Production of anti-tumor compounds by plant cell cultures

trends in production of secondarymetabolites from higher plantsOrgan cultures for secondary metabolite production - In vitro organ culture of Fritillaria unibracteata rapidly propagates, directly from small cuttings of the bulb. The growth rate of 3050 times higher than that under natural wild growth conditions is seen. The content of alkaloid and beneficial microelements in the cultured bulbs were higher than found in the wild bulb.Precursor addition for improvement of secondary metabolite production - Coniferyl alcohol in the form of complex with -cyclodextrin was used as precursor for podophyllotoxin accumulation in P. hexandrum cell suspension cultures. - The involvement of amino acids (Val, Ile) in the biosynthesis of hyperforin and adhyperforin was reported in H. perforatum shoot cultures.Elicitation of in vitro products - The addition of selenium for ginseng production. - Chitosan, a biotic elicitor polysaccharide increases production of anthraquinone in Rubia akane cell culture.

Hairy root cultures as a source of secondary metabolites - Inoculation of plant with Agrobacterium rhizogenes produces infection in roots, exuding out the secondary metabolites with higher yields.Genetic manipulation in hairy root culture for secondary metabolite production - Hairy root cultures of Nicotiana rustica with ornithin decarbosylase gene from yeast, and Peganum harmala with tryptophane decarboxylase gene from C. roseus have been shown to produce increased amounts of the secondary metabolites nicotine and serotonin when expressing transgenes from yeast.Role of endophytes in in vitro production of secondary metabolitesBioreactors scaling upImmobilization scaling up of secondary metabolite accumulation - Cell cultures of Plumbago rosea immobilized in calcium alginate and cultured in MS basal medium containing 10 mM CaCl2 for the production of plumbagin showed 3 fold increase as compared to the control.

Production of anti-tumor compounds by plant cell culturesBy:Mugdha NigamME Biotech2011H129007H

Cancer is one of the deadliest diseases known through ages and has become the leading cause of death worldwide in 2010.

Cancer cells divide much faster than normal cells. Therefore, two modes of chemotherapy are possible: - compounds that stop cell division - cytotoxic compounds that destroy cells directly.

The use of plant drugs for medical treatment is possible since plants have evolved bioactive secondary metabolites (SM) that have been selected during evolution as a defence system against, for example, microbes and herbivores.

Due to their complex structure with several chiral centres important anticancer agents are still extracted from plants and not synthesized chemically on a commercial scale.

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Common Anti-tumor compoundsVinblastine and Vincristine Source - Madagascar periwinkle, Catharanthus roseus Parts used - pure alkaloids are extracted from aerial parts as important medicines to treat breast, uterine cancer and Hodgkins as well as non-Hodgkins lymphoma. - Only organized shoot cultures have produced trace amounts of these dimeric alkaloids.Camptothecin

Source - mainly obtained from the happy tree, Camptotheca acuminata and related species (Cornaceae). Parts used Stem bark and young leaves - The callus cultures grown on MS medium containing 4 mg/l NAA, show accumulation of camptothecin upto 0.998 mg/l. - Best results had been obtained with hairy root cultures of O. pumila with a production of approximately 1mg/g dry wt of CPT.

Podophyll0toxin (ptox)P. hexandrum Royle or the Indian Podophyllum and P. peltatum L. or the American Podophyllum of are only commercially exploitable plant sources of PodopyllotoxinThe rhizome and roots of P. hexandrum contains up to 6% of a resin podophyllin, which is a rich source (up to 50%) of podophyllotoxin.

Podophyllotoxin is a pharmaceutically active form of lignans.

It is used as a precursor for the synthesis of important antitumor drugs like etoposide and teniposide , used in the treatment of lung cancer, testicular cancer, a variety of leukemias and other solid tumours

SIDE EFFECTS - Podophyllotoxin itself has got severe gastrointestinal side effects and is too toxic for therapeutic purposes.

MODE OF ACTION - Inhibition of the assembly of microtubules. Hence, cells are arrested in the metaphase stage of mitosis and cell growth stops. The derivatives of podophyllotoxin are topoisomerase II inhibitors arresting cells in the late S or early G2 phase of the cell cycle.

PROBLEMS ASSOCIATED WITH CONVENTIONAL PROPAGATION - - P. hexandrum is an endangered species in India. - P. peltatums podophyllotoxin content in the rhizome is much smaller than that in P. hexandrum. - 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.

USE OF IN-VITRO TECHNIQUESCell cultures of P. peltatum for production of podophyllotoxin was first attempted by Kadkade et al.

To increase the yield of podophyllotoxin, Woerdenberg et al. used a complex of a precursor, coniferyl alcohol and b-cyclodextrin to P. hexandrum cell suspension cultures. The addition of 3 mM coniferyl alcohol complex yielded 0.013% podophyllotoxin on a dry weight basis, but the cultures without the precursor produced only 0.0035%. Van Uden et al. initiated podophyllotoxin producing callus cultures from in vitro plantlets of the Indian Podophyllum on B5 and MS media supplemented with growth regulators, showing dark-grown cultures accumulated upto 0.3% podophyllotoxin (dry weight basis).

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

Suspension cultures from other species tried: - Callitris drummondii - Linum album - L. nodiflorum - L. mucronatum spp. armenum

Anbazhagan et al. established embryogenic cell and adventitious root culture systems in P. peltatum and revealed that adventitious roots contained higher podophyllotoxin than embryogenic cell clumps through HPLC.

Establishment of l. album hairy root and cell cultures

taxol / PACLITAXELTaxol is a complex diterpene alkaloid found in the bark of the Pacific yew (Taxus brevifolia Nutt.)

Paclitaxel has been isolated from various parts of 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.

It is an antineoplastic agent, being widely used majorly for the treatment of advanced, progressive and drug refractory ovarian cancer and breast cancer along with the derivative taxases.

It is also effective against noncancerous conditions like polycystic kidney diseases and has shown promising results in multiple sclerosis and AIDS related Karposis sarcoma.

MODE OF ACTION Taxol promotes the polymerization of microtubules but inhibits depolymerisation. This unusual stability blocks the cells ability to disassemble the mitotic spindle during cell division; blocking it in the G2/M phase and eventually leading to cell death.

PROBLEMS ASSOCIATED WITH CONVENTIONAL PROPAGATION - T. Brevifolia is slow growing and is rarely found. - The quantity of taxol obtained is in trace amounts (0.01% of dry weight of the bark).

USE OF IN-VITRO TECHNIQUESChristen et al. reported the production of taxol by Taxus cell cultures.

Fett-Neto et al. studied the effect of nutrients and other factors on paclitaxel production by T. cuspidata cell cultures (0.02% yield on dry weight basis).

Paclitaxel production by T. baccata cell suspension cultures was studied (1.5 mg/l).

Maximum taxol production in T. cuspidata cultures obtained by adding Phenylalanine.

Propagation of taxus seeds by embryo culture reported for T. brevifolia and other species.

Ketchum and Gibson showed addition of carbohydrate during the growth cycle increased the production rate of paclitaxel in suspension cultures (14.5 mg/l).

The production can be enhanced 1719 fold by methyl jasmonate and various other biotic and abiotic stress factors up to 35mg/l.

STRATEGIES FOR IMPROVING THE IN VITRO PRODUCTIONOptimization of culture conditions - Quantity and intensity of light affects Podophyllotoxin accumulation. - Low oxygen content, temperature between 24-29 C and high osmotic pressure enhance taxol production.Optimization of culture mediumCell cloning and selection Cross species culture - Coculture of Linum flavum hairy roots and P. hexandrum cell suspension increased Podophyllotoxin accumulation. - Cell suspensions of Taxus chinensis var. mairei cocultured with its endophytic fungi, Fusarium mairei, showed 38-fold higher than that by uncoupled culture.Elicitation - Salicyclic acid used as elicitor for podophyllotoxin. - Methyl jasmonate used for eliciting taxol production.

Large scale production - Commercial production of podophllotoxin is yet to be achieved. - Batch cultivation of T. baccata var. fastigata and T. wallichiana suspension cultures has been carried out in 20 litre airlift bioreactor. - 100-500 litre BTBBs have also been used for culture of T. cuspidata. After a period of 27 days the yield of paclitaxel and total taxanes was 3mg l-1 and 74mg l-1 respectively. - Reactors with stirrers having low shear forces of 70,000 litre volume are used of Taxol production (Phyton Biotech, Germany).

REFRENCESSustainable bioproduction of phytochemicals by plant in vitro cultures: anticancer agents, Wink et al, 2009Studies on the production of some important secondary metabolites from medicinal plants by plant tissue cultures, Vanisree et al, Bot. bull Acad. Sin. 2004.Biotechnological approaches for the production of potential anticancer leads Podophyllotoxin and Paclitaxel: An overview, Anrini Majumder, Sumita Jha, eJournal of Biological Sciences, 2009.A review on trends in production of secondary metabolites from higher plants by in vitro tissue, organ and cell cultures, S. Karuppusamy, Journal of Medicinal Plants Research, 2009.