxic mechanism of other chalcones was also associated with GSH oxidation. The cytotoxic mechanisms examined here are likely to be applicable to the observed anticancer effects of chalcones that have previously been reported. Our laboratory is currently investigating chalcone toxicity and potential anticancer activity towards Hep G2 cells, a human hepatoma cell model using a clonogenic assay and are finding that hydroxychalcones are much more toxic to Hep G2 cells than isolated rat hepatocytes suggesting that these hydroxychalcones may prove useful as anticancer agents (G. Galati et al., in preparation).

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References [1] B.A. Bohm, Chalcones and aurones, in: P.M. Dey, J.B. Harborne (Eds.) Plant Phenols, Academic Press, London (1989) pp. 237–282. [2] D.N. Dhar, The chemistry of chalcones and related compounds, Wiley, New York (1981). [3] F.A. Tomas-Barberan, M.N. Clifford, Flavones, chalcones and dihydrochalcones—nature occurrence and dietary burden, J. Sci. Food Agric. 80 (2000) 1073–1080. [4] J.W. Wegener, H. Nawrath, Cardiac effects of isoliquiritigenin, Eur. J. Pharmacol. 326 (1997) 37–44. [5] K. Satyanarayana, M.N.A. Rao, Anti-inflammatory, analgesic and anti-pyretic activities of 3-(4-(3-(4-dimethylaminophenyl)-1-oxo-2-propenyl) phenyl)sydnone, Indian Drugs 30 (1993) 313–318. [6] J.F. Ballesteros, M.J. Sanz, A. Ubeda, M.A. Miranda, S. Iborra, M. Paya, M.J. Alcaraz, Synthesis and pharmacological evaluation of 2 -hydroxychalcones and flavones as inhibitors of inflammatory mediators generation, J. Med. Chem. 38 (1995) 2794–2797. [7] H.K. Hsieh, L.T. Tsao, J.P. Wang, C.N. Lin, Synthesis and anti-inflammatory effect of chalcones, J. Pharm. Pharmacol. 52 (2000) 163–171. [8] C.C. Yit, N.P. Das, Cytotoxic effect of butein on human colon adenocarcinoma cell proliferation, Cancer Lett. 82 (1994) 65– 72. [9] Y. Satomi, Inhibitory effects of 3 -methyl-3-hydroxychalcone on proliferation of human malignant tumor cells and on skin carcinogenesis, Int. J. Cancer 55 (1993) 506–514. [10] R.J. Anto, K. Sukumaran, G. Kuttan, M.N. Rao, V. Subbaraju, R. Kuttan, Anticancer and antioxidant activity of synthetic chalcones and related compounds, Cancer Lett. 97 (1995) 33–37. [11] S. Ducki, R. Forrest, J.A. Hadfield, A. Kendall, N.J. Lawrence, A.T. McGown, D. Rennison, Potent antimitotic and cell growth inhibitory properties of substituted chalcones, Bioorg. Med. Chem. Lett. 8 (1998) 1051–1056. [12] K. Iwashita, M. Kobori, K. Yamaki, T. Tsushida, Flavonoids inhibit cell growth and induce apoptosis in B16 melanoma 4A5 cells, Biosci. Biotechnol. Biochem. 64 (2000) 1813– 1820. [13] M.L. Edwards, D.M. Stemerick, P.S. Sunkara, Chalcones: a new class of antimitotic agents, J. Med. Chem. 33 (1990) 1948–1954. [14] M. Kobori, H. Shinmoto, T. Tsushida, K. Shinohara, Phloretin-induced apoptosis in B16 melanoma 4A5 cells by inhibition of glucose transmembrane transport, Cancer Lett. 119 (1997) 207–212. [15] M. Kobori, K. Iwashita, H. Shinmoto, T. Tsushida, Phloretininduced apoptosis in B16 melanoma 4A5 cells and HL60 human leukemia cells, Biosci. Biotechnol. Biochem. 63 (1999) 719–725. [16] J. Ma, N.-Y. Fu, D.-B. Pang, W.-Y. Wu, A.-L. Xu, Apoptosis induced by isoliquiritigenin in human gastric cancer MGC803 cells, Planta. Med. 67 (2001) 754–757.

[17] M. Baba, R. Asano, I. Takigami, T. Takahashi, M. Ohmura, Y. Okada, H. Sugimoto, T. Arika, H. Nishino, T. Okuyama, Studies on cancer chemoprevention by traditional folk medicines XXV. Inhibitory effect of isoliquiritigenin on azoxymethane-induced murine colon aberrant crypt focus formation and carcinogenesis, Biol. Pharm. Bull. 25 (2002) 247–250. [18] S. Yamamoto, E. Aizu, H. Jiang, T. Nakadate, I. Kiyoto, J.C. Wang, R. Kato, The potent anti-tumor-promoting agent isoliquiritigenin, Carcinogenesis 12 (1991) 317–323. [19] S. Shibata, Anti-tumorigenic chalcones, Stem Cells 12 (1994) 44–52. [20] L.W. Wattenberg, J.B. Coccia, A.R. Galbraith, Inhibition of carcinogen-induced pulmonary and mammary carcinogenesis by chalcone administered subsequent to carcinogen exposure, Cancer Lett. 83 (1994) 165–169. [21] H. Makita, T. Tanaka, H. Fujitsuka, N. Tatematsu, K. Satoh, A. Hara, H. Mori, Chemoprevention of 4-nitroquinoline 1oxide-induced rat oral carcinogenesis by the dietary flavonoids chalcone, 2-hydroxychalcone and quercetin, Cancer Res. 56 (1996) 4904–4909. [22] A.T. Dinkova-Kostava, M.A. Massiah, R.E. Bozak, R.J. Hicks, P. Talalay, Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 3404–3409. [23] C.L. Miranda, G.L.M. Aponso, J.F. Stevens, M.L. Deinzer, D.R. Buhler, Prenylated chalcones and flavanones as inducers of quinone reductase in mouse Hepa 1c1c7 cells, Cancer Lett. 149 (2000) 21–29. [24] H. Fiander, H. Schneider, Dietary ortho phenols that induce glutathione S-transferase and increase the resistance of cells to hydrogen peroxide are potential cancer chemopreventives that act by two mechanisms: the alleviation of oxidative stress and the detoxification of mutagenic xenobiotics, Cancer Lett. 156 (2000) 117–124. [25] G. Galati, S. Teng, M.Y. Moridani, T.S. Chan, P.J. O’Brien, Cancer chemoprevention and apoptosis mechanisms induced by dietary polyphenolics, Drug Metab. Drug Interact. 17 (2000) 311–349. [26] M.Y. Moridani, A. Siraki, P.J. O’Brien, Quantitative structure toxicity relationships for phenols in isolated rat hepatocytes, Chem. Biol. Interact. 145 (2003) 213–223. [27] A.G. Siraki, T. Chevaldina, M.Y. Moridani, P.J. O’Brien, Quantitative structure-toxicity relationships by accelerated cytotoxicity mechanism screening, Curr. Opin. Drug Discov. Devel. 7 (2004) 118–125. [28] P. Moldeus, J. Hogberg, S. Orrenius, Isolation and use of liver cells, Methods Enzymol. 52 (1978) 60–71. [29] D.J. Reed, J.R. Babson, P.W. Beatty, A.E. Brodie, W.W. Ellis, D.W. Potter, High-performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides, Anal. Biochem. 106 (1980) 55–62. [30] J. Huang, S. Khan, P.J. O’Brien, The glutathione dependence of inorganic sulfate formation from L- or D-cysteine in isolated rat hepatocytes, Chem. Biol. Interact. 110 (1998) 189–202.

O. Sabzevari et al. / Chemico-Biological Interactions 148 (2004) 57–67 [31] B.S. Andersson, T.Y. Aw, D.P. Jones, Mitochondrial transmembrane potential and pH gradient during anoxia, Am. J. Physiol. 252 (1987) C349–C355. [32] J.E. Estabrook, Mitochondrial respiratory control: polarographic measurement of ADP-O ratios, Methods Enzymol. 10 (1967) 41–47. [33] C.D. Selassie, A.J. Shusterman, S. Kapur, R.P. Verma, L. Zhang, C. Hansch, On the toxicity of phenols to fast growing cells. A QSAR model for a radical-based toxicity, J. Chem. Soc. Perkin Trans. 2 (1999) 2729–2733. [34] G. Galati, O. Sabzevari, J.X. Wilson, P.J. O’Brien, Prooxidant activity and cellular effects of the phenoxyl radicals of dietary flavonoids and other polyphenolics, Toxicology 177 (2002) 91–104. [35] K. Zhang, E.B. Yang, W.Y. Tang, K.P. Wong, P. Mack, Inhibition of glutathione reductase by plant polyphenols, Biochem. Pharmacol. 54 (1997) 1047–1053. [36] A.J. Elliott, S.A. Scheiber, C. Thomas, R.S. Pardini, Inhibition of glutathione reductase by flavonoids: a structure-activity study, Biochem. Pharmacol. 44 (1992) 1603–1608.

67

[37] S. Awasthi, U. Pandya, S.S. Singhal, J.T. Li, V. Thiviyanathan, W.E. Siefert, Y.C. Awasthi, G.A.S. Ansari, Curcuminglutathione interactions and the role of human glutathione S-transferase P1-1, Chem. Biol. Interact. 128 (2000) 19–38. [38] M.Y. Moridani, G. Galati, P.J. O’Brien, Comparative quantitative structure toxicity relationships for flavonoids evaluated in isolated rat hepatocytes and HeLa tumor cells, Chem. Biol. Interact. 139 (2002) 251–264. [39] P. Ravanel, M. Iissut, R. Douce, Uncoupling activities of chalcones and dihydro-chalcones on isolated mitochondria from potato tubers and mug bean hypocotyls, Phytochemistry 21 (1982) 2845–2850. [40] W.F. Hodnick, F.S. Kung, W.J. Roettger, C.W. Bohmont, R.S. Pardini, Inhibition of mitochondrial respiration and production of toxic oxygen radicals by flavonoids—a structureactivity study, Biochem. Pharmacol. 35 (1986) 2345– 2357.