The Promising Therapeutic and Preventive Properties of Anthocyanidins/Anthocyanins on Prostate Cancer

As water-soluble flavonoid derivatives, anthocyanidins and anthocyanins are the plants pigments mostly rich in berries, pomegranate, grapes, and dark color fruits. Many bioactivity properties of these advantageous phytochemicals have been reported; among them, their significant abilities in the suppression of tumor cells are of the promising therapeutic features, which have recently attracted great attention. The prostate malignancy, is considered the 2nd fatal and the most distributed cancer type in men worldwide. The present study was designated to gather the preclinical and clinical studies evaluating potencies of anthocyanidins/anthocyanins for the treatment and prevention of this cancer type for the first time. In general, findings confirm that the anthocyanins (especifically cyanidin-3-O-glucoside) indicated higher activity against prostatic neoplasms compared to their correlated anthocyanidins (e.g., delphinidin); in which potent anti-inflammatory, apoptosis, and anti-proliferative activities were analyzed. Complementary anti-prostate cancer assessment of diverse naturally occurred anthocyanidins/anthocyanins and their synthetically optimized derivatives through preclinical experiments and eventually confirmed by clinical trials can promisingly lead to discover natural-based chemotherapeutic drug options.


Introduction
Prostate cancer (PC) is known as the 4th commonly diagnosed cancer (7.3% of total cases) in human and the 2nd mortal cancer type in men; moreover, in accordance with the global cancer burden (GLOBOCAN 2020), PC is reported as the most frequent cancer in men prevailing in 112 countries [1].
In the recent years, some systemic chemicals have been developed for the treatment of metastatic castration-resistant PC. Besides mitoxantrone, as the first cytotoxic chemotherapy approved by the US Food and Drug Administration (FDA), cabazitaxel, abiraterone acetate, enzalutamide, pembrolizumab, sipuleucel-T, as well as radioisotopes (e.g., samarium-153 and strontium-89) have been developed as successful PC therapeutic options [2]. Although chemotherapeutic drugs with possible side effects can suppress several tumors types, they phosphorylate and regulate various transcription factors, leading to change in gene expression [26,27].
The retinoblastoma (Rb) tumor suppressor protein plays a role in the control of the cell cycle, terminal differentiation, control the DNA damage response and promotes cell cycle arrest in G1. Its function is reverted by Rb phosphorylation by cyclin D-CDK, which inactivates Rb and promotes E2F1-mediated transcription [28][29][30]. This situation causes constitutive activation and the induction of uncontrolled cell proliferation.
Chemopreventive effect is stated as potency of natural, synthetic, or biological compounds to hinder cancer incidence. These substances are able to protect DNA damage, specifically by reversing or blocking proliferation of these nonmalignant cells [31]. In the clinical aspect, dietary phytochemicals and non-steroidal anti-inflammatory drugs (NSAID) can be consumed as chemopreventive agents in the populations with high cancer risk and the normal populations [32].
The following sections provide evidence describing effects of the plant extracts rich in these phenolic compounds as well as the pure forms on the treatment and prevention of prostate cancer cells, which have been evaluated through various preclinical (in vitro, in vivo) studies. Figure 3 renders a general information concerning the mechanism of action of ACDs/ACNs and plant extracts towards prostate cancer cells.

Rich Anthocyanidins/Anthocyanins Plant Extracts
As documented in Table 1, most of the anticancer assessments of ACDs/ACNs in pure form and the enriched plant samples have been conducted in vitro against PC3, PCai1, CRPC (castration resistant cancer), LNCaP (androgen-independent prostate cancer), and DU145 (androgen-resistant tumoral prostatic) human cell lines. Among different evaluated plants, cranberry, jaboticaba peel, strawberry and wild blueberry (35% ethanol fraction) revealed higher capacities to target cancerous cell lines in order to reduce cell survival and induction of apoptosis compared to the rest of the tested extracts. Overal, the proposed mechanism of actions might involve down-regulation of cyclin-dependent kinase, enhanced expression of p21, activation of caspase-3, inhibition of MMP (matrix metalloproteinase) activity, and lessening Snail and pSTAT3 (phosphorylated-Signal transducer and activator of transcription-3) expression. Muscadine grape skin extract LNCaP The extract decreased Snail and pSTAT3 expression and abrogated Snail-mediated CatL activity, migration, and invasion [34] ARCaP-E Brazilian native fruits, pitanga (red and purple) and araçá (yellow and red), as well as strawberry cultivars Albion, Aromas, and Camarosa, blackberry cultivar Tupy, and blueberry cultivar Bluegen

DU145
No cytotoxicity was found [35] Vaccinium myrtillus berry extract LNCaP Apoptotic rate (early and late) was statistically higher in tested cell lines exposed to Vaccinium myrtillus berry extract compared to control. [36] PC3 and DU-145 Anchorage-dependent and anchorage-independent growth inhibition were seen American cranberry (Vaccinium macrocarpon) extract DU145 10, 25 and 50 µg/mL of the extract significantly decreased the cellular viability of DU145 cells. [37] The extract at 25 and 50 µg/mL also lessened the proportion of cells in the G2-M phase of the cell cycle and increased the proportion of cells in the G1 phase after 6h in prostate carcinoma.

Anthocyanin Source Cell Line Effect Ref
Lowbush blueberry DU145 Inhibition of MMP9 activity at 0.5 and 1.0 mg of crude fraction/mL was seen. Also, 1.0 mg/mL extract decreased the gelatinolytic activity of the activated isoforms of MMP-2 and complete inhibition of the pro-MMP-2, with an increase in TIMP-1 and TIMP-2 action. [50] (Vaccinium angustifolium): Jaboticaba peel

PC3
The non-polar extract was the most active agents against prostate cancer cells with GI 50 = 13.8 µg/mL. [51] Sweet potato (Ipomoea batatas)

C4-2
The extract significantly inhibited cell proliferation of all prostate cancer cells with IC 50 values in the range of 145-315 µg/mL. IC 50 of extract in normal prostate epithelial cells (PrEC and RWPE-1) was between 1000 and 1250 µg/mL.

Purple corn LNCaP
Purple corn color at 50 and 100 ppm inhibited the proliferation of LNCaP cells by decreasing the expression of cyclin D1 and inhibiting the G1 stage of the cell cycle. [53] Acanthopanax senticosus (Siberian Ginseng) LNCap Cyanidin-3-O-(2"-O-xylosyl) glucoside showed potent anticancer effects with IC 50 of 5.2 µg/mL [54] The IC 50 viability of the compound against DU145 cells was about 361.58 µg/mL. The main anthocyanin monomer also inhibited cell proliferation, induced apoptosis, and promoted cell cycle arrest at the S phase. [55] Cranberries (Vaccinium macrocarpon Ait.)

Rice
The potential activities of the methanolic and dichloromethane extracts of three purple rice (Oryza sativa) in Thailand, named Doisaket, Nan, and Payao combined with chemotherapeutic drugs on human cancer cells have previously been investigated. Methanolic extract of Payao was the most potent cytotoxic agent, via inhibition of LNCaP cell growth. Taking into account that through a quantitative analysis by utilization of HPLC, the methanolic extract of Payao indicated the highest ACN content (5.80 mg/g), thus these compounds are supposed to be the main bioactive constitiuents [33].
In another study, anti-proliferation action of the plant extracts of Opuntia ficus Indica flower and fruit of Oryza sativa (black rice) and their synergistic action on BPH-1 cells (epithelial cells of benign prostatic hypertrophy), LNCaP cells (androgen-dependent tu- moral prostatic cells), and DU145 cells (androgen-resistant tumoral prostatic cells) were investigated. The flower extract of O. ficus Indica (10 µg/mL) and the extract of black rice (100 µg/mL) were individually capable to induce the reduction of cellular vitality of the BPH-1, LNCaP, and DU145 cell lines with the geometric mean of 70% and 80% cell viability, respectively. The treatment of the same cell lines with the combination of two extracts (210 µg/mL) could significantly lessen the cellular vitality (geometric mean of~36%) with respect to the single extracts confirming a synergistic anti-proliferative effect [59].
Previously, the rice bran fraction enriched in ACNs was separated and processed until the major compound was acquired. The sub-fraction of the rice bran and the isolate cyanidin 3-O-glucoside (C3G) (Figure 4) were studied against PC3 using immunohistochemicalstaining and immuno-blotting approaches. Both the extract and purified compound displayed cell viability with an IC 50 value of 168 µM on PC3 cells. C3G reduced the expression of Smad/Snail signaling molecules at 100 µM and enhanced the expression of E-cadherin as cell surface protein by reducing the tumor transformation. This ACN was also able to inhibit MMP-9, in addition to reduction the nuclear factor kappa B (NF-κB) expression levels and the activity of the enzyme in PC3 cells leading to decelerate cell migration and invasion process [47]. In another study, insoluble fraction (1% hexane) of the purple rice ethanolic extract on transgenic rats against adenocarcinoma of the prostate showed lessening effect of adenocarcinoma in the lateral lobes of the prostate as well as prostatic intraepithelial neoplasia. Also, such diet significantly suppressed the tumor growth in PCai1 cells (castration-resistant prostate cancer xenograft model). In vitro evaluations on LNCaP and PCai1 cells recorded the suppression of the cell proliferation and induction of G0/G1 cell-cycle arrest. Interestingly, the cyclin D1, cdk4, androgen receptor (AR), and fatty acid synthase expression were down-regulated after treatment while expression of p38 mitogen-activated protein kinase and activation of AMP-activated protein kinase-α occurred in the treated PC cells, rat prostate tissues, and CRPC (Castration-resistant prostate cancer) tumors. Noteworthy, the chemical profile analysis of the extract characterized C3G as the active compound; subsequently the abovementioned potencies can be mainly correlated to this ACN [60].

Grape
Grape (Vitis vinifera L.) is a natural source of phenolic compounds specifically stilbenes and flavonoid derivatives including ACDs. Many bioactivities have been reported for this fruit, whereas the following properties can be highlighted: antioxidant, antimicrobial, cardio-, hepato-, neuro-, and skin-protective effects, as well as anti-tumor activities [61]. Few studies evaluated the preventive/treatment effects of this ACD-rich fruit on PC.
Burton et al. assessed the effects of muscadine grape skin extract containing ACNs as the main bioactive components on the prostate (LNCaP, ARCaP-E) and breast (MCF-7) cancer cells overexpressing snail transcription factor. It was documented that the snail overexpression led to increase cathepsin L expression/activity and pSTAT-3 which increased the invasion and migration. Muscadine grape skin extract at 5 µg/mL and 20 µg/mL inhibited snail overexpression leading to decrease cell invasion and migration. Phytochemical analysis showed cyanidin 3,5-diglucoside ( Figure 5), ellagic acid, and ellagic acid precursors presented in the extract; however, the responsible bioactive compound has not been determined [34].

Cranberry
The tremendous health beneficial impacts of cranberry (Vaccinium macrocarpon) has been correlated to its phenolic contents such as flavonoids, proanthocyanidins, ACDs, and ACNs [62].
In a study performed by Déziel et al., the cell cycle arrest effect of American cranberry was confirmed by modulating the expression of cell cycle regulators. The treatment of DU145 with the cranberry extract at 25 and 50 mg/mL for 6 h decreased the proportion of cells in the G2/M phase and increased the proportion of cells in the G1 phase. Molecular analysis showed that the extract decreased the expression of CDK4, cyclin A, cyclin B1, cyclin D1, and cyclin E, in addition, increased the expression of p27. Moreover, the decrease in p16INK4a and an increase in pRBp107 protein expression levels were evident, although the changes were not statistically significant compared to the control [37].
In another study, 200 µg/mL of total cranberry extract and all fractions including sugars-, organic acids-, and polyphenols-enriched extract were evaluated against prostate (RWPE-1, RWPE-2, 22Rv1) cancer cell lines. The crude extract and all fractions represented at least 50% anti-proliferative activity against prostate cancer cells except the sugars-rich fraction [56], confirming the high potency of natural acids and polyphenols including ACDs/ACNs in the extract.

Rhoeo discolor
The antiproliferative potency of 10 different crude extracts of Rhoeo discolor as well as the most bioactive subfractions have been tested against PC3 prostate cancer cell line, as well as a control fibroblast cell line NIH3T3. Results showed that mainly aqueous, methanolic, and ethanolic extracts were the most effective ones. Ten µg/mL of the crude extracts and 50 µg/mL of the experimented fractions significantly inhibited proliferation up to 61.8% in PC3. These natural compounds showed no significant ability to induce damage to the control cell line with less than around 28% apoptosis induction [63].
Several studies reported red maize (Zea mays L.) as a valuable source of water-soluble pigments such as flavonoids and ACNs [64]. In a previous study, the chemical profile assessment of ACNs via HPLC-ESI-MS identified 20 compounds, characterizing cyanidin 3-p-hydroxy-benzoyl sophoroside-5-glucoside, C3G (Figure 4), and cyanidin-3-O-(6′-acetyl-arabinoside) ( Figure 6) as major compounds. MTT assessments exerted significant antiproliferative activity and apoptosis effect in cell lines DU145 at 1000 μg/mL [65]. Antiproliferative effects of blue maize as a natural source of phenolic acids and ACNs were evaluated against PC3 cancer cell. HPLC analysis showed ferulic acid and C3G ( Figure 4) as the major phenolic and the most abundant anthocyanin compound, Antiproliferative effects of blue maize as a natural source of phenolic acids and ACNs were evaluated against PC3 cancer cell. HPLC analysis showed ferulic acid and C3G ( Figure 4) as the major phenolic and the most abundant anthocyanin compound, respectively. Among four different tested extracts, the acetone extract of native blue maize with just 30% cell viability was the most cytotoxic sample. In comparison, among the ACN compounds, cyanidin malonyl-glucoside ( Figure 7) showed the best antiproliferative effects with the strongest correlation with the reduction of cell viability [41]. Preparative column chromatography was formerly used to purify ACNs from blue corn and tortilla. Results revealed that both of the ACNs rich extracts especially the tortillas extract was able to decrease the LNCaP cell viability and arrest the cell cycle in the G1 phase at 500 to 1000 µg/mL. Further assessments exhibited induction of apoptosis behavior on LNCaP cells when the ACN rich extract of tortilla was co-treated with 2-amino-N-quinolin-8-yl-benzenesulfonamide [66].
Purple maize at 100 mg/L was able to inhibit the proliferation of LNCaP cells by decreasing the expression of cyclin D1 and inhibiting the G1 stage of the cell cycle. Eight weeks diet with 10% purple corn decreased the incidence of adenocarcinoma in the lateral prostate and slowed down the progression of PC through decrease the expression of cyclin D1, and downregulation of the Erk1 ⁄2 and p38 MAPK activation. Search for active compounds in purple corn color resulted in determination C3G (Figure 4), pelargonidin-3-O-glucoside, and peonidin-3-O-glucoside ( Figure 8) as the major compounds. Further biological results suggested that C3G and pelargonidin-3-O-glucoside are the active compounds against LNCaP cells [53].
In a former investigation, phytochemical analysis of strawberry extracts identified C3G ( Figure 4), pelargonidin, pelargonidin-3-O-glucoside, pelargonidin-3-O-rutinoside, aglycone and glycosylated flavonoids (Figure 9), as well as phenolic acids as the main compounds [39]. Quercetin, ellagic acid, 3,4,5-trihydroxyphenyl-acrylic acid, and C3G, as pure compounds at 100 µg/mL effectively inhibited the growth of the human prostate (LNCaP, and DU145) cancer cell lines, compared to the rest of pure compounds, while, anthocyanin and tannin-rich extracts of strawberry at 250 µg/mL demonstrated the best potency against these two cancer cells [39]. A polyphenol-rich extract plus anthocyanin or tannin-rich sub-fractions derived from a strawberry sample at 1.5 to 50 µg/mL were experimented against P21 (human prostate epithelial cell line), LNCaP and PC3 (tumor cell line). The strawberry extract (5 µg/mL) was cytotoxic on normal, and the tumor cell lines. The findings showed that the tannin-rich fraction was considerably more toxic to all tested cells than the ACN-rich fraction. As predicted, the prostate (LNCaP and PC3) tumor cell lines were more resistant to the extracts with around 50 µg/mL required for 50% survival reduction [40].

Cherry
The sweet cherry (Prunus avium L.) is characterized for its rich phytoconstituents particularly ACNs, hydroxybenzoic and hydroxycinnamic acids, carotenoids, and flavonoids [69]. In a previous study, the amounts of ACNs in the methanolic extracts of different sweet cherry samples were examined and the "late harvest" extract highly enriched in ACNs was selected for the proliferation, and apoptosis assessments on the neoplastic (LNCaP and PC3) and non-neoplastic (PNT1A) human prostate cells. HPLC-analysis confirmed that the most abundant phenolic compounds in the extract were the flavonoid quercetin-3-4-di-O-glucoside (24.61 ± 0.42 mg/100 g fresh fruit) and the anthocyanin C3G ( Figure 4) (22.03 ± 0.74/100 g fresh fruit) [43].
The biological results exhibited that the cherry extract-treatment diminished the viability of neoplastic cells mostly at 2 µg/mL and non-neoplastic cells at 20 and 200 µg/mL, whereas it enhanced apoptosis in LNCaP. This was confirmed through caspase-9 increasing in LNCaP cells and decreasing in PC3 cells after the cherry extract treatment. In addition, such treatment did not significantly change Bax (proapoptotic, Bcl-2-associated X protein) protein expression in all cell lines. Anti-apoptotic protein Bcl-2 (B-cell lymphoma 2) was significantly decreased in PNT1A and LNCaP cells after exposuring to the cherry extracts with no remarkable alterations observed in PC3 cells. Besides, the glucose consumption was decreased in LNCaP cells (43%) and increased in PC3 (113%) when treated with the cherry extract. The PFK1 protein levels were significantly reduced in PNT1A-and PC3-treated cells, while LNCaP showed a significant increase in PFK1 protein expression. Moreover, the activity of lactate dehydrogenase (LDH) was improved to~27% in the treated PNT1A cells while LDH activity was decreased in both neoplastic cells, LNCaP and PC3 to 49% and 60%, respectively [43].

Ixora coccinea
In a previous study, the phytochemical screening showed that Ixora coccinea (jungle geranium) contains total phenolic, flavonoid, and anthocyanin contents with 128 mg, 20.63 mg, and 30 mg/100 g fresh weight, respectively. Although the analytical analysis demonstrated sinapic acid (21.96 mg) and myricetin (0.13 mg) as the major phenolics, the significant ACN content can not be ignored. According to the MTT evaluations, the fruit crude extract exhibited good anti-cancer potential against LNCaP.FGC cells with an IC 50 value of 34.09 mg/mL after 24 h exposure [44].

Potato
Phytochemical studies showed that the Solanum spp. is rich in phenolic compounds including flavonoids such as kaempferol, naringenin, rutin, catechin, and epicatechin, however ACNs can be found in purple flesh varieties abundantly [70]. Some investigations have been designed in order to assess the potato extract's capacities to affect PC cells.
In an experiment, the effects of the potato phenolic extract as well as organic acid, phenolic acid, and ACN-rich fractions against LNCaP and PC-3 were investigated. Potato extract and an ACN-rich fraction (at 5 mg chlorogenic acid eq/mL) were more potent compared to the rest of the extracts, where it inhibited cell proliferation, increased the cyclin-dependent kinase inhibitor p27, and induced apoptosis in both LNCaP and PC3 cells. Mitogen-activated protein kinase, c-Jun N-terminal kinase activation, as well as stimulated caspase-independent apoptosis through nuclear translocation of endonuclease G (Endo G) and apoptosis-inducing factor were observed [46].

Lycium ruthenicum
The fruit of Lycium ruthenicum commonly is famed as black wolfberry. ACNs have been reported as the main chemical ingredient, apart from phenolic acids, flavonoids, and alkaloids [71]. The extracted monomer ACN ( Figure 11
Anticancer potencies of the H. sabdariffa Linne leaf, as a waste product, has been investigated. The extract exhibited high content of polyphenols, including catechin (4.25%) and ellagic acid (28.20%) with 2% total ACN. The leaf extract at 0.5, 1.0 and 2.5 mg/mL induced anticancer potential against PC3 and DU145 especially LNCaP. Furthermore, the extract and ellagic acid demonstrated IC 50 values of 2.5 mg/mL and 100 µM against LNCaP cells, respectively. Molecular assessment of LNCaP cells after treatment with the H. sabdariffa L. leaf extract proposed the two death pathways including intrinsic (Bax/cytochrome c-mediated caspase 9) and extrinsic (Fas-mediated caspase 8/t-Bid) apoptotic pathways [49].

Blueberry
Elaboration of anti-tumor activities of Blueberry (Vaccinium L.), as one of the main sources of ACDs and ACNs, has been targeted by many researchers. The mechanism to downregulate MMP activity in DU145 human PC during treatment with the crude extract, anthocyanin-enriched and proanthocyanidin-enriched fractions from lowbush blueberry were examined at 0.1, 0.5, and 1.0 mg/mL. The downregulation of MMPs activity and up-regulation of tissue inhibitors of metalloproteinases (TIMP) activity in DU145 cells were evinced in three tested extracts; however, proanthocyanidin-enriched were more potent compared to the other extracts tested. Multiple mechanisms in down-regulating MMPs involve an increase in TIMP-1 and TIMP-2 activities via inhibition of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase pathways without affecting PI-3 kinase [50].
In another study, phytochemical profiles of araca (yellow and red), and pitanga (red and purple), as well as strawberry, blackberry, and blueberry were analysed. Among the tested extracts, blueberry showed the highest total ACN content with 1202 mg/C3G kg fresh fruit, while strawberry exhibited the highest total phenolic content (13.55 g equivalent of gallic acid/kg fresh fruit). Strong positive correlations were detected between the total ACN content of the tested extract and antioxidant potential but not with total phenolic content. Screening of the extracts at maximum concentration of 0.5 mg/mL showed no cytotoxicity against DU145 cancer cell line [35].
The V. myrtillus extract and recombined standard mixture of its main native polyphenols have shown beneficial effects against hormone-dependent and hormone-independent PC cell lines. The extract at 1/800 (v/v) corresponding to 14.15 C3G equivalents/mL of culture medium decreased the proliferation of anchorage-dependent prostate cancer cell lines in a dose-dependent manner. Moreover, the V. myrtillus extract at 1/100 (v/v) demonstrated a significant growth inhibitory effect towards PrEC and DU145 cells with high apoptotic rates compared to control. The recombined standard mixture of its main native polyphenols also showed the same activity as the extract with induction of growth-inhibitory effect under hypoxia [36].

Blackberry and Raspberry
Blackberry (Rubus fruticosus) belonging to Rosaceae family is enriched in phenolic compounds mostly flavonoids including flavonols, ACNs, and ellagitannins [73]. It has previously been reported that this fruit due to the presence of these phenolics, is a potent natural agent for the treatment of the age-related neurodegenerative diseases, apart from its high antioxidant effect [74].
Ellagic acid and ACN constituents including urolithin A and protocatechuic acid of black raspberry as well as the black raspberry (Rubus occidentalis) extract have been evaluated for the interference with chemotherapy docetaxel and cabazitaxel for castrateresistant prostate cancer (CRPC). None of the extracts altered the enzyme responsible of CYP3A4 for catabolizing taxanes. Also, protocatechuic acid and the black raspberry extract were not able to enhance the effects of taxane chemotherapeutics, taxane metabolism, or mechanisms of resistance; however, the microtubule polymerizing effect of ellagic acid in vitro might interfere with microtubule polymerization induced by taxane drugs which reduce their effectiveness, although dietary ellagic acid did not alter the tumor growth inhibition by docetaxel of xenografted 22Rv1 cells. It seems that consumption of black raspberry during taxane chemotherapy could not provide a therapeutic benefit, it is safe and not likely to reduce drug effectiveness or increase toxicity while potential adverse interaction between taxane chemotherapy and ellagic acid was detected [75].
Previously, it has been reported that constitutive NF-κB activation is observed in androgen-independent PC. In this context, the pomegranate extract effect was evaluated towards the progression of PC. The pomegranate extract mediated NF-κB blockade and decreased the cell viability of PC cell lines in a dose-dependent fashion in vitro. Importantly, the increase in NF-κB activity during the transition from androgen dependence to androgen independence in the xenograft model was overturned by the extract [45].

Black Carrots
Studies showed that the dark color of black carrot (Daucus carota ssp. sativus var. atrorubens Alef.) is due to the ACNs richness [78]. In a previously performed experiment, the PC3 cancerous cell lines have been treated with the extracts of black carrot specimens in 6.25, 12.5, 25, 50, and 100 µg/mL doses of the extracts. MTT assessments recorded moderate cell viabilities with the values of 58 and 77% at a concentration of 100 µg/mL [48].

In Vitro Screening of Pure Anthocyanidin/Anthocyanin Derivatives on Prostate Cancer Cell Lines
In few studies potential effects of delphinidin and cyanidin-3-O-β-glucopyranoside (C3G) on prostate malignancy have been analyzed; however due to the remarkable activities of the extracts as above overviewed, further studies can complement those effects. Delphinidin Delphinidin ( Figure 12, 3,3 ,4 ,5,5 ,7-hexahydroxyflavylium), an ACD compound, which can be found abundantly in berries and red wine demonstrated a variety of bioactivities, dominantly antioxidant, anti-inflammatory, and anti-tumor properties [79]. In line with anticancer assessments of this compound, some researchers have also evaluated its capacity to suppress prostatic cancer cells. In a previous study, the apoptotic properties of delphinidin against human prostate cancer cells LNCaP, C4-2, 22RN1, and PC3 were analyzed, without bearing any substantial effect on normal human prostate epithelial cells. Delphinidin decreased the phosphorylation of IKB kinase (NEMO), phosphorylation of nuclear factor-KB (NF-κB) inhibitory protein IKBA, phosphorylation of NF-κB/p65 at Ser536 and NF-κB/p50 at Ser529, plus reducing NF-κB/p65 nuclear translocation and NF-κB DNA binding activity in vitro [58].
In another study conducted by Malik et al., the treatment of delphinidin (30-300 µM) on PC3 cells for 48 h resulted in a dose-dependent inhibition of cell growth and/or cell viability through induction of cyclin kinase inhibitors p21/WAF1 and p27/KIP1. Also, the downregulation of cyclin E, D1, and D2 and cyclin-dependent kinase 2, 4, and 6 were recorded. Apoptosis induction during treatment with delphinidin was validated via decreasing level of anti-apoptotic protein Bcl-2 and increasing the pro-apoptotic protein Bax. Supportively, significant activation of caspases and procaspase-3, -6, -8, and -9 as well as the release of cytochrome c from the mitochondria to the cytosol were measured. Finally, flow cytometry and morphological changes confirmed the apoptosis induction via treatment with delphinidin [80].

In Vivo Experiments Evaluating the Effectiveness of Anthocyanidins/Anthocyanins on Prostate Cancer
Preclinical in vivo experimental data was gained from rodent models in mice including athymic nude mice implanted with PC3 and stress provoked benign prostate hyperplasia (BPH); however, animal models in rats include BPH induced by testosterone derivatives such as testosterone enanthate or testosterone propionate. Followings describe the animal studies evaluating the impacts of ACDs/ACNs and natural sources of these compounds on PC cells (Table 2).

Mice Model Experiments
Diet with delphinidin suppressed the tumor growth in the athymic nude mice implanted with PC3 cells. In addition, it controlled the protein levels of Bax and Bcl-2, and inhibited the protein levels of cyclin D1 and NF-κB. Furthermore, it suppressed PCNA and Ki67 expression (markers of proliferation) in PC3 originated tumors in athymic nude mice; suggesting the anti-proliferative efficacy of delphinidin [81]. Supportively, the delphinidin administration (2 mg, i.p. thrice weekly) to athymic nude mice implanted with PC3 cells showed a significant decrease in the expression of NF-κB/p65, Bcl-2, Ki67, and PCNA [58].
Oral administration of sweet potato greens extract (SPGE) significantly inhibited growth and progression of prostate tumor xenografts by 69% in athymic nude mice, as shown by non-invasive real-time bioluminescent imaging and tumor volume measurements. The SPGE treatment reduced the cyclin D, E and D1 expression, and rose the p21 expression in tumor cells. Also, the caspase-3/7 activity and cleaved caspase-3 expression was higher in SPGE-treated tumors in comparison with control, while it proposes the in vivo anti-proliferative response of the SPGE. Moreover, this extract showed no toxicity to quickly divide normal tissues such as bone marrow and gut [52]. Sweet potato extract also inactivated Bcl-2 as well as upregulated BAX, cytochrome c release, and triggered downstream apoptotic signaling. Oral administration of 400 mg/kg extract inhibited growth and progression of prostate tumor xenografts by~70% in nude mice [52].
A supplementary diet with the rich ACNs extract (AE) of bilberry (Vaccinium myrtillus L.), administered to stress-provoked BPH in mice, had the protective effects against stressinduced oxidative injury by reducing lipid peroxidation content, elevating glutathione content, oxygen radical absorbance capacity, as well as glutathione peroxidase and superoxide dismutase activities. These results showed the antioxidative activity of AE involved in the amelioration of BPH [84].
Wang et al. investigated the antitumor effect of anthocyanin C3G extracted from Chinese bayberry fruit (Myrica rubra Sieb. et Zucc.), on a nude mouse tumor xenograft model. C3G inhibited the growth of SGC-7901 tumor xenografts. The average tumor volume on the 18th day were measured 771.5 mm 3 , 211.7 mm 3 , 384.1 mm 3 and 276.0 mm 3 , in the control group, tegafur-treated group (positive control), C3G treatment groups with low dose and high dose, respectively. Moreover, C3G revealed ability to inhibit the cell cycle of SGC-7901 tumor xenografts in the C3G treatment groups by obvious positive p21 staining in cell nuclei in a dose-dependent manner. The mRNA expression and protein expressions of KLF6 and p21 were significantly augmented in the C3G treatment group in comparison with normal group, representing the vital role of KLF6 in the tumor xenograft inhibition in vivo. However, the p53 protein did not alter, but Cyclin D1 and CDK4 mRNA and proteins levels were reduced [91].
Ha et al. evaluated the apoptosis and tumor growth of ACNs extracted from the black soybean on DU-145 tumor xenografts established in athymic nude mice. Black soybean (Glycine max L. Merr) has been known for its phenolic rich content mainly flavonoids (i.e., ACDs), stilbenes, lignans, and phenolic acids. It has demonstrated a variety of biological potencies including anti-tumor, anti-diabetic, and for the treatment of cerebrovascular, neurodegenerative, and cardiovascular diseases [92]. It has been exhibited that 2 weeks oral treatment with daily 8 mg/kg of ACN increased apoptosis significantly in a dosedependent manner. Also, the decrease in p53 and Bcl-2 expressions with the increased Bax expression, as well as a lessening in PSA and AR co-expression was observed [87].
In another study, Lamas et al. evaluated the impact of two doses of the patented jaboticaba peel extract (PJE) on inflammation and oxidative stress in the prostate of high-fat-fed (HFD) aging or aging mice. The PJE decreased the contents of oxidative-stress parameters (4-hydroxynonenal, glutathione peroxidase 3, glutathione reductase, and catalase), inflammatory mediators phosphorylated signal transducers and activators of transcription 3 (pSTAT-3), tumor necrosis factor α (TNF-α), cyclooxygenase 2 and CD3 + T cells' number, which were correlated to preservation of the glandular morphological integrity of prostate in HFD-fed aging and aging mice. Nevertheless, merely the high PJE dose ameliorated the toll-like receptor 4 (TLR4), and nuclear factor κB (NF-κB) contents in aging mice, and IL-1β, SOD2, and IL-6 levels in HFD-aging mice. Authors stressed that PJE had a dose-dependent impact on regulating oxidative-stress and inflammation in HFD-fed aging and aging mice prostate [90].

Rat Model Experiments
Oral administration of the ACNs extracted from black soybeans significantly reduced prostate weight in a rat model of BPH induced by testosterone propionate (TP). The mean prostate weight for the control, BPH-induced rats, and rats receiving 40, 80, and 160 mg/kg ACNs were 674.17 ± 28.24 mg, 1098.33 ± 131.31 mg, 323.00 ± 22.41 mg, 324.00 ± 26.80 mg, and 617.50 ± 31.08 mg, respectively. The average prostate weight in the ACNs-treated groups were markedly lower rather than in the BPH-induced rat, while the apoptotic body counts were significantly higher than in the BPH-induced animal. These findings propose that ACNs may be useful in reducing the volume and inhibiting the proliferation of the prostate [82].
In another study, Jang et al. showed that oral administration of an ACN extracted from the seed coat of the black soybean prevented apoptosis in the prostate cells in andropause animal model by bilateral orchiectomy. Despite reporting no remarkable changes in prostate weight between andropause and andropause plus anthocyanin-treated groups, the apoptotic index was meaningfully lesser in andropause plus anthocyanin-treated groups compared to the andropause group. The oxidative stress in the ACN-administered group was reduced rather than in the andropause group [83].
A diet with ACNs extracted from black soybean may be useful in treating chronic bacterial prostatitis (CBP) rat model. The ACNs plus ciprofloxacin group indicated a significant reduction in improvement of the prostatic inflammation and bacterial growth in comparison with the ciprofloxacin group; suggesting that ACNs may have antimicrobial and anti-inflammatory effects [85].
Seoritae is a black soybean that contains high isoflavone and anthocyanin compounds. Oral administration of seoritae extract (SE) previously demonstrated beneficial effects on the loss of prostate weight and prostate proliferation in the BPH induced by testosterone enanthate in a rat model. The SE treatment groups indicated a significant reduction in prostate weight, apoptosis (caspase-3), oxidative stress markers (8-hydroxy-2-deoxyguanosine and SOD), and 5-α reductase activity compared to the non-treated BPH group [86].
In another research, oral administration of polymerized anthocyanin (PA) from grape skin was able to reduce the prostate weight in the TP-induced BPH rats. PA ameliorated serum dihydrotestosterone (DHT) levels, and the 5-α reductase type 2, androgen receptor, steroid receptor coactivator 1, proliferating cell nuclear antigen (PCNA), prostate-specific antigen, and cyclin D1 expression in prostate tissues. Furthermore, PA downregulated and upregulated the expressions of Bcl-2 and Bax, respectively, in the prostate tissues of rats with BPH. Authors stated that PA may be a possible natural agent for BPH treatment [88].
A diet with the extract of Aronia melanocarpa in TP-induced BPH Wistar rats attenuated prostate enlargement and decreased the levels of 5 α-reductase and DHT in prostate tissue and serum. The PCNA expression in prostate tissue was significantly suppressed in the A. melanocarpa-treated group. These results displayed that the extract (including ACNs) weakened the progression of testosterone-induced prostatic hyperplasia, and recommended that A. melanocarpa has beneficial potential to treat prostate enlargement and BPH [89].
A diet with 1% hexane insoluble fraction from a purple rice ethanolic extract (PRE-HIF) indicated a reduction in the prevalence of adenocarcinoma in the lateral lobes of the prostate and markedly higher percentage of low-grade prostatic intraepithelial neoplasia in transgenic rat for adenocarcinoma of prostate model. In addition, 1% PRE-HIF diet meaningfully repressed the tumor growth in a rat CRPC xenograft model of the PCai1 cells. Additionally, cyclin D1, androgen receptor, fatty acid synthase, and cdk4 expression were reduced while AMP-activated protein kinase α activation, and p38 mitogen-activated protein kinase upregulated in the prostate tissues of PRE-HIF-treated rats and CRPC tumors. Authors stated that the PRE-HIF can inhibit the progression of CRPC and PC by blocking the cell proliferation and metabolic pathways [60].

The Efficacy Assessment of Anthocyanidins/Anthocyanins on Prostate Cancer through Clinical Trials
In three clinical trials, the efficacy of ACDs or ACDs rich fruits have assessed, while 198 prostatic cancer men were targeted in those studies (Table 3). In a clinical trial, the effect of black raspberry (Rubus occidentalis) in different concentrations were evaluated on 56 men newly diagnosed with resectable prostate cancer. According to the results, during 8 weeks of black raspberry storage, no significant changes in bioactivities were observed, whereas with both dosages of 10 and 20 g black raspberry/day in nectar and confection products were accepted to be considered for further prostate cancer clinical trials [93].
Inflammation of the bladder which is called acute radiation cystitis, is defined as a prevalent side effect of external beam radiation therapy, used for the treatment of prostate cancer [94]. In two double-blinded randomized placebo-controlled trials, the impact of ACNs on cystitis of the patients during radiation therapy treatment have been investigated.
Hamilton et al. selected 41 patients with normal mainly New Zealand European diet, who were receiving image guided intensity modulated radiation therapy for the treatment of prostate cancer. The patients were taken one capsule of cranberry (Vaccinium macrocarpon) per day after breakfast for two weeks post radiation therapy treatment. Consequently, 65% and 30% of patients taking the capsules developed cystitis and severe cystitis, thus taking of cranberry is suggested for patients receiving radiation therapy, specifically with low hydration regimens or baseline urinary symptoms [94].
In another clinical trial, 101 men were chosen, who were receiving beam radiation therapy for the treatment of their prostate or prostate bed. After taking two capsules containing cranberry (Vaccinium macrocarpon) daily, during radiation therapy, no remarkable effect on RICAS (novel radiation induced cystitis assessment scale) was observed in comparison with the taking capsules and placebo groups. Therefore, it is suggested that analysis of RICAS can be implemented for measuring severe cystitis in prostate cancer therapy [95]. Since the fruits analysed in the abovementioned studies are well-known as rich sources of ACDs/ACNs, thus the effects can accordingly be related to those compounds, however further studies might be interesting to assess their effects in pure forms.

Conclusions and Perspectives
Several successful anti-tumor drugs have been derived and developed from natural sources, more effective than synthetic counterparts. So far, paclitaxel and the analogs docetaxel and cabazitaxel, vincristine, vinblastine and their derivatives vindesine and vinorelbine, camptothecin and analogs belotecan, topotecan, and irinotecan, besides podophyllotoxin and analogs etoposide and teniposide are the well-known phytochemical drugs for the remedy of diverse cancer types. In addition, the other natural sources have further proven be worth to investigate their secondary metabolites; notably doxorubicin, epirubicin, daunorubicin, bleomycin, and dactinomycin have been isolated and developed from bacteria, prescribed as potent natural antitumor medications [96,97]; therefore discovering potent antitumor natural products is still a promising approach for researchers in diverse fields.
Through a comprehensive literature overview, the present study described the beneficial effects of ACDs/ACNs, and the plants rich in these compounds on the treatment and prevention of prostate cancer. The preclinical including in vitro and animal studies confirmed the valuable effects of these secondary metabolites. Overall, the ACNs indicated higher anti-prostate cancer activities compared to the aglycone derivatives (ACDs) and plant crude extracts, whilst among them cyanidin-3-O-glucoside is the most studied compound possessing highest potency, which can abundantly be found in rice, maize, cherry, berries, etc.
Through animal studies, these compounds and their rich content extracts have further demonstrated potent effects on suppressing prostatic malignancy. The major pathways including anti-inflammatory (via reducing NF-κB/p65 and PCNA expression), and apoptosis induction (via reducing Bcl-2 and increasing Bax expression), as well as physical effects on tumor cells i.e., attenuating the tumor weight and prostate hyperplasia can be highlighted as effective traits of these natural products.
Fruits are inseparable part of the human diet, while wide range of them are as sources of ACDs/ACNs. Through preclinical and clinical studies, the preventive effects of these natural class of compounds against various cancer types have been confirmed. Lin et al. overviewed their high cancer chemopreventive potencies in accordance with the remarkable related biological effects (e.g., anti-inflammation, antioxidant, anti-proliferation, antiinvasion, inducing cell cycle arrest, apoptosis, autophagy, anti-metastasis, etc.) [9]. As a consequence of the present review, aside from potent activities of ACDs/ACNs for the treatment of PC, their abilities to prevent this malignancy are also acknowledged.
Since most of the studies have evaluated the crude extracts, further in vitro and in vivo experiments can potentially discover promising bioactive derivatives of these natural compounds, as adjuvant or merely consumption for the prevention and treatment of prostate cancer, while they might be of interest by phyto-pharmaceutical and food industries. Moreover, the efficacy assesments through clinical trials as well as toxicological studies of them in pure forms can confirm their potential effects on prevention and treatment of prostate cancer.

Acknowledgments:
The authors wish to thank the support of the Vice-Chancellor for Research of Shiraz University of Medical Sciences (Grant No: 25062). This agency was not involved in the design of the study and collection, analysis, and interpretation of data as well as in writing the manuscript.

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