Combined Curcumin and Luteolin Synergistically Inhibit Colon Cancer Associated with Notch1 and TGF-β Signaling Pathways in Cultured Cells and Xenograft Mice

Abstract

This study aimed to select a combination of curcumin and luteolin, two phytochemicals from food, at lower concentrations with a higher inhibitory effect on colon cancer growth and investigate possible molecular mechanisms of this anti-colon cancer effect. By pairwise combination screening, we identified that the combination of curcumin (CUR) at 15 μM and luteolin (LUT) at 30 μM (C15L30) synergistically suppressed the proliferation of human colon cancer CL-188 cells, but the individual chemicals had a little inhibitory effect at the selected concentrations. This result was also confirmed in other colon cancer DLD-1cells, suggesting that this synergistic inhibitory effect of C15L30 applies to different colon cancer cells. The combination C15L30 synergistically suppressed the wound closure (wound healing assay) in CL-188 cells. We also found that the combination of CUR and LUT (at 20 mg/kg/day and 10 mg/kg/day, respectively, IP injection, 5 days for 2 weeks) synergistically suppressed tumor growth in CL-188 cell-derived xenograft mice. Western blot results showed that protein levels of Notch1 and TGF-β were synergistically reduced by the combination, both in CL-188 cells and xenograft tumors. Tumor pathological analysis revealed that combined CUR and LUT synergistically increased necrosis, but the individual treatment with CUR and LUT had no significant effect on tumor necrosis. Therefore, combined curcumin and luteolin synergically inhibit colon cancer development by suppressing cell proliferation, necrosis, and migration associated with Notch1 and TGF-β pathways. This study provides evidence that colon cancer may be prevented/treated by consuming foods having high levels of luteolin and curcumin in humans.

Keywords: TGF-β-signaling pathways; colon cancer; combination; curcumin; kinases; luteolin; notch1; synergistic; xenograft mice.

Figure 1

Combined curcumin and luteolin synergistically inhibited cell proliferation in colon cancer CL-188 cells and DLD-1cells: (A). The time course (24 h, 48 h, and 72 h) of cell proliferation by individual or combination of luteolin (LUT) and curcumin (CUR) in CL-188 and DLD-1 cells. (B). Heat map of combined CUR and LUT indicating inhibitory effects of the agents in CL-188 cells. (C). Quantitative plot of fraction affected-combination index showing the combinatorial doses of CUR and LUT in CL-188 cells. Each point represents the growth inhibition rate and fraction of affected cells. Points with a combination index (CI) > 1 indicates an antagonistic effect, CI = 1 indicates an additive effect, and CI < 1, a synergistic effect. (D). Representative images of CL-188 cells treated with LUT 30 µM, CUR at 10 or 15 µM, and their combinations for 72 h. (E). Bar graph showing growth inhibition of individual chemical CUR at 10 or 15 µM, LUT at 30 µM, or combinations in CL-188 cells. (F). Representative images of DLD-1 cells treated with LUT 30 µM, CUR at 10 or 15 µM, and their combinations for 72 h. (G). Bar graph showing growth inhibition of individual chemical CUR at 10 or 15 µM, LUT at 30 µM, or combinations in DLD-1 cells. *, Significant difference between DMSO and group; +, Significant difference between C10L30 and C10, L30 or C15L30 and C15 or L30; #, Significant difference between C10L30 and C15L30. *, + p < 0.05, **, ++, or ## p < 0.01, ***, +++ p < 0.001. Results of A, E, and G were from the average of 3–4 separate repeats.

Figure 2

Combined curcumin (CUR) and luteolin (LUT) synergistically suppressed wound healing process in CL-188 cells: (A). Representative images of CL-188 cells treated with LUT 30 µM, CUR at 10 or 15 µM, and their combinations for 0 h, 24 h, and 72 h. (B). Bar graph showing area of wound closed in CL-188 cells using agents CUR at 10 or 15 µM, LUT at 30 µM, or combinations after 24 h. (C). Bar graph showing area of wound closed in CL-188 cells using agents CUR at 10 or 15 µM, LUT at 30 µM, or combinations after 72 h. Data were expressed in mean ± SEM of at least four independent experiments. *, Significant difference between DMSO and group, +, Significant difference between C10L30 and C10, L30 or C15L30 and C15 or L30; #, Significant difference between C10L30 and C15L30. *, + p < 0.05, **, ++, or ## p < 0.01, *** p < 0.001.

Figure 3

Combined curcumin (CUR) and luteolin (LUT) synergistically inhibited protein expression of Notch 1 and TGF-β in CL-188 cells: (A). Bar graph indicating the level of Notch1 in CL-188 cells treated with the CUR and LUT and their combinations after 72 h. (B). Bar graph indicating the level of TGF-β in CL-188 cells treated with the CUR and LUT and their combinations after 72 h. Data were expressed in means ± SEM of at least four independent experiments. *, Significant difference between DMSO and group, +, Significant difference between C10L30 and C10, L30 or C15L30 and C15 or L30; #, Significant difference between C10L30 and C15L30. *, +, # p < 0.05, **, or ++, p < 0.01, ***, p < 0.001. Please see original WB images in Supplementary Figure S1.

Figure 4

Combination of curcumin (CUR) and luteolin (LUT) suppressed colon cancer tumor growth in CL-188 cell-derived xenograft mice; 100 µL of CL-188 cells mixed with Matrigel and HBSS in ratio 1:1 was injected into the flank of BALB/C nude mice. Tumor volume was monitored thrice a week. Mice with similar body weight and tumor volume were assigned into one of four groups (average tumor volume of 200 mm³/group) to receive vehicle (VEH, 5% DMSO, 5% Tween20, 90% PBS), LUT (10 mg/kg/day), CUR (20 mg/kg/day) or the combination (CURLUT, LUT at 10 mg/kg/day + CUR at 20 mg/kg/day) via intraperitoneal injection daily, allowing 2 days rest for 2 weeks (14 days). (A). A line showing tumor volume with time across all treatment groups of VEH, LUT, CUR, and CURLUT. (B). Illustration of tumor sizes from all groups. (C). A bar graph showing the average tumor volume in mm³ across all treatment groups. (D). A bar graph showing the average tumor weight of mice expressed in grams (g) between groups. (E). Bar graph indicating the level of Notch1 in tumors of mice treated. (F). Bar graph indicating the level of TGF-β in tumors. Data were expressed as means ± SEM of the animals, n = 9 mice/group. * Significant difference between group and VEH group, +, significant difference between group and LUT group, #, significant difference between group and CUR group. *, +, # p < 0.05, ***, p < 0.001. Please see original WB images in Supplementary Figure S2.

Figure 5

Combination of curcumin (CUR) and luteolin (LUT) synergistically induced necrosis in xenograft colon tumors: (A). Representative images of all groups. Areas of necrosis are pink-orange (asterisks); Intact tumor areas are dark pink-purple. 20× magnification. (B). A box plot showing necrosis of tumors (percentage of total area) within groups. + Significant difference between group and LUT group. ++ p < 0.01. n = 7–9.