Phytochemicals mediated signalling pathways and their implications in cancer chemotherapy: challenges and opportunities in phytochemicals based drug development : A review

Cancer is considered as major leading cause for death in India and around world. Modern drug-targeted therapies have undoubtedly improved treatment to the cancer patients but also evoke severe side effects. Organ failure and immunosuppression is also a reason for death of cancer patients. However, advanced metastasized stage of cancers remained untreatable at present. So, there is need for the safer and more effective treatment for the improvement of efficiency and to lower the treatment cost to treat the disease. To readdress the above mentioned issues, phytochemical (s) based therapies are being advocated. Phytomedicine is an emerging strategy for the prevention, delaying, impeding the occurrence of cancer and curing the patients. The active herbal compounds of plants induce cytoprotective enzymes by the modulation of molecular targets of cancer while acting in co-ordination to detoxify and remove reactive substances formed by carcinogenic agents. The plant based principles have been reported to possess anti-carcinogenic, anti-proliferative and anti-mutagenic properties and hence exhibit potential to induce and stimulate cell death by genotoxic damage and reduction-oxidation imbalance in cells. These herbal compounds may inhibit or reverse multi-stages of cancer proliferation. This review summarizes an updated account of research in cancer chemoprevention and treatment strategy using phytochemical agents from medicinal plants. The underlying molecular mechanisms of actions of phytochemicals, the challenges in developing phytochemicals as effective anticancer drugs and possible solutions are also illustrated.


Introduction
Cancer is recognised as an abnormal growth of cells. It is originated due to lack of proper regulation in cell cycle. Cancer develops through an accumulation of genetic changes or mutations which could emerge due to different factors which could be physical (such as UV and other radiations), chemical (such as chewing and smoking of tobacco, chemical pollutants/mutagens), biological (such as viruses) and in some cases it may be hereditary. The types of damages within DNA may be induced by free radicals including strand breaks (single or double strand breaks), various forms of base damage in DNA (such as 8-hydroxyguanosine, thymine glycol, damage to deoxyribose sugar as well as DNA protein cross links) result into a heritable change in the DNA (mutations) thus causing cancer in the germ cells or malformations in fetus (somatic cells). Different types of free radicals have been reported to react with the biomolecules by (i) electron donation and electron acceptance (ii) hydrogen abstraction, (iii) addition reactions, (iv) self-annihilation reactions and (v) by disproportionation [1], leading to the production of reactive oxygen species (ROS) and reactive nitrogen species (NOS) which are linked to onset of cancer and other severe diseases [2]. The consequences of free radical induced oxidative stress are presented in Figures 1 and 2. 2 doi: 10.7243/2052-9341-5-2 worldwide estimate of new cancer cases and cancer deaths to be about 12.7 million and 7.6 million, respectively [4]. Several workers have reported that the cancer rates in successive generations of migrants shifts in the direction of prevailing rates in host country, suggesting that the variations in cancer rates largely reflect differences in environmental risk factors such as lifestyle and culture etc. rather than their genetic differences [5]. A list of gender based common diagnosed cancers along the world has been listed in Table 1. In general, the life style have been known to be the major factors for cancer development and as an infectious agents in developing countries. While prevalence of smoking is declining in developed countries [6], it is increasing in some developing countries which may be a main cause of increase in the burden of cancer in the developing countries [7]. However, the complete effect of these unhealthy lifestyle changes on the cancer burden in developing countries are likely to take decades to be realized [8,9].

Review Prevention and treatment for cancer by phytochemicals
Prevention of a disease is always considered superior approach than its cure. A large number of medicinal herbal plants have been reported to prevent and treat various diseases for thousands of years [10]. The naturally occurring bioactive chemical components derived from plants have been reported to be exerting their beneficial effects, and have also been confirmed for their anti-cancerous activities ( Table 2) [11 -145]. The available experimental and epidemiological data have shown that a variety of nutritional factors including vitamin A, C, E, beta-carotene and micronutrients and different phytochemicals found in edible and non-edible plants can act as anti-cancer agents and inhibit the process of cancer development. Extensive studies on anti-cancer phytochemicals has been done by Wang et al. (2012) [10]. The name and properties of these herbal compounds are shown in Table 2.

Interaction of phytochemicals with signaling pathways involved in apoptosis of cancer cells
Many phytochemicals used as anti-inflammatory or anti-viral reagents target the apoptosis pathways in cancer [146]. Based on practical experiences of applications of traditional Chinese medicines, the involvement of apoptosis pathways was deciphered [146]. Apoptosis is the process of programmed cell death that may occur in multicellular organisms which includes blebbing, cell shrinkage, and nuclear fragmentation. The apoptosis mechanism involves several signalling pathways. Apoptotic proteins cause mitochondrial swelling and increase the permeability of the mitochondrial membrane through membrane pores and leak out the apoptotic effectors [147].
Small mitochondrial derived activators of caspases (SMACs) are released from mitochondria into cytosol. These activators bind to inhibitor of apoptosis proteins (IAPs), inactivate IAPs and prevent them from arresting the apoptotic processes. Caspases, which carry out the cell degradation and are suppressed by IAPs, proceed for cell apoptosis process [148]. Cytochrome c released from mitochondria due to the formation of mitochondrial apoptosis-induced channel (MAC) in the outer membrane of mitochondria and binds with apoptotic protease activating factor-1 (Apaf-1) and ATP. This assembly then binds to pro-caspase-9 followed by the formation of an apoptosome and cleaves pro-caspase and release active caspase-9, which is then followed by the activation of caspase-3 [149]. Bcl-2 family proteins regulate MAC and Mitochondrial Outer Membrane Permeabilization Pore (MOMPP) complex. The anti-apoptotic Bcl-2, Bcl-xL or Mcl-1 inhibits the formation of the pore [150]. When binding of Tumor Necrosis Factor (TNF), a cytokine mainly produced by activated macrophages, with its receptor takes place, the cell survival and inflammatory responses are initiated. The interaction of FasL (a trans-membrane protein of the TNF family) and Fas receptor (Apo-1 or CD95) forms Fas-associated death domain protein (FADD), caspase-8, and caspase-10 complex, also called death-inducing signaling complex (DISC) [151]. In mammalian cells, a balance between pro-apoptotic (BAX, BID, BAK, or BAD) and anti-apoptotic (Bcl-2 and Bcl-Xl) proteins of the Bcl-2 family is maintained. Caspase activators (such as cytochrome c and SMAC) can be released from the mitochondrial membrane when the pro-apoptotic homodimers are formed in the outer-membrane of the mitochondrion. Inhibitor caspases (caspase 2, 8, 9 and10) may require certain adaptor proteins. The effector caspases (caspases 3, 7 and 6) are activated by the active initiator caspase via proteolytic cleavage and degradation of intracellular proteins to promote the cell death process. Some of the cancer and phytochemicals associated apoptotic signalling mechanisms are discussed in more detail in the following sections.

Cyclooxygenases-2 (COX-2)
Cyclooxygenases are bi-functional membrane-bound enzymes [152,153]. Housekeeping function mediated by COX-1 and COX-2 is low in most cells but is constitutively elevated in colorectal and other cancers [152]. COX-2 has been reported to be associated in colorectal cancers with larger tumour size and poor survival of the cells [154] therefore the expression of COX-2 has been proposed to be a nutritional target for colon cancer [155]. COX-2 may be induced at very early  stage of cancer development therefore the prevention of its aberrant expression may prevent the formation of cancer [156]. COX-2 contained a number of upstream regulatory sequences specific for binding with a variety of transcription factors, such as NF-κB, SP-1 transcription factor and activator protein-1 (AP-1) [157]. These transcription factors could be the final executors for a number of intracellular signaling pathways which make the COX-2 transcriptional regulation highly complicated.

Hedgehog signaling pathway (HSP)
Hedgehog signaling pathway (HSP) has been reported to be involved in providing the instructions to the cells for their proper development. The abnormal activation of this pathway may give rise to cancer through transformation of adult stem cells into cancer stem cells. Therefore, the researchers are looking for specific inhibitors of this pathway to devise an efficient cancer therapy [158]. In vertebrates, when sonic hedgehog (SHH) binds to the Patched-1 (PTCH1) receptor, the downstream protein Smoothened (SMO) which is inhibited by PTCH1, resulting in SHH activation leading to the activation of GLI transcription factors [159]. The accumulation of activated GLI in the nucleus controls the transcription of hedgehog target genes. Therefore the activation of hedgehog signaling pathway results into the increases of angiogenic factors and the decreases of apoptotic genes [160,161]. Hedgehog signaling pathway has been extensively reviewed as a target pathway for cancer treatment [162]. Therefore the approaches to regulate the hedgehog signaling pathway have been used to inhibit cell growth and promote apoptosis in prostate cancer by modulating SMO, PTCH and Gli3 (5E1) [163].

NF-κB pathway
NF-κB is a family of rapid-acting primary transcription factors. They are present in inactive state inside cells and do not require new protein synthesis to get activated which allows them to be the first responder to harmful stimuli. Free radicals such as reactive oxygen species (ROS), lipopolysaccharide (LPS), TNF alpha and IL-1 beta are some examples of NF-κB inducers. The NF-κB dimmers are sequestered in the cytoplasm by a family of IκBs. The ankyrin repeat domains of IκBs mask the nuclear localization signals (NLS) of NF-κB. IκBs are modified by ubiquitination via IκB kinases (IKK). NF-κB is then free to enter in to the nucleus where it may turn on the expression of specific genes. The NF-κB turns on expression of its own repressor, IκB alpha, which in turn reinhibits NF-κB, which results in oscillating levels of NF-κB activity [164]. Blocking NF-κB may cause tumor cells to stop proliferating, become more sensitive to the action of anti-cancer agents and to die [165].

STAT 3 pathway
STAT 3 (Signal transducer and activator of transcription 3) is a transcription factor that plays a key role in cell growth and apoptosis. STAT3 is activated through phosphorylation of tyrosine 705 and serine 727 residues in response to cytokines and growth factors then form homo-or heterodimers that translocate to the cell nucleus. The constitutive STAT3 activation has been associated with poor prognosis, anti-apoptotic and proliferative effects in cancer cells [175].

Wnt pathway
Wnt proteins are involved in normal physiological process of adult animals as well as in embryogenesis and cancers [176]. These proteins activate various pathways in the cell including canonical and noncanonical Wnt pathways and exert their effect in cell differentiation, embryonic development and generation of cell polarity [177]. In canonical pathway, the Wnt proteins bind to cell-surface receptors, causing the activation Dishevelled (DSH) family proteins and ultimately change in the amount of β-catenin that reaches into the nucleus. DSH complex inhibits axin, GSK-3 and APC complex proteins which normally promotes the proteolytic degradation of β-catenin. The inhibition of β-catenin destruction allows cytoplasmic β-catenin stabilization and entering the nucleus to interact with TCF/LEF family transcription factors to promote specific doi: 10.7243/2052-9341-5-2 expression of a gene. Therefore, the modifications in Wnt, APC, axin, and TCF are associated with carcinogenesis. The non-steroidal anti-inflammatory drugs (NSAIDs) that interfere with β-catenin signaling have been shown to prevent colorectal cancer [178]. Besides the mechanisms listed above, there are several other mechanisms for apoptosis such as the extra-virgin olive oil may target the human epidermal growth factor receptor (HER2) in breast cancer [179], resveratrol may reduce hypoxia-induced factor-1α, MMP-9 expression in colon cancer, lycopene may alter mevalonate pathway and many others [180].

Pharmaceutical challenges and opportunities in developing phytochemicals based drugs in therapy for cancer
Many natural dietary phytochemicals have been studied for cancer prevention and treatment. These native phytocompounds and/or their synthetic analogues have guided continuing research to bring them into the market as anti-cancer agents. Applying phytochemicals to cancer patients for chemoprevention encounters an immediate challenge in terms of their effects on human, as it is not feasible to design a clinical study to prove that the suppression of cancers in patient is due to the intake of phytochemicals. Though the chemical structures of some of the potential phytochemicals are well understood, but their physicochemical properties are not well documented yet and needs detailed investigation. Bioavailability of phytochemicals is another challenge that needs to be addressed. The nanotechnology, liposomes, micelles and phospholipids complexes have been applied to increase the water solubility of phytochemicals to enhance their bioavailability. Phytochemicals are generally considered as non-toxic materials but they may exert their toxicities to animals or humans at certain situation (drug-drug interaction and concentration), which may delay their application in the clinical studies and application in cancer treatment. This may be due to the synergistic effects existing in natural doi: 10.7243/2052-9341-5-2 compounds consumed as a whole rather than a single extracted/ purified compound. Using recently developed new technologies; some novel natural plant based compounds may be identified and developed as anticancer agents for chemopevention of the disease. Such phytochemicals may prove to be cost effective, safe and more potential with enhanced efficacy against cancer [181]. However, a thorough study of these phytocompounds and their pharmacological effects may generate insights for their drugability as well as transition from laboratory to the patients.

Conclusion
Phytochemicals in cancer chemoprevention are considered as the cheapest option in cancer treatment. Phytochemicals have been widely used in preclinical cancer prevention and treatment studies. Phytochemical chemopreventive agents are believed to play significant roles in controlling, inhibiting, and blocking signals which can cause translation of normal cells to cancer cells. The chemoprevention of cancer using phytochemicals have been such an attractive approach therefore efforts should be made to thoroughly understand their potencies, pharmacokinetics, pharmacodynamic responses, metabolisms, toxicities, drug-drug interactions, polymorphisms, formulations dose and to explore the molecular mechanisms of phytochemicals in cancer treatment more clearly. Genomic instability provides a means for selective targeting of cancer cells over normal cells. Among the various cancers chemopreventive agents many cause changes in chromatin conformation, disrupt the intracellular redox balance and deregulate DNA repair proteins. Thus, these compounds might activate the DNA damage response in cancer cells as compared to the normal cells. It is hoped that the improved understanding of these mechanisms will provide a more rational basis for combining specific dietary compounds and radiation therapy or chemotherapy approaches. More studies should be focused on dose-dependent responses and toxicity of the phytochemicals to ascertain their safety before usage.

Competing interests
The authors declare that they have no competing interests.