Apoptosis-Inducing Activity of HPLC Fraction from Voacanga globosa (Blanco) Merr. on the Human Colon Carcinoma Cell Line, HCT116

Alvin Resultay Acebedo,Evangeline Cancio Amor,Sonia Donaldo Jacinto

doi:10.7314/apjcp.2014.15.2.617

Abstract

Voacanga globosa (Blanco), a plant endemic to the Philippines, is traditionally used especially by indigenous people of Bataan in the treatment of ulcers, wounds and tumorous growths. This study aimed to provide scientific evidence to therapeutic properties by determining cytotoxic and pro-apoptotic activity of HPLC fractions from leaves on HCT116 human colon carcinoma and A549 human lung carcinoma cell lines. Ethanolic extraction was performed on V globosa leaves followed by hexane and ethyl acetate partitioning. Silica gel column chromatography and high performance liquid chromatography (HPLC) produced MP1, MP2 and MP3 fractions. Cytotoxic activity of the fractions was determined through MTT assay against the cancer cell lines HCT116 and A549 and the non-cancer AA8 Chinese hamster ovarian cell line. Pro-apoptotic activities of the most active fractions were further assessed through DAPI staining, TUNEL assay and JC-1 mitochondrial membrane potential assay with HCT116 cells. While the MP1 fraction exerted no significant activity against all cell lines tested, MP2 and MP3 fractions demonstrated high toxicity against HCT116 and A549 cells. The MP3 fraction induced formation of apoptotic bodies, condensed DNA and other morphological changes consistent with apoptosis of HCT116 cells and TUNEL assay showed significant increase in DNA fragmentation over time. In these cells, the MP3 fraction also induced mitochondrial membrane destabilization, which is generally associated with the beginning of apoptosis. Phytochemical analysis demonstrated the presence only of saponins and terpenoids in the MP3 fraction. The results indicate that the MP3 fraction exerts cytotoxic activity on HCT116 cells via induction of apoptosis triggered by loss of mitochondrial membrane potential crucial for cell survival.

Highlights

During carcinogenesis, certain mutations suppress apoptosis allowing the continued survival of cancer cells (Hanahan and Weinberg, 2000; Varfolomeev and Vucic, 2011)
Pro-apoptotic activities of the most active fractions were further assessed through DAPI staining, transferase dUTP nick end labelling (TUNEL) assay and JC-1 mitochondrial membrane potential assay with HCT116 cells
The results indicate that the MP3 fraction exerts cytotoxic activity on HCT116 cells via induction of apoptosis triggered by loss of mitochondrial membrane potential crucial for cell survival

Summary

Introduction

Certain mutations suppress apoptosis allowing the continued survival of cancer cells (Hanahan and Weinberg, 2000; Varfolomeev and Vucic, 2011). It is widely accepted that induction of apoptosis plays a role in killing cancer cells in vitro and is an attractive target for cancer therapy (Blagosklonny, 2004; Strasser et al, 2011). Leaves of Voacanga globosa were investigated for possible cytotoxicity against cancer cells. A previous study has demonstrated that extracts from V globosa leaves exhibit high toxicity against two cancer cell lines, HCT116 human colon carcinoma and A549 human lung carcinoma (Canoy, 2011). Using MTT assay, the study aimed to test HPLC fractions from the V globosa leaf extract for cytotoxicity against the HCT116 human colon carcinoma, A549 human lung carcinoma and the non-cancer AA8 Chinese hamster ovarian cell lines. Possible pro-apoptotic activity was assessed using DAPI nuclear staining, TUNEL assay and the JC-1 mitochondrial membrane potential assay

Objectives

Methods

Results

Conclusion