Nghiên cứu hoạt tính gây độc một số dòng tế bào ung thư của các hợp chất phân lập từ ba loài san hô mềm sinularia nanolobata, sinularia leptoclados, sinularia conferta thu thập ở vùng biển trung bộ việt nam TT TA

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY - Ninh Thi Ngoc STUDY ON CYTOTOXIC ACTIVITY OF SOME CANCER CELL LINES OF ISOLATED COMPOUNDS FROM THREE SOFT CORALS SINULARIA NANOLOBATA, SINULARIA LEPTOCLADOS, SINULARIA CONFERTA COLLECTED FROM THE SEA AREA OF CENTRAL VIETNAM Major: Biochemistry Code: 9.42.01.16 SUMMARY OF BIOLOGY DOTORAL THESIS Ha Noi - 2021 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 Hoai Nam Advisor 2: PhD Tran My Linh Reviewer 1: Assoc Prof PhD Nguyen Dinh Thang Reviewer 2: Assoc Prof PhD Tran Thu Huong Reviewer 3: PhD Bui Thi Thuy Luyen This thesis will be defended at Graduate University of Science and Technology - Vietnam Academy of Science and Technology at hour date month 2021 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 The urgency of the thesis In recent years, many countries have exploited bioactive substances from marine organisms to serve researches to find drugs to treat dangerous diseases such as cancer, hepatitis, and diseases infections and viruses Up to this point, a number of pharmaceuticals derived from marine organisms have reached the users' hands, such as Cytarabine, Vidarabine, Eribulin, Trabectedin To achieve this achievement, research institutes around the world has screened the biological activity of millions of compounds from marine species, and invested financial resources and time in pre-clinical and clinical research phases for potential compounds With the advantage of owning a long coastline of over 3.260 km along with many islands and bays, Vietnam has great potential for exploiting a diverse marine resources, rich in both species composition and storage amount However, up to now, 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 drug-cell interaction mechanisms cancer The studies are currently in the early stages compared with other countries in the region and far behind the advanced countries The reason is that there are still some difficulties such as: surveying and collecting samples of marine organisms requires modern equipment, compounds isolated from marine organisms often have very small concentrations, complex structures impurities, some compounds that are easy to decompose right in the analysis process 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 Genus Sinularia is one of the genus of soft corals that is interested in research by many scientists around the world Up to now, there have been many studies on the chemical composition and biological activities of many compounds isolated from soft corals belonging to this genus However, studies on many soft corals of the genus Sinularia such as S nanolobata, S leptoclados, S conferta in Vietnam are few and almost no systematic and methodical studies on these species mentioned above Stemming from the above fact, I chose the thesis topic "Study on cytotoxic activity of some cancer cell lines of isolated compounds from three soft corals Sinularia nanolobata, Sinularia leptoclados, Sinularia conferta collected from the sea area of Central Vietnam" was chosen The objectives of the thesis - Determine the chemical composition of three soft coral species S nanolobata, S conferta, S leptoclados collected in the central sea of Vietnam - Detecting active substances with cytotoxic activity in soft corals studied, orienting their application for biomedical and pharmacological studies The main contents of the thesis - Determine the scientific names of three soft corals collected from the sea area of Central Vietnam by molecular markers - Isolation and Determination of chemical structures of isolated compounds from three soft corals S nanolobata, S conferta, S leptoclados - Evaluation of cytotoxic activity in vitro of isolated compounds - Evaluation of cytotoxicity mechanism of some typical compounds 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 soft corals and genus Sinularia, about the chemical composition and biological activity of soft corals genus Sinularia 1.1 Test to evaluate cytotoxic activity 1.2 Natural compounds from marine organisms in cancer treatment 1.3 Introduction to soft corals 1.3.1 Characteristics of soft corals 1.3.2 Overview of soft corals of the genus Sinularia 1.3.3 Application of molecular indicators in the classification of soft corals 1.3.4 Study on the biological activity of isolated compounds from soft corals of the genus Sinularia Statistics of published studies show that compounds isolated from soft corals genus Sinularia mainly include sesquiterpen, diterpen and steroid compounds Many of these compounds exhibit interesting biological activity such as cytotoxic, anti-inflammatory, antibacterial, antiviral, neuroprotective and antioxidant activities CHAPTER 2: SUBJECTS AND METHODS 2.1 Subjects Sample of S nanolobata Verseveldt, 1977 was collected at Lang Co, Hue, Vietnam (4/2015) Sample of S leptoclados Ehrenberg, 1834 was collected at Con Co, Quang Tri, Vietnam (5/2016) Sample of S conferta Dana, 1846 was collected at Con Co, Quang Tri, Vietnam (5/2015) 2.2 Methods 2.2.1 Determination of scientific name of soft coral samples by molecular markers (msh1 and 28S RNA) 2.2.1.2 Amplification and sequencing of marker DNA fragments M SN SLE SCO M SN SLE SCO A M B M Figure 2.5 PCR product electrophoresis image cloning indicator gene segments from studied soft coral samples (A) 28S rRNA gene segment using 28SF and 28SR primers (B) msh1 gene segment using MSHF and MSHR primers M: GeneRulerTM 1kb DNA ladder 10 11 12 13 14 M 15 16 17 18 19 20 21 Hình 2.6 Electrophoresis of PCR-colony products from some colonies after transformation of vector pTZ57R/T at 28S rRNA of soft coral samples SN (well No 1-7), SLE (well No 8-14), SCO (well No 15-21) M: GeneRulerTM 1kb DNA ladder M 22 23 24 25 26 27 28 29 30 31 32 33 34 M 35 36 37 38 39 40 41 42 Hình 2.7 Electrophoresis of PCR-colony products from some colonies after transformation of vector pTZ57R/T with msh1 gene attached to soft coral samples SN (well No 22-28), SLE (well No 29-35), SCO (well No 36-42) M: GeneRulerTM 1kb DNA ladder 2.2.2 Methods for isolation of secondary metabolites 2.2.2.1 Isolation of compounds from S nanolobata Hình 2.8 Isolation of compounds from S nanolobata 2.2.2.2 Isolation of compounds from S leptoclados Hình 2.9 Isolation of compounds from S leptoclados 2.2.2.3 Isolation of compounds from S conferta Hình 2.10 Isolation of compounds from S conferta 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 species by molecular makers 3.1.1 Sequencing 28S rARN and msh1 gene fragments of soft corals - Nucleotide sequence msh1 gene segment of SN sample with length 639 bp, including 190 A, 113 C, 121 G, 215 T - Nucleotide sequence msh1 gene segment of SLE sample with length 639 bp, including 189 A, 113 C, 119 G, 218 T - Nucleotide sequence msh1 gene segment of SCO sample with length 639 bp, including 189 A, 113 C, 122 G, 215 T - Nucleotide sequence of 28S rRNA gene segment of SN sample with length 695 bp, including 139 A, 194 C, 228 G, 134 T - Nucleotide sequence of 28S rRNA gene segment of SLE sample with length 709 bp, including 145 A, 197 C, 231 G, 136 T - Nucleotide sequence of 28S rRNA gene segment of SCO sample with length 709 bp, including 144 A, 199 C, 228 G, 138 T 3.1.2 Sequence comparison of soft corals by BLAST program The results of analyzing the msh1 and 28S rRNA gene sequences of the study samples with the reference sequences on the NCBI international gene bank showed that the sequences of the indicator genes of the study samples have similarities high with the corresponding sequences on the gene bank, namely: - Sequence of msh1 gene fragment of SN sample had 100% similarity with the corresponding sequence (code FJ621451.1) of the sample S nanolobata vourcher RMNH coel 38441 on the NCBI gene bank - Sequence of msh1 gene fragment of SLE sample has 100% similarity with the corresponding sequence (code KC542857.1) of the sample S leptoclados vourcher ZMTAU: CO35308 on the NCBI gene bank - Sequence of msh1 gene fragment of SCO sample has 100% similarity with corresponding sequence (code FJ621389.1) of sample S conferta vourcher NTM C13972 on NCBI gene bank - Sequence of 28S rRNA gene fragment of SN sample has 99.8% similarity with corresponding sequence (code KF915519.1) of Sinularia sp RMNH voucher: Coel.41326 on the NCBI gene bank - Sequence of 28S rRNA gene fragment of SLE sample has 100% similarity with the corresponding sequence (code KC542837.1) of the sample S leptoclados voucher ZMTAU: CO34095 on the NCBI gene bank - Sequence of 28S rRNA gene fragment of SCO sample has 99.6% similarity with corresponding sequence (code MF817932.1) of Sinularia sp on the NCBI gene bank Table 3.1 The results of species identification of the soft coral samples studied based on analysis of the similarity (%) of the sequence of the marker DNA segments (genes 28S rARN and msh1) of the study samples with the reference sequences on gene bank msh1 maker 28S rRNA maker Name Species SN SLE SCO Sinularia nanolobata Sinularia leptoclados Sinularia conferta Ident Species Ident 100% Sinularia sp ≥ 99.8% 100% Sinularia leptoclados 100% 100% Sinularia sp ≥ 99.6% The general conclusion is based on DNA markers Sinularia nanolobata Sinularia leptoclados Sinularia conferta 3.2 Determination of chemical structure of the compounds 3.2.1 Determination of chemical structure of compounds from S nanolobata 28 21 22 18 11 19 13 H HO 10 17 16 14 26 24 23 29 12 20 25 27 OH 15 H H Figure 3.2 Chemical structure of isolated compounds from S nanolobata 3.2.2 Determination of chemical structure of compounds from S leptoclados 10 Table 3.2 Results of cytotoxic activity of SN compounds Compounds KB SKLU-1 HepG2 SN - - - SN - - SN 30 1.26± 0.20 2.07± 0.33 Elipticine* 64.35± 7.00 1.95± 0.28 IC50 (µM) MCFLNCaP SKMel-2 - - - - - - 2.36± 0.24 2.07± 0.28 1.46± 0.20 HL-60 89.02± 9.93 33.53± 4.25 1.91± 0.37 SW480 71.02± 4.00 2.24± 0.16 *: Positive control; "-": No activity The results of evaluation of 10 SN compounds showed that: The new compound SN showed an average cytotoxic activity on the line of cells HL-60, HepG2 and SW480 (IC50 from 33.53-71.02 µM) Compound SN exhibits toxic activity on the HL-60 cell line The remaining compounds did not show activity 3.3.2 Evaluation of cytotoxic activity of compounds isolated from S leptoclados Table 3.3 Results of cytotoxic activity of SLE compounds Compou -nds IC50 (µM) SKLU-1 51.80± 31.22 KB HepG2 MCF-7 SLE 10 34.95± 4.21 21.13± 0.70 38.92± 6.26 SLE 20 - - - 19.03± 2.92 32.86± 3.46 21.79± 2.20 39.54± 4.90 17.29± 1.91 36.97± 2.24 21.21± 1.47 49.13± 4.74 SLE 30 - - - - Ellipticine* 1.79± 0.28 1.38± 0.28 1.34± 0.16 1.26± 0.28 SLE 27 SLE 28 - 62.07± 10.88 SW480 28.65± 1.53 SKMel-2 59.35± 4.23 92.96±8 29 83.84± 3.72 - 13.45± 1.81 20.53± 2.26 82.80 ± 3.65 1.91± 0.12 14.4± 1.88 26.6± 1.59 HL-60 2.03± 0.16 29.01± 3.21 33.87± 3.82 72.32 ± 1.30 1.91± 0.20 LNCaP 64.12± 1.71 89.80± 3.34 17.13± 1.81 40.55± 3.63 1.95± 0.20 *: Positive control; "-": No activity The results of 15 SLE compounds showed that: compounds SLE 10, SLE 27 and SLE 28 (IC50 in the range of 1.78 to 78.33 µM) showed cytotoxic activity on cancer cell lines test letter Compounds SLE 20 and 11 SLE 30 showed activity in 2-3 tested cancer cell lines The remaining compounds did not show activity 3.3.3 Evaluation of cytotoxic activity of compounds isolated from S conferta Table 3.4 Results of cytotoxic activity of SCO compounds Compound SCO 27 SCO 35 SCO 37 Etoposide * Camptothecin* *: Positive control; A-549 3.64±0.18 78.73±2.11 27.12±1.69 IC50 (µM) Hela 19.34±0.42 30.5±0.77 24.64±1.28 27.99±2.01 PANC-1 1.78±0.69 9.35±0.37 20.51±2.72 1.17±0.42 12.65±1.01 The results of evaluation of 12 SCO compounds showed that compounds SCO 27, SCO 35 and SCO 37 exhibited significant cytotoxic activity in all experimental cancer cell lines The remaining compounds did not show activity 3.3.4 Study on cytotoxicity mechanism of compounds SLE 27 and SCO27 3.3.4.1 Study on cytotoxicity mechanism of compounds SCO 27 in lung cancer cell line A549 a Evaluation of the effect of SCO 27 on morphological change in cancer cells Figure 3.5 Cell morphology A549 under the influence of SCO 27 at different concentrations and positive control (camptothecin µM) Arrows indicate cells in a state of apoptosis 12 b Evaluate the ability of SCO 27 to stimulate production of the enzyme caspase Figure 3.6 The ability to stimulate production of caspase of SCO 27; sco-10 µM; sco-5 µM and sco-2,5 µM: The analyzed samples were supplemented with SCO 27 at the respective concentrations Control: Analytical sample with no added SCO 27 compound Camptothecin: analytical sample supplemented with camptothecin c Determination of the ability of SCO 27 to induce apoptosis in lung cancer cell line A549 A C B D Figure 3.7 Effects of SCO 27 on apoptosis of cell line A549 at 24 h at different concentrations of µM (C), 10 µM (D), negative control (A) and positive control (B) using the Method of dyeing Annexin V/PI 13 Table 3.4 Apoptosis rate under the influence of SCO 27 on lung cancer cell line A549 Compounds The rate of survival cells (%) Control SCO 27 - µM SCO 27 - 10 µM Camptothecin-5µM 89.08 88.50 83.33 79.17 The rate of early apoptosis cells (%) 3.89 4.90 4.76 15.49 The rate of late apoptosis cells (%) 5.18 5.72 8.93 1.57 The rate of necrotic cells (%) 1.85 0.88 2.99 3.77 3.3.4.2 Study on cytotoxicity mechanism of compound SLE 27 in breast cancer cell line MCF-7 a Evaluation of the effect of SLE 27 on morphological change in cancer cells Figure 3.8 Effects of compound SLE 27 at concentrations of 10, 30 and 100 µM on morphology of breast cancer cell line MCF-7 Arrows indicate cells in a state of apoptosis 10X and 20X magnification Control: negative control - MCF-7 cells not supplemented with SLE 27 compound b Determination of induction apoptosis of breast cancer cell line MCF-7 of compound SLE 27 Table 3.5 Apoptosis rate under the influence of SLE 27 on the breast cancer cell line MCF-7 14 Compounds The rate of survival cells (%) The rate of early apoptosis cells (%) The rate of late apoptosis cells (%) The rate of necrotic cells (%) Control SLE 27-10 µM SLE 27-30 µM SLE 27-100 µM 97.08 97.39 86.08 39.10 0.67 1.15 1.72 3.71 1.93 1.28 7.16 23.02 0.32 019 5.04 34.16 c Evaluation of the effect of compound SLE 27 on breast cancer cell cycle MCF-7 Figure 3.10 Effect of compound SLE27 (concentration 10; 30; 100 µM) on cell cycle MCF-7 at 48 h, using the Flow cytometer Novocyte system Table 3.6 The rate (%) of MCF-7 cells in G0/G1, S, G2/M phases and apoptosis (sub-G1) after 48 hours of treatment with SLE 27 compound Compounds Control SLE 27 - 10 µM SLE 27 - 30 µM SLE 27- 100 µM The rate of cells in all phases of the cell division cycle (%) sub-G1 G0/G1 S G2/M phase phase phase phase 0.54 41.21 41.22 14.81 0.23 36.57 33.41 17.78 0.76 40.59 27.19 15.86 10.24 66.08 15.96 4.42 15 CHAPTER 4: DISCUSSIONS 4.1 Determination of species of soft coral based on DNA makers After cloning and sequencing of marker gene segments from soft coral samples, the DNA sequences of Sinularia leptoclados (MW077896, MW077906); Sinularia conferta (MW077897, MW077907) and Sinularia nanolobata (MW077898, MW077908) are registered on the NCBI Sinularia-abruptaMF817864 Sinularia-slieringsiMH516803 Sinularia-penghuensisJX991181 Sinularia-molestaJX991172 30 Sinularia-abrubtaFJ621374 Sinularia-abruptaJX991168 37 Sinularia-leptocladosKC542857 66 SLE 46 Sinularia-compactaFJ621384 Sinularia-bisulcaFJ621378 99 Sinularia-acutaFJ621375 46 Sinularia-verseveldtiKC542859 Sinularia-robustaFJ621473 Sinularia-diffusaFJ621399 Sinularia-sp.KF915757 64 Sinularia-abhishiktaeFJ621373 Sinularia-tumulosaFJ621482 50 Sinularia-siaesensisFJ621478 Sinularia-polydactylaKU230374 90 Sinularia-confertaFJ621389 99 43 SCO Sinularia-peculiarisJX023274 64 Sinularia-ornataJX991173 Sinularia-nanolobataFJ621451 65 90 SN Sinularia-brassicaKF915724 48 47 0.01 Figure 4.1 The results of classification analysis by the NJ (NeighborJoining) method on MEGA6 of soft coral samples based on msh1 gene sequence polymorphism of the studied samples and related soft corals samples on NCBI The tributary numbers are the Bootdtrap values that represent the reliability of the genetic branching of the sequences and groupings Analysis of genotypes of the msh1 gene segment based on the nucleotide sequence was performed on study sequences and 23 reference sequences on NCBI The results in Figure 4.1 show that the SLE sample has a high genetic similarity with the species that has been sequenced in the world gene bank, S leptoclados KC542857 (bootstrap 66%) The SCO sample 16 has a high genetic affinity with S conferta FJ621389 (bootstrap 90%) SN pattern has a high genetic similarity with S nanolobata species FJ62621451 (bootstrap 90%) Similarly, genotyping analysis based on 28S rARN gene segment nucleotide sequence was performed on study sequences and 21 reference sequences on NCBI The results in Figure 4.2 show that the SLE sample has a high genetic similarity with the species that have been sequenced on the world gene bank, namely S leptoclados KC542857, S leptoclados MF817912, S densa KC542829, S abrupta KC542822, S australiensis KC542825, Sinularia sp MF817932 (bootstrap 100%) SCO samples have high genetic relationships with species that have been sequenced on the world gene bank such as S bisulca KC542826, S slieringsi MK333594, S penghuensis KC542842, S robusta KC542843, S verseveldti KC542845, S eilatesis KC542832, S corpulentissima KC542827, S maxima KC542839 (bootstrap 100%) The SN pattern has a highly similar genetic relationship with the species Sinularia sp KC915519 and S polydactila KF915515 (bootstrap 98%) Thus, the synthesis of the results comparing the sequences of msh1 and 28S marker genes of soft corals are presented in Table 3.1 Sample SN was identified scientifically as S nanolobata, SLE sample was identified scientifically as S leptoclados, SCO sample was identified scientifically as S conferta This result also coincided with the results identified by analyzing the initial morphological characteristics of Prof Dr Do Cong Thung, Institute of Marine Environmental Resources This is an initial research result that will partly help in the search for specific molecular markers to support the fast and accurate scientific name determination of marine species 17 4.2 Determination of chemical structure of compounds from S nanolobata From the soft coral S nanolobata, 10 compounds were isolated These include: new compounds named: 24(S),28-epoxyergost-5-ene-3β,4α-diol (SN 6), 3β,4α-dihydroxyergosta-5,24(28)-diene (SN 8), nanolobatol B (SN 20), nanolobatol A (SN 30) and known compounds: 16α- Hydroxysarcosterol (SN 3), sarcophytosterol (SN 4), sinularianin B (SN 10), sinularianin D (SN 11), Cholesta-5,24(28)-dien-3β-ol-7-one (SN 16), dissesterol (SN 17) The compounds isolated were structurally in the sterol and sesquiterpen classes 4.3 Determination of chemical structure of compounds from S leptoclados From the soft coral S leptoclados has isolated and determined the structure of 15 compounds: all 15 compounds belong to the sterol class, of which new compounds are named: Leptosteroid (SLE 10), 5β,6βepoxygorgosterol (SLE 21) and 13 known compounds: sarcophytosterol (SLE 13), Ergosta-24(28)-en-3β-ol (SLE 19), Ergosta-5,22,24(28)-trien-3βol (SLE 20), 3β,4α-Dihydroxyergosta-5,24(28)-die (SLE 22), Ergosta5,24(28)-dien-3β-ol-7-one (SLE 23), 7-oxogorgosterol (SLE 25), Ergosta5-en-3β-ol-7-one (SLE 26), Ergosta-5,24(28)-dien-3β,7β-diol (SLE 27), Ergosta-5-en-3β,7β-diol (SLE 28), 7β-hydroxygorgosterol (SLE 29), Ergosta-5,24(28)-dien-3β,7α-diol (SLE 30), Ergosta-5-en-3β,7α-diol (SLE 31), 7α-hydroxygorgosterol (SLE 32) 4.4 Determination of chemical structure of compounds from S conferta From the soft coral S conferta has isolated and determined the structure of 12 compounds: all 12 compounds belong to the sterol class, of which new compounds are named: 7α-Methoxygorgosterol (SCO 29), 7αMethoxy-ergosta-5-ene-3β-ol (SCO 30), 3β-Hydroxyergosta-4-ene-6-one 18 (SCO 44), 3β-Hydroxyergosta-4,24(28)-diene-6-one (SCO 32), 24- methylenecholestane-3β,5α,6β-triol-3-monoacetate (SCO 42) and known compounds: 3β-Hydroxy-24-methylenecholest-5-en-7-one (SCO 26), 24methylenecholestane-3β,5α,6β-triol-6-monoacetate (SCO 27), 7- methoxyergosta-5,24(28)-diene-3 -ol (SCO 31), 24-methylenecholestane3β,5α,6β-triol (SCO 35), Ergosta-3β,5α,6β-triol (SCO 37), 3β,7αDihydroxyergosta-5,24(28)-dien (SCO 34.1) (24S)-ergost-5-en-3β,7αdiol (SCO 34.3) 4.5 Evaluation of cytotoxic activity of isolated compounds 4.5.4.1 Study on cytotoxicity mechanism of compounds SCO27 in lung cancer cell line A549 a Evaluation of the effect of SCO 27 on morphological change in cancer cells The results of morphological analysis of A549 lung cancer cells (Figure 3.5) showed that cells in samples treated with SCO 27 showed apoptosis in some cells with characteristic morphology changes cells such as: condensed cells, fragmented nuclei The number of apoptosis cells increases with increasing concentration of SCO 27 reagent from 2.5 to 10 µM The positive control (camptothecin) was stable, the cells after treatment also had a homogeneous, bright, round and bright dye nucleus Thus, compound SCO 27 has the effect of changing cell morphology, this is the basis for conducting further studies to determine the ability of this compound to cause apoptosis b Evaluate the ability of SCO 27 to stimulate production of the enzyme caspase The results of Figure 3.6 show that SCO 27 is capable of inducing A549 cells to produce caspase This activity is only evident at a concentration of 10 µM with an increase of 1.74 times compared to the negative control The camptothecin positive control was stable in the experiment with a caspase 19 activity at a concentration of 2.86 times that of a negative control (P