Accumulating evidence suggests that propolis and its flavonoids have a wide range of molecular targets either through direct interactions or through modulation of gene expression. Oršolić and Bašić ^[28][3] summarized knowledge of the various molecular mechanisms of chemopreventive or therapeutic action of flavonoids and other polyphenols in the prevention of chemical carcinogenesis in animal models. These molecular mechanisms can be divided into several categories: (i) mechanisms involved in the prevention of metabolic activation of carcinogens, (ii) mechanisms that inhibit proliferation of tumor cells through inactivation or downregulation of prooxidant enzymes and signal transduction enzymes, (iii) mechanisms involved in the initiation of cell death (apoptosis), and (iv) mechanisms that inhibit mitochondrial functions. When considering molecular targets relevant for cancer prevention, it has been shown that the flavonoids quercetin and kaempferol interact with the transcription factor aryl hydrocarbon receptor, which has a prominent role in the development of chemical-induced cancer ^[15][13].

Molecular targets modulated by propolis and its flavonoids include transcription factors, growth factors and their receptors, cytokines, enzymes, and genes participating in the regulation of cell proliferation, and apoptosis ^{[1][2][3][5][12][13][14][15][28][29][30]}[3,4,5,6,7,9,10,11,12,13,14].

Growing data ^{[1][2][3][15][28][29][30]}[3,4,5,6,7,13,14] suggest that propolis and its polyphenolic/flavonoid compounds are multitarget agents in cancer prevention. As indicated, the modulation of oncogenes, tumor suppressor genes, and intracellular signaling pathways inhibits cell proliferation and induces transformation, angiogenesis, and apoptosis, which have been proposed as the key mechanisms of the chemopreventive action of diverse polyphenolic compounds.

Various flavonoids demonstrate powerful antioxidant properties in vitro and in vivo, directly scavenging a broad spectrum of reactive oxygen, nitrogen, and chlorine species, such as superoxide anion, hydroxyl radical, peroxyl radical, hypochlorous acid, and peroxynitrous acid. In addition, they act as metal ion chelators, thus reducing their prooxidant capacity via Fenton chemistry ^{[26][28][31][32][33]}[3,41,42,43,62]. Furthermore, the antioxidant action of flavonoids is achieved through the inhibitory activity of certain enzymes that participate in ROS production (e.g., xanthine oxidase, PKC, ascorbic acid oxidase, cyclooxygenases, lipoxygenases, Na⁺/K⁺ ATPase, and cAMP phosphodiesterase), or by potentiating the action of other cellular antioxidants. By scavenging free radicals, chelating metal ions (mainly iron and copper), and stimulating endogenous antioxidant defenses, propolis and its flavonoids directly attenuate the generation of ROS, reduce lipid peroxidation and oxidative DNA damage, decrease levels of oxidized glutathione and increase the pool of reduced glutathione, restore activities of antioxidative enzymes, such as catalase, superoxide dismutase, glutathione S-transferase, and glucose 6-phosphate dehydrogenase, modulate signal transduction pathways, such as ERK-Nrf2-HO1 pathways, and regulate expression and activity of proteins involved in redox regulation such as glutamate-cysteine ligase regulatory subunit (GCLM) and thioredoxin reductase (TrxR1) ^{[15][27][30][34][35][36][37][38][39][40]}[13,14,63,64,65,66,67,68,69,70].

In addition, flavonoids and other phenolic compounds may inhibit activities of telomerase ^{[27][34][35][36][37][38]}[63,64,65,66,67,68], matrix metalloproteinases ^{[1][2][3][28][29][41][42]}[3,4,5,6,7,71,72], angiotensin-converting enzyme ^[43][73], and sulfotransferase ^[44][74]. They also may interact with sirtuins ^[45][75] and cellular drug transport systems ^[46][47][48][76,77,78], compete with glucose for transport across the membrane ^[49][50][51][79,80,81], interfere with cyclin-mediated cell cycle regulation ^{[28][29][52][53]}[1,2,3,4], and modulate platelet function ^[54][82]. Furthermore, isoflavones and lignans found in propolis act as phytoestrogens and modulate hormone-dependent carcinogenesis in animals ^[55][56][57][83,84,85]. Finally, it must be emphasized that flavonoids are recognized as xenobiotics, which are visible by their rapid metabolism, and their detrimental effects have been observed in vitro and in vivo ^[40][58][59][70,86,87].

Therapeutic effects of propolis on numerous diseases, such as infections, hypertension, cardiovascular disorders, diabetes, allergies, asthma, and cancer, are assigned to its anti-oxidative, immuno-modulatory, and anti-inflammatory properties ^{[4][5][12][13][14][15][28][30][31][32][33][60][61][62][63][64][65][66][67][68][69][70]}[3,8,9,10,11,12,13,14,15,16,17,34,35,36,37,38,39,40,41,42,43,88]. These diseases are accompanied by mitochondrial dysfunction and increased ROS production and the enzymatic and non-enzymatic oxidation of polyunsaturated fatty acids (PUFAs). Fenton and Haber–Weiss reactions, mainly catalyzed by iron ions, generate ^•OH (hydroxyl radicals) from H₂O₂ (hydrogen peroxide) and superoxide anions (^•O₂⁻). During normal metabolism and NADPH oxidation, ROS/RNS (e.g., OH•, O₂^•−, nitric oxide NO, nitrogen dioxide NO₂, and peroxyl ROO^• and lipid peroxyl LOO• radicals) are continuously produced, and are significantly involved in cellular aging, mutagenesis, and carcinogenesis. At physiological concentrations, ROS are important signaling molecules involved in the regulation of cell growth, the adhesion of cells toward other cells, differentiation, senescence, and apoptosis, while prolonged and enhanced ROS production is considered essential for the progression of inflammatory disease. Inflammation is a critical component of tumor progression. ROS-sensitive signaling pathways are persistently activated in many types of cancers, contributing to cell growth/proliferation, differentiation, protein synthesis, glucose metabolism, cell survival, and inflammation ^[71][89]. Oxidative DNA damage considerably contributes to mutagenesis and cancer. If unrepaired, ROS-induced DNA damage may result in mutations. Moreover, oxidative damage of oncogenes or tumor suppressor genes may accelerate cancer transformation. Thus, ROS participate in both the development and progression of cancer ^[72][90]. ROS may also regulate cell motility and tumor cell metastasis by increasing vascular permeability. Furthermore, ROS can induce intracellular signaling pathways by reversible oxidation of phosphatases, such as phosphatase and tensin homolog (PTEN) or protein tyrosine phosphatase (PTP), at cysteine residues in their active sites, or by direct oxidation of kinases such as Src. This results in the activation of several signaling pathways such as Src/PKD1-mediated activation of NF-κB, the mitogen-activated protein kinases (MAPKs) (ERK1/2, p38, and JNK) signaling pathways, and the phosphatidylinositide 3-kinases/protein kinase B (PI3K/Akt) signaling cascade. However, another mechanism by which ROS initiate cellular signaling is based on the activation of redox-regulated transcription factors such as activating protein-1 (AP-1) or forkhead transcription factors (FOXO) ^{[71][72][73][74]}[89,90,91,92].

It has to be emphasized that cancer development is a multistage process that may be induced by environmental hazards, inflammatory agents, and modulators of transcription factors (e.g., NF-κB, STAT3, and AP-1) that mediate communication between cancer cells and inflammatory cells. Overexpression of proinflammatory transcription factors NF-κB, STAT3, and AP-1 results in the release of cytokines and chemokines, and pre-existing inflammation plays a critical role in immunosuppression via the STAT3 pathway, which stimulates the expression of programmed death ligands 1 and 2 (PD-L1 and PD-L2) in cancer cells and suppresses the activity of immune cells ^[75][93].

2.2. Regulation of Cell Proliferation by Propolis and Its Polyphenolic/Flavonoid Compounds

Propolis and flavonoids are chemopreventive compounds that affect the proliferation, differentiation, and apoptosis of cancer cells, and their effect is especially pronounced in direct contact with cancer cells, for example, in the gastrointestinal system ^[3][28][3,7]. Antitumor properties of propolis and its phenolic constituents have been reported in various culture cell lines of human origin, including leukemia cells (HL-60, U937, MOLT-4, NALM-6, K562, CI41, HL-205, K-562-J, RS4;11, CEM, NB4, NALM-16, SUP-B15, and REH), colon cancer cells (SW480, HCT116), cervical cancer cells (ME180, Hela), glioblastoma cells (U87MG), lung carcinoma cells (A549), hepatocellular carcinoma cells (HepG2, SNU449), pancreatic cancer cells (PANC-1, BxPC-3), and mammary carcinoma cells (MCF-7) ^{[76][77][78][79][80][81][82][83][84][85][86][87][88]}[103,104,105,106,107,108,109,110,111,112,113,114,115]. Direct cytotoxic effects of propolis and polyphenolic/flavonoid compounds were confirmed in mammary carcinoma cells ^[21][60][15,61] and primary culture of human papillary urothelial carcinoma cells ^[30][85][14,112]. ThWe researchers aand others have shown that propolis and its flavonoids inhibit the proliferation of cancer cells by activating cytotoxic and apoptotic mechanisms ^{[17][23][24][25][34][36][86][87][88][89][90][91][92]}[18,47,48,49,57,64,66,113,114,115,116,117,118]. It seems that the cytotoxicity of flavonoids depends on their chemical structure, concentration, and type of leukemic cells. Reynal et al. ^[93][119] found that genistein induces a time- and dose-dependent effect on both myeloid and lymphoid leukemic cell lines. Plasma concentrations of genistein (1–20 µM) after a soy-enriched diet results in a loss of clonogenicity in leukemic cells. In leukemic mice fed on a diet enriched with 0.5% genistein, survival time was significantly increased in comparison with mice on a normal diet. The moderate antileukemic effect of genistein in vivo is probably due to its very rapid inactivation (15 min) in mice. In another study, genistein did not exceed 1 µM concentration in the blood of mice fed with a 0.6% soy extract supplement ^[94][120]. The antitumor activity of curcumin, epigallocatechin gallate (EGCG), or indole-3-carbinol (I3C) in MDA-MB-231 breast cancer cells was confirmed through the inhibition of clonogenic growth by 55% to 60%, an increase in basal caspase-3/7 activity by 1.5 to 2 times, and a prolonged doubling time, while genistein did not show an anticancer effect on the same cells ^[95][121]. Similarly, animal studies did not show the inhibition of tumor growth in the MDA-MB-231 cell xenograft with the ~1 μM concentration of genistein in serum ^[96][122]. This probably indicates the lack of a protective effect of genistein in estrogen receptor-α-negative tumors ^[97][123]. CAPE inhibited the proliferation of the colorectal cell line SW480 by downregulating the expression of β-catenin, c-myc, and cyclin D1 ^[98][124]. The dose-dependent effect of CAPE on the proliferation of LNCaP, DU-145, and PC-3 human prostate cancer cells was confirmed in a xenograft model using LNCaP cells and nude mice ^[99][125]. In addition, propolis and CAPE in a dose-dependent manner inhibited self-renewal of cancer stem cells, progenitor formation, and clonal growth.

Key Mechanisms of Propolis and Its Polyphenolic/Flavonoid Compounds Involved in Inhibition of Cell Proliferation

Several mechanisms have been implicated in the inhibition of cell proliferation: (i) inhibition of the activity of tyrosine-specific protein kinases ^{[100][101][102]}[126,127,128], such as inhibition of PKC, which is one of the key enzymes in the regulation of cellular proliferation and tumor growth; (ii) induction of the differentiation of carcinoma cells ^{[15][28][100][101][102][103]}[3,13,126,127,128,129]; (iii) transcriptional changes in cell cycle- and apoptosis-related genes such as NF-κB, Bcl-X(L) and COX-2; (iv) binding to the estrogen receptor and inhibition of an estrogen receptor-positive human breast cancer cells ^[104][105][130,131]; (v) induction of apoptosis, (vi) downregulation of the expression of mutated H-Ras and p53 tumor suppression gene ^{[15][28][100]}[3,13,126], (vii) modulation of gene methylation and re-expression of tumor suppressors or other genes silenced by aberrant DNA methylation ^{[93][104][105]}[119,130,131], (viii) inhibition of pro-oxidative enzymes xanthine oxidase, cyclooxygenases, or lipooxygenases ^{[15][28][100][101]}[3,13,126,127], and (ix) some flavonoids are a direct poison for topoisomerase II (TopoII), through the stabilization of double strand breaks in the TopoII-DNA cleavage ^{[5][12][13][14][15][29]}[4,9,10,11,12,13]. Flavonoids exhibit a wide range of activities that orchestrate signal transduction. They downregulate growth factors (epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF)), modulate survival signaling pathways (ERK, JNK, AP-1, and NF-κB), and reduce the expression of cell cycle regulators (cyclinD1, cdk4/6, p21, and p53) and apoptosis regulators (PARP, ceramides, and caspases) ^{[15][23][24][25][28][52][53][100]}[1,2,3,13,47,48,49,126]. Cell cycle progression is a sequential and highly regulated process that drives the division of mammalian cells through the G1, S, G2, and M phases. G1-S or G2-M transitions are regulated by checkpoints that may arrest cell cycle progression in environmental stress conditions. As balanced interactions between cyclins, cyclin-dependent kinases (CDK), and CDK inhibitors (CDI) regulate progression through the cell cycle ^[106][132], altered expression of the cell-cycle-specific proteins by dietary components potentially may sustain the proliferation of neoplastic cells and serve as “effect” biomarkers of their cytotoxic activity. The phenolic compounds of propolis, such as genistein, quercetin, kaempferol, myricetin, luteolin, chrysin, and apigenin, have been confirmed to modulate cell proliferation through an effect on the cell cycle by inducing CDI (p21 and p27) and inhibiting CDK4, CDK2, cyclin D1, and cyclin E ^{[107][108][109][110][111][112][113]}[133,134,135,136,137,138,139]. Kabała-Dzik et al. ^[25][49] compared caffeic acid (CA) and CAPE activity on triple-negative human Caucasian breast adenocarcinoma cell line (MDA-MB-231). They showed that CAPE displays higher cytotoxic and apoptotic activity compared to CA, and that CAPE induces cell cycle arrest in the S phase (time and dose-dependently), while CA demonstrated this effect only at 50 and 100 μM concentrations. Algerian propolis and its combination with doxorubicin decreased cell viability, prevented cell proliferation and cell cycle progression, induced apoptosis by activating caspase-3 and -9 activities, and increased the accumulation of chemotherapeutic drugs in MDA-MB-231 cells by blocking P-gp function and cell cycle arrest in the S phase ^[110][136]. Jiang and co-authors also demonstrated that Chinese propolis selectively inhibits the proliferation of human gastric cancer SGC-7901 cells by triggering both death receptor- and mitochondria-induced apoptosis, and cell cycle arrest in the S phase ^[111][137]. In the study by Zang et al. ^[112][138], it was shown that flavones (luteolin, chrysin, and apigenin) and flavonols (quercetin, kaempferol, and myricetin) can induce cytotoxicity to OE33 cells (a human esophageal adenocarcinoma cell line) by causing G₂/M arrest and induction of apoptosis. At low μM doses, polyphenols activated hormetic signaling pathways involving various kinases and transcription factors ^[112][138]. These pathways, in turn, activate the expression of genes encoding stress resistance proteins, such as antioxidant and detoxifying enzymes, protein chaperones, neurotrophic factors, and other cytoprotective proteins. Of note, one such pathway is the Nrf2/ARE pathway ^{[113][114][115]}[139,140,141]. At low concentrations, polyphenolic compounds are not cytotoxic, but cytostatic. They usually arrest the cell cycle in the S/G2 and mitotic phases, leading to cell death. In addition, they act on multiple kinases implicated in cancer pathogenesis ^[116][142]. Propolis, as well as flavonoids derived from propolis, such as galangin, is a potent COX-2 inhibitor ^{[15][23][24][25][28][52][53][100][117]}[1,2,3,13,47,48,49,126,143]. Flavonoids and caffeic acid analogs are also effective xanthine oxidase inhibitors ^{[15][23][24][25][28][52][53][100][117]}[1,2,3,13,47,48,49,126,143]. COX-2 and xanthine oxidase are ROS-producing pro-oxidant enzymes that contribute to inflammation. There is considerable evidence suggesting that angiogenesis and chronic inflammation are mutually interdependent. Inhibition of angiogenesis has an anti-inflammatory effect. For example, EEP and components of propolis, such as CAPE, quercetin, resveratrol, and genistein, exhibit anti-inflammatory and anti-angiogenic properties in urothelial cancer ^{[15][23][24][25][28][52][53][100][117]}[1,2,3,13,47,48,49,126,143]. Some flavonoids from propolis, such as galangin, genistein, baicalein, hesperetin, naringenin, and quercetin, suppressed the proliferation of an estrogen receptor (ER)-positive human breast cancer cell line MCF-7 ^{[15][28][52][53][118][119][120]}[1,2,3,13,144,145,146]. Flavonoids possess a binding affinity for ER-α estrogen receptors expressed in the breast, endometrium, and ovaries, and for ER-β estrogen receptors in the brain, blood vessels, lungs, and bones. CAPE greatly contributes to the estrogenic activity of propolis and shows a higher affinity for ER-β than to ER-α. Studies have shown that CAPE is a selective agonist of the ER-β. As ER-β does not contribute to the estrogenic effect on the ER-positive cancer cells, it is assumed that CAPE acts as a modulator of the estrogen receptor ^[120][146]. In addition, chrysin, yet another active compound from propolis, blocks the conversion of androgens into estrogens by inhibiting aromatase, hence increasing testosterone levels. Besides chrysin, galangin, as well as flavones such as naringenin, deplete estrogen synthesis via aromatase inhibition. Flavonoids and flavonoid derivatives possess two structural features that contribute to the aromatase inhibitory activity. Firstly, the A and C rings of the flavonoids are structurally similar to the D and C rings of the aromatase substrate androstenedione, and secondly, the presence of oxo-group at the C4 position, which is essential for binding iron atoms of the heme group of aromatases ^[121][147]. Similar activity was also observed for apigenin ^{[122][123][124][125]}[148,149,150,151]. In breast carcinoma cells, the antiproliferative effects of genistein were mediated by an intracellular metabolite, which inhibited cell cycle progression. In addition, genistein may affect apoptosis, angiogenesis, and metastatic processes. Various molecular effects modulated by genistein include the downregulation of growth factors (EGF and VEGF), and modifications of survival signaling pathways (ERK, JNK, AP-1, and NF-κB), cell cycle regulators (cyclinD1, cdk4/6, p21, and p53), and regulators of apoptosis (PARP, ceramides, and caspases) ^{[126][127][128]}[152,153,154]. Regarding propolis, it has been shown that it inhibits cell cycle progression by suppressing expressions of cyclin A, cyclin B, and Cdk2 and by stopping proliferation at the G2 phase, by increasing levels of p21 and p27 proteins, and through the inhibition of telomerase reverse transcriptase (hTERT), leading to telomere shortening and cancer cell death ^{[5][12][13][14][15][28][29][52][53][129][130]}[1,2,3,4,9,10,11,12,13,155,156].

2.3. Epigenetic Regulation by Propolis and Its Polyphenolic/Flavonoid Compounds

Growing evidence suggests that flavonoids may affect tumor progression by acting at chromatin remodeling and interfering with epigenetic alterations. Chromatin remodeling refers to chemical modifications of DNA and histones, including DNA methylation and multiple histone modifications, such as acetylation, phosphorylation, sumoylation, and ubiquitination. Post-translational modifications of histones are important for the epigenetic regulation of gene expression. In that regard, two important modifications of histones are processes of acetylation and deacetylation. Histone acetylation is catalyzed by the histone acetyltransferases (HATs), whereas 18 different histone deacetylases (HDACs) catalyze histone deacetylation. HDACs are divided into four classes based on their structural similarity ^{[15][131][132]}[13,157,158], and are distributed in the cytoplasm, mitochondria, and nucleus. Seven members of HDACs are known as sirtuins (SIRTs—SIRT1—SIRT7) ^[131][132][157,158]. Noncoding RNAs, such as microRNAs (miRNAs) and short interfering RNAs (siRNAs), as well as some other epigenome constituents, are also implicated in the control of epigenetic mechanisms that regulate gene expression. The genetic mutations along with the epigenetic alterations can be found in cancer cells. Several dietary and nutritional compounds have been reported to affect epigenetic modifications and gene expression of tumor suppressors and other cancer-related genes. The epigenetic alterations are reversible, and under certain conditions may be induced by natural polyphenols ^[131][157]. It is known that enzymes regulating epigenetic processes, DNA methyltransferases (DNMTs) and HDACs, are aberrantly upregulated in both developed cancer and in early phases of carcinogenesis ^[132][158]. The network of SIRT1-modulated signals is very complex and involves direct interactions of SIRT1 with proteins involved in cell survival, DNA repair, and cell cycle/apoptosis ^{[131][132][133][134][135]}[157,158,159,160,161]. If the tight homeostatic balance between the acetylation and deacetylation changes due to the abnormal activities of either HATs or HDACs, this will end in pathological situations, as seen in cancer ^[132][158]. Not surprisingly, HDAC inhibitors have been in the focus as therapeutic options in cancer chemotherapy. On the other hand, dietary polyphenols from propolis may act as HDAC inhibitors, which might be of great importance in the prevention and therapy of cancer and other diseases ^{[131][132][133][134][135][136][137][138][139][140]}[157,158,159,160,161,162,163,164,165,166]. It has been shown that specific enzymes and methylated DNA binding proteins may suppress the expression of tumor suppressor genes, leading to tumor formation and progression. Genes participating in cell cycle regulation, DNA repair, angiogenesis, and apoptosis are inactivated by hypermethylation of their respective 5′CpG islands. Some key regulatory genes influenced by DNA hypermethylation and histone acetylation and deacetylation are genes coding E-cadherin, pi-class glutathione S-transferase, the tumor suppressor cyclin-dependent inhibitor 2A (CDKN2A), PTEN, and insulin-like growth factor (IGF-II), among others. Accordingly, there are growing efforts to discover and develop compounds capable to act as DNA demethylating agents and histone deacetylases inhibitors (HDACi) ^[136][162]. One potential approach to prevent hypermethylation of DNA and inactivation of tumor suppressor genes is to use natural polyphenols from propolis, such as genistein, several isothiocyanates, catechin, lycopene, and quercetin.

2.4. Regulation of Cell Death by Propolis and Its Polyphenolic/Flavonoid Compounds

The growth rate of pre-neoplastic or neoplastic cells is higher than that of normal cells due to disturbed functioning of their cell-growth and cell-death mechanisms. Many studies have revealed that apoptosis- and/or autophagy-related signaling pathways are modulated by propolis and polyphenols. The complex network of functional interconnections between apoptosis and autophagy in polyphenol-mediated cancer therapy may be the key factor involved in the actions of polyphenols (Figure 1).