1-(2,6-dihydroxy-4-methoxyphenyl)-2-(4-hydroxyphenyl) ethanone-induced cell cycle arrest in G₁/G₀ in HT-29 cells human colon adenocarcinoma cells

1-(2,6-Dihydroxy-4-methoxyphenyl)-2-(4-hydroxyphenyl) ethanone (DMHE) was isolated from the ethyl acetate fraction of Phaleria macrocarpa (Scheff.) Boerl fruits and the structure confirmed by GC-MS (gas chromatography-mass spectrometry) and NMR (nuclear magnetic resonance) analysis. This compound was tested on the HT-29 human colon adenocarcinoma cell line using MTT (method of transcriptional and translational) cell proliferation assay. The results of MTT assay showed that DMHE exhibited good cytotoxic effect on HT-29 cells in a dose- and time-dependent manner but no cytotoxic effect on the MRC-5 cell line after 72 h incubation. Morphological features of apoptotic cells upon treatment by DMHE, e.g., cell shrinkage and membrane blebbing, were examined by an inverted and phase microscope. Other features, such as chromatin condension and nuclear fragmentation were studied using acridine orange and propidium iodide staining under the fluorescence microscope. Future evidence of apoptosis/necrosis was provided by result fromannexin V-FITC/PI (fluorescein-isothiocyanate/propidium iodide) staining revealed the percentage of early apoptotic, late apoptotic, necrotic and live cells in a dose- and time-dependent manner using flow cytometry. Cell cycle analysis showed G0/G1 arrest in a time-dependent manner. A western blot analysis indicated that cell death might be associated with the up-regulation of the pro-apoptotic proteins Bax PUMA. However, the anit-apotptic proteins Bcl-2, Bcl-xL, and Mcl-1 were also found to increase in a time-dependent manner. The expression of the pro-apoptotic protein Bak was not observed.