**4.2.1 Prevention of skin cancer**

There is an urgent need to develop mechanism-based approaches for the prevention/therapy of lethal skin cancer (non-melanoma). Skin is the organ most accessible to sunlight, and directly suffers from the deleterious effects of ultraviolet (UV)1 radiation, that is known to accelerate aging changes, causing fine and coarse wrinkling, rough skin texture, dryness, telangiectasia and dyspigmentation, resulting in skin cell DNA damage (Afaq, 2011). The increase in incidinces of skin cancer due to constant exposure of skin to environmental carcinogens, such as chemical agents and ultraviolet radiation, provides a strong basis for chemoprevention (Gupta & Mukhtar, 2002). There is a considerable attention on the use of naturally occurring botanicals for their potential preventive effect against UV-mediated damages referred to as *photochemopreventive* effects (Afaq et al., 2005). In general, skin carcinogenesis, being a stepwise process of all three distinct stages, is an effective model for cancer chemoprevention (Richmond & Viner, 2003).

Hora et al. (2003) investigated pomegranate seed oil (PSO) stated that PSO appears to be a natural product with potential as a topical chemopreventive agent against skin cancer, through inhibition of PG biosynthesis and ornithine decarboxylase. PSO treatment did not delay the appearance of tumors, but significantly decreased the rate of tumor development, skin tumor multiplicity, and ornithine decarboxylase activity during 20 weeks of promotion. They stated that PSO, being rich in punicic acid, has inhibitory effect on PG biosynthesis, as well as inhibiting upstream eicosanoid enzyme, phospholipase A2.

Murthy et al. (2004) studied the wound healing activity of phenol-rich methanolic extract of dried pomegranate peel on the skin of Wistar rats. Following the application of the extract, formulated as a water-soluble gel, the animals treated with 5% gel showed complete healing

cell-based assay in order to reflect bioavailability of the test compound to the cells, and the antioxidant activity is evaluated in the cellular environment and in terms of inhibition of intracellular generation of reactive oxygen species. They found that urolithins exhibited a significant antioxidant activity correlated with the number of hydroxyl groups as well as

The critical success factor in cancer chemoprevention is the capacity of the agent to selectively inhibit proliferation and/or induce apoptosis in malignant cells, preserving normal cells. Pomegranate, consumed as whole fruit, juice, or any form of derivates, possess anti-proliferative, pro-apoptotic, and/or anti-angiogenic effects superior to those observed with their isolated active compounds, suggesting therapeutic strategies that may depart from preference for pure single agents. There are several publications on the anticarcinogenic effects of pomegranate (Ahmed et al., 2005; Jeune et al., 2005; Malik et al., 2005; Syed et al., 2007; Lansky & Newman, 2007; Hajimahmoodi et al., 2008; Jurenka, 2008, Sartippour et al. 2008; Adams et al., 2010; Adhami et al., 2010; Faria & Calhau, 2010,2011; Miguel et al., 2010). Therefore, due to the vast explosion of interest in pomegranate as a functional food and therapeutic source the present work is launched to make a review that highlights anticarcinogenic activity of pomegranate and its products of recently published

There is an urgent need to develop mechanism-based approaches for the prevention/therapy of lethal skin cancer (non-melanoma). Skin is the organ most accessible to sunlight, and directly suffers from the deleterious effects of ultraviolet (UV)1 radiation, that is known to accelerate aging changes, causing fine and coarse wrinkling, rough skin texture, dryness, telangiectasia and dyspigmentation, resulting in skin cell DNA damage (Afaq, 2011). The increase in incidinces of skin cancer due to constant exposure of skin to environmental carcinogens, such as chemical agents and ultraviolet radiation, provides a strong basis for chemoprevention (Gupta & Mukhtar, 2002). There is a considerable attention on the use of naturally occurring botanicals for their potential preventive effect against UV-mediated damages referred to as *photochemopreventive* effects (Afaq et al., 2005). In general, skin carcinogenesis, being a stepwise process of all three distinct stages, is an

Hora et al. (2003) investigated pomegranate seed oil (PSO) stated that PSO appears to be a natural product with potential as a topical chemopreventive agent against skin cancer, through inhibition of PG biosynthesis and ornithine decarboxylase. PSO treatment did not delay the appearance of tumors, but significantly decreased the rate of tumor development, skin tumor multiplicity, and ornithine decarboxylase activity during 20 weeks of promotion. They stated that PSO, being rich in punicic acid, has inhibitory effect on PG biosynthesis, as

Murthy et al. (2004) studied the wound healing activity of phenol-rich methanolic extract of dried pomegranate peel on the skin of Wistar rats. Following the application of the extract, formulated as a water-soluble gel, the animals treated with 5% gel showed complete healing

effective model for cancer chemoprevention (Richmond & Viner, 2003).

well as inhibiting upstream eicosanoid enzyme, phospholipase A2.

lipophilicity of the molecules.

**4.2.1 Prevention of skin cancer** 

works.

**4.2 Anticarcinogenic/antitumoral activity** 

after 10 days, whereas in rats treated with 2.5% gel, healing was observed on day 12, in contrast to the positive control animals receiving the blank gel, which took 16–18 days for complete healing. The animals treated with 2.5% gel showed moderate healing (55.8% and 40.8% healing compared with negative and positive controls, respectively), whereas the group treated with 5.0% gel showed good healing (59.5% and 44.5% healing compared with negative and positive controls, respectively). Histopathological studies supported the wound healing increased on application of the gels.

Afaq et al. (2005) showed that pretreatment of mouse skin with pomegranate fruit extract modulated the activation of mitogen-activated protein kinase (MAPKs) and nuclear factor kappa B (NF-κB), in the 12-*O*-tetradecanoylphorbol 13-acetate (TPA)-induced or ultra violet-B induced skin carcinogenesis model. Aslam et al. (2006) assessed the cosmeceutical value of pomegranate where aqueous fraction of the peel was shown to stimulate type I procollagen synthesis and inhibit MMP-1 production by human dermal fibroblasts. Syed et al. (2006) reported the remarkable photochemopreventive effects of pomegranate fruit extract (PFE) against UVA using normal human epidermal keratinocytes (NHEK) as a test system. PFE, extracted edible part of fruit with acetone, treatment was shown to inhibit UVA-induced phosphorylation of STAT3, ERK1/2 and AKT1 in human epidermal cells. In addition, the inhibitory effect of PFE on UVA-mediated phosphorylation of mTOR and p70S6K may have a regulatory effect on the rate of protein synthesis and activation of tumor cell proliferation.

In a study pretreatment of EpiDerm with pomegranate juice, oil or by-product resulted in marked inhibition in the number of cyclobutane primidine dimers (CPDs) and 8-hydroxy-2 deoxyguanosine (8-OHdG) positive cells, ultimately, showing a protective effect of against UVB-mediated DNA damage. UVB irradiation results in the induction in metalloproteinases (MMPs) which degrade extracellular matrix proteins, and eventually, cause skin wrinkling. It was shown that all three components of pomegranate were able to inhibit UVB-induced expressions of MMPs as well as MMP-2 and MMP-9 activity in the EpiDerm (Zaid et al., 2007). Cell culture and animal studies have also supported that intake of pomegranate is associated with decreased skin cancer risk (Pacheco-Palencia et al., 2008). Afaq et al. (2009) found that pretreatment of human reconstituted skin (EpiDermTM FT-200) with pomegranate-derived products inhibited UVB-induced CPDs and 8-OHdG as well as protein oxidation and proliferating cell nuclear antigen (PCNA) protein expression. In addition they reported an inhibition of UVB-induced metalloproteinases (collagenase, gelatinase, stromelysin, marilysin, elastase and tropoelastin).

George et al. (2011) examined the chemopreventive efficacy of pomegranate fruit extract (PFE) and diallyl sulfide (DAS), alone and in combination, using 2-stage mouse skin tumorigenesis model. PFE alone delayed onset and tumor incidence by 55%, while in PFE+DAS combination at low doses synergistically decreased tumor incidence more potentially (84%). In addition, regression in tumor volume was seen with continuous combinatorial treatment (*p* < 0.01). Mechanistic studies revealed that this inhibition was associated with decreased expression of phosphorylated ERK1/2, JNK1 and activated NFκB/p65, IKKα, IκBα phosphorylation and degradation in skin tissue/tumor. Histological and cell death analysis also confirmed that combined PFE and DAS inhibit cellular proliferation and markedly induce apoptosis than the single agents.

The Therapeutic Potential of Pomegranate and Its Products for Prevention of Cancer 343

ellagitannin-rich extract and whole juice extract on the expression of genes for key androgen-synthesizing enzymes [HSD3B2 (3β-hydroxysteroid dehydrogenase type 2), AKR1C3 (aldo-keto reductase family 1 member C3) and SRD5A1 (steroid 5α reductase type 1)] and AR in LNCaP, LNCaP–AR and DU-145 human prostate cancer cells. Pomegranate polyphenols inhibited gene expression and AR most consistently in the LNCaP–AR cell line. Therefore, inhibition by pomegranate polyphenols of gene expression involved in androgensynthesizing enzymes and the AR may be of particular importance in androgenindependent prostate cancer cells and the subset of human prostate cancers where AR is up-

Since the anticarcinogenic activity of ellagic acid, the main polyphenol in the pomegranate, has been shown on several cancer types Malik et al. (2011) evaluated the effect of ellagic acid treatment on the cell viability of human prostate cancer cells. They observed that ellagic acid (10-100 mol/L) treatment (48 h) of human prostate carcinoma PC3 cells resulted in a dose dependent inhibition of cell growth/cell viability. Ellagic acid caused cell growth inhibition which was accompanied by induction of apoptosis, as assessed by the cleavage of poly (ADP-ribose) polymerase (PARP) and morphological changes. Further, ellagic acid treatment was also found to result in significant activation of caspases, as shown by the dose dependent decrease in the protein expression of procaspase-3, -6, -8 and -9. This ellagic acidmediated induction of apoptosis was significantly (80-90%) inhibited by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-FMK).

In a study, Koyama et al. (2010) investigated the relationship between pomegranate-induced apoptosis in human prostate cancer cells and the insuline-like growth factor (IGF)/IGF binding protein (IGFBP) system, as the IGF axis is critical for the regulation of apoptosis in many human cancer cell lines and IGFBPs in serum are responsible for regulation of IGF action, inhibition of cell proliferation and enhancement of apoptosis in many cell types, including prostate (Rajah et al., 1997) and breast (Gucev et al., 1996; Kim et al., 2004) cancers. They concluded that there are novel interactions between the IGF system and pomegranateinduced apoptosis, and pomegranate products modulate the tumor production and responsiveness to IGFs and the IGFBPs. Treatment of LAPC-4 prostate cancer cells with 10 g/mL pomegranate extract, standardized to ellagitannin content (37% punicalagins by HPLC), resulted in inhibition of cell proliferation and induction of apoptosis. Co-treatment with pomegranate extract and IGFBP-3 revealed synergistic stimulation of apoptosis and additional inhibition of cell growth. The researchers also investigated the relationship between IGF-1 and pomegranate-induced apoptosis in 22RV-1 prostate cancer cells. Cotreatment with 100 ng/mL IGF-1 completely blocked apoptosis induction by pomegranate extract. In contrast, IGF-I failed to inhibit pomegranate-induced apoptosis in R- cells, suggesting the importance of IGF-IR. POMx-treatment decreased Igf1 mRNA expression in a dose-dependent manner indicating that its actions also involve tumor-specific suppression

Along with enthusiastic efforts in early diagnosis, aggressive surgical treatment and application of additional non-operative modalities, the prognosis of breast cancer is stil chaotic. Pomegranate has been the target of several work in laboratories and cancer centres and known to have inhibition properties against diverse types of cancers. Recent review

regulated.

of IGF-1.

**4.2.3 Prevention of breast cancer** 

### **4.2.2 Prevention of prostate cancer**

Prostate cancer is the second-leading cause of cancer-related deaths in men in the World. *In vitro* studies stated several pomegranate products inhibit prostate cancer cell growth, induce apoptosis of several prostate cancer cell lines, suppress invasive potential of PC-3 cells, and decrease proliferation of DU-145 prostate cancer cells (Lansky et al., 2005a,b; Pantuck et al., 2006). Albrecht et al. (2004) showed that pomegranate seed oil (PSO) as well as polyphenols present in the pericarp and fermented juice suppress proliferation and invasion of several human prostate cancer cells, LNCaP, PC-3 and DU-145 across the matrigel matrix. Supraadditive, complementary and synergistic effects were proven in all models. Lansky et al (2005b) found equally combined amounts of pomegranate fermented juice, pomegranate pulp juice, cold-pressed pomegranate seed oil extracts resulted in a 99% suppression of DU-145 prostate cancer cell invasion across a matrigel matrix. Ellagic acid, caffeic acid, luteolin and punicic acid, important components of pomegranate significantly inhibited *in vitro* invasion of human PC-3 prostate cancer cells when employed individually.

Malik et al. (2005) showed that pomegranate fruit extract exhibited significant antiproliferative and pro-apoptotic activity against highly aggressive human PC-3 cells. The cell growth inhibition was dose-dependent, and alterations were in the regulatory molecules responsible in the G1 phase of the cell cycle. Another molecular mechanism through which pomegranate fruit extract is capable of inducing apoptosis in prostate cancer cells may be up-regulation of Bax and down-modulation of Bcl-2. PFE intake was observed to significantly slow the progression of tumor growth in athymic nude mice implanted with androgen-responsive CWR22R-1 cells. Importantly, this tumor growth inhibition followed a significant decrease in the serum levels of PSA.

Pantuck et al. (2006) studied the effects of pomegranate juice consumption on prostatespecific antigen (PSA) progression in men with a rising PSA following primary therapy. A phase II, Simon two-stage clinical trial for eligible men patients with rising PSA after surgery or radiotherapy was conducted. The eligible patients had previous surgery or radiation therapy for prostate cancer, Gleason score ≤7, rising PSA value of 0.2-5.0 ng/mL, no prior hormonal therapy, and no evidence of metastases. Patients were treated with 8 ounces of pomegranate juice daily until disease progression. Mean PSA doubling time significantly increased with treatment from a mean of 15 months at baseline to 54 months posttreatment. *In vitro* assays comparing pretreatment and posttreatment patient serum on the growth of LNCaP showed a 12% decrease in cell proliferation and a 17% increase in apoptosis, a 23% increase in serum nitric oxide, and significant reductions in oxidative state and sensitivity to oxidation of serum lipids, after versus before pomegranate juice consumption.

Prostate cancer is dependent on circulating testosterone in its early stages and is treatable with surgery, radiation therapy, stereotactic radiosurgery, and proton therapy. Both androgen and androgen receptor (AR) are recognized risk factors in the development of prostate cancer (Heinlein & Chang, 2004). Reduction of circulating levels of androgens and suppression of AR are crucial for the treatment of prostate cancer as an elevated level of androgen causes enhancement of prostate cancer (Attard et al., 2006). Pomegranate extracts has been shown to inhibit both androgen-dependent and androgen-independent prostate cancer cell growth. Since androgen and AR play central roles throughout prostate cancer development Hong et al. (2008) examined the effects of pomegranate polyphenols,

Prostate cancer is the second-leading cause of cancer-related deaths in men in the World. *In vitro* studies stated several pomegranate products inhibit prostate cancer cell growth, induce apoptosis of several prostate cancer cell lines, suppress invasive potential of PC-3 cells, and decrease proliferation of DU-145 prostate cancer cells (Lansky et al., 2005a,b; Pantuck et al., 2006). Albrecht et al. (2004) showed that pomegranate seed oil (PSO) as well as polyphenols present in the pericarp and fermented juice suppress proliferation and invasion of several human prostate cancer cells, LNCaP, PC-3 and DU-145 across the matrigel matrix. Supraadditive, complementary and synergistic effects were proven in all models. Lansky et al (2005b) found equally combined amounts of pomegranate fermented juice, pomegranate pulp juice, cold-pressed pomegranate seed oil extracts resulted in a 99% suppression of DU-145 prostate cancer cell invasion across a matrigel matrix. Ellagic acid, caffeic acid, luteolin and punicic acid, important components of pomegranate significantly inhibited *in vitro*

Malik et al. (2005) showed that pomegranate fruit extract exhibited significant antiproliferative and pro-apoptotic activity against highly aggressive human PC-3 cells. The cell growth inhibition was dose-dependent, and alterations were in the regulatory molecules responsible in the G1 phase of the cell cycle. Another molecular mechanism through which pomegranate fruit extract is capable of inducing apoptosis in prostate cancer cells may be up-regulation of Bax and down-modulation of Bcl-2. PFE intake was observed to significantly slow the progression of tumor growth in athymic nude mice implanted with androgen-responsive CWR22R-1 cells. Importantly, this tumor growth inhibition followed a

Pantuck et al. (2006) studied the effects of pomegranate juice consumption on prostatespecific antigen (PSA) progression in men with a rising PSA following primary therapy. A phase II, Simon two-stage clinical trial for eligible men patients with rising PSA after surgery or radiotherapy was conducted. The eligible patients had previous surgery or radiation therapy for prostate cancer, Gleason score ≤7, rising PSA value of 0.2-5.0 ng/mL, no prior hormonal therapy, and no evidence of metastases. Patients were treated with 8 ounces of pomegranate juice daily until disease progression. Mean PSA doubling time significantly increased with treatment from a mean of 15 months at baseline to 54 months posttreatment. *In vitro* assays comparing pretreatment and posttreatment patient serum on the growth of LNCaP showed a 12% decrease in cell proliferation and a 17% increase in apoptosis, a 23% increase in serum nitric oxide, and significant reductions in oxidative state and sensitivity to oxidation of serum lipids, after versus before pomegranate juice

Prostate cancer is dependent on circulating testosterone in its early stages and is treatable with surgery, radiation therapy, stereotactic radiosurgery, and proton therapy. Both androgen and androgen receptor (AR) are recognized risk factors in the development of prostate cancer (Heinlein & Chang, 2004). Reduction of circulating levels of androgens and suppression of AR are crucial for the treatment of prostate cancer as an elevated level of androgen causes enhancement of prostate cancer (Attard et al., 2006). Pomegranate extracts has been shown to inhibit both androgen-dependent and androgen-independent prostate cancer cell growth. Since androgen and AR play central roles throughout prostate cancer development Hong et al. (2008) examined the effects of pomegranate polyphenols,

invasion of human PC-3 prostate cancer cells when employed individually.

**4.2.2 Prevention of prostate cancer** 

significant decrease in the serum levels of PSA.

consumption.

ellagitannin-rich extract and whole juice extract on the expression of genes for key androgen-synthesizing enzymes [HSD3B2 (3β-hydroxysteroid dehydrogenase type 2), AKR1C3 (aldo-keto reductase family 1 member C3) and SRD5A1 (steroid 5α reductase type 1)] and AR in LNCaP, LNCaP–AR and DU-145 human prostate cancer cells. Pomegranate polyphenols inhibited gene expression and AR most consistently in the LNCaP–AR cell line. Therefore, inhibition by pomegranate polyphenols of gene expression involved in androgensynthesizing enzymes and the AR may be of particular importance in androgenindependent prostate cancer cells and the subset of human prostate cancers where AR is upregulated.

Since the anticarcinogenic activity of ellagic acid, the main polyphenol in the pomegranate, has been shown on several cancer types Malik et al. (2011) evaluated the effect of ellagic acid treatment on the cell viability of human prostate cancer cells. They observed that ellagic acid (10-100 mol/L) treatment (48 h) of human prostate carcinoma PC3 cells resulted in a dose dependent inhibition of cell growth/cell viability. Ellagic acid caused cell growth inhibition which was accompanied by induction of apoptosis, as assessed by the cleavage of poly (ADP-ribose) polymerase (PARP) and morphological changes. Further, ellagic acid treatment was also found to result in significant activation of caspases, as shown by the dose dependent decrease in the protein expression of procaspase-3, -6, -8 and -9. This ellagic acidmediated induction of apoptosis was significantly (80-90%) inhibited by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-FMK).

In a study, Koyama et al. (2010) investigated the relationship between pomegranate-induced apoptosis in human prostate cancer cells and the insuline-like growth factor (IGF)/IGF binding protein (IGFBP) system, as the IGF axis is critical for the regulation of apoptosis in many human cancer cell lines and IGFBPs in serum are responsible for regulation of IGF action, inhibition of cell proliferation and enhancement of apoptosis in many cell types, including prostate (Rajah et al., 1997) and breast (Gucev et al., 1996; Kim et al., 2004) cancers. They concluded that there are novel interactions between the IGF system and pomegranateinduced apoptosis, and pomegranate products modulate the tumor production and responsiveness to IGFs and the IGFBPs. Treatment of LAPC-4 prostate cancer cells with 10 g/mL pomegranate extract, standardized to ellagitannin content (37% punicalagins by HPLC), resulted in inhibition of cell proliferation and induction of apoptosis. Co-treatment with pomegranate extract and IGFBP-3 revealed synergistic stimulation of apoptosis and additional inhibition of cell growth. The researchers also investigated the relationship between IGF-1 and pomegranate-induced apoptosis in 22RV-1 prostate cancer cells. Cotreatment with 100 ng/mL IGF-1 completely blocked apoptosis induction by pomegranate extract. In contrast, IGF-I failed to inhibit pomegranate-induced apoptosis in R- cells, suggesting the importance of IGF-IR. POMx-treatment decreased Igf1 mRNA expression in a dose-dependent manner indicating that its actions also involve tumor-specific suppression of IGF-1.

#### **4.2.3 Prevention of breast cancer**

Along with enthusiastic efforts in early diagnosis, aggressive surgical treatment and application of additional non-operative modalities, the prognosis of breast cancer is stil chaotic. Pomegranate has been the target of several work in laboratories and cancer centres and known to have inhibition properties against diverse types of cancers. Recent review

The Therapeutic Potential of Pomegranate and Its Products for Prevention of Cancer 345

androgens to estrone. Ellagic acid seems to exhibit apoptosis, inhibits activation of inflammatory pathways, and inhibits angiogenesis. However, these assays being performed

Grossmann et al. (2010) found that punicic acid inhibited the proliferation of estrogen insensitive breast cancer cell line (MDA-MB-231) and an estrogen sensitive cell line developed from the MDA-MB-231 cells (MDA-ER 7), as well as induced apoptosis in both type of cells 86% and 91% respectively. They stated that punicic acid also disrupted mitochondrial membrane potential of both cell lines. Such antiproliferative effect of punicic acid on human breast cancer cells was due to lipid peroxidation of cells and activation of

Tran et al. (2010) evaluated pomegranate seed linolenic acid isomers as selective estrogen receptor modulators (SERMs) *in vitro*. Punicic acid and -eleostearic acid present in seed oil of pomegranate inhibited the IC50 estrogen receptors ER and ERβ depending on the dose. At lower doses of punicic acid acted as agonist for both receptors and antagonist at higher concentrations. Both acids were effective in producing effective inhibition of cancer cell proliferation: MCF-7 (ER-positive human breast cancer cells) and MDA-MB-231 (ER-

Current treatment options in colorectal cancer such as surgical intervention and adjuvant chemotherapy have several limitations in counteracting the disease. Furthermore, at advanced stages the patients might be unresponsive to any form of treatment. In this regard, an optimal model for primary and secondary prevention in colon cancer, given the availability of effective screening procedures and a well-defined multi-step carcinogenic pathway, can be thought as the development of new cancer chemopreventive agents that could be employed to inhibit tumor development without causing systemic toxicity such as increasing the consumption of food containing anticarcinogenic compounds. Phytochemicals from pomegranate have been shown to inhibit colon cancer cell proliferation and apoptosis through the modulation of cellular transcription factors and signaling proteins (Mertens-Talcott & Percival, 2005; Seeram et al., 2006; Khan, 2009;

Kohno et al. (2004a,b) reported that dietary administration of pomegranate seed oil rich in conjugated linolenic acid markedly inhibited the development of azoxymethane-induced colonic adenocarcinomas in male F344 rats without causing any adverse effects. This was associated with an increased content of conjugated linoleic acid in the colon and liver and/or increased expression of peroxisome proliferator-activated receptor (PPAR)-protein

There is considerable evidence that the anticarcinogenic effect of pomegranate ellagitannins is mainly due to ellagic acid, which induces apoptosis in human colon cancer cell line via the intrinsic pathway with release of cytochrome *c* into the cytosol, activation of initiator caspase 9 and effector caspase 3 and down-regulation of B-cell lymphoma-extra large (Bcl-XL). In addition, pomegranate treated Caco-2 cells showed arrest in the S phase of the cell cycle, down-regulation of cyclins A and B1 and upregulation of cyclin E (Larossa et al.,

in animal models need to be confirmed in humans.

negative human breast cancer cells and are SERMs.

**4.2.4 Prevention of colon cancer** 

Kasimsetty et al., 2010).

2006).

in the non-tumor colon mucosa.

protein kinase C (PKC).

articles reported the laboratory and clinical evidence of cancer chemoprevention or treatment of pomegranate fruit, pomegranate juice, pomegranate seed and seed oil on prostate, breast, skin, colon, lung, oral and leukaemia cancers, through antioxidant, antiproliferation, antiangiogenesis and antiinflammatory mechanisms of action (Adhami et al., 2009,2010; Amin et al., 2009; Faria & Calhau, 2011; Johanningsmeier & Harris, 2011). They all reported that extracts of pomegranate or the juice are generally more active than individual or purified compounds.

The antiangiogenic potential of pomegranate was evaluated by Toi et al. (2003) where VEGF, interleukin-4 and migration inhibitory factor (MIF) were measured in the conditioned media of estrogen sensitive MCF-7, estrogen resistant MDA-MB-231 human breast cancer cells and MCF-10A immortalized human breast epithelial cells, grown in the presence or absence of pomegranate seed oil or fermented juice polyphenols. Polyphenols from fermented pomegranate juice, pericarp and oil were shown to inhibit endogenous active estrogen biosynthesis with subsequent inhibition of aromatase activity. VEGF was strongly downregulated in MCF-10A and MCF-7 cells, and MIF upregulated in MDAMB-231 cells, representing a marked potential for downregulation of angiogenesis by pomegranate fractions. Mehta & Lansky (2004) examined the effects of pomegranate fermented juice, cold pressed pomegranate seed oil extract and an HPLC-isoIated peak (from the fruit extract-peak B), using the mouse mammary organ culture, an animal model of breast cancer having >75% accuracy to predict *in vivo* carcinogenesis. They showed that the purified chromatographic peak of pomegranate fermented juice polyphenols and pomegranate seed oil possesses greater chemopreventive potential than that previously reported by Kim et al. (2002). While fermented juice polyphenols effected a 42% reduction in the number of DMBA-induced cancerous lesions compared with control, purified compound, peak B, and pomegranate seed oil each effected an 87% reduction. Peak B is believed to be a phenolic compound with potent chemopreventative properties. Combination treatment of MCF-7 breast cancer cells with both pomegranate extracts and genistein was found to be more effective on inhibition and cytotoxicity than with single treatments (Jeune et al., 2005).

In an ethnobotanical study of medicinal plants in Chandauli District, Singh & Singh (2009) were able to document 40 medicinal plants belonging to 27 families by semi-structured interviews, field observations, preference and direct matrix ranking with traditional medicine practitioners. Pomegranate was found to be an ingredient of a powder for external treatment of breast cancer along with whole plant of *Vernonia cinerea* Less. (AS38) and leaves of *Crataeva nurvala.* 

Epidemiological studies have demonstrated that elevated serum levels of the estrogens, mainly estrone and estradiol, and lower levels of sex hormone binding globulin, after menopause substantially increased the risk of breast cancer. After menopause, most circulating estrogen is derived from the conversion of adrenal androgens to estrone, and some of the estrone is further converted to estradiol, the most active estrogen in breast tissue. Sturgeon & Ronnenberg (2010) described the *in vitro* cell culture studies, animal studies and available data about the property of pomegranate to prevent breast cancer as well as the possible mechanisms involved. They reported that cyclooxigenase inhibition by the constituents of the pomegranate fruit, seed oils or pure compounds induce the decrease of PGE2 that known to downregulate aromatase expression, that converts adrenal androgens to estrone. Ellagic acid seems to exhibit apoptosis, inhibits activation of inflammatory pathways, and inhibits angiogenesis. However, these assays being performed in animal models need to be confirmed in humans.

Grossmann et al. (2010) found that punicic acid inhibited the proliferation of estrogen insensitive breast cancer cell line (MDA-MB-231) and an estrogen sensitive cell line developed from the MDA-MB-231 cells (MDA-ER 7), as well as induced apoptosis in both type of cells 86% and 91% respectively. They stated that punicic acid also disrupted mitochondrial membrane potential of both cell lines. Such antiproliferative effect of punicic acid on human breast cancer cells was due to lipid peroxidation of cells and activation of protein kinase C (PKC).

Tran et al. (2010) evaluated pomegranate seed linolenic acid isomers as selective estrogen receptor modulators (SERMs) *in vitro*. Punicic acid and -eleostearic acid present in seed oil of pomegranate inhibited the IC50 estrogen receptors ER and ERβ depending on the dose. At lower doses of punicic acid acted as agonist for both receptors and antagonist at higher concentrations. Both acids were effective in producing effective inhibition of cancer cell proliferation: MCF-7 (ER-positive human breast cancer cells) and MDA-MB-231 (ERnegative human breast cancer cells and are SERMs.
