Cytotoxicity and apoptosis induction of sea cucumber Holothuria atra extracts

Abstract

Context: Cancer is one of the major causes of death. Sea cucumbers are marine invertebrate that is widely used and utilized in traditional medicine. One of the most studied of sea cucumbers efficacy is its anticancer property. The anticancer property of sea cucumbers is much related to the content of an active compound called saponin. Objective: The objective of this research is to determine the cytotoxicity and apoptosis induction of sea cucumber Holothuria atra ethanol extract and to study its anticancer active compound. Materials and Methods: Sea cucumber of H. atra was taken from Halmahera waters, North Maluku Indonesia. The extraction was conducted by maceration method using ethanol 96% then continued with liquid-liquid partition and separation using C18 column. The investigation of active compound content was conducted by phytochemical and spectroscopic analysis using liquid chromatography-ion trap-time of a flight-mass spectrophotometer (LC-IT-ToF-MS). The cytotoxicity test was performed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide method against human breast ductal carcinoma cell (T47D), human breast adenocarcinoma (MCF7), human colorectal adenocarcinoma cell (WiDr), and human cervix adenocarcinoma cell lines (HeLa). The apoptotic induction test was performed using flow cytometry using Fluorescein isothiocyanate (FITC) Annexin-V-Apoptosis and caspase-3 detection using FITC Active Caspase-3 kit. Results: Phytochemical test showed that ethanol extract of H. atra contained alkaloids, flavonoids, steroids-triterpenoids, phenols, and saponins. The extract showed cytotoxic activity against the 4 cell lines with Inhibition concentration 50 values ranging from 9.6 to 14.3 μg/ml. Flow cytometry analysis showed that the T47D cell population underwent apoptosis after treated with ethanol extract. The extract also activated caspase-3 on the T47D cells. The results of LC-IT-ToF-MS analysis showed that the active fraction from C18 column separation contained saponin and identified as Cucumechinol and Philinopgenin B. Conclusions: The results of this study indicated that H. atra active extract has good cytotoxicity and has potential to be developed as an anticancer agent. Abbreviations used: MNPs: Marine natural products; LC-IT-ToF-MS: Liquid chromatography-ion trap-time of flight-mass spectrophotometer; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; T47D: Human breast ductal carcinoma cell; MCF7: Human breast adenocarcinoma cell; WiDr: Human colorectal adenocarcinoma cell; HeLa: Human cervix adenocarcinoma cell; RPMI: Roswell park memorial institute medium; FBS: Fetal bovine serum; FITC: Fluorescein isothiocyanate; PBS: Phosphate buffer saline; BD: Becton Dickinson; IC50: Inhibition concentration 50; PS: Phosphatidylserine; PI: Propidium iodide.