Camptothecin Production by In Vitro Cultures and Plant Regeneration in Ophiorrhiza Species
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Camptothecin
Camptothecin-derived compounds are widely used for clinical treatment of human cancer. They are synthesized from natural camptothecin, which is obtained by extraction from intact plants. For the feasible production of camptothecin, tissue cultures of Ophiorrhiza liukiuensis and O. kuroiwai, an interspecies hybrid of O. liukiuensis and O. pumila, have been investigated. The aseptic plants and hairy roots of O. liukiuensis and O. kuroiwai were established in addition to the previously-established O. pumila. The camptothecin production by O. kuroiwai was better than that by O. liukiuensis. 10-Methoxycamptothecin was accumulated in tissue cultures of O. liukiuensis and O. kuroiwai but not in O. pumila. Methyl jasmonic acid slightly enhanced the production of camptothecin in the O. liukiuensis hairy roots. These results indicate that the tissue cultures of O. liukiuensis and O. kuroiwai would be the feasible ways for production of camptothecin and related alkaloids.
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Euphorbia motuogensis M. T. Li, X. Z. Lan, H. P. Deng & W. L. Zheng, sp. nov., a new species from Motuo, Tibet, China, is described and illustrated here. It is closely similar to Euphorbia sikkimensis in having terete root, alternate leaves, well-developed pseudoumbellate inflorescence, cyathium, smooth and glaborus capsule, but Euphorbia motuogensis is clealy distinguishable by its pilose stems, involucral leaves color, secondary involucral leaves absent, cyathophylls number and color, and five similar glands. Furthermore, molecular phylogenetic analyses of sequences from both nuclear ribosomal ITS confirm that this species is distinct from morphologically similar species in this subgenus.
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In a soil bioassay, adult Deroceras reticulatum (Stylommatophora: Limacidae) and three different weight-classes of young Arion lusitanicus (Stylommatophora: Arionidae) were exposed to a single dosage (170 dauer larvae per g of soil) of the nematode Phasmarhabditis hermaphrodita monoxenically associated with the bacterium Moraxella osloensis. Groups of 10 slugs were continuously exposed to nematodes for 4 days, and then transferred individually to Petri-dishes containing a disc of Chinese cabbage as food. Food consumption—measured by image analysis—and slug mortality were recorded daily for 10 days. Food consumption was inhibited in both slug species tested. D. reticulatum stopped feeding 6 days after the start of nematode treatment, while all A. lusitanicus continued to feed. However, in the three weight-classes of A. lusitanicus (0.15 g, 0.24 g, 0.45 g), food consumption was reduced by at least 50 %. The greatest reduction in feeding, nearly 90 %, was noted in the smallest A. lusitanicus. The nematodes successfully killed D. reticulatum but were less efficient at killing young A. lusitanicus. At the end of the experiment, mortality was highest in D. reticultatum (98 %) and the smallest weight-class of A. lusitanicus (47 %). There was almost no mortality in the largest weight-class of A. lusitanicus treated with nematodes. P. hermaphrodita associated with M. osloensis can thus be considered as a biological control agent for young stages of A. lusitanicus for its effect as a feeding inhibitor, rather than for its ability to kill the slugs.
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Camptothecin is a strong anti-tumor compound isolated from Camptotheca acuminata. One of the most important way for the production of Camptothecin is by cell cultures of Camptotheca acuminata. The effect of Cu~(2+) on camptothecin accumulation in Camptotheca acuminata cell line was described in this paper. The results showed that the optimum CuCl-2 concentration in B5 medium was 0.008mg/mL, which increased camptothecin production for 30 times compare to the control while has no inhibitive effects on cell growth, at the same time, the peroxidase activity was increased and the anthocyanidin accumulation was inhibited. The promotive effects of Cu~(2+) on camptothecin accumulation in light was higher than that in dark.
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The concentration of camptothecin was determined in different tissues of Camptotheca acuminata seedling. The concentrations of camptothecin in new leaves and roots were significantly higher than in other tissues. However, the concentration of camptothecin declined with leaves becoming old. The induction of callus and cell suspension cultures from younger leaves of Camptotheca acuminata was observed. Cell lines were selected with improved camptothecin production as 0.02%.
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Camptothecin is an anticancer and antiviral alkaloid produced by the Chinese tree Camptotheca acuminata (Nyssaceae). Despite previous reports of low levels of anticancer activity in leaves of Camptotheca acuminata, we have discovered that camptothecin accumulates to approximately 0.4% of the dry weight of young leaves. This level is 1.5-fold higher than that of the seeds and 2.5-fold higher than that of the bark, the two currently used sources of the drug. As the leaves mature, the concentration and absolute amount of camptothecin decreases rapidly. The high levels of camptothecin in young leaves could provide an easily harvested, non-destructive source of this important drug.
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Camptothecin is an anti-cancer alkaloid. Acetylsalicylic acid (ASA) treatment was evaluated as a potential tool for increasing camptothecin accumulation (concentration and yield) in Camptotheca acuminata. Increasing ASA concentration increased leaf camptothecin concentration, but stunted growth and decreased total leaf camptothecin yield. Thus, ASA treatment does not enhance camptothecin yield. Key words: Hydroponic culture, plant secondary metabolite
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HLA-B-associated transcript 3 (BAT3) was originally identified as one of the genes located within human major histocompatibility complex. It encodes a large proline-rich protein with unknown function. In this study, we found that a fragment of the BAT3 gene product interacts with a candidate tumor suppressor, DAN, in the yeast-based two-hybrid system. We cloned the full-length rat BAT3 cDNA from a fibroblast 3Y1 cDNA library. Our sequence analysis has demonstrated that rat BAT3 cDNA is 3617 nucleotides in length and encodes a full-length BAT3 (1098 amino acids) with an estimated molecular mass of 114,801 daltons, which displays an 87.4% identity with human BAT3. The deletion experiment revealed that the N-terminal region (amino acid residues 1-80) of DAN was required for the interaction with BAT3. Green fluorescent protein-tagged BAT3 was largely localized in the cytoplasm of COS cells. Northern hybridization showed that BAT3 mRNA was expressed in all the adult rat tissues examined but predominantly in testis. In addition, the level of BAT3 mRNA expression was more downregulated in some of the transformed cells, including v-mos- and v-Ha-ras-transformed 3Y1 cells, than in the parental cells.
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In order to understand the high efficacy of camptothecin derivatives against human colon tumor xenografts in nude mice, we have studied the transport properties of camptothecin derivatives across cellular membranes of MDR1-overexpressing cells. MDR1 overexpression was shown to have little effect on camptothecin cytotoxicity; camptothecin was equally cytotoxic to both the drug-sensitive parental cell line, KB 3-1, and its multidrug-resistant derivative, KB V1. The ability of camptothecin to overcome MDR1-mediated resistance is most likely due to unimpaired accumulation of camptothecin in MDR1 cells as suggested from the following experiments: (a) cytotoxicity of camptothecin against KB V1 cells was not altered by the known MDR1-reversing agent, verapamil; (b) camptothecin was ineffective as compared with vinblastine in competing with [3H]azidopine for photoaffinity labeling of MDR1; (c) camptothecin was equally efficient in trapping cellular topoisomerase I molecules on chromosomal DNA in the form of cleavable complexes in both KB 3-1 and KB V1 cells. The mechanism by which camptothecin overcomes MDR1-mediated resistance has been further studied using a number of uncharged and charged camptothecin derivatives. In contrast to the uncharged camptothecin derivatives, such as 9-amino-camptothecin and 10,11-methylenedioxy-camptothecin, the charged camptothecin derivative, topotecan, showed reduced cytotoxicity against MDR1-overexpressing KB V1 cells. The reduced cytotoxicity of topotecan in KB V1 cells was due to the overexpression of MDR1 in KB V1 cells since verapamil restored both topotecan accumulation and cytotoxicity. These results suggest that the charge on camptothecin can affect the drug's sensitivity to MDR1. The possible effect of membrane permeability in determining drug selectivity of MDR1 is discussed.
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Camptothecin is one of the clinically used anticancer compounds derived from plants. We have established a hairy root culture Ophiorrhiza pumila , which efficiently produces camptothecin. The strictosidine synthase cDNA was obtained from O. pumila , and its properties were characterized using recombinant protein expressed in Escherichia coli . The mechanisms of camptothecin transport and self-resistance of producing plant cells have also been investigated. These studies offer a basis for pathway engineering of camptothecin in the future.
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