Taxifolin, a natural flavonoid interacts with cell cycle regulators causes cell cycle arrest and causes tumor regression by activating Wnt/ β -catenin signaling pathway

Abstract

Background: New approaches for the prevention of colon cancer perseveres an essential necessity. Though, resistance to existing chemo-preventive drugs is moderately predominant in colon carcinogenesis. Taxifolin (dihydroquercetin) is a flavononol, have shown virile biological activities against few cancers. The current study was designed to investigate and equate antitumor activity of Taxifolin (TAX) in colorectal cancer cell lines and in HCT116 xenograft model in a comprehensive approach.

Methods: Two human colorectal cancer cell lines HCT116 and HT29, were used. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide (MMT) protocol was performed to elucidate the impact of TAX and β- catenin inhibitor (FH535) on the viability of HCT116 and HT29 cell lines. Apoptosis /cell cycle assay was performed. Data interpretation was done with a FACScan (Becton Dickinson, NJ). About 1 × 10⁴ cells per sample were harvested. Histograms of DNA were analyzed with ModiFitLT software (verity Software House, ME, USA). Western blotting and RT-PCR were performed for protein and gene expression respectively in in vitro and in vivo.

Results: We found that TAX induced cytotoxicity in colorectal cells in a dose-dependent manner and time dependent approach. Further, our data validated that administration of TAX to human colorectal cancer HCT116 and HT29 cells resulted in cell growth arrest, variation in molecules controlling cell cycle operative in the G2 phase of the cell cycle and apoptosis in a concentration dependent approach. Further our results concluded that TAX administration decreases expression of β-catenin gene, AKT gene and Survivin gene and protein expression in in vitro and in vivo.

Conclusion: Our findings proposed that targeting β-catenin gene may encourage the alterations of cell cycle and cell cycle regulators. Wnt/β-catenin signaling pathway possibly takes part in the genesis and progression of colorectal cancer cells through regulating cell cycle and the expression of cell cycle regulators.

Keywords: Colorectal cancer; Taxifolin and cell cycle; Wnt/ β-catenin.

Fig. 1

a and b Inhibition of cell growth in colorectal cancer. 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide assay was carried out on two colorectal cancer cell lines HCT116 and HT29 cells in order to gauge the effect of TAX and FH535 administration on growth of these colorectal cancer cell lines ***, P < 0.001 and **, P < 0.01 vs. control. % cell viability of HCT116 and HT29 cells were determined. c and d Inhibition of cologenecity in colorectal cancer cells by TAX (Clonogenic assay; 7 days). TAX administration (40 and 60 μM) of HCT116 and HT29 cells inhibiting colony formation. Each value represents a mean ± SD (n = 3)

Fig. 2

a and b TAX treatment on HCT116 and HT29 cells resulted in accumulation of cells at the G2-phase. After 24 h incubation of TAX treated cells and staining with propidium iodide, DNA content was analyzed by flow cytometry. Percentage of cell population in G2-phase of the cell cycle is shown. c, d and e TAX treatment on HCT116 and HT29 cells resulted in growth inhibition and apoptosis, after 24 h incubation of TAX treated cells and staining with propidium iodide, Cells were analyzed by flow cytometry. Percentage of apoptotic cells is shown. Experiments were performed in triplicate

Fig. 3

Effect of TAX treatment of HCT116 and HT 29 cells on protein expression of WAF1/p21 and KIP1/p27, cdk 2, 4 and 6, cyclin D, B1 and A. Experiments were performed in triplicate

Fig. 4

Effect of TAX on induction of apoptosis. Effect of TAX on protein expression of active Caspase 3, 7 and 9 and truncation of Bid in HCT116 and HT29 cells. Cells were treated with TAX (20, 40 and 60 μM), 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 verified by stripping the Immunoblot and again probing it for Actin. The immunoblots shown here are representative of three individual experiments with similar results

Fig. 5

Effect of TAX on protein expression of PI3K and phosphorylation of Akt at Ser473 in HCT116 and HT29 cells. Also the effect of treatment of TAX on HCT116 and HT29 cell lines on protein expression of Bax, Bcl2, Bak, Bcl-X_L and PARP. Total cell lysate were prepared and 40 μg of 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 Actin

Fig. 6

a Immunoblot analysis of β-catenin expression of HCT116 and HT29 in TAX administrated group as compared to control group (b, c) qPCR analysis of TAX administrated HCT116 and HT29 cells for changes in β-catenin mRNA levels. The data expressed as fold change represent the mean ± standard errors experiment performed in triplicate where*p < 0.05,**p < 0.01 was considered significant vs control

Fig. 7

Imunofluorescence staining of HCT116 (b) and HT29 cells (d) demonstrating expression of β-catenin in both TAX treated (40 mol/L) as compared to control HCT116 (a) and HT29 (c) cells (untreated). Alexa fluor staining of TAX of both cell lines (green fluorescence) and counter stained with DAPI (blue fluorescence) were seen

Fig. 8

Imunofluorescence staining of HCT116 (A1) and HT29 cells (B1) demonstrating expression of p-AKT in both TAX treated as compared to control HCT116 (a) and HT29 (b) cells (untreated). Alexa fluor staining of p-AKT of both cell lines (green fluorescence) and counter stained with DAPI (blue fluorescence) were observed

Fig. 9

Consequence of TAX administration on HCT116 tumor growth in athymic nude mice. a Average tumor volume of water fed, 15 & 25 kg/mg taxifloin injected mice plotted over days after tumor cell inoculation. Values represent mean ± SD of six mice. *, p < 0.01 (25 mg/kg); **, p < 0.05 (15 mg/kg) vs water fed normal control mice ***, p < 0.001. b H&E staining of taxifolin administrated xenograft tumors vs control. c and d Immunoblotting and Immunohistochemistry of cleaved Caspase 3 of TAX administrated tumor xenograft vs untreated control tumor xenografts. The data are representative of three independent experiments with similar results

Fig. 10

TAX hampers proliferation in xenograft tumors in athymic nude mice. a Effect of TAX administration (15 and 25 mg) on protein expression of cyclin D of HCT116 implanted xenograft tumors in athymic nude mice. The data are representative of three independent experiments with similar results. b Immunohistochemistry of cyclin D tumor xenograft vs untreated control tumor xenografts

Fig. 11

Effect of TAX administrated treatment on HCT116 xenograft tumors for protein expression of β-catenin. a Expression of β-catenin and survivin protein by immunobloting in TAX administrated and control group, experiment performed in triplicate. b qPCR analysis of TAX administrated for changes in β-catenin mRNA levels. The data expressed as fold change represent the mean ± standard errors experiment performed in triplicate where*p < 0.05, **p < 0.01 was considered significant v/s control. c Effect of TAX administrated as detected by immunohistochemical staining. DAB staining of β-catenin (brown) and counter stained with hematoxylin (blue). d Effect of TAX administrated on protein expression of Akt and control as detected by immunohistochemical staining. DAB staining of Akt (brown) and counter stained with hematoxylin (blue)