Rutamarin, an active constituent from Ruta angustifolia Pers., induced apoptotic cell death in the HT29 colon adenocarcinoma cell line

Abstract

Background:Ruta angustifolia Pers. is a perennial herb that is cultivated worldwide, including Southeast Asia, for the treatment of various diseases as traditional medicine.Objective:The purpose of the study was to identify an active principle of R. angustifolia and to investigate its effect on the HT29 cell death.Materials and Methods:The methanol and fractionated extracts (hexane, chloroform, ethyl acetate, and water) of R. angustifolia Pers. were initially investigated for their cytotoxic activity against two human carcinoma cell lines (MCF7 and HT29) and a normal human colon fibroblast cell line (CCD-18Co) using sulforhodamine B cytotoxicity assay. Eight compounds including rutamarin were isolated from the active chloroform extract and evaluated for their cytotoxic activity against HT29 human colon carcinoma cell line and CCD-18Co noncancer cells. Further studies on the induction of apoptosis such as morphological examinations, biochemical analyses, cell cycle analysis, and caspase activation assay were conducted in rutamarin-treated HT29 cells.Results:Rutamarin exhibited remarkable cytotoxic activity against HT29 cells (IC50 value of 5.6 μM) but was not toxic to CCD-18Co cells. The morphological and biochemical hallmarks of apoptosis including activation of caspases 3, 8, and 9 were observed in rutamarin-treated HT29 cells. These may be associated with cell cycle arrest at the G0/G1 and G2/M checkpoints, which was also observed in HT29 cells.Conclusions:The present study describes rutamarin-induced apoptosis in the HT29 cell line for the first time and suggests that rutamarin has the potential to be developed as an anticancer agent.SUMMARY Rutamarin was cytotoxic to HT29 colon cancer cells but exerted no damage to normal colon cellsRutamarin induced morphological and biochemical hallmarks of apoptosis in HT29 cellsRutamarin induced cell cycle arrest at the G0/G1 and G2/M checkpoints in a dose-dependent manner in HT29 cellsRutamarin activated caspases 3, 8, and 9 in a dose-dependent manner in HT29 cells. Abbreviations used: ACN: Acetonitrile, ANOVA: One-way analysis of variance, BrdU: Bromodeoxyuridine, 13C-NMR: Carbon-13 Nuclear magnetic resonance, CAD: Caspase-activated endonuclease, CCD-18Co: Human colon normal, DLD1: Human Duke's type C colorectal adenocarcinoma, DMRT: Duncan's multiple range test, DMSO: Dimethyl sulfoxide, DNA: Deoxyribonucleic acid, DR4/5: Death receptor 4/5 protein, EMEM: Eagle's minimum essential media, FBS: Fetal bovine serum, FITC Annexin V: Annexin V conjugated with fluorescein isothiocyanate, FITC-DEVD-FMK: Fluorescein isothiocyanate conjugate of caspase inhibitor Asp-Glu-Val-Asp-fluoromethyl ketone, FITC-IETD-FMK: Fluorescein isothiocyanate conjugate of caspase inhibitor Ile-Glu-Thr-Asp-fluoromethyl ketone, FITC-LEHD-FMK: Fluorescein isothiocyanate conjugate of caspase inhibitor Leu-Glu-His-Asp-fluoromethyl ketone, G0: Quiescent phase of cell cycle, G1: Gap 1 phase of cell cycle, G2: Gap 2 phase of cell cycle, GC-MS: Gas chromatography-mass spectrometry, HeLa: Human cervical aden...

Cytotoxic effect of methanolic extract, alkaloid and terpenoid fractions of Stachys pilifera against HT-29 cell line

“…The untreated cells in 1% DMSO were used as a negative control. The changes in cellular morphology were observed using phase contrast microscopy at ×10 magnification (Olympus, Japan)( 21 ).…”

Expression and Purification of Recombinant Bowman–Birk Trypsin Inhibitor from Foxtail Millet Bran and Its Anticolorectal Cancer Effect In Vitro and In Vivo

“…Cell Culture and MTT Assay. These CRC cell lines were classified according to the stage of the tumor from which they were established: HCT-116 14 as Dukes' stage A, HT-29 15 as Dukes' stage B, SW620 16,17 and DLD-1 18 as Dukes' stage C. CRC cells (SW620, DLD-1, HT-29 and HCT-116) and normal colonic epithelial cells (FHC) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and cultured in an incubator at 5% CO 2 at 37 °C.…”

Cytotoxic, Anti-Hemolytic, and Antioxidant Activities of Ruta chalepensis L. (Rutaceae) Extract, Fractions, and Isolated Compounds

“…There are a variety of studies where the extracts, fractions, and major components of plants of the Rutaceae family, showed antioxidant and cytotoxicity against a variety of tumor cells [12,16].To date, there are several studies with furanocoumarins, including CHL (also known as xylotenin) and rutamarin (RTM; also known as chalepin acetate), that report cytotoxic, antiproliferative, antimicrobial, antiparasitic, anti-inflammatory, and anticancer properties, among others [6,15,[17][18][19][20]. Another important family of compounds are the alkaloids, such as graveoline (GRV), which possess antibacterial, antifungal, cytotoxic, apoptotic, and autophagic activity [21][22][23].…”