Association between reproductive factors and Colorectal Cancer; Identification and Characterization of β-catenin (CTNNB1) gene and modulatory potential of Taxifolin and Nano-particle with vitamin D (NVD) against colorectal cancer in Pakistani population

Abstract

Colorectal cancer (CRC) is categorized by alteration of vital pathways such as β- catenin (CTNNB1) mutations, WNT signaling activation, tumor protein 53 (TP53) inactivation, BRAF, Adenomatous polyposis coli (APC) inactivation, KRAS, dysregulation of epithelial to mesenchymal transition (EMT) genes, MYC amplification etc. The assorted molecular outlines of colorectal cancer (CRC) and the necessity to categorize patients which could efficiently take clinical benefit from combined chemotherapies ignited the categorization of the mechanisms accountable for sensitivity and confrontation to treatments. More than the precedent two decades, the question of whether vitamin D has a role in cancer frequency, development, and transience has been premeditated fully. Colorectal, breast, and prostate cancers have been a scrupulous spot of center, in concert, these three malignancies report for approximately 35% of cancer cases and 20% of cancer demises in the United States, and as such are a chief public health apprehension. In the present study an attempt was made to screen CTNNB1 gene in colorectal cancer samples from Pakistani population and investigated the association of CTNNB1 gene mutations in the development of colorectal cancer. Data on 25-OH VD concentrations and the associated factors in colorectal cancer (CRC) patients are scarce and need to be investigated and t o evaluate and equate antitumor activity of taxifolin (TAX) and nanoparticle plus vitamin D (NVD) in colorectal cancer cell lines and HCT116 xenograft model in a comprehensive approach. Materials and Methods: 200 colorectal tumors approximately of male and female patients with sporadic or familial colorectal tumors and normal tissues were included. DNA was extracted and amplified through Polymerase chain reaction (PCR) and subjected to exome sequence analysis. Immunohistochemistry was done to study protein expression. Molecular dynamic (MD) simulations of CTNNB1WT and mutant S33F and T41A were performed to evaluate the stability, folding, conformational changes and dynamic behaviors of CTNNB1 protein. A total of 200 CRC patients participated in this cross-sectional study conducted in Pakistan. Socio- demographic and other health data were collected in a pretested questionnaire. Serum measurements of Vitamin D (1, 25(OH)2 D3) levels and hormones were performed. Association of age, sex, primary site, and stage of disease and effects of hormone therapy and selected reproductive health indicators on vitamin D status were initially investigated by univariate analysis. Two human colorectal cancer cell lines HCT116 and HT29 XII (gained from College of Pharmacy, King Saud University, KSA were grown. 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide protocol were performed to show the impact of TAX, NVD and β- catenin inhibitor (FH535) on the viability of HCT116 and HT29 cell lines. Apoptosis /cell cycle assay was performed. Analysis was done with a FACScan (Becton Dickinson, NJ). About 10,000 cells per sample were harvested and 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: Sequence analysis revealed two activating mutations (S33F and T41A) in Exon 3 of CTNNB1 gene involving transition of C.T and A.G at amino acid position 33 and 41 respectively (p.C33T and p.A41G). Immuno-histochemical staining showed the accumulation of β-catenin protein both in cytoplasm as well as in the nuclei of cancer cells when compared with normal tissue. Further molecular modeling, docking and simulation approaches revealed significant conformational changes in the N-terminus region of normal to mutant CTNNB1 gene critical for binding with Glycogen synthase kinase 3-B (GSK3) and transducin containing protein1 (TrCp1). The blood samples of 200 colorectal cancer patients, males and females were taken under consideration. The mean age of the population was 55.3 years (±15.6; Range: 20-90 years). Estradiol concentration were significantly higher in young female as compared to young male patients (p<0.001). The concentrations of FSH, LH testosterone and estradiol were significantly lower in post-menopausal female CRC patients as compared to their male counterparts of old age (p, for all trends <0.05). Both LH and FSH showed significant gender difference only in older ages. Level of estrogen is markedly decreased in older post-menopausal CRC patients compared to premenopausal CRC cohorts, which might be associated with CRC progression. In the group of women who ever used hormone therapy had differences of statistical significance (p, for all trends <0.05) in their mean serum 25-OH VD concentrations, while in the group of women who never used hormone therapy had non-significant differences in their mean serum 25-OH VD concentrations (p, for all trends>0.05). High 25-OH VD concentrations were observed in women who had their menarche at the age 15 years or more. Nulliparous women had the highest mean 25-OH VD concentrations as compared to unparious or multiparious women. Women having their menopause at 40-44 years of age had the highest 25-OH VD concentrations, although the difference was not significant (p=0.08). Women who never used any oral contraceptive had higher 25-OH VD concentrations as compared to those who ever XIII used oral contraceptives. We found that TAX and NVD induced cytotoxicity in colorectal cells in a dose-dependent manner and time dependent approach. Further, our data validated that TAX and NVD administration of human colorectal cancer HCT116 and HT29 cells resulted in cell growth arrest, alteration in molecules regulating cell cycle operative in the G2 phase of the cell cycle and apoptosis in a dose 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: Screening of Pakistani population revealed association of two non-synonymous polymorphisms in CTNNB1 gene with colorectal cancer. These genetic variants led to the accumulation of CTNNB1, a hallmark of tumor development. Also analysis of structure to function alterations in CTNNB1 gene is crucial in understanding downstream biological events. The results of this analysis support a role for hormones in 25-OH VD concentrations. Further prospective studies that directly evaluate levels of circulating hormones and hormone therapy in women in relation to 25-OH VD concentrations as well as their possible role in colorectal cancer risk would be highly informative. Also our findings suggest 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: Wnt/ β-catenin; Taxifolin, cell cycle. CTNNB1, Colorectal cancer, Immunohistochemistry, Vitamin D (1, 25(OH)2 D3), sex hormones.