**4.2.5 Prevention of other cancer types**

346 Cancer Prevention – From Mechanisms to Translational Benefits

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

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

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

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

proliferation and clonogenic efficiency of HT-29 cells.

(Seeram et al., 2005).

cachexia.

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 and antiinflammatory mechanisms of action.

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

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 cytotoxicity of conjugated inoleic acids.

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

The Therapeutic Potential of Pomegranate and Its Products for Prevention of Cancer 349

pomegranate ingestion in healthy subjects, whereas in colon inflammation, the effects could

Structure–activity relationships of natural products have been found to influence the various pharmacological functions. *In vitro* and *in vivo* antiinflammatory effects of *Punica granatum* Linne, a high phenolic content fruit, widely used as an antipyretic analgesic in Chinese culture, were investigated by Lee et al. (2010). Pomegranate has shown potential nitric oxide (NO) inhibition in liposaccaharide (LPS)-induced RAW 264.7 macrophage cells, with significant decrease in carrageenan-induced mice paw edema. Hydrolysable tannins, punicalagin, punicalin, strictinin A, and granatin B, inhibited NO production and inductible nitric oxide synthase (iNOS) expression in RAW 264.7 cells. Granatin B showed the strongest iNOS and COX-2 inhibitory effects, and exhibited these effects in the inhibition of paw

Inflammatory disorders are due to excessive production of pro-inflammatory mediators such as TNFa, GM-CSF, IL-1, IL-6, IL-8, leukotriene B4 and PAF, the activity of inflammatory cells such as neutrophils, monocytes and macrophages, and excessive production of reactive oxygen species (ROS) (Nathan, 2006). Bachoual et al. (2011) investigated the effect of pomegranate peel aqueous extract (PPAE) on human neutrophil reactive oxygen species (ROS) production *in vitro* and on LPS-induced lung inflammation *in vivo* in mice. PPAE, in a concentration-dependent manner, inhibited luminol-amplified chemoluminescence of resting neutrophils and N-formyl-methionylleucyl- phenylalanine (fMLF)- or phorbol myristate acetate (PMA)-stimulated neutrophils. On the contrary, had no significant effect on superoxide anion generation, suggesting that it does not directly inhibit NADPH oxidase activity or activation pathways, or scavenge superoxide anions. *In vivo* studies showed that PPAE also attenuated LPS-induced lung inflammation in mice. Consequently PPAE found to inhibit neutrophil myeloperoxidase activity and attenuates

Cardiovascular diseases (CVDs) are a leading cause of death and disability worldwide. Hypertension and atherosclerosis, a chronic inflammatory disease characterized by plaque formation in the large arteries, are major risk factors for CVDs, such as stroke, myocardial infarction and heart failure. In addition to genetic factors, age, body weight, blood pressure, dyslipidemia, physical inactivity and behavioural risk factors such as tobacco or alcohol use, diets that include high fat, salty food are thought to play an important role in the development of cardiovascular disease. Epidemiological data have clearly shown that independent risk factors for CVD are serum total cholesterol and low-density lipoprotein cholesterol (LDL-C) (Kannel et. al., 1986; Mirmiran et al., 2009). A large number of clinical trials have demonstrated in order to prevent these cardiovascular diseases from occurring, control of a patient's blood pressure is necessary, either by lifestyle modifications, medication(s) such as use of cholesterol-lowering statins, antihypertensive drugs and

While modification of dietary patterns and increased physical activity constitute the primary preventive intervention in lowering coronary heart disease (CHD) and stroke, the role of plant-based bioactive compounds or phytochemicals has attracted much attention since

swelling and the prostaglandin (PG) E2 level in carrageenan-induced mice.

be due to the nonmetabolized ellagitannin-related fraction.

LPS-induced lung inflammation in mice.

**4.3.2 Cardiovascular health** 

antiplatelet agents or both.

cell differentiation and proliferation. *In vitro* assays confirmed that both the pomegranate fermented juice and pericarp extracts strongly promoted cellular differentiation and inhibited proliferation in HL-60 cell cultures; the effect of pomegranate juice on cellular differentiation was less significant. In view of the observations the authors stated the hypothesis of another mechanism by which pomegranate constituents impart an anticarcinogenic effect.

Mertens-Talcott & Percival (2005) investigated the interactions of ellagic acid and quercetin with resveratrol, with the hypothesis that the selected polyphenols would interact synergistically in the induction of apoptosis and reduction of cell growth in human leukemia cells (MOLT-4). They found significant interaction for the combination of ellagic acid with resveratrol, and alterations in cell cycle kinetics induced by single compounds and combinations were also observed. The authors concluded that the anticarcinogenic potential of foods containing polyphenols may not be based on the effects of individual compounds, but may involve a synergistic enhancement of the anticancer effects.
