Mechanism of Resistance to Camptothecin, a Cytotoxic Plant Secondary Metabolite, by Lymantria sp. Larvae.[Pubmed: 29797164]

Camptothecin (CPT), a monoterpene indole alkaloid, is a potent inhibitor of eukaryotic topoisomerase I (Top 1). Because of this property, several derivatives of CPT are widely used as chemotherapeutic agents. The compound is produced by several plant species, including Nothapodytes nimmoniana (Family: Icacinaceae) presumably as a deterrent to insect pests. Here, we report, a lepidopteran larva, Lymantria sp. of Lymantriidae family which feeds voraciously on the leaves of N. nimmoniana, without any adverse consequences. Larval body weight and molting period were unaffected despite captive feeding of the larva with CPT enriched leaves. Mass spectrometric analysis indicated that nearly 46% of the ingested CPT was excreted while the rest was sequestered predominantly in the exuviae and setae (~35%). Although most of the CPT was in the parental form as found in the plant, traces of inactive, sulfated forms of CPT were recovered from the larva. Compared to that in plant, there were no critical mutations at the CPT binding domain of the insect's Top 1. The gut pH of the larva was alkaline (pH 10.0). The alkaline gut environment converts CPT from its active, lactone form to inactive, carboxylate form. It is likely that such conversion might help the larva to reduce the overall burden of CPT in its gut. We discuss the results in the context of the mechanisms of resistance adapted by insects to plant toxins.

De novo transcriptome analyses reveals putative pathway genes involved in biosynthesis and regulation of camptothecin in Nothapodytes nimmoniana (Graham) Mabb.[Pubmed: 29270891]

Comprehensive transcriptome analysis of leaf and root tissues of Nothapodytes nimmoniana unravels several putative pathway genes, transcription factors and CYPs related to camptothecin (CPT) biosynthesis. Additionally, post-transcriptional suppression by artificial microRNA (aMIR) of NnCYP76B6 (geraniol 10-hydroxylase) suggests its role in CPT biosynthesis. Tissue-specific LC-MS/MS analysis revealed the presence of secologanin as the central intermediate of MIA pathway in N. nimmoniana. Nothapodytes nimmoniana is a rich source of potent anticancer drug camptothecin (CPT) whose biosynthetic pathway is unresolved due to the lack of genomic and transcriptomic information. Present investigation entails deep transcriptome analysis of N. nimmoniana which led to identification of putative pathway genes and regulatory components involved in CPT biosynthesis. Using Illumina HiSeq 2500 sequencing platform a total of 31,172,889 (6.23 Gb) and 31,218,626 (6.24 Gb) raw reads were generated from leaf and root wood, respectively. These were assembled de novo into 138,183 unique contigs. Additionally, 16 cytochrome P450 transcripts related to secondary metabolism were also identified. Further, transcriptome data pool presented 1683 putative transcription factors of which transcripts corresponding to WRKY TFs were the most abundant (14.14%). A total of 2741 transcripts were differentially expressed out of which 478 contigs showed downregulation in root wood and 2263 contigs were up-regulated. Further, comparative analyses of 17 genes involved in CPT biosynthetic pathway were validated by qRT-PCR. On basis of intermediates, two distinct seco-iridoid pathways are involved in the biosynthesis of monoterpene indole alkaloids either through multiple isomers of strictosidinic acid or strictosidine. Tissue-specific LC-MS/MS analysis revealed the presence of secologanin as the central intermediate of MIA pathway in N. nimmoniana. Geraniol-10 hydroxylase (NnCYP76B6) an important enzyme in CPT biosynthesis which specifically shunts geraniol into the secologanin pathway was also cloned from the trancriptome resource. In planta transient expression of NnCYP76B6 showed a significant enhancement in mRNA transcript levels coincident with enhanced CPT accumulation. Further, artificial microRNA (aMIR) mediated downregulation of NnCYP76B6 resulted in reduction of mRNA transcript levels as well as CPT content in comparison to control. These empirical results suggest a plausible regulatory role for NnCYP76B6 in CPT biosynthesis and also establish a valuable repository for deciphering various structural, rate limiting and regulatory genes of CPT biosynthetic pathway.

Topoisomerases inhibitory activities and DNA binding properties of 9-methoxycamptothecin from Nothapodytes nimmoniana (J. Graham) Mabberly.[Pubmed: 29130341]

We have reported previously that 9-methoxycamptothecin (MCPT) showed significant antitumor activity in vitro. Here, agarose gel electrophoresis experiments were performed to evaluate MCPT's unwinding ability toward plasmid DNA and inhibitory activities against topoisomerases (Topo) I and II. Binding properties of MCPT to calf thymus DNA (CT-DNA) were evaluated by UV-vis, melting temperature, fluorescence, circular dichroism methodologies and molecular docking technique. Results showed that MCPT at 100 μM inhibited Topo I activity, but had no effect on Topo II. Studies on the binding properties indicated that minor groove binding was the most probable binding mode of MCPT to DNA. The abilities of MCPT to act as Topo I inhibitor and minor groove binding agent may be related to its strong antitumor activity.

Ultrasound-assisted rapid extraction and kinetic modelling of influential factors: Extraction of camptothecin from Nothapodytes nimmoniana plant.[Pubmed: 28427671]

Ultrasound-assisted extraction (UAE) of commercially important natural product camptothecin (CPT) from Nothapodytes nimmoniana plant has been investigated. The influences of process factors such as electric acoustic intensity, solid to liquid ratio, duty cycle, temperature and particle size on the maximum extraction yield and kinetic mechanisms of the entire extraction process have been investigated. The kinetics results showed that increasing the intensity, duty cycle, solid to liquid ratio and decreasing the particle size lead to substantial increase in extraction yields compared to classical stirring extraction. Different kinetic models were applied to fit the experimental data. The second order rate model appears to be the best. The extraction rate constant, initial extraction rate and the equilibrium concentration for all experimental conditions have been calculated. SEM analysis of spent plant material clearly showed hollow openings on cell structure, which could be directly correlated to explosive disruption by the action of ultrasound waves. Overall 1.7-fold increase in extraction yields of CPT (0.32% w/w) and decrease in time from 6h to 18min was observed over the stirring method.

Camptothecine production by mixed fermentation of two endophytic fungi from Nothapodytes nimmoniana.[Pubmed: 27268247]

Two endophytic fungi isolated from the endangered plant Nothapodytes nimmoniana (Grah.) Mabb. were found to effectively synthesize CPT independent of their host plant under submerged fermentation conditions. Molecular characterization of fungi revealed their identity as Colletotrichum fructicola SUK1 (F1) and Corynespora cassiicola SUK2 (F2). Mixed fermentation experiments were carried out to study the effect of microbial signalling between the two fungal species on camptothecine production. Effect of culture parameters on CPT production was studied for both mono-cultures (F1 and F2) separately as well as for the mixed fermentation (F1 + F2). Further the most influencing ones were optimized statistically using response surface methodology. Statistical model based optimized parameters were whey (70 %), agitation rate (110 rpm), temperature (30 °C), and incubation period (7 d) for the mixed fermentation. Monocultures of the two fungal species F1 and F2 yielded CPT up to 33 ± 1.1 mg l(-1)and 69 ± 1.1 mg l(-1), respectively; while their mixed fermentation under the optimized conditions yielded up to 146 ± 0.2 mg l(-1). HPLC and LC-MS/MS techniques were used to analyze the products obtained.

Transcriptome analysis of stem wood of Nothapodytes nimmoniana (Graham) Mabb. identifies genes associated with biosynthesis of camptothecin, an anti-carcinogenic molecule.[Pubmed: 26949094]

Camptothecin (CPT), a monoterpene indole alkaloid, is a potent inhibitor of DNA topoisomerase I and has applications in treating ovarian, small lung and refractory ovarian cancers. Stem wood tissue of Nothapodytes nimmoniana (Graham) Mabb. (family Icacinaceae) is one of the richest sources of CPT. Since there is no genomic or transcriptome data available for the species, the present work sequenced and analysed transcriptome of stem wood tissue on an Illumina platform. From a total of 77,55,978 reads, 9,187 transcripts were assembled with an average length of 255 bp. Functional annotation and categorization of these assembled transcripts unraveled the transcriptome architecture and also a total of 13 genes associated with CPT biosynthetic pathway were identified in the stem wood tissue. Four genes of the pathway were cloned to full length by RACE to validate the transcriptome data. Expression analysis of 13 genes associated with CPT biosynthetic pathway in 11 different tissues vis-a-vis CPT content analysis suggested an important role of NnPG10H, NnPSLS and NnPSTR genes in the biosynthesis of CPT. These results indicated that CPT might be synthesized in the leaves and then perhaps exported to stem wood tissue for storage.

Micropropagation and validation of genetic and biochemical fidelity among regenerants of Nothapodytes nimmoniana (Graham) Mabb. employing ISSR markers and HPLC.[Pubmed: 28330243]

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Restoration of camptothecine production in attenuated endophytic fungus on re-inoculation into host plant and treatment with DNA methyltransferase inhibitor.[Pubmed: 26289161]

Fungal endophytes inhabit living tissues of plants without any apparent symptoms and in many cases are known to produce secondary metabolites similar to those produced by their respective host plants. However on sub-culture, the endophytic fungi gradually attenuate their ability to produce the metabolites. Attenuation has been a major constraint in realizing the potential of endophytic fungi as an alternative source of plant secondary metabolites. In this study, we report attempts to restore camptothecine (CPT) production in attenuated endophytic fungi isolated from CPT producing plants, Nothapodytes nimmoniana and Miquelia dentata when they are passed through their host plant or plants that produce CPT and when treated with a DNA methyl transferase inhibitor. Attenuated endophytic fungi that traversed through their host tissue or plants capable of synthesizing CPT, produced significantly higher CPT compared to the attenuated fungi. Attenuated fungus cultured in the presence of 5-azacytidine, a DNA methyltransferase inhibitor, had an enhanced CPT content compared to untreated attenuated fungus. These results indicate that the attenuation of CPT production in endophytic fungi could in principle be reversed by eliciting some signals from plant tissue, most likely that which prevents the methylation or silencing of the genes responsible for CPT biosynthesis.

Structural insight of DNA topoisomerases I from camptothecin-producing plants revealed by molecular dynamics simulations.[Pubmed: 25733498]

DNA topoisomerase I (Top1) catalyzes changes in DNA topology by cleaving and rejoining one strand of the double stranded (ds)DNA. Eukaryotic Top1s are the cellular target of the plant-derived anticancer indole alkaloid camptothecin (CPT), which reversibly stabilizes the Top1-dsDNA complex. However, CPT-producing plants, including Camptotheca acuminata, Ophiorrhiza pumila and Ophiorrhiza liukiuensis, are highly resistant to CPT because they possess point-mutated Top1. Here, the adaptive convergent evolution is reported between CPT production ability and mutations in their Top1, as a universal resistance mechanism found in all tested CPT-producing plants. This includes Nothapodytes nimmoniana, one of the major sources of CPT. To obtain a structural insight of the resistance mechanism, molecular dynamics simulations of CPT- resistant and -sensitive plant Top1s complexed with dsDNA and topotecan (a CPT derivative) were performed, these being compared to that for the CPT-sensitive human Top1. As a result, two mutations, Val617Gly and Asp710Gly, were identified in O. pumila Top1 and C. acuminata Top1, respectively. The substitutions at these two positions, surprisingly, are the same as those found in a CPT derivative-resistant human colon adenocarcinoma cell line. The results also demonstrated an increased linker flexibility of the CPT-resistant Top1, providing an additional explanation for the resistance mechanism found in CPT-producing plants. These mutations could reflect the long evolutionary adaptation of CPT-producing plant Top1s to confer a higher degree of resistance.