Brief Article
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Nov 21, 2013; 19(43): 7726-7734
Published online Nov 21, 2013. doi: 10.3748/wjg.v19.i43.7726
Events associated with apoptotic effect of p-Coumaric acid in HCT-15 colon cancer cells
Saravana Kumar Jaganathan, Eko Supriyanto, Mahitosh Mandal
Saravana Kumar Jaganathan, Eko Supriyanto, IJ N-UTM Cardiovascular Engineering Centre, Faculty of Bioscience and Medical Engineering, Universiti Teknologi Malaysia, Johor Bahru 81310, Malaysia
Saravana Kumar Jaganathan, Department of Research and Development, PSNA College of Engineering and Technology, Dindigul 624622, India
Mahitosh Mandal, School of Medical Science and Technology, Indian Institute of Technology, Kharagpur 721302, West Bengal, India
Author contributions: Jaganathan SK designed and performed the research experiments; Jaganathan SK, Supriyanto E analyzed the data; Jaganathan SK wrote the paper; Mandal M contributed the new reagents for the study; Supriyanto E and Mandal M provided suggestions and edited the paper.
Supported by Universiti Teknologi Malaysia, Malaysia for providing Visiting Research Fellowship
Correspondence to: Dr. Saravana Kumar Jaganathan, IJ N-UTM Cardiovascular Engineering Centre, Faculty of Bioscience and Medical Engineering, Universiti Teknologi Malaysia, Johor Bahru 81310, Malaysia. jaganathaniitkgp@gmail.com
Telephone: +91-607-5558548 Fax: +91-607-5558553
Received: May 14, 2013
Revised: July 24, 2013
Accepted: August 20, 2013
Published online: November 21, 2013
Processing time: 217 Days and 19.2 Hours
Abstract

AIM: To investigate the events associated with the apoptotic effect of p-Coumaric acid, one of the phenolic components of honey, in human colorectal carcinoma (HCT-15) cells.

METHODS: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tertazolium-bromide assay was performed to determine the antiproliferative effect of p-Coumaric acid against colon cancer cells. Colony forming assay was conducted to quantify the colony inhibition in HCT 15 and HT 29 colon cancer cells after p-Coumaric acid treatment. Propidium Iodide staining of the HCT 15 cells using flow cytometry was done to study the changes in the cell cycle of treated cells. Identification of apoptosis was done using scanning electron microscope and photomicrograph evaluation of HCT 15 cells after exposing to p-Coumaric acid. Levels of reactive oxygen species (ROS) of HCT 15 cells exposed to p-Coumaric acid was evaluated using 2’, 7’-dichlorfluorescein-diacetate. Mitochondrial membrane potential of HCT-15 was assessed using rhodamine-123 with the help of flow cytometry. Lipid layer breaks associated with p-Coumaric acid treatment was quantified using the dye merocyanine 540. Apoptosis was confirmed and quantified using flow cytometric analysis of HCT 15 cells subjected to p-Coumaric acid treatment after staining with YO-PRO-1.

RESULTS: Antiproliferative test showed p-Coumaric acid has an inhibitory effect on HCT 15 and HT 29 cells with an IC50 (concentration for 50% inhibition) value of 1400 and 1600 μmol/L respectively. Colony forming assay revealed the time-dependent inhibition of HCT 15 and HT 29 cells subjected to p-Coumaric acid treatment. Propidium iodide staining of treated HCT 15 cells showed increasing accumulation of apoptotic cells (37.45 ± 1.98 vs 1.07 ± 1.01) at sub-G1 phase of the cell cycle after p-Coumaric acid treatment. HCT-15 cells observed with photomicrograph and scanning electron microscope showed the signs of apoptosis like blebbing and shrinkage after p-Coumaric acid exposure. Evaluation of the lipid layer showed increasing lipid layer breaks was associated with the growth inhibition of p-Coumaric acid. A fall in mitochondrial membrane potential and increasing ROS generation was observed in the p-Coumaric acid treated cells. Further apoptosis evaluated by YO-PRO-1 staining also showed the time-dependent increase of apoptotic cells after treatment.

CONCLUSION: These results depicted that p-Coumaric acid inhibited the growth of colon cancer cells by inducing apoptosis through ROS-mitochondrial pathway.

Keywords: Honey; Apoptosis; Rhodamine-123; Sub-G1; Merocyanine; p-Coumaric acid; Reactive oxygen species

Core tip: This article describes apoptotic effect of p-Coumaric acid, one of the phenolic components of honey, against colon cancer cells. p-Coumaric acid treatment resulted in the inhibition of proliferation and colony forming ability of human colorectal carcinoma (HCT-15) and HT 29 cells. Major events associated with growth-inhibition are increasing reactive oxygen species generation, increasing lipid layer breaks and a fall in Mitochondrial membrane potential. Further, membrane blebbing and shrinkage of p-Coumaric acid exposed HCT 15 cells insinuated apoptosis. Hence our results depicted that p-Coumaric acid is a prospective candidate for chemoprevention of colon cancer.