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Potential Chemopreventive Mechanisms of Chalcones
4.4
Potential Chemopreventive Mechanisms of Chalcones C. Gerhauser, E. Heiss, C. Herhaus and K. Klimo GERMAN CANCER RESEARCH CENTER, DIVISION O F TOXICOLOGY AND CANCER RISK FACTORS (C0200), IM NEUENHEIMER FELD 280,69120 HEIDELBERG, GERMANY
1 Introduction Chalcones are ring-open biosynthetic precursors of flavonoids and contain an a$-unsaturated carbonyl moiety flanked by two phenyl groups as a common structural feature. The chemical structure of the unsubstituted chalcone is shown in Figure 1. Chalcones have been reported to inhibit skin, oral, pulmonary and mammary carcinogenesis in rodent models. These effects have been attributed to a variety of anti-carcinogenic properties including antiinflammatory,Iy2 anti-pr~liferative,~anti-oxidant4 and radical-scavenging5 mechanisms as well as induction of detoxification enzymes.6 As an approach to further determine chemopreventive mechanisms, we have performed a detailed analysis of modulatory effects of selected chalcones on xenobiotic metabolism. Induction of NAD(P)H:quinone oxidoreductase (QR) as a model phase 2 detoxification enzyme was tested in Hepa lclc7 murine hepatoma The mode of induction was determined using northern blotting and transient transfection techniques.8 In addition, we have investigated the influence of chalcones on induction of CyplA as a phase 1 cytochrome P450 enzyme and tested the potential of chalcones to inhibit CyplA activity.'
Figure 1 Schematic representation of the chalcone structure
189
Potential Chemopreventive Mechanisms of Chalcones
190
Excessive production of nitric oxide (NO) and prostaglandins in inflammation is thought to be a causative factor of cellular injury and cancer. Elevated levels of the inducible forms of nitric oxide synthase (iNOS) and cyclooxygenase (Cox-2) have been implicated in the pathogenesis of many disease processes including acute and chronic neuro-degenerative diseases, rheumatoid arthritis and carcinogenesis.’&12Thus, we have determined the potential of chalcones to suppress lipopolysaccharide (LPS)-mediated NO formation in murine Raw macro phage^.'^ Western blot analyses were performed to examine the influence on iNOS and Cox-2 protein levels.
2 Results and Discussion We have tested a series of about twenty synthetic chalcones with hydroxy-, methoxy-, methyl- and bromo-substitution for their potential to induce QR in cultured Hepalclc7 cells (Herhaus et al., in preparation). These compounds were identified as potent inducers of QR activity and displayed C D values (concentration required to double the specific activity of QR) below 1 pM. Results obtained with a selection of six chalcones Chl to Ch6 are summarised in Table 1. The QR-inducing potential of two 2’-hydroxy-substituted chalcones Chl and Ch2, which were additionally found to potently elevate GSH levels in Hepa lclc7 cell culture, was analysed in more detail. Northern blotting experiments revealed transcriptional regulation of QR induction in a time- (0 to 15 h) and dose-dependent (0.2 to 25 p M ) manner (data not shown). To determine the mode of induction, namely monofunctional or bifunctional mechanisms, transient transfection experiments with HepG2 cells were performed, using a construct containing all of the known regulatory elements of the rat QR gene (pDTD- 1097-CAT) and a XRE (xenobiotic responsive element)-CAT construct, respectively. After treatment with 10 pM of Chl and Ch2 for 48h, chloramphenicol acetyltransferase (CAT) levels (determined by CAT-ELISA,
Table 1 Summary of potential chemopreventive activities of selected chalcones Chalcone
QR induction
Name Substitution
CDa/IC5,b
Ch 1 Ch 2 Ch 3 Ch 4 Ch 5 Ch 6
0.4114.4 0.3117.3 0.6p1.2
2’-OH, 2,6-OMe 2’-OH, 2-OMe 2’-OMe, 2-OMe 2’-OMe, 2,6-OMe 2’,6’-OMe 2’,6‘-OMe, 2-OMe
0.5/9.0 0.818.2 0.2/11.4
CYPIA
induction CD
0.2 0.2 No induction 0.47 No induction No induction
CYPlA inhibition
NO
ICSO
inhibition ICS,
0.3 0.2 0.4 0.2 >5 0.4
3.6 8.9 2.1 1.1 0.7 2.1
” CD: Concentration required to double the specific activity of QR (in p M ) .
* ICso: Half-maximal inhibitory concentration of cell viability, CyplA activity or NO production (in PM).
C . Gerhauser et al.
191
Boehringer Mannheim) were significantly induced about 10-fold ( P > 0.0001) in comparison with a solvent control in cultures transfected with either construct. This indicated an Ah (aryl hydrocarbon) receptor-mediated and thus bifunctional mode of induction, i.e. concomitant induction of Ah-receptordependent phase 1 and 2 enzymes. Consequently, weak induction of CyplA activity by Chl, Ch2, and Ch4 was also observed in intact Hepa lclc7 cells by fluorimetric determination of the time-dependent dealkylation of 3-cyano-7ethoxycoumarin (CEC) to 3-cyano-7-hydroxycoumarin,9although the compounds have no structural similarity to known ligands of the Ah-receptor (e.g. polychlorinated biphenyls, polyaromatic arylhydrocarbons). Interestingly, Cypl A-inducing potential was abolished by replacement of the 2’-OH-group by a methoxy-group (Ch2 vs. Ch3). In addition to CyplA induction, however, these compounds were also identified as potent inhibitors of CyplA enzyme activity using lysates of P-naphthoflavone-induced rat hepatoma (H4IIE) cells as an enzyme source. The results are summarised in Table 1. To further investigate chalcone-mediated chemopreventive mechanisms, effects on NO production” and on the expression of iNOS and Cox-2 were examined. Chalcones were found to potently inhibit LPS-mediated NO production in Raw 264.7 macrophages with ICso values ranging from 0.7 to 2 pM (Table 1). Western blot analyses revealed that Chl time- and dose-dependently (0 to 12 h incubation; dose range 0.4 to 25 pM) suppresses LPS-mediated upregulation of iNOS and Cox-2 protein levels in Raw macrophages (data not shown). This effect was measurable when the compound was added up to 4 h post LPS treatment, indicating a rather early target (activation of NF-KB?) in the signal transduction pathway from LPS stimulation to protein expression. These novel results suggest that the known anti-inflammatory activity of chalcones may not only be mediated by inhibition of relevant enzyme activities like Cox-1,’ but also by inhibition of the expression of enzymes involved in inflammatory reactions. Taken together, our investigations provide additional data to demonstrate potential chemopreventive activity of chalcones by multiple anti-initiating and -promoting effects.
3 References 1 C.N. Lin, T.H. Lee, M.F. Hsu, J.P. Wang, F.N. KO and C.M. Teng, J. Pharm. Pharmacol., 1997,49,530-538. 2 F. Herencia, M.L. Ferrandiz, A. Ubeda, J.N. Dominguez, J.E. Charris, G.M. Lob0 and M.J. Alcaraz, Bioorg. Med. Chem. Lett., 1998,8, 1169-1174. 3 S. Ducki, R. Forrest, J.A. Hadfield, A. Kendall, N.J. Lawrence, A.T. McGown and D. Rennison, Bioorg. Med. Chem. Lett., 1998,8, 1051-1056. 4 R.J. Anto, K. Sukumaran, G. Kuttan, M.N. Rao, Subbaraju and R. Kuttan, Cancer Lett., 1995,97, 33-31. 5 A.T. Dinkova-Kostova, C. Abeygunawardana and P. Talalay, J . Med. Chem., 1998, 41,5287- 5296. 6 L.L. Song, J.W. Kosmeder 11, S.K.Lee, C. Gerhauser, D. Lantvit, R.C. Moon, R.M. Moriarty and J.M. Pezzuto, Cancer Res., 1999,59, 578-585. 7 H.J. Prochaska and A.B. Santamaria, Anal. Biochem., 1988,169,328-336.
192
Potential Chemopreventive Mechanisms of Chalcones
8 C. Gerhauser, M. You, J. Liu, R.M. Moriarty, M. Hawthorne, R.G. Mehta, R.C. Moon, and J.M. Pezzuto, Cancer Res., 1997,57,272-278. 9 C.L. Crespi, V.P. Miller and B.W. Penman, Anal. Biochem., 1997,248, 188-190. 10 T.P. Misko, J.L. Trotter and A.H. Cross, J. Neuroimmunol., 1995,61, 195-204. 1 1 N.A. Simonian and J.T. Coyle, Annu. Rev. Pharmacol. Toxicol., 1996,36,83-106. 12 M. Takahashi, K. Fukuda, T. Ohata, T. Sugimura and K. Wakabayashi, Cancer Res., 1997,57, 1233-1237. 13 A.H. Ding, C.F. Nathan and D.J. Stuehr, J. Immunol., 1988,14,2407-2412.
Potential Chemopreventive Mechanisms of Chalcones C. Gerhauser, E. Heiss, C. Herhaus and K. Klimo GERMAN CANCER RESEARCH CENTER, DIVISION O F TOXICOLOGY AND CANCER RISK FACTORS (C0200), IM NEUENHEIMER FELD 280,69120 HEIDELBERG, GERMANY
1 Introduction Chalcones are ring-open biosynthetic precursors of flavonoids and contain an a$-unsaturated carbonyl moiety flanked by two phenyl groups as a common structural feature. The chemical structure of the unsubstituted chalcone is shown in Figure 1. Chalcones have been reported to inhibit skin, oral, pulmonary and mammary carcinogenesis in rodent models. These effects have been attributed to a variety of anti-carcinogenic properties including antiinflammatory,Iy2 anti-pr~liferative,~anti-oxidant4 and radical-scavenging5 mechanisms as well as induction of detoxification enzymes.6 As an approach to further determine chemopreventive mechanisms, we have performed a detailed analysis of modulatory effects of selected chalcones on xenobiotic metabolism. Induction of NAD(P)H:quinone oxidoreductase (QR) as a model phase 2 detoxification enzyme was tested in Hepa lclc7 murine hepatoma The mode of induction was determined using northern blotting and transient transfection techniques.8 In addition, we have investigated the influence of chalcones on induction of CyplA as a phase 1 cytochrome P450 enzyme and tested the potential of chalcones to inhibit CyplA activity.'
Figure 1 Schematic representation of the chalcone structure
189
Potential Chemopreventive Mechanisms of Chalcones
190
Excessive production of nitric oxide (NO) and prostaglandins in inflammation is thought to be a causative factor of cellular injury and cancer. Elevated levels of the inducible forms of nitric oxide synthase (iNOS) and cyclooxygenase (Cox-2) have been implicated in the pathogenesis of many disease processes including acute and chronic neuro-degenerative diseases, rheumatoid arthritis and carcinogenesis.’&12Thus, we have determined the potential of chalcones to suppress lipopolysaccharide (LPS)-mediated NO formation in murine Raw macro phage^.'^ Western blot analyses were performed to examine the influence on iNOS and Cox-2 protein levels.
2 Results and Discussion We have tested a series of about twenty synthetic chalcones with hydroxy-, methoxy-, methyl- and bromo-substitution for their potential to induce QR in cultured Hepalclc7 cells (Herhaus et al., in preparation). These compounds were identified as potent inducers of QR activity and displayed C D values (concentration required to double the specific activity of QR) below 1 pM. Results obtained with a selection of six chalcones Chl to Ch6 are summarised in Table 1. The QR-inducing potential of two 2’-hydroxy-substituted chalcones Chl and Ch2, which were additionally found to potently elevate GSH levels in Hepa lclc7 cell culture, was analysed in more detail. Northern blotting experiments revealed transcriptional regulation of QR induction in a time- (0 to 15 h) and dose-dependent (0.2 to 25 p M ) manner (data not shown). To determine the mode of induction, namely monofunctional or bifunctional mechanisms, transient transfection experiments with HepG2 cells were performed, using a construct containing all of the known regulatory elements of the rat QR gene (pDTD- 1097-CAT) and a XRE (xenobiotic responsive element)-CAT construct, respectively. After treatment with 10 pM of Chl and Ch2 for 48h, chloramphenicol acetyltransferase (CAT) levels (determined by CAT-ELISA,
Table 1 Summary of potential chemopreventive activities of selected chalcones Chalcone
QR induction
Name Substitution
CDa/IC5,b
Ch 1 Ch 2 Ch 3 Ch 4 Ch 5 Ch 6
0.4114.4 0.3117.3 0.6p1.2
2’-OH, 2,6-OMe 2’-OH, 2-OMe 2’-OMe, 2-OMe 2’-OMe, 2,6-OMe 2’,6’-OMe 2’,6‘-OMe, 2-OMe
0.5/9.0 0.818.2 0.2/11.4
CYPIA
induction CD
0.2 0.2 No induction 0.47 No induction No induction
CYPlA inhibition
NO
ICSO
inhibition ICS,
0.3 0.2 0.4 0.2 >5 0.4
3.6 8.9 2.1 1.1 0.7 2.1
” CD: Concentration required to double the specific activity of QR (in p M ) .
* ICso: Half-maximal inhibitory concentration of cell viability, CyplA activity or NO production (in PM).
C . Gerhauser et al.
191
Boehringer Mannheim) were significantly induced about 10-fold ( P > 0.0001) in comparison with a solvent control in cultures transfected with either construct. This indicated an Ah (aryl hydrocarbon) receptor-mediated and thus bifunctional mode of induction, i.e. concomitant induction of Ah-receptordependent phase 1 and 2 enzymes. Consequently, weak induction of CyplA activity by Chl, Ch2, and Ch4 was also observed in intact Hepa lclc7 cells by fluorimetric determination of the time-dependent dealkylation of 3-cyano-7ethoxycoumarin (CEC) to 3-cyano-7-hydroxycoumarin,9although the compounds have no structural similarity to known ligands of the Ah-receptor (e.g. polychlorinated biphenyls, polyaromatic arylhydrocarbons). Interestingly, Cypl A-inducing potential was abolished by replacement of the 2’-OH-group by a methoxy-group (Ch2 vs. Ch3). In addition to CyplA induction, however, these compounds were also identified as potent inhibitors of CyplA enzyme activity using lysates of P-naphthoflavone-induced rat hepatoma (H4IIE) cells as an enzyme source. The results are summarised in Table 1. To further investigate chalcone-mediated chemopreventive mechanisms, effects on NO production” and on the expression of iNOS and Cox-2 were examined. Chalcones were found to potently inhibit LPS-mediated NO production in Raw 264.7 macrophages with ICso values ranging from 0.7 to 2 pM (Table 1). Western blot analyses revealed that Chl time- and dose-dependently (0 to 12 h incubation; dose range 0.4 to 25 pM) suppresses LPS-mediated upregulation of iNOS and Cox-2 protein levels in Raw macrophages (data not shown). This effect was measurable when the compound was added up to 4 h post LPS treatment, indicating a rather early target (activation of NF-KB?) in the signal transduction pathway from LPS stimulation to protein expression. These novel results suggest that the known anti-inflammatory activity of chalcones may not only be mediated by inhibition of relevant enzyme activities like Cox-1,’ but also by inhibition of the expression of enzymes involved in inflammatory reactions. Taken together, our investigations provide additional data to demonstrate potential chemopreventive activity of chalcones by multiple anti-initiating and -promoting effects.
3 References 1 C.N. Lin, T.H. Lee, M.F. Hsu, J.P. Wang, F.N. KO and C.M. Teng, J. Pharm. Pharmacol., 1997,49,530-538. 2 F. Herencia, M.L. Ferrandiz, A. Ubeda, J.N. Dominguez, J.E. Charris, G.M. Lob0 and M.J. Alcaraz, Bioorg. Med. Chem. Lett., 1998,8, 1169-1174. 3 S. Ducki, R. Forrest, J.A. Hadfield, A. Kendall, N.J. Lawrence, A.T. McGown and D. Rennison, Bioorg. Med. Chem. Lett., 1998,8, 1051-1056. 4 R.J. Anto, K. Sukumaran, G. Kuttan, M.N. Rao, Subbaraju and R. Kuttan, Cancer Lett., 1995,97, 33-31. 5 A.T. Dinkova-Kostova, C. Abeygunawardana and P. Talalay, J . Med. Chem., 1998, 41,5287- 5296. 6 L.L. Song, J.W. Kosmeder 11, S.K.Lee, C. Gerhauser, D. Lantvit, R.C. Moon, R.M. Moriarty and J.M. Pezzuto, Cancer Res., 1999,59, 578-585. 7 H.J. Prochaska and A.B. Santamaria, Anal. Biochem., 1988,169,328-336.
192
Potential Chemopreventive Mechanisms of Chalcones
8 C. Gerhauser, M. You, J. Liu, R.M. Moriarty, M. Hawthorne, R.G. Mehta, R.C. Moon, and J.M. Pezzuto, Cancer Res., 1997,57,272-278. 9 C.L. Crespi, V.P. Miller and B.W. Penman, Anal. Biochem., 1997,248, 188-190. 10 T.P. Misko, J.L. Trotter and A.H. Cross, J. Neuroimmunol., 1995,61, 195-204. 1 1 N.A. Simonian and J.T. Coyle, Annu. Rev. Pharmacol. Toxicol., 1996,36,83-106. 12 M. Takahashi, K. Fukuda, T. Ohata, T. Sugimura and K. Wakabayashi, Cancer Res., 1997,57, 1233-1237. 13 A.H. Ding, C.F. Nathan and D.J. Stuehr, J. Immunol., 1988,14,2407-2412.