10.1. INTRODUCTION

Eating a diet rich in fruits and vegetables may lead to a reduction in the risk of common forms of cancer and may be useful in cancer prevention. For pomegranate (Punica granatum L.), both basic and clinical evidence of the benefits of particular classes of bioactive substances have been developed specifically for the juice and juice extracts. Since ancient times, pomegranate has been used for medicinal purposes. Extensive research on bioactive substances in the pomegranate extract has shown potential applications in the chemoprevention of common forms of cancer. This work has progressed in cell culture, human studies, and in some clinical research demonstrating the preventive potential of pomegranate.

Pomegranates have been shown to contain 124 different phytochemicals, and some of them act in concert to exert antioxidant and anti-inflammatory effects on cancer cells. Ellagitannins are bioactive polyphenols present in pomegranate. Pomegranate juice obtained by squeezing the whole fruit has the highest concentration of ellagitannins than any commonly consumed juice and contains the unique ellagitannin, punicalagin. Punicalagin is the known largest molecular weight polyphenol. Pomegranate ellagitannins are not absorbed intact into the blood stream but are hydrolyzed to ellagic acid over several hours in the intestine. Ellagitannins are also metabolized into urolithins by gut flora, which are conjugated in the liver and excreted in the urine. These urolithins are also bioactive and inhibit prostate cancer cell growth. Research on basic mechanisms of action in cell culture, animal model systems, and limited clinical research in prostate cancer patients has been carried out with pomegranate juice. This chapter discusses the evidence for the bioactivity of pomegranate ellagitannins, along with the progress in defining the pharmacokinetics and metabolism of ellagitannins in pomegranate juice in humans.

10.2. BIOACTIVITY OF POMEGRANATE POLYPHENOLS AND METABOLITES

Ellagitannins are a family of bioactive polyphenols in fruits and nuts such as pomegranates, black raspberries, raspberries, strawberries, walnuts, and almonds (Amakura et al. 2000; Clifford and Scalbert 2000). Squeezing whole pomegranate fruit (P. granatum L.) yields the richest source of ellagitannins among other fruit juices. This juice has been used for centuries in ancient cultures for medicinal purposes (Longtin 2003). Commercial pomegranate juice, which has recently become popular in the United States, has more potent antioxidant properties than other common fruit juices, and this is attributed to its high content of polyphenols. Emerging science has demonstrated anticancer effects, with the most impressive data so far in prostate cancer. However, the inhibition of subcellular pathways of inflammation triggered by nuclear factor κB (NF-κB), angiogenesis under hypoxic conditions triggered by the hypoxia-inducible factor 1α (HIF-1α), and cellular proliferation along with the stimulation of apoptosis suggests that ellagitannins can act through multiple pathways and may be used as a dietary agent for preventing and treating many common forms of cancer.

The most abundant type of polyphenols in pomegranate juice are ellagitannins that are hydrolyzable tannins releasing ellagic acid on hydrolysis (Gil et al. 2000) and form urolithins such as urolithin A following metabolism by gut flora (FIGURE 10.1). Punicalagin is unique to pomegranate and is part of a family of ellagitannins, which also include minor tannins such as punicalin and gallagic acid (structures not shown here). All these ellagitannins have in common the ability to be hydrolyzed to ellagic acid, resulting in a prolonged release of ellagic acid into the blood following the ingestion of pomegranate juice.

FIGURE 10.1

Chemical structures of punicalagin isomers, the major ellagitannins present in pomegranate juice and its metabolites, dimethylellagic acid glucuronide, ellagic acid, and urolithins A and B.

Among the pomegranate ellagitannins, punicalagin, which is the largest polyphenol, having a molecular weight of greater than 1000, is reported to be responsible for more than half the potent antioxidant activity of the juice (Gil et al. 2000). Punicalagin is most abundant in the fruit husk as opposed to the juicy seeds (arils) found within the fruit. By pressing the whole fruit during the squeezing process, ellagitannins are extracted into pomegranate juice in significant quantities, reaching levels of >2 g/L juice (Gil et al. 2000). Pomegranate juice also contains other polyphenols, such as anthocyanins (cyanidin, delphinidin, and pelargonidin glycosides) and flavonols (quercetin, kaempferol, and luteolin glycosides; Gil et al. 2000). In all, about 124 phytochemicals have been identified in the pomegranate (Seeram, Schulman, and Heber 2006).

Following metabolism by gut flora, urolithins A and B are formed and conjugated in the liver prior to excretion in the urine over 12–56 hours after a single administration of 8 oz (250 mL) of pomegranate juice. These urolithins circulate in the blood and can reach many of the target organs where the effects of pomegranate ellagitannins are noted.

10.3. CANCER PREVENTIVE POTENTIAL OF POMEGRANATE POLYPHENOLS

Results from studies in cells, animals, and humans clearly point to the importance of following ellagitannin metabolites as markers of pomegranate juice intake and studying them in detail to explain the effects of pomegranate juice on the inhibition of prostate cancer cell growth in vitro and in severe combined immunodeficient (SCID) mice with orthotopically transplanted human prostate cancer cells. Our group and others have also shown that pomegranate fruit extract and its purified ellagitannins inhibit the proliferation of human cancer cells and modulate the inflammatory subcellular signaling pathways and apoptosis (Afaq et al. 2005; Seeram et al. 2005; Adams et al. 2006).

Based on the observation that a pomegranate fruit extract inhibited prostate cancer growth in athymic nude mice, some authors have proposed that anthocyanins are the major phytochemical group responsible for the observed anticancer effects (Malik et al. 2005). Although anthocyanins do contribute to the observed antioxidant activity of pomegranate juice, it is unlikely that anthocyanins account for the complete profile of activities observed with pomegranate juice and extracts. In fact, pomegranate extracts containing ellagitannins without anthocyanins have previously been shown to exhibit in vitro and in vivo anticarcinogenic properties, including induction of cell-cycle arrest and apoptosis, inhibition of tumor formation, and xenograft tumor growth (Castonguay et al. 1997). The bioavailability of ellagitannins from pomegranate extract is equivalent to that observed after the administration of pomegranate juice, so that this point is highly relevant to the potential utility of pomegranate extracts as dietary supplements (Seeram, Zhang et al. 2008).

Inflammation is a hallmark of prostate cancer and is observed in prostate tissue at the time of prostatectomy. Inflammation has also been implicated in colon cancer, breast cancer, and other common forms of cancer. Indeed, the proliferative inflammatory atrophy may be a precursor to Prostatic Intraepithelial Neoplasia (PIN) and prostate cancer (De Marzo et al. 2003). Multiple molecular targets are related to the inflammatory pathway in prostate and other types of cancer cells (Figure 10.2). Inflammatory cells are found in prostate tissue at the time of prostatectomy and the NF-κB expression is increased in more advanced lesions. In fact, nuclear localization of NF-κB is a risk factor for prostate cancer recurrence following the prostatectomy (Fradet et al. 2004). In other cancers also, this transcription factor is found to be of central importance. It is fair to say that without this transcription factor, there is no inflammation. It is unknown whether this is the only pathway mediating the effects of ellagitannins, and it is entirely possible that other interacting pathways reviewed in this section may well be involved.

FIGURE 10.2

NF-κB activation in inflammatory cells can lead to oxidant stress and inflammation in prostate epithelial cells. Constitutive activation of NF-κB is then found in prostate cancer cells and may be the result of the interaction of inflammatory (more...)

NF-κB activation leads to immune activation, inflammation, and cell proliferation (Biswas et al. 2000; Kim, Sovak, and Zanieski 2000). NF-κB can also upregulate the transcription of genes that produce collagenases, cell adhesion molecules, and inflammatory cytokines, including tumor necrosis factor α (TNF-α) and interleukins (IL) 1, 2, 6, and 8 (Conner and Grisham 1996; Allison 1997; Winyard and Blake 1997). NF-κB regulates genes involved in the immune and inflammatory responses, as well as the cell-cycle control and cell death in response to proinflammatory cytokines such as IL-1 and TNF-α (Rayet and Gelinas 1999; Mayo and Baldwin 2000). NF-κB is also associated with the transcription of genes involved in cell survival, such as Bcl_x and inhibitors of apoptosis. Constitutive activation of NF-κB is identified in prostate cancers (Domingo-Domenech et al. 2005). Interestingly, the genes coding for the NF-κB proteins p52 and p65 have been mapped to sites of frequent rearrangement and amplification, which give rise to many cancers. PC3 and DU145 prostate cancer cell lines and prostate carcinoma xenografts have demonstrated constitutive NF-κB activity through constitutive activation of IκB kinase α (IKKα) protein complex (Palayoor et al. 1999; Gasparian et al. 2002; Suh et al. 2002).

Activation of NF-κB also regulates a number of downstream genes, including cyclooxygenase-2 (COX-2). COX-2 is the key enzyme regulating the production of prostaglandins, the central mediators of inflammation. The COX-2 expression is induced by several extracellular signals, including proinflammatory and growth-promoting stimuli. COX-2 mRNA expression is regulated by several transcription factors, including the cyclic-AMP response element-binding protein (CREB), NF-κB, and the CCAAT-enhancer-binding protein (C/EBP). COX-2 is also affected posttranscriptionally at the level of mRNA stability. Inflammatory cells such as macrophages and mast cells release angiogenic factors and cytokines, such as TNF-α, IL-1, and vascular endothelial growth factor (VEGF; O’Byrne and Dalgleish 2001), which signal cell growth and proliferation.

Ellagitannins and their hydrolysis product, ellagic acid, inhibit prostate cancer cell growth through cell-cycle arrest and stimulation of apoptosis (Castonguay et al. 1997; Albrecht et al. 2004; Lansky et al. 2005). In addition, they inhibit the activation of inflammatory pathways including, but not limited to, the NF-κB pathway. Inhibition of angiogenesis has also been demonstrated both in vitro and in vivo for prostate cancer. The universal nature of these mechanisms in common forms of cancer suggests that pomegranate ellagitannins, which have been tested in both prostate and colon cancer cells by our group, may also be useful dietary agents for the prevention and treatment of other forms of cancer, such as breast cancer.

10.4. MECHANISTIC INSIGHTS FROM CELL CULTURE AND ANIMAL STUDIES

In cell culture, combinations of ellagitannins are more potent than any single compound (Narayanan et al. 1999; Losso et al. 2004; Seeram et al. 2005; Sartippour et al. 2008). They have activity in combination against both prostate and colon cancer cells. While punicalagin is the most active of ellagitannins, it is possible to design experiments that demonstrate the additional effects of other phytochemicals found in pomegranate juice.

Overall, the significance of these in vitro findings is in doubt because only ellagic acid (along with the urolithins) is found in the blood circulation after the ingestion of pomegranate juice. Urolithins are formed by the action of gut bacteria on ellagitannins and are recirculated through the liver prior to excretion in the urine. Urolithins inhibit the growth of both androgen-dependent and androgen-independent prostate cancer cell lines, with IC₅₀ values lower than that of ellagic acid. Future studies to evaluate the mechanistic basis for the antiproliferative effects of urolithins are required.

Angiogenesis is critical to tumor growth and is stimulated by tissue hypoxia due to poor oxygen delivery. Cellular hypoxia leads to angiogenesis via the induction of HIF-1α and VEGF at a cellular level. Pomegranate juice and extracts, which are rich sources of ellagitannins, have been shown to have chemopreventive potential against prostate cancer, but there have been no studies to date on the effects of an ellagitannin-rich pomegranate extract on angiogenesis directly. However, in the study of human prostate cancer cells (LNCaP) and human umbilical vein endothelial cells (HUVEC) incubated in vitro with a pomegranate extract standardized to ellagitannin content (POMx), the proliferation of LNCaP and HUVEC cells was significantly inhibited under both normoxic and hypoxic conditions, and HIF-1α and VEGF protein levels were decreased by POMx under hypoxic conditions (Sartippour et al. 2008).

Recently, there have been a number of reports on antiproliferative, proapoptotic, and antiangiogenic activity by pomegranate polyphenols, as well as on the inhibition of NF-βP activity and xenograft growth (Albrecht et al. 2004; Afaq et al. 2005; Lansky et al. 2005). In a recent study by Sartippour et al. (2008), human prostate cancer cells (LAPC4) were injected subcutaneously into SCID mice, and the effects of the oral administration of POMx on tumor growth, microvessel density, and HIF-1α and VEGF expressions were determined after 4 weeks of treatment. POMx decreased prostate cancer xenograft size, tumor vessel density, VEGF peptide levels, and HIF-1α expression (Sartippour et al. 2008). These results demonstrate that an ellagitannin-rich pomegranate extract can inhibit tumor-associated angiogenesis as one of the several potential mechanisms for slowing the growth of prostate cancer in chemopreventive applications. Further studies in humans are needed to confirm that angiogenesis can be inhibited by an ellagitannin-rich pomegranate extract administered orally as a dietary supplement.

Constitutive NF-κB activation is observed in androgen-independent prostate cancer and represents a predictor for biochemical recurrence after the radical prostatectomy. Pomegranate extract inhibited NF-κB activation and cell viability of prostate cancer cell lines in a dose-dependent fashion in vitro. Importantly, maximal pomegranate extract-induced apoptosis was dependent on pomegranate extract-mediated NF-κB blockade. In the LAPC4 xenograft model, pomegranate extract also delayed the emergence of LAPC4 androgen-independent xenografts in castrated mice through an inhibition of proliferation and induction of apoptosis. Moreover, the observed increase in NF-κB activity during the transition from androgen dependence to androgen independence in the LAPC4 xenograft model was abrogated by pomegranate extract. These observations suggest that pomegranate extract may be active in the prevention of the emergence of androgen independence that is driven in part by heightened NF-κB activity (Rettig et al. 2008).

The androgen independence of prostate cancer is characterized by intracellular androgen synthesis. In men treated with antiandrogen therapy, blood levels of testosterone are at castrate levels, whereas intracellular testosterone levels are about 50% the normal levels. Pomegranate extract was shown to inhibit the expression of the genes coding for the major androgen-synthesizing enzymes in vitro. This may be another mechanism through which pomegranate ellagitannins inhibit androgen-independent prostate cancer cell proliferation (Hong, Seeram, and Heber 2008).

The aromatase enzyme found in fat tissue and some breast tumors converts adrenal androgens into estrogen and represents the primary source of estrogens in postmenopausal women. Recent studies have demonstrated that in cell culture, urolithins, the metabolites of pomegranate ellagitannins, can inhibit the proliferation of MCF-7 breast cancer cells with transgenic overexpression of aromatase (MCF7aro cells). In addition, the isolated enzyme is inhibited by urolithin A in vitro. Our laboratory is currently studying the relevance of these observations in animal and human studies relevant to the prevention of breast cancer and the potential interaction of diet and exercise for breast cancer prevention (Adams et al. 2010).

Finally, insulin-like growth factor 1 is known to stimulate prostate cancer cell proliferation, whereas insulin-like growth factor binding protein 3 (IGFBP-3) induces apoptosis of prostate cancer cells. Recently, pomegranate extract was shown to enhance the proapoptotic effects of IGFBP-3 (Koyama et al. 2010).

10.5. EVIDENCE OF BIOACTIVITY FROM HUMAN CLINICAL STUDIES

Plasma prostate-specific antigen (PSA) is a biomarker of prostate cancer progression. A clinical study in men with increasing levels of PSA after surgery or radiotherapy was begun in January 2003. Eligible patients had a PSA level > .2 ng/mL and < 5 ng/mL, and a Gleason score of 7 or less. The Gleason score is a pathological grading of tumors that predicts to some extent the future clinical course and guides therapy. The score is a composite of the predominant histology. The transition between the aggressive biology and the more commonly encountered biology is a score of 7. About 30% of men with a Gleason score of 7 have biochemical recurrence after primary therapy by surgery or radiation. Patients were provided with 8 oz (250 mL) of pomegranate juice (wonderful variety) to consume daily. Each 8 oz serving contained 570 mg of total polyphenols, quantitated as gallic acid equivalents. Interim results were published by Pantuck et al. (2006) and showed a significant increase in mean PSA doubling time following the treatment from 15 months at baseline to 54 months post-treatment (p < .001). In this study, 85% of patients had a decrease in the rate of PSA rise, which is being secreted solely under these conditions by prostate cancer cells that have proliferated after the removal of the primary tumor and all normal prostate tissue.

The PSA doubling time is a predictor of survival in prostate cancer patients with recurrent disease. The study was amended to allow patients to continue the pomegranate juice treatment and to undergo evaluation at 3-month intervals until clinical disease progression, defined as the decision by their urologist to use androgen blockade. In an ex vivo mitogenic bioassay, serum obtained from these pomegranate juice-treated patients, who were given 8 oz (250 mL) of pomegranate juice daily for 2 years, inhibited proliferation and stimulated apoptosis of LNCaP prostate cancer cells in vitro when the patient’s serum was substituted for fetal calf serum in the cell-culture medium (Aronson et al. 2010). Further studies have been designed to determine whether ellagic acid, urolithins, and related metabolites in patient sera are responsible for these antiproliferative and proapoptotic effects.

10.6. DETAILED STUDIES OF BIOAVAILABILITY AND METABOLISM

Because the bioavailability of phytochemicals is critical to their bioactivity, it was studied in 18 volunteers to quantitate the plasma appearance and disappearance rates of ellagic acid hydrolyzed from ellagitannins in administered pomegranate juice (Seeram, Lee, and Heber 2004). In addition, it was demonstrated that the absorbed ellagic acid from the hydrolysis of pomegranate juice punicalagin is converted to dimethylellagic acid glucuronide (DMEAG) in plasma and urine on the day of administration of pomegranate juice (Seeram, Lee, and Heber 2004; Seeram, Henning et al. 2006). Urolithins derived from ellagic acid appeared in human urine after the disappearance of DMEAG about 12 hours after the administration of pomegranate juice.

Additional bioavailability data on DMEAG and urolithins were obtained in mice in support of our planned studies on the effects of pomegranate juice on orthotopically transplanted LNCaP cells in SCID mice (Seeram et al. 2007). Studies in rats and humans have shown that ellagitannins are hydrolyzed to ellagic acid in the gut, and this is metabolized by the colon microflora to form urolithins A and B. Urolithins can be absorbed into the enterohepatic circulation and can be excreted in urine and feces (Cerda et al. 2003; Cerda et al. 2004; Espin et al. 2007). Ellagic acid and urolithins can accumulate in the intestine and prostate (Larrosa, Tomás-Barberán, and Espin 2006; Seeram et al. 2007). Ellagitannins, ellagic acid, and urolithin A exhibit cancer chemopreventive activities in various cell and animal models. Oral administration of the pomegranate extract to wild-type mice led to increased plasma levels of ellagic acid, but ellagic acid was not detected in the prostate gland. On the other hand, intraperitoneal administration of pomegranate extract led to tenfold higher ellagic acid levels in the plasma and detectable and higher ellagic acid levels in the prostate, intestine, and colon relative to other organ systems. The detectable ellagic acid levels in prostate tissue following intraperitoneal, but not oral, administration were likely due to higher plasma levels attained after the intraperitoneal administration.

Intraperitoneal and oral administration of synthesized urolithin A led to uptake of urolithin A and its conjugates in prostate tissue, and levels were higher in prostate, colon, and intestinal tissues relative to other organs. It is unclear why pomegranate ellagitannins metabolites localize at higher levels in the prostate, colon, and intestinal tissues relative to the other organs studied. Importantly, the predilection of bioactive pomegranate ellagitannins metabolites to localize in prostate tissue, combined with clinical data demonstrating the anticancer effects of pomegranate juice, suggests the potential for pomegranate products to play a role in prostate cancer chemoprevention. Whether uro-lithins in human prostate tissue can be used as a biomarker following the long-term administration of pomegranate juice or pomegranate extract remains to be determined.

10.7. CONCLUSIONS

The ellagitannins found in pomegranate fruit are very potent antioxidants, and pomegranate juice exceeds the in vitro antioxidant potency of other common commercial fruit juices (Seeram, Aviram et al. 2008). There are limited treatment options for prostate cancer patients who have undergone primary therapy such as radical prostatectomy with curative intent but who have progressive elevations in their PSA. Pomegranate juice given daily for 2 years to 40 prostate cancer patients with increasing PSA levels provides evidence for the possible utilization of a nontoxic option for prevention or delay of prostate carcinogenesis. It is remarkable that 85% of patients responded to pomegranate juice in this study. Both in vitro and in vivo investigations in prostate cancer models of the molecular mechanisms that may account for these pomegranate juice effects have been explored.

Although researchers focus on the idea of a single pathway, it is evident that pomegranate ellagitannins, like other phytochemicals, work through multiple targeted pathways. Nonetheless, evidence that NF-κB activation is associated with heightened proliferation, increased neoangiogenesis, and resistance to apoptosis suggests that the antitumor action of pomegranate juice polyphenols are mainly mediated through their NF-κB inhibitory effects. This hypothesis is given added support by the recent implication of NF-κB as an independent risk factor for PSA rise (i.e., the sign of biochemical recurrence of prostate cancer) after the prostatectomy. Studies of the effects of pomegranate juice and dietary supplements made from pomegranate extract in patients prior to prostatectomy and after biochemical recurrence with increasing PSA are ongoing and should provide further information on the prostate cancer prevention and treatment potential of the juice of this ancient fruit. Further studies on other forms of cancer, including colon cancer and breast cancer, may reveal additional potentials for pomegranate juice in cancer chemoprevention.

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