Abstract B73: The association of caffeine, trigonelline, and chlorogenic acid, active components from coffee, enhances caffeine-induced cytotoxicity in hepatocellular carcinoma cells

Abstract

Abstract Background: A cup of traditional coffee is a complex mixture of biologically active components, including caffeine (CAF), trigonelline (TRI), chlorogenic acid (CGA), and many others (1). Daily coffee consumption reduces by ~40% the risk of fibrosis/cirrhosis and hepatocellular carcinoma (HCC) in humans while, in general, decaffeinated coffee consumption does not (2-4). However, it is not clear whether this beneficial effect is attributed to the CAF alone or its association with other coffee constituents. Aim: This work evaluated the modifying effects of CAF alone or associated to CGA and/or TRI in human HCC and hepatic stellate cells (HSC). Experimental Procedures: Human C3A (clonal derivative of HepG2 cells) and LX2 (HSC) cells were grown in Eagle's Minimum Essential Medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37oC. C3A and LX2 cells were seeded in 96-well plates at a density of 7 x 104 and 1 x 105 cells/mL, respectively, and treated with CAF alone (20, 40, 80, or 160 μM) or combined to TRI (10, 20, 40, or 80 μM) and/or CGA (10, 20, 40, or 80 μM) for 24 and 48 h. To select the CAF concentration, we considered the human serum peak (~35 µM) after the ingestion of ~450 mg of CAF, corresponding to 2-3 cups of coffee, and the toxic limit established for plasma concentration in humans (~400 μM) (5,6). For CGA and TRI, abundant in coffee beverages, we considered the 1:2 proportion (CGA or TRI:CAF) found in traditional coffee (1). We evaluated the cell viability (MTT assay) of LX2 and the cytotoxicity (lactate dehydrogenase [LDH] assay) of C3A cells. Results: All tested CAF concentrations significantly increased cytotoxicity in C3A cells at 48 h compared to untreated cells (p<0.001). The CAF+TRI+CGA combinations (20, 10, and 10 µM and 40, 20, and 20 µM, respectively) enhanced cytotoxicity at 24 and 48 h when compared to the control, CAF alone, and other associations (p<0.001). Notably, these combinations enhanced caffeine-induced cytotoxicity at 48 h (p<0.001). All treatments did not alter the viability of LX2 cells at both 24 and 48 h. Conclusion: In general, the combination of CAF with other common compounds found in filtered and espresso coffee, TRI and CGA, enhanced CAF-mediated cytotoxicity on HCC cells at physiologically applicable concentrations. These findings suggest a synergistic effect of coffee compounds in exerting a chemopreventive effect and reducing the risk for HCC. The potential mechanisms related to these modifying effects will be further evaluated. Financial support: CNPq (140251/2016-2) and FAPESP (16/14420-0).