Cell culture :

1. METHODS AND MATERIALS INTRODUCTION # # Fig 1 : Structure of curcumin and tetrahydrocurcumin # RESULTS DISCUSSION AND CONCLUSION REFERENCES Comparative cytoprotective effect of curcumin and tetrahydrocurcumin in doxorubicin-induced cytotoxicity in normal hepatocyte cell line SomparnN1, KuklongviriyapanV 2 1 Department of Preclinical Science, Faculty of Medicine, Thammasat University, Rangsit Campus, Pathum Thani, 12120,Thailand, 2 Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002,Thailand ABSTRACT Curcumin (CUR) and its major metabolite, tetrahydrocurcumin (THC), have been shown to have some similar pharmacological effects. However, there are only few reports comparing cytoprotection and antioxidant properties of CUR with THC. In this present study, we investigated the cytoprotective effect and antioxidant genes response by CUR and THC against doxorubicin (DOX)-induced cytotoxicityin Chang liver cells. The exposure to DOX (0.3-3 uM for 24 hr) induced cell killing in dose dependent manner. Pre-treatment with CUR or THC (1, 3, 6 uM for 24 hr) significantly increased cell survival up to 80% or 68%, respectively when compared to DOX-treated controls. To examine whether the cytoprotective effect of both compounds were involved with induction of antioxidant and cytoprotective enzymes, cultured cells were pretreated with CUR or THC for 24 h, followed by DOX. Treatments with a combination of CUR and DOX significantly induced NQO1 and HO-1 (p<0.05), but not SOD2 and GCLC mRNA expression (p>0.05). On the other hand, a combination of DOX and THC did not alter the expression. The study suggested that although CUR and THC processes antioxidant effect, they are different in ability to induce antioxidant and cytoprotective genes. The mechanism of cytoprotection of both compounds remains to be further investigated. Cell culture : Chang liver cells were were routinely cultured in Ham’s F12 media, supplemented with, 100 U/mL penicillin 100 μg/mL streptomycin sulfate and 10% fetal calf serum. Sulforhodamine B (SRB) assay : The SRB assay was used for cell viability determination . Cells were seeded onto 96-well plates. After overnight incubation, the culture medium was removed and treatment with different concentrations of CUR or THC. After 24 h of incubation, cells were treated with DOX at different concentrations for 24 hr. After that cells were fixed in 10% trichloroacetic acid for 1 h at 4 °C and stained with 0.4% SRB in 1% acetic acid for 30 min. The excess dye was removed by washing repeatedly with 1% acetic acid. The protein-bound dye was dissolved in 10 mM Tris base solution and optical density was determined at 510 nm using a microplate reader. Curcumin (CUR) is a phenolic compound from Curcuma longa. A variety of pharmacology effects of curcumin have been reported, including anti-inflammatory andantioxidant 1-3. In common with several other diet-derived polyphenols, curcumin precess low systemic bioavalibility4 . The poor absorption of curcumin in both humans and animals has raised several concerns that this may limit its clinical impact. Therefore, leading to suggestion that in vivo efficacy may come from a more bioavalibility and/or potent metabolism. Recently, Trtrahydrocurcumin (THC), a reduced derivative of curcumin and one of the major metabolite of curcumin, has been focused on for its antioxidant and chemopreventive activity. In addition, tetrahydrocurcumin has been shown to exhibits physiology and pharmacological properties of protective effect on oxidative stress similar to those of curcumin in vivo5-6. The role of CUR and THC in antioxidant activity have been implicated and their possible mechanism and efficacy are still controversial. Therefore, In this present study, we investigated the cytoprotective effect and antioxidant genes response by CUR and THC against doxorubicin (DOX)-induced cytotoxicityin Chang liver cells. Real-time polymerase chain reaction : Cells were seeded in 6 well-plates. After overnight incubation, the culture medium was removed and treatment with 3 mM of CUR or THC. After 24 h of incubation, cells were treated with DOX at 1 mM for 24 hr.Total RNA wasisolated and reverse transcribed to single-stranded cDNAusing a previously described method7.The reverse transcription products served as a template for real-time PCR. To quantify the relative expressionofNQO1, HO-1,GCLC, SOD2 genes,the relative quantitation using standard curve method was used. The amount ofthosemRNAwereexpressed as a ratio toβ-actin mRNA. Statistical Analysis Data are expressed as mean + SEM of three separated experiments. An analysis of variance was used to determine significant differences between each experimental group. The level of significance was set at P<0.05 DOX is an anthracycline drug most widely applied in chemotherapy of various cancers. DOX is a potent pro-oxidant. Its undergoes redox cycling and reacts withenzymes of mitochondrial respiration. The exposure to DOX induced oxidative damage to cells as well as damage to proteins and DNA. The exposure to DOX induced cell killing in dose dependent manner and pre-treatment with CUR or THC significantly increased cell survival. Several Nrf2-dependent genes such as GST, NQO1, SOD, GCL, and HO-1 are known to mediate detoxification and/or to exert antioxidant functions thereby protecting cells from genotoxic damage8. The identification of curcumin-regulated Nrf2-dependent genes provides potential novel insights into the biological effects of curcumin on global gene expression and chemoprevention9. Whereas, the anti-oxidant role of THC has been implicated in many studies such as chloroquine-mediated oxidative damage in rat kidney10 , ferric nitrilotriacetate (Fe-NTA)-induced oxidative renal damage in male ddY mice11 and streptozotocin-nicotinamide induced diabetic rats 12 . In summary ,the study suggested that although CUR and THC possess antioxidant effect, they are different in ability to induce antioxidant and cytoprotective genes. The mechanism of cytoprotection of both compounds remains to be further investigated. Fig 3 : The antioxidant genes response by CUR and THC against doxorubicin (DOX)-induced cytotoxicity in Chang liver cells # Significantly different from normal control and DOX group; p<0.05 NQO1 = NAD(P)H quinone oxidoreductase-1 HO-1= Heme oxygenase 1 SOD2 = Superoxide dismutase 2 GCLC = Gamma-glutamate cysteine ligasecatalytic subunit 1. Aggarwal BB, Sundaram C, Malani N, Ichikawa H. Curcumin: the Indian solid gold. Adv Exp Med Biol 2007;595:1-75. 2. Jurenka JS. Anti-inflammatory properties of curcumin, a major constituent of Curcuma longa: a review of preclinical and clinical research.Altern Med Rev 2009;14(2):141-53. 3. Jain SK, Rains J, Jones K. Effect of curcumin on protein glycosylation, lipid peroxidation, and oxygen radical generation in human red blood cells exposed to high glucose levels. Free Radic Biol Med 2006;41(1):92-6. 4. Garcea G, Jones DJ, Singh R, Dennison AR, Farmer PB, Sharma RA, et al. Detection of curcumin and its metabolites in hepatic tissue and portal blood of patients following oral administration. Br J Cancer 2004;90(5):1011-5. 5. Kurathong S, Lerdverasirikul P, Wongpaitoon V, Pramoolsinsap C, Kanjanapitak A, Varavithya W, et al. Opisthorchis viverrini infection and cholangiocarcinoma. A prospective, case-controlled study. Gastroenterology 1985;89(1):151-6. 6. Sripa B, Kaewkes S, Sithithaworn P, Mairiang E, Laha T, Smout M, et al. Liver fluke induces cholangiocarcinoma. PLoS Med 2007;4(7):e201. 7. Buranrat B, Prawan A, Kukongviriyapan U, Kongpetch S, Kukongviriyapan V. Dicoumarol enhances gemcitabine-induced cytotoxicity in high NQO1-expressing cholangiocarcinoma cells. World J Gastroenterol. 2010;16(19):2362-70. 8. Lee, J. S. and Y. J. Surh. "Nrf2 as a novel molecular target for chemoprevention." Cancer Lett .2005 ;224(2): 171-184. 9. Yang C, Zhang X, Fan H, Liu Y. Curcumin upregulates transcription factor Nrf2, HO-1 expression and protects rat brains against focal ischemia. Brain Res 2009;1282:133-41. 10. Pari, L. and P. Murugan. "Effect of tetrahydrocurcumin on blood glucose, plasma insulin and hepatic key enzymes in streptozotocin induced diabetic rats. J Basic Clin Physiol Pharmacol 2005;16(4): 257-274. 11. Okada, K., C. Wangpoengtrakul, et al.. "Curcumin and especially tetrahydrocurcumin ameliorate oxidative stress-induced renal injury in mice. J Nutr 2001;131(8): 2090-2095. 12. Murugan, P. and L. Pari . Effect of tetrahydrocurcumin on plasma antioxidants in streptozotocin-nicotinamide experimental diabetes. J Basic Clin Physiol Pharmacol 2006;17(4): 231-244. ACKNOWLEDGEMENT Fig 2 : The cytoprotective effect by CUR and THC against doxorubicin (DOX)-induced cytotoxicity in Chang liver cells. This research was financially supported by Thailand Research Fund.