Biotechnological Approaches for the Production of Anticancer Taxol

e Journal of Biological Sciences Volume: 1, Issue 1 (December 2009)

ISSN: 2076-9946, EISSN: 2076-9954 ©All rights reserved.

46

BIOTECHNOLOGICAL APPROACHES FOR THE PRODUCTION

OF POTENTIAL ANTICANCER LEADS PODOPHYLLOTOXIN

AND PACLITAXEL: AN OVERVIEW

Anrini Majumder, Sumita Jha*

Centre of Advanced Study in Cell and Chromosome Research, Department of Botany,

University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019, India

*E-mail: [email protected]; [email protected]

Telephone no.: 91-33-2461-5445

Fax: 91-33-2461-4849

ABSTRACT

A number of plant-derived compounds with diverse chemical structures have played significant roles in the

development of several potent anticancer drugs. Podophyllotoxin, a lignan and paclitaxel, a diterpenoid are two such

anticancer lead molecules. Compounds like etoposide, teniposide, taxotere etc. have emerged as successful

antineoplastic agents upon modification of these two natural leads. The original sources of podophyllotoxin and

paclitaxel have become scarce in occurrence due to overexploitation and their own biological characteristics and

synthetic production of these compounds is still commercially unacceptable. Biotechnological approaches for the

production of these two anticancer leads, involving plant cell and organ cultures, have been considered to be

attractive alternatives. To meet the growing demand by pharmaceutical industries these compounds have been

isolated from sources other than the original plants and different strategies have been adapted to improve their yield

in in vitro cultures. Although large scale production of paclitaxel is reported, production of podophyllotoxin on a

commercial scale is yet to be achieved. This review focuses on these two anticancer lead molecules and summarizes

the different biotechnological approaches applied to improve their productivity.

Key words: Diterpenoids, lignan, Podophyllum, Taxus

Abbreviations: Amsl: above mean sea level; B5: Gamborg‟s basal medium (1968); BA: 6-benzyladenine; cDNA:

complementary DNA; GA3: gibberellic acid; IAA: indole-3-acetic acid; IBA: indole-3-butyric acid; m.p.: melting

point; mol. wt.: molecular weight; MS: Murashige and Skoog‟s basal medium (1962); NAA: α-Napthaleneacetic

acid; NADP: nicotinamide adenine dinucleotide phosphate, NADPH: reduced nicotinamide adenine dinucleotide

phosphate 2,4-D: 2,4-Dichlorophenoxyacetic acid

1. INTRODUCTION

Cancer is one of the deadliest diseases known through ages and is projected to become the leading cause of

death worldwide in 2010 [according to a new edition of the World Cancer Report from the International Agency for

Research on Cancer (Medscape Medical News © 2008)]. At present, cancer is the second most common cause of

death in the US, exceeded only by heart disease. In the US, cancer accounts for 1 of every 4 deaths; the estimated

number of deaths in the US alone in 2008 was 565,650 (American Cancer Society, Cancer Facts & Figures 2008,

and Atlanta: American Cancer Society; 2008). Cancer cells divide at a fast pace and thus compounds inhibiting cell

division or cytotoxic compounds destroying cells directly are the two most commonly applied modes of

chemotherapy [1]. The search for natural products as potential anticancer agents dates back to the Ebers papyrus in

1550 BC, but the scientific period of this search is much more recent, beginning in 1950s with the discovery and

development of the vinca alkaloids, vinblastine and vincristine, and the isolation of the cytotoxic podophyllotoxins

[2, 3]. Some of the world‟s best known lead antineoplastic agents are still derived from plants as the complex

structures of these compounds prohibit their synthesis on an industrial scale [1]. These include vinblastine,

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vincristine from Catharanthus roseus, paclitaxel (Taxol®) from Taxus sp., camptothecin from Camptotheca

acuminata and podophyllotoxin from Podophyllum sp. [1]. Extensive research by pharmaceutical companies have

led to the development of several potential anticancer drugs like vinorelbine, vindesine, topotecan (Hycamtin®),

irinotecan (CPT-11, Camptostar®), docetaxel (Taxotere®), etoposide, teniposide, etc. upon modification of the

natural leads and many more are yet to come. Several other plant-derived compounds are currently in clinical and

preclinical trials.

Production of a plant-based pharmaceutical is always not upto the desired level. It may be produced only at a

specific developmental stage or under specific environmental condition, stress or nutrient availability [4]; the plants

may be very slow growing taking several years to reach a size suitable for product extraction. Indiscriminate

collection of medicinal plants from the wild for extraction of products of interest has led to the extinction of certain

number of species. Thus biotechnological approaches involving plant cell and organ cultures appear to be attractive

alternatives. Such cultures provide a continuous and reliable supply of plant natural products regardless of the state

of the plant in the wild or geographical, political, seasonal or environmental influences [5, 6].

Different strategies have been used with the aim of increasing the production of bioactive secondary metabolites

in plant cell cultures. These include screening and selection of high producing cell lines, optimization of nutrient

media for growth and production, organ culture, culture of immobilized cells etc. [4]. Another promising tool to

improve product yield in cell culture is the use of biotic and abiotic elicitors [7]; feeding of biosynthetic precursors

is yet another effective technique. A novel strategy developed is the in vitro cross-species coculture, through which

metabolites produced by one species can be taken up by another species for biochemical conversion [8, 9, and 10].

Large scale culture of plant cells has been shown to be feasible for industrial production and is an attractive

alternative to the traditional method of cultivation. It ensures controlled supply of phytochemicals and provides a

well defined production system which results in improved yields and consistent quality of the product [11]. Since

the first report on cultivation of plant cells in a large volume [12] considerable progress has been made in the field of

scale up of cell cultures and plant cells have been successfully cultivated in bioreactors of upto 75,000 litres capacity

[13]. But in spite of the progress that has been made, production on an industrial level is limited to only a few

phytochemicals- shikonin [14], ginseng saponins [15], berberine [16] and paclitaxel [17]. Efforts are being

continuously made to optimize conditions for large scale plant cell cultures.

This review focuses on two important plant-derived anticancer leads – podophyllotoxin and paclitaxel and

summarises the different biotechnological approaches that have been applied to improve their yield.

2. PODOPHYLLOTOXIN

Podophyllotoxin (Figure 1a) is a pharmaceutically active natural product belonging to the chemical group of

lignans. It is used as a precursor for the synthesis of important antitumour drugs like etoposide (VP-16-213) (Figure

1b) and teniposide (VM-26) (Figure 1d) which are used in the treatment of lung cancer, testicular cancer, a variety

of leukemias and other solid tumours [18, 19, 20]. NK611, TOP 53, GL 331 and azatoxin are some other promising

antitumour derivatives. Podophyllotoxin, endowed with potent cytotoxicity, acts by inhibiting the assembly of

microtubules. It binds to α/β-tubulin dimer giving podophyllotoxin-tubulin complex; this stops the polymerisation of

microtubules at one end but does not stop the disassembly at the other end leading to the degradation of

microtubules; cells are arrested in the metaphase stage of mitosis and thus cell growth stops [21, 22].

Podophyllotoxin itself has got severe gastrointestinal side effects and thus too toxic for therapeutic purposes. So

with an aim to develop more potent and less toxic anticancer agents several glycosides and aglycon derivatives were

prepared by the chemists in the pharmaceutical research department of Sandoz (Switzerland) and this ultimately led

to the discovery of etoposide in 1966, which received FDA (Food and Drug Administration) approval in 1983. In

order to overcome the limitations associated with poor water solubility of etoposide, etopophos or etoposide

phosphate (Figure 1c) was introduced by Bristol-Myers Squibb Co., USA which was approved by the FDA in 1996.

Teniposide is another derivative [20]. These derivatives of podophyllotoxin are topoisomerase II inhibitors [22, 23]

arresting cells in the late S or early G2 phase of the cell cycle [24, 25]. Podophyllotoxin is also the precursor of other

derivatives such as CPH82 (Reumacon®) that has entered phase III of clinical trials against rheumatoid arthritis in

Europe [26]. It has also been applied topically for the treatment of veneral warts (condyloma acuminata) caused by

papilloma virus [27] and was found to inhibit the replication of measles and herpes simplex type I viruses [28].

Because of its tubulin binding property it disrupts the cellular cytoskeleton and thereby interferes with viral

replication [29].


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2.1 BIOSYNTHESIS OF PODOPHYLLOTOXIN

Podophyllotoxin (C22H22O8, mol. wt. 414.41, m.p.183.3-184°C) is a member of the group of lignans which are

dimers of phenylpropanoid pathway intermediates linked by the central carbons of their side chain [30, 31]. Total

biosynthetic pathway of podophyllotoxin has not yet been elucidated, a hypothetical scheme is proposed. The

pathway, like most other plant secondary metabolite pathways, is branched at several places. The early steps of

lignan biosynthesis until matairesinol were elucidated by Lewis and Davin and their coworkers in Forsythia species

[32]. The biosynthesis of lignans originates from phenylalanine and tyrosine [33] which are converted via a series of

intermediates to coniferyl alcohol, a key precursor in the biosynthetic pathway of podophyllotoxin [34]. Two

molecules of coniferyl alcohol are stereospecifically coupled to (+)-pinoresinol, the first lignan of the pathway by a

protein system pinoresinol synthase, consisting of a radical-forming oxidase and a 78 kDa dirigent protein [35]. (+)-

pinoresinol is reduced by NADPH dependant pinoresinol/lariciresinol reductase to (–)-secoisolariciresinol via (+)-

lariciresinol [36, 37] which is oxidized to matairesinol by NADP dependant secoisolariciresinol dehydrogenase [32,

38, 39]. The first cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductase were isolated from

Forsythia intermedia [37, 40]. cDNAs encoding secoisolariciresinol dehydrogenase were isolated not only from

Forsythia intermedia but also from Podophyllum peltatum [39]. Biosynthetic steps from matairesinol to

deoxypodophyllotoxin are not yet known; the steps include the formation of methylenedioxy bridge leading to ring

A, hydroxylation and methylation in ring E and closure of ring C [21]. Yatein may be an intermediate [41, 42].

Deoxypodophyllotoxin is hydroxylated in position 4 (ring C) by deoxypodophyllotoxin 4-hydroxylase to

podophyllotoxin [43].


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TABLE 1. PHYSICOCHEMICAL PROPERTIES OF PODOPHYLLOTOXIN [219, 220]

IUPAC systematic name (7α,7'α,8α,8'β)-7-hydroxy-3',4',5'-trimethoxy-4,5-

methylenedioxy-2,7'-cyclolignano-9',9-lactone

Molecular formula C22H22O8

Molecular weight 414.41

Melting point 183.3-184°C

Solubility in water 120 mg l-1

at RT

Optical rotation[α]² 0

D 132,5 (C 0,2 CHCl3)

2.2 PODOPHYLLOTOXIN FROM PLANT SOURCES

Podophyllotoxin has been reported to occur in both gymnosperms and angiosperms. In gymnosperms its

presence is noted only in some of the members of the family Cupressaceae while in angiosperms few members

belonging to families Berberidaceae, Polygalaceae, Lamiaceae and Linaceae contain this compound [22] (Table 2).

TABLE 2. PLANT SOURCES OF PODOPHYLLOTOXIN [22]

Family Plant species

Gymnosperms Cupressaceae Callitris drummondii

Juniperus sabina

J. sabina var. tamariscifolia

J. virginiana

J. chinensis

J. lucayana

J. scopulorum

Angiosperms Berberidaceae Dyphylleia cymosa

D. grayi

Podophyllum hexandrum

P. peltatum

P. pleianthum

P. versipellis

Polygalaceae Polygala polygama

Lamiaceae Hyptis verticillata

Linaceae Linum album

L. capitatum

L. flavum

L. arboretum

L. campanulatum

L. elegans

L. pamphylicum

Commercially exploitable plant sources of podopyllotoxin are few and this compound is currently extracted for

drug use from the roots and rhizomes of two species of Podophyllum – P. hexandrum Royle or the Indian

Podophyllum and P. peltatum L. or the American Podophyllum of family Berberidaceae. P. peltatum as a source of

podophyllotoxin is commercially inferior to P. hexandrum [44] as the levels of podophyllotoxin in P. peltatum are

lower than P. hexandrum and purification procedures are complicated by the presence of similar levels of the related

lignans – α and β peltatin [45].

P. hexandrum is a perennial rhizomatous herb growing in the subalpine forests of the Himalayas [46, 47]. The

rhizome is the chief source of a resin known as podophyllin [42, 48] which is a mixture of several lignans with

antitumour properties, podophyllotoxin being the most active cytotoxic compound [49]. Podophyllotoxin content of

rhizomes ranges between 0.36-1.08% (on dry weight basis) [50].


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The demand for podophyllotoxin is ever increasing. But the occurrence of P. hexandrum is scarce due to its

long juvenile phase and poor fruit setting ability [51]. Overexploitation and lack of organised cultivation have made

the plant „critically endangered‟ [52, 53]. Owing to its immense clinical importance new routes for total synthesis of

podophyllotoxin have been discovered [54, 55]. But these are low yielding processes and not economically feasible.

Thus isolation from plant sources continues to be the only viable option [29]. A lot of effort has been put in the past

several years to improve its production from different podophyllotoxin producing plant species.

2.3 PROPAGATION OF Podophyllum SPECIES BY CONVENTIONAL AND IN VITRO TECHNIQUES

There are several problems associated with the isolation of compounds for production of pharmaceuticals from

biomass collected from wild populations of plants. Destruction of plant populations due to over exploitation or

natural calamities affects drug supply and the content of bioactive secondary metabolite in the plant. Moreover, wild

populations may be represented by various genotypes growing under different environmental conditions which may

affect drug profile leading to problems in the purity of the final product. Thus cultivation of suitable clones would

ensure a reliable supply of the material with consistent quality. Seeds of P. hexandrum remain dormant for about 10

months under natural conditions [56]. Strategies towards conventional and in vitro propagation of Podophyllum sp.

have been developed. [56] reported 44% germination upon sowing P. hexandrum seeds with fruit pulp immediately

after collection. Cultivation trials using rhizome as the planting material at higher altitudes (3600m amsl) and seeds

at a relatively lower altitude (1150m amsl) have been reported [57, 58]. Nadeem et al. [48] reported 5 fold

improvement in seed germination of P. hexandrum following pretreatment with sodium hypochlorite and also

improved vegetative multiplication using rhizome segments treated with IBA. The authors also reported in vitro

multiplication of the Indian Podophyllum via multiple shoot formation from zygotic embryos and subsequent

rooting. Plant regeneration via somatic embryogenesis has been reported [59]. Callus derived from zygotic embryos

differentiated globular somatic embryos which developed into plantlets. In our laboratory, we observed occasional

shoot organogenesis in mature root derived callus cultures maintained on MS basal medium supplemented with BA

and NAA for 12 weeks. Nodular structures developed on such calli after 12 weeks which developed into bud

primordia like structures within another 3 weeks of culture. Further development of bud primordia into adventitious

shoot buds was found to occur after 2 weeks of culture on the same medium. Shoot buds developed upto 1-2cms

with leaf differentiation when transferred onto fresh medium under 16/8 hrs (light/dark) photoperiod but did not

grow further even after trials with other combination and concentration of different cytokinins with or without auxin

[60]. In vitro propagation of P. peltatum by using rhizome tips as explants has been reported by Moraes-Cerdeira et

al. [61]. Different types of buds were induced from a terminal bud derived from rhizome tips of P. peltatum which

developed into plantlets.

2.4 PRODUCTION OF PODOPHYLLOTOXIN BY PLANT CELL AND ORGAN CULTURES

Since total chemical synthesis of podophyllotoxin is complicated and expensive biotechnological approaches

particularly plant cell and tissue cultures appear to be attractive alternatives for the production of this

pharmaceutically important lignan. Induction of callus culture from P. peltatum and detection of podophyllotoxin

from such cultures was first reported by Kadkade [62, 63]. Van Uden et al. [49] initiated podophyllotoxin producing

callus cultures from in vitro plantlets of the Indian Podophyllum; dark-grown cultures accumulated upto 0.3%

podophyllotoxin (dry weight basis). After several tedious trials we initiated and established callus cultures of the

Indian Podophyllum from in vitro grown axenic seedling explants and roots and rhizomes isolated from 1 year old

mature plants on B5 and MS media supplemented with growth regulators [64]. Podophyllotoxin could be detected

from all the callus lines that survived after 1 year of initiation, induced from different juvenile and mature explants

[64]. Callus cultures producing podophyllotoxin have also been initiated from needles of Callitris drummondii [65],

leaves of Juniperus chinensis [66].

Suspension cultures have been proposed to be a viable alternative for the production of economically important

phytochemicals. Such cultures have a relatively fast growth rate and are easy to manipulate. Suspension cultures

producing podophyllotoxin were initiated from different plant species which are tabulated below (Table 3).


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TABLE 3. PODOPHYLLOTOXIN PRODUCTION IN CELL SUSPENSION CULTURES OF DIFFERENT

PLANT SPECIES

Species References

Callitris drummondii [65]

Linum album [221]

L. nodiflorum [222]

L. mucronatum spp. armenum [223]

Podophyllum hexandrum [49, 60, 224]

P. peltatum [225, 226]

In a recent study, Anbazhagan et al. [67] established embryogenic cell and adventitious root culture systems in

P. peltatum and high performance liquid chromatography analysis revealed that adventitious roots contained higher

podophyllotoxin than embryogenic cell clumps.

2.5 STRATEGIES FOR IMPROVING THE IN VITRO PRODUCTION OF PODOPHYLLOTOXIN

Several studies have been carried out for improving the accumulation of podophyllotoxin in in vitro cultures by

optimizing culture conditions and nutrient levels, addition of elicitors and precursors, immobilization etc. with

limited success.

Culture conditions had a direct effect on biomass yield and in vitro accumulation of podophyllotoxin. Light

inhibited podophyllotoxin accumulation in callus cultures of P. hexandrum [49]. Cultures grown in light contained

3-4 times less podophyllotoxin than dark grown cultures. Similarly, dark conditions favoured cell growth and

podophyllotoxin accumulation in suspension cultures of P. hexandrum [68]. The accumulation of podophyllotoxin

in callus cultures of P. peltatum was also strongly affected by the quality and intensity of light [63]; total

podophyllotoxin level was promoted by red light at an intensity of 750µW/cm2. The pH of the culture medium

proved to be another critical factor; a medium pH of 6.0 was conducive for high biomass production and

podophyllotoxin accumulation in cell cultures of P. hexandrum [69]. Manipulation of the hydrodynamic stress is yet

another important parameter for enhancing secondary metabolite production in in vitro cultures. Plant cells are

sensitive to shear forces which may be due to large plant cell sizes, presence of a cell wall and vacuoles and

aggregation patterns [70]. P. hexandrum cells were also found to be sensitive to hydrodynamic stress [68]. Higher

rotational speed (>150 rpm) reduced cell viability and thereby had an inhibitory effect on podophyllotoxin

production.

Optimization of the culture medium is an important parameter to improve productivity. To optimize

podophyllotoxin production the effects of major medium components (carbon source, nitrogen and phosphate) and

different types of basal media in P. hexandrum cell suspension culture have been studied [68, 71]. It was found that

podophyllotoxin content could be correlated to the amount and type of sugar used and to the amount of nitrogen and

phosphate present in the culture medium. Glucose was a better carbon source than sucrose for both cell growth and

podophyllotoxin production, the optimum concentration being 60g l-1

. The optimum level of nitrogen was 60mM

with ammonium salts and nitrate in the ratio of 1:2 and that of phosphate was 1.25mM. Also from a wide range of

basal media tested MS medium was found to be most suitable for growth and production of podophyllotoxin [71].

Contrary to these observations, among different basal media tested, we found B5 basal medium to be most suitable

for establishing cell suspension cultures of the Indian Podophyllum [64]. Kadkade [63] showed that the growth of

callus cultures of P. peltatum as well as accumulation of podophyllotoxin in such cultures were also dependant on

the type and concentration of plant growth regulators and other complex supplements (yeast extract, casamino acid,

corn steep liquor and peptone) used. A combination of IAA and/or NAA in the presence of kinetin and casamino

acid promoted callus growth while 2,4-D yielded maximum podophyllotoxin accumulation.

Individual cells of a heterogenous cell suspension culture often differ in their ability to synthesize secondary

metabolites [72]. Cell cloning provides a promising way of screening and selecting high producing cell lines [5].

This approach has been found to be successful for the production of quite a few important plant derived compounds

like shikonin from Lithospermum erythrorhizon [14], berberine from Coptis japonica [73], 10-deacetylbaccatin III

from Taxus baccata [74], forskolin from Coleus forskohlii [75]. Cell plating is a simple technique to select cell lines

with improved growth rates and productivity. Selection of the best performing cell line, its maintenance and

stabilization are necessary prerequisites for the production of compounds in bioreactors and subsequent scale up of


52

the cultivation process to the industrial level. Extensive screening programmes were employed in our laboratory

with an aim of obtaining fast growing cell lines of P. hexandrum with high podophyllotoxin producing capacity.

Indeed, cell lines differed distinctly in growth rates and podophyllotoxin content and selection of fast growing cell

lines and recloning of such lines led to establishment of cell lines capable of optimum growth accumulating

optimum levels of podophyllotoxin [64].

Exogenous supply of a biosynthetic precursor is another interesting alternative to increase the level of a desired

product. Since podophyllotoxin is a product of the phenylpropanoid pathway compounds from the pathway

(phenylalanine, tyrosine, cinnamic acid, caffeic acid, coumaric acid, ferulic acid and coniferin) and one related

compound 3,4-methylenedioxycinnamic acid were added to suspension cultures of P. hexandrum [76]. Of these

precursors only coniferin significantly increased the level of podophyllotoxin – a 12.8 fold increase in content was

observed. We also studied the effect of different concentrations of direct (phenylalanine, tyrosine, trans-cinnamic

acid, para-coumaric acid) and one indirect precursor (tryptophan) of podophyllotoxin on cell suspension cultures of

the Indian Podophyllum [60]. In contrast to observations made by van Uden et al. [76], maximum accumulation of

podophyllotoxin was noted after the addition of para-coumaric acid which was 4.5 fold more than that accumulated

by untreated control cultures. Significant enhancement (2 to 4 fold over control) in podophyllotoxin content was

achieved after adding tyrosine, tryptophan and phenylalanine to cell suspension cultures [60]. Addition of coniferyl

alcohol, the poorly water soluble precursor, complexed with β-cyclodextrin resulted in enhanced podophyllotoxin

accumulation in P. hexandrum cell suspension cultures [77]. Podophyllotoxin content increased 11 fold when

phenylalanine was added to callus cultures of J. chinensis induced from leaves of young trees on Schenk and

Hildebrandt medium [66].

Coniferin is a precursor of podophyllotoxin, but it is not available commercially. Reports indicate that cross

species coculture system using Linum flavum hairy roots (a natural source of coniferin) and P. hexandrum cell

suspensions was an effective strategy for improving podophyllotoxin accumulation. Coniferin generated by L.

flavum hairy roots was effectively used by P. hexandrum cells for in vitro production of podophyllotoxin [10].

Coculture of cell suspensions of P. hexandrum with Beta vulgaris hairy roots (another natural source of coniferin)

revealed that podophyllotoxin was exuded in the culture medium [60]. The study demonstrated the feasibility of

cross species coculture of B. vulgaris hairy roots and cells of the Indian Podophyllum for release of podophyllotoxin

into the culture medium, thereby recycling cells for future use.

Among the manipulative techniques applied to promote the accumulation of secondary metabolites, elicitation

results in significant increases in product yield [78, 79, 80, 81, 82]. In nature plants produce secondary metabolites

as a defense response to pathogen attack or as a wound response and the use of elicitors is usually intended to mimic

these responses in plants. Elicitors are signal molecules triggering the formation of secondary metabolites by

activating novel genes encoding enzymes in different biosynthetic pathways [5, 83, 84]. Addition of elicitors with an

aim of improving the accumulation of bioactive secondary metabolites is well documented [78, 79, 80, 85, 86, 87,

88, 89, 90, 91, 92, 93, 94, 95]. However, there are very few reports on enhancement of podophyllotoxin

accumulation following elicitation. A 15 fold increase in podophyllotoxin accumulation by callus cultures of J.

chinensis was noted after the addition of chito-oligosaccharides [66]. In contrast addition of laminaran enzyme

hydrolysates to the same culture resulted in only a 3.5 fold increase in podophyllotoxin content over the control.

Significant enhancement (10 fold) in podophyllotoxin accumulation was noted when methyl jasmonate was added to

suspension cultures of Linum album line X4SF [96]. We analysed the effect of two well known elicitors (salicylic

acid and methyl jasmonate) on cell suspension cultures of the Indian Podophyllum and a very significant increase in

podophyllotoxin accumulation was noted after the addition of salicylic acid [60].

Another approach of improving productivity by plant cell cultures is the immobilization of plant cells in which

improved cell to cell interaction may lead to improved productivity [4]. Reports also indicate that addition of

precursors to immobilized plant cells resulted in improved accumulation of secondary metabolites than freely

suspended cells under the same condition [97, 98]. However, entrapment of P. hexandrum cells in calcium alginate

as such or addition of precursors to immobilized cells did not improve podophyllotoxin accumulation [76].

Bioreactor studies represent the final step leading to commercial production of economically important

phytochemicals from plant cell cultures. Although a number of economically important compounds have been

produced by plant cell and tissue culture techniques, production of compounds on an industrial scale is still

restricted. Major constraints of industrial or commercial production of compounds of interest have been slow growth

of plant cells, susceptibility to microbial contamination, oxygen needs and susceptibility to shearing stress due to

large cell size [99]. Plant cell cultures often undergo spontaneous genetic variation in terms of secondary metabolite

accumulation resulting in a heterogenous population. For large scale production of any compound selection of a high

yielding and genetically stable cell line is desirable. Also, a multitude of other parameters like growth and

production medium, physicochemical conditions, type of reactor, inoculum density can affect scale up of plant cell


53

cultures. Batch cultivation of P. hexandrum for production of podophyllotoxin was conducted in a 3 litre stirred tank

bioreactor using optimized medium which resulted in biomass yield of 21.4g l-1

in 24 days and podophyllotoxin

concentrations of 13.8mg l-1

in 26 days [100]. In another study, upto 0.2% podophyllotoxin (on dry weight basis)

was obtained when L. album cell suspensions were cultured in a 20 litre bioreactor [42]. Commercial production of

podophyllotoxin using plant cell cultures is yet to be achieved.

2.6 PODOPHYLLOTOXIN PRODUCTION IN TRANSGENIC CULTURES

The formation of secondary metabolites is correlated with the degree of organization of cell structures and is

often low and unstable in undifferentiated callus and suspension cultures [101]; for example, several alkaloids which

are scarcely synthesized in undifferentiated cultures are produced at higher levels in cultured roots thereby

correlating alkaloid production with root organogenesis [102]. But the major constraint associated with in vitro root

cultures is their slow growth rate. On the other hand, hairy roots transformed with Agrobacterium rhizogenes have a

fast growth rate, are genetically stable and produce secondary metabolites at levels comparable to or greater than

that of intact plants [103, 104, 105]. There is a single report on transformation of P. hexandrum by different strains

of A. rhizogenes (A4, 15834 and K599) in which transformed calli obtained from embryo were reported to contain 3

fold more podophyllotoxin compared to controls [106]. However, till date establishment of hairy roots following

transformation using A. rhizogenes has not been reported.

2.7 ALTERNATIVE SOURCES OF PODOPHYLLOTOXIN

Apart from plant sources some endophytic fungi have also been reported to produce podophyllotoxin. Eyberger

et al. [107] reported that two endophytic fungi, both strains of Phialocephala fortinii isolated from the rhizomes of

P. peltatum produced podophyllotoxin at measurable amounts in the culture broth. In another study Kour et al. [108]

reported that an endophytic fungus Fusarium oxysporum isolated from Juniperus recurva produced

podophyllotoxin. Although optimization studies to increase the production by the cultured fungal endophytes are in

progress, these organisms can be promising candidates for large scale production of podophyllotoxin.

3. PACLITAXEL

Paclitaxel (Taxol®: is the registered trade name of a formulated drug based on the chemical paclitaxel and

marketed by Bristol-Myers Squibb Co., USA) is a novel complex diterpenoid originally isolated from the stem bark

of Pacific yew (Taxus brevifolia Nutt.) (Taxaceae) [109]. It is of immense importance as an antineoplastic agent,

being widely used for the treatment of advanced, progressive and drug refractory ovarian cancer [110, 111, 112] and

breast cancer [113]. Since 1992 it was approved by the FDA for the treatment of ovarian cancer; approval for

treatment of breast cancer followed in 1994 [114]. Clinical use of paclitaxel has increased steadily since then and

now it is also used for the treatment of lung cancer [115], head and neck cancer [116], renal, prostrate, colon, cervix,

gastric and pancreatic cancers [111, 117, 118, 119]. Apart from being an excellent antineoplastic agent it is also

effective against noncancerous conditions like polycystic kidney diseases [120] and has shown promising results in

multiple sclerosis [121] and AIDS related Karposi‟s sarcoma [122].

Paclitaxel has a unique mode of action. Unlike other antimicrotubule agents like podophyllotoxin,

colchicine, vinca alkaloids, combretastatin which inhibit microtubule assembly, paclitaxel stabilises microtubules

against depolymerisation; it promotes the polymerization of microtubules but inhibits depolymerisation [123, 124].

This unusual stability blocks the cell‟s ability to disassemble the mitotic spindle during cell division; cells are

blocked in the G2/M phase of the cell cycle [124] and this finally leads to cell death. Paclitaxel thus represents a new

class of antineoplastic agents [125].

The original source, T. brevifolia Nutt., is also the only FDA approved source of paclitaxel [126]. However, the

supply of paclitaxel from the bark is limited because the plant is not abundantly found in nature [127], it grows

slowly taking several decades to increase a few inches in diameter [128] and contains trace amounts of paclitaxel

(0.01% of dry weight of the bark) [129]. 1kg paclitaxel is produced after extraction from 10,000kg bark [130]. Also,

the removal of the bark results in the death of the tree [131]. Total synthesis of paclitaxel has been achieved [132,

133], but the process is complicated and not economically feasible. Thus, pharmaceutical companies still rely

heavily on plant sources. High demand, combined with such low yields from such slow-growing trees has prompted

researchers to explore alternative sources of paclitaxel.


54

TABLE 4. PHYSICOCHEMICAL PROPERTIES OF PACLITAXEL [130]

IUPAC systematic name [2aR-(2a,4,4a,6,9,11,12,12a,12b)]

-Benzoylamino-hydroxy benzenepropionic acid 6,

12b-bis(acetyloxy)-12-(benzoyloxy)-2a,3,4,4a,5,6,

9,10,11,12,12a,12b-dodecahydro-4,11-dihydroxy-

4a,8,13,13-tetramethyl-5-oxo-7,11-methano-1H-

cyclodeca[3,4]benz[1]ester

Molecular formula C47H51NO14

Molecular weight 853.92g mol-1

Melting point 213-216°C

Solubility in water 4mg l-1

at RT

% Composition C(66.11%), H(6.02%), N(1.64%), O(26.23%)

Specific rotation -49º in methanol

UV max 227, 273nm in methanol

Mass spec M+H calculated for C47H51NO14=854.3388

3.1 OTHER NATURAL SOURCES OF PACLITAXEL

Paclitaxel has been isolated from various parts of several other species of Taxus like T. baccata, T. cuspidata, T.

canadensis, T. chinensis, T. x media, T. floridana, T. yunannensis, T. mairei, T. sumatrana and T. wallichiana [79,

131, 134, 135, 136, 137, 138, 139, 140, 141, 142]. However significant variation in taxane content exists among and

within population and species [135]. The authors also demonstrated that paclitaxel levels in shoot tissues of T.

brevifolia may exceed or equal that in the bark tissue of the same plant. Studies by Witherup et al. [143] and ElSohly

et al. [144] also revealed that the needles of Taxus species represent a rich and renewable source for paclitaxel and

other related taxanes. The needles of T. wallichiana (the Himalayan yew) are particularly good sources of 10-

deacetylbaccatin III which can be used as a precursor for the semisynthesis of paclitaxel and docetaxel (taxotere®)

[145], an analogue of paclitaxel [146]. Kwak et al. [131] detected paclitaxel from seeds of T. baccata, T. brevifolia,

T. cuspidata and T. cuspidata var. latifolia, the content varied according to different stages of seed maturation. It has

also been isolated from needles and bark of Austrotaxus spicata and Torreya grandis [147].

3.2 ALTERNATIVE SOURCES OF PACLITAXEL

The increase in the use of paclitaxel for cancer chemotherapy and basic research warrants efforts to improve the

existing production processes. Semisynthesis is the major route for the production of paclitaxel and related taxoids

[148, 149, 150, 151]. It involves a limited number of steps to convert paclitaxel pathway intermediates (viz. baccatin

III isolated from Taxus needles) to the target compound. But purification of semisynthetic precursors from plant

tissues is required to separate the desired intermediates from phenolics, lipids and other contaminants occurring in

planta.

Few organisms producing paclitaxel include a group of endophytic fungi of Taxus species viz. Taxomyces

andraenae, Fusarium sp. Alternaria sp. [152, 153], an unknown fungus from Taxus yunannensis [154],

Pestalotiopsis microspora and Phoma sp. isolated from T. wallichiana [155, 156]. In a recent study, genes coding

for 10-deacetylbaccatin III-10-O-acetyl transferase and C-13 phenylpropanoid side chain-CoA acyltransferase were

used as molecular markers for screening of taxol-producing endophytic fungi. Using PCR, three out of ninety

endophytic fungi, isolated from T x media and T. yunnanensis, gave positive results. These 3 strains, when grown in

300 ml potato/dextrose liquid medium at 25°C for 10 days, contained 100-160 mg taxol g-1

dry wt of mycelium

[157]. A paclitaxel producing bacterium Erwinia sp. was reported from T. canadensis [158].

3.3 BIOSYNTHESIS OF PACLITAXEL

Paclitaxel (C47H51NO14, m.p.213~216°C, mol. wt. 853.92g mol-1

) (Figure 2) belongs to the class of taxane

diterpenoids which currently has over 300 known members [159]. The structure of paclitaxel differs from the other

members in having a complex β-phenylisoserine side chain esterifying the C-13 position and has the unusual

http://en.wikipedia.org/wiki/10-Deacetylbaccatin

http://en.wikipedia.org/wiki/Transferase

http://en.wikipedia.org/wiki/Phenylpropanoid

http://en.wikipedia.org/wiki/Acyltransferase

http://en.wikipedia.org/wiki/Genetic_marker

http://en.wikipedia.org/wiki/Endophyte

http://en.wikipedia.org/wiki/Fungus

http://en.wikipedia.org/wiki/Polymerase_chain_reaction

http://en.wikipedia.org/wiki/Endophyte

http://en.wikipedia.org/wiki/Fungus

http://en.wikipedia.org/wiki/Glucose

http://en.wikipedia.org/wiki/Liquid

http://en.wikipedia.org/wiki/Paclitaxel

http://en.wikipedia.org/wiki/Mycelium


55

oxetane ring [114]. The biosynthetic pathway is complex involving several enzymatic steps and is not totally

elucidated. The taxane skeleton is synthesized from geranylgeranyl diphosphate. The committed step in the

biosynthesis of paclitaxel and related taxoids is the cyclization of the universal diterpenoid precursor geranylgeranyl

diphosphate to taxa-4(5),11(12)-diene by taxadiene synthase. A cDNA encoding taxadiene synthase (tasy) from T.

brevifolia was isolated by [160]. Hydroxylation of taxa-4(5),11(12)-diene at C-5 to taxa-4(20),11(12)-dien-5α-ol

catalysed by cytochrome P450 taxadiene 5α-hydroxylase is probably the next step in the biosynthetic pathway. The

following steps include acetylation of taxa-4(20),11(12)-dien-5α-ol to taxa-4(20),11(12)-dien-5α-yl acetate by taxa-

4(20),11(12)-dien-5α-ol-O-acetyltransferase and hydroxylation of taxa-4(20),11(12)-dien-5α-yl acetate to taxadien-

5α,10β-diol monoacetate by cytochrome P450 taxane 10β-hydroxylase. Biosynthetic steps from taxadien-5α,10β-

diol monoacetate to 2-debenzoyltaxane are not yet known. Bezoylation of 2-debenzoyltaxane to 10-

deacetylbaccation III by a taxane 2α-O-benzoyltransferase and acetylation of 10-deacetylbaccatin III to baccatin III

by a 10-deacetylbaccatin III-10-O-acetyltransferase are the next steps. Phenylisoserine derived from phenylalanine

by phenylalanine aminomutase, is condensed with baccatin III by baccatin III-10-O-phenylpropanoyltransferase to

3'-N-debenzoyl-2-deoxy-taxol. N-debenzoyl-deoxytaxol N-benzoyltransferase adds a benzoyl-CoA moiety to the 2-

deoxytaxol and a subsequent benzamidation produces paclitaxel. Of the several genes involved in the biosynthesis

of paclitaxel, those encoding taxadiene synthase, taxadien-5α-ol-O-acetyltransferase, cytochrome P450 taxanedienyl

acetate 10β-hydroxylase, 10-deacetylbaccatin III-10β-O-acetyltransferase and taxane 2α-O-benzoyltransferase have

been isolated, expressed and characterized [161, 162, 163, 164]. A thorough understanding of the pathway and its

rate limiting steps is required to improve the biological production of paclitaxel and related taxoids. With the

corresponding enzymes identified and assays developed, the role of each enzymatic step in the control of pathway

flux can be assessed by in vitro and in vivo studies and suitable strategies can be developed to isolate and

overexpress the corresponding genes in Taxus cell cultures for improving the production of paclitaxel.

3.4 PROPAGATION OF Taxus SPECIES BY CONVENTIONAL AND IN VITRO TECHNIQUES

Species of Taxus are propagated by seeds, cuttings and grafting [165]. Propagation by cuttings is well

documented for T. brevifolia, T x media, T. cuspidata [166] and T. baccata [167]. Cutting and grafting techniques

have been employed in the propagation of Himalayan yew T. wallichiana [168, 169]. Of the different treatments

applied to vegetatively propagate the Himalayan yew using branch cuttings, IBA dip treatment proved to be the best

method for induction of roots in stem cuttings [170].

Propagation of Taxus sp. by in vitro techniques is also reported. Taxus species are found to be recalcitrant

towards direct regeneration from axillary buds, nodal, internodal and leaf explants [171]. Taxus seeds have a lengthy

seed dormancy period of nearly 1.5-2.0 years [172] which can be overcome by embryo culture techniques [173].

Propagation by embryo culture technique has been reported for T. brevifolia, T. x media, T. cuspidata, T. baccata, T.

baccata stricta, T. mairei, T. wallichiana [128, 172, 174, 175, 176, 177].

Plant regeneration via direct organogenesis from zygotic embryos has been reported in T. brevifolia[178] and by

indirect organogenesis in T. chinensis [179]. Chee in 1996 [180] reported regeneration of T. brevifolia from


56

immature zygotic embryos via somatic embryogenesis. Regeneration of T. wallichiana plants via shoot

organogenesis from callus cultures derived from zygotic embryos is also reported [181]. In a recent study, Datta and

Jha [182] reported regeneration of complete plantlets of the Himalayan yew via somatic embryogenesis from zygotic

embryo derived callus cultures.

3.5 IN VITRO PRODUCTION OF PACLITAXEL AND RELATED TAXANES

Cell cultures of Taxus represent a potential alternative source of paclitaxel and related taxanes. Rohr [183] first

established callus cultures from different explants of T. baccata, including microspores while production of

paclitaxel using cell cultures of Taxus was first reported by Christen et al. [184]. Since then, cell cultures have been

reported from different explant sources of different Taxus species with detectable amount of taxanes. Almost every

Taxus species has been studied in terms of media optimization to yield maximum biomass [185]. Fett-Neto et al.

[134] reported induction of callus cultures from different types of explants of T. cuspidata and T. canadensis and

paclitaxel in amounts comparable to T. brevifolia bark was detected from callus cultures of T. cuspidata. Paclitaxel

was also produced by immobilized cell cultures of T. cuspidata. Improved growth and paclitaxel yield in calli of T.

cuspidata were obtained by several sequential modifications of the culture medium viz. addition and/or variation in

the concentration of sucrose, B5 organic supplements, GA3, combinations of 2,4-D/kinetin ratios, media salts and

organic supplements, phenylalanine, casein hydrolysate and variation of medium pH [186]. Wickremesinhe and

Arteca [187] reported induction of callus cultures from stems and needles of T. brevifolia, T. cuspidata and cultivars

of T. baccata and T. x media using different concentrations of 2,4-D, IBA or NAA in combination with kinetin;

paclitaxel could be detected from the callus lines. The authors also reported establishment of cell suspension cultures

from T. x media cv. Hicksii callus cultures after optimizing the carbon source in the medium and detected paclitaxel

from such cultures [188]. Jha and Jha [174] reported establishment of fast growing cell lines of the Himalayan yew

by selection and cloning; though the level of paclitaxel was low, high levels of 10-deacetylbaccatin III were detected

in suspension cultures and culture media, thereby representing a potential source of the precursor of paclitaxel. In a

similar study, screening of cultures derived from different types of explants collected from a number of T.

wallichiana trees growing naturally in different regions of Darjeeling led to the establishment of high paclitaxel

yielding cell lines [189]. Recently, taxane analysis in embryogenic and non embryogenic calli derived from zygotic

embryos of the Himalayan yew revealed that paclitaxel accumulation was higher in embryogenic calli than in non

embryogenic calli [182].

For efficient production of paclitaxel, the effects of cultivation environment and various precursors and

elicitors were studied. Mirjalili and Linden [190, 228] and their co-workers investigated the effects of different

concentrations and combinations of oxygen, carbon dioxide and ethylene on cell growth and paclitaxel production in

cell suspension cultures of T. cuspidata. They found that a low headspace oxygen concentration promoted early

production of paclitaxel while a high carbon dioxide concentration was inhibitory. The effect of temperature shift on

cell growth and paclitaxel production was studied in suspension cultures of T. chinensis [191]. Cell growth was

optimum at 24°C while paclitaxel production reached its maximum at 29°C. The effect of osmotic pressure on

paclitaxel production was investigated in cell suspension cultures of T. chinensis [192]. High osmotic pressure

conditions generated by mannitol, sorbitol and polyethylene glycol enhanced paclitaxel production.

The accumulation of paclitaxel and related taxanes in Taxus is thought to be a biological response to specific

external stimuli [78]. Supplementation of biotic and abiotic elicitors in cell cultures of different Taxus species has

shown to affect both cell growth and paclitaxel accumulation. The use of fungal elicitors and other selected

compounds for the production of paclitaxel by suspension cultures of T. brevifolia was first demonstrated by

Christen et al. [193]. Ciddi et al. [194] demonstrated improved taxoid production in cell suspension cultures of

Taxus sp. after the addition of cell extracts and culture filtrates of Penicillium minioluteum, Botrytis cinerea,

Verticillium dahliae and Gilocladium deliqucescens. Aspergillus niger, an endophytic fungus isolated from the inner

bark of T. chinensis, resulted in a two fold increase in paclitaxel yield when added as an elicitor in the late

exponential growth phase of T. chinensis cell suspension cultures [195]. Reports indicate methyl jasmonate to be an

effective elicitor for the in vitro production of paclitaxel and related taxanes [78, 79, 80, 88]. Probably jasmonates

act as signal compounds in the elicitation process leading to de novo transcription and translation of genes and

ultimately to the biosynthesis of secondary metabolites in plant cell cultures [79]. Yukimune et al. [78] suggested

that methyl jasmonate contributes to paclitaxel production by activating the biosynthetic steps from geranylgeranyl

diphosphate to baccatin III and subsequent steps leading to the formation of paclitaxel from baccatin III. Mirjalili

and Linden [190] claimed that a higher level of paclitaxel was obtained in cell suspension cultures of T. cuspidata

when ethylene was added in combination with methyl jasmonate compared to treatment with methyl jasmonate


57

alone. Paclitaxel accumulation in cell suspension cultures of T. media was improved by adding methyl jasmonate

together with mevalonate and N-benzoylglycine [196]. Another study showed that the application of ethylene

inhibitors to Taxus cell cultures before elicitor treatment was an effective strategy to enhance elicitor-induced

paclitaxel production suggesting that the control of ethylene production is important for the elicitation of paclitaxel

biosynthesis in cultured cells [197]. Yu et al. [198] reported that a T. chinensis culture treated with a fungal elicitor

in combination with salicylic acid achieved higher biomass and paclitaxel production than when treated with the

fungal elicitor alone. The effects of several other elicitors like arachidonic acid [199], silver ion [200], chitosan [200,

201] and La 3+

ion [202] on paclitaxel production have also been studied.

Brassinolide, a member of brassinosteroid group of steroidal lactones, increased paclitaxel content in cell

suspension culture of T. chinensis [203]. Clinton et al. [204] reported that a strong correlation exists between

paclitaxel and abscisic acid recovered from yew and suggested that an increase in abscisic acid concentration might

lead to an increase in paclitaxel production. Indeed, Luo et al. [205] demonstrated that abscisic acid could stimulate

paclitaxel accumulation in cell suspension cultures of T. chinensis and the stimulating effect depended on the dosage

of abscisic acid as well as the stage of cell growth.

Paclitaxel yields in both callus and cell suspension cultures of T. cuspidata were improved after the addition of

phenylalanine and other potential paclitaxel side chain precursors at different concentrations [206]. The promotion

of paclitaxel accumulation in in vitro cultures by phenylalanine might be due to its involvement as a precursor for

the C13 N-benzoylphenylisoserine side chain of paclitaxel. Significant enhancement in the level of paclitaxel was

noted in a cell line of T. wallichiana by supplementation of the basal medium with IAA conjugates like IAA-

phenylalanine and IAA-glycine [207]. Using three different cell lines of T. wallichiana the authors demonstrated

that 2,4-D and IAA-phenylalanine when present alone favoured cell growth and paclitaxel production but when

combined enhanced biomass to a maximum without increasing paclitaxel accumulation, suggesting that a two stage

culture might be needed to optimize paclitaxel accumulation in cell cultures of T. wallichiana. Use of a two stage

culture method for optimum growth and taxane production was also suggested by Furmanowa et al. [79]. In

another study, callus cultures established from young leaf explants of the Himalayan yew showed the presence of

taxanes viz. paclitaxel, 10-DAB and baccatin III [208]. Different precursors and growth retardants like

phenylalanine, sodium benzoate, hippuric acid, leucine, ancymidol, 2-chloroethyl phosphonic acid and

chlorocholine chloride improved the accumulation of taxanes in these cultures.

Expósito et al. [209] conducted an interesting study using cell cultures of T. baccata. A T. baccata cell line

growing for 20 days in a selected growth medium was treated at the beginning of the experiment with different

concentrations of taxol (25, 50, 100 and 200 mg l−1

). Compared with an untreated control, these exogenous taxol

concentrations stimulated cell-associated taxol content (up to 32.7 times in the presence of 200 mg l−1

exogenous

taxol), although higher concentrations significantly depressed cell viability. On the basis of RT-PCR expression

analysis of genes encoding taxadiene synthase (ts) and 1-deoxy-d-xylulose-5-phosphate synthase (dxs) from treated

and nontreated T. baccata cell line cultures, it was observed that exogenous taxol clearly induced the mRNA levels

of both taxane-related enzymes. Exogenous taxol also caused a considerable increase in taxadiene synthase activity.

Cross species coculture proved to be another effective technique for successful production of paclitaxel. In a

recent study, cell suspensions of Taxus chinensis var. mairei were cocultured with its endophytic fungi, Fusarium

mairei, in a 20 litre co-bioreactor, resulting in a production of 25.63 mg l-1

paclitaxel within 15 days; it was

equivalent to a productivity of 1.71 mg l-1

per day and 38-fold higher than that by uncoupled culture [210].

3.6 GENETIC TRANSFORMATION OF Taxus FOR PACLITAXEL PRODUCTION

Genetic transformation of T. baccata and T. brevifolia by Agrobacterium tumefaciens strains Bo542 and C58 is

reported [211]. Strain Bo542 was more efficient in inducing galls on shoot segments of mature yew trees than strain

C58. Although tumours were induced on both the species, T. baccata was more susceptible to transformation than T.

brevifolia. In contrast to untransformed callus cultures, the gall cell lines proliferated on phytohormone free

medium. Paclitaxel and related taxanes were detected in the transgenic cell lines.

In another study, A. rhizogenes strain LBA9402 was used to develop hairy root cultures of T. x media var.

Hicksii [212]. These cultures accumulated twice the amount of paclitaxel than that in the bark of T. brevifolia after

treatment with methyl jasmonate.


58

3.7 LARGE SCALE PRODUCTION OF PACLITAXEL

Cell cultures of Taxus species have been grown in different types of bioreactors for large scale production of

taxoids. Zhong et al. [213] demonstrated that the lag phase of cell cultures of T. chinensis was shortened when

cultured in a novel low-shear centrifugal impeller bioreactor. However, pneumatically agitated bubble columns and

airlifts have been more commonly used for taxoid production than stirred reactors [214]. Batch cultivation of T.

baccata var. fastigata and T. wallichiana suspension cultures was carried out in 20 litre airlift bioreactor [215]. In

another study, fast growing cell lines of T. cuspidata cells were inoculated in 20 litre bioreactors of different types of

which balloon type bubble bioreactor (BTBB) was most efficient in promoting cell growth. The cells were then

cultured in 100-500 litre BTBBs; more than 70% of the cells were viable at the time of harvest. After a culture

period of 27 days the yield of paclitaxel and total taxanes was 3mg l-1

and 74mg l-1

respectively [216]. Cusido et al.

[196] reported a two stage culture and addition of elicitors and precursors to T. media cells in 5 litre stirred reactor

which resulted in a paclitaxel production of 21.1mg l-1

. Cell suspension cultures of the Himalayan yew, cultivated in

20 litre airlift bioreactor resulted in a 20.84 mg l-1

yield of paclitaxel on the 24th

day [217]. Production of paclitaxel

in flasks in industrially interesting levels has also been recorded in cell cultures of different Taxus species (Table 5).

TABLE 5. PRODUCTION OF PACLITAXEL IN COMMERCIALLY INTERESTING LEVELS IN CELL

CULTURES OF DIFFERENT Taxus SPECIES

Species Production References

(mg l−1

)

T. canadensis 117 (day 12) [88]

T. media 110.3 (day 14) [78]

T. chinensis 153 (day 42) [229]

67 (day 35) [227]

78.5 (day 35) [192]

137.5 (day 42) [191]

Paclitaxel is one of the few examples of pure chemicals so far produced on an industrial scale by plant cell

cultures. As early as 1994, Phyton (Ithaca, NY, USA) and ESCAgenetic (San Carlos, CA, USA) cultured Taxus

suspensions in 75,000 litre and 2500 litre bioreactor respectively [17]. The commercial production of paclitaxel

using plant cell suspension cultures was achieved by Samyang Genex (Taejon, Korea) in 2001 [218].

TABLE 1. PHYSICOCHEMICAL PROPERTIES OF PODOPHYLLOTOXIN [219, 220]

IUPAC systematic name (7α,7'α,8α,8'β)-7-hydroxy-3',4',5'-trimethoxy-4,5-

methylenedioxy-2,7'-cyclolignano-9',9-lactone

Molecular formula C22H22O8

Molecular weight 414.41

Melting point 183.3-184°C

Solubility in water 120 mg l-1

at RT

Optical rotation[α]² 0

D 132,5 (C 0,2 CHCl3)


59

4. CONCLUSIONS

Podophyllotoxin and paclitaxel are two potent plants derived antitumour compounds. Supply of these

compounds from traditional sources is limited either due to the dwindling condition of plant sources or due to trace

amounts of compounds present in the plants. So several alternative strategies have been adapted to increase the

production of these compounds. Plant tissue culture represents a sustainable and contained production system which

is constantly improving with respect to reliability and capacity. Indeed, recent years have seen great achievements in

the production of these compounds using plant cell culture systems. Although large scale production of paclitaxel is

reported, production of podophyllotoxin on a commercial scale is yet to be achieved; technological developments

are required. Development of transgenics and metabolic engineering are other important tools. Genes encoding

enzymes for the biosynthesis of paclitaxel and podophyllotoxin can be expressed in fast growing microorganisms.

Genes controlling slow steps in the pathways can be overexpressed or synthesis of unwanted metabolites can be

suppressed by various sense and antisense technologies. However, more basic knowledge about the pathways to

paclitaxel and podophyllotoxin and their regulation is required. The challenge remains to elucidate the complete

biosynthetic pathways, thereby exploiting cell cultures more rationally for the production of these compounds so

much in need by the pharmaceutical industry.

Acknowledgement

A. M. thanks Council of Scientific and Industrial Research, New Delhi, for the award of Senior Research

Fellowship. The financial assistance from Department of Biotechnology, Ministry of Science and Technology, India,

is gratefully acknowledged.

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Biotechnological Approaches for the Production of Anticancer Taxol

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