THE POTENTIAL OF NATURAL PRODUCTS IN CANCER TREATMENT clusters or combining them with genes of other pathways to obtain new compounds (combi- natorial biosynthesis) could be a further advantage over traditional natural product discovery methodologies. Study of ecological interactions among organisms and between them and their environ- ment, mediated by secondary metabolites that organisms produce (chemical ecology), e.g. plants poisonous to animals, has long been a successful strategy in discovering bioactive natural products (Harborne, 2001). One example of how this field changes under the light of findings from cellular and molecular biology is the search not for general cytotoxic com- pounds, but for effective nuclear factor (NF)-kB inhibitors in marine organisms (Folmer et al., 2007). Several bacteria and viruses have been reported to modulate NF-«B activity in host cells in order to increase their chances to survive as parasites within the host. Pancer et al., (1999) have shown that sea urchins use a NF-«B analogue to protect themselves against apoptosis-inducing compounds released by the diatoms on which they graze, and to respond to bacterial infection and other pathogens. From the evolutionary point of view, one interest- ing potential explanation for the finding of NF-kB inhibitors in marine organisms is the fact that marine invertebrates and fish, no matter how distantly related they appear to be, possess, in many cases, NF-«B or closely related analogues (Folmer et al., 2007). 1.4 Methodologies of Lead Compound or New Drug Identification The tools for evaluating the ‘anticancer’ potential of natural products are rapidly increasing. Preclinical evaluation involves in vitro assays of the effect of natural products on specific molecular targets involved in apoptosis, mitosis, cell cycle control or signal transduction, in vitro evaluation of cytotoxicity or other mechanisms of action in cultured cancer cells and other normal cells, and evaluation of the antitumour activity in animal models (Mishra et al., 2007). Bioactivity evaluation should also incorporate methods for evaluating the immunomod- ulating, anti-invasion or angiosuppressive potential of natural products. This vast array of available bioassays necessitates a strategic decision of which will be the first-line assays, which will determine the natural products that are candidates for the next round of bioactiv- ity evaluation; it is obvious that the complete preclinical evaluation of all natural products is not possible not only because of incredibly high cost but also of ethical considerations. The recent overemphasis on the molecular targets is criticized as simplistic and reductionist, and the study of the effect at the cellular level is reappraised (Houghton et al., 2007; Subrama- nian et al., 2006). The case of the discovery of vincristine (9) and vinblastine (10), anticancer drugs approved in the early 1960s, which led to the semisynthesis of vinorelbine and vinde- sine, poses other interesting implications. Catharanthus roseus (former Vinca rosa) is used in ayurvedic medicine for the treatment of diabetes mellitus (Figure 1.5). In search for effec- tive hypoglycaemic agents, Robert Noble and Charles Beer were surprised to observe that intravenous administration of the C. roseus extract to experimental mice resulted in a rapidly falling white blood count, granulocytopenia, and profoundly depressed bone marrow (Duffin, 2002). This chance observation led to the isolation and identification of vinblastine and vin- cristine as potent therapeutic agents and novel lead compounds. Bioactivity evaluation is performed on isolated natural products and/or on extracts and/or purified fractions of those. The classic phytochemical approach has the risk of missing natural products that are in trace amounts, and, thus, rediscover known compounds, —$— _ 1.4. METHODOLOGIES OF LEAD COMPOUND OR NEW DRUG IDENTIFICATION at vincristine, 9 vinblastine, 10 Figure 1.5 Catharanthus roseus was traditionally used for treatment of diabetes but by chance discov- ery it was shown to possess anti cancer activity. The useful anticancer agents, vincristine and vinblastine, were isolated from C. roseus. Photo by Conrado, retrieved from http://en.wikipedia.org on December 1 2007 and under GNU Free Documentation license eg. polyphenolics, in high abundance. Bioassay-guided fractionation is the most commonly used strategy for the identification of the bioactive lead compounds. Fractionation reduces complexity, increases the titer of low abundance components and removes ‘nuisance’ substances. The strategy followed for the isolation of camptothecin is shown in Figure 1.6. Bioassay-guided fractionation can lead to ‘strange’ findings (Pieters and Vlietinck, 2005). The bioactivity of the extract might be higher than that of the isolated compounds or frac- tions and this may be attributed to synergy of the phytochemicals present or decomposition and/or oxidation of the phytochemicals due to the lower amounts of antioxidants present in the fractions and the materials/solvents used in the fractionation process. The possibility of the presence of not one active compound but of several is very strong. On the other hand, the bioactivity of the isolated compounds might be higher than 100 % of the extract due to com- petition of the phytochemicals present in the extract. The pharmacokinetics of the fractions may also be different from that of the extract (better or worse) since it has been shown that certain natural products affect absorption, e.g. tannins decrease absorption from intestine. These procedures may lead to the re-isolation and re-identification of known compounds as the bioactive constituents, which is regarded as a considerable loss of time and funds in the search for novel bioactive lead compounds. Thus, de-replication is necessary at an early stage of the discovery process, preferably in the primary extracts, so as to allow the priori- tization of work and concentration on those sources that produce novel compounds. Liquid chromatography—mass spectroscopy (LC-MS)/MS coupled with the on-line acquisition of UV/vis spectra and the construction of libraries is a tool for correct structure identification of phytochemicals in an extract (Fredenhagen et al., 2005). Bordczky et al. (2006) suggested a simple gas chromatography-based method using cluster analysis as a data-mining tool to select samples of interest for further analysis of lipophilic extracts. Furthermore, the con- struction of natural product-focused spectral libraries of nuclear magnetic resonance data of isolated compounds allow for the rapid structural elucidation and thus an early de-replication (Dunkel et al., 2006; Lopez-Pérez et al., 2007). Once identified and the results of preclinical evaluation are good, the bioactive natural product will be directed to clinical evaluation. Results of clinical phase | and II trials will determine if the compound will be evaluated in phase III trials, will be sent back to the laboratory for optimization, or abandoned (Connors, 1996). On the way to the marketplace, the crucial problems of supply and large-scale production must be solved (McChesney et al., —$— _ 12 CH 1 EXPLORING THE POTENTIAL OF NATURAL PRODUCTS IN CANCER TREATMENT Plant Material 19 kg (dry material) Heptane (Hot) Heptane Extract Plant Material Concentrate Ethanol Marc-rejected Treat with 4 volumes chloroform +1/4 volume Precipitate Concentrate 5% aqueous ethanol 10 g, F127 468 g, F123, Inactive Inactive Chloroform Aqueous 222 g, F124 Highly active Continuous CHCl, extraction Chloroform Aqueous 32 g, F125 F126 Highly active Slightly active Figure 1.6 Bioassay-guided fractionation of Camptotheca acuminata wood plus wood bark. Bioac- tivity of the fractions was evaluated by the in vivo L1210 mouse life prolongation activity. Most of the chloroform phase after concentration was subjected to an 11-stage preparative Craig countercurrent distribution. The bioactive fractions were then combined and further purified by chromatography on a silica gel column and crystallization. The pure bioactive compound was proved to be camptothecin. Reproduced from Wall ME, Wani MC. Camptothecin and taxol: discovery to clinic — thirteenth Bruce F. Cain Memorial Award Lecture. Cancer Res 1995, 55(4), 753-60, with permission from the American Association of Cancer Research 2007). Medicinal chemists play a key role in the generation of structural analogues and introduction of ‘drug-like’ features. Apart from the traditional chemoenzymatic approach, combinatorial chemistry of a natural product-lead compound is often involved in construc- tion of libraries whereas combinatorial biosynthesis holds a great promise in the field (Boldi, 2004; Harvey, 2007). Combinatorial biosynthesis can be defined as the application of genetic engineering to modify biosynthetic pathways to natural products in order to produce new and altered structures using nature's biosynthetic machinery (Floss, 2006; Julsing et al., 2006). Theintroduced structural alterations range from simple reactions such as glycosylation, oxida- tions and reductions, methylations, isoprenylations, halogenations and acylations to the gen- eration of complex hybrid ‘unnatural’ compounds. The screening of natural product libraries and extracts usually yields a substantially higher percentage of bioactive hits than that of —$— oe 1.6. CONCLUDING REMARKS. 43 chemical libraries; a recent review (Berdy, 2005) estimates an approximately 100-fold higher hit rate for natural products. 1.5 Chemoprevention — A New Area for Natural Product Research Epidemiological studies, showing that increased intake of fruits and vegetables is associated with reduced risk of cancer, triggered research on the identification and characterization of the biological properties of the natural products in edible plants and the creation of anew sci- entific field, that of chemoprevention (Reddy et al., 2003). Chemoprevention, as a scientific field, may be considered still at its infancy, and includes the use of natural or pharmacological agents to suppress, arrest or reverse carcinogenesis, at its early stages. Studies, mainly in vitro, have shown that most dietary natural products exhibit pleiotropy; they affect several biolog- ical processes (even opposing functions) and act on a multitude of molecular targets (Reddy et al., 2003; Russo, 2007). The‘ antioxidant’ effect is put forward by most scientists and helps unify the positive effects on different systems, e.g. cardiovascular, neurodegenerative dis- eases and cancer. Natural products, like genistein, resveratrol, curcumin, retinoic acid and epigall ocatechin-3-gallate, became the focus of intense research and public interest. In paral- lel, alot of dietary supplements, functional or medical foods, became available to the public and this created alot of concerns about the safety, the quality, the efficacy and the legislative status of these products. The field of the study of natural products as chemopreventive agents has to address many problems and challenges. A major problem is the confusion in the literature; from experiments in cell cultures with concentrations of natural products equal to or even higher than those appropriate for pharmacological agents and with no knowledge or study of the absorption and the bioavailability, some scientists jump to conclusions about anticancer or chemopreventive potential (Russo, 2007). Other important questions are ‘when’ the chemopreventive interven- tion must take place to show efficacy and what happens if the antioxidant treatment does not occur at the‘ appropriate time’ and especially what happens when it takes place with standard chemotherapy (Russo, 2007). The clinical evaluation of the chemopreventive properties of anatural product is particularly challenging due to the time involved, the lack of appropriate biomarkers and the fact that it involves healthy people. However, the fact that selective oestro- gen receptor modulators, like tamoxifen and raloxifene, do decrease the incidence of breast cancer in post-menopausal women suggests a bright future for chemoprevention; raloxifene especially is a multifunctional medicine that was approved for reducing the risk of invasive breast cancer in postmenopausal women with osteoporosis and in postmenopausal women at high risk for invasive breast cancer (Jordan, 2007). Thus, it is possible that natural products, analogues or combinations of these will be used as chemopreventive agents. Furthermore, the public attention paid to dietary chemoprevention can only be viewed as positive since it increases the awareness of people of the significance of well-balanced diet rich in fruits and vegetables. 1.6 Concluding Remarks Plants and microorganisms have been sources of a significant percentage of potent anticancer drugs used nowadays, although a small portion of these have been studied. Further exploration —$— oe 14 CH 1 EXPLORING THE POTENTIAL OF NATURAL PRODUCTS IN CANCER TREATMENT of those, of marine organisms and of other novel sources will certainly reveal new drugs, novel lead compounds and new mechanisms of action. However, this process is time-consuming since it involves several steps: selection of the sources, screening and identification of bioac- tive drugs or lead compounds, in vitro and in vivo studies of the toxicity and mechanism of action, production or synthesis in high quantities, preclinical and clinical evaluation, approval and development of analogues with better characteristics which enter again the same cycle of drug development. Recognition that the biological diversity of the earth and, thus, the chemi- cal diversity is rapidly diminishing is a very important stimulus for natural products research inthe face of irreversibleloss of sources of potential drugs. Moreover, in the light of increasing cancer rates, the area of cancer prevention using natural products is very important. Progress can only be realized with sufficient funds. The immediate co-operation of univer- sities, institutes, big pharmaceutical companies and small biotechnology firms is necessary in order to meet the demands for effective pharmaceuticals. Each sector can contribute in a different way; large-scale random high-throughput screening and clinical development can take place in pharmaceutical companies and in large institutes; universities and institutions can take on research directions that require lengthy procedures and are not so expensive, e.g. screening according to ethnopharmacology. The involvement of scientists from all fields in natural products research has and will further transform the field and the techniques involved in order to meet the demands of modern drug discovery and development.
Key Takeaways
- Natural products can be used as lead compounds for new cancer drugs.
- Bioassay-guided fractionation is a common method to identify bioactive natural products.
- Combinatorial biosynthesis holds promise in generating new and altered structures.
Practical Tips
- Utilize bioassay-guided fractionation techniques to increase the efficiency of identifying bioactive compounds.
- Consider combining natural products with other pathways or genes for potential synergistic effects.
- Screen a wide range of natural sources, including plants, microorganisms, and marine organisms.
Warnings & Risks
- Be cautious of re-isolation and re-identification of known compounds as it can be time-consuming and costly.
- Ensure proper bioavailability and absorption studies are conducted to avoid false positives in bioactivity evaluation.
- Understand the complex interactions between natural products and their mechanisms of action.
Modern Application
The exploration of natural products for cancer treatment remains a critical area, with modern techniques enhancing the efficiency and accuracy of identifying potential lead compounds. While traditional methods like bioassay-guided fractionation still hold value, advancements in combinatorial biosynthesis offer new possibilities for generating novel drug candidates. The integration of these historical practices with contemporary scientific approaches ensures that this knowledge continues to be relevant in today's rapidly evolving pharmaceutical landscape.
Frequently Asked Questions
Q: What is the significance of natural products in cancer treatment?
Natural products have been a significant source of potent anticancer drugs, as they often contain bioactive compounds with unique structures that can target specific molecular pathways involved in cancer. For example, vincristine and vinblastine from Catharanthus roseus were discovered through traditional medicine practices and later developed into effective anticancer agents.
Q: How does combinatorial biosynthesis contribute to the development of new drugs?
Combinatorial biosynthesis allows for the modification of natural product pathways using genetic engineering techniques, enabling the production of new and altered structures. This approach can generate complex hybrid compounds that may offer improved therapeutic properties compared to their natural counterparts.
Q: What are some challenges in evaluating the bioactivity of natural products?
Evaluating the bioactivity of natural products is challenging due to factors such as the presence of multiple active compounds, potential synergistic effects, and the need for comprehensive absorption studies. Additionally, there can be false positives or negatives if proper bioavailability and pharmacokinetics are not considered.