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An Alternative Approach for Anticancer Compounds Production Through Plant Tissue Culture Techniques

  • Hari Gajula
  • Kumar Vadlapudi
  • Poornima D. Vijendra
  • J. Rajashekar
  • Torankumar Sannabommaji
  • Giridhara Basappa
  • T. U. Santhosh
Chapter

Abstract

Higher plants produce various anticancer secondary metabolites (colchicine, camptothecin, combretastatin, paclitaxel, plumbagin, podophyllotoxin, psoralen, vincristine, vinblastine, etc.). The indiscriminate harvesting of these plants from the wild for the metabolites and inadequate efforts for cultivation led to a decrease in natural populations. However, these metabolites/compounds exist in low quantities, and it is economically not feasible to obtain them in large scale. Moreover, the accumulation of these metabolites varies from its geographical and environmental conditions. Alternatively, economically feasible production strategies should be investigated in order to overcome these problems and to overproduce the metabolites of therapeutic importance. To this perspective, advances in plant cell and tissue cultures, mainly culturing of cells/tissues, suspension cultures, precursor feeding, hairy root cultures, and bioreactors using cell suspensions/hairy root cultures are evaluated as the feasible and cost-effective alternative means for the production of economically important compounds. The present chapter summarizes the latest techniques/strategies for the overproduction of anticancer metabolites using plant cell and tissue culture approaches.

Keywords

Anticancer compounds Bioreactors Cambial meristematic cells Hairy root cultures Plant tissue cultures 

22.1 Introduction

Cancer is one of the leading causes of death in the world. It has been reported by the World Health Organization (WHO) that about 8.8 million people died worldwide in 2015 due to cancer, nearly 1 in 6 of all the deaths globally. In 2016, unfortunately 595,690 people died due to cancer out of 1,685,210 new cases of cancer diagnosed. Currently, there are many types of treatments available for cancer. However, a patient receives the treatment based on the cancer type and how advanced it is. The available treatments include chemotherapy, radiation therapy, immunotherapy, surgery, etc. However, these treatments are not free from side effects. For example, radiation therapy affects healthy cells also along with cancer cells, while plant-based drugs are successfully employed for the treatment of cancer with lesser side effects (Somasundaram et al. 2010). In 1960s vinblastine and vincristine are the two natural anticancer compounds introduced into the market to treat Hodgkin’s lymphoma and acute childhood leukemia, respectively. Likewise another plant-based anticancer compound paclitaxel is used to treat breast cancer and ovarian cancer, while camptothecin is used to treat gastric, rectal, colon, and bladder cancers. Another important natural compound, podophyllotoxin confers a great efficacy against lymphosarcoma and Hodgkin’s disease (Nissen et al. 1972). Discovery and introduction of these compounds to the market supported further drug discovery programs utilizing natural resources or products. However, the cost of these compounds in the market appears to be very high. For example, vincristine 1 kg costs around 20,000 US$, and annual world market cost for this compound is about 5 million US$. Similarly, the demand and cost of camptothecin and podophyllotoxin (etoposide, a semisynthetic derivative of podophyllotoxin) seem to be in the range of vincristine (Khani et al. 2012; Atanas et al. 2015; Shiv et al. 2016).

From the past decades, mankind depends on plants as a source of pharmaceuticals to treat various cancers. Plant-based drugs (natural products) play a significant role in the treatment of cancer. However, these compounds are produced in plants at very minute quantities, and hence, there is no adequate supply of these compounds owing to increased number of cancer incidences in recent years. Thus, there is a high demand for these plant-based anticancer compounds globally. Biotechnological approaches using plant cell cultures are the attractive and alternative means for the product enhancement of plant-derived anticancer compounds (Ramachandra Rao and Ravishankar 2002). These approaches offer a defined production system, which ensures the production of novel compounds and stable supply of products without any alteration in the quality and yield. To overcome this supply crisis, many scientists have adopted various plant biotechnology protocols/techniques for the enhanced production of anticancer compounds (Table 22.1). In fact, there have been many plant biotechnology techniques used for mass production of anticancer compounds (Fig. 22.1). Presently, the demand for plant-derived anticancer compounds is steadily increasing, but their production in parent plants in the wild is at below the required level (Fig. 22.2). Therefore, plant cell and tissue cultures will be a promising, cost-effective, and viable alternative for the increased production of anticancer compounds in a commercial level. The present chapter summarizes the latest techniques/strategies for the overproduction of anticancer metabolites using plant cell and tissue culture approaches.
Fig. 22.1

Strategies to increase anticancer compounds through plant cell cultures

Table 22.1

Summary of various techniques used to enhance the production of anticancer compounds

Plant name

Anticancer compound

Culture type

Yield

References

Taxus cuspidata

Taxol

Cell suspension cultures

12.2 mg/l

Mirjalili and Linden (1995)

T. baccata

Paclitaxel

Bioreactor cultures

1.5 mg/l

Srinivasan et al. (1995)

T. media

Paclitaxel; baccatin III

5 l Stirred reactor

21.1 mg/l

Cusido et al. (2002)

56.0 mg/l

Camptotheca acuminata

Camptothecin

Hairy root culture

1 mg/g DW

Lorence et al. (2004)

Taxus media

Paclitaxel

Cell suspension cultures

1.25 mg/g DW

Baebler et al. (2005)

T. baccata

Paclitaxel

Airlift bioreactor (4 l);

12.03–20.79 mg/l;

Bentebibel et al. (2005)

Baccatin III

Wave bioreactor (2 l);

Stirred bioreactor (5 l)

7.78 mg/l;

5.06 mg/l

T. baccata

Taxol

Precursor feeding

13.75 mg/l

Khosroushahi et al. (2006)

P. peltatum

Podophyllotoxin

Adventitious roots

0.588 mg/g DW

Anbazhagan et al. (2008)

O. prostrata

Camptothecin

Adventitious root cultures

0.19%

Martin et al. (2008)

Liunm mucronatum

Podophyllotoxin

Hairy roots

5.78 mg/g DW

Samadi et al. (2014)

O. mungos

Camptothecin

In vitro regeneration

0.0768%

Namdeo et al. (2012)

Psoralea corylifolia

Psoralen

Precursor feeding

2.5 mg/g FW

Mohammad Parast et al. (2014)

Plumbago rosea

Plumbagin

Embryogenic cell suspension cultures

1 mg/g DW

Silja et al. (2014)

Plumbago rosea

Plumbagin

Adventitious root cultures

1.23%

Silja and Satheeshkumar (2015)

Catharanthus roseus

Vinblastine and vincristine

Callus

0.5623 and 0.1651 μg/g DW

Iskandar and Iriawati (2016)

Ophiorrhiza mungos

Camptothecin

Cell suspension cultures

0.8 mg/g DW

Deepthi and Satheeshkumar (2016)

22.2 Approaches to Overproduce Anticancer Compounds Production Using Plant Cell Culture

22.2.1 Highly Productive Cell Lines Selection and Screening

Plant cells in vitro culture systems exhibit genetic variation and heterogeneity in which the physiological and expression profiles of the respective natural products can be different. Hence, highly productive cell line selection for the establishment of stable and prolific plant metabolites can be imperative. Detection of highly productive cell lines is generally done through visual selection, mutant selection, and clonal selection (Remotti et al. 1997; Shiba and Mii 2005; Liang et al. 2006). These are the techniques for the detection of highly productive cell lines. The accumulation patterns of natural products in the similar plant can be genotype specific, and hence, the selection of a high-yielding clone appears to be critical. Berlin and Sasse (1985) had surveyed various techniques utilized for describing the variants recovered from cell and organ cultures. There would be a possibility of decreased or complete loss of the secondary metabolite-producing ability during repeated subcultures. This might appear because of the genetic instability associated with the somaclonal variation. Deepthi and Satheeshkumar (2016) had isolated high-yielding cells through an aggregate cloning method in cell cultures of Ophiorrhiza mungos L. for the enhancement of camptothecin. Formation of a new cell wall further substantiates the consideration of the produced clones as true colonies (Dougall 1987).

22.2.2 Nutrient Manipulation to Increase Yield of Anticancer Compounds

For the production enhament of secondary compounds in cell and tissue cultures, many approaches are in use, including manipulating the composition of media (Karwasara and Dixit 2011), augmentation of plant growth regulators (Karwasara et al. 2011), and slight changes in the concentrations of carbon source, nitrogen source, nitrate-to-ammonia ratio, phosphate, and micronutrients. These manipulations bring out tremendous results with respect to cell growth and accumulation of desired phytochemical content. Further, developing favorable culture conditions is of prime importance for the growth and biosynthetic efficiency of the in vitro cultured cells. Basic knowledge about a particular secondary metabolite biosynthesis and regulation that influence the productivity of cultured cells is required to standardize culture conditions for optimizing the production. This involves standardization of plant growth regulator(s) concentration and composition and also composition of culture medium.

22.2.2.1 Carbon Source

Sugars such as glucose, fructose, and maltose are tried in plant tissue culture media to induce cell growth as well as secondary metabolite production. Among these, sucrose is considered as the efficient source of carbon for inducing cell proliferation, as the disaccharide sucrose is a balanced carbon source. Sucrose supplemented in the medium not only supports for cell proliferation and growth but also influences the biosynthesis and accumulation of plant metabolites in culture (Wang and Weathers 2007). For the production of camptothecin in suspension culture of Nothapodytes nimmoniana, 5% (w/v) sucrose was found to be optimal at the tested concentration range of 2–7% (w/v) (Karwasara and Dixit 2013). Similarly, Rajesh et al. (2014) evaluated the effect of different carbon sources on podophyllotoxin production in adventitious root culture of Podophyllum hexandrum. Among the carbon sources tested, 6% sucrose resulted in a maximum accumulation of podophyllotoxin (4.8 mg/g DW). Supplementation of sucrose 20 g/l to cultures at a later stage improves the taxane production along with cell growth and concluded that an addition of 30 g/l sucrose at the initial stage of culture improved the cell growth and even the cell growth reached to stationary phase and significantly increased the taxane production (Wang et al. 1999). Addition of 30 g/l sucrose at day 20, 36 mg/l taxol is produced in a two-stage culture of T. chinensis (Wang et al. 2001).

22.2.2.2 Nitrogen Source and Nitrate-to-Ammonia Ratio

Nitrogen has a greater influence on the metabolism of the plant cell and greatly contributes for the growth and development of a plant (Chen et al. 2003). It is useful in the biosynthesis of nitrogen-containing substances such as amino acids, proteins, nucleic acids, and other nonprotein nitrogen metabolites. There are different types of media that contain nitrogen source in the form of nitrate and ammonium. The ratio of nitrate to ammonia has a significant influence on the cell proliferation, growth and development, biosynthesis, and product accumulation of metabolites. High nitrate (NO3) and low ammonium (NH4) supplemented in the medium at a ratio of 10:20 resulted in maximum podophyllotoxin (5.8 mg/g DW) accumulation in adventitious root culture of P. hexandrum and induced maximum adventitious roots (24.3/explant) and biomass accumulation (5.1 g FW) after 2 months of culture (Rajesh et al. 2014). Whereas, in cell suspension cultures of N. nimmoniana, increasing the concentration of nitrate and ammonium (NO3/NH4+) at a ratio of 1:5 (with 60 mM total nitrogen) promoted camptothecin production (48.7 μg/g DW), while increased nitrate favored cell growth only and increased ammonium favored camptothecin biosynthesis (Karwasara and Dixit 2013). In the root cultures of O. mungos Linn., it was reported that low NO3/NH4+ ratio (10:50 mM) is required for maximum camptothecin production, while for biomass production high ratio of NO3/NH4+(40:20 mM) is required (Deepthi and Satheeshkumar 2017). This shows that nitrate-to-ammonia ratio in the medium plays an important role in metabolite accumulation and cell growth. So in order to attain product enhancement of anticancer compounds in plant cell and tissue cultures, standardization of nitrate-to-ammonia ratio is one of the prerequisites.

22.2.2.3 Phosphate

Phosphate is one of essential inorganic nutrients in plant cell culture medium for the growth and production of metabolites. For example, lower phosphate concentration in the medium not only decreased the cell growth but also camptothecin accumulation (Deepthi and Satheeshkumar 2017). A similar effect of low phosphate in the medium for adventitious root culture of Podophyllum hexandrum was also observed for the production of podophyllotoxin (Rajesh et al. 2014). Phosphate (KH2PO4) supplemented at 1.25 mM concentration induced 27.1 adventitious roots per explant with biomass 5.7 g FW and 1.42 g DW. Whereas, increased phosphate concentration (0.5 mM) impeded the biomass accumulation and camptothecin production in N. nimmoniana cell cultures (Karwasara and Dixit 2013).

22.2.2.4 Plant Growth Regulators

Providing stress to the culture media in the form of plant growth regulator concentration and combination shows positive influence on the accumulation of plant metabolites. Generally plant growth regulators influence the callus formation, modulation of callus initiation, dedifferentiation of cells, cell division, and secondary metabolite production. It has been reported that one of the plant growth regulators 2,4-D supplemented in the medium reduces the secondary metabolite content; hence it is supplemented at very low concentrations for secondary metabolite production. However replacing 2,4-D with NAA showed better results on secondary metabolite accumulation. For example, cultures of Nothapodytes foetida in presence of 2,4-D resulted in low levels of camptothecin accumulation, while in the presence of NAA increased the amounts of camptothecin and its accumulation (Fulzele et al. 2001). NAA at higher concentrations in O. rugosa var. decumbens cultures increased the camptothecin production, while the combination of NAA with 6-BAP induced more camptothecin production rather than when they have supplemented alone (Vineesh et al. 2007). Increasing the BAP concentration to NAA inhibited the camptothecin production in N. nimmoniana callus cultures. However other auxin IBA had shown the positive influence on camptothecin accumulation at higher concentrations (Isah and Mujib 2015). The superiority of NAA over 2,4-D on plumbagin production was also reported in Plumbago indica hairy roots (Gangopadhyay et al. 2011). In contrast to this, plumbagin production was affected in the presence of NAA in Drosophyllum lusitanicum (Nahalka et al. 1996). The negative influence of NAA on camptothecin production in Catharanthus roseus cell cultures was also reported by Whitmer et al. (1998). High cytokinin (BAP) presence in the medium decreased the plumbagin content, while at lower concentrations the same cytokinin (BAP) showed better results for the plumbagin production. Abscisic acid supplemented in the medium induced about five times more paclitaxel production in cell suspension cultures of T. chinensis compared to control cultures with no abscisic acid (Luo et al. 2001). Plant growth regulators show significant effect on plant metabolite production in a concentration-dependent manner. Sometimes higher concentrations of NAA gives better results for secondary metabolite formation in comparison to 2,4-D. In conclusion, for the production/enhancement of plant metabolites (anticancer compounds), plant growth regulator(s) concentration and combination is one of the critical factors that plays a major role in cell cultures. So, the ideal concentration and combination of plant growth regulators has to be standardized in order to increase the production of anticancer compounds in plant cell and tissue cultures.

22.2.2.5 Precursor Feeding

Another important approach for the product enhancement of anticancer metabolites is the addition of intermediate metabolites (precursors) of the respective biosynthetic pathway to the cell cultures. A precursor is generally an intermediate that is enzymatically converted into another compound or desired metabolite. Therefore the addition of these intermediates (precursors) to the cell cultures might result in increasing of the desired metabolite. Exogenous supplementation of precursors to the medium induces a subset of genes associated with secondary metabolite biosynthesis, modulates their expression, and promotes the accumulation of desired metabolites. Addition of phenylalanine to the medium resulted in a 5.6-fold increase in taxol (13.75 mg/l) production compared to the respective control cultures (Khosroushahi et al. 2006). Similarly, in Taxus baccata cell cultures, taxol content was increased from 5.2 to 13.1 μg/g (DW) with the addition of 1 mM phenylalanine (Cusido et al. 1999). Similarly addition of phenylalanine in cell cultures of Linum flavum resulted in fivefold increase in 6-methoxypodophyllotoxin (Uden van et al. 1990). Coniferyl alcohol supplemented in combination with cyclodextrin resulted in fourfold increase in podophyllotoxin in P. hexandrum cell cultures (Woerdenbag et al. 1990). With the addition of 2.5 mg/l cinnamic acid, psoralen content was increased from 1.93 to 2.50 mg/g FW in callus cultures of Psoralea corylifolia (Mohammad Parast et al. 2014).

22.2.3 Optimizing the Culture Environment

It is known that basal medium composition, pH, temperature, and light have a major influence on cell growth and secondary metabolite accumulation in plant cell tissue cultures. Therefore, optimizing the culture environment will certainly enhance the biomass and metabolite accumulation in plant cell cultures.
Fig. 22.2

Some important plant-derived anticancer compound structures

22.2.3.1 pH

One of the crucial factors for anticancer compounds production is pH of the culture medium. Changes/manipulation of pH determines the release of intracellular alkaloids into the culture medium (Asada and Shuler 1989). Alkaloids are stored in the vacuole when synthesized in cell suspension cultures. It has been showed that culture medium pH and intravacuolar pH determine the storage capacity of alkaloids (Neumann et al. 1983). In general cell culture medium pH is adjusted in between 5 and 6 units; however, during sterilization by autoclaving medium, pH drops by 0.6–1.3 units. For the production of podophyllotoxin and cell growth in P. hexandrum cell culture, the optimal pH is 6.0 (Chattopadhyay et al. 2002b). Similarly, Rajesh et al. (2014) also reported that pH in the range of 5–6 favored the production of podophyllotoxin and biomass accumulation in adventitious root culture of P. hexandrum.

22.2.3.2 Temperature

Temperature is another important factor that influences the production of plant metabolites. Generally plant cell cultures are maintained at a temperature of 25 ± 2 °C. However, increasing the temperature to 29 °C favored the paclitaxel production (Choi et al. 2000). Interestingly temperature maintained at 24 °C favored the biomass accumulation in suspension culture of Tsuga chinensis but at 29 °C inhibited cell growth. Therefore, to reduce the negative influence of temperatures on cell growth initially, the temperature was maintained at 24 °C, and later cultures were shifted to 29 °C. Maintaining these temperatures, paclitaxel production was optimized for a maximum yield of 137.5 mg/l and an average production of 3.27 mg/l over a period of 42 days.

22.2.3.3 Light

Light intensity and regime is another important factor that influences the production of biomass and metabolites in culture. In T. cuspidate cell cultures, white light strongly inhibited the production of taxol and baccatin III. While in dark-grown callus and suspension cultures, three times higher yield of these metabolites was observed compared to cultures maintained in the presence of light (Fett-Neto et al. 1995). Similarly, podophyllotoxin accumulation favored dark conditions in cultures of P. hexandrum and P. peltatum (Kadkade 1982; Chattopadhyay et al. 2002a; Anbazhagan et al. 2008). Likewise, camptothecin concentrations in Camptotheca acuminata leaves significantly increased under blue light and 50% shading treatments (Liu et al. 2015; Hu et al. 2016).

22.2.3.4 Aeration and Agitation

Aeration and agitation of cultures appear to play a crucial role in the production of anticancer compounds. Increasing either aeration rate or negligible gas transfer has a negative influence on growth and anticancer compounds production. So, it is essential to evaluate the adequate amount of oxygen requirement of the cell cultures (Zhong et al. 1992). Gases such as oxygen (O2), carbon dioxide (CO2), and ethylene are also important in plant cell cultures (Lee et al. 2010). Respiration and metabolism are carried out by O2; the main metabolic component in cell growth is CO2, while ethylene is believed to be a gas component produced by the plant in response to environmental stress (Khani et al. 2012). The importance of nutrients, inoculum density, and oxygen supplementation for the production of paclitaxel in Linum album cell cultures has been suggested by Garden (2004). Linum album cell cultures maintained in a bioreactor (stirred tank with a capacity of 5 l) by optimizing aeration conditions, 183.6 mg/l podophyllotoxin yield was achieved (Baldi et al. 2008). Likewise, Luo et al. (2001) reported that 40–60% dissolved oxygen allowed a 7.2 mg/l paclitaxel production in a bioreactor with a six-flat-bladed turbine in cell suspension cultures of T. chinensis. Increased CO2 (10%) levels inhibited the production of paclitaxel while at low headspace oxygen concentration promoted the paclitaxel in T. cuspidate cell cultures (Mirjalili and Linden 1995). An optimized combination of these three gases for the production of paclitaxel is 5 ppm ethylene, 10% (v/v) O2, and 0.5% (v/v) CO2 as evaluated and suggested by Mirjalili and Linden (1995). Camptothecin production was achieved in cultures of O. mungos Linn. maintained at 90 rpm continuous agitation (Deepthi and Satheeshkumar 2016).

22.2.4 Elicitation

In nature, plants create a vast defense system by producing secondary metabolites against various environmental stresses (pathogen attack). Similarly, in in vitro conditions, plant cells and tissues respond to trigger the synthesis of secondary metabolites when challenged with similar elicitors from pathogens or of synthetic origin (Giri and Zaheer 2016). Elicitors are the signaling molecules; depending on their molecular origin, they may be either biotic or abiotic and physical or chemical factors (Angelova et al. 2006). At low concentrations, elicitors evoke the production of enhanced levels of commercially important metabolites. Elicitation of important natural products through cell cultures leads to not only marking down the process time but also enhance the desired product yield. From past decades, many authors have reported the effect of various elicitors such as oligosaccharides, biotic or abiotic, glycoproteins, yeast, and plant signaling molecules on plant metabolite production. For example, 13.3-fold increase in camptothecin was observed in O. mungos Linn. cell cultures elicited with yeast when compared to the control cultures (Deepthi and Satheeshkumar 2016). Likewise the treatment of C. acuminata cell suspension cultures with sorbitol resulted in increased production of camptothecin (5 mg/l) (Yang et al. 2017). About 1.130 mg/g DW plumbagin was produced in Plumbago rosea L. cultures after challenging with NAA (Silja et al. 2014) and in adventitious root cultures established from leaf explants of P. rosea (Silja and Satheeshkumar 2015). Taxol is a plant diterpenoid, a well-known anticancer compound as it is a unique poison of microtubular cell system (Woo et al. 1994). To avoid its supply crisis, many people have focused toward elicitation strategies for the synthesis of taxol and taxol-like derivatives. Methyl jasmonate, a plant stress signaling molecule synthesized through octadecanoid pathway, tangled in the production of anticancer plant metabolites through plant cell and tissue cultures. Suspension cultures of T. chinensis var. mairei elicited with 100 μM methyl jasmonate accumulated more amounts of taxol (Wang et al. 2004). Similarly, paclitaxel was overproduced from 0.4 to 48 mg/l with 100 μM methyl jasmonate (Yukimune et al. 2000). Taxol production through methyl jasmonate is well known, but the mechanism of how methyl jasmonate is involved in the synthesis of taxol is not clearly understood. Even though different elicitors induce different types of anticancer compounds with different amounts, one has to consider the concentration of a suitable elicitor, time of addition and interaction of the elicitor in the cultures, and age of the cultures (Namdeo 2007). These parameters play a significant role in the production of anticancer compounds in plant cell cultures using elicitors.

22.2.5 Permeabilization

The metabolites produced by the cell culture systems are stored in the vacuoles of the cells. Therefore for the ease of recovery and purification, it is essential to release the produced metabolite into the culture medium (Cai et al. 2012). This product removal further facilitates the continuous production of the metabolite by relieving the feedback inhibition, and thus improvement in the consistent production can be expected. Till date there have been numerous attempts for the permeabilization of plant cells in cultures for the release of metabolites (Trejo-Tapia et al. 2007; Siu and Wu 2014). To achieve the desired results there are different strategies followed, like change of culture medium pH, transferring culture to medium that contains no phosphate, electroporation and using the permeabilizing agents like chitosan and dimethyl sulfoxide that affect cell membrane permeability. Using these strategies the metabolites produced by the cells in cultures are made to get released into the culture medium. Moreover, definite concentration and time period of addition of permeabilizing agents to the media are the few factors to be considered so as to avoid the inhibition of cell growth. Utilization of 10% dibutyl phthalate as a permeabilizing agent was found superior for the release of taxol into the medium compared to hexadecane and decanol. While combining the selective permeabilizing agent with sucrose, feeding selectively increased the taxol production to sixfold higher (Wang et al. 2001).

22.2.6 In Situ Adsorption and Two-Phase Culture System

In general, a decrease in product formation is observed in the intact cell cultures. Further, storage of the produced metabolite at internal sites such as vacuoles also makes the situation very critical for the extraction and becomes economically not feasible. In addition, the following factors such as higher volatile nature of the metabolites produced, feedback inhibition, and product degradation because of lower stability also limit the maximum accumulation of plant metabolites in cell cultures (Berger 1995). A concomitant strategy is adopted for the product removal continuously either through solvent extraction or using an adsorbent makes it feasible for harvesting or isolating the desired metabolites from the culture containers. This strategy not only decreases the intrusion between growth of the cell and product accumulation but also reduces the product degradation from the producing cell lines due to the release of enzymes, and environmental conditions, and finally minimizes the downstream processing events (Freeman et al. 1993; Malik et al. 2013). Harmonious effect of in situ product removal with other general methods of enhanced metabolite production such as immobilization, elicitation, precursor feeding, and permeabilization can further potentiate the product enhancement of desired metabolites through cell and tissue cultures of plants.

Through in situ adsorption using styrene-divinylbenzene resin (Diaion® HP-20), plumbagin production has been enhanced by precursor feeding in root cultures and obtained 1.4- and 1.6-fold, respectively, higher than L-alanine alone-fed cultures or untreated control root cultures (Jaisi and Panichayupakaranant 2017). Taxol production in T. cuspidata can be enhanced from 40 to 70% by utilizing a nonionic adsorbent, Amberlite® XAD-4 (Kwon et al. 1998). Likewise, Pavao et al. (1996) had established two-phase culture systems in T. brevifolia for partitioning taxol released in the second phase. Tricaprylin, an eight-carbon triglyceride, partitioned the secreted taxol into the culture medium.

22.3 Organ Cultures

Product enhancement through differentiated cultures has an advantage over undifferentiated cells. It has been said that during plant development presence of some of the desired metabolites is confined to a particular tissue or organ. Production of anticancer compounds through in vitro regenerated plants has tremendous importance because of high production in differentiated tissues and more stability in organ cultures (Roja 1994). A study by Sankar-Thomas and Lieberei (2011) suggested the low profile of camptothecin levels in undifferentiated calli compared to differentiated tissues of C. acuminata organ cultures. Thus biosynthesis of camptothecin requires tissues or organs of differentiated stages. The study supported the use and advantages of organ cultures over undifferentiated cultures. Similarly, higher levels of camptothecin (66–111%) accumulated in the regenerated O. pumila plants as compared with that in the wild plant. Likewise, 15 μg/g tissue dry weight vinblastine was produced in multiple shoots of C. roseus (Miura et al. 1988). Two-month-old plantlets produced 0.11%–0.36% taxol in T. canadensis and 0.01%–0.1% in T. baccata (Zhiri et al. 1994), while T. baccata shoot tip cultures produced taxol from 0.03 to 0.46 mg/kg (Jaziri et al. 1990). In addition to shoot cultures, root cultures are also promising to produce plant metabolites. The highest amount of camptothecin was obtained from the roots of early-stage seedlings of C. acuminata Decaisne (Nyssaceae) and also reported the decrease in camptothecin content in aged seedlings (Valletta et al. 2007). Similarly, Kaushik et al. (2015) evaluated the camptothecin yield in micropropagated plants of O. mungos and found that tissue culture-raised clones had 0.0438 ± 0.18% of camptothecin content, while the mother plant contained 0.043 ± 0.16%. This showed a similar chemical profile between wild-grown and micropropagated plants. More recently, maximum camptothecin content (0.12% w/w) was found to occur in the root samples of in vitro regenerated N. nimmoniana. Further, it has been reported that camptothecin content varied in plant parts (leaves, stems, and roots). The amount of camptothecin in leaves and stem was reported to be 0.0013% w/w and 0.026% w/w, respectively (Prakash et al. 2016). Moreover, the compounds produced through root cultures are very difficult to harvest and show slower growth. Therefore, an alternative method is required to produce root-derived compounds, and as of now, hairy root cultures are the best system to produce root-derived compounds which are described in the following sections.

22.4 Adventitious Root Cultures

Adventitious root system is an alternative to the conventional tissue culture methods for studying the biochemical events occurring during the secondary metabolite production and also to understand the effect of various factors on the normal metabolic pathways of the culture system. They can be produced under natural or various stress conditions and also induced by mechanical damage or tissue regeneration. Hence, it can be considered as more natural for not containing any chimeric DNA. Adventitious roots can be produced using any plant part (node, stem, leaf, non-pericycle tissues, or any other organ) as an explant. Adventitious root cultures have more genetic stability than any other culture systems (Casson and Lindsey 2003). They can also be used as a source of food storage organs and site of synthesis and accumulation of produced natural products. Adventitious roots exhibit higher multiplication and biomass accumulation and show an elevated secondary metabolite biosynthesis (Murthy et al. 2014). Product enhancement of desired metabolites depends on changes in the culture conditions by using different elicitors and growth promoters, precursors. etc. The metabolites thus produced by adventitious cultures can be cost-effective and used as antioxidants, food ingredients, pharmaceuticals, and therapeutic agents. An efficient culture system for the product enhancement in Andrographis paniculata adventitious roots has been evaluated for the andrographolide production (Praveen et al. 2009). Compared to callus suspension cultures, adventitious root cultures showed an increase in andrographolide amount. A cytotoxic quinoline alkaloid, camptothecin, and its derivatives were used for the treatment of cancer. The anticancer property of camptothecin is exhibited by its reversible binding to the topoisomerase-1 enzyme and inhibits the DNA replication by stabilizing the enzyme/DNA complex (Deepthi and Satheeshkumar 2016). Martin et al. (2008) successfully established the adventitious root culture system which has been evaluated as a promising approach for the production of camptothecin in O. prostrata. In root cultures of O. mungos, 3.4-fold higher amount of camptothecin has been produced by changing the medium composition and plant growth regulators (Deepthi and Satheeshkumar 2016). Differentiated cultures like hairy roots and adventitious roots of O. pumila produce higher amounts of camptothecin (Saito et al. 2001).

22.5 Hairy Root Cultures

Camptothecin is a monoterpenoid indole alkaloid produced in the plants which belong to Rubiaceae, Apocynaceae, Nyssaceae, and Loganiaceae (Cui et al. 2015). In a study reported by Ochoa-Villarreal et al. (2016), hairy root cultures were established infecting plant explants with A. rhizogenes, a Gram-negative soil bacterium that transfers its plasmid DNA that carries the genes which encode enzymes responsible for auxin biosynthesis which induce hairy roots. Thus accumulated auxin at the site of A. rhizogenes infection influences the surrounding cells mitotically to produce hairy roots. The differentiated hairy roots exhibit high growth rate, independent of growth hormones, high genetic stability, lateral branching, and lack of geotropism. These attributes make the hairy roots to accumulate the desired anticancer plant metabolites.

An efficient hairy root culture was established for the enhancement of camptothecin in O. pumila (Saito et al. 2001). Significant improvements in the camptothecin production were achieved by releasing into the adsorption medium using Diaion HP-20 (a polystyrene resin). Nearly a twofold improvement in plumbagin was observed with combined elicitation of methyl jasmonate and chitosan in Plumbago indica hairy root cultures (Gangopadhyay et al. 2011). Likewise there are Ophiorrhiza pumila, N. foetida, C. acuminata, O. liukiuensis, and O. kuroiwai (Lorence et al. 2004) are some of the few potential plant species which can be explored with genetic transformation to improve camptothecin production in the future. From these observations, it could be speculated that hairy root culture could be a sustainable strategy for the product enhancement of anticancer compounds and also useful for the understanding and elucidation of metabolic pathway and factors responsible for the metabolite synthesis.

22.6 Immobilizations

Slow growth rate and limited metabolite production can further offer the utilization of an alternative system for the product enhancement of anticancer compounds in vitro. In this context, immobilized plant cell and tissue cultures offer the continuous production of the desired metabolites. Immobilization is advantageous in the following contexts: (a) improved viability of cells in stationary phase, facilitating ease of maintenance and recovery, (b) simplified downstream processing (in case of secretary products), (c) cost-effective and lesser chance of contamination obtained through utilization of high cell density in small-scale bioreactors, (d) low shear stress for shaking cultures, (e) increased product accumulation, (f) improves flow rates by utilizing flow through reactors, and (g) knocks down the problems associated with mixing and aeration such as fluid viscosity in suspension cultures (Dicosmo and Misawa 1995). Matrices like agar, agarose, carrageen, polyurethane foam, alginate, and vanadium sulfate are used for the immobilization of plant cells and tissues and release of produced metabolites (anticancer compounds) into the medium (van der Heijden et al. 1989). It has been suggested that the adsorption method of immobilization is superior over cell entrapment (Dicosmo and Misawa 1995). The immobilization process ensures the sustenance of cells for a longer period of time and reduces the cost incurred on production of desired anticancer compounds (Moreno et al. 1995).

The surface immobilization of cultured cells of Catharanthus roseus, Nicotiana tabacum, and Glycine max has been achieved for the production of useful metabolites. Komaraiah et al. (2003) produced the plumbagin 21 times higher than control cultures by using immobilization followed by elicitation with chitosan. Increased production of baccatin and paclitaxel was reported changing alginate concentration (Bentebibel et al. 2005). Taxane production was improved with 2% calcium alginate in shake flasks and in bioreactor cultures (Bentebibel et al. 2005). Release of taxol and baccatin III from the plant cells into the culture medium was enhanced by 2.5- and 3.6-fold, respectively, using vanadium sulfate (Cusido et al. 1999).

22.7 Bioreactor and Scale-Up Process

Bioreactors are devices or systems that are used to carry out biological processes to produce the desired products. Sudo et al. (2002) have produced camptothecin in cultures of O. pumila in a 3 l bioreactor. A total yield of 22 mg was obtained after 8 weeks of culture period. An approximate amount of total camptothecin released into the culture medium was found to be 17% (3.6 mg). Additionally, for the easy purification, the excreted camptothecin could be adsorbed with polystyrene resin Diaion HP-20. Taxol production by cell suspensions of T. baccata in bioreactors was reported by Srinivasan et al. (1995). Paclitaxel and baccatin III were enhanced in different types of bioreactors by combining with an immobilization event (Bentebibel et al. 2005). Among them, stirred bioreactor potentially induced 43.43 mg/l paclitaxel at 16 days of culture period which is equal to 2.71 mg/l per day. Likewise, Navia-Osorio et al. (2002) established an efficient culture system for the production of taxol and baccatin III in T. baccata var. fastigiata and T. wallichiana suspension cultures using 20 l airlift bioreactor. When both the cell line at their maximum growth stage of accumulating the metabolites (28 days) T. wallichiana (21.04 mg/l) was superior to the T. baccata (12.04 mg/l) in the accumulation patterns of taxol and baccatin III. Further, they observed the release of taxol (40%) and baccatin III (67%) into the release of taxol and baccatin III into the growth medium. A commercial producer, rootec GmbH, Seuzach, Switzerland, has established an efficient bioreactor for the production of camptothecin in hairy roots production at large scale (Wildi et al. 2003). In this system, culture medium is sprayed continuously to grow roots on racks, and then continuous and semi-continuous modes are used to harvest the camptothecin.

22.8 Cambial Meristematic Cells as a Source of Anticancer Compound Production

Cambial meristematic cells provide an attractive alternative to traditional dedifferentiated cells for the synthesis of anticancer compounds. For example, elicitation of T. cuspidata cambial meristematic cells (CMCs) with methyl jasmonate induced paclitaxel production (Lee et al. 2010). Contrarily, the production of paclitaxel in T. cuspidata dedifferentiated cell lines (DDCs) derived from either needle or embryos was recorded to be reduced; aggregation of plant cells in cell suspension cultures was confirmed as the negative sign for the product yield (Joshi et al. 1996). Pronounced cell aggregates are typically found in DDCs in this context, and the reduced aggregation size of CMCs is observed between two and three cells per cluster. Using a 3 l airlift bioreactor, T. cuspidata CMCs produced 98 mg/kg of fresh cell weight (FCW) after 10 days of elicitation with MeJA, whereas needle- or embryo-derived DDCs produced only 11 and 13 mg/kg of FCW. In a 20 l airlift bioreactor, 268 mg/kg of paclitaxel is synthesized by CMCs. Contrast to this, no paclitaxel production was detected by either T. cuspidata needle- or embryo-derived DDC lines. Apart from paclitaxel, CMCs produced higher amounts of taxamarin A and C which are abietane tricyclic diterpenoids derivatives from Taxus species (Yang et al. 1999). Traditional DDCs which show some limitations for the synthesis of natural products such as fresh cell weight, cell aggregation, product yield, and sensitivity to shear stress represent significant obstacles. These problems associated with DDCs can be overcome by CMCs that provide a sustainable and cost-effective production of plant anticancer compounds.

22.9 Conclusions and Future Prospects

In conclusion, plant cell and tissue cultures can serve as an alternative means for the enhanced production of anticancer compounds. In this chapter, the strategies and approaches that are employed for the product enhancement of anticancer compounds are described. Every strategy and approach has got its own advantages and disadvantages over each other. Hence, there is a need for the substantial improvement for the product enhancement in lifesaving anticancer compounds on a commercial scale in order to bring down the cost of these compounds. Elucidation and understanding of the biosynthesis pathways of anticancer compounds and their regulation and seasonal influence on the production of anticancer compounds in respective plants in the wild are the prime concerns that help to overcome the difficulties experienced by various scientific groups for the overproduction of anticancer plant metabolites using plant cell and tissue cultures. Identification of suitable precursors, optimization of elicitors, medium composition growth regulators and culture conditions are the important parameters that are associated with product enhancement in plant cell cultures. A comprehensive knowledge of these factors and conditions will be of great importance for manipulating the desired metabolite production through product/metabolic engineering. Application genetic tools or advanced molecular techniques could be helpful to overproduce anticancer compounds through heterologous production of desired metabolites in lower organisms like bacteria and yeast. Paucity of information and knowledge about functions of enzymes, their regulation, respective genes sequence information and their expression associated with anticancer compounds are the setbacks in product enhancement by metabolic engineering, although there are some success stories reported pertaining to anthocyanin biosynthesis. Future studies pertaining to production of anticancer compounds using plant cell and tissue cultures should focus on addressing these aforementioned issues so as to resolve the complexities associated with plant secondary metabolites over production. With the knowledge gained, the attempts should focus on desired metabolite product enhancement through heterologous gene expression and metabolic engineering in lower organisms to develop cost-effective technologies for the large-scale production of anticancer compounds so as to ensure their availability at low prices affordable to the common man.

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Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • Hari Gajula
    • 1
  • Kumar Vadlapudi
    • 1
  • Poornima D. Vijendra
    • 1
  • J. Rajashekar
    • 1
  • Torankumar Sannabommaji
    • 1
  • Giridhara Basappa
    • 1
  • T. U. Santhosh
    • 1
  1. 1.Department of BiochemistryDavangere UniversityDavangereIndia

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