RESEARCH ARTICLE Xylocarpus Moluccensis Induces Cytotoxicity In
Total Page：16
File Type：pdf, Size：1020Kb
DOI:10.31557/APJCP.2021.22.S1.17 Xylocarpus Moluccensis Induces Cytotoxicity in Human Hepatocellular Carcinoma HepG2 Cell Line RESEARCH ARTICLE Editorial Process: Submission:12/11/2019 Acceptance:07/06/2020 Xylocarpus Moluccensis Induces Cytotoxicity in Human Hepatocellular Carcinoma HepG2 Cell Line via Activation of the Extrinsic Pathway Gul-e-Saba Chaudhry1*, Nur Khairina Ahmed Sohimi1, Habsah Mohamad1, Muhammad Naveed Zafar2, Aziz Ahmed3, Yeong Yik Sung1, Tengku Sifzizul Tengku Muhammad1* Abstract Objective: Liver cancer is one of the most common causes of cancer death, with reduced survival rates. The development of new chemotherapeutic agents is essential to find effective cytotoxic drugs that give minimum side effects to the surrounding healthy tissues. The main objective of the present study was to evaluate the cytotoxic effects and mechanism of cell death induced by the crude and diethyl ether extract of Xylocarpus mouccensis on the human hepatocellular carcinoma cell line. Methods: The cytotoxicity activity was measured using the MTS assay. The mode of cell death determined by the apoptosis study, DNA fragmentation analysis done by using the TUNEL system. The pathway study or mechanism of apoptosis observed by study caspases 8, 9, 3/7 Glo-caspases method. Results: In this study, the methanol extracts prepared from leaf Xylocarpus mouccensis leaf produced cytotoxicity effect with IC50 (72hr) < 30µg/ml. The IC50 value at 72 hours exerted by diethyl ether extract of Xylocarpus moluccensis leaf was 0.22 µg/ml, which was more cytotoxic than to that of crude methanol extract. The results obtained by the colorimetric TUNEL system suggest that methanol crude extract of Xylocarpus moluccensis (leaf), diethyl ether extract of Xylocarpus moluccensis (leaf) and methanol extract of Xylocarpus granatum (bark) induced DNA fragmentation in the HepG2 cell line. Besides, the caspase-Glo assay demonstrated that diethyl ether leaf extract of Xylocarpus moluccensis triggered apoptotic cell death via activation of caspases -8, and -3/7 However, no visible activation was noticed for caspase -9. Furthermore, TLC indicates the presence of potential metabolites in an extract of Xylocarpus moluccensis. Conclusion: Thus, the present study suggests the remarkable potential of active metabolites in the extract of Xylocarpus moluccensis as a future therapeutic agent for the treatment of cancer. Keywords: Apoptosis- cytotoxicity- DNA fragmentation- extrinsic pathway- HepG2- Mangroves- Xylocarpus moluccensis Asian Pac J Cancer Prev, 22, Anticancer Activity of Natural Compounds: HOW’s on Methods and Reports Suppl, 17-24 Introduction liver is supplied with cytotoxic drugs directly to the fiver and kills the cancer cells. Many drugs in chemotherapy Cancer is one of the significant causes of death around treatment are plant-derived anticancer compounds the world. Despite the progress in cancer treatment, (Valli et al., 2012; Cragg and Newman, 2005). Natural cancer continues to affect patients and is the leading compounds derived from a variety of medicinal plants cause of death worldwide. There is still a need for finding led to the development of anticancer drugs with reduced a solution to cure the complex disease, especially for adverse side effects (Desai et al., 2008). Many studies aggressive and metastasized cancer (Chaudhry et al., demonstrated the existence of compounds in traditional 2020b; Chaudhry et al., 2019b; Nithya et al., 2014; Mou herbs that can inhibit cancer growth. et al., 2011). Liver cancer is the second leading cause Mangroves trees have been used in traditional of global cancer death 788,000 mortality (Ferlay et al., medicinal practice as they show healing properties 2015). About 90% of primary liver cancer is hepatocellular on various diseases. Previous studies show that the carcinoma (HCC) Treatment for advanced liver cancer methanolic extract of Xylocarpus moluccensis exhibited may involve chemotherapy, radiation therapy, or both Cytotoxicity on gastric and breast ductal carcinoma cells (Kleinsmith, 2007). During chemotherapy treatment, the (Uddin et al., 2011). The biologically active compounds; 1Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030 Terengganu, Malaysia. 2Department of Chemistry, Quaid-i-Azam University, Islamabad, 45320, Pakistan. 3School of Fundamental Science, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia. *For Correspondence: [email protected], [email protected] Asian Pacific Journal of Cancer Prevention, Vol 22 17 Gul-e-Saba Chaudhry et al limonoids, gedunin, and la-hydroxy-1,2-dihydrogedunin became colorless, indicating that most of the compounds isolated from the tree bark of Xylocarpus granatum extracted out. The extracts filtered through Whatman’s No. of Bangladeshi Sundarban were reported to exhibit 1 filter paper. The solvent dried under reduced pressure by cytotoxicity effects toward cancer cells, in vitro (Uddin using a rotary evaporator at 30-35°C. Furthermore, four et al., 2007, Wangensteen et al., 2006). extracts, methanol, diethyl ether, butanol, and aqueous The cancer cell typically involves two distinct forms extract used in this study. of cell death; Apoptosis and necrosis. (Jan and Chaudhry, 2019). Necrosis is generally known as the ‘accidental cell Cytotoxicity study by using MTS assay death caused by severe physical or chemical interruption, CellTiter 96TM Aqueous Non-Radioactive Cell and physiologically shows severe inflammatory responses Proliferation Assay (MTS assay) was used to evaluate (Kaloger, 2012). Apoptosis is the “normal cell death by the cytotoxicity activity of extract on the HepG2 cell suicide that removes unwanted or useless cells, causes line (Hudaya et al., 2017; Chaudhry GS et al., 2020a; no inflammatory responses (Fuchs and Steller, 2011). Chaudhry GS et al., 2020c). The human hepatocellular Apoptosis is control by different groups of the executioner carcinoma cell line, HepG2, was cultured in complete and regulatory molecules (Mukhopadhyay et al., 2014). medium (RPMI), supplemented with 10% (v/v) fetal A series of biochemical events include; activation of bovine serum (FBS), 1% (v/v) essential amino acid, 1% caspases, cytochrome c release, phosphatidylserine (v/v) sodium pyruvate, and 1% (v/v) of antibiotic solution, externalization, and fragmentation of internucleosomal penicillin-streptomycin. For the negative control, the cells DNA (Jan and Chaudhry, 2019). Biochemical alterations treated with 1% (v/v) DMSO without extracts. Vincristine include DNA fragmentation and activation of cysteine sulfate (Sigma, USA) used as positive control (Song et aspartyl proteases, called caspases, which leads to the al., 2008). After that, CellTiter 96TMAQueous Solution destruction of the cell (Nikoletopoulou et al., 2013). Reagent (20μl) was added into each well for 3hr at 37°C. Caspases are central in apoptosis for the induction of Finally, the absorbance recorded using ELISA 96-well apoptosis by individual effectors (Li and Yuan, 2008). plate reader at 490nm. Therefore, the significant caspases study done to reveal the pathway of apoptosis events. The present study conducted Determination of apoptotic cell death (DNA fragmentation) to determine the of the Xylocarpus molucconsis induce by TUNEL assay Cytotoxicity and mode of cell death in (HepG2) cell line. The Terminal deoxynucleotidyl transferase UTP nick end labeling (TUNEL) assay used to study DNA Materials and Methods fragmentation in HepG2 cells. The cells were cultured in the labtek chamber slides at a cell density of 2×104 cells/ Materials chamber and incubated at 37ºC in a humidified atmosphere Human hepatocellular carcinoma cell line (HepG2) in the presence of 5% (v/v) CO2 for 24hr. As previously purchased from American Type Cell Culture, USA. reported (Chaudhry et al., 2019a; Zafar et al., 2019; Mahar Roswell Park Memorial Institute (RPMI) 1640 et al., 2019) with slight modifications as in this study, the medium was purchased from Nacalai Tesque, Japan. colorimetric reagent used. After treatment, the cells fixed Penicillin-streptomycin solution, Fetal Bovine Serum by using 4% paraformaldehyde solution in PBS (pH 7.4) (FBS), and other cell culture supplies obtained from for 25 mins at 4°C. The fixed cells then permeabilized by Gibco, USA. CellTiter 96TM Aqueous Non-Radioactive immersing the slides in 0.2% (v/v) Triton X-100 solution Cell Proliferation Assay kit (MTS), Calorimetric (prepared in PBS). For negative control, the cells treated Apoptosis Detection Kit (TUNEL), and Caspases-GloTM with 1% (v/v) DMSO. The slides were mounted with kit -3/7, 8 and 9 purchased from Promega, USA. All other DPX mounting medium and allowed to dry overnight. chemical solvents purchased from Merck, Germany. The slides observed under the light microscope and the images captured. Preparation of extracts and partition extracts The mangrove plants (Xylocarpus moluccensis) Caspase assay collected from Umbai, Malacca. Xylocarpus granatum The levels of active caspases-3/7, -8, -9 were leaves obtained from Umbai, Malacca GPS coordinates determined by using Caspase Glo™ Assay (Promega, (N 02°09’. 330”), stem bark and root GPS coordinates USA) (Chaudhry et al., 2019b). Human hepatocellular (E 102°20’.192”). Xylocarpus moluccensis leaves carcinoma cell line, HepG2 was treated with extracts at a collected from Umbai, Malacca GPS coordinates (N concentration of IC50 (72hr). The cells were incubated for 02°09’.335”) stem bark and root GPS coordinates (E different time points (0hr, 1hr, 3hr, 6hr,