Fisetin and 5-fluorouracil: Effective combination for PIK3CA-mutant colorectal cancer

Abstract

The normal colon epithelium is transformed into its neoplastic counterpart through a series of genetic alterations in driver genes including activating mutations in PIK3CA. Treatment often involves surgery followed by 5-fluorouracil (5-FU) based therapy, which has limited efficiency and serious side effects. We sought to determine whether fisetin, a dietary flavonoid, alone or in combination with 5-FU affected tumorigenesis in the mammalian intestine. We first determined the effect of fisetin, 5-FU or their combination on PIK3CA-mutant and PIK3CA wild-type colon cancer cells by assessing cell viability, colony formation, apoptosis and effects on PI3K/AKT/mTOR signaling. Treatment of PIK3CA-mutant cells with fisetin and 5-FU reduced the expression of PI3K, phosphorylation of AKT, mTOR, its target proteins, constituents of mTOR signaling complex and this treatment increased the phosphorylation of AMPKα. We then determined whether fisetin and 5-FU together or singly affected tumorigenesis in Apc^Min/+ mice that also express constitutively active PI3K in the distal small intestine and colon. Tumor incidence was markedly lower in fisetin-treated FC¹ 3K¹ Apc^Min/+ mice that also express constitutively active PI3K in distal small intestine and colon, as compared to control animals, indicating that fisetin is a strong preventive agent. In addition, the combination of fisetin and 5-FU also reduced the total number of intestinal tumors. Fisetin could be used as a preventive agent plus an adjuvant with 5-FU for the treatment of PIK3CA-mutant colorectal cancer.

Keywords: PIK3CA; 5-fluorouracil; PI3K/AKT/mTOR signaling; colorectal cancer.

Figure 1.

Effect of fisetin, 5-FU or their combination on cell growth and colony formation in human colon cancer cells. (a) Effect of fisetin, 5-FU or their combination on cell growth. As described in Materials and Methods, SW480, HCT116 and HT29 cells were treated with fisetin (30–90 μM), 5-FU (50 μM) and their combination for 48 hr and the viability of cells was determined by the MTT assay. The data is expressed as the percentage of cell viability and represent the mean ± SEM of three experiments in which each treatment was performed in multiple wells. Treatment groups of each cell line were compared to the respective control group of each cell line (*p < 0.0001). Effect of fisetin, 5-FU and their combination on colony formation in (b) HCT116 and (c) HT29 cells. The cells were seeded in six-well plates and treated with fisetin (30–90 μM), 5-FU (50 μM) or their combination as described in Materials and Methods. At the end of the experiment (14 days), colonies were washed with 1× phosphate-buffered saline, stained with crystal violet and photographed. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2.

Effect of fisetin, 5-FU or their combination on apoptosis by annexin-V-FLUOS staining in (a) HCT116 cells and (b) HT29 cells. The cells were treated with fisetin (30–90 μM), 5-FU (50 μM) or their combination and resuspended in Annexin-V-FLUOS labeling solution. The data was collected on a Becton-Dickinson FACSCalibur (San Jose, CA) and analyzed in FlowJo, Version 9.7 (FLowJo, LLC, Ashland, OR). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3.

Effect of fisetin, 5-FU or their combination on the protein expression of PI3K, phosphorylation of AKT, mTOR and AMPKα in PIK3CA-mutant human colon cancer cells. (a) Effect of fisetin, 5-FU or their combination on the protein expression of PI3K and phosphorylation of AKT in HCT116 cells, and (b) HT29 cells. (c) Effect of fisetin, 5-FU or their combination on the phosphorylation of mTOR and AMPKα in HCT116 cells, and (d) HT29 cells. As described in the in Materials and Methods, the cells were treated with fisetin (30–90 μM), 5-FU (50 μM) and their combination for 48 hr and then harvested. Total cell lysates were prepared and 40 μg protein was subjected to SDS-PAGE followed by immunoblot analysis and chemiluminescence detection. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for vinculin.

Figure 4.

Effect of fisetin, 5-FU or their combination on the phosphorylation of mTOR target proteins and constituents of the mTOR signaling complex in PIK3CA-mutant human colon cancer cells. (a) Effect of fisetin, 5-FU or their combination on the phosphorylation of mTOR target proteins in HCT116 cells, and (b) HT29 cells. (c) Effect of fisetin, 5-FU or their combination on the protein expression of constituents of the mTOR signaling complex in HCT116 cells, and (d) HT29 cells. As described in the Materials and Methods, the cells were treated with fisetin (30–90 μM), 5-FU (50 μM) and their combination for 48 hr and then harvested. Total cell lysates were prepared and 40 μg proteins was subjected to SDS-PAGE followed by immunoblot analysis and chemiluminescence detection. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for vinculin.

Figure 5.

Effect of the treatment of fisetin, 5-FU or their combination on colorectal tumorigenesis in FC¹3K¹Apc^Min/+ mice. (a) Effect of the treatment of fisetin, 5-FU or their combination on colon tumor incidence in FC¹3K¹Apc^Min/+ mice that had no visible tumors at the beginning of treatment. (b) Effect of the treatment of fisetin, 5-FU or their combination on tumor growth (early) in FC¹3K¹Apc^Min/+ mice. The size of each tumor at each colonoscopy visit was calculated by determining what percentage of the fully inflated lumen was occluded by the tumor and plotted with each different colored line representing a distinct tumor. (c) The calculation was made from still images of each tumor that were collected during each colonoscopy visit. NA, not available, because the control became moribund and was euthanized prior to the fourth colonoscopy visit. (d) Effect of the treatment of fisetin, 5-FU or their combination on tumor multiplicity in FC¹3K¹Apc^Min/+ mice. When mice became moribund or the study ended, experimental mice in all groups were euthanized and necropsied to score the number of intestinal tumors. The small intestine was divided into four equal segments with S1 being closest to the stomach and S4 being closest to the cecum; S1 was combined with S2 and S3 was combined with S4 because the FC transgene is expressed only in S3, S4 and the colon. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6.

Effect of the treatment of fisetin, 5-FU or their combination on tumor growth (late) in FC¹3K¹Apc^Min/+ mice. Treatment with fisetin and 5-FU seemed to extend the life of experimental animals. To determine whether the drugs were effective on tumors that were more advanced, a second set of experimental mice received drugs starting between 45 and 53 days of age. Mice were given up to five rounds of treatment and euthanized when moribund or when the study was completed. Tumor growth was monitored by colonoscopy and plotted. [Color figure can be viewed at wileyonlinelibrary.com]