Nghiên cứu thành phần hóa học và hoạt tính gây độc tế bào ung thư từ hai loài sên biển Aplysia dactylomela và Dendrodoris fumata ở vùng biển miền trung Việt Nam.Nghiên cứu thành phần hóa học và hoạt tính gây độc tế bào ung thư từ hai loài sên biển Aplysia dactylomela và Dendrodoris fumata ở vùng biển miền trung Việt Nam.Nghiên cứu thành phần hóa học và hoạt tính gây độc tế bào ung thư từ hai loài sên biển Aplysia dactylomela và Dendrodoris fumata ở vùng biển miền trung Việt Nam.Nghiên cứu thành phần hóa học và hoạt tính gây độc tế bào ung thư từ hai loài sên biển Aplysia dactylomela và Dendrodoris fumata ở vùng biển miền trung Việt Nam.Nghiên cứu thành phần hóa học và hoạt tính gây độc tế bào ung thư từ hai loài sên biển Aplysia dactylomela và Dendrodoris fumata ở vùng biển miền trung Việt Nam.Nghiên cứu thành phần hóa học và hoạt tính gây độc tế bào ung thư từ hai loài sên biển Aplysia dactylomela và Dendrodoris fumata ở vùng biển miền trung Việt Nam.Nghiên cứu thành phần hóa học và hoạt tính gây độc tế bào ung thư từ hai loài sên biển Aplysia dactylomela và Dendrodoris fumata ở vùng biển miền trung Việt Nam.Nghiên cứu thành phần hóa học và hoạt tính gây độc tế bào ung thư từ hai loài sên biển Aplysia dactylomela và Dendrodoris fumata ở vùng biển miền trung Việt Nam.
MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY - PHAM THI MAI HUONG STUDY ON CHEMICAL COMPOSITION AND CYTOTOXIC ACTIVITY OF CANCER CELLS FROM TWO SPECIES OF SEA SLUGS APLYSIA DACTYLOMELA AND DENDRODORIS FUMATA COLLECTED FROM THE SEA AREA OF CENTRAL VIETNAM Major: Biochemistry Code: 9.42.01.16 SUMMARY OF BIOLOGY DOTORAL THESIS Ha Noi - 2022 This thesis was completed at: Graduate university of Science and Technology - Vietnam Academy of Science and Technology - Institute of Biotechnology - Institute of Marine Biochemistry Advisor 1: PhD Nguyen Van Thanh Advisor 2: Assoc Prof PhD Do Thi Thao Reviewer 1: Reviewer 2: Reviewer 3: This thesis will be defended at Graduate University of Science and Technology - Vietnam Academy of Science and Technology at hour date month 2022 The thesis can be found in: - The Library of Graduate University of Science and Technology, Vietnam Academy of Science and Technology - National Library of Vietnam - Institute of Biotechnology INTRODUCTION As society develops, people pay more and more attention to health, and disease prevention has become an essential need Humans are facing many dangerous diseases, especially cancer Therefore, the search for specific drugs as well as supportive drugs and chemical prophylactic agents is very necessary and urgent Screening for active ingredients that kill cancer cells from nature is the first step but is imperative in the process of developing new drugs By the end of 2020, nine marine-based anticancer drugs have been placed on the market and many compounds are undergoing experimental studies [2] It can be seen that the source of marine medicinal herbs contains potential and prospects for the research and creation of products for the community [3, 4] With the advantage of sea area of 1,000,000,000 km2 and owning a long coastline of over 3.200 km along, Vietnam has great potential for exploiting a diverse marine resources However, so far, there are not many studies searching for valuable active ingredients from Vietnamese marine organisms and limited in the in vivo activity test step and research on drugcell interaction mechanisms cancer Therefore, an urgent requirement for our country is to develop research to step by step systemize the chemical composition and biological activity of marine species Sea slugs are a group of marine animals that many scientists around the world are interested in researching Up to now, there have been many studies on the chemical composition and biological activities of compounds isolated from sea slugs However, in Vietnam with species diversity, there is only one initial study on compounds isolated from sea slugs Stemming from the above fact, the thesis topic "Study on chemical composition and cytotoxic activity of cancer cells from two species of sea slugs aplysia dactylomela and dendrodoris fumata collected from the sea area of central Vietnam " was chosen The objectives of the thesis - Determine the chemical composition of three sea slugs of two species of in the central sea of Vietnam - Detection of cytotoxic active substances from isolated compounds and study of their effects on cancer cells in vitro Research subjects - Aplysia dactylomela Rang, 1828 (were collected at Lang Co island, Thua Thien Hue and Hon Me island, Thanh Hoa), Dendrodoris fumata Ruppell & Leuckart, 1831 (were collected at Hon Me island, Thanh Hoa) The main contents of the thesis - Isolation of isolated compounds from three sea slugs of two species A dactylomela D fumata - Determination of chemical structures of isolated compounds - Evaluation of cytotoxic activity in vitro of isolated compounds - Evaluation of the apoptosis-inducing effects of some typical compounds on some experimental cancer cell lines CHAPTER 1: OVERVIEW This chapter provides an overview domestic and international studies on the extraction of natural compounds from marine organisms in cancer treatment, research on cytotoxic activity, general characteristics of the sea slugs and genus Aplysia and Dendrodoris, about the chemical composition and biological activity of sea slugs and genus Aplysia and Dendrodoris 1.1 Cancer and the relationship between cancer cells and apoptosis Presenting the concept of cancer, the concept of apoptosis, the importance of apoptosis, the relationship between aoptososis and cancer cells 1.2 Several assays to evaluate cytotoxic activity Present some tests to evaluate cytotoxic activity 1.2.1 The assay determines cytotoxic activity by MTT technique 1.2.2 The assay determines the ability to induce apoptosis using Hoechst 33342 1.2.3 The assay determines the ability to induce caspase-3 enzyme 1.2.4 The assay evaluates cell apoptosis by flow cytometry 1.3 Introduction to sea slugs In addition to searching for biologically active substances from traditional medicinal resources (terrestrial plants and animals), marine mollusks, especially sea slugs, are now widely used in research and development experiments around the world of interest Currently, cancer drugs containing derivatives of compounds isolated from sea slugs and many compounds isolated from sea slugs have cytotoxic activity v.v 1.3.1 Biological characteristics of sea slugs 1.3.2 Some compounds with cytotoxic activity from sea slugs 1.3.3 Overview of sea slugs of the genus Aplysia 1.3.3 Overview of sea slugs of the genus Dendrodoris 1.3.4 Research status of sea slugs in Vietnam Statistical publications show that compounds isolated from sea slugs Aplysia and Dendrodoris are mainly sesquiterpenes and steroids compounds Many of these compounds exhibit interesting biological activities such as cytotoxic activity etc CHAPTER 2: SUBJECTS AND METHODS 2.1 Subjects and materials Sample of A dactylomela Rang, 1828 was collected at Lang Co, Hue, Vietnam (5/2016) Sample of A dactylomela Rang, 1828 was collected at Hon Me, Thanh Hoa, Vietnam (8/2017) Sample of D fumata Ruppell & Leuckart, 1831 was collected at Hon Me, Thanh Hoa, Vietnam (5/2016) Materials Human cancer cell lines: KB, LNCaP, SK-LU-1, SK-Mel-2, HepG2, MCF-7, HL-60, SW480 Cell culture medium: DMEM or MEME, with L-glutamine, sodium pyruvate, NaHCO3, penicillin/streptomycin, 10% FBS, Trypsin-EDTA (0.05%) of Invitrogen Plastic 96-well plate, 6-well plastic cell culture plate, pipette, 96-well ELISA reader Reference substance: Camptothecin 2.2 Methods 2.2.1 method for extraction methanol from sea slugs 2.2.2 Methods for isolation of secondary metabolites 2.2.2.1 Extraction and isolation of compounds from sea slugs a A dactylomela collected in Lang Co, Thua Thien Hue, Viet Nam Fig 2.4 Schematic diagram of fractionation from A dactylomela Fig 2.5 Isolation of compounds from A dactylomela (ASP) b A dactylomela collected in Hon Me, Thanh Hoa, Viet Nam Fig 2.7 Isolation of compounds from A dactylomela (AD) 2.2.2.3 Isolation of compounds from S conferta Fig 2.8 Schematic diagram of fractionation from D fumata Fig 2.9 Isolation of compounds from D fumata 2.2.3 Methods for determination of chemical structure of compounds 2.2.4 Methods of assessment of activity and mechanism of cytotoxicity CHAPTER 3: RESULTS 3.1 Determination of chemical structure of compounds from A.dactylomela (Lang Co, Thua Thien Hue) Fig 3.1 Chemical structure of isolated compounds from A.dactylomela (Lang Co, Thua Thien Hue) 3.2 Determination of chemical structure of compounds from A dactylomela (Hon Me, Thanh Hoa) Fig 3.2 Chemical structure of isolated compounds from A dactylomela (Hon Me, Thanh Hoa) 3.3 Determination of chemical structure of compounds from D fumata (Hon Me, Thanh Hoa) Fig 3.3 Chemical structure of isolated compounds from D fumata (Hon Me, Thanh Hoa) 3.4 Evaluation of cytotoxic activity of compounds isolated from sea slugs) 3.4.1 Evaluation of cytotoxic activity of compounds isolated from A dactylomela (Thua Thien Hue) 11 3.4.4 Evaluation of the apoptosis-inducing activity in cancer cells of compound DN01 and compound AD02 3.3.4.1 Evaluation of the apoptosis activity in HepG2 cancer cells of AD02 compound a Determination of AD02 compound's ability to induce apoptosis in HepG2 cancer cells through changes in cell morphology Fig 3.4 Effects of AD02 at different concentration (A: µg/mL (0 µM); B: 0.685 µg/mL (2.5 µM), C: 1.73 µg/mL (5 µM) on the morphological changes of HepG2 cells after 48 h treatment; The microscope was observed under 40× magnification; Arrows showed the dead cells with apoptotic bodies The results showed that the compound AD02 changed the cell morphology from normal cells to cells with apoptosis state b Evaluation of the ability to induce apoptosis using annexin V Fig 3.5 Apoptotic effect of AD02 at different concentrations (0.685µg/mL and 1.73 µg/mL) in HepG2 cells analyzed with FIT Annexin-V/Dead Cell Apoptosis Kit with FICT Annexin-V and PI, using flow cytometer after 48 h of treatment; Q2-1 necrosis, Q2-2 late apoptosis, Q2-4 early apoptosis, and Q2-3 viable cell To validate the apoptotic potential of 2, the cellular apoptosis population induced by treatment with was assessed by the flow cytometry with 12 annexin V-FITC and propidium iodide (PI) double staining The number of viable treated cells decreased compared to untreated cells After 48 h of treatment, the percentage of viable cells decreased from 96.03% to 76.70% (Fig 7) Flow cytometry revealed that µM of compound induced apoptosis in HepG2 cells with 14.94% early apoptotic cells The number of necrotic cells also increased (7.20%) compared to the negative control (2.89%) The overall cell population shift indicated that induced cell death via both apoptosis and necrosis c Caspase inducible effects of AD02 Subsequently, to understand the apoptotic mechanisms the effect of on caspase activity was examined In agreement with the FACS analysis, the caspase inducible activities of increased significantly compared to the control (P < 0.01) (Fig 3.7) Fig 3.7 Caspase inducible effects of AD02 at different concentration (0.685µg/mL and 1.73 µg/mL) in HepG2 cells after 24 h treatment; Normal saline was served as the negative control; Each value represents the mean ± SD; **P