Effect of Different Coffee Brews on Tryptophan Metabolite-Induced Cytotoxicity in HT-29 Human Colon Cancer Cells

Abstract

Coffee consumption positively influences colon health. Conversely, high levels of tryptophan metabolites such as skatole released from intestinal putrefactive fermentation in the presence of excessive dietary animal protein intake, and gut microbiota alterations, may have several adverse effects, including the development of colorectal cancer. Therefore, this study aimed to elucidate the potential protective effects of coffee in the presence of different skatole levels. The results showed that skatole exposure induced reduced cell viability and oxidative stress in the HT-29 human colon cancer cell line. However, co-treatment of cells with skatole and coffee samples was able to reduce ROS production (up to 45% for espresso) compared to cells not treated with coffee. Real-time PCR analysis highlighted that treating HT-29 cells with skatole increased the levels of inflammatory cytokines and chemokines TNF-α, IL-1β, IL-8, and IL12, whereas exposure to coffee extracts in cells that were pretreated with skatole showed anti-inflammatory effects with decreased levels of these cytokines. These findings demonstrate that coffee may counteract the adverse effects of putrefactive compounds by modulating oxidative stress and exerting anti-inflammatory activity in colonocytes, thus suggesting that coffee intake could improve health conditions in the presence of altered intestinal microbiota metabolism.

Keywords: ROS; anti-inflammatory activity; chlorogenic acids; coffee; polyphenols; skatole.

Figure 1

Evaluation of cell viability in HT-29 cells. The effect of treatment with different types of coffee extract: instant (A), Americano (B), and espresso (C), at 0.250 and 0.500 mg/mL on cell viability was evaluated using the MTT assay after 24 h with respect to the mock control. The MTT test was used to determine the impact of skatole treatment (D) on cell viability after 24 h at the concentrations of 250, 500, 750, and 1000 µM compared with control cells (treated only with DMSO). The graph represented the mean and SD of three separate experiments. * p-value ≤ 0.05 and *** p-value ≤ 0.001 compared to the control group (calculated as fold-change relative to control cells, arbitrarily set at 100%).

Figure 2

Evaluation of intracellular ROS level in HT-29 cells. The effect on the generation of intracellular ROS levels after treatment with a blank control (resulting from in vitro digestion) and the different types of coffee extract (instant, Americano, and espresso) at 0.250 and 0.500 mg/mL was evaluated using the H₂DCF-DA assay after 24 h of treatment, and compared with the mock control (untreated cells) (A). The effect of skatole treatment (B) on the production of intracellular ROS was estimated by fluorometric assay after 24 h at the concentrations of 250 and 500 µM and compared to the control cells (treated with DMSO only). Cells treated only with H₂O₂ were used as a positive control. The graphs represent the mean and SD of three separate experiments. * p-value ≤ 0.05, ** p-value ≤ 0.01 and *** p-value ≤ 0.001 compared to the control group (calculated as fold-change relative to control cells, arbitrarily set at 100%).

Figure 3

Evaluation of intracellular ROS levels under challenging conditions in HT-29 cells. The effect of treatment with different types of coffee extract (instant, Americano, and espresso) at 0.250 mg/mL after skatole treatment of 250 µM (A) and 500 µM (B) on the production of intracellular ROS levels was determined by H₂DCF-DA assay after 24 h of treatment, and compared to the mock control. Cells treated only with H₂O₂ were used as a positive control. The graphs represent the mean and SD of three separate experiments. * p-value ≤ 0.05, ** p-value ≤ 0.01 and *** p-value ≤ 0.001 compared to untreated control (calculated as fold-change relative to control cells, arbitrarily set at 100%). ## p-value ≤ 0.01 and ### p-value ≤ 0.001 skatole versus coffee treatment.

Figure 4

Evaluation of mRNA expression levels in HT-29 cells. The effect of treatment with the different types of coffee extract (instant, Americano and espresso) at 0.250 mg/mL after stimulation with 250 µM skatole on the expression level of TNF-α (A), IL-1β (B), IL-8 (C), and IL12 (D) was performed using real-time PCR analysis after 24 h of treatment, and compared to the control group. Cells treated only with LPS were used as a positive control. The graph represented the mean and SD of three separate experiments. * p-value ≤ 0.05, ** p-value ≤ 0.01 and *** p-value ≤ 0.001 compared to untreated control. # p-value ≤ 0.05, ## p-value ≤ 0.01 and ### p-value ≤ 0.001 skatole versus coffee treatment.

Figure 4

Evaluation of mRNA expression levels in HT-29 cells. The effect of treatment with the different types of coffee extract (instant, Americano and espresso) at 0.250 mg/mL after stimulation with 250 µM skatole on the expression level of TNF-α (A), IL-1β (B), IL-8 (C), and IL12 (D) was performed using real-time PCR analysis after 24 h of treatment, and compared to the control group. Cells treated only with LPS were used as a positive control. The graph represented the mean and SD of three separate experiments. * p-value ≤ 0.05, ** p-value ≤ 0.01 and *** p-value ≤ 0.001 compared to untreated control. # p-value ≤ 0.05, ## p-value ≤ 0.01 and ### p-value ≤ 0.001 skatole versus coffee treatment.