Production Of Clinically Used Antineoplastic Compounds

A. Catharanthus Alkaloids

The dimeric indole alkaloids, vincristine and vinblastine (Fig. 3), are clinically used as antileukemic agents, which are produced in Catharanthus ro-seus (periwinkle). These dimeric alkaloids are formed by condensation of vindoline and catharanthine, which are both derived from strictosidine as the common intermediate for all monoterpenoid indole alkaloids (Fig. 3). A number of studies have been performed to produce antineoplastic compounds in cell cultures of C. roseus. These include callus, cell suspension, and transformed hairy roots (102,103). Although substantial amounts of monomeric indole alkaloids can be produced in these cell cultures, production of the dimeric alkaloids is not fully successful.

Feeding of biosynthetic precursors, secologanin and loganin, resulted in increased accumulation of ajmalicine and strictosidine (104). Cell differ-

H3cooc/;

R = CHO vincristine R = CH3 vinblastine

H3cooc/;

R = CHO vincristine R = CH3 vinblastine

camptothecin

coch3

paclitaxel

h3co y och3

och3 podophyllotoxin h3co y och3

och3 podophyllotoxin

Figure 3 Chemical structures of clinically used antineoplastic compounds from plants.

entiation also induced the production of the alkaloids (105). Since the genes encoding tryptophan decarboxylase and strictosidine synthase were isolated, the effects of overexpression of these two genes in C. roseus cell culture were elucidated (106). The cell lines with enhanced strictosidine synthase activity accumulated over 200 mg/L of the indole alkaloids. A recent study has partially clarified the signal transduction cascade of strictosidine synthase gene expression, which involves the octadecanoid pathway and protein phosphorylation (107). The specific transcriptional factor for inducible gene expression of tryptophan decarboxylase and strictosidine synthase has been isolated (108). These molecular factors for regulation of indole alkaloid biosynthesis are promising for future application to engineered cell culture production.

B. Camptothecin

Camptothecin is a pentacyclic quinoline alkaloid, which was isolated from Camptotheca acuminata in 1966 (109) (Fig. 3). It exhibits potent anticancer activity that is due to the strong inhibitory action on DNA topoisomerase I. Irinotecan is a semisynthetic derivative of camptothecin improved by decreasing the cytotoxicity of camptothecin and increasing the water solubility. Irinotecan is a prodrug that is metabolically activated by esterase to an ultimate form for anticancer action.

Although the final skeleton of camptothecin belongs to the group of quinoline alkaloids, it is synthesized via strictosidine, a common intermediate of monoterpenoid indole alkaloids. However, the biosynthetic pathway after strictosidine and strictosamide is not yet clear (110). Camptothecin has been found not only in C. acuminata (Nyssaceae) but also in several other plant species of different plant families, i.e., Icacinaceae, Rubiaceae, and Apo-cynaceae.

Because of its potent antitumor activity and the unique mechanism of action, a number of attempts at production by cell culture have been made (Table 4). These are mostly with C. acuminata. The first trial of production by cell culture of C. acuminata was performed in 1974 by Misawa's group (111). Although they established a rapidly growing cell suspension culture, the productivity was not satisfactory. A shoot culture instead of a cell suspension of C. acuminata produced a high amount (0.1% dry weight) of camptothecin, but the growth rate was low (112). High producibility (0.2% dry weight) was reported with a few cell lines of C. acuminata callus (113), although the growth rate of these lines was not measured. With C. acuminata cell cultures, the addition of yeast extracts and jasmonates increased camptothecin production (114).

Species in the genus Ophiorriza (Rubiaceae) are receiving growing attention as a target for cell cultures producing camptothecin instead of C. acuminata. Although a callus culture of O. pumila did not produce camptothecin, the regenerated aspetic plantlets accumulated camptothecin and its derivatives (115). More interestingly, a hairy root culture stimulated by infection of a pathogenic soil bacterium, Agrobacterium rhizogenes, showed rapid proliferation and also good camptothecin producibility (0.1% dry weight) (116,117). A substantial amount of camptothecin was excreted into the liquid medium. Because camptothecin is cytotoxic and exhibits inhibitory activity toward plant growth, there should be a particular mechanism of sequestering camptothecin into vacuoles or excretion to outside the cells.

Table 4 Camptothecin Production in Cell Culture

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