Non-communicable diseases including cancer are the major cause of global deaths. According to a recent estimate, 18.1 million people have been newly diagnosed for cancer and 9.6 million deaths being recorded globally in 2018. This high rate of mortality can be prevented if detected early. As the treatment strategy varies with the pattern, type and form of cancer, management of this disease requires further development of better therapeutics and preventive strategies. Broadly, cancer shows the following seven characteristic features: uncontrolled proliferation, self-sufficient growth factors, contact-independent growth, absence of apoptosis, abnormal angiogenesis, increased inflammatory response and prevalence of metastasis and invasion. The abnormal cellular physiology of cancerous cells is due to altered molecular patterns at genomic, epigenomic, transcriptomic, proteomic and metabolomic levels. Unlike genetic control which was described as the trigger for evolutionary mechanisms, epigenomics deals with the study of a set of processes (above the DNA) involving DNA methylation, histone modifications and remodeling, and miRNA expressions. These epigenetic modifications play a vital role in chromatin remodeling for the regulation of gene expression. Altered epigenetic patterns have been strongly associated with cancer related events (1, 2). Recent studies have confirmed that mitochondria regulate epigenome either by controlling the concentration of the cofactors or by the redox-mediated alteration of the activities of epigenetic modifiers (3-5). However, finding potent epigenetic targets for therapeutic intervention intended for cancer prevention is an emerging aspect where different epigenetic inhibitors/modulators are being discovered, validated and verified (6-8). Over the centuries, many herbal and medicinal plants and their active constituents have been analyzed as possible remedies for several chronic and metabolic diseases. Secondary metabolites from plants, such as polyphenols including alkaloids, flavonoids, and stilbenes, which are present in small quantity have been tested for preventive management of several non-communicable diseases such as diabetes, cardiovascular diseases, asthma, neurological disorders and cancer (9-11). Interestingly, these molecules not only possess anti-oxidant, anti-inflammatory, cyto-protective and geno-protective properties but also modulate epigenomic re-programming through a mitochondrial mediated pathway (12-14).

Mitochondria not only control cellular oxidation via oxidative phosphorylation but also regulate programmed cell death, calcium concentration, metabolite concentrations, and diverse signaling pathways. These multifaceted roles make it dynamic controllers of cellular health and disease. Moreover, mitochondria may directly influence genome methylation, covalent histone modification, and expression of miRNA arrays to eventually alter the expression of target nuclear genes (15-17). However, the therapeutic outcome of flavonoids is limited due to reduced bioavailability as a result of limited solubility, poor permeability and pre-systematic metabolic effects. The other mechanisms involved in restricting the application of flavonoids include metabolism by gut micro flora, absorption across the intestinal wall, active efflux, and susceptibility to modification by environmental factors such as temperature, pH and light. In addition, recent studies have suggested that mitochondria are the central target of mechanistic basis of flavonoid’s mode of action, thus modulating the epigenetic machinery and complex cancer signaling, thereby suppressing the cancer progression programming. Therefore making a nanocarrier based system targeting mitochondria would offer a suitable strategy (Figure 1). Our previous studies have also explored numerous anti-cancer biological activities of the flavonoid-rich contents of an important medicinal herb Selaginella bryopteris (Sanjeevani) (11, 14). Our recent preclinical study demonstrated how nano-engineered flavonoid rich fraction isolated from S. bryopteris (NP.SB) act through mitochondria to alter the epigenetic landscape from the tumorigenesis route to normal cellular physiology (14). This contemporary approach offers a promising strategy to treat cancer with minimal side effects.

Figure 1.

An outline sketch of the proposed nano-engineered flavonoid based approach for effective mitochondrial targeting, maintenance of epigenomic machinery and induction of protective anti-cancer effects.

Epigenetic modifications play a vital role in gene regulation beyond the genetic material and exhibit stable meiotic and mitotic inheritance. Such epigenetic processes include covalent methylation of 5-cytosine of DNA, covalent modifications of N-terminal amino acid moieties of nucleosomal histone cores, and regulation through non-coding miRNA expressions. These modifications are important, intricate underlying mechanisms for maintenance of chromatin remodeling status. Epigenetic modifications occur commonly in all cell types which instruct genes either to turn off or on. Thus, cells with same genetic material respond differentially depending upon the internal and external environmental cues. Basically, epigenetic modifications are catalyzed by epigenetic modifiers known as writers (that add a specific modifying moiety); erasers (that remove a specific modifying moiety); and readers (that recognize and bind to specific modified moieties). Several distress points observed in the above mechanisms are implicated in various forms and types of cancers (18-22).

miRNAs are small non-coding RNAs transcripts of 21 to 23 nucleotides, which play a major role in gene expression regulation. Unlike regulation by DNA methylation and histone modifications at the genetic and epigenetic levels, microRNA functions at the post-transcriptional level. Molecular mechanisms of miRNAs functions involve binding to its complimentary mRNA where complete binding induces mRNA degradation and partial binding causes mRNA silencing (45). miRNAs are the important regulators of various fundamental processes such as growth, differentiation, stress response, and cellular homeostasis. miRNAs are known to interact with oncogenes and tumor suppressor genes and are thus involved in the control of tumor development and progression, invasion, metastasis, and in epithelial–mesenchymal transition of several cancers (46). Analysis of miRNA profiling suggests that some of miRNA are upregulated, and some are down-regulated in different sets of cancer (47).

Nutraceuticals are bioactive molecules with nutritional value such as dietary food components including phytochemicals. Phytochemicals are a group of secondary metabolites such as polyphenols, flavonoids, terpenoids, xanthones, organo-sulfur compounds, phytosterols, alkaloids, and carotenoids, which are known to have curative properties for diverse human ailments. Presence of hydroxyl groups and aromatic ring in polyphenols categorizes them into following subclasses: flavonoids, phenolic acids, stilbenes, and lignans. Flavonoids are widely distributed cluster of seven subgroups categorized on the basis of their chemical structures. There are several representative bioactive compounds in each subgroup of flavonoids (see Figure 2). These are synthesized via phenyl propanoid pathway, which converts phenylalanine into 4-coumaroyl-CoA to produce flavonoids. All subgroups of flavonoids consist of the same structural backbone of two phenolic rings A and B connected by the third oxygen-containing heterocyclic C ring (see the ring structures in Figure 3 and 4). These subgroups are named as flavones, flavonols, flavanones or dihydroflavones, flavanonols or dihydroflavonols, isoflavones or phytoestrogens, flavanols or catechins or anthocyanins, and proanthocyanins (Figure 3 and 4).

Figure 2.

The flow diagram showing detailed classification of polyphenols.

Figure 3.

A combined figure showing structures of different flavonoids (a) Flavones that consists of 4H-chromen-4-one with a phenyl substituent at position 2 and are found in fruits and vegetables, including onions, apples, broccoli, and berries, thyme, parsley, celery and capsicum pepper. (b) Flavanones or dihydroflavones which consists of flavan bearing an oxo substituent at position 4. It is derived from a hydride of a flavan and is generally found in grapes, orange and lemon juice. (c) Flavonols, a monohydroxyflavone that comprises a class of compounds with 3-hydroxy derivative of flavone. It is a conjugate acid of a flavonol (1-) and is found in green and black tea, yellow onions, apples, broccoli, black grapes, tomato, red wine, carrot, cherry, tomato, curly kale, leek, lettuce, nuts, walnuts, ginger, and blueberry.

Figure 4.

Figure showing structural representation of (a) flavanonols or dihydroflavonols are a class of flavonoids with 3-hydroxy-2,3-dihydro-2-phenylchromen-4-one backbone and are generally isolated from sources such as lemon, sour orange, cocoa, cocoa beverages, and chocolates. (b) Isoflavones or phytoestrogens are a class of polyphenolic compounds which consists of 4H-chromen-4-one ring in which the hydrogen at position 3 is replaced by a phenyl group and are generally found in sources such as soya bean, legumes, red clover chickpeas, peanuts, soy cheese, soy flour and tofu. (c) Flavanols or flavan-3-ol is a hydroxyflavonoid which are found in various sources such as green and black tea, apple, chocolate, beans, apricot, cherry, grapes, peach, red wine, cider, and blackberry. (d) Anthocyanidins, which are the salt derivatives of the 2-phenylchromenylium cation, also known as flavylium cation and most commonly found in blue berries, black grapes, and red wine, blackcurrant, cherry, rhubarb, plum, red cabbage, and cocoa.

These molecules have the potential to alter the health status as they have molecular targets within the cell, however, timing of food consumption, absorption, metabolism, and excretion are vital along with the presence of other factors like the use of tobacco and alcohol. Being a part of our ancient system of medicine, phytochemicals-based therapy is emerging as one of the most efficient therapies not only against cardiovascular and metabolic diseases, but also in cancer treatment (Table 1). Previous preclinical studies have demonstrated the therapeutic aspects of these bioactive molecules. Anti-oxidative, anti-inflammatory, anti-proliferative, cyto-protective and geno-protective effect are the potential therapeutic outcome of these flavonoids (11, 14, 47, 48). These therapeutic properties of flavonoids are due to their interaction with the intermediate of cell signaling proteins. Major regulatory mechanisms of phytochemicals involve modulation of redox reactions, enzyme activities, mitochondrial-retrograde responses, and cellular signaling pathways that significantly alter the metabolome and gene expression or vice-versa (Figure 5) (49, 50). Flavonoids possessing epigenetic restoration property have also been reported (51, 52).

Figure 5.

The diagrammatic representation of different mechanistic aspects of flavonoids

Epigenetic modifications are greatly affected with the change in the environmental conditions and act as interface between the gene and environment. Defined concentration of polyphenols in the diet can modulate epigenome-mediated genomic expressions essentially by controlling the metabolic fate of a cell hence influencing the mechanisms underlying human health and disease (9, 51, 53-55). Accumulative literature on epigenetically active polyphenols suggests that these molecules have anti-cancer effects as these can potentially manipulate the activities of epigenetic modifiers such as writers (DNMT, HAT, and HMT) and erasers (HDAC, KDM) (52). A diet containing epigenome-modifying and chemo preventive nutraceuticals influence the level of bioactive metabolites that in turn modulate cellular pools of methyl donor groups, acetyl-CoA, NAD+, and ATP, resulting in altered DNA methylation pattern and histone and non-histone protein acetylation/methylation/phosphorylation (3-5). For instance, alteration in DNA and histone methylation level is associated with the imbalance in the SAM/SAH ratio. A significant change in the SAM/SAH ratio level has been reported following ingestion of flavanol-rich diets (56-59). In vitro and in vivo tumor/cancer models treated with specific polyphenolic bioactive components have revealed efficient antitumor potential by stimulating the signaling pathway for programed cell death mediating epigenetic machinery (60). Such observations are enabling the modifiers of epigenetic mechanisms to emerge as anti-cancer therapeutics. Therefore, currently a powerful tenet is to explore how dietary molecules may influence prevention and treatment of various chronic diseases based on nutri-epigenomic intervention. Recently, the anti-cancer therapeutic potential of some bioactive compounds with the epigenetic reversal ability have been tested in clinical trials (see Table 2) (61-64).

Flavonoids or mitochondria-targeted drugs act through redox-dependent and redox-independent regulation of nuclear gene expression. Although many studies have emphasized the anti-oxidative properties of flavonoids, their effect on mitochondria in a redox-independent manner has not been extensively studied (65). However, individual cancer type has different cellular and molecular disorientation due to abnormal expression of susceptible genes. Therefore, a tumor cannot be treated effectively by targeting a distinct gene or protein or a single signal transduction pathway (11, 14, 47, 66). Since, several intra- and extracellular signaling pathways are governed by mitochondria and therefore, it is the primary site of action for flavonoids and also promotes apoptosis and inhibition of cell growth (Figure 6). Various mitochondria-targeted flavonoid compositions have been analyzed for their anti-cancer effects. Numerous mitochondria-targeted drugs have also been identified and validated for their anti-cancer effects (67-69) and based on their mode of action, these mitochondria-targeted anti-cancer drugs as classified as mitocans. However, (68) Gorlach et al., described the molecular mechanisms of each mitocan underlying anti-cancer activity.

Figure 6.

Scheme illustrating the vital role of mitochondria in regulation of major cellular processes involved in signaling, cell cycle, autophagy, apoptosis, angiogenesis, and epigenetic modifications.

Further, harboring healthy mitochondria greatly impact the redox status and related processes. Most observation favors the flavonoid induced upregulation of mitochondrial biogenesis through increase in the PGC-1α level. Recent reports have shown that (-)-epicatechin increase biogenesis by G-protein coupled estrogen receptor, fisetin enhance biogenesis by downregulation of glycogen synthase kinase 3β activity, and digitoflavone upregulate biogenesis by enhancing AMPK phosphorylation (70-72). However, results are conflicting for quercetin, as it can both increase and attenuate this process (73).

Recent experiments have uncovered the intricate role of mitochondria in the epigenetic regulation of several processes involved in human health and disease (13, 16, 74). There are two important routes through which mitochondria regulate the epigenetic machinery. Firstly, as the hub for the metabolic processes, the mitochondrion holds several tiny co-factors and substrates that are utilized in the epigenetic modification processes, such as the methyl group from the SAM (indirect source of methionine) for DNA and histone methylation, the acetyl group from acetyl-CoA for histone acetylation, and ATP for histone phosphorylation (15). Second, being the center for the oxidative mechanisms, mitochondria can oxidatively modify the catalytic properties of the mitochondrial and cellular enzymes and proteins thereby affecting the epigenetic process and redox signaling. For instance, mitochondria alter the activity of sirtuins, which are a class of NAD-dependent deacetylases. Sirtuin 6 has been implicated in ageing, many metabolic diseases and explored for therapeutic interventions (75).

Flavones such as apigenin, chrysin, and baicalein are potential anti-neoplastic and anti-carcinogenic agents. The anti-cancer activity of these flavonoids is mediated through activation of mitochondria-dependent apoptotic pathway, which has been validated through in vitro and in vivo studies (76-81). Induction of mitochondria-dependent apoptotic pathway in peripheral blood lymphocytes from B-chronic lymphocytic leukemia patients and leukemia cell lines have been observed following chrysin treatment (78). Chrysin disturbs the mitochondrial membrane potential that causes release of cytochrome C, up-regulate the pro-apoptotic Bax and caspase-3 and down-regulate the anti-apoptotic Bcl-2 protein that in turn result in apoptosis/cell death (78). Additionally, silibinin, in breast cancer, triggers mitochondria-mediated cell death. Basically, silibinin induces ROS-dependent mitochondrial dysfunction leading to ATP depletion by involving BNIP3 to initiate autophagy (79). Similarly, apigenin also causes mitochondrial dysfunction leading to mitochondria-mediated apoptosis in hepatocytes of HCC rats (80). Apigenin-loaded PLGA nanoparticles targeted at the nucleus damage DNA and subsequently induce mitochondria-mediated apoptosis in tumor cells (76). Moreover, baicalein follows a similar path in the gastric cancer cell line where it induces S phase arrest, down-regulate Bcl-2, up-regulate Bax and disrupt mitochondrial membrane potential in dose dependent manner (81). Additionally, mitochondriotropic nanoemulsified genistein-loaded carriers have shown pro-apoptotic anti-cancer effects due to mitochondrial damage through depolarization-mediated cytochrome C release (82). Moreover, a pro-oxidant action upon treatment of colorectal cancer with 5-Hydroxy-7-methoxyflavone was observed due to ROS triggered mitochondria-mediated apoptosis (77).

Several preclinical evidences have suggested that flavonoids possess anti-cancer properties; however, proper exploitation of the associated health benefits is limited. Primarily, limitations are due to poor bioavailability as a result of low solubility, insignificant permeability, first-pass metabolic effects, metabolism by gut micro flora, and absorption across the intestinal wall, active efflux mechanism, and easy modification by environmental factors such as temperature, pH, and light. Moreover, differences in isolation, purification, treatment strategy, interpretation, and analysis create an additional level of challenge in using flavonoids as anti-cancer agents.

To allow clinical applicability of dietary flavonoids, exploration of various approaches has been reviewed.

Extensive work in the recent times have been done for the development of a delivery system with ability to surpass biological barriers; avoidance of premature deactivation of bioactive molecules; and elevation of the intracellular availability of the drugs at their target site (123). Multifaceted cellular regulatory mechanisms mark mitochondria as a critical target for the cancer-prevention strategy. Specific characteristics of mitochondria include mitochondrial trans-membrane electrochemical gradient, protein import machinery, pH difference, and fusion process. These advantages are also being exploited for developing targeted strategies, which are known as mitochondria medicine (124). For mito-targeting of therapeutic agents, approaches involve either loading of drugs into surface-modified nanoparticles or conjugation of the targeting moiety with model drugs. In this context, specialized nano-delivery systems are being utilized such as delocalized lipophilic cations, polymeric NPs, mitochondrial targeting peptide, carbon nanostructures, blended nanoparticles, metal nanoparticles, and mesoporous silica nanoparticles (Figure 7). Uptake of mitochondria targeted nano-carrier is shown in the Figure 8.

Figure 7.

Detailed classification of different nanocarriers based strategies for effective flavonoid delivery and mitochondrial targeting.

Figure 8.

Schematic representation of the delivery mechanism of different mitochondria-targeted nanocarriers

Several groups have illustrated the combinatorial effects of flavonoids with anti-cancer agents in reducing drug resistance, tumor recurrence and metastasis through nano-engineered systems. The mitochondrial targeting strategy through flavonoids may serve as an effective therapeutic approach to combat cancer stem cells (CSCs). For instance, one such combinatorial synergistic approach using doxorubicin and quercetin was demonstrated by Fang et al., using hyaluronic acid tethered mesoporous silica nanoparticles for gastric carcinoma chemotherapy (138). Signaling pathways such as Hh, Wnt/β-catenin and Notch pathways playing an important role in differentiation of normal stem cells offer an attractive approach to target CSCs. Wu et al., in their earlier study demonstrated anti-proliferative and apoptosis inducing activity of 2’ Hydroxyflavanone (2HF) via blockage of the Akt/STAT3 signaling pathway in prostate cancer. The subsequent studies further investigated the effect of 2HF on EMT, and cell migration and invasion using Wnt/β catenin signaling pathway by suppressing GSK 3 β phosphorylation, β catenin expression and transactivation (104). There have been several reports where the anti-tumor and anti-metastatic effects of flavonoids have been demonstrated. Dai et al., (139) reported the anti-carcinogenic effect of bacailin using tumor xenograft model reported antitumor activity of silybin in human HCC HepG2 using both in vitro and in vivo conditions (140). The results suggested a decrease in the expression of the Notch1 intracellular domain (NICD), RBP-Jκ, and Hes1 proteins, upregulation of apoptosis pathway-related protein Bax, and downregulation in Bcl2, survivin, and cyclin D1. Lim et al., illustrated caused a dose-dependent growth inhibition of various brain tumor cell cultures and neurospheres through curcumin encapsulated in polymeric nanoparticles, effectively blocking the CSC-associated Hh pathway and reducing IGF and STAT3 levels (141).

Several evidences suggesting the use of plant-derived phytoconstitutents in the treatment of chemo-resistant cells have reported. Mohammadian et al., reported significant inhibitory effect on AGS human gastric cell line than free chrysin (142). The study also has elucidated successful restoration of tumor suppressor miRNA expression levels. Minimization or reversion of drug resistance using MCF-7/ADR tumor was well demonstrated by Lv et al., using biotin conjugated doxorubicin and quercetin co-loaded polymeric nanoparticles. The resultant nanoparticle system reported a significant inhibition of the activity and expression of P-glycoprotein and in MCF-7/ADR cells both in vitro and in vivo conditions (143). A synergy in the combinantion therapy of paclitaxel and difluorinated curcumin was observed by Gawde et al., against gynaecological tumors (144).

Mitochondrial stress associated oxidative radical injury may alter cellular epigenome which in turn may initiate the process of carcinogenesis, therefore, an effective protective agent should be capable of maintaining epigenetic stability. Analysis of post-translational histone modifications showed that NP.SB potentially protects B/CMBA cells from epigenetic injury following exposure to pro-oxidants (14). In recent study, we observed that in addition to protecting mitochondria from pro-oxidants induced alterations, encapsulated SB fraction (NP.SB) significantly protects cellular epigenome. Exposure of cells to pro-oxidants resulted in significant epigenomic alterations, as evident from increased methylation of H3K9me1 and H4K20me3 and phosphorylation of H3 and H2A histones. While hyperubiquitination of uH2A/uH2B and hypoacetylation of AcH3 further highlighted the seriousness of these alterations following pro-oxidants exposure. In contrast, no such alterations were observed among cells pre-treated with NP.SB. Altered histone modification pattern modulates and affect DNA methylation machinery, which thus alters the expression of different genes important for cellular integrity and polarity. In addition, studies have shown that dysregulated mitochondrial functioning results in the activation of retrograde signalling cascade which consequently affects nuclear gene arrangements (145, 146). In a recent study, NP.SB pre-treatment regulated DNA methylation at p16 promoter regions even after pro-oxidants treatment. Furthermore, the un-altered levels of let-7a and let-7b in NP.SB pre-treated cells showed the ability of this encapsulated fraction to protect cells from transcriptional dysregulations due to altered histone profile, which were significantly reported among pro-oxidants alone treated cells.

Nutri-epigenomics is an emerging field in terms of dosage, targeting, tissue distribution, bioavailability, molecular and/or cellular interactions that limits the clinical utility of several plant secondary metabolites including flavonoids. Since, cancer is characterized by both genetic and epigenetic instability; it requires multifaceted protective strategies for effective tumour cell targeting while conferring better protection of normal cells from adverse drug-induced toxicity. In this regard, nano-engineered systems encapsulating bioactive flavonoids holds great promise for improved delivery and selective tumour cell targeting with better efficacy and pharmacokinetic properties. Recently, several nanocarrier based approaches, including polymeric nanoparticles, SLNs, liquid crystals, liposomes, and microemulsions have been employed to overcome the limitations associated with clinical use of flavonoids. Overall, nano-engineered systems offer attractive opportunities for the safe, effective, sustained and targeted delivery of flavonoids to reshape the mitochondrial mediated epigenetic regulation for cancer therapeutics. However, challenges such as inquesting their targeting efficiency, meeting up the regulatory international standards and feasibility of scale-up are few concerns which warrants attention for effective bench-to bedside translation of flavonoids as broad spectrum cancer protective agents.

The authors are thankful to Council of Scientific and Industrial Research (CSIR) and University Grant Commission (UGC), Government of India, New Delhi for providing necessary assistance. The award of Junior and Senior Research Fellowships to NB by Department of Biotechnology (DBT), Government of India, New Delhi is gratefully acknowledged.