Rutin Potentially Binds the Gamma Secretase Catalytic Site, Down Regulates the Notch Signaling Pathway and Reduces Sphere Formation in Colonospheres

Abstract

Rutin, a natural flavonol, can modulate molecular signaling pathways and has considerable potential in cancer treatment. However, little is known about the effect of rutin on the notch signaling pathway (NSP) in cancer and cancer stem-like cells. In this study, we explored the effect of rutin on gamma secretase (GS, a putative notch signaling target) inhibition mediated NICD (Notch Intracellular Domain) production in colon cancer cells. Molecular docking, MM-GBSA, and Molecular dynamics (MD) simulation experiments were performed to check rutin’s GS catalytic site binding potential. The HCT-116 colon cancer and cancer stem-like cells (colonospheres) were utilized to validate the in silico findings. The NICD production, notch promoter assay, expression of notch target genes, and cancer stemness/self-renewal markers were studied at molecular levels. The results were compared with the Notch-1 siRNA transfected test cells. The in silico study revealed GS catalytic site binding potential in rutin. The in vitro results showed a decreased NICD formation, an altered notch target gene (E-cad, Hes-1, and Hey-1) expression, and a reduction in stemness/self-renewal markers (CD44, c-Myc, Nanog, and SOX2) in test cells in a time and dose-dependent manner. In conclusion, rutin inhibits the notch signaling pathway and reduces the stemness/self-renewal property in colon cancer cells and the colonospheres by targeting gamma secretase. The clinical efficacy of rutin in combination therapy in colon cancer may be studied in the future.

Natural Steroidal Lactone Induces G1/S Phase Cell Cycle Arrest and Intrinsic Apoptotic Pathway by Up-Regulating Tumor Suppressive miRNA in Triple-Negative Breast Cancer Cells

“…Withaferin A (WA) was procured from Sigma-Aldrich (89910-10MG) and dissolved in DMSO (0.1%). The cell proliferation inhibition potential of WA was studied using an MTT assay as per the method described elsewhere [ 20 ]. Briefly, the cells (1 × 10 4 cells/well) were seeded in the 96-well microtiter plates in triplicate.…”

“…Formazan crystals were solubilized with 100 μL DMSO, and the absorbance was measured at 590 nm using a microplate reader (BioTek Instruments, Inc., Santa Clara, CA, USA). The percentage cell proliferation inhibition potential was calculated as per a formula described elsewhere [ 20 ]. The 50% inhibitory concentration (IC 50 ) for both the cell lines was calculated by GraphPad Prism 5.0 software using a regression analysis tool.…”

“…The final concentration of the miRNA mimic and negative control was 60 nm. The cells were transfected for 24, 48, and 72 h. To assess the transfection efficacy, the effect (gain of function) of miRNA mimic transfection was measured compared to negative control group [ 20 ]. For this, the total miRNA was isolated using a mirVanaTM miRNA isolation kit (Catalog# P/N 15604, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and a SYBER green-based qRT-PCR analysis was performed as per the methodology discussed in Section 2.8 .…”

“…GAPDH was used as the endogenous control to normalize the expression level. The cycle of threshold (Ct) value of each sample was calculated by using the 2 −∆∆ CT method for the fold-change expression of mRNA expression [ 12 , 20 , 24 ]. The primer sequence of test genes (caspase 3, caspase 8, BAX, PAPR, Bcl2, and GAPDH) is provided in Supplementary Table S2 .…”

“…The bands were developed with the enhanced chemiluminescence (ECL) system (Thermo Fisher Scientific). The signals were captured through the ChemiDoc imaging instrument (Bio-Rad), Hudson MA, USA and the image was processed using image lab software 6.0.1 Bio-Rad, Hudson MA, USA [ 12 , 20 ].…”