Synergistic chemopreventive effects of nobiletin and atorvastatin on colon carcinogenesis

Abstract

Different cancer chemopreventive agents may act synergistically and their combination may produce enhanced protective effects against carcinogenesis than each individual agent alone. Herein, we investigated the chemopreventive effects of nobiletin (NBT, a citrus polymethoxyflavone) and atorvastatin (ATST, a lipid-lowering drug) in colon cancer cells/macrophages and an azoxymethane (AOM)-induced colon carcinogenesis rat model. The results demonstrated that co-treatments of NBT/ATST produced enhanced growth inhibitory and anti-inflammatory effects on the colon cancer cells and macrophages, respectively. Isobologram analysis confirmed that these interactions between NBT and ATST were synergistic. NBT/ATST co-treatment also synergistically induced extensive cell cycle arrest and apoptosis in colon cancer cells. Oral administration of NBT (0.1%, w/w in diet) or ATST (0.04%, w/w in diet) significantly decreased colonic tumor incidence and multiplicity in AOM-treated rats. Most importantly, co-treatment of NBT/ATST at their half doses (0.05% NBT + 0.02% ATST, w/w in diet) resulted in even stronger inhibitory effects on colonic tumor incidence and multiplicity than did NBT or ATST alone at higher doses. Statistical analysis confirmed that the enhanced chemopreventive activities against colon carcinogenesis in rats by the NBT/ATST combination were highly synergistic. Our results further demonstrated that NBT/ATST co-treatment profoundly modulated key cellular signaling regulators associated with inflammation, cell proliferation, cell cycle progression, apoptosis, angiogenesis and metastasis in the colon of AOM-treated rats. In conclusion, for the first time, our results demonstrated a strong synergy in inhibiting colon carcinogenesis produced by the co-treatment of NBT and ATST, which provided a scientific basis for using NBT in combination with ATST for colon cancer chemoprevention in humans.

Figure 1.

(A) Chemical structures of NBT and ATST. (B) Growth inhibitory effect of NBT, ATST and their half dose combinations on HT29 human colon cancer cells. HT29 cells were treated with serial concentration of NBT, ATST and their combinations for 72 h. Viable cells were measure by cell viability assay as described in Materials and methods. (C) Percentage of inhibition on nitric oxide (NO) production by NBT, ATST and their half dose combinations in LPS-stimulated RAW 264.7 macrophages. RAW 264.7 cells were treated with serial concentration of NBT, ATST and their combinations for 24 h (cell viability ≥ 90%). NO production was measure by NO assay as described in Material and methods. Data were shown as mean ± SD (n = 6).

Figure 2.

Median effect and interaction index plots of NBT, ATST and their combinations in inhibiting (A) the growth of HT29 human colon cancer cells and (B) NO production in LPS-stimulated RAW 264.7 cells. The median effect plot and interaction index plot were constructed by using modified Chou and Talalay’s method as described in Materials and methods. The median effect plot demonstrated that the linear regression model used in the isobologram analysis was well-fitted to the dose-response relationship of NBT, ATST and their combinations with the concentration ranges exploited herein. Synergistic effect of two agents was defined as interaction index lower than 1.0. Error bars in interaction index plots represented 95% confidence interval and were calculated using delta method.

Figure 3.

Effects of NBT (36 μM), ATST (18 μM) and their half dose combination (NBT 18 μM + ATST 9 μM) on cell cycle progression (A) and apoptosis (B) of HT29 human colon cancer cells. Cells were seeded in six-well plates for 24 h, and then treated with NBT, ATST and their half dose combination. After another 24 or 48 h of treatments, cells were harvested and subject to cell cycle and apoptosis analysis, respectively. Data were shown as mean ± SD. Different notations in the bar charts indicated statistical significance (P < 0.01, n = 3) by ANOVA.

Figure 4.

Effects of NBT, ATST and their half dose combination on cancer related proteins in the whole cell lysate (A) or the membrane fraction lysate (B) of the colonic mucosa in the AOM-treated rats. Colon tissues samples were subjected to Western Blot analysis as described in Materials and methods. The numbers underneath of the blots represented band intensity (normalized to that of β-actin, means of three independent experiments) measured by ImageJ software. The SDs (all within ±15% of the means) were not shown. β-Actin was served as an equal loading control. *Indicated statistical significance in comparison with the control (P < 0.05, n = 3).

Figure 5.

(A) Effects of NBT, ATST and their half dose combination on mRNA levels of IL-1β, IL-6 and TNF-α in the colonic mucosa of AOM-treated rats. Colon tissues samples were subjected to qRT-PCR analysis as described in Materials and methods. Data were shown as the mean ± SD of three independent experiments. The amount of IL-1β, IL-6, TNF-α mRNA expression was normalized to that of β-actin. (B) Effects of NBT, ATST and their half dose combination on COX-2 expression in colonic tissue of AOM-treated rats. Colonic tissues were embedded in paraffin and COX-2 expression level was visualized by immunohistochemical analysis using COX-2 antibody (×150 magnification). (C) Quantification of COX-2-labeling index (%) was determined in five different image fields for each rat by ImageJ (Samples from five rats were analyzed). Different notations in the bar charts indicated statistical significance (P < 0.01) by ANOVA.