*2.2. Anticancer Properties*

Cancer is the principal cause of death in both developed and developing countries. The mortality rate of this pathology is superior among low and middle-income populations. The higher mortality rates in less well-o ff countries are mainly due to the lack of adequate health systems [33–36]. Reactive oxygen species' (ROS) overproduction is held liable to be one of the key factors for the development of several diseases, including cancer. Tumor biology has revealed that most neoplasms have a much higher amount of reactive oxygen species than healthy ones, such as superoxide anion, H2O2, and hydroxyl radicals [37]. These reactive oxygen-containing chemicals react with nucleic acids, proteins, and lipids, contributing significantly to tumor cells proliferation, DNA alterations, apoptosis, metastasis, and angiogenesis [38–41].

As a consequence of the troubles produced by the existing chemotherapeutic agents, nowadays there is an increasing concern in the search for herbal formulation with cancer preventive e ffect. Indeed, studies are focusing particularly on fruits rich in polyphenols due to their anticancer potential [42].

In order to investigate the e ffectiveness of pomegranate and its derivatives as anti-proliferative, anti-invasive, and pro-apoptotic agents, several studies have been conducted on di fferent cell lines, such as breast cancer lines (MCF-7 and MDA-MB-231), uterine cancer lines (HeLa and Ishikawa), colorectal adenocarcinoma lines (RKO), and animal models [43–45]. Some research groups have shown that the simultaneous use of skin, seed, and pomegranate juice extracts has a synergistic action in inhibiting cell proliferation in several in vitro models, such as: Human breast MCF-7, uterine HeLa, human prostate DU 145, and PC-3 cancer cells. [46,47]. This result has been confirmed by Hong et al. [48], who demonstrated that pomegranate extracts and juice components have a more potent action than the individual isolated polyphenols, suggesting that it is a synergistic and additive e ffect of many phytochemical compounds, among which proanthocyanidins, anthocyanins, flavonoids, and ellagitannins. The proanthocyanidins are strong antioxidant compounds that, after acid hydrolysis, can release catechins as (+)-(2R,3S)-catechin (1). They act synergistically with ascorbic acid, which gives therapeutic potential against neoplastic and cardiovascular diseases, since it is able to suppress the action of free radicals, as well as to protect the body from the development and metastasis of cancer (which in part depends on the damage caused to the DNA). The anthocyanins are derivatives of phenyl-4H-benzopyran-4-one and are present in the vacuoles of the epidermal cells of many plants [49]. These are characterized by a high chemical reactivity and very low toxicity. They have, in fact, an anti-free radical action and they modulate the arachidonic acid cascade by inhibiting cAMP-phosphodiesterase [49]. The main anthocyanins present in the pomegranate juice are: cyanidin (2), delphinidin (3), and pelargonidine-3-O-glucoside (4). It was shown that anthocyanins decrease the proliferation of colon cancer cells, HT-29, in a dose-dependent manner [5].

The flavonoids, present in the bark and skin responsible for the red color, have a very high antioxidant activity and are useful for blood circulation [50]. The main flavonoids are quercetin (5), which, in addition to an antioxidant action [51], also showed antiviral and cardioprotective effects [52,53], kaempferol (6), with anticancer properties, and the rutin (7), a molecule with antithrombotic properties [54]. Among the ellagitannins, ellagic acid (8), a highly thermostable molecule, can be extracted from pomegranate skin and possesses important biological activities including antitumor, antiviral, and antimicrobial [55]. In addition, it was shown that ellagic acid (8) induces cell lysis, apoptosis, and thus decreases cell viability due to DNA breakage and alterations in the cell cycle. Gonzalez–Sarrias et al. [56] have demonstrated that ellagic acid and its secondary products can contribute to the prevention of colon cancer by regulating the expression of multiple genes implicated in crucial processes linked to the development of cancer.

Another tannin, the punicalagin (9), is present almost exclusively in the skin. It has several pharmacological properties, among which are anti-inflammatory, anti-proliferative, pro-apoptotic, and anti-genotoxic [57]. It also induces apoptosis in colon cancer cells (cell lines: HT-29, HCT116) and prostate cancer cells at a concentration of 100 mg/mL [5]. A study with 46 patients with prostate cancer under experimentation showed, for 16 of them, a considerable decrease in PSA (prostate-specific antigen) during treatment with pomegranate juice [58].

Koyama et al. [59] report that treatment of LAPC4 prostate cancer cells with 10 μg/mL pomegranate extract, obtained from seedless arils and skin, and standardized to a 37% ellagitannin content in punicalagin, blocks cell proliferation and triggers apoptosis. Furthermore, pomegranate skin extract (PoPx) with a high concentration of ellagitannins provokes apoptosis in human breast cancer cells (MCF-7), estrogen receptor (ER)-positive (ER+).

In previous investigations, PoPx and genistein have shown significant inhibitory effects on the proliferation of breast cancer cells MCF-7. In addition, PoPx can impede cell proliferation and the expression of angiogenesis markers, phosphorylation of p38, and C-Jun protein kinases activated by mitogens, and the activation of pro-survival signaling pathways. PoPx inhibits the nuclear factor NF-kB gene expression, connected with proliferation, invasion, and motility in aggressive breast cancer phenotypes [60]. Aiyer et al. reported that treatment at a dose of 300 mg/mL of pomegranate fruit extract (PoMx) in combination with 1 μM tamoxifen is able to reverse the resistance [61]. Moreover, recently Peng et al. identified PoMx as an interesting agen<sup>t</sup> to cope up with oral cancer metastasis. Indeed, it was able to block wound healing migration, transwell migration, and matrix gel invasion. Analyzing the molecular mechanism, they also demonstrated that PoMx was able to downregulate matrix metalloproteinase MMP-2 and MMP-9 activities and expressions as well as epithelial-mesenchymal transition (EMT) signaling in HSC-3 cells [62].

PoPx prevent melanocyte proliferation and melanin synthesis by suppressing tyrosinase activity (IC50 = 182.2 mg/mL). The amount of inhibition is comparable to arbutin, a glycoside with isoquinoline structure capable of being hydrolyzed in glucose and hydroquinone. An extensive amount of research has confirmed the ability of PoPx and ellagitannins to block the generation of free radicals in Ultraviolet (UV)A- and B-irradiated human skin, thus defending it from DNA fragmentation, from skin burns and depigmentation [63,64].

In 2010, Dai et al. reported that pomegranate extract (PE) is able to inhibit in a time- and concentration-dependent manner (already at 10 μg/mL) the viability and proliferation of a mouse mammary cancer cell line (WA-4, resulting from mouse MMTV-Wnt-1 mammary tumors) characterized by the presence of several cells possessing stem cells features. In particular, the arrest of cellular growth in the G0/G1 phase and an increased level of caspase 3 enzyme was observed, suggesting the mechanism of cell death by apoptosis.

Further investigations assessed the outcomes of individual phytochemicals derived from PE. Indeed, ellagic acid, ursolic acid (10), and luteolin (11) could contribute to the inhibitory potential of PE, with an IC50 value of 10 μM. Instead, caffeic acid seemed to be inefficient [65].

Subsequently, in 2011, the same research group verified the arrest of cell growth due to PE but in the G2 phase by using as an in vitro model the human pancreatic cells PANC-1 and AsPC-1. The inhibition of cellular growth was already observed with an IC50 amounting to 50 μg/mL in both the lines, but individual phytochemicals were slightly responsible for it. However, data showed increased proportion levels of cells lacking of CD44 and CD24 (connected with high tumor-initiating ability), demonstrating that PE can modify cell phenotypes, reducing the tumorigenicity [66].

Motaal and Shaker assessed the antioxidant and anticancer properties of di fferent PEs, highlighting that the peel extract possesses the best antioxidant activity with an IC50 value of 0.50 ± 0.9 mg/mL, and promising anticancer action toward MCF-7 breast cancer cells and HCT-116 colon cancer with IC50 values of 7.7 ± 0.01 and 9.3 ± 0.06 μg/mL, respectively [67].

In 2015, El-Awady and co-workers investigated the possible antitumor activity of two di fferent extracts of *Punica Granatum* grown in Saudi Arabia. Their findings evidenced a good percentage of cellular growth inhibition at the maximum concentration tested (100 μg/mL) from both seeds and husks extracts, tested on hepatocellular carcinoma HepG2 (values amounting to 95.8 and 98.3%, respectively) and colon cancer CACO cells (values amounting to 99 and 97%, respectively). The whole cytotoxic profile exhibited a dose-dependent trend. The percentages of cellular growth inhibition permitted to determinate the corresponding IC50 values. They were 45 and 40 μg/mL on both HepG2 and CACO cells for pomegranate seeds and husks extracts, respectively [68].

Another study, performed by Modaeinama et al. in 2015, documented that the peel methanolic extract possesses a potent anti-proliferative action toward several tumor cell lines as: MCF-7 (breast adenocarcinoma), A549 (lung non-small cell cancer), SKOV3 (ovarian cancer), and PC-3 (prostate adenocarcinoma). The cytotoxicity has been investigated at di fferent concentrations; the best antitumor effect was detected on MCF-7, with a percentage of survival inhibition amounting to 83.7% at doses of 5 μg/mL. Instead, for the other cell lines, this percentage was 77.87, 76.54, and 63.41% for PC3, A549, and SKOV3, respectively, at the same dosage used for MCF-7 [69].

Research based on the possible e ffect of a PE on chronic myeloid leukemia surveys that it is able to suppress cell growth by activating apoptosis and inducing cell cycle arrest. The findings of the research group led by Asmaa, outlined that the peel extract of pomegranate could inhibit K562 cell growth in a dose-dependent manner, with total cell death at the maximum concentration tested and an IC50 value of 100 ± 0.05 μg/mL. Further investigations have assessed the ability of the extract to induce the cell cycle arrest in the G2/M phase and apoptosis. Indeed, an upregulation of proapoptotic proteins caspases 9, 7, 3, and cytochrome c, and a downregulation of antiapoptotic protein Bcl-2 were detected [70].

In depth studies of genetics appraised the possible influence of PE on immunoregulatory-related miRNAs level of adipose-derived MSCs (Ad-MSCs) obtained from adipose tissue. Data suggested that the principal mechanism of action could be due to the capability to decrease the expression of PI3K\AKT1\NF-kB genes involved in inflammatory pathways via miRNAs expression. It plays a role in the immune modulation in Ad-MSCs. In other words, PE could possess a key role in regulating the immunomodulatory function of stem cells [71].

A new approach in the use of phytochemicals for the cancer fighting lies in the knowledge of preparing metal-based nanoparticles from the extract of di fferent plant parts, using green synthesis procedures. Indeed, silver nanoparticles possess the capability to up- and downregulate several cellular mechanisms, and this has propelled to the forefront in investigations to use them as a possible delivery system for other chemicals or as real control systems for the cellular growth [72–74].

In 2018, Sarkar and Kotteeswaran carried out the green synthesis of silver nanoparticles from the aqueous leaf extract of pomegranate, in order to test their capability as anticancerous toward human cervical cancer cells (HeLa). Their findings have proven that this kind of delivery system is able to inhibit cell growth in a dose-dependent manner with an IC50 value of 100 μg/mL. Moreover, they assessed the ability of these pomegranate nanoparticles to induce necrosis and apoptosis by detecting increased levels of lactate dehydrogenase and DNA fragmentation [75].

Recently, Yusefi et al. employed four di fferent weight % of *Punica granatum* fruit peel extract as green stabilizers to produce iron oxide nanoparticles (IONPs) and investigated their potentiality as anticancer agents. Among all the cell lines tested, IONPs have been shown to have a powerful inhibitory e ffect against nasopharyngeal carcinoma (NPC) cell line, HONE1. In particular, the most active IONPs, containing 2 and 4 wt% of peel extract, exhibited IC50 values amounting to 197.46 and 85.06 mg/mL respectively [76].

All the data reported contribute to a vision of the state of the art in this field, remarking the potential antiproliferative e ffect of di fferent parts of this fruit on a large panel of tumoral cells. However, in vivo studies, nowadays lacking, could be important to upgrade the researches.
