Keywords

3.1 Introduction

Alkaloids are nitrogen-containing basic compounds known from about 20 % of all plant species. Many alkaloids are pharmacologically active and have been used traditionally in the form of medicinal plant extracts as treatments for various diseases [1]. A few dozen pharmacologically active alkaloids, including camptothecin, are widely used in modern medicine, and worldwide sales of alkaloid-containing drugs were projected to exceed US$ 4 billion in 2002 [2].

Camptothecin (1) is a well-known monoterpenoid indole alkaloid and was originally identified in the extracts of the Chinese tree Camptotheca acuminata (Nyssaceae) [3]. Camptothecin exhibits antitumor activity , which is due to its ability to kill cancer cells via topoisomerase I poisoning [4]. At present, the semi-synthetic water-soluble camptothecin derivatives, topotecan (2) and irinotecan (3), are used worldwide as clinical antitumor agents against cancers of the lung, cervix, ovaries, colon [5], and other organs [6] (Fig. 3.1). In addition, a number of reports are available announcing the therapeutic values of camptothecin derivatives against acquired immunodeficiency syndrome (AIDS) [7] and falciparum malaria [8]. Consequently, the demand for camptothecin will continue to increase in the future.

Fig. 3.1
figure 1

Camptothecin (1) and its clinically used derivatives, topotecan (2) and irinotecan (3). (With permission from Ref. [38])

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Despite the rapid growth of the pharmaceutical market for this compound, camptothecin is still supplied exclusively from intact plants, mainly C. acuminata and Nothapodytes foetida [9]. However, the extraction of this compound from intact plants is problematic because of the shortage of natural resources and the resultant environmental concerns. Thus, the production of secondary metabolites by genetically engineered plant cell cultures, particularly for compounds such as camptothecin , has become a keen issue [10].

Camptothecin-related alkaloids have been reported to be produced in a relatively wide array of plant species, besides C. acuminata and N. foetida [11]. For instance, Merrilliodendron megacarpum [12], Pyrenacantha klaineana (Icacinaceae) [13], Ervatamia heyneana (Apocynaceae) [14], Mostuea brunonis (Loganiaceae) [15], Ophiorrhiza mungos [16], and O. filistipula (Rubiaceae) [17] have been reported to produce camptothecin-related compounds. Moreover, the results of phytochemical studies of the genus Ophiorrhiza have shown that camptothecin also accumulates in some Ophiorrhiza species (e.g., O. pumila) distributed in Japan [18, 19].

The genus Ophiorrhiza is widely distributed around tropical and subtropical Asia and comprises about 150 species [20]. Moreover, some of these species produce indole alkaloids [21]. With regard to the chemical constituents of Ophiorrhiza species distributed in Japan, O. pumila accumulated camptothecin and related alkaloids [18, 22] and O. japonica accumulated β-carboline-type alkaloids, such as lyalosidic acid and harman [23, 24]. Meanwhile, O. liukiuensis [25] and O. kuroiwai [26], which was shown to be an interspecies hybrid of O. pumila and O. liukiuensis, accumulated both camptothecin-related alkaloids and β-carboline-type alkaloids (Fig. 3.2). Therefore, these Ophiorrhiza species are important as resources for the production of various alkaloids, including camptothecin.

Fig. 3.2
figure 2

The genus Ophiorrhiza species distributed in Japan

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In this chapter, we describe the production of camptothecin-related alkaloids and the elucidation of the mechanisms of camptothecin biosynthesis by use of plant cell and tissue cultures.

3.2 In Vitro Cultures of Camptothecin-Producing Plants

3.2.1 Establishment of In Vitro Cultures

Cell and tissue cultures of several camptothecin-producing plants have been investigated as alternative sources for camptothecin production [27]. Sakato et al. [28] reported the first establishment of a rapidly growing cell suspension culture of C. acuminata, although the camptothecin productivity was insufficient (0.002 mg g−1 dry weight) for practical use. Callus cultures of C. acuminata established by Wiedenfeld et al. [29] produced comparatively adequate amounts of camptothecin (2 mg g−1 dry weight). These callus cultures were also reported to contain 10-hydroxycamptothecin, from trace amounts up to 0.08–0.1 mg g−1 dry weight [29]. Callus cultures of N. foetida were found to accumulate small amounts of camptothecin and 9-methoxycamptothecin [30–32], but the level of alkaloid production was 100- to 1000-fold lower than that from soil-grown plants. Callus cultures of O. pumila produced no camptothecin-related alkaloids but accumulated only anthraquinones [33, 34].

Since alkaloid biosynthesis and accumulation are under the strict control of cell developmental and environmental factors [35], establishing cultures of cell types suitable for the production of the camptothecin is important. Accordingly, aseptic plants and hairy roots of Ophiorrhiza species have been established as an effective means of producing camptothecin (Fig. 3.3) [36–38].

Fig. 3.3
figure 3

Established tissue cultures of Ophiorrhiza liukiuensis, O. kuroiwai, and O. pumila. a Aseptic plants cultured for 5 weeks on 1/2 MS medium containing 1 % sucrose and 0.2 % gellan gum in test tubes. b Hairy roots cultured for 4 weeks in B5 liquid medium containing 2 % sucrose. (With permission from Ref. [38])

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3.2.2 Camptothecin Production and Metabolite Profiles in Tissue Cultures of Ophiorrhiza Species

In shoots and roots of established aseptic plants of Ophiorrhiza species, camptothecin production per tissue weight was the highest in the roots of O. pumila. On the other hand, the production per tube was the highest in O. kuroiwai because it showed the higher growth rate of the two species. The concentration and total amount of camptothecin in O. liukiuensis were lower than those of O. kuroiwai and O. pumila.

Camptothecin accumulated to higher levels in hairy root lines of O. pumila than in those of O. liukiuensis and O. kuroiwai [38]. Camptothecin accumulation and increased growth rate of O. pumila hairy roots have the best results in the reports of camptothecin production by in vitro tissue cultures [37, 39] .

The patterns of secondary metabolite production in the aseptic plants and hairy roots of Ophiorrhiza species were profiled by high-performance liquid chromatography–diode array detection–electrospray ion trap tandem mass spectrometry (Fig. 3.4 and Table 3.1) [38]. The metabolite profiles of O. liukiuensis and O. kuroiwai were highly similar in the shoot and root. 10-Methoxycamptothecin (5) and lyalosidic acid (6) were detected in the roots and shoots, respectively, of both O. liukiuensis and O. kuroiwai but not in those of O. pumila. Moreover, 3(S)- and 3(R)-deoxypumilosides (9, 10) were detected only in O. pumila. Camptothecin (1), 9-methoxycamptothecin (4), strictosamide (7), pumiloside (8), strictosidinic acid (11), and 3-O-caffeoylquinic acid (13) were detected in all three species. The metabolite profiles of the hairy roots were not identical to those of aseptic plants .

Fig. 3.4
figure 4

Chemical structures of secondary metabolites detected in tissue cultures of Ophiorrhiza species. (With permission from Ref. [38])

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Table 3.1 Alkaloids and anthraquinones detected in tissue cultures of Ophiorrhiza species
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3.3 Biosynthesis of Camptothecin

3.3.1 Camptothecin Biosynthetic Genes

Monoterpenoid indole alkaloids , including camptothecin, are derived from strictosidine, which is a common intermediate formed by condensation of the indole tryptamine with the iridoid glucoside secologanin by the enzyme strictosidine synthase (STR) [40–42] (Fig. 3.5) . The intramolecular cyclization of strictosidine results in strictosamide, which is an intermediate peculiar to camptothecin biosynthesis, as proven by the incorporation of radiolabeled precursor [43]. The remaining details between strictosamide and camptothecin are not completely defined. However, camptothecin has been postulated to be formed potentially from strictosamide by three transformations: (1) oxidation–recyclization of the B- and C-rings, (2) oxidation of the D-ring and removal of the C-21 glucose moiety, and (3) oxidation of ring E [43]. Plausible camptothecin precursors, such as pumiloside and 3(S)-deoxypumiloside, were isolated from Ophiorrhiza species [18, 19]. Pumiloside has been found also in C. acuminata [44].

Fig. 3.5
figure 5

Predicted camptothecin biosynthetic pathway in O. pumila. The enzymes are as follows: TDC, tryptophan decarboxylase; G10H, geraniol 10-hydroxylase; CPR, NADPH:cytochrome P450 reductase; SLS, secologanin synthase; STR, strictosidine synthase. Plausible intermediates of camptothecin biosynthesis are provided in parentheses

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The cloning of complementary DNAs (cDNAs) from O. pumila hairy roots has been successfully performed to isolate the genes encoding the biosynthetic enzymes involved in the upper part of camptothecin biosynthesis, including STR, tryptophan decarboxylase (TDC) [45], and nicotinamide adenine dinucleotide phosphate, reduced form (NADPH):cytochrome P450 reductase (CPR), in this species [46] (Fig. 3.5). The full-length STR cDNA sequence isolated from O. pumila (OpSTR) contained a 1,056-bp open reading frame (ORF) encoding a protein of 351 amino acids with a molecular mass of 38.9 kDa. The deduced amino acid sequence of OpSTR exhibited 55 % and 51 %identities with STRs from Rauwolfia serpentina [41] and Catharanthus roseus [47], respectively. OpSTR most likely localizes to the vacuole, as predicted by the PSORT program. Southern blot analysis suggested that a single STR-encoding gene is present in the genome of O. pumila. The highest OpSTR expression occurred in hairy roots , followed by the root, and the stem, whereas OpSTR was apparently not expressed in leaves. STR enzymatic activity was detected in the protein extracts of stems, roots, and hairy roots; however, no activity was detected in leaf and callus extracts. The distribution of STR activity correlated with the messenger RNA (mRNA) accumulation pattern and the camptothecin concentrations in O. pumila tissues, with the exception of the young leaves, suggesting that roots and stems are the main tissues for camptothecin biosynthesis [34] .

Tryptamine, a precursor of strictosidine, is formed by the decarboxylation of tryptophan by the enzyme TDC. The cDNA clone encoding TDC was first isolated from C. roseus [48]. The full-length TDC cDNA sequence isolated from O. pumila (OpTDC) contained a 1,521-bp ORF encoding a protein of 506 amino acids with a molecular mass of 56.6 kDa. The deduced amino acid sequence of OpTDC showed high identity to TDCs from C. acuminata [49] and C. roseus [48] (71 and 67 %, respectively). Southern blot analysis suggested that at least TDC-encoding genes are present in the genome. The expression patterns of OpSTR and OpTDC were nearly the same.

The enzyme CPR is essential for the activity of cytochrome P450 monooxygenases, such as geraniol 10-hydroxylase (G10H) and secologanin synthase (SLS), which are involved in camptothecin biosynthesis [50] (Fig. 3.5). The full-length CPR cDNA sequence isolated from O. pumila (OpCPR) contained a 2,073-bp ORF encoding a protein of 690 amino acids with a molecular mass of 76.6 kDa. The deduced amino acid sequence of OpCPR showed high identity with Arabidopsis thaliana, Petroselinum crispum, Pisum sativum, and Triticum aestivum CPRs (72, 66, 65, and 67 %, respectively). Southern blot analysis suggested that only a single CPR-encoding gene was present in the genome of O. pumila. Mirroring the general importance of the enzyme, OpCPR was expressed in all tissues.

Studies have been performed to investigate the effects of stress compounds, such as methyl jasmonate (MeJA), salicylic acid (SA), and yeast extract (YE), on the expression of OpSTR, OpTDC, and OpCPR in O. pumila hairy roots [46]. The changes in the expression patterns of OpSTR and OpTDC in response to these various compounds were highly similar. In particular, OpSTR and OpTDC expression was repressed by SA and YE treatments but unaffected by MeJA. Meanwhile, no treatment resulted in the induction or repression of OpCPR transcripts. In addition, no change in STR activity was observed after treatment with either stress compounds or phytohormones .

3.3.2 In Silico and In Vitro Tracer Studies with [1-13C]glucose

Both the mevalonate (MVA) pathway [51] and the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway [52–54] have been recognized for their role in the formation of isopentenyl diphosphate, the precursor of terpenoid biosynthesis. Yamazaki et al. [55] investigated the incorporation of [1-13C]glucose into camptothecin in the hairy roots of O. pumila by in silico computation using the Atomic Reconstruction of Metabolism (ARM) [56] program and by in vivo tracer experiments. The 13C-nuclear magnetic resonance (13C-NMR) analysis clearly showed that the secologanin moiety of camptothecin was synthesized via the MEP pathway . Furthermore, in O. pumila hairy root cultures, treatment with fosmidomycin, a specific inhibitor of the MEP pathway, resulted in a significant decrease in camptothecin production. These results support the conclusion that the secologanin moiety of camptothecin is derived from the MEP pathway .

3.4 Metabolic Modification in Hairy Roots of O. pumila by RNA Interference

A detailed understanding of camptothecin production, including the enzymatic pathway for its biosynthesis, will be essential to the ultimate goal of the metabolic engineering of this compound. In Papaver somniferum (opium poppy), genetic approaches using antisense RNA [57, 58] or RNA interference (RNAi)-mediated silencing [59] of biosynthetic enzymes have been performed, leading to rapid progress in the metabolic engineering of benzylisoquinoline alkaloids. Therefore, it is considered that RNAi technology is an effective strategy for investigating camptothecin biosynthesis. In our study, the production of camptothecin, strictosidine, and camptothecin-related alkaloids was suppressed in a TDC expression-dependent manner in RNAi hairy roots . Among the hairy root-specific peaks correlated with TDC expression in the liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) analysis, two unknown peaks with a positive correlation were annotated as alkaloids and six unknown peaks with a negative correlation, as flavonoids. The exact mass of several non-annotated peaks was similar to those of predicted intermediates in camptothecin biosynthesis, suggesting that most peaks that positively correlated with TDC expression could be intermediates in camptothecin biosynthesis [60].