Computational protocol: Dietary mastic oil extracted from Pistacia lentiscus var. chia suppresses tumor growth in experimental colon cancer models

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[…] The potential synergy of α-pinene and myrcene, regarding the inhibition of Caco-2 cell proliferation, was assessed using combination index (CI) and isobolographic analysis based on Lowe additivity. The IC20 values for 72 h of α-pinene and myrcene on Caco-2 cells, are 0.0523 and 0.4204 mg/ml, respectively. The IC30 values are 0.0661 and 0.5171 and the IC50 values are 0.0720 and 0.6300 mg/ml for α-pinene and myrcene, respectively. IC values were determined by the results of the SRB assay (Fig. ). Caco-2 cells were treated for 72 h with the IC20 of α-pinene combined with different concentrations of myrcene and vice versa. Viability of cells was assayed with the SRB method and data were analyzed with SigmaPlot v11. For each series of combinations, the IC50 and IC30 values were determined and plotted along with the IC30 and IC50 values of α-pinene and myrcene (Fig. ). The theoretical additive effect of the two compounds is depicted by the dashed lines that connect the two points, representing the iso-effective concentrations of α-pinene and myrcene (either of red color for IC50, or green for IC30). If the experimentally estimated IC50 and IC30 values of the different combinations of the examined compounds are plotted by data points that lie below the respective dashed line, we can assume that the compounds act synergistically. On the other hand, if the data points lie above the dashed line, the two compounds are antagonistic. Moreover, we determined α-pinene’s and myrcene’s combination index (CI) whose value indicates the degree of synergism or antagonism between two compounds. More specifically CI < 1, = 1, or > 1 indicates synergistic, additive or antagonistic effect, respectively.CI was calculated using the equation:2CIx=C1,x/ICx,1+C2,x/ICx,2where CIx stands for the combination index based on the effect of x% cell growth inhibition (either 50% or 30% here), C1,x and C2,x represent the concentrations of compounds 1 and 2 (α-pinene and myrcene), used in combination for inducing the same x% inhibition, and ICx,1 and ICx,2 represent the iso-effective concentrations of the same compounds that, when used individually, induce the same x% cell growth inhibition as their combination. Representative results of at least three independent experiments are being presented. [...] CT26, Caco-2 and HT29 cells were seeded in 35-mm culture dishes with IBIDI silicon inserts (IBIDI GmbH) consisting of two reservoirs separated by a 500 μm wall. 3 × 105 cells/ml were seeded in 70 μl of standard DMEM culture medium per reservoir. One insert was used per dish, and two dishes were seeded per cell line. After an overnight incubation at 37 °C and 5% CO2, the IBIDI insert was removed creating a 500 μm wide wound. In order to exclude the possibility that the wound healing process is attenuated due to the growth inhibitory effects of MO, we used non-toxic concentrations for the treatment of the cells that did not inhibit cell growth. Cells were treated with MO (0.015 mg/ml for CT26, 0.020 mg/ml for HT29 and 0.004 mg/ml for Caco-2) or DMSO (control, DMSO concentration ≤ 0.1% v/v) and photographed at indicated time points with a ZEISS Primovert light microscope (Zeiss, Göttingen, Germany) equipped with a digital camera (Axiocam ERc 5 s). Multiple photographs per time point were analyzed with ImageJ software (NIH, USA) and the average % wound area (% open image area) was calculated. […]

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