Gallic acid induced apoptotic events in HCT-15 colon cancer cells.
OBJECTIVE
To investigate the inhibitory action of diet-derived phenolic compound gallic acid (GA) against HCT-15 colon cancer cells.


METHODS
The antiproliferative effect of GA against colon cancer cells was determined by performing thiazolyl blue tetrazolium bromide (MTT) assay. The colony forming ability of GA treated colon cancer cells was evaluated using the colony forming assay. The cell cycle changes induced by GA in HCT-15 cells were analyzed by propidium iodide staining. Levels of reactive oxygen species (ROS) and mitochondrial membrane potential of HCT-15 exposed to GA was assessed using 2',7'-dichlorfluorescein-diacetate and rhodamine-123 respectively, with the help of flow cytometry. Morphological changes caused by GA treatment in the colon cancer cells were identified by scanning electron microscope and photomicrograph examination. Apoptosis was confirmed using flow cytometric analysis of GA treated HCT-15 cells after staining with Yo-Pro-1.


RESULTS
MTT assay results illustrated that GA has an inhibitory effect on HCT-15 cells with IC50 value of 740 μmol/L. A time-dependent inhibition of colony formation was evident with GA treatment. Cell cycle arrest was evident from the accumulation of GA treated HCT-15 cells at sub-G1 phase (0.98 ± 1.03 vs 58.01 ± 2.05) with increasing exposure time. Flow cytometric analysis of GA treated HCT-15 cells depicted early events associated with apoptosis like lipid layer breakage and fall in mitochondrial membrane potential apart from an increase in the generation of ROS which were in a time dependent manner. SEM and photomicrograph images of the GA-treated cells displayed membrane blebbing and cell shrinking characteristics of apoptosis. Further apoptosis confirmation by Yo-Pro-1 staining also showed the time-dependent increase of apoptotic cells after treatment.


CONCLUSIONS
These results show that GA induced ROS dependent apoptosis and inhibited the growth of colon cancer cells.
Aruna Priyadharshni Subramanian, Saravana Kumar Jaganathan, Eko Supriyanto, Ida Idayu Muhamad, IJN-UTM Cardiovascular Engineering Centre, Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, Johor Bahru 81310, Malaysia.
Aruna Priyadharshni
AP
Subramanian
Aruna Priyadharshni Subramanian, Saravana Kumar Jaganathan, Eko Supriyanto, Ida Idayu Muhamad, IJN-UTM Cardiovascular Engineering Centre, Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, Johor Bahru 81310, Malaysia.
Saravana Kumar
SK
Jaganathan
Aruna Priyadharshni Subramanian, Saravana Kumar Jaganathan, Eko Supriyanto, Ida Idayu Muhamad, IJN-UTM Cardiovascular Engineering Centre, Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, Johor Bahru 81310, Malaysia.
Mahitosh
M
Mandal
Aruna Priyadharshni Subramanian, Saravana Kumar Jaganathan, Eko Supriyanto, Ida Idayu Muhamad, IJN-UTM Cardiovascular Engineering Centre, Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, Johor Bahru 81310, Malaysia.
Eko
E
Supriyanto
Aruna Priyadharshni Subramanian, Saravana Kumar Jaganathan, Eko Supriyanto, Ida Idayu Muhamad, IJN-UTM Cardiovascular Engineering Centre, Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, Johor Bahru 81310, Malaysia.
Ida Idayu
II
Muhamad
D000230:Adenocarcinoma / Q000188:drug therapy* / Q000378:metabolism / Q000648:ultrastructure; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000648:ultrastructure; D004305:Dose-Response Relationship, Drug; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects; D005707:Gallic Acid / Q000494:pharmacology*; D006801:Humans; D020128:Inhibitory Concentration 50; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D013997:Time Factors
D000970:Antineoplastic Agents; D017382:Reactive Oxygen Species; D005707:Gallic Acid
Apoptosis; Cell cycle; Colon cancer; Gallic acid; Lipid layer break; Reactive oxygen species