Role of Reactive Oxygen Species in Carcinogenesis and Polyphenols as an Emerging Therapeutic Intervention

Abstract

Reactive oxygen species (ROS) are generated in the body as a by-product of cellular enzymatic reactions. Under normal conditions, an antioxidant defense mechanism in the body regulates the level of ROS produced and maintains a redox balance. However, in cases of metabolic disorder, chronic inflammation, or prolonged exposure to xenobiotics and environmental stressors, this balance is disturbed and leads to the generation of oxidative stress. ROS can attack the structural integrity of the major macromolecules of the body such as nucleic acids, lipids, and proteins leading to the generation of pathologies including cancer. Polyphenols have emerged as potent nutraceuticals that can not only augment the body’s antioxidant defense system to combat the generated oxidative stress but can also selectively act as pro-oxidants in cancer cells, a dichotomous phenomenon that is being actively studied for implementation in cancer therapeutics. This chapter will present in a comprehensive manner the role of ROS in the pathogenesis of cancer and the application of pro-oxidant nature of polyphenols as chemotherapeutics.

1. Introduction

Free radicals are molecules with an unpaired electron in their electronic configuration. As a consequence, they act as strong oxidizing agents and thereby react with or attack the major biomolecules of the body and take up electrons from them. The molecule, thus robbed of an electron, itself gets converted to a free radical which either initiates a chain reaction of oxidation-reduction or gets converted to another form with altered functionality and characteristics [1]. Oxygen-based free radicals and reactive non-radicals comprise reactive oxygen species (ROS) and have been listed in Table 1. Moderate amount of ROS is necessary for the maintenance of normal physiological processes like cellular proliferation and programmed cell death, redox signaling, wound healing, male fertility, immune responses, and aging to name a few [6, 7]. Antioxidants are compounds that keep in check the ROS content of the body either by directly quenching ROS by supplying electrons or indirectly by mediating reactions that facilitate reduction [8]. Endogenous antioxidants (enzymatic and non-enzymatic) are present inside the cell and also circulate in plasma. Edibles like diet and supplements can also provide antioxidants and are categorized as exogenous sources [8]. However, in case the level of ROS increases in cells in the presence of biotic or abiotic stimulants or in case of any pathological condition, an excess amount of ROS can harm the major biomolecules of the cell which leads to the development of several pathological conditions. Further increase in ROS level beyond a particular threshold induces activation of signaling pathways leading to cellular death such as apoptosis, autophagy, ferroptosis, and pyroptosis [9, 10]. In cancer therapeutics, one of the strategies to combat cancer is based on this phenomenon and involves the induction of cancer cell death by increasing the concentration of ROS in them.

ROS species	Physiological sources
Superoxide anion; (O2^.−)	Reduction of molecular oxygen. Spontaneous (leakage of an electron in electron transport chain or in the presence of reduced metal ion) or via enzymes
Perhydroxyl radical; (HOO^.)	Cytosolic protonation of O2^.−
Hydrogen peroxide; (H2O2)	Dismutation of superoxide anions (spontaneous or enzyme-mediated) or double reduction of molecular oxygen by enzymes
Hydroxyl radical; (OH^−)	Reduction of H2O2 in the presence of Fe^2+/ Cu^+ (Fenton reaction) or in the presence of O2^.− (Haber-Weiss reaction)
Hypochlorous acid; (HOCl)	Forms when H2O2 reacts with Cl^− in the presence of enzyme myeloperoxidase
Singlet oxygen; (^1O2)	Forms when H2O2 reacts with HOCl or with O2^.−. Also released as by-products of biochemical reactions involving primary or secondary reactive species
Ozone; (O3)	Oxidation of water or deoxidation of oxidized biomolecules by ^1O2
Alkoxyl radical, peroxyl radical, organic peroxide	Formed when primary reactive species react with cellular macromolecules/organic compounds

Table 1.

Major reactive oxygen species.

Source: [1, 2, 3, 4, 5].

Polyphenols are a group of phytochemicals, well known for their health advantages in humans [11]. The commercialization of polyphenol-based products and the application of polyphenols in various ailments is based on their potency as antioxidants. However, as will be discussed in this chapter, under certain conditions, polyphenols can act as pro-oxidants. This creates an avenue for utilizing polyphenols with a different perspective. In cancer cells, polyphenols may act as pro-oxidants, and this characteristic of these compounds is inspiring researchers to develop strategies so that they can be used either in isolation or in combination with traditional approaches to treat cancer. Knowledge about this phenomenon along with the various new approaches with which this aspect is being utilized by researchers to develop novel strategies for the treatment of cancer will be highly beneficial. Keeping that in mind, this chapter will discuss the role of ROS in the development of cancer and then in subsequent sections, findings from existing literature will be analyzed that will give an idea about the underlying phenomenon and information about a few novel interventions that are being researched upon at current times, utilizing polyphenols for cancer therapeutics.

2. Sources of reactive oxygen species

Sources of ROS generation can be categorized as endogenous or exogenous. When ROS is generated as a by-product of regular cellular metabolism, then it is considered an endogenous pathway of ROS generation. Normally, ROS is generated in electron transport chains, lipid metabolism, purine metabolism, metabolism of amino acids, protein folding, chronic infection or inflammation, or any kind of physical or mental stress. However, if ROS is generated in a cell as a response to an external stimulus (xenobiotics, ionizing radiation, etc.), then the source is termed exogenous [12, 13].

Several enzymes generate ROS as by-products of their activity, and they have a wide range of localization inside cells including the membrane, cytosol, or organelles. The most prominent ROS generating membrane-associated enzyme is nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase. The organelles involved in ROS production are mainly mitochondria, endoplasmic reticulum, peroxisome, and lysosomes. In mitochondria, apart from the electron transport chain, several enzymes (for example, monoamine oxidase, pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, α-glycerophosphate dehydrogenase, glycerol 3-phosphate dehydrogenase, aconitase, and CYP enzymes) are involved in producing ROS. In the endoplasmic reticulum, xenobiotic metabolizing enzymes of the microsomal monooxygenase family, enzymes involved in the protein folding process (endoplasmic reticulum oxidoreductin-1) and membranous NADPH oxidase are involved in ROS generation. In peroxisomes, the activity of various enzymes (for example, xanthine oxidoreductase, D-aspartate oxidase, acyl CoA oxidases, D-amino acid oxidase, etc.) along with an electron transport chain in the membrane leads to ROS generation. Lysosomes are also a storehouse of ROS. An electron transport chain (involved in the maintenance of high pH concentration) and a high concentration of iron ions assist ROS generation [1, 2, 12].

The major exogenous sources of free radicals are xenobiotics (metals, drugs, cosmetics, food additives, pesticides and insecticides, and polycyclic aromatic hydrocarbons) and radiation. Once cells are exposed to xenobiotics, they are metabolized by various xenobiotic metabolizing enzymes (like cytochrome P-450 and monoamine oxidases) so that they can be readily eliminated from the body. However, in the process, ROS is generated [9].

3. Oxidative stress and effects on major macromolecules

Under normal circumstances, the ROS produced in cells is neutralized or acted upon by a plethora of antioxidant molecules (from endogenous and exogenous sources) which maintains the level of ROS to an optimum level that is required for the proper functioning of cells. However, if the balance between oxidants and antioxidants is perturbed with an overall rise in ROS concentration, oxidative stress is generated. Under conditions of oxidative stress, ROS owing to their reactive nature causes detrimental effects on the major biomolecules of the cell like lipids, nucleic acids, and proteins. The effects of oxidative stress on the major biomolecules of the body are discussed below.

The major effect of ROS on lipids is the oxidation of polyunsaturated fatty acids (PUFA), and the phenomenon is termed lipid peroxidation [14]. Lipid peroxidation is majorly caused by hydroxyl and hydroperoxyl radical and membrane lipids such as glycolipids, phospholipids, and cholesterol are majorly attacked [15]. Generated reactive intermediates undergo further structural modifications to produce toxic aldehydes such as 4-hydoxy-2-nonenal (4-HNE), malondialdehyde (MDA), along with γ-ketoaldehydes [14]. Oxidation of membrane lipids leads to characteristic changes of lipid membrane-like organizational alterations, changes in thermal phase behavior, thickness, symmetry, polarity, and permeability, which might lead to aberration in the interaction between lipids and membrane proteins, ultimately leading to the pathogenesis of diseases [16]. A schematic representation regarding the lipid peroxidation of arachidonic acid, a major component of membrane phospholipids, leading to the formation of MDA (a marker of lipid peroxidation) has been presented in Figure 1. Arachidonic acid peroxidation can lead to the formation of several by-products via several pathways. After initial H abstraction by reactive species, the lipid radical generated gives rise to a peroxyl radical in the presence of oxygen. This peroxyl radical in one of its metabolic pathways undergoes subsequent cyclization reactions with simultaneous formation of intermediate radicals, which ultimately give rise to bicyclic endoperoxide. Bicyclic endoperoxides also have several fates, one of which is to disintegrate to form MDA and heptadecatrienoic acid [17].

Figure 1.

Lipid peroxidation of arachidonic acid. Adapted from [17].

Reactive oxygen species (ROS) can attack both the sugar and the base of a nucleotide. On reaction with the sugar, the resultant point of abstraction being radicalized leads to cyclization with adjacent base pair leading to the formation of altered nucleosides like 8,5′-cyclopurine-2′-deoxynucleoside [18]. ROS can oxidize DNA bases (both purine and pyrimidines) to form modified bases. Purines are modified to form 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 4,6-diamino-5-formamidopyrimidine, 8-hydroxy adenine, 2-hydroxy adenine, 8-hydroxy guanine, and oxazolone, whereas pyrimidines are oxidatively modified to form 5-hydroxycytosine, cytosine glycol, 5-hydroxydeoxy uridine, uracil glycol, and thymine glycol to name a few [19]. The presence of a modified base pair may lead to mutation, formation of DNA strand breaks, and instability of genetic integrity. A schematic representation of the oxidative modification of guanine to form 8-hydroxy guanine (a marker for oxidative stress in DNA) is represented in Figure 2A.

Figure 2.

Schematic representation for (A) Reactive oxygen species-mediated modification of guanine to 8-Hydroxyguanine (adapted from [18]); (B) Reactive oxygen species-mediated oxidation of arginine to form carbonylated by-products (adapted from [20]).

Amino acids such as methionine, proline, cysteine, threonine, tyrosine, histidine, arginine, and lysine are prone to be modified by ROS to produce several compounds including carbonyl derivatives [21, 22]. The formation of carbonylated derivative (a marker for protein oxidation) from arginine has been illustrated in Figure 2B. ROS attacks peptides resulting in the formation of carbon-based radicals, which in the presence of oxygen forms peroxyl radical that can undergo further reactions to give rise to different types of by-products. In one pathway, it abstracts hydrogen to form hydroperoxide and subsequently alkoxyl radical on reduction. Alkoxyl radicals can disintegrate to form several by-products. However, in the absence of oxygen, the initial alkyl radical forms cross-link with another adjacent carbon-based radical to form aggregates [23]. A schematic illustration of oxidative stress-mediated damage to peptide chains has been presented in Figure 3. Attack by ROS brings about effects such as structural modification, denaturation, aggregation of proteins, and loss/abnormal functioning of the protein and enzymes.

Figure 3.

Oxidative damage of peptide backbone by reactive oxygen species. Adapted from [23, 24].

4. Role of oxidative stress in the pathogenesis of cancer

Carcinogenesis is a multi-step process in which genetic alterations, oncogene activation, tumor suppressor gene deactivation, epigenetic modifications, and subsequent perturbations of signal transduction pathways bring about uncontrolled and abnormal growth, proliferation, and differentiation of cells [25]. Oxidative stress is found to be involved in the genesis of several types of cancer [26]. ROS-induced modifications in nucleic acids may cause mutation of genes and damage to DNA strands. These in turn can lead to catastrophic events like oncogene activation and tumor suppressor gene deactivation, which are primary events leading to carcinogenesis.

Oxidative stress can also modulate the signal transduction pathways of a cell by either directly modifying a key molecule that initiates a pathway or by activating/deactivating a molecule which in turn activates a signal transduction pathway. Signaling pathways activated by ROS such as mitogen-activated protein kinase (MAPK) pathway, phosphatidylinositol-4,5-bisphosphate 3-kinase-protein kinase B (PI3K-AKT) pathway, Kelch-like ECH associating protein 1-nuclear factor erythroid 2-related factor 2 (Keap 1-Nrf2) pathway, Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway, wingless-related integration site-β-catenin (Wnt-β-cat) pathway, p53 pathway, SRC pathway (non-receptor tyrosine kinase initiated pathway), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway lead to the activation of several downstream targets like transcription factors which promote cellular proliferation and survival, inflammation, epithelial-mesenchymal transition (EMT), invasion and angiogenic capacity [27, 28]. Structural alteration via oxidation of crucial amino acid residues in receptors (leading to activation of a pathway even in the absence of ligand), downstream kinases and inhibitor proteins (that otherwise keep the key signal transducer in an inactive form) may be involved behind erroneous activation of signaling pathways [29].

Keap-1-Nrf2 pathway is activated when ROS oxidatively deactivates Keap-1, which under normal conditions keeps the transcription factor Nrf-2 in an inactive form. Once activated, Nrf2 moves to the nucleus and activates transcription of oxidative stress-responsive genes, which upregulate the endogenous antioxidant level of the cell [30, 31]. In cancer cells, ROS levels are higher than in normal cells due to chronic inflammation and altered metabolism. In the presence of excessive ROS beyond a certain level, programmed cell death mechanisms are initiated. To maintain survival, cancer cells constitutively express an antioxidant defense system [30]. The PI3K-AKT pathway is normally inhibited by the phosphatase and tensin homolog (PTEN) by dephosphorylation of PI3K. ROS has been found to inhibit PTEN activity by forming a disulfide bridge between cysteine residues, leading to the activation of the pathway [32]. AKT regulates the expression of an array of cellular proteins supporting cell survival and proliferation, metabolism, and angiogenesis [33]. MAPKs are a family of kinases with members like extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, which are involved in the mediation of life processes such as cellular proliferation, differentiation, apoptosis, and immunological activities [27]. They are majorly activated when a signaling ligand binds to its receptor, but ROS can activate the receptor even in the absence of the ligand which leads to the activation of the signaling cascade [34]. Initiation of signal transduction pathways by ROS leads to the activation of several transcription factors like NF-κB, hypoxia-inducible factor-1α (HIF-1α), activator protein- 1(AP-1), p53, STAT-3, Nrf-2, etc. [27, 28, 35]. Apart from activation by signaling pathways as downstream effectors, ROS can activate both NF-κB and HIF-1α by deactivating their inhibitors directly. ROS can also modify the inhibitory component of NF-κB, inhibitors of NF-κB (IκBs), or activate NF-κB-inducing kinase (NIK), thereby leading to activation of the transcription factor [27, 36]. Activated NF-κB is involved in the induction of genes for cellular survival and proliferation, inflammatory mediators like cytokines and chemokines, invasion and metastatic potential, angiogenesis, and cellular metabolism and also mediates the induction of changes at genetic and epigenetic level in cells, thereby setting the base for carcinogenesis [37]. HIF-1α is activated by ROS via oxidation-based deactivation of its inhibitor, prolyl hydroxylase domain protein 2 (PHD2) [38]. Activated HIF-1α is involved in the transcription of genes that support angiogenesis, metabolism, cellular survival, and proliferation, which augments carcinogenesis [39]. AP-1 on being activated regulates expression of genes related to cellular growth and death, angiogenesis, and upregulated metastatic potential [40]. Transforming growth factor-β (TGF-β) is a crucial cytokine that promotes EMT (via transcription factors like Snail and Slug), angiogenesis, and evasion of immune response in cancer cells. In normal cells, TGF-β exhibits a tumor-suppressor role, but in transformed cancer cells, it manifests pro-carcinogenic activity. It is normally secreted by cells in a latent form, associated with a latency-associated peptide (LAP), and is sequestered in the ECM. On being activated by any form of stimulus, it binds to its receptor and initiates either canonical (SMAD-mediated) or non-canonical pathways (MAPK, PI3-AKT, etc.). A dynamic interplay occurs between ROS and TGF-β in cancer cells where ROS not only increases the expression of TGF-β but also diffuses out of the cell and activates latent TGF-β by modifying the associated LAP domain. In turn, TGF-β acts to increase the ROS content in cells via both direct ROS generation (mediated by NOX-4) or by downregulating the cellular antioxidant status. Moreover, ROS also mediates several pathways induced by TGF-β leading to EMT, like the NF-κB pathway [41, 42]. Therefore, several factors and phenomena induced by ROS act in synergy to induce carcinogenesis in cells. A schematic representation of oxidative stress generation and its carcinogenic effects in a cell has been presented in Figure 4.

Figure 4.

Schematic illustration depicting the carcinogenic effects of reactive oxygen species and its sources in a cell.

Another important aspect regarding ROS is its ability to modulate the tumor microenvironment (TME) that promotes tumor aggressiveness and cancer metastasis. Tumor microenvironment is composed of several types of cells—cancer-associated fibroblasts (CAFs), tumor-infiltrating leukocytes (like myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) and T cells), mesenchymal stem cells, endothelial cells, lymph and blood vessels, and extracellular matrix (ECM) components [43]. ROS, inflammatory cytokines, and chemokines released by cancer cells trigger infiltration of immune cells to the tumor site and modulate characteristics of cells in the immediate environment. Infiltrated immune cells further aggravate the oxidative stress load of TME via the activity of their intracellular pro-oxidant enzymes like NOX, MPO, etc. [44]. ROS (like H₂O₂) released by cancer cells can diffuse into the extracellular environment and modulate the differentiation of CAFs into subtypes like myofibroblasts, which can modulate TME and promote cancer. Myofibroblasts are a type of CAF that secrete ECM modulators and can therefore promote cellular migration [45]. Cancer cell-generated ROS drives stress generation in CAFs and metabolically re-wires them to switch to a glycolytic mode of energy generation and undergo autophagy and subsequently provide substrates like pyruvate and lactate to cancer cells for enhanced survival [46].

Immune cells summoned by tumor cells into TME act to suppress immunoreactivity against tumor, which facilitates cancer metastasis and promotes resistance to therapy [47]. ROS has been found to impede cytokine production by T cells and induce their hypo-responsivity [48, 49]. ROS in TME regulates the immunosuppressive activity by T_reg cells. Under conditions of microenvironmental oxidative stress, T_regs undergo apoptosis and the apoptotic T_regs secrete large amounts of ATP and convert it into adenosine with the help of membrane molecules. Adenosine binds to A_2a receptors in nearby T cells and suppresses their cytokine production leading to subdued T-cell reactivity [50]. ROS generated by tumor cells via the activity of NOX-4 facilitates TAM polarization to pro-tumorigenic phenotype (M2), which facilitates malignancy [51]. In response to oxidative stress generated by tumor cells, activated immune cells too generate oxidative stress intracellularly, which not only induces their differentiation into pro-carcinogenic phenotype but also influences the activity of other immune cells in the vicinity. ROS generated by MDSCs suppresses the proliferation and function of T cells and promotes cancer cell growth [52, 53]. In TME, immature myeloid cells are induced to produce ROS that in turn restricts their differentiation into mature immune cells like macrophages, dendritic cells, and granulocytes [54]. High levels of ROS generation in TAMs upregulate the secretion of tumor necrosis factor-α that facilitates cancer metastasis [55]. When cultured in tumor cell-conditioned media, activation of myofibroblasts was triggered via enhanced intracellular ROS generation [56]. Therefore, a dynamic interplay exists between cancer cells and the surrounding cells in TME (both immunological or non-immunological), where ROS plays a significant role as a mediator. ROS acts both as an initiator and perpetuator of cancer and, as we will discuss in subsequent sections, can also be employed in cancer therapeutics. A minimal amount of ROS is required for normal cell functioning, whereas a high level of oxidative stress leads to carcinogenesis as already discussed. However, further increase in ROS level beyond a threshold triggers cell death pathways like apoptosis and autophagy [27].

5. Polyphenols: potential candidates as chemotherapeutics

Polyphenols are plant-based active compounds that are originally secondary metabolites of plants, present in their fruits, leaves, and seeds. As the name suggests, their structure characteristically contains phenol rings with specific substitutions at various positions. Polyphenols are a huge family of phytochemicals with more than 8000 known compounds [57]. From the functional point of view, polyphenols have been found to have antioxidant and anti-inflammatory effects on cells and thereby can be used as a preventive agent against the development of disorders arising from the state of chronic inflammation like cardiovascular disease, neurodegenerative disorders, atherosclerosis, diabetes, hypertension, and cancer to name a few [58, 59].

On the basis of their structure, polyphenols are broadly categorized into four major groups, namely, flavonoids, phenolic acids, stilbenes, and lignans [60]. Apart from these, a category of polyphenols encompasses miscellaneous members like curcuminoids (http://www.phenol-explorer.eu).

5.1 Polyphenols as pro-oxidants

Just like the two sides of a coin, polyphenols in addition to their antioxidant capacity also act as pro-oxidants under conditions of high concentration and pH and in the presence of transition metal ions (Cu^2+/Fe³⁺) [61, 62]. Polyphenols reduce metal ions and themselves get converted to a phenoxyl radical. In the presence of molecular oxygen, the phenoxyl radical gets converted to a quinone, and a superoxide anion is generated. This superoxide anion then forms hydrogen peroxide. Hydrogen peroxide in the presence of transition metal ions forms hydroxyl radicals via Fenton reactions or Haber-Weiss reaction in the presence of superoxide anion [61]. Several in vitro studies have found that polyphenolic compounds caused DNA damage in cells when transition metals are present [63, 64, 65, 66]. Polyphenols reduce copper ions (Cu²⁺) bound to chromatin, and it leads to the generation of ROS, as discussed above, in very close vicinity to DNA molecules, and the ROS generated thereby causes DNA damage [64]. Cancer cells, owing to their metabolically re-programmed condition and chronic inflammatory status, survive in a state containing higher oxidative stress, altered pH level, and high concentration of transition metal ions [67, 68]. As a high ROS level beyond a certain threshold level activates signaling pathways leading to programmed cell death, the pro-oxidative feature of polyphenols in cancer cells further increases the oxidative stress load in them, leading to the induction of cell death. This characteristic is exploited in the usage of polyphenols as chemotherapeutic agents. A schematic diagram of pro-oxidant activity of phenols in high oxidative stress and metal ion concentration in cancer cells has been illustrated in Figure 5.

Figure 5.

Pro-oxidant activity of phenols in cancer cells under conditions of high oxidative stress and transition metal ion concentration. (I) Phenols react with metal ions to form phenoxyl radical and reduced metal ions, (II) Phenol reacts with superoxide anion to form phenoxyl radical and hydrogen peroxide, (III) Phenoxyl radical in presence of molecular oxygen undergoes further oxidation and forms quinone and superoxide anion, (IV) Superoxide anion undergoes spontaneous dismutation in presence of cellular proton to form hydrogen peroxide and oxygen, (V) Reduced metal ion and hydrogen peroxide react to generate hydroxyl radical. Adapted from [61].

Several phenomena lead to the generation of ROS by flavonoids and it includes their transition metal or pH-dependent auto-oxidation, oxidation in the presence of both molecular oxygen and transition metals, oxidation of cellular NADH (nicotinamide adenine dinucleotide hydrogen) and antioxidants by the intermediate phenoxyl radicals, and impairment of mitochondrial respiration [69]. Pro-oxidant activity is also a function of the concentration and structure of flavonoids. At higher concentrations, flavonoids exert a pro-oxidant effect instead of acting as an antioxidant [70]. The number and position of hydroxyl groups in the second phenol ring and the occurrence of the double bond in the pyran ring are important for pro-oxidant activity of flavonoids [61].

The direct pro-oxidant effect of phenolic acids is prominent in the presence of transition metal ions and is also a function of their structure and oxidation potential. In the presence of Cu²⁺ ions, hydroxycinnamic acid derivatives (caffeic acid, chlorogenic acid, sinapic acid, and ferulic acid) induced oxidative stress-mediated DNA damage. The presence of orthohydihydroxyl group and 4-hydroxyl-3-methoxl group in the compounds markedly enhanced their pro-oxidant functionality. Monohydroxylated derivatives like 3-hydroxycinnamic acid and 4-hydroxycinnamic acid did not depict DNA damaging activity even in the presence of Cu²⁺ ions. Moreover, compounds with lower oxidation potential were found to have greater pro-oxidant ability [71]. Structural dependence of phenolic acids for pro-oxidant ability was also observed in the study conducted by Khan and Hadi, where it was found that the DNA cleaving potential of syringic acid was lower than that of gallic acid due to methylation of the hydroxyl groups, whereas decarboxylation of gallic acid leads to enhancement of DNA damaging potential as is observed in pyragallol [72]. Rosmarinic acid has also been found to induce oxidative stress-mediated DNA damage in the presence of transition metals (Cu/Fe) [73]. Tannic acid, ellagic acid, and gallic acid were found to have antioxidant activity in the Chinese hamster fibroblast cell line, but the effect was reduced in the presence of hydrogen peroxide and Cu²⁺ ions. Moreover, these phenolic acids induced thiol oxidation, protein carbonylation, DNA damage, and apoptosis in tested cell lines individually (at higher concentrations) and the effect was more conspicuous in the presence of Cu²⁺ ions [74, 75].

In the case of stilbenes too, structure, concentration, and presence of transition metals play an important part in facilitating pro-oxidant effects. Resveratrol, oxyresveratrol, and piceatannol (hydroxylated derivatives of trans-stilbene) caused ROS generation and ds break in plasmid DNA in the presence of Cu²⁺, whereas this effect was not observed in the case of trans-stilbene. Moreover, the DNA-damaging activity was observed only at higher concentrations of oxyresveratrol [65, 76]. Resveratrol and piceatannol have also been observed to induce DNA damage in lymphocytes in the absence of added Cu²⁺, possibly via mobilization of intracellular Cu²⁺. However, in that case, much higher dosage of the compounds was required to be administered [77]. Structure-activity analysis study conducted with resveratrol and seven of its synthetic structural analogs has found that the presence of orthodihydroxyl group or 4-hydroxyl group containing compounds has higher pro-oxidant activity along with the enhanced capacity for inducing DNA damage [78].

6. Polyphenols in cancer therapeutic research: a brief overview of select few

Polyphenols exhibit antioxidant and anti-inflammatory effects, which accentuate their chemopreventive roles. However, they also act as pro-oxidants in cancer cells because of the altered cellular environment, an attribute that is harnessed for facilitating anti-tumorigenicity or anti-carcinogenicity in chemotherapeutics. In recent times, novel chemotherapeutic strategies are being designed to incorporate polyphenols into mainstream treatment regimens either as an individual component or as an adjunct with traditional chemotherapeutic drugs like doxorubicin, paclitaxel, etc. In this section, a few polyphenols will be discussed which are known to have potent chemotherapeutic properties, whose structures (along with the basic structures of the major groups of polyphenols) have been depicted in Figure 6.

Figure 6.

(I–VI) Basic structures of polyphenols. I. Flavonoids; II–III., Phenolic acids, Hydroxybenzoic acid, and Hydroxycinnamic acid, respectively; IV. Stilbene; V. Curcuminoid (R1, R2: H/OCH3); VI. Lignan. (VII– X) Structures of discussed polyphenols. VII. Curcumin; VIII. Caffeic acid; IX. Resveratrol; X. Podophyllotoxin.

6.1 Curcumin

Curcumin (diferuloylmethane, Cur), a curcuminoid, is obtained from the rhizome of Curcuma longa, generally called turmeric. Cur can act as an antioxidant molecule by virtue of its structure (the β-diketo group, carbon-containing double bonds, and phenyl ring with hydroxyl and methoxy substituents). The antioxidant role of Cur in normal cells protects it from ailments resulting from oxidative stress. In a study conducted by Dange et al., it was observed that feeding mice Cur (post-irradiation) prevented the development of radiation-induced thymic lymphoma [79]. However, in radio-resistant pancreatic cancer cell line, Cur was found to induce radio-sensitivity leading to DNA damage induction, cell cycle arrest, and apoptosis on irradiation [80]. This contrasting role of Cur provides evidence supporting its differential activity in normal and cancerous cells. Moreover, the dosage of Cur used in therapeutic procedures may also decide the pro- or anti-oxidant role of Cur [81].

Several in vitro studies have observed a pro-oxidant role of Cur in the presence of Cu²⁺ ions, and the generated ROS led to DNA damage and cellular death [63, 82]. The presence of a conjugated beta-diketone moiety in the structure of Cur is speculated to be accountable for the pro-oxidant effect [63]. As presented in Table 2, Cur has shown toxicity in cancer cells originating from various organs by increasing the oxidative stress level. Cur induces senescence or cell death in exposed cells in the form of apoptosis, autophagy, ferroptosis, and pyroptosis by increasing the level of oxidative stress in them. Both the mitochondrial dysfunction-mediated intrinsic pathway and Toll-like receptor-mediated extrinsic pathway of apoptosis have been found to be induced by Cur, based on the type of cancer cell [87]. Some mechanisms by which it mediates pro-oxidative anticancer effects include the downregulation of cellular antioxidant enzymes, induction of damage to DNA and cellular lipids, induction of mitochondrial dysfunction, inhibition of DNA repair proteins, induction of pro-apoptotic proteins, and repression of anti-apoptotic proteins, modulation of the activity of cell cycle regulatory molecules and pathways like JAK-STAT3, ERK, p38, and p53. Cur has also been found to be suitable as an adjunct to traditional chemotherapeutic drugs. It acts synergistically with the drugs to induce anti-tumorigenicity and sensitizes chemo-resistant cells to chemotherapeutic drugs [92, 93].

Compound	Test model	Effect	Ref.
Curcumin	Osteosarcoma cell lines	ROS generation, anti-proliferative, cell cycle arrest, apoptosis/ ferroptosis, anti-invasive	[83]
	Mouse model xenografted with cancer cells	Anti-tumorigenic
	Non-small-cell lung cancer cell line	ROS generation, anti-proliferative, DNA damage, G2/M cell cycle arrest, apoptosis, inhibits colony formation	[84]
	Hepatocellular carcinoma cell line	ROS generation, anti-proliferative, apoptosis/pyroptosis	[85]
	Colorectal cancer line	ROS generation, anti-proliferative, anti-migratory, apoptosis/ senescence, activation of ROS-Nrf2-miR 34a/b/c signaling cascade.; Potentiated activity of 5-fluorouracil	[86]
	Mouse xenograft model	Anti-tumorigenic
	Liver cancer cell line	ROS generation, anti-proliferative, apoptosis	[87]
	Lung cancer cell line (chemo-resistant)	ROS generation, anti-proliferative, apoptosis, p38 activation	[88]
	Cervical cancer cell line	ROS generation, anti-proliferative, G2/M cell cycle arrest, apoptosis, autophagy, cellular senescence (via p53-p21 induction)	[89]
CUR and Cisplatin: Nano-Liposome encapsulated	Hepatocellular carcinoma cell line	ROS generation, anti-proliferative (synergistic effect), apoptosis (synergistic effect), ERK, and p53 activation	[90]
	Mice model with xenografted tumor	Increased circulation and residence time, anti-tumorigenic, increased bioavailability, reduced toxicity.
WZ26 (CUR analog)	Cholangiocarcinoma cell line	More potent activity than Cur itself, ROS generation, anti-proliferative, inhibits migration, apoptotic, G2/M cell cycle arrest, inhibits STAT3 signaling	[91]
	Mice model with xenografted cancer cells	Anti-tumorigenic
CUR in combination with Cisplatin	Papillary thyroid cancer cell lines	ROS generation, anti-proliferative, apoptotic, anti-migratory, inhibition of matrix metalloproteinases, synergistic effects with cisplatin, decrease stemness of cancer stem cells, inhibit JAK-STAT3 pathway	[92]
	Breast cancer cell line	ROS generation, inhibition of DNA repair protein, sensitized resistant triple negative breast cancer cells to carboplatin, apoptosis	[93]
Caffeic acid (CA)	Colon cancer cell line	ROS generation, anti-proliferative, apoptosis	[94]
	Breast cancer cell lines	ROS generation, apoptosis	[95]
	Chronic myeloid leukemia cell line	ROS generation, anti-proliferative, apoptosis, activation of transglutaminase type 2	[96]
CA N-butyl ester	Lung cancer cell line	ROS generation, DNA damage, anti-proliferative, necrosis	[97]
CA-phenyl ester	Myeloma cell line	Oxidative stress, DNA damage, anti-proliferative, apoptosis	[98]
	Normal peripheral blood cells	Non-toxic
	Medulloblastoma cell line	ROS generation, anti-proliferative, apoptosis, anti-invasive, inhibition of NF-κB; Radio-sentitization	[99]
CA-3,4-dihydroxy-phenethyl ester	Breast cancer cell lines	ROS generation, anti-proliferative, apoptosis	[100]
CA-Bortezomib-Iron-conjugated nanomedicine	Colon carcinoma cells (Mice)	ROS generation, DNA damage, Inhibit NF-κB. Complement apoptotic action of the drug	[101]
	Normal fibroblast cell	Nominal toxicity
CA, TRF, nano-emulsion Cisplatin	Lung cancer cell line; Hepatocellular carcinoma cell line	ROS generation, apoptosis.; Complements the chemotherapeutic action of Cisplatin by reducing the dosage required and toxicity	[102]
	Normal human embryonic kidney cells	Lowered toxicity. Viability significantly high in comparison to only cisplatin treatment
Poly CA-Bortezomib-Folic acid coated Au-NP; nanomedicine	Squamous cell carcinoma cell line	ROS generation, anti-proliferative, apoptosis, inhibits NF-κB	[103]
	BALB/c nude mice xenografted with cancer cells	Anti-tumorigenic
Resveratrol (RSV)	Cervix cancer cell lines	ROS generation, anti-proliferative, autophagy/mitophagy	[104]
	Normal cell line	Non-toxic (very high IC50)
	Melanoma cell line	ROS generation, ER stress, cell cycle arrest, anti-proliferative, apoptosis, Induction of p38 and p53	[105]
	Colon cancer cell line	ROS generation, anti-proliferative, autophagy, apoptosis	[106]
	Ovarian cancer cell lines	ROS generation, apoptosis, inhibit Notch1 and AKT	[107]
	Anaplastic thyroid cancer cell line	ROS generation, apoptosis	[108]
	Prostate cancer cells (Mice)	ROS generation, anti-proliferative, anti-invasion, apoptosis, induction of p53 and HIF-1α	[109]
	Pancreatic cancer cell lines	ROS generation, apoptosis (downregulated NAF-1), induction of Nrf-2; Sensitize cells non-responsive to Gemcitabine	[110]
	Breast cancer cell lines; Lung cancer cell line	ROS generation, DNA damage, anti-proliferative, senescence (via induction of deleted in liver cancer-1)	[111]
	Normal breast epithelial cell line	Non-toxic
	Cancer stem cells derived from the human ovarian cancer cell line	ROS generation, apoptosis. Reduced Sox, Nanog expression in surviving cells. Reduced cell renewal capacity in surviving cells	[112]
	Human normal fibroblast	Non-toxic
RSV-nanomedicine *,#	Colorectal cancer cells	ROS generation, anti-proliferative, lipid peroxidation, reduced invasive capability, ferroptosis	[113]
	Mice (nude) xenografted with colorectal cancer cells	Anti-tumorigenic
Podophyllotoxin (PT)	Colorectal Cancer Cells	ROS generation, ER stress, G2/M cell cycle arrest, anti-proliferative, inhibition of anchorage-independent growth, p38 activation, apoptosis	[114]
PT acetate	Non-small cell lung cancer	Radio-sensitization, increased apoptosis in combination with radiation, ROS generation, inhibits ERK, activates p38	[115]
	Mice (nude) xenograft model	Anti-tumorigenic
Ptox^DPT	Hepatocellular carcinoma cell lines	ROS generation, anti-proliferative, anti-invasion, inhibit matrix metalloproteinases, expression, downregulate expression of vimentin, Snail and Slug, upregulate expression of E-cadherin and p53, inhibition of PI3K/AKT/mTOR pathway	[116]
PT-based PPM-DDS with cucurbitacin B (micelles)	Multi-drug-resistant lung cancer cell lines; Mice (nude) model xenografted with cancer cells	ROS generation, anti-proliferative, anti-tumorigenic	[117]
DPT	Prostate cancer cell lines (chemoresistant)	ROS generation, G2/M cell cycle arrest, apoptosis	[118]
	Non-small cell lung cancer cells (Gefitinib-resistant)	ROS generation, ER stress, G2/M cell cycle arrest, anti-proliferative, inhibit anchorage-independent growth, apoptosis. Chemo-sensitization by reducing activity of receptor tyrosine kinases (EGFR and MET and downstream kinases ERK and AKT)	[119]
DPMA	Non-small cell lung cancer cells	ROS generation, hamper microtubule formation, induce p53 expression, apoptosis	[120]
	Mesenchymal stem cells	Lower level of toxicity
	Human umbilical vein endothelial cells	Anti-angiogenic
PPT	Esophageal Squamous Cell Carcinoma Cell line	ROS generation, ER stress, G2/ M cell cycle arrest, anti-proliferative, inhibit anchorage independent growth, apoptosis, activation of JNK-p38 pathway	[121]
	Colorectal Cancer Cells	ROS generation, ER stress, cell cycle arrest, anti-proliferative, apoptosis, p38 activation	[122]

Table 2.

Compilation of studies showing chemotherapeutic effects of polyphenols and their derivatives alone or in combination with traditional anticancer drugs.

Abbreviations (not mentioned in text): WZ26: (1E,4E)-1-(3-bromo-4-hydroxyphenyl)-5-(4-hydroxy-3-methoxyphenyl)penta-1,4-dien-3-one; TRF: tocotrienol; ER: endoplasmic reticulum; NAF-1: nutrient-deprivation autophagy factor-1; PPM-DDS: polymeric prodrug micellar-based drug delivery systems; *: RSV-loaded poly (ε-caprolactone)-poly (ethylene glycol) (PCL-PEG) nanoparticles (NP) encapsulated with red blood cell (RBC) membrane (RSV-NPs@RBCm); #: RSV-NPs@RBCm co-delivered with a tumor-penetrating peptide iRGD (RSV-NPs@RBCm&iRGD); Ptox^DPT: combinatorial compound of 4 ′-demethylepipodophyllotoxin and dithiocarbamate (di-2-pyridineketone hydrazone dithiocarbamate S-propionate podophyllotoxin ester); DPT: deoxypodophyllotoxin (analog of PT); DPMA: 2,6-dimethoxy-4-(6-oxo-(5R,5aR,6,8,8aR,9-hexahydrofuro[3’,4’:6,7]naphtho[2,3-d][1,3] dioxol-5-yl)phenyl ((R)-1-amino-4-(methylthio)-1-oxobutan-2-yl) carbamate (derivative of DPT); EGFR: epithelial growth factor receptor; MET: mesenchymal epithelial transition; PPT: picropodophyllotoxin (epimer of PT).

Due to constraints like its poor solubility in water and low bioavailability, its concentration level in plasma does not reach the values that are required for it to be a potential therapeutic agent. Hence, newer drug delivery systems are being designed like metal-organic frameworks, stimuli-sensitive nanocarriers with added ligands for target-specific homing of drugs, or encapsulation in nano-sized liposomes, which carry curcumin alone or in combination with other chemotherapeutic drugs. These techniques lead to enhanced drug retention, improved drug targeting to tumor site, better uptake by tumor cells, and higher efficacy in inducing anti-tumorigenic response along with reduced toxicity [90, 123, 124]. Moreover, several analogs of Cur are either being isolated or synthesized, e.g., terpene-conjugated analogs (bisabolocurcumin ether, demethoxybisabolocurcumin ether) and WZ26, which have shown better potential as anti-cancer agents in comparison to Cur [91, 125].

6.2 Caffeic acid

Caffeic acid (3, 4-dihydroxycinnamic acid, CA) is a hydroxycinnamic acid derivative and is found in a variety of vegetables, fruits, and beverages like coffee. Several compounds are derived from CA including its phenyl-, methyl-, ethyl-, butyl-, and benzyl ester, chlorogenic acid, methyl caffeate, methyl dihydrocaffeate, octyl caffeate, ferulic acid, and cichoric acid. CA is a potent antioxidant involved in the direct scavenging of reactive species [126]. However, a high concentration of caffeic acid in the presence of transition metal ions can lead to the generation of its pro-oxidant nature [127]. As already mentioned in the previous section, CA can act as a pro-oxidant when transition metal ion like Cu²⁺ is present and the ROS generated thereby induces DNA damage [71]. A low level of ROS generation by CA might also facilitate their pro-survival or antioxidant effects in normal cells by induction of pro-survival pathways like ERK [128].

The potential of both CA and its derivatives has been investigated in several studies and it has been found that in cancerous cells, it exerts a pro-carcinogenic effect, whereas in normal cells, they are either non-toxic or have nominal toxicity (Table 2). Therefore, they have a selective action profile which asserts their candidature as potent anti-cancer agents. The mechanisms by which CA and its derivatives induce pro-oxidative anticancer effects mainly include induction of oxidative stress by either ROS generation and/or depletion of cellular antioxidants, DNA damage, apoptosis, mitochondrial dysfunction, and inhibition of pro-survival pathways like NF-κB (data compiled in Table 2).

Caffeic acid (CA) as nanomedicine or as nano-emulsions has been tested with chemotherapeutic drugs like bortezomid and cisplatin [102, 103]. CA-Bortezomid not only have selective toxic effects on cancer cells, these have also shown anti-tumorigenic effects in mice cancer model that has been xenografted with tumor cells [103]. Chemotherapeutic drugs have severe side effects and sometimes cancer cells become unresponsive to treatment. Such conjugated nano-medicines overcome this problem, resulting in increased uptake of drugs, lessening the amount of effective dosage, and diminishing systemic toxic responses. CA was observed to be effective in inducing cell cycle arrest and apoptosis in chronic myeloid leukemia cells, which are resistant to Imatinib mesylate [129]. In addition to CA, its natural derivatives have also been found to induce anticancer effects in cancer cell lines. Therefore, there is scope in areas of active drug designing too, where novel CA derivatives can be designed and newer anticancer treatment options can be investigated.

6.3 Resveratrol

Resveratrol (trans-3,4′,5-trihydroxystilbene, RSV) is the most abundantly studied component of the stilbene family of polyphenols. RSV has been extensively studied as an anticancer molecule in cancer cell lines and in in vivo models (Table 2). It selectively induces toxicity in cancer cell lines and is non-toxic in normal cells. The mechanisms by which RSV induces pro-oxidative anticancer effects include induction of oxidative stress either by depleting the existing reserve of antioxidant enzymes or by inhibiting their activity, induction of mitochondrial dysfunction and endoplasmic reticulum stress, induction of cell cycle arrest by inducing changes in expression level of cell cycle regulatory molecules, causing interference in pathways involved in generation of energy (glycolysis and oxidative phosphorylation) [104], modulation of signaling pathways involving transcription factors and kinases like p38, p53, NF-κB, HIF-1α, Sox, Nanog, AKT, and Notch signaling, induction of apoptosis, autophagy, ferroptosis, and cellular senescence (data compiled in Table 2).

Studies have shown that RSV reduces the self-renewal ability of cancer stem cells and even sensitizes resistant cells to chemotherapeutic drugs like gemcitabine [110, 112]. Development of nano-medicines using RSV is also in the process, where RSV-containing nanoparticles are enabled to escape cellular immune defense and more efficiently reach the target site [113]. These nanoparticles have also been found to efficiently execute their anti-tumorigenic activity in mice model. In a few studies conducted in pancreatic cell line, it was observed that RSV counteracted hypoxia and hyperglycemia induced ROS-driven metastasis [130, 131]. These observations depict the chemo-preventive role of RSV. In a study conducted by Zheng et al., it was found that between two cell lines of thyroid cancer, one showed susceptibility to RSV treatment, while the other showed resistance [108]. The resistant cell lines encode higher levels of enzymes involved in the metabolism of RSV. These studies highlight the fact that the type of effect a polyphenol will adopt depends on the characteristics and type of malignancy and it is crucial that the treatment approach be tailored accordingly.

6.4 Podophyllotoxin and related compounds

Podophyllotoxin (PT) is a lignan present in the root and rhizome of the herb Mayapple (Podophyllum peltatum). It is a cyclo-lignan of the tetra-aryl group that exerts anti-tumor activity by inhibiting microtubule assembly and DNA topoisomerase II [114]. Synthetic anticancer drugs like etiposide and teniposide are used in conventional chemotherapeutic practices, whose structure is based on podophyllotoxin. Etiposide is known to act by inhibiting the activity of DNA-topoisomerase II [120]. Apart from PT itself, several structurally related compounds like picropodophyllotoxin (epimer of PT) and deoxypodophyllotoxin (an analog of PT) have also been found to have anticancer activity in in vitro studies (Table 2). Synthetic compounds have been designed by modifications of the basic structure of PT or conjugation of PT with other compounds, to derive novel chemical structures with anticancer activity that improves bioavailability, reduces systemic toxic effects, and even counters multi-drug resistance developed by cancer cells [115, 116, 132]. Apart from that, nanomedicines are being designed and synthesized which are modified in a way that facilitates better drug circulation, enhanced drug uptake by tumor cells, and more efficient anti-tumor activity. Two such studies have reported the development of nanomedicines (prodrug loaded nanoparticles), which utilize the oxidatively stressed and acidic environment of cancer cells as cues for site-specific activation and release of pro-drugs, and ultimately induce apoptosis by augmenting the oxidative stress of cancer cells [117, 133]. Liang and Zhou [133] studied the anti-tumor effects of nanoparticles formed from conjugation of homodimers of PT and vitamin K3 into a biocompatible polymer (Pluronic F127). Intracellular metabolism of vitamin K3 increased the oxidative load of cells, which further triggers the release of active PT and acts in synergy to induce cellular apoptosis. In normal cells, due to a lower concentration of ROS, the release of pro-drug is not sufficient to induce cellular death. Xenografted mice model too showed higher anti-tumor activity of the nanodrug due to its higher bioavailability and lesser systemic toxicity [133]. The mechanisms by which PT, its derivatives, and PT-based drugs induce pro-oxidative anticancer effects include induction of oxidative stress and endoplasmic reticulum stress, cell cycle arrest (majorly a G2/M arrest), mitochondrial dysfunction, microtubular damage, modulation of transcriptional factors and signaling pathways like p38, p53, epithelial cadherin, vimentin, snail and slug, inhibition of PI3K-AKT-mTOR pathway, ERK, and induction of oxidative stress-mediated apoptosis (data compiled in Table 2).

7. Conclusion

Reactive oxygen species (ROS) can act as both a cause and a cure for cancer. In the present chapter, the prime focus was directed toward discussing the mechanisms by which polyphenolic compounds exert their anti-cancer effects by increasing the oxidative stress level in cancer cells. However, polyphenols can also employ other mechanisms to combat cancer. In a study conducted by Win et al., it was observed that genistein inhibited DNA damage induced by hydrogen peroxide and Cu²⁺, whereas resveratrol at similar concentration range potentiated the effect and itself caused strand breaks in the presence of metal ions [134]. It indicates that polyphenols can work via different mechanisms to have anti-tumorigenic effects and some polyphenols act as pro-oxidants to deliver the job. Structural characteristics enhancing the antioxidant properties of polyphenols also govern their pro-oxidant roles, which are switched on majorly when metal ions are present or in altered pH conditions. The presence of a metal redox cycle (e.g., Cu²⁺/Cu¹⁺), formation of polyphenol-metal intermediate complex, or stabilization of intermediate radical are prominent factors that govern the dynamics of the pro-oxidation effect of polyphenols [78]. Orally administered polyphenols undergo extensive metabolism by gut microbial population and phase I and II metabolizing enzymes in the liver and the intestines. The metabolites thus generated may have altered bio-potential. The concentration of polyphenols in systemic circulation is therefore restricted, which may further be subjected to selective absorption in cells due to the presence of membrane efflux pumps. Owing to these factors, it is difficult to achieve a proper effective concentration of polyphenols required to have therapeutic effects, if administered orally. Moreover, the administration of polyphenols in high dosages may lead to the generation of systemic toxicity [135]. Therefore, instead of oral administration, targeted delivery is desirable to manifest anticancer effects. To overcome issues like low bioavailability, reduced circulation time, and improper drug release, nano-sized drug delivery systems (micelles, liposomes, emulsions, metal-, polymer-, and cyclodextrin-based nanoparticles) along with homing ligands and pH/oxidative stress-responsive pro-drug delivery systems are being tested and developed that ensures targeted drug delivery and enhanced anti-tumorigenic response, as already discussed in previous sections. Moreover, the implementation of polyphenol-based chemotherapeutic approaches, either as a mono-therapeutic agent or as an adjunct, is subjective to the cancer type and cellular metabolic profile, as a vast range of heterogeneity exists between different types of cancer. Therefore, extensive in vivo and clinical trials are required before the implementation of polyphenol-based anticancer drugs in chemotherapy. This area of research demands a continuous influx of knowledge, ideas, and scientific improvisations which is the need of the hour.