Chim Kei Chan1, Hadi Supriady1, Bey Hing Goh2, Habsah Abdul Kadir3. 1. Biomolecular Research Group, Biochemistry Program, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. 2. Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Jalan Lagoon Selatan, Bandar Sunway, 46150 Selangor, Malaysia. 3. Biomolecular Research Group, Biochemistry Program, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. Electronic address: habsah@um.edu.my.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Elephantopus scaber also known as Elephant's foot (Asteraceae family) has a plethora of traditional applications including dysuria, diarrhea, dysentery, leukemia and cancer. This study aimed to investigate the apoptosis inducing effects of E. scaber and the underlying mechanisms in HCT116 colorectal cell line. METHODS: The MTT assay was used to determine the IC50 values on cancer cell lines by the ethanol, hexane, ethyl acetate and water fractions. Apoptosis was detected by cell morphologic observation through Hoechst 33342/PI dual staining, phosphatidylserine externalization by Annexin V/PI staining and DNA fragmentation by TUNEL assay. The caspase activity, Bcl-2 family and p53 proteins were determined by flow cytometric analysis. The cleaved PARP protein expression was assessed by western blot analysis RESULTS: The ethanol extract of E. scaber and its fractions significantly inhibited the growth of HCT116 and HT-29 cells and induced apoptosis. The E. scaber ethyl acetate fraction (ESEAF) was the most potent on HCT116 cell line with the IC50 value of 1.42 ± 0.10 µg/mL. The induction of apoptosis was marked by nuclear shrinkage accompanied with chromatin condensation, DNA fragmentation and phosphatidylserine externalization. The results showed that ESEAF-induced apoptosis was associated with an upregulation of proapoptotic Bax, elevation of Bax/Bcl-2 ratio, dissipation of mitochondrial membrane potential, activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). In addition, a compromised mitochondrial membrane potential and overproduction of ROS demonstrated the involvement of the mitochondrial signaling pathway. Mechanistic studies further revealed that ESEAF caused the augmentation of the intracellular ROS, subsequently incited the increase in p53 protein expression and led to oligomerization of Bax, depolarization of mitochondrial membrane potential and caspases cascade (caspase-3/7 and -9) in a time-dependent manner. The attenuation of intracellular ROS level by N-acetyl-l-cysteine (NAC) preserved the integrity of mitochondrial membrane and rescued the cells from cell death. Furthermore, caspase cascade results in the cleavage of PARP which ultimately activated DNA fragmentation and eventually apoptosis. CONCLUSION: Taken together, cumulative evidences in this study suggest that ESEAF induces apoptosis through ROS-dependent mitochondrial signaling pathway and holds potential therapeutic effect for colorectal cancer.
ETHNOPHARMACOLOGICAL RELEVANCE: Elephantopus scaber also known as Elephant's foot (Asteraceae family) has a plethora of traditional applications including dysuria, diarrhea, dysentery, leukemia and cancer. This study aimed to investigate the apoptosis inducing effects of E. scaber and the underlying mechanisms in HCT116 colorectal cell line. METHODS: The MTT assay was used to determine the IC50 values on cancer cell lines by the ethanol, hexane, ethyl acetate and water fractions. Apoptosis was detected by cell morphologic observation through Hoechst 33342/PI dual staining, phosphatidylserine externalization by Annexin V/PI staining and DNA fragmentation by TUNEL assay. The caspase activity, Bcl-2 family and p53 proteins were determined by flow cytometric analysis. The cleaved PARP protein expression was assessed by western blot analysis RESULTS: The ethanol extract of E. scaber and its fractions significantly inhibited the growth of HCT116 and HT-29 cells and induced apoptosis. The E. scaberethyl acetate fraction (ESEAF) was the most potent on HCT116 cell line with the IC50 value of 1.42 ± 0.10 µg/mL. The induction of apoptosis was marked by nuclear shrinkage accompanied with chromatin condensation, DNA fragmentation and phosphatidylserine externalization. The results showed that ESEAF-induced apoptosis was associated with an upregulation of proapoptotic Bax, elevation of Bax/Bcl-2 ratio, dissipation of mitochondrial membrane potential, activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). In addition, a compromised mitochondrial membrane potential and overproduction of ROS demonstrated the involvement of the mitochondrial signaling pathway. Mechanistic studies further revealed that ESEAF caused the augmentation of the intracellular ROS, subsequently incited the increase in p53 protein expression and led to oligomerization of Bax, depolarization of mitochondrial membrane potential and caspases cascade (caspase-3/7 and -9) in a time-dependent manner. The attenuation of intracellular ROS level by N-acetyl-l-cysteine (NAC) preserved the integrity of mitochondrial membrane and rescued the cells from cell death. Furthermore, caspase cascade results in the cleavage of PARP which ultimately activated DNA fragmentation and eventually apoptosis. CONCLUSION: Taken together, cumulative evidences in this study suggest that ESEAF induces apoptosis through ROS-dependent mitochondrial signaling pathway and holds potential therapeutic effect for colorectal cancer.