Phyto-factories of anti-cancer compounds: a tissue culture perspective
Beni-Suef University Journal of Basic and Applied Sciences volume 11, Article number: 43 (2022)
Cancer is one of the most critical but ubiquitous causes of death grappled from past decades. Widely used chemotherapy with cytotoxic activity blocks/ kills the cancer cell. The compounds targeted for anticancerous activity are either derived synthetically or naturally (through plants or microbial origin). Current day, versatile role of plants in medicinal field has been attributed to the secondary metabolites it produces, known for their anticancer activity. Therefore, discovery, identification and commercial production of such novel anticancer drugs is escalated and are centerpiece for pharmaceuticals.
A biotechnological approach, principally tissue culture, leads the candidacy to be an alternative method for production of anticancer compounds. A wide range of bioactive agents like alkaloids, steroids, phenolics, saponins, flavonoids, and terpenoids are in huge demand commercially. Plant tissue culture applications are constructively more advantageous over conventional methods in terms of their continuous, controlled, aseptic production, large scale and de novo synthesis opportunity. Various bioreactors are used for mass cultivation of bioactive compound at commercial level. For example: stirred tank reactors are used for production of shikonin from Lithospermum erythrorhizon, vincristine from Catharanthus roseus, podophyllotoxin from Podophyllum etc. Strategies like callus culture, suspension culture and hairy root culture are opted for mass cultivation of these bioactives.
This review summarizes plant tissue culture as a promising strategy proven to be a colossal breakthrough in reliable and continuous production of existing and novel anticancer compounds and help in combating the increasing future demands.
Cancer is defined as uncontrolled growth of a group of cells, which invade surrounding tissues/organs and lead to destruction of normal cells. It is one of the most fatal diseases worldwide and is the second major cause of death after heart diseases. According to Global cancer statistics in 2018, 18.1 million new cases of cancer were reported with 9.6 million deaths. Lung cancer was most common and leading cause of death (18.4%) followed by breast cancer (11.6%), prostate cancer (7.1%) and colorectal cancer (6.1%) . Treatments using chemo-preventive measures and radiation therapy are common in use but are painful to tolerate. These treatments should be highly effective but in the present scenario, adverse side effects and resistance to radiation or drug treatments have overpowered its benefits. Hence, there is an urge for targeting an alternative strategy which can slow down, prevent or cure carcinoma. Plants and their derived compounds are thoughtful substituent in relation to cancer with respect to their high bio-accessibility, less side effects, safer in use and importantly are cost effective . There are more than 1500 plant derived anticancer compounds which can be developed as potential drugs are under research and about five hundred drugs are under clinical trials. Thus, it is an imperative prerequisite to develop potent and less noxious drugs for fighting cancer with its ever increasing demand. As the plant-based compounds are obtained from natural sources, which in turn lead to extinction of those sources.
Nowadays, tools in biotechnology-based techniques offer production of bio-active compounds from plants in laboratories without disturbing the natural ecosystem. It has opened a pavement for production of these active compounds directly via, using plant cells, tissues or organs by aseptically growing and through genetic improvement to obtain desirable product . This review summarizes, present scenario of naturally derived anticancer compounds and plant cell culture techniques as an alternative strategy for the production of same.
Typically, about 80% of global human population is driven by using traditional medicines for its primary health preventive measures back from three decades. Plants, therefore, are proven sources of best medicinal practices; extensive research is en-route to find natural aspects for control and cure of cancer. The continuous and enormous use of herbal remedies has widely been escalated with domination in countries like the UK, Europe, North America, Australia, Africa, India as well as other developing countries are also seeking for these alternatives over the chemical drugs . The current status on market of plant-based medicines forecast the uplift by 5.88% (annual growth rate) in period of 2018–2023 with inclination of consumers demand toward source of plant-based medicinal products. The report provides outlooks into the major leading companies which play eminent role in herbal medicine sector are: Bayer AG, ZeinPharma Germany GmbH, Arkopharma, Dasherb Corp, Hishimo Pharmaceuticals, Blackmores, BEOVITA, Dr. Willmar Schwabe India Pvt. Ltd., Schaper&Brummer, Himalaya Global Holdings Ltd, Venus Pharma GmbH and Arizona Natural Products .
Till now the studies in field of cancer, had focused to rule out the deleterious effects of cancer via targeting the vital mechanism playing role behind regulation of cancer cells which affect locally or systemically. And finally through identification, isolation, characterization, studying their effects, a particular anticancer agent is selected and undergoes clinical trials.
1.1 Anticancer compounds
In general anticancer compounds by definition are the compounds or agents with potent activity to act against or arrest the growth of cancer. In an overview from 1980s and till now, around 174 potent anticancer compounds have been approved commercially, with 53% (93 drugs) accounted for natural based or derived products . Normally, the anticancer compounds are categorized into two types: chemical compounds and natural compounds.
1.1.1 Chemical compounds
Compounds such as alkylating agents (cisplatin), anti-metabolites (5-Florouracil, 6-mercaptpurine), antibiotics (bleomycin, dactinomycin) etc which are used in chemo-treatments usually affecting the dividing cancer cells as well as normal cells during the course of treatment. Mostly all chemical cytotoxic agents have severe side effects, majority of them targets bone marrow, gastrointestinal tract, gonads (sex organs) and skin (hair follicle cells) etc.. Also, these conventional drugs limits its usability by their unsuitable oral route application, less solubility in water, inadequate specificity, severe side effects and short half-life of compounds in body .
1.1.2 Natural compounds
Compounds which are derived from plants, microbes and animals are referred as natural compounds. These compounds provided naturally, have an interesting perspective towards development of novel outcomes for cancer treatment. Interestingly drugs developed from natural compounds such as vinblastine and vincristine (Catharanthus roseus), camptothecin (Camptotheca), paclitaxel/Taxol (Taxus sp.) and podophyllotoxin (Podophyllum sp.) and others are proven anticancer targets. In actual fact, almost half of the globally accepted drugs are nature-based, -derived or -small molecules .
Commercially available anticancer drugs are broadly classified into various categories based on their mechanism of action: alkylating agents, antimetabolites, antitumor antibiotics and topoisomerase inhibitors, hormone and hormone antagonists, natural therapy and other medications (Fig. 1) .
1.2 A better outlook with natural medicines!
The traditional uses of natural medicines for healing purposes are mentioned since ancient times. Common applications of these medicines included curing fever, cough and cold, irritable bowel syndrome, menstrual problems, wounds, stings, burns, ulcers, anxiety and depression . Scientists have extensively researched for extracting potential anti-cancer compounds from plants which could be applied in preparation of drugs . World health organization (WHO) has reported that, there are certain populations that rely completely on medicines from tropical plants as major source for treatment of diseases . Boy et al.  reported that, around 35,000–70,000 plant species are targeted as drugs. Hence, the usage of plants for isolation of anticancer drugs had played a pivotal role in drug discovery. It is also critical to ensure that plant derived cytotoxic compound has a combination of desired effects such as: maximum potency against cancer and minimum deleterious effects on normal cells.
1.3 Methodology for anti-cancer drug development
Conventionally anticancer drug discovery and development were primarily based on cytotoxic agents which were basically chemical compounds, as majority of other drugs. For example, the use of folate analogues for therapy of acute lymphoblastic leukemia was developed in 1948 . On the other hand, the underlying mechanism of action playing role in inhibition of dihydrofolate reductase, was observed by Osborn et al. in 1958 . Likewise, for nitrogen mustard, mustine, was already in use before its mode of action was even known. Recently the process of drug discovery and development has become very systemic and methodological, focusing on the insights of cancer at molecular level and specifically on target-based drugs. They are pre-processed for modification / inhibition of selected molecular targets. In recent years there have been various novel drugs approved by Food and Drug Administration (FDA) enlisted in Table 1 from 2010 to 2019. Anticancer compounds, as like other drugs, also undergo different stages in developmental paradigm before their marketing approval by the regulatory. The stages for potential drug development are as follows (Fig. 2):
Identification of potential anticancer compounds: It is accomplished through chemical synthesis or by natural extraction of targeted compound. This stage comprises identification of promising compounds, exploring its physical properties (such as solid state form, stability, melting point and solubility) and testing its potential under conditions of physiological stress at cellular level as expected in malignancy. Various other studies like ADME (absorption, distribution, metabolism and excretion) profile and those defining the best dose for the compound, toxicological profile, interaction profile with other drugs and its effectiveness are being conducted. Shortlisted compounds based on pre-mentioned criterion are scaled up in further developmental phases for formulation, which is guided by selected mode of administration in humans and its subsequent commercialization post approval.
Drug screening and preclinical trials: This stage involves confirmation of safety aspects of a drug for use in humans and detailed toxicological profile. Studies are being carried out in vivo (within the organism) and in vitro (a setting outside the organism) during the preclinical trials. This phase focuses mainly upon the safety aspects of the drug and its levels which can prove to be toxic to humans. After keen review of preclinical trial results, the decision is made, whether the new compound can be tested in humans or not .
Clinical trials: They are a type of research study which explores the safety and efficacy of a treatment, strategy or a device by following strict regulations with a purpose of protecting the patients, parallelly producing reliable results which could be easily reproduced. Clinical trials comprise of various phases:
Phase 0 (Especially carried out for cytotoxic drugs)-These are typically “first in human studies”. Study population is targeted to cancer patients, approx. 10-15 subjects per study. Sub therapeutic doses of the drug are administered for a maximum up-to 7 days. Focus of phase 0 is to select the ‘lead candidate’ by mainly looking at the bio-distribution (pharmacokinetics) and confirming whether the effects on the molecular target replicate the ones produced by the preclinical studies.
Phase I—For drugs other than cytotoxic agents, these are “first in human” studies. Phase-1 established recommended phase 2 dose of a drug, (RP2D) and collects data about the adverse effects in human subjects. It also includes details about pharmacokinetics (effect of body on the drug) and pharmaco-dynamics (effects of drug on the body) of the drug. Generally, the study population includes normal adult volunteers but occasionally the studies may target specific patient populations. It establishes MTD (maximum tolerated dose) and the dose range for a drug. Interestingly, phase-1 cancer trials include specific dose escalations in addition to the above-mentioned details. The escalations are basically of 3 types: 3+3 cohort (traditional), accelerated titration, and model guided (continuous reassessment). Typically, phase one for any drug consists of 20-100 subjects and as per an estimate 70% of drugs move on to phase 2.
Phase II—These are initial pilot studies involving several hundreds of subjects. Basic goal is to achieve preliminary efficacy data and details of side effects in human subjects (patient population). They can be further classified into phase 2a and phase 2b studies. 2a studies are pilot studies to understand drug efficacy in selected populations. Dose range of a drug is determined by testing multiple doses. While, 2b studies are well controlled efficacy trials with a definitive dose and provide a “Go/No go” decision. As per an estimate 33% of the phase 2 drugs move on to phase 3.
Phase III—These are actual pivotal trials carried out in larger population (300–3000 subjects) depending on diseased state. The main aim of phase 3 is to confirm the efficacy in large population and to monitor all the adverse effects encountered. Typically phase 3 studies last from one to several years. Again, it is estimated that 25% of drugs move on to next phase.
Phase IV—These studies are better known as ‘post marketing surveillance’. Goal of this phase is to monitor the long-term side effects of a drug. Data are collected from consumers post approval and marketing .
2 Plant tissue culture: a new vision for cancer drug development
Plant tissue culture is a technique of plant propagation under controlled environment (also known as micropropagation). The primary objective of the micropropagation is to establish aseptic conditions so that only plant cell will grow and other biotic factors such as bacteria, yeasts and fungi can be eliminated. Due to its intrinsic potential for growth (known as totipotency), any plant cell from any organ of the plant can be nurtured under in vitro conditions, which ultimately gives rise to whole new plant. This is achieved through the maintenance of physical factors such as light, humidity, pH of the medium and temperature and chemical factors such as macro and micro-nutrients, sugars, vitamins, plant growth regulators and gelling agents. The process of micropropagation starts with establishment of axenic culture (obtained from any plant parts) and ends with the hardening of the tissue culture raised plantlets. In between these two process there are steps of organogenesis. Organogenesis can be divided in to two type's viz., direct organogenesis in which the new shoot is emerged from the meristamatic cells such as shoot tips or nodes, or non-meristamatic cells such as leaf or internode; and indirect organogenesis in which the new shoot is emerged from the undifferentiated mass (callus) or through somatic embryogenesis. The whole process ends up with the development of true-to-type plants ready to transfer to the field. Now the basic question would arise that why the need of tissue culture in plant production when they can be cultivated (and subsequently the product can be harvested) on field? Here, the advantages of micropropagation are that the process is season independent (where on field it needs the right time to cultivate); it can be done in relatively less space and time; and the production of disease free superior quality plantlets . However, the process for the plant production is costly so that it should be noted that not all the plants are suitable for the commercial production, rather, the selection of plant species to be micropropagated should be based on the issue they are facing during vegetative or seed-based propagation. These issues include limited numbers of offshoots as in case of Banana ; dioecious nature of the plants in cases of Date palm and Papaya [63, 66]; and less viabilities of the seeds in case of Stevia .
Plants have always been most effective and lifesaving entity in the world of drugs for human population. Commonly, the bioactive compounds or secondary metabolites from plants are used as pigments, dyes, food additives, drugs, cosmetics, pesticides, perfume etc. . Generally, plants modulate themselves by using their bizarre feature of plasticity when they fall victim to biotic and abiotic stresses. This lead plant to synthesize secondary bioactive molecules through activation of elicitors which evoke mechanism for production of these kind of secondary biomolecule. Mainly, for biotic stress: elicitors are categorized as exogenous elicitors such as chitin, glucans, chitosan which are pathogen induced, whereas the endogenous elicitors are pectin, pectic acid, polysaccharides induced by plants due pathogen attack. In contrast, there are abiotic elicitors such as: physical agents—heat, cold, atmospheric pressure, UV light; chemical agents: antibiotics, fungicides, pesticides, salts, heavy metals, ethylene. Thus elicitors regulates the gene expression, induces enzyme synthesis, promote formation of various secondary metabolites like alkaloids, flavonoids, phenylpropanoid, terpenoids, thionins and polypeptides .
In plants these compounds are not only vital for adaptation in relation to environmental fluctuations, but also have significant role as pharmaceutical products. Many plant species in nature are empowered with a range of curative properties, which are now being unraveled for plethora of applications in medical background. Numerous compounds in nature are already identified and proven to be effective against cancer. But the future demands of increasing population and a fewer availability of other alternatives has led to a potential and more eco-friendly approach (i.e. tissue culture) for production of anticancer drugs from plants.
Recent advances in biotechnology aided with plant tissue culture methodology have provided new insights into commercial production of anticancer drugs . Various strategies in tissue culture can be utilized for enhancing production of natural cytotoxic compounds like: screening and selection of high production cell lines, optimization of media for growth and development of targeted bioactive compound etc. Plant tissue culture is the technique through which such important anti-cancer compounds can be produced under controlled conditions with less occupancy of space. Not only these bio-active compounds but also some of the therapeutic proteins have been produced commercially with the use of plant tissue culture. As compared to other cell-based production system for the production of therapeutic proteins, plant cell-based system has advantages with low to medium production costs; high scale-up capacity; highly purified products; very low contamination risk; and comparatively less cost for the product purification [39, 108]. Production of recombinant antibodies such as Human HIV antibody in Tobacco BY-2 cells ; recombinant proteins like Human epidermal growth hormone in Tobacco ; and recombinant vaccine such as Hepatitis B in Potato  have been reported using plant cell culture system.
2.2 Screening and selection of high production cell lines
Exploring preparatory material (initial material from the plant for procuring high production cell lines) which is directly correlated with highest anticancer activity (anticipated/studied) is the initial prerequisite in this strategy. The screening techniques are principally based on two conventional methods:
2.2.1 Cell-based bio-assay
The National Cancer Institute (NCI) screening was based on human tumor cell lines to search, compounds that obstruct cell growth. The program was initiated for plants, back in 1956. It covers novel active compounds, their botanical classification and its relationship with antitumor activities . During initial years, the program was based on in vitro cytotoxicity assay which mainly involved three murine tumors (carcinoma 755, L1210 leukemia and sarcoma 180). But it had certain flaws like lack of identification of targeted compounds and high incidence of cells losing its viability. Hence, improved methods were developed which were primarily based on in vivo screening by P388 leukemia and later by 9ASK astrocytoma in in vitro condition . Further advanced system was developed with human cell line-KB (human epidermoid carcinoma of the nasopharynx) cell culture , a primary indicator for determining the potent activity of compounds. As a matter of fact, human cell lines have higher sensitivity as compared murine leukemia cell lines (L1210 and P388, or in vivo assessment).
2.2.2 High-throughput screening
It involves compounds that hinder specific enzymes or proteins in vitro (phosphatases, kinases etc.). This approach is grounded on establishment of in vitro assay based on significance of specific protein. For, e.g. kinases as substrates are ideal system for screening, because there are many well designed chemical libraries already available for identification of kinase inhibitors. Pengo and coworkers identified kinases and phosphatases that regulate ATG4B activity by siRNA which could be helpful in developing a therapeutic anticancer strategy .
3 Structure-based classification of anticancer compounds in plants
Plants for their normal growth and development synthesizes mainly; primary or secondary metabolites. Carbohydrates, proteins and lipids are primary and have direct association with regular ongoing necessary mechanisms in them, whereas, secondary metabolites are biosynthetically driven from the primary metabolites and are involved in functions such as resistance towards disease, protection, species interactions and competition . These compounds on the basis of their biosynthetic origins are classified into three main groups: phenolics, terpenoids and nitrogen/sulfur-containing compounds . They are further classified on the basis of their structure as shown in Table 2 . A few examples of anticancer secondary metabolites in regular use and under clinical trials: vincristine, paclitaxel, homoharringtonine, ingenolmebutate, curcumin, betulinic acid . On a different perspective every year, number of new cytotoxic secondary metabolites are identified and with new sources aiming with innovative solutions against deadly cancer. Thus, bringing modifications in these chemical structures can provide a new-fangled strategy for more specificity in action of anti-cancerous drug development [41, 111].
3.1 Plant tissue culture: a doorway with multiple prospects!
Plant tissue culture is considered as basic technique, as a substitute to vegetative propagation in plants and in various commercial applications. It is an indispensable module in mass multiplication of elites, developing true-to-type plant cultures and in regeneration of genetically engineered novel plants. Basically, these are the culture systems with explants as plant cell, tissues or organs grown on artificial media (offers nutrient for its growth) in vitro. Explant obtained from plant tissues gradually transforms into cell mass that appears to be amorphous and colorless to pale brown, in sterile in vitro condition. Passaging callus on fresh media and growth condition at 25 ± 2 °C helps them to maintain indefinitely and when need, by supplementing growth regulators (auxins/cytokinines) and appropriate growth chamber conditions (16 h light, 8 h dark) can again re-differentiated into a whole plant. Callus in general resembles the non-differentiated meristematic cells. They consists small vacuoles but lack photosynthetic apparatus: chloroplast. By nature, they exist in compact, friable and semi friable form. Friable callus on slowly shaken condition can give rise single cell cultures .
Plant tissue culture was pioneered by Gottlieb Haberlandt (1902), who entrenched first callus culture in roots or embryo cultures, in the beginning of the twentieth century . The period of 1940s and 1960s led to advancements in tissue culture techniques, studies in details of cell behavior: metabolism, cytology, morphogenesis, nutrition, embryogenesis and pathology; regenerated plants which were free from pathogen, clonal propagation and germplasm storage. Also during this phase (end of 1960s) plant tissue culture was considered to be a promising tool for the study and production of various bioactive compounds as the alliance of various gene-based innovative molecular methodologies were developed and were implicated in callus culture and other plant tissue culture techniques [31, 101].
The true search for natural products as potential anticancer compounds dates back to Ebers papyrus in 1550 BC , but scientific period of this search began in 1950s with extensive research, identification and isolation of potent cytotoxic compounds like alkaloid vinca (vinblastine and vincristine) and podophyllotoxins [19, 29]. These technical innovations led United States National Cancer Institute (NCI) to initiate worldwide collection of plants in tropical areas during 1960s which in turn helped in discovery of potential novel cytotoxic drugs like campothecins and taxanes . Unfortunately, in 1982 the program of plant collection was terminated, but the screening program in plants for investigation of new anticancer drug was continued in 1986 at tropical and sub-tropical regions .
3.2 Tissue culture approaches for the production of anticancer compounds
As its name suggests, medicinal plants are, more or less, used for almost all the types of medical conditions due to their properties of bearing number of secondary chemical. Plant tissue culture of medicinal plants may be proven as promising strategy for the production of such chemicals . Looking into the strategy in details, it involves cell level production (cell culture), tissue level or organ level (organ culture) productions. Before we discuss about the production, first we should know how micropropagation strategy operates.
Plant tissue culture starts with very basic property of any plant cell, i.e. totipotency which states that any plant cell, if nurtured in suitable environment, can regenerate into a new plant. This includes steps like dedifferentiation and re-differentiation of cells and tissues synthesized. In the process of morphogenesis, plant cell undergoes unorganized mass phase which is known as callus (indirect organogenesis) or a new organ, e.g. roots, stems (direct organogenesis) (see Fig. 3). Callus is further converted into new organ by maintaining supplied auxin to cytokinin ratio. Most techniques of organogenesis end up with plant reproduction or crop improvement. However, in case of callus (and cell suspension) culture and hairy root cultures, secondary metabolites can be harvested systematically, and thus, these techniques can be used for the production of plant secondary metabolites . Here, we describe both the techniques in details with case studies of production of anti-cancer compounds.
3.2.1 Callus and cell suspension culture
Callus culture is generally achieved through culturing plant parts like leaf, cotyledon internode on callus inducing medium (CIM). CIM contains higher level of auxins such as NAA or 2,4-D and low levels of cytokinins. This may result in either friable or compact callus. Compact calli are generally green and used for the organogenesis purpose. Friable calli are used for the cell suspension culture. In cell suspension culture system, cells are maintained in their proliferation state with all the physical and chemical factors associated with the growth. After their maintenance, elicitors such as fungal cell walls, modulators such as plant growth regulators and signaling molecules such as jasmonic acid are added which enhance the secondary metabolism. By this means, production of commercially and medically important secondary metabolites is synthesized in simple and easily cultivable cells .
3.2.2 Biotransformation: hairy root culture (a propitious approach)
Plants produce numerous chemicals known as phytochemicals and secondary metabolites. Certainly, these chemicals have applications in mainly pharmaceuticals, food and flavor industries. As far as plant growth and survival are concerned, these chemicals help plant to combat in unusual environmental conditions known as stress. However, these chemicals are synthesized in very minute amount but still are very effective; humans are “hungry” for such chemicals with the goal of commercialization. Thus, attempts have been made to escalate these bioactive chemicals using biotransformation methodology such as hairy root culture  technique in plant tissues. In current scenario hairy root culture has evolved as a good alternative for production of secondary metabolites with desirable remodeling in bioactive plant metabolites. Hairy roots are form of differentiated organs developed by infection of Agrobacterium rhizogenes. These differentiated organs (roots) displays ability to fabricate and release complex active glycoprotein as well as facilitate plants to produce specialized biomolecules by altering its biosynthetic pathways. The growth of roots is rapid and vigorous add on to rapid establishment of the hairy root culture. Numerous secondary metabolites can be obtained from these cultures. Molecular mechanism of this is a transformation of genetic material from bacterium to plant cell. Agrobacterium rhizogenes contain Ri (Root inducing) plasmid having T-DNA region. These Ri plasmid can be modified and gene of interest can be inserted in the T-DNA region. After infection, bacterium transfers this T-DNA into chromosome of the plant cell. Thus, this technique can be used as a tool for transformation as well as secondary metabolite and its enhanced production inside the roots . As seen in Fig. 4, it starts from explant collection and agro-infection under in vitro conditions which later on results with the formation of hairy roots (highly bulkier than seedling roots). With these roots, some optimization can be carried out such as precursor feeding, elicitation or metabolic engineering to get maximum amount of secondary metabolites.
3.3 Strategies used for production
Biotechnological interventions in field of plant cells and tissues have offered different methodologies such as callus cultures, cell suspension cultures, organ cultures and hairy root cultures (as discussed above) for production of bioactive compounds. The two breakthrough techniques namely: cell cultures and clonal propagation are in wide use. Cell culture study is based on callus initiation via in vitro raised cultures on best suitable medium. Usually such cultures undergo somaclonal variation on frequent sub-culturing. Therefore, callus is first screened and selected (high genetic stability and highly productive cell lines), then taken further for cell suspensions. The final step involves the bioreactor which may probably lead to commercial production of bioactive compound. Transition from shaker flask to bioreactors is the most critical step while scaling up the production .
Bioreactors now have become one of the crucial contrivances in cell-based strategic productions. They are functioning as a biological factory to produce bioactive compounds and research from past many years conclude for its variable benefits such as: homogeneity, controlled cultural and physical environment, reproducible yields under controlled growth conditions, better control for scale up, simple and fast harvest of cells, easier separation of targeted compound etc. There are different bioreactor designs made for specific growth conditions for profuse cell growth and suitable secondary metabolite production. Bioreactors types are classified as (1) Stirred tank bioreactor, (2) Airlift bioreactor, (3) Fluidized bed bioreactor, (4) Packed bed bioreactor, (5) Photo-bioreactor and (6) Membrane bioreactor. Scale up strategy in general, starts from lab scale and reproducing it as nearly as possible for production at larger number of products. An archetypal scale-up series in plant cell and tissue culture studies involve initiation with simple jars, to 1 L shake flasks, then 1–10 L glass bioreactors, after which it is scaled up through stainless steel vessels of varying size from 30–150 to 1000 L (see Fig. 5) (Sarkar et al. 2018).
Further to increase production, different viewpoints were used to carry out:
Standardization of culture medium: Culture media is one of the important constituent in bioreactor system. Optimal conditions can be found by changing the chemical and physical factors of the culturing system. These factors include chemical components or phyto-hormones in the medium, pH, aeration, temperature, light, addition of antifoams etc. .
Elicitors or Precursors: Occasionally precursors and elicitors are added in culture system to enhance its production level; however the timing of addition into media and type or nature of precursor chosen is important. For, e.g. the study conducted by Sivanandhan and his coworkers concluded that, in bioreactor they obtained 1.66 fold higher concentration of total withanolides as compared to control Withaniasomnifera cell suspension culture via addition of elicitors such as: aluminium chloride, chitosan and cadmium chloride; and precursors: cholesterol, mevalonic acid and squalene .
Cell lines selection: The variation in production entity from cell to cell in a heterogeneous cell mass provides an opportunity in selection of high production strain. Selected strains are normally cloned and plated on agar medium to form colonies. Then, amount of active compound is measured through radioimmunoassay (RIA) or HPLC (High Performance Liquid Chromatography) methods for selecting colony of high production yield. For selection criteria there are chiefly three ways:
Visual—It is on the basis of visual marking such as color of product.
Nutritional requirement or chemical resistance—The selection by growing cells on a selective media such as specific nutrient deficient or in presence of some chemical inhibitor.
Production analysis: cells with high production yield can be selected by developing a rapid, sensitive method to analyze the metabolites.
Mutation: Mutagenesis using nitrosoguanidine, radiation (UV, X-ray, or ϒ-ray) or others can sometimes produce the cells to increase the productivity of metabolites, though in plants this method is not easy, since most of the vegetative cells are diploid. Baskaran et al.  reported two induced EMS macro-mutants (necrotic leaf and nerium leaf) of periwinkle which result increased content in root and leaf alkaloids and anticancer leaf alkaloids, vincristine and vinblastine than the parental variety. Kannabiran et al.  concluded that gamma radiation (40 Kr) and chemical mutagen (EMS-30 mM) increased content by 0.37% and 0.32% respectively.
Natural Products Genomics: A novel approach intended to access the plants own genomic capacity to boost yields and transform complex bioactive metabolite. This technology unifies the gain of functional mutagenesis and selection to (a) imitate the development of novel compounds in plants, and (b) to increase yield of known bioactive compounds. And carry out selection at pace during cell culture level in mutants consisting of large population .
Morphological Variation: There are some evidences describing the role of undifferentiated  and differentiated cells in production of secondary metabolites at higher levels.
The strategy of hairy root culture was reported back in 1985 by Flores and Filner, they introduced hairy root cultures induced by Agrobacterium rhizogenes have potential to increase productivity. It is also a useful method to induce the function of the plants to produce the metabolites when the cells grown in a proliferating medium are transferred to a medium for its differentiation. Hanafy et al.  showed the production of indole alkaloids (i.e vinblastine, vincristine and catharanthine) in hairy roots cultures of Catharanthus roseus L. in leaf explants and stems.
Immobilized Cells: It refers to confinement or localization of cells into a defined region. Immobilization helps to combat problems of cell aggregation and low shear resistance. The surface immobilization and cell aggregation technique are in wide use, wherein cells are entrapped in particular gel or combinations of different gels. Generally alginate, carrageenan, agar, agarose, polyacrylamide, etc. are used to immobilize the cells. This technique has several benefits such as: high cell density in small scale bioreactor which are cost efficient and with less risk of contamination, easy and simple downstream processing and extension of cell viability in stationary phase, higher product accumulation. But still, there is dilemma regarding, extracting out the metabolites from cells and to prevent cell damage by different treatments are need of investigation. There are few cases which reports that plant cells can secrete metabolites exterior to cells in media provided. The initial reports were production of indole alkaloids or digoxin using immobilized cells by .
4 Case studies on tissue culture-based production of anticancer compounds
The recent investigations and in-depth knowledge about novel natural structures with important biological activity and mechanisms have remarkable influence on pharmaceutical business. The discoveries of various anticancer compounds in association with plant tissue culture hold excellent new possibilities to tackle the disease ‘cancer’. The tissue culture operations used for production of cytotoxic secondary metabolites have potentially led to production of wide variety compounds of like terpenoids, alkaloids, flavanoids, steroids and amino acids. Few successful attempts are discussed here and enlisted in Table 3.
4.1 Taxol (Paclitaxel)
A complex diterpene alkaloid present in the bark of the Taxus tree, is one of the most potent anticancer compounds due to its mode of action on the micro tubular cell system. It is in market, back since 1993 with trade name Taxol® . The two important factors which are noteworthy with increasing demand of taxols are: trees take years to grow and mature, and majorly the taxol concentration in tissues is very less. To overcome these situations, plant tissue culture prompts to be a potential, stable and long-term method to produce taxoids at commercial scale. Taxol production in Taxus baccata callus culture was carried out with salicylic acid pretreatment to enhance the resistance against water stress and oxidative stress. One of the most important taxanes, taxol, was also elevated 5.1 times higher as compared to control calli. Total taxanes was increased up to 3.5 fold . Salehi et al.  worked out in modifying media as M10 medium (MS medium with pH 6.0 and supplemented with 1000 mg L−1 spirulina powder, 1000 mg l−1 casein hydrolysate and 3 g L−1 Gelrite) for improved callus growth and improved taxol production (106.6 µg L−1) as compared to control (77.7 µg L−1) in Hazel (Corylus avellana L.) plant. Filová and Krivosudská reported Gamborg’s B5 medium supplemented with 2,4-dichlorophenoxiacetic acid (2 mg L−1), kinetin (0.5 mg L−1) and gibberellic acid (0.25 mg L−1) for callus growth and elevated taxane production in Taxusbaccata L. .
It is anaryltetralin (2, 7 Cyclolignan) lignin occurring in Podophyllum peltatum and Podophyllum hexandrum. It also serves as an initial material for preparation of its semisynthetic derivatives, teniposide and etoposide, commonly used in anti-tumor therapy . Slow growth rate of plants and practice of using rare wild plants for healthcare, results in its limited supply against the booming demand. This condition has called for alternative methods for production of podophyllotoxin. First attempt of using tissue culture technique was carried out by Kadkade in 1982  to increase yield of podophyllotoxin. Although it was difficult to enhance this anticancer compound in cell suspension culture, Lalaleo et al.  optimized the culture method with respect to organogensis from callus and found maximum level of podophyllotoxins in dark treated culture which again showed morphogenic structures and they established clear relationship between concentration of these metabolites and organogenic capacity. Hence, nurturing the callus mass for organogenic pathway may be recommended to get higher content of podophyllotoxin and its derivatives. Taking an advantage of both the hairy roots and elicitation, Tashackori et al.  attempted the gene expression level of secondary metabolites, mainly podophyllotoxin and its derivatives (lignans) using cell wall of Piriformospora indica on hairy roots of Linum album and found significantly higher expression of genes involved in secondary metabolites such as phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, cinnamoyl-CoA reductase and pinoresinol–lariciresinol reductase. As a result, podophyllotoxin was increased from 64.14 µg G−1 of dry roots to 124.46 µg G−1 (1.94 times) in 1% v/v concentration of elicitor.
4.3 Vincristine and vinblastine
These compounds are dimeric indole alkaloids obtained from Catharanthus roseus. They have great antitumour activity against various solid tumors and leukemias. Commercially, the procurement of these compounds from Catharanthus roseus consumes large quantity of these plants and also the intact plant contains low concentrations of these compounds, about 0.0005%. Alternative strategy pioneered is plant tissue culture. Mishra and coworkers established a culture system in Catharanthus roseus with B5 medium, in addition with 3% sucrose, 0.5 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mg L−1 Kinetin (KIN), 2 mg L−1 α-naphthalene acetic acid (NAA) and obtained enhanced alkaloid production by 5.67 mg G−1 dry weight . Also the addition of elicitor “chitosan” leads to enhanced production of vincristine and vinblastin in cell culture of Catharanthus roseus (L.) G. Don as studied by Pliakong and their coworkers. They reported the highest amount of accumulation of vincristine and vinblastin at 4.15 and 5.48 µg Mg−1 cell dry weights respectively in cell suspension culture at 14th day .
It is a cytotoxic quinone alkaloid which inhibits enzyme DNA topoisomerase I and is isolated from bark and stem of Camptotheca acuminate (Happy tree)  and is used as anticancer drug worldwide. Lower yield of camptothecin from whole plant, poor seed germination and deficit viable method for production has been opted for tissue culture for its production through various available plants like Merriliodendron megacarpum, Eravatamia heyneana, Nothapodytes foetida and Ophiorrhiza acuminata, to meet the enormous demand of camptothecin . Attempts were also carried out by Wetterauer et al.  to produce camptothecin from Ophiorrhizasa mungos through infection of Agrobacterium rhizogenes. They cultured 25 different lines of Ophiorrhiza mungos to produce hairy roots and found maximum of 3.2 mg of camptothecin/g of dry weight. With their experiments, they made this production feasible and scalable so that it can be used by pharmaceutical industries. Deepti and Sateeshkumar  reported enhanced production of camptothecin in cell suspension cultures of Ophiorrhiza mungos Linn. through elicitor (silver nitrate and yeast extract (YE) treatment. About 13.3-fold enhanced production and threefold increments in cell growth was obtained in cell cultures elicited with 50 mg L−1 YE on 10th day.
Curcumin, a part of polyphenol superfamily a bioactive component of turmeric (an Indian spice-obtained from dried rhizomes of Curcuma longa plant) shows anticancer activity , via inhibition of cancer cell proliferation, metastasis, induction of cell cycle arrest and apoptosis. The rhizomes of turmeric were studied for their anticancer activity in vitro using tissue culture methods, showed cytotoxic effect on Chinese Hamster Ovary (CHO) cells at a concentration of 0.4 mgML−1 .
4.6 Other promising anticancer compounds
The novel findings in research have brought forth many potential and promising compounds against cancer. Such as sanguinarine , flavanoids , polyphenols , glucosinolates , quercitin , caffeine, chlorogenic acid, caffeic acid [69, 82] etc. with details are mentioned in Table 3.
A huge amount of research is also targeted on plants and plant-derived natural products. With recent investigations, new plant species with potent anticancer activity either in vivo or in vitro, have added to our knowledge. Few examples plant species with promising anti-cancer activity are: Achyranthes aspera, Allium sativum, Andrographis paniculata, Annona muricata, Bidens pilosa, Astralagus hedysarum, Bolbostemma paniculatum, Cannabis sativa, Centaureaainetensis,Camellia sinensis, Gossypium hirsutum, Daphne mezereum, Mangifera indica, Hypericumper foratum, Nerviliafordii, Hydrocotyle asiatica, Oroxylum indicum, Salvia miltiorrhiza, Scutellaria, Rubia cordifolia, Picrorrhiza kurroa, Silybum marianum, Smilax china, Withania somnifera, Strychnos nuxvomica, Zingiber officinale, Taraxacum officinale etc. (Table 4).
5 Advantages of plant tissue culture technique in anticancer compound production
The evident role of plant tissue culture in production and identification of novel active anticancer compounds has performed excellent over these past years. As there are many difficulties in procuring these compounds in bulk quantities directly from plants. The practises using callus culture, cell suspension culture and hairy root system has eminent advantages over whole plant cultivation, enlisted below:
The targeted compound can be obtained in regulator free media, are easily culturable with less growth time also with more stability in genotypic and biochemical level.
The cultured cells within system would be free of microbes, thus contamination rate and losses are minimized.
The cells of any plant (rare or endangered species) can easily be managed to produce secondary metabolites as a continuous source, once a system is defined.
It is easier to study the active metabolites from cells with known production system and also manage it through robotics, leading to reduced cost and improved productivity.
The recent advances and developments of research have given various scopes in fighting cancer. Most reliable and effective sources were found to be natural bioactive compounds. There are various novel compounds actively being discovered such as taxol, camptothecin, vinblastin and vincristin etc. But the availability and amount of compound procurement has always been the question. The progress in bio-techniques, particularly in area of tissue culture has satisfied the question of daily requirement and its availability for the burning demand of population and industrial commencement. The use of plant tissue culture strategy has enhanced a progressive channel for reliable and continuous source of these compounds. Further the insight into the biosynthetic pathways of important anticancer compounds in plants as well as cultures are still unknown, and henceforth these applications are in demand to unravel information grounded at cellular and molecular level to bring forth at industrial level.
Availability of data and materials
No additional data and material other than the manuscript is to be produced.
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I would like to show gratitude to my mentor and colleagues in the Department of Plant Tissue Culture and Biotechnology, Anand Agricultural University.
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Patel, P., Patel, V., Modi, A. et al. Phyto-factories of anti-cancer compounds: a tissue culture perspective. Beni-Suef Univ J Basic Appl Sci 11, 43 (2022). https://doi.org/10.1186/s43088-022-00203-5