Effects Of Camptothecin On Top Ⅰin Spodoptera Exigua

DNA topoisomerase I (Top I) is one of the enzymes that participate in DNA strand nicking-closing reactions and solve the torsional problems in a series of genetic processes, such as DNA replication, transcription, DNA repair and DNA recombination. Since the demonstration of camptothecin as an inhibitor of eukaryotic Top I in1988, a plethora of studies have indicated that camptothecin and its derivatives hamper cell growth mainly by targeting the transient Top I-DNA cleavable complex (Top Icc) in DNA replication and transcription. In our previous studies, we verified that0.1-30μM camptothecin treatment to IOZCAS-Spex-Ⅱ cells established from Spodoptera exigua led to cell apoptosis with possible Topol-induced DNA breaks, which seemingly characterized the mentioned mechanism above. Based on the similar mechanism of camptothecin toxity against insects and tumor cells, it intrigued us to explore camptothecin as the lead compound of a pest-controlled agent in the development of biorational pesticides, and to investigate its possibility to develop a new Top I-based botanical insecticide. Here, the paper aimed at revealing such specific mechanisms as growth inhibition and cell death of camptothecin toxicity against the target insect at three levels of Top I gene organization, Top I mRNA expression and Top I enzyme activity. The whole process included:determing the inhibitory effects of camptothecin and hydroxycamptothecin on DNA relaxation by Top I from IOZCAS-Spex-II cell extracts in vitro; Top I gene cloning and recombinant Top I expression; determing the inhibitory effects of camptothecin and hydroxycamptothecin on DNA relaxation by purified recombinant Top I in vitro; determing Top I mRNA expression by real-time PCR and Top I enzyme activity in IOZCAS-Spex-II cells treated with camptothecin and hydroxycamptothecin in vivo. The results available in this study were listed as follows.The Top I protein of Spodoptera exigua is natively camptothecin-sensitive based on its amino acid residues associated with the camptothecin-resistant type. Top1gene sequence was amplified by RT-PCR and RACE methods. The core regions in Top I gene sequence were amplified stepwise with a final length of1626bp. To obtain the complete Top I cDNA,5’and3’RACE were performed respectively to find terminal regions. The total size of Top I gene sequence was3743bp, where5’UTR (un-translated region) and3’UTR (un-translated region) was80bp and870bp respectively flanking by the open reading frame (ORF). The nucleotide sequence in ORF was2793bp, which encoded deduced930amino acids with another stop codon UAA. Protein multiple sequence alignment among eukaryotic Top Is with Clustalx software revealed clearly that the Top I protein of Spodoptera exigua exhibited high homology (65.25%identity) with the Top I protein of B. mori and shared over42.58%identity with all other species listed in the paper. The great genetic divergence of the Top Is of Camptotheca acuminata and Spodoptera exigua illustrated by the phylogenic tree produced by MEGA5.0verified that the amino acid polymorphism was related to camptothecin sensitivity/resistance. Meanwhile, such key camptothecin-associated residues as E418, Q421, L617, R621, A653, E710, N722, S729(numberd according to human Top I) in Spodoptera exigua were not substituted correspondingly by those of Camptotheca acuminate in situ, concluding that the Top I of Spodoptera exigua was in a camptothecin-sensitive state from the point of molecular evolution.To investigate whether in vitro the Top I protein of Spodoptera exigua was sensitive to camptothecin as expected, Top I-based supercoiled DNA relaxation method was recommended to determine the inhibitory effects of on camptothecin and hydroxycamptothecin on DNA relaxation by Top I from IOZCAS-Spex-II cells and the truncated protein TOP70(335amino acids truncated from the amino end) expressed by the PGEX-TOP70expression vector in BL21(DE) strains. The results indicated that the inhibitory effect of CPT and HCPT on DNA relaxation by Top I in two forms was both dosage-dependent in a similar "S" curve. Camptothecin showed higher inhibitory effects than hydroxycamptothecin when the concentration ranged from1.00to7.50μM, but no significant difference between them was observed at0.01-1.00μM and7.50-100μM respectively. The inhibitory effects of camptothecin and hydroxycamptothecin on DNA relaxation by TOP70was higher than those by Top I in cell extracts, which indicated that protein purification excluded the interference with Top I enzyme activity by unknown proteins, and accordingly the effects of those drugs on Top I was identical in virtue but variable in quantity.The Top I enzyme activity of Spodoptera exigua in vivo was also sensitive to camptothecin, which was instant, time-dependent but not dosage-dependent. The enzyme activity was determined by two-fold dilution method. The result indicated that no significant changes of Top I enzyme activity in untreated cells were observed in48hours (the relative specific activity in the untreated cells was set as1). At the time point2h,4h,6h,12h,24h, IOZCAS-Spex-II cells treated with10.0μM camptothecin and10.0μM hydroxycamptothecin showed similar gradually reduced Top I enzyme activity with no significant difference. But especially at48h, CPT-treated cells showed much less Topol enzyme activity than HCPT-treated cells with nearly no detectable DNA relaxation. Meanwhile, no dosage-dependent effects were observed in IOZCAS-Spex-II cells treated with both camptothecin and hydroxycamptothecin respectively at different dosages for24h treatment. Both drugs at all specified dosages (0.10,0.50,1.00,5.00,10.0,50.0,100μM) except0.50μM hydroxycamptothecin reduced Topol enzyme activity greatly.Top I mRNA level in vivo was less sensitive to camptothecin, which was also time-dependent but not dosage-dependent. Top I mRNA levels in each sample were determined by real time PCR with2-△△Ct method. The result told us that no significant changes of Top I mRNA level in the untreated cells were observed in48hours (the relative expression quantity in the untreated cells was set as1). At the time point12h,24h and48h, Top I mRNA level was both gradually upregulated in IOZCAS-Spex-II cells treated with10.0μM camptothecin and10.0μM hydroxycamptothecin respectively. But at48h, Top I mRNA level in camptothecin-treated cells was higher than that in hydroxycamptothecin-treated cells. Meanwhile, Top I mRNA levels were all upregulated in IOZCAS-Spex-II cells treated with both camptothecin and hydroxycamptothecin respectively at all specified dosages for24h (0.10,0.50,1.00,5.00,10.0,50.0,100μM) except50.0and100μM hydroxycamptothecin.Taken together, camptothecin and hydroxycamptothecin may inhibit IOZCAS-Spex-II cell growth and induce cell death by targeting its Top I in a multiple manner. Based on gene sequence and cell responses to those camptothecin-derived drugs, we proposed that the multiple effects of camptothecin on Top I of Spodoptera exigua were regulated in a transcription-related manner rather than in a replication-related manner.