Synergistic Cytotoxic Effect of Anonna muricata and Caesalpinia sappan Nanoparticles via Expression of BAD Pro-Apoptotic Protein in HeLa Cervical Cancer Cells doi.org/10.26538/tjnpr/v5i12.11
Main Article Content
Abstract
Annona muricata and Caesalpinia sappan are among the most important traditional medicinal plants, which contain numerous chemicals with various pharmacological properties. Although extracts from both plants have demonstrated anticancer activity, there has been no report on the anticancer effect of the combination of A. muricata and C. sappan nanoparticles particularly on cervical cancer (HeLa) cells. This study aimed to determine the optimum combination dose of both nanoparticles that showed a synergistic effect to induce apoptosis in HeLa cells. The nanoparticles were prepared using A. muricata leaves and C. sappan heartwood using the glass ionic method. Immunostaining was performed to evaluate the expression of pro-apoptotic marker BAD (BCL2 associated agonist of cell death) protein. Cytotoxicity effect was tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The size of the nanoparticles ranged between 237-453 nm and showed polydispersity index of 0.354 (A. muricata) and 0.486 (C. sappan), which means that the level of nanoparticle size distribution is quite uniform. BAD acts as an essential mediator of intrinsic apoptosis, as shown by enhanced expression of BAD in the group of cells treated with the nanoparticles. The employment of two nanoparticles dose combinations showed a synergistic cytotoxic effect (Combination Index<1) when each nanoparticle concentration was given at ½ IC50 (C. sappan) and ¼ IC50 (A. muricata), resulting in 52.51% of cell viability. The synergistic effect exhibited by A. muricata and C. sappan nanoparticles suggests a possible different target or signaling pathway that results in a reduction of required nanoparticles concentrations for individual sample.
Downloads
Article Details
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
References
Choudhari A, Snehal S, Ruchika K-G. The aqueous extract of Ficus Religiosa induces cell cycle in human cervical cancer cell lines SiHa (HPV-16 Positive) and apoptosis in HeLa (HPV-18 Positive). PLoS One. 2013; 8(7):e70127.
Singh S, Dhasmana DC, Bisht M, Singh PK. Pattern of adverse drug reactions to anticancer drugs: A quantitative and qualitative analysis. Indian J Med Paediatr Oncol. 2017; 38(2):140-145.
Monsuez J-J, Charniot J-C, Vignat N, Artigou J-Y. Cardiac side-effects of cancer chemotherapy. Int J Cardiol. 2010; 144(1):3-15.
Astirin OP, Prayitno A, Artanti AN, Herawati E, Saad ANA, Firstlia AD. Single-dose and combined-dose of nanoparticles from soursop leaves (Annona muricata L.) and sappan wood (Caesalpinia sappan L.) induced apoptosis and necrosis in HeLa cells. Pharmacogn J. 2021;13(5):1134-1142.
Astirin OP, Prayitno A, Artanti AN, Fitria MS, Witianingsih DA, Pranatami DA, Putra ST. The expression of p53 and hsp70 proteins after treatment with Annona muricata Linn leaf for activating apoptotic and lead to homeostasis program of Raji cells. Int J Cancer Ther Oncol.
; 2(2):02028. 6. Bukke AN, Hadi FN, Babu KS, Shankar PC. In vitro studies data on anticancer activity of Caesalpinia sappan L. p
heartwood and leaf extracts on MCF7 and A549 cell lines. Data Brief. 2018; 19:868-877.
Artanti AN, Astirin OP, Prayitno A. Cytotoxic activity of non polar fraction from Annona Muricata L. leaves on Hela and Raji Cell Line. J Pharm Sci Clin Res. 2016; 1(2):112-118.
Najmuddin SUFS, RomLi MF, Hamid M, Alitheen NB, Rahman NMANA. Anti-cancer effect of Annona muricata Linn leaves crude extract (AMCE) on breast cancer cell line. BMC Compl Altern Med. 2016; 16(1):1-20.
Juang SH, Chiang CY, Liang FP, Chan HH, Yang JS, Wang SH, Lin YC, Kuo PC, Shen MR, Thang TD, Nguyet BT, Kuo SC, Wu TS. Mechanistic study of tetrahydrofuranacetogenins in triggering endoplasmic reticulum stress response-apoptosis in human nasopharyngeal carcinoma. Sci Rep. 2016; 6:39251.
Han B, Cao Yx, Li Zm, Wu Zx, Mao Yq, Chen Hl, Yao Zj, Wang Ls. Annonaceous acetogenin mimic AA005 suppresses human colon cancer cell growth in vivo through downregulation of Mcl-1. Acta Pharmacol Sin. 2019; 40:231-242.
Kim BS, Chung TW, Choi HJ, Bae SJ, Cho HR, Lee SO, Choi JH, Joo JK, Ha KT. Caesalpinia sappan induces apoptotic cell death in ectopic endometrial 12Z cells through suppressing pyruvate dehydrogenase kinase 1 expression. Exp Ther Med. 2021; 21(4):357.
Hung TM, Dang NH, Dat NT. Methanol extract from Vietnamese Caesalpinia sappan induces apoptosis in HeLa cells. Bio Res. 2014; 47(1):1-5.
Yang X, Ren L, Zhang S, Zhao L, Wang J. Antitumor effects of purified protosappanin B extracted from Lignum Sappan. Integr Cancer Ther. 2016; 15(1):87-95.
Wang SL, Cai B, Cui CB, Zhang HF, Yao XS, and Qu GX. Apoptosis induced by Caesalpinia sappan L. extract in leukemia cell line K562. Chin J Cancer. 2001; 20(12):1376-1379.
NgamwonglumLert L, Devahastin S, Chiewchan N, Raghavan VGS. Colour and molecular structure alterations of brazilein extracted from Caesalpinia sappan L. under different pH and heating conditions. Sci Rep. 2020; 10:12386.
Tao L, Li J, Zhang J. Brazilein overcame ABCB1-mediated multidrug resistance in human leukaemia K562/AO2 cells. Afr J Pharm Pharmacol. 2011; 5(16):1937-1944.
Czabotar PE, Lessene G, Strasser A, Adams JM. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat Rev Mol Cell Biol. 2014; 15(1):49-63.
Luna-Vargas MP and Chipuk JE. The deadly landscape of pro-apoptotic BCL-2 proteins in the outer mitochondrial membrane. Federation Eur Biochem Soc J. 2016; 283(14):2676-2689.
Yu Y, Zhong Z, Guan Y. The downregulation of BclxL/Bcl-2-associated death promoter indicates worse outcomes in patients with small cell lung carcinoma. Int J Clin Exp Pathol 2015; 8(10):13075-13082.
Birkinshaw RW and Czabotar PE. The BCL-2 family of proteins and mitochondrial outer membrane permeabilisation. Semin Cell Dev Biol. 2017; 72:152-162.
Pawlowski J and Kraft AS. Bax-induced apoptotic cell death. Proc Nat Acad Sci of the USA. 2000; 97(2):529-531.
Xing X, He X, Peng J, Wang K, Tan W. Uptake of silicacoated nanoparticles by HeLa cells. J Nanosci Nanotech. 2005; 5(10):1688-1693.
Kravanja G, Primožič M, Knez Ž, Leitgeb M. Chitosanbased (nano)materials for novel biomedical applications. Molecules. 2019; 24(10):1-23.
Jiménez-Gómez CP and Cecilia JA. Chitosan: A natural biopolymer with a wide and varied range of applications. Molecules. 2020; 25(17):3981.
Domenico L, Mikhail AK, Maria TC. Smart nanoparticles for drug delivery application: Development of versatile nanocarrier platforms in biotechnology and nanomedicine. J Nanomaterials. 2019(12); 1-26.
Rubiyanto D. Basic Techniques of Chromatography. Yogyakarta: Deepublish; 2016; 130p.
Oksal E, Pangestika I, Muhammad TST, Mohamad H, Amir H, Kassim MNI, Andriani Y. In vitro and in vivo studies of nanoparticles of chitosan-Pandanus tectorius fruit extract as new alternative treatment for hypercholesterolemia via Scavenger Receptor Class B type 1 pathway. Saudi Pharm J. 2020; 28(10):1263-1275.
Buxton. Design Expert. [Online]. 2007 [cited 2020 Jul 31]. Available from: https://www.lboro.ac.uk/media/media/schoolanddepar
tments/mLsc/downloads/Design-Expert-7.pdf
Cancer Chemoprevention Research Center. In vitro assay protocols. Cancer Chemoprevention Research Center UGM. [Online]. 2010 [cited 2019 Sept 26 ] Available from: http://ccrc.farmasi.ugm.ac.id/?page_id=240.
Reynolds CP and Maurer BJ. Evaluating response to antineoplastic drug combinations in tissue culture models.Methods Mol Med. 2005; 110:173-183.
Michurina SV, Kolesnikov SI, Bochkareva AL, Ishchenko IY, Arkhipov SA. Expression of apoptosis regulator proteins Bcl-2 and Bad in rat ovarian follicular apparatus during recovery after extreme hypothermia. Bull Exp Biol Med. 2019; 168(2):205-209.
Abubakar AR and Haque M. Preparation of medicinal plants: Basic extraction and fractionation procedures for experimental purposes. J Pharm Bioallied Sci. 2020;12(1):1-10.
Hung TM, Dang NH, Dat NT. Quality evaluation using TLC methods with reference to brazilin content of Caesalpinia sappan heartwoods in Thailand. Songklanakarin J Sci Tech. 2020; 42(1):196-202.
Yemirta. Identifikasi kandungan senyawa antioksidan dalam kayu secang (Caesalpinia Sappan). Jurnal Kimia dan Kemasan. 2010; 32(2):41-46.
Melot A, Fall D, Gleye C, Champy P. A polar Annonaceous acetogenins from the fruit pulp of Annona muricata. Molecules. 2009; 14(11):4387-4395.
Lee DY, Lee MK, Kim GS, Noh HJ, Lee MH. Brazilin inhibits growth and induces apoptosis in human glioblastoma cells. Molecules. 2013; 18(2):2449-2457.
Chaudhary S, Garg T, Murthy R, Rath G, Goyal AK. Development, optimization and evaluation of long chain nanolipid carrier for hepatic delivery of silymarin through lymphatic transport pathway. Int J Pharm. 2015; 485(1-2):108-121.
Arthur FKN, Woode E, Terlabi EO, Larbie C. Evaluation of acute and subchronic toxicity of Annona Muricata (Linn.) aqueous extract in animals. Eur J Exp Biol. 2011; 1(4):115-124.
Nugroho YA and Soeradi DO. Toksisitas akut dan efek pemberian ekstrak etanol kayu Secang (Caesalpinia sappanL) terhadap struktur anatomi tubulus seminiferus testis tikus putih. Jurnal Bahan Alam Indonesia. 2002; 1(1):35-39.
Prabha S, Arya G, Chandra R, Ahmed B, Nimesh S. Effect of size on biological properties of nanoparticles employed in gene delivery. Artif Cells Nanomed Biotechnol. 2016; 44(1):83-91.
Avadi MR, Sadeghi AMM, Mohammadpour N, Abedin S, Atyabi F, Dinarvand R, Rafiee-Tehrani M. Preparation and characterization of insulin nanoparticles using chitosan and Arabic gum with ionic gelation method. Nanomed: Nanotech Biol Med. 2010; 6(1):58-63.
Cosgrove T. Colloid science principles, methods and applications. (2nd ed). New York: Wiley. 2010; 363p.
Wulandari V. Association between oral contraceptives use and sexual activity with cervical cancer. Jurnal Berkala Epidemiologi. 2016; 4(3):432-442.
Antony P and Vijayan R. Acetogenins from Annona muricata as potential inhibitors of antiapoptotic proteins: A molecular modeling study. Drug Des Dev Ther. 2016; 10:1399-1410.
Wu FE, Zhao GX, Zeng L, Zhang Y, Schwedler JT, McLaughlin JL. Additional bioactive acetogenins, annomutacin and (2,4-Trans and Cis)-10R-Annonacin-AOnes, from the leaves of Annona muricata. J Nat Prod. 1995; 58(9):1430-1437.
Torres MP, Rachagani S, Purohit V, Pandey P, Joshi S, Moore ED, Johansson SL, Singh PK, Ganti AK, Batra SK. Graviola: A novel promising natural-derived drug that inhibits tumorigenicity and metastasis of pancreatic cancer cells in vitro and in vivo through altering cell metabolism. Cancer Lett. 2012; 323(1):29-40.
Goswami CP, Cheng L, Alexander PS, Singal A, Li L. A new drug combinatory effect prediction algorithm on the cancer cell based on gene expression and dose-response curve. CPT: Pharmacometrics Syst Pharmacol. 2015; 4(2):80-90.
Wong KH, Lu A, Chen X, Yang Z. Natural IngredientBased Polymeric Nanoparticles for Cancer Treatment. Molecules. 2020; 25(16):3620.