**4.2.4 Prevention of colon cancer**

344 Cancer Prevention – From Mechanisms to Translational Benefits

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

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

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

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

individual or purified compounds.

treatments (Jeune et al., 2005).

of *Crataeva nurvala.* 

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; Kasimsetty et al., 2010).

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 in the non-tumor colon mucosa.

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., 2006).

The Therapeutic Potential of Pomegranate and Its Products for Prevention of Cancer 347

concluded that urolithins produced in the colon from ellagitannins present in pomegranate are inhibitors of the canonical Wnt signalling pathway at physiologically relevant

Eventhough there are advances in detection and therapy of cancer, the severe morbidity rate from cancer have not improved. Various parts of the pomegranate plant have been stated to exert selective antiproliferative effects on a lung, leukemia, stomach, bladder, oesaphagus and oral cancers (Lansky & Newman, 2007; Syed et al., 2007; Heber, 2008; Jurenka, 2008; Rahman et al., 2010; Khan & Mukhtar, 2010; Faria & Calhau 2011), through antioxidant, antiproliferation (growth inhibition, cell cycle disruption and apoptosis), antiangiogenesis

*In vitro* and *in vivo* studies revealed that pomegranate fruit extract (PFE) have chemopreventive/therapeutic potential of against lung cancer models (Khan et al., 2007a,b,2008). Normal human bronchial epithelial cells (NHBE) and human lung carcinoma A549 cells, in mice, were treated with pomegranate fruit extract (50–150 µg/ml) for 72 h. There was a significant decrease in the viability of A549 cells, however, only minimal effects were observed on NHBE cells. Pomegranate fruit extract treatment of A549 cells resulted in dose-dependent arrest of cells in G0/G1 phase of the cell cycle, which was associated with induction of WAF1/p21 and KIP1/p27 and accompanied by decrease in the expression of

The effect of oral consumption of a human achievable dose of pomegranate fruit extract on tumor growth, progression and signaling pathways involved, was studied further in two other mouse lung tumor protocols. Benzo(a)pyrene [B(a)P] and N-nitroso-trischloroethylurea (NTCU) were used to induce lung tumors, and PFE was given in drinking water to A/J mice. Lung tumor yield was examined on the 84th day and 140 days after B(a)P dosing and 240 days after NTCU treatment. Mice treated with PFE and exposed to B(a)P and NTCU had statistically significant lower lung tumor multiplicities than mice treated with carcinogens only. Tumor reduction was 53.9% and 61.6% in the B(a)P+PFE group at 84 and 140 days, respectively, compared with the B(a)P group. The NTCU+PFE group had 65.9% tumor reduction compared with the NTCU group at 240 days. PFE treatment also resulted in inhibition of NF-κB, MAPK, and PI3K/Akt signaling. Treatment with B(a)P and NTCU caused increased phosphorylation of mTOR at Ser2448, whereas PFE administration resulted in inhibition of phosphorylation of mTOR. This observation was significant since the mTOR integrates mitogenic signals and intracellular nutrient levels to activate 4EBP1 and p70S6K

that control protein translation and cell cycle progression (Khan et al., 2007b).

Suzuki et al. (2001) investigated cytotoxicity of pomegranate seed oil and other plant seed oils containing conjugated linoleic acids in mouse tumors and human monocytic leukemia cells. They stated the cytotoxic effect of pomegranate seed oil (containing 9c,11t,13c-18:3), as well as tung seed oil (containing 9c,11t,13t-18:3) and catalpa seed oil (containing 9t,11t,13c-18:3), was much stronger than that of pot marigold seed oil (containing 8t,10t,12c-18:3), suggesting that the position of the double bond could be an important determinant for the

Kawaii & Lansky (2004) examined the effect of flavonoid-rich pomegranate juice, pomegranate fermented juice and pomegranate pericarp extracts on HL-60 human leukemia

concentrations.

**4.2.5 Prevention of other cancer types** 

and antiinflammatory mechanisms of action.

cytotoxicity of conjugated inoleic acids.

downstream cell cycle regulatory proteins (Khan et al., 2007a).

Adams et al. (2006) examined the effects of pomegranate juice on inflammatory cell signaling proteins in the HT-29 human colon cancer cell line. In HT-29 colon cancer cells, at a concentration of 50 mg/L pomegranate juice significantly suppressed TNFalpha-induced COX-2 protein expression by 79%, total pomegranate tannin extract (TPT, 55%), and punicalagin 48% Cyclooxygenase-2 (COX-2) expression is increased via activation of nuclear factor kappa-B (NFKB) by tumor necrosis factor-alpha (TNF-Ot), an inflammatory cell signaling process that may be a cause of cancer initiation and progression. Additionally, pomegranate juice reduced phosphorylation of the p65 subunit and binding to the NFkappaB response element 6.4-fold. TPT suppressed NFK binding 10-fold, whereas punicalagin 3.6-fold. It was shown that inflammatory enzymes in colon cancer cells were inhibited by the pomegranate juice components. Ellagic acid, punicalagin and TPT failed to induce apoptosis in HT-29 and HCT-116 cells when treated at doses equivalent to found in pomegranate juice. They were only effective when treated at equivalent doses of 100μg/mL (Seeram et al., 2005).

In a seperate study, Boateng et al. (2007) examined the effect of pomegranate juice given access to 20%, before and after treatment with colon-specific chemical carcinogen, azoxymethanein to F-344 rats for 17 weeks. Pomegranate fruit juice reduced the number of aberrant cryptic foci (ACF) of the colon by 91% in male rats. Histopathology of rat colon after 17th week of treatment revealed a remarkable decrease in the number of large crypts in pomegranate juice-fed rats, and the number of crypts/ACF was also low. When compared to water melon and cranberry juices, pomegranate juice was found to be superior as an inhibitor of ACF in rat colon. Increase in weight gain and feed intake was observed in pomegranate fruit juice-fed rats, suggesting a possible protective effect against cancer cachexia.

Kasimsetty et al. (2010) investigated the colon cancer chemopreventive properties of pomegranate ellagitannins and their intestinal bacterial metabolites, urolithins, in HT-29 human colon cancer cells, and stated that the ellagitannins and urolithins released in the colon upon consumption of pomegranate juice in considerable amounts could potentially reduce the risk of colon cancer development, by inhibiting cell proliferation and inducing apoptosis. Ellagitannins and urolithins inhibited endocrine disrupter 2,3,7,8 tetrachlorodibenzo-*p*-dioxin (TCDD)-induced CYP1-mediated ethoxyresorufin-O-deethlyase (EROD) activity *in vitro* with IC50 values ranging from 56.7 M for urolithin A to 74.8 M for urolithin C. These compounds exhibited dose- and time-dependent decreases in cell proliferation and clonogenic efficiency of HT-29 cells.

In colon cancer, a large percentage of the tumour arises from activating mutations in the Wnt protein pathway. Sharma et al. (2010) studied the effects of urolitinins, ellagic acid and ellagitannin-rich fruit extracts on Wnt signalling in a human 293T cell line using a luciferase reporter of canonical Wnt pathway-mediated transcriptional activation. In the canonical Wnt pathway, the signal produced by the binding of Wnt ligands to cell surface receptors is transmitted through a cytoplasmic protein called disheveled (Dvl) to inhibit the activity of a complex of cellular proteins that phosphorylate *β*-catenin, and target it for destruction. Therefore, Dvl-mediated inhibition of the *β*-catenin destruction complex results in increased levels of cellular β-catenin and translocation of *β*-catenin into the nucleus, where *β*-catenin activates transcription factors of the LEF/TCF families and initiates transcription of target genes effective on tissue proliferation, differentiation and tumorigenesis. The researchers concluded that urolithins produced in the colon from ellagitannins present in pomegranate are inhibitors of the canonical Wnt signalling pathway at physiologically relevant concentrations.
