Cancer Therapy Challenge: It Is Time to Look in the “St. Patrick’s Well” of the Nature

Cancer still remains a leading cause of death despite improvements in diagnosis, drug discovery and therapy approach. Therefore, there is a strong need to improve methodologies as well as to increase the number of approaches available. Natural compounds of different origins (i.e., from fungi, plants, microbes, etc.) represent an interesting approach for fighting cancer. In particular, synergistic strategies may represent an intriguing approach, combining natural compounds with classic chemotherapeutic drugs to increase therapeutic efficacy and lower the required drug concentrations. In this review, we focus primarily on those natural compounds utilized in synergistic approached to treating cancer, with particular attention to those compounds that have gained the most research interest.


Introduction
Cancer is considered, in our century a leading cause of death and the single most significant obstacle to increasing life expectancy in every country in the world.
So, cancer remains a worldwide challenge with significant influence, not only on human health, but also on the global economy.
According to World Health Organization (WHO) data, cancer is reported to be the first-or second-most common cause of death in people under 70 years of age in 91 of 172 countries, and the third-or fourth-most common in an additional 22 countries [1]. Neoplasm incidence and mortality are fast rising all around the world, reflecting both the population's increasing growth and age.
In addition, the escalating rise of tumours as a cause of death is nearly equal to the noticeably declining mortality rates for coronary heart disease and stroke in many countries. It is interesting, today, to notice that changes in tumour incidence are most pronounced in emerging economies. There, history is repeating itself in the shift from poverty-and infection-related malignancies to those diseases that are already dominant in developed areas (e.g., in North America and Europe). These kinds of cancers are often described as "caused by the westernization of lifestyle", however, the differing cancer profiles in several countries and regions show that marked geographic disparities still exist; in fact, there are local risk factors persisting in populations at quite different phases of social and economic transition. Table 1. Natural products, their effects on cancer cells and their synergy with chemotherapeutics.

Common Sources Mechanisms of Action Chemotherapy Drug and Synergistic Effects
Ascorbate // Ascorbate's toxicity on cancer cells is due to hydrogen peroxide formation with ascorbate radical as an intermediate state [15].
Ascorbate showed synergistic effects in in vitro [15] and in vivo experiments with common anti-cancer drugs [16]. Ascorbate is used in synergistic approaches at mM concentrations.

Neem extract Azadirachta indica
Neem components' modify the tumour environment, decreasing vessel formation [22]. They are employed against cervical, breast, and ovarian cancer.
Neem extract showed synergistic effects with [23]: -Cisplatin -Genduin Capsaicin Red and chili peppers The antitumor mechanism of capsaicin increases apoptosis and cell cycle arrest [24] Capsaicin (at µM concentrations) showed synergistic activities with other agents, such as resveratrol and genistein [25].
It is possible to be dosed up with 5-FU [42].
It has shown suppression activity on colon cancer carcinoma cells (HT-29), and also inhibited colon cancer growth in a xenograft model. Its activity is related to the ability to arrest cell cycles in the G0/G1 phase and to induce apoptosis. GLT allows the formation of autophagous vacuoles and increases the expression of some proteins, like Beclin-1 and LC-3. Autophagy is facilitated by p38 MAPK inhibition [43].
It has shown anti-growth effects on two tumoral cell lines (HCT116 and DLD-1), however, cannabidiol derivatives are ineffective against healthy cells' proliferation [50]. In vivo, CBD extract proved effective in reducing pre-neoplastic lesions and polyps (induced through azoxymethane) as well as in xenograft models [50]. In addition, CBD displayed chemopreventive effect on HCT116 and Caco-2 cell lines, protecting them from oxidative damage and decreased cell proliferation through CB1, TRPV1, and PPARg [47].

Flavonoids
Several plant organs and compounds.
Flavonoids are able to promote a protective effect on cells against cancer evolution in several ways [13].

Epigallocatechin (EGCG) Camellia Sinensis
It acts on cell signalling. In fact, it can stop both the growth and migration of CRC cells. These effects are due to the inhibition of the TF/VIIa/PAR2 signalling pathway, which is very important for inducing ERK1/2 phosphorylation and activation of NF-kB. In particular, EGCG reduces NF-kB transcription factor activity, induces increased expression of caspase-7, and decreased expression of MMP-9 [51]. Moreover, epigenetic process could be regulated by EGCG, it allows the ubiquitination of colorectal cells sensitive to methylation, helping in DNMT3A (DNA methyltransferase 3A) and HDAC (histone deacetylases) degradation [52,53] EGCG showed synergistic effect with 5-FU [54]. In the HT-29 cell line, genistein up-regulates the expression of Bax, p21 proteins and glutathione peroxidase expression. However, it can also inhibit some molecules like NF-kβ, topoisomerase II and MMP2 (this last function helps to prevent mestastases in CRC patients) [55,56] Genistein showed synergistic effects with [57]: For in vivo studies mice were injected 50 mg genistein/kg body weight.

Apigenin Several plants species
Apigenin induces colorectal cell growth inhibition, decreases angiogenesis, promotes cell arrest and apoptosis in vitro [58]. In both in vitro and in vivo studies the expression of NAG-1, P53 and p21 (cell cycle inhibitor) are increased by apigenin, reducing intestinal tumour load and number [59]. Apigenin is able to inhibit ABC receptors that increase the efflux of a chemotherapeutic agent in cancer cells, on the other hand it increases their bioavailability [55].
Chrysin honey, propolis, chamomile and martyrs (Passiflora caerulea) Chrysin has shown, in HCT116, DLD-1 and SW837 cells, the ability to induce cell apoptosis. This compound increases TNFα and TNFβ genes, activating the TNF and AHR (aryl hydrocarbon receptor) signalling pathways [61]. Moreover, chrysin can induce a cell cycle arrest at the G2/M transition phase, as seen in a study on the colorectal cell line SW480 [62].
Isoliquiritigenin has shown a synergistic effect with cisplatin [64].

Kaempferol Propolis and several plants
Kaempferol increases chromatin condensation and DNA fragmentation. It is also able to increase the cleavage of caspase-9, caspase-3, and caspase-7. Together, this is how this compound induces apoptosis [65].

Quercetin
Many types of vegetables and fruits.
Quercetin is an antiproliferative compound. It prevents the activation of RAS and inhibits migration and invasion [66,67].

Artesunate
Artemisia annua Artesunate displays anti-proliferative activity, for these reasons it is a potential anticancer agent. It is cytotoxic, allowing it to induce cell cycle arrest in the G1 phase. Furthermore, Artesunate has shown the ability to reverse immunosuppression in the cancer microenvironment [68].

Natural Product Common Sources Mechanisms of Action Chemotherapy Drug and Synergistic Effects
Gossypol Gossypium In tumour cells, Gossypol can inhibit proliferation, inducing apoptosis [75]. It was tested on CRC and prostate cancer [75].

Cruciferous vegetables
It has an important antitumoral effect, blocking cytochromes P450, and also inducing the phase II detoxification enzymes glutathione S-transferases, and promotes the elimination of carcinogens from the organism [76]. Cell metastasis and its tumoral properties (migratory and invasive mechanisms) can be inhibited by allyl isothiocyanate [77].

Allysulfates Alliaceae
They suppress proliferation and induce apoptosis by increasing the production of ROS in cancer cells.

Ethanol extract of Aaptos suberiotides Aaptos suberiotides
The ethanol extract of Aaptos suberiotides inhibits cell proliferation and migration in HER2-Positive breast cancer [80].
It can reduce resistance to Trastuzumab [80].

Ethanol Extract of Marine Sponge
Stylissa carteri Stylissa carteri This extract inhibits growth and migration, and also induces apoptosis in breast tumour cells [82].
It has shown a synergistic effect with [82]: Cecropins show cytotoxic activity only against cancer cells (leukaemia, colon carcinoma, stomach, small cell lung and ovarian cancer with a multi-drug resistant phenotype) [83].

Mycalamide A/B and Onamide extracted from sponges
Mycale sp. and Theonella sp.
These inhibit the cell-free translation of RNA in cancer cells (leukaemia) [84].
Emericellipsin A

Coriolus vescicolor
PSK inhibits the growth of various types of tumours (fibrosarcoma, colon adenocarcinoma). Data suggest that PSK-induced immunity is tumour-specific and that T lymphocytes play an important role in antitumor memory functions [86].
It has shown a synergistic function with 5-FU [86].

Common Sources Mechanisms of Action Chemotherapy Drug and Synergistic Effects
AraC

Cryptotethya crypta
It induces apoptosis through many mechanisms. AraC, and its different metabolites, contribute to its cytotoxicity, the including incorporation of AraCTP into DNA and AraUMP into RNA, inhibition of polymerase α and β, and the impairment of repair mechanisms [87]. Besides this DNA synthesis impairment, AraC causes several signalling events, the including activation of PKC and MAPK [88] and the upregulation of AP-1 and NF-κB [89][90][91].
It has shown a synergistic effect with [92]: cantharidin

Blister beetles
It causes apoptosis by a p53-dependent mechanism in leukaemia cells. Cantharidin induces both DNA single-and double-strand breaks [96].
It has shown synergy with Tamoxifen [97].

Natural Compounds and Sinergy
After the extensive review of many natural compounds of different origins, in the present work we want to focus, in particular, on those compounds that, over the years, have collected the greatest interest from researchers, demonstrating synergistic behaviours with some drugs used in the therapies of different tumors ( Table 2).

Ascorbic Acid
Ascorbate is an important redox cofactor and catalyst for many biochemical reactions. In humans, it cures or prevents scurvy (this word comes from skjoerberg or skorbjugg that, in the Scandinavian language, mean 'rough skin') [98][99][100].
Vitamin C is contained in several well-known plants and fruits, but also in animal organs (brain, kidney, liver), yeasts and prokaryotes (but not in cyanobacteria) [101]. In superior plants, vitamin C is obtained from D-glucose, and it is involved in many metabolic processes, for example the scavenging of H 2 O 2 [102], the maintenance of the α-tocopherol pool [103] and, also, it behaves like violaxanthin deoxidase cofactor [104].
In animals, ascorbic acid is synthetized from glucose by enzyme L-gluconolactone oxidase, and has been found in the kidneys of reptiles but, in mammals, it is found in the liver. Most animals can synthesize ascorbic acid, but some species (humans, other primates, guinea pigs, Passeriformes birds and flying mammals) must get it from their diets. This condition is due to a deficiency in gluconolactone oxidase that does not allow the conversion of L-gluconolactone to 2-keto-L-gluconolactone [105]. Table 2. Natural compounds currently used in cancer treatment and described in the chapter "Natural Compounds and Sinergy".

Natural Product Synergy
Ascorbic acid Some studies have demonstrated the synergistic effects of ascorbate with other classic anti-cancer drugs.

Curcumin
Phase II clinical studies have suggested that a combination of gemcitabine and curcumin is a conceivable treatment for pancreatic cancer patients.

Resveratrol
Resveratrol exhibits synergistic effects with anti-cancer drugs, such as doxorubicin, cisplatin and vinorelbine.

Capsaicin
Some studies have suggested combinational use of capsaicin with other chemotherapeutics drugs or dietary molecules.
Vitamin C, as highlighted in Vitamin C and the Common Cold [106], is linked to the immune system. This is confirmed by the rapid decrease in ascorbate and leucocytes during stress and infection [106]. Ascorbate is also an antiviral agent; it acts against viruses aiding in the degradation of their nucleic acids [107][108][109]. Furthermore, Vitamin C accelerates the destruction of histamine, a molecule that mediates allergy and cold symptoms, reducing it by 30-40% [110][111][112].
Recently, Vitamin C was studied on some chronic diseases, in particular, atherosclerosis. An ascorbate deficiency (<0.2 mg/dL) is inversely related to this condition [113,114]. Atherosclerosis is related to the oxidation of low-density lipoprotein, but the consumption of Vitamin C by hospitalized patients can reduce myocardial infarction [115] and, in acute smokers, which have two-fold LDL oxidation levels, ascorbate supplementation can reverse LDL peroxidation [116][117][118].
The poor intake of Vitamin C and E is related to an increase in fractures, especially in female smokers, up to five-fold. However, among women smokers with a high intake of both vitamins (>200 mg/day), the probability of fractures is not increased [119].
The use of ascorbate as a cancer therapy is under controversy. Seventy years ago William McCormick [120] described how tumour patients often showed low blood levels of vitamin C and featured scurvy-like symptoms, leading him to assume that vitamin C might protect against cancer by increasing collagen synthesis. In 1972, Ewan Cameron theorised that ascorbate could suppress cancer development by inhibiting hyaluronidase, weakening the extracellular matrix and enabling tumours to form metastases. In 1976, Cameron and Pauling published a study of 100 patients with terminal neoplasms treated with ascorbate. Even though the study was not guided by modern clinical standards, mainly because they lacked a placebo control group, their results revealed that ascorbate-treated patients showed improved quality of life of life and increased mean survival time [121,122]. Other clinical trials have independently indicated similar results. So, interest in the potential of ascorbate for tumour treatment grew.
However, double-blind randomized clinical trials directed by Charles Moertel of the Mayo Clinic failed to show any positive effects of high-dose vitamin C in cancer patients [123]. So, the enthusiasm for the results obtained by the Cameron-Pauling trials was dampened by these data and the research on ascorbate was silenced for many years.
At the beginning of the 2000s, some studies at the National Institute of Health (NIH) established dietary recommendations for ascorbate [124,125]. When people received oral doses, low plasma concentrations of ascorbate were achieved (around 100-200 µM), while intravenous administration allowed 100-fold higher concentration than oral (around 15 mM) [126]. This is due to partial intestinal absorption, excretion and renal re-absorption during oral administration. Intravenous administration evades this control, allowing high plasmatic concentrations [126]. So, a high ("pharmacologic") ascorbate level is achievable only with intravenous administration, not with oral administration ("physiologic" level). So, while only pharmacologic vitamin C level could be considered as a drug, the attention for using ascorbate as an anti-tumour agent has re-emerged.
After the basic information about ascorbate pharmacokinetics was understood, some studies described the effects of ascorbate on cancer cells. The in vitro analyses showed that ascorbic acid, at around 20 mM concentration, is able to selectively kill cancer cells, without affecting normal cell lines [127]. Additionally, other authors found that ascorbate toxicity in cancer cells was due to hydrogen peroxide formation, with ascorbate radical as an intermediate [128,129].
Some studies also explored the intravenous administration of vitamin C in cancer patients. Padayatty and co-workers and Hoffer and colleagues demonstrated that intravenous ascorbate, at high doses, is well tolerated by patients with different cancer types [126,130]. Other studies have highlighted that ascorbate. administered intravenously, improves quality of life of life in cancer patients [131].
Some studies concentrated their attention on the intravenous effect of ascorbate in cancer patient's survival. Ascorbate treatment could increase quality of life and decrease chemotherapy-related side effects in cancer patients [130,132].
Moreover, in vitro tests [15,133] and in vivo xenografts have also demonstrated the synergistic effects of ascorbate with other classic anti-cancer drugs [16]. Recently, some clinical studies are demonstrating that ascorbate, in combination with chemotherapeutic drugs, displays promising clinical efficacy [134,135].

Curcumin
Curcumin has fascinated humankind since ancient times due to its numerous biological effects, including anti-antioxidant, inflammatory and antitumor abilities [136]. Curcumin is obtained from the rhizome of Curcuma longa (ginger family) and recognised, from a chemical point of view, as 1,7-bis-(4-hydroxy-3-methoxyphenyl)-hepta-1,6-diene-3,5dione. Curcuma longa extracts and its components have been utilized in traditional Chinese medicine for thousands of years.
A huge number of experiments, both in vitro and in vivo, have established that curcumin could impede various cancers' growth including ovarian, gastric and colorectal neoplasms, by activating apoptosis.
Although it is well tolerated by patients, curcumin is poorly adsorbed by organisms, therefore, it is very difficult to utilize. Some approaches have tried to improve its bioavailability; in particular, the main strategies are its combination with adjuvants, the utilization of chemical analogues and the development of novel delivery approaches.
The pharmacodynamics data available for humans are limited and there is debate as to whether its efficacy is due to some curcumin components, or to other mechanisms, acting indirectly. At present, several clinical trials (phase I or II) are ongoing to investigate the benefits of curcumin as a chemo-preventive and chemotherapeutic agent in a variety of tumours [137][138][139].
A clinical study (phase I) on twenty-five patients with different lesions both (premalignant and high-risk lesions) revealed that oral curcumin can be chemopreventive [140,141]. The Cleveland Clinic carried out research with five patients with familial adenomatous polyposis, who were treated three times a day with a combination of curcumin and quercetin for a mean duration of 6 months. Data showed that polyps' number and size were decreased in all patients, compared with controls [142].
Carrol et al. [143], in a recent phase II clinical trial, investigated curcumin and its potential activity for prevention of colorectal neoplasia in smokers with aberrant crypt foci (ACF). The results showed a significant reduction of ACF number by a 4-g dose curcumin at the level and indicated that curcumin may have cancer-preventive effects against pre-invasive neoplastic lesions [143].
Unfortunately, interpretation of this study is limited, asACF is a controversial biomarker of colon carcinogenesis.
Human pancreatic cancer treatment with curcumin has been evaluated in a phase II clinical trial. Curcumin (8 g) was administered to 25 patients orally, daily, with restaging every two months, of whom 21 were evaluable for response [139].
Some phase II clinical studies suggested that a gemcitabine and curcumin combination is a conceivable treatment for pancreatic cancer patients [144].
In a study by Bayet-Robert et al. [145], 14 advanced and metastatic breast cancer patients were treated with curcumin and docetaxel combined therapy. The research confirmed that this combination reduced the level of vascular endothelial grow factor (VEGF) with optimistic results [146].
EGCG represents more than 50% of the total catechins, is one of the best-studied constituents of green tea and appears to be the most effective one. EGCG seems promising for chemoprevention according to in vitro, animal, clinical and epidemiological studies. EGCG can induce the reduction of some cancer cell lines' growth and apoptosis in vitro [127,147], inhibiting tumour incidence in in vivo experiments, for example colon, liver, lung, mammary glands, pancreas, prostate, and skin neoplasm models [148].
Anticancer effects ascribed to EGCG are: antioxidant activities, apoptosis induction, carcinogen metabolism modification, cell cycle arrest, DNA damage prevention, metastasis inhibition modulation of multiple pathways of signal transduction and proteasome blocking [149].
However, the EGCG antitumoral effect observed in animals is not confirmed, at moment, for green tea consumption in humans. Probably, these epidemiological studies' inconsistent results were caused by various confounding factors, for example the quantity and the quality of the tea consumed or, possibly, the effect of caffeine [151].
Moreover, EGCG tissue and plasmatic concentrations obtained by the oral intake of tea are lower than the effective concentrations utilized in in vitro experiments (10-100 µmol/L). To avoid these kinds of problems, better-designed clinical studies have been designed; for example, EGCG-enriched fractions such as polyphenon E, a well-defined green tea catechin (GTC) extract, or highly purified EGCG, which have been provided by pharmaceutical companies [148].
Systemic bioavailability analyses in human volunteers of orally administered catechins have been already performed. Chow et al. examined the tolerability, safety, and pharmacokinetics of EGCG and polyphenon E, administered at doses ranging from 200 to 800 mg [152,153].
Some recent trials have confirmed EGCG's chemopreventitive and chemotherapeutic role, offering more details on its action in the human body. For example, Ahn et al. described that oral treatment with polyphenon E or purified EGCG (200 mg daily for 3 months) was effective in patients with cervical lesions infected by human papilloma virus (HPV) [154].
In Japan, green tea extract's (GTE) effect on metachronous colorectal adenomas was evaluated. GTE, administered orally (1.5 g/d for 12 months) in addition to a tea-drinking lifestyle, has been shown to be useful in reducing the incidence of metachronous adenoma in people 1-year post-polypectomy.
Another application of catechins is the chemoprevention of prostate cancer (oral GTCs). Sixty volunteers with high-grade prostate neoplasia received either 600 mg of GTCs or a placebo, daily, for 1 year. After 1 year of follow-up, only 3% of patients that had received oral GTCs showed a prostate tumour, while 30% of patients in the placebo group developed cancer. According to these observations, treatment with GTCs was able to reduce prostate cancer diagnoses by almost 80% [155].
From several clinical studies it is emerging that EGCG is an active cancer suppressor, with limited side effects and high safety. In addition, it has shown synergistic effects with other anticancer drugs like chrysin, curcumin, erlotinib, etoposide, 5-fluorouracil, tamoxifen and temozolomide [156,157]. However, it can also inhibit some anticancer treatments (bortezomib and other proteasome inhibitors) [158,159].

Resveratrol
Resveratrol is a polyphenol, isolated for the first time in 1940. It is an ingredient of white hellebore roots (Veratrum grandiflorum O.Loes) contained in various food sources including grapes, mulberries, red wine and peanuts. In 1963, resveratrol was recognised as the active element in Polygonum cuspidatum roots, a plant used in Japanese and Chinese traditional medicine.
It can inhibit tumorigenesis at multiple phases, including initiation, as well as during a cancer's progression [160]. Several other studies confirmed the strong chemo-preventive resveratrol efficacy in in vivo carcinogenesis models. In mice and rats, the oral or local application of resveratrol reduced DMBA-initiated and 12-otetradecanoylphorbol-13-acetate-promoted skin cancers, repressed DMBA-induced mammary carcinogenesis, inhibited 1,2-dimethylhydrazine-induced carcinogenesis of the colon epithelium and Nnitrosomethylbenzylamine-induced esophageal tumors [161].
Moreover, extensive study has suggested that resveratrol might be an important candidate for cancer therapy because it could act by interfering with many signalling pathways playing pivotal roles for cell growth, cell death, inflammatory process, angiogenic mechanisms and metastasis formation.
Besides, resveratrol was also described to show exhibit synergistic effects with other classic anti-cancer drugs, such as doxorubicin, cisplatin and vinorelbine.
The first clinical trial of resveratrol in colon cancer patients was performed with the aim to assess low dose effects of a plant-derived resveratrol formulation and resveratrolcontaining freeze-dried grape powder (GP). Treatment was administrated to 8 patients received for 14 days until the day prior to surgery for colon cancer resection.
The two compounds showed significant ability to inhibiting Wnt pathway targets on normal colon mucosa, whereas GP treatment augmented some Wnt target genes expression in colon cancer. Therefore, resveratrol may show more clinical utility as colon cancer prevention agent rather than for established colon cancer treatment [162,163].
A randomised, double-blind, phase I, clinical trial, showed the SRT501 (micronized resveratrol) effects in colorectal cancer and hepatic metastases patients. In malignant hepatic tissue following SRT501 treatment, a marker of apoptosis, i.e., cleaved caspase-3, was significantly increased by 39% compared to tissue from placebo-treated patients [164].
Moreover, a recent study suggested that resveratrol could attenuate the paclitaxel's anticancer efficacy in some breast cancer cell lines and in vivo experiments [165], but it has also shown synergistic effects with other anticancer drugs like cisplatin, doxorubicin and vinorelbine [166,167].

Capsaicin
Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is a homovanillic acid derivative and represents the major spicy component in red and chili peppers.
Capsaicin has been studied in the past for medical applications such as in anti-oxidant, anti-inflammatory and analgesic compounds [24].
Recently, some researchers have explored the benefits of capsaicin as an anti-cancer agent, with a detailed analysis of the molecular mechanisms induced by its exposure. In fact, capsaicin is able to influence the expression of some genes, in different types of tumor models, that are directly involved in cell growth, apoptosis, metastatization and angiogenesis processes [168].
The interest in capsaicin is also due to its possible combinational use with other chemotherapeutics drugs or dietary molecules, highlighting its synergistic antitumor activities.
Capsaicin combined with resveratrol was able to induce apoptotic pathways by nitric oxide (NO) elevation in a p53-dependent way [25].
Capsaicin has shown an interesting synergistic behavior in association with genistein, acting in breast tumor cell lines through AMPK and cyclooxygenase 2 regulation [169].
Moreover, capsaicin and brassinin, an indole obtained from cruciferous vegetables, possess synergistic antitumor abilities in regulating matrix metalloproteinases, thereby reducing migration and the invasion of prostate carcinoma cell lines [170].

Natural Compounds as Epigenetic Modulators
An increasing number of scientific reports highlight the implication of genetic and epigenetic alterations that can lead to the alteration of transcription factors, oncogenes' overexpression, tumor suppressor genes' inactivation, producing a deregulation of signaling pathways, and, finally, tumor occurrence [170,171].
The term "epigenetics" has been utilized to include the heritable changes in DNA and protein alterations that lead to a disturbed expression of genes involved in cell growth and cell cycle progression, cell death, metabolism, etc. [172].
Epigenetic alterations are hypothetically reversible; so, they are interesting for the development of new anti-tumor strategies [173].
Drugs able to target epigenetic mechanisms could represent the frontier of a new chemotherapeutic approach, and natural compounds have demonstrated their great potential [174].
It has been demonstrated that a vegetables-and fruits-rich diet can significantly reduce the risk of tumor growth. This is mainly due to the presence of some phytochemicals that are able to modulate oncogenes' expression and tumor suppressor genes [175].
In fact, some natural compounds have been described as being able to influence some of the epigenetic processes involved in carcinogenesis, such as the modification of histone proteins (acetylation and methylation), DNA methylation and microRNA expression [176].
The natural compounds most studied in epigenetic processes in tumorigenesis are EGCG, curcumin and resveratrol. In particular, EGCG could epigenetically reactivate p21/waf1, Bax and PUMA in prostate cancer cell lines, promoting the block of cell cycle and cell death induced by degradation at the proteasome of histone deacetylases (HDACs) [177]. EGCG is also able to repress the androgen receptor (AR) hormone response by the reduction of AR acetylation. This phenomenon determines a reduction of prostate cancer cells' growth, promoting apoptosis [178]. Moreover, EGCG has been also described as a potential epigenetic modifier of HDACs, restoring epigenetically silenced genes in cervical and skin tumors. EGCG could also reactivate the WIF (Wnt inhibitory factor-1) expression by demethylation of the gene promoter, inducing cell growth arrest and influencing the Wnt pathway in A549 and H460 lung tumor cell lines [179].
Moreover, EGCG reactivates the expression of WIF-1 (Wnt inhibitory factor-1) through promoter demethylation and inhibits cell growth by downregulating the Wnt canonical pathway in H460 and A549 lung cancer cell lines [171].
EGCG sensitizes ERα-negative cancer cells to respond to 17β-estradiol, and the antagonist tamoxifen. EGCG associated with trichostatin A (TSA, a HDAC inhibitor) reactivates the ERα expression in MDA-MB213 cells (a triple-negative breast cell line) by influencing histone methylation and acetylation, thus remodeling chromatin assembly [180].
Resveratrol induces, in p53-wild type and p53-mutant prostate tumor cells, the downregulation of metastasis-associated protein 1 (MTA1), promoting the destabilization of its nucleosome, remodeling deacetylation co-repressor complex. This complex is able to mediate the histone and non-histone post-translational modifications inducing transcriptional repression [176].
Taking these data together, natural compounds express a real potential effect for cancer therapies due to their reverting effects on epigenetic modifications in the oncogenes and tumor-suppressor genes involved in cancer's development and growth.

Conclusions
In the current century, cancer appears to be the most challenging pathology to treat; therefore, new, well-tolerated and effective therapeutic approaches are necessary.
The primary problem in cancer therapy is drug resistance; a huge number of cellular mechanisms are involved in this resistance [172] and no molecule is excluded from acquiring a resistant phenotype. In this regard, the risk of resistance could be minimized by combined therapies; if tumour cells gain resistance against one of the drugs, other components of the mixture can still have an impact on them.
Even though natural compounds and their extracts, sometimes, can exhibit resistance phenomena, pure natural compounds can be utilized in combination with another agent to reduce the development of resistance, but natural extracts are, themselves, a blend, acting as a synergic therapy, and contributing to the reduction of drug-resistant phenotypes [173].
Natural extracts may share some disadvantages with classical cancer drugs. Resistance is not the only problem, in fact, poor bioavailability is a common problem due to their very different structures. This results in poor absorption, high metabolism rates and a rapid excretion process. In all these cases, low plasma concentrations are reached. On the other hand, some natural compounds are absorbed rapidly and completely, entering the plasma in their native form, thus reaching significant plasma concentrations.
Therefore, the poor bioavailability of some natural extracts really hinders these natural product's potential to be developed into a clinically approved drug. In fact, this poor bioavailability requires a long-term dosing strategy. Another problem is due to the wide patient population required, representing significant drug exposure variability for natural extracts. So, bioavailability represents an important concern for the possible use of natural compounds as drug candidates.
Therefore, a new strategy is these of drug delivery systems to precisely target given body parts. This option might solve these critical issues [174]. Nanotechnology could play a noteworthy role for advanced drug preparations, controlling both drug release and delivery. The green chemistry-design approach of for the loading of nanoparticles with drugs can also be very useful in minimizing the hazardous constituents of the biosynthetic process. Thus, these green drug-delivery nanoparticles could reduce the side-effects of medications [175]. However, drug's precise release at determined sites, assessments of their effects at the cellular and tissue levels, and the required predictive mathematical modelling have not yet been developed [176].
Moreover, another interesting application of natural compounds is that the onset of resistance is made more difficult by the use of natural extract, thanks to their polypharmacological properties and, as has happened with common drugs, bioavailability problems can be solved with novel approaches, such as encapsulation [177], nanoparticles [178], liposomes [179] or emulsions [180]. These approaches ameliorate the bioavailability of hydrophilic compounds with poor absorption or low stability and increase the solubility of highly hydrophobic compounds and extracts [172,181].
The issues of reproducibility and the complexity of natural mixtures are the most important drawbacks thereof. Furthermore, positive in vitro data are not directly correlated to positive in vivo data due to poor solubility and, consequently, lesser accumulation at the target site, leading to a significant increase in systemic toxicity.
In conclusion, it will be crucial to understand the signaling pathways involved, as well as bioavailability and true cytotoxicity of these natural products, in answering the growing demand for the evaluation of natural products in clinical trials.
Funding: This research received no external funding.

Conflicts of Interest:
The authors declare no conflict of interest.