Cytotoxicity Of Camptothecin Analogues Against Spodoptera Frugiperda Cultured Cell Line Sf9 And Molecular Docking With Topo?

Camptothecin is a pentacyclic alkaloids, which was isolated from Camptotheca acuminata. Due to it's significant anti-tumor or anti-viral activities in the field of pharmaceutical researches and outstanding insecticidal activity in the filed of pesticide, camptothecin and its analogues arose tremendous attention. 27 camptothecin analogues on the article were based on not only oxgen atom of the D-ring was replaced by sulfur atom but also 7 site of the B-ring was modified, which including 7-acyl thiocamptothecin analogues(CPTA-1 to CPTA-12), 7-acyl camptothecin analogues(CPTA-1 to CPTA-27) and thiocamptothecin(CPTA-13). Cytotoxicity to Spodoptera frugiperda cultured cell line Sf9 was measured by MTT assay. The induced-apoptosisactivities were assayed by flow cytometry. In addition, through a series of physiological,and biochemical and molecular biology techniques to study identifying their potential target.Moreover, its mechanism was explored by molecular docking technology. The specific research results listed as follows.Part I. Antiproliferative activities of CPT analogues.The result of antiproliferative activities of the 27 CPT analogues on Sf9 cells showed that all of the candidate 27 CPT analogues have antiproliferative activities on Sf9 cell lines and the cell growth was significantly inhibited in a dose and time-dependent manner.Compared with 7- acyl camptothecin analogues, 7- acyl thiocamptothecin analogues had higher cytotoxicities. When Sf9 cells were treated by thiocamptothecin and 7-acyl thiocamptothecin analogues with 10 ?g/m L for 24 h, the antiproliferative rate ranged from50% to 95%. In addition, the IC50 of CPTA-3, 4, 6, 7, 8, 11, 13, 26 are lower compared with the IC50 of camptothecin, which suggested the antiproliferative activities of these CPT analogs were better than that of camptothecin.Part II. Apoptosis in Sf9 induced by CPT analoguesBy inverted phase contrast microscope observation, the results showed that there were four camptothecin analogues(CPTA- 1, 3, 9, 11) could induce apoptosis in Sf9 cells. When the Sf9 cells were treated by CPTA- 1, 3, 9, 11 respectively, a large number of apoptotic body gathered around the cell, chromatin condensed and the nucleus offset, visible cavity structure could be observed in cells. With the drug concentration increasing, the number of apoptotic body increased. Sf9 cell were treated by CPTA- 1, 3, 9, 11 and CPT, respectively.through DNA electrophoresis, DNA ladders and DNA fragmentation stripe can be clearly observed, which suggested CPTA- 1, 3, 9, 11 can make DNA degrade. In adddtion, cells double dyed by Annexin V- FITC/PI can be seen green and red fluorescence in fluorescent microscope. What's more, a strong blue fluorescence could be observed when the cells were dyed by Hoechst 33342, which suggested that CPTA-1, 3, 9, 11 could induce phosphatidylserine(phosphatidyl serine, PS) ectropion, nuclear chromatin condensation,membrane permeability changes and eventually induce apoptosis in Sf9 cells.When the Sf9 cells were treated by 2.5 ?g/m L CPTA-1, 3, 9, 11 for 24 h respectively,the apoptosis rate ranged from 25.1%,45.9%,19.9%,36.2% to 23.3% respectively showed by Annexin V-FITC/PI and FCM, which indicated that the apoptosis effect induced by CPTA-3 and CPTA- 11 were more significant than CPT, CPTA – 1, and basically the same as CPT, while CPTA- 9 was less than CPT. These result was consistent with the results of DNA fragmentation. The cell cycle change and the apoptosis rate were analyzed by flow cytometry. PI and FCM pointed that the cell-cycle changed when Sf9 cells treated by CPTA-1, 3, 9, 11, the ratio of cultured cells increased dramatically in the S phase.Besides, the expression levels of Gene Cyt C and Casepase 9 were also examined by quantitative real time PCR. When Sf9 cells were treated by CPTA-1, 3, 9 and 11 with1?g/m L for 12 h respectively, the m RNA expression levels of Cyt C and Casepase 9 had a significant increasing and reached 3 to10 times, which suggested that mitochondrial pathways could be involved in this apoptosis process.Part III. Inhibition activity to Topo?by CPT analoguesIn order to study whether the 27 kinds of camptothecin analogues could inhibit theTopo?, a series of experiments were conducted. Firstly, Topoisomerase?which extracted from Sf9 cell lines were treated by the 27 CPTA. Secondly, p BR322 DNA was treated by these Topo?and then PBR322 DNA were observed by gel electrophoresis. From the results of gel electrophoresis, the unwinding p BR322 DNA expression was suppressed by Topo?which treated by 27 CPTA or CPT. Therefore, our results showed the 27 camptothecin analogues could significantly inhibit Topo?activity, thereby block DNA unwinding.Part IV. Cloning and sequence analysis of Sf9 Topo?The coding region of Sf9 Topo?contains 2790 bases, coded 929 amino acids, and with protein molecular weight of 108.04 KD and the isoelectric point is 8.91. Sequence analysis results showed that it had no signal peptide and no transmembrane area, and most amino acids were hydrophilic. In addition, Topo?contained 96 phosphorylation sites,which including 77 Ser phosphorylation sites, 10 Thr phosphorylation sites and 9 Tyr phosphorylation sites. What is more, multiple sequence alignments and phylogenetic analysis suggested that Topo?had a high conserved sequence in the C-terminal among different insects. The Topo?in Sf9 shared highest similarity with Lepidoptera insects such as S. exigua, Papilio machaon, P. xuthus, P. polytes, but showed significant difference from Wasmannia auropunctata.Part V. Molecular docking of the ternary complex(camptothecin analogues-Topo?homology modeling protein- DNA).The molecular docking was carried out through molecular docking results from CDOCKER of Discover studio 4.5. The Topo I active areas included amino acids. such as ARG530, ASP698, LYS697, THR887, ASN518, ARG654, HIS797, ILE700, ASN886. And the bases of DNA are mainly DT, DG 11, C: DC 112, C: DA 113, DT9, DT12, etc. These residues binded with camptothecin analogues, and then formed hydrogen bond, such as ?-?,?-Alkyl, and the roles of ?-Anion, ?-sigma, ?--S bond, and van der Waals force, etc.CPTA-1 to CPTA-13 could form PI-S bond with residue DT10 or C: DA113, because the atoms were replaced by sulfur atoms in the D ring oxygen, but 7- acyl camptothecinanalogues can not form ?-S bond. Binding toxicological result showed that electron donating groups like a heterocyclic benzene, benzene, propyl, methoxy phenyl were induced at the 7-position acyl thiocamptothecin, and these groups could participate in the formation of hydrogen bonds, or participate in the formation of ?-Sigma, ?-Alkyl and van der Waals forces, which could increase the stability and cytotoxicity of complexes.Therefore, this thesis focuses on selected compounds from 27 camptothecin analogues, some compounds such as CPTA-3, 4, 6, 7, 8, 11, 13, 26 have good cytotoxic effects and some compounds such as CPTA-1, 3, 9, 11 can induce cell apoptosis.CPTA-3,11 noly only have high antiproliferative activities but also can iduce Sf9 cell lines apoptosis. In addition, the toxicology mechanism of camptothecin analogues were explored by molecular docking technology. The results can provide a reference for the transformation and modification of camptothecin as a new type of botanical pesticides.