CANCER TREATMENT _CHEMOTHERAPY_ALTERNATIVES Compiled and Edited By G Vijaya Raghaqvan, CEO, DVS BioLife Ltd, HYDERABAD. CONTENTS: 1. INTRODUCTION 2. CHEMOTHERAPY 3. COMMON CANCER DRUGS 4. ROLE OF UNUSUAL PLANT DERIVATIVES INCLUDING ALKALOIDS FROM POMEGRANATE 5. ROLE OF MICROBES LIKE E COLI IN DIAGNOSIS AND IN TREATMENT 6. COMPLEMENTARY APPROACHES 1. INTRODUCTION:In the year 2000, malignant tumours were responsible for 12 per cent of the nearly 56million deaths worldwide from all causes. In many countries, more than a quarter ofdeaths are attributable to cancer. In 2000, 5.3 million men and 4.7 million womendeveloped a malignant tumour and altogether 6.2 million died from the disease. Thereport also reveals that cancer has emerged as a major public health problem indeveloping countries, matching its effect in industrialized nations.Every year about 85,0000 new cancer cases are diagnosed in India resulting in about58,0000 cancer related death every year.Bladder Cancer, Melanoma, Breast Cancer, Non-Hodgkin Lymphoma, Colon and RectalCancer, Pancreatic Cancer, Endometrial Cancer, Prostate Cancer, Kidney (Renal Cell)Cancer, Skin Cancer (Nonmelanoma), Leukemia, Thyroid Cancer and Lung Cancerare the common types of Cancer found worldwide.Cancer management practices involve in screening, diagnostics, surgery,chemotherapy, radiation, alternative and complimentary therapies.Cancer management is partly based on weighing risk factors attributed to noninfectiousagents, human genes and epigenetic factors. Infectious disease causation has largelybeen restricted to genes directly responsible for causing cancer after sustaining damagei.e. oncogenes. Lately, evidence has emerged linking infectious agents to a number ofchronic diseases. These studies have recognized the influence that acute, atypical,latent and chronic infections may play in tricking the immune system and affectingdisease etiology. Similar evidence is emerging in model systems with respect to the roleof infectious agents in gastrointestinal, liver and lung cancers. Although viruses havebeen found in association with breast cancer, skepticism remains about a role for otherinfectious agents, notably microbes in the disease etiology. Improved experimentaldesigns employed in different cancer studies and a less rigid definition of infectiouscausation may aid in confirming or refuting a microbe-breast cancer connection. Cancerrecurrence could potentially be minimized and treatment options further tailored on acase by case basis if microbes/microbial components/strain variants associated with
breast cancer are identified; probiotics are employed to reduce treatment side-effectsand if microbes could effectively be harnessed in immunotherapy. 2. ChemotherapyChemotherapy is a cancer treatment that uses drugs to destroy cancer cells.Chemotherapy can be used to: • Destroy cancer cells • Stop cancer cells from spreading • Slow the growth of cancer cellsChemotherapy can be given alone or with other treatments. It can help other treatmentswork better.Chemotherapy can be given in these forms: • An IV (intravenously) • A shot (injection) into a muscle or other part of the body • A pill or a liquid that can be swallowed • A cream that is rubbed on the skin • Other ways:Chemotherapy is achieved by one or more of the following. • Alkylating Agents • Antimetabolites • Cytotoxic Agents • Plant Derivatives • MicrobialThe following are the common drugs used in Cancer. 1. Acarbose 2. Acivicin 3. Aclarubicin 4. Acodazole 5. Acronine 6. Actinomycin D 7. Adenine Phosphate 8. Adenosine 9. Adozelesin 10. Adriamycin 11. Adrucil 12. Alanosine 13. Aldesleukin 14. Alemtuzumab 15. Alestramustine 16. Alfacalcidol 17. Alitretinoin
386. Rapamycin 387. Razoxane 388. Resveratrol 389. Retigabine 390. Riboprine 391. Risedronic acid 392. Ristocetin A 393. Ritrosulfan 394. Rituximab 395. Rocuronium Bromide 396. Roflumilast 397. Rogletimide 398. Ropinirole HCL 399. Rosuvastatin Calcium 400. Rubitecan 401. Sarcolysin 402. Sargramostim 403. Sebriplatin 404. Semustine 405. Sermorelin 406. Simtrazene 407. Sitagliptin Phosphate 408. Sizofiran 409. Sobuzoxane 410. Sodium Borocaptate[10B] 411. Solaziquone 412. Sonermin 413. Sorafenib 414. Spiclomazine 415. Spirogermanium 416. SpiromustineFollowing are some excerpts from public domain.Conventional Alkylating AgentsAlkylating agents are one of the earliest and most commonly used chemotherapy agentsused for cancer treatments. Their use in cancer treatments started in early 1940s.Majority of alkaline agents are active or dormant nitrogen mustards, which are poisonouscompound initially used for certain military purposes. Chlorambucil, Cyclophosphamide,CCNU, Melphalan, Procarbazine, Thiotepa, BCNU, and Busulfan are some of thecommonly used alkylating agents.They are more effective in treating slow-growing cancers such as solid tumors andleukemiaTraditional AntimetabolitesStructure of antimetabolites (antineoplastic agents) is similar to certain compounds suchas vitamins, amino acids, and precursors of DNA or RNA, found naturally in humanbody. Antimetabolites help in treatment cancer by inhibiting cell division thereby
hindering the growth of tumor cells. These agents get incorporated in the DNA or RNA tointerfere with the process of division of cancer cells. They are commonly used to treatgastrointestinal tract, breast, and ovary tumors.Methotraxate, which is a commonly used antimetabolites chemotherapy agent, iseffective in the S-phase of the cell cycle. It works by inhibiting an enzyme that isessential for DNA synthesis.6-mercaptopurine and 5-fluorouracil (5FU) are two other commonly usedantimetabolites. 5-Fluorouracil (5-FU) works by interfering with the DNA components,nucleotide, to stop DNA synthesis. This drug is used to treat many different types ofcancers including breast, esophageal, head, neck, and gastric cancers. 6-mercaptopurine is an analogue of hypoxanthine and is commonly used to treat AcuteLymphoblastic Leukemia (ALL).Other popular antimetabolite chemotherapy drugs are Thioguanine, Cytarabine,Cladribine. Gemcitabine, and Fludarabine.AnthracyclinesAnthracyclines are daunosamine and tetra-hydronaphthacenedione-basedchemotherapy agents. These compounds are cell-cycle nonspecific and are used totreat a large number of cancers including lymphomas, leukemia, and uterine, ovarian,lung and breast cancers.Anthracyclines drugs are developed from natural resources.Daunorubicin is developed by isolating it from soil-dwelling fungus Streptomyces.Doxorubicin, which is another commonly used anthracycline chemotherapy agent, isisolated from mutated strain of Streptomyces.Doxorubicin is more effective in treating solid tumors.Idarubicin, Epirubicin, and Mitoxantrone are few of the other commonly usedanthracycline chemotherapy drugs.Anthracyclines work by forming free oxygen radicals that breaks DNA strands therebyinhibiting DNA synthesis and function.These chemotherapeutic agents form a complex with DNA and enzyme to inhibit thetopoisomerase enzyme.Topoisomerase is an enzyme class that causes the supercoiling of DNA, allowing DNArepair, transcription, and replication.Antitumor antibioticsAntitumor antibiotics are also developed from the soil fungus Streptomyces. Thesedrugs are widely used to treat and suppress development of tumors in the body. Similarto anthracyclines, antitumor antibiotics drugs also form free oxygen radicals that result inDNA strand breaks, killing the growth of cancer cells. In most of the cases, these drugsare used in combination with other chemotherapy agents.Bleomycin is one of the commonly used antitumor antibiotic used to treat testicularcancer and hodgkin’s lymphoma.Monoclonal antibodiesThe treatment is known to be useful in treating colon, lung, head, neck, and breastcancers. Some of the monoclonal drugs are used to treat chronic lymphocytic leukemia,acute myelogenous leukemia, and non-Hodgkins lymphoma.Monoclonal antibodies work by attaching to certain parts of the tumor-specific antigensand make them easily recognizable by the host’s immune system. They also preventgrowth of cancer cells by blocking the cell receptors to which chemicals called ‘growthfactors’ attach promoting cell growth.
Monoclonal antibodies can be combined with radioactive particles and other powerfulanticancer drugs to deliver them directly to cancer cells. Using this method, long termradioactive treatment and anticancer drugs can be given to patients without causing anyserious harm to other healthy cells of the body.PlatinumPlatinum-based chemotherapy agents work by cross-linking subunits of DNA. Theseagents act during any part of cell cycle and help in treating cancer by impairing DNAsynthesis, transcription, and function.Cisplatin, although found to be useful in treating testicular and lung cancer, is highlytoxic and can severely damage the kidneys of the patient. Second generation platinum-complex carboplatin is found to be much less toxic in comparison to cisplatin and hasfewer kidney-related side effects. Oxaliplatin, which is third generation platinum-basedcomplex, is found to be helpful in treating colon cancer. Although, oxaliplatin does notcause any toxicity in kidney it can lead to severe neuropathies. Platinum-based drugsare often used for treatment of mesothelioma. Role of UNUSUAL Plant DerivativesThey are primarily categorized into four groups: topoisomerase inhibitors, vincaalkaloids, taxanes, and epipodophyllotoxins.Topoisomerase inhibitors are chemotherapy agents are categorized into Type I andType II Topoisomerases inhibitors and they work by interfering with DNA transcription,replication, and function to prevent DNA supercoiling. Type I Topoisomerase inhibitors: These chemotherapy agents are extracted from the bark and wood of the Camptotheca accuminata. • Type II Topoisomerase inhibitors: These are extracted from the alkaloids found in the roots of May Apple plants. • Amsacrine, etoposide, etoposide phosphate, and teniposide are some of the examples of type II topoisomerase inhibitors.Vinca alkaloidsVinca alkaloids are derived from the periwinkle plant, Vinca rosea (Catharanthusroseus) and are useful in treating leukemias. They are effective in the M phase of thecell cycle and work by inhibiting tubulin assembly in microtubules.Vincristine, Vinblastine, Vinorelbine, and Vindesine are some of the popularly used vincaalkaloid chemotherapy agents used today. Major side effect of vinca alkaloids is thatthey can cause neurotoxicity in patients.TaxanesTaxanes are plant alkaloids that are isolated from the bark of the Pacific yew tree,Taxus brevifolia.. Paclitaxel and docetaxel are commonly used taxanes. Taxanes workin the M-phase of the cell cycle and inhibit the function of microtubules by binding withthem. Taxanes are used to treat a large array of cancers including breast, ovarian, lung,head and neck, gastric, esophageal, prostrate and gastric cancers. The main side effectof taxanes is that they lower the blood counts in patients.
EpipodophyllotoxinsEpipodophyllotoxins chemotherapy agents are extracted from the American May Appletree (Podophyllum peltatum).Etoposide and Teniposide are commonly used epipodophyllotoxins chemotherapyagents which are effective in the G1 and S phases of the cell cycle. They prevent DNAreplication by stopping the cell from entering the G1 phase and stop DNA replication inthe S phase.Phytoestrogens and Cancer TreatmentPhytoestrogens are polyphenol compounds of plant origin that exhibit a structuralsimilarity to the mammalian steroid hormone 17β-oestradiol. In Asian nations the stapleconsumption of phyto-oestrogen-rich foodstuffs correlates with a reduced incidence ofbreast cancer. Human dietary intervention trials have noted a direct relationship betweenphyto-oestrogen ingestion and a favourable hormonal profile associated with decreasedbreast cancer risk. However, these studies failed to ascertain the precise effect of dietaryphyto-oestrogens on the proliferation of mammary tissue. Epidemiological and rodentstudies crucially suggest that breast cancer chemoprevention by dietary phyto-oestrogencompounds is dependent on ingestion before puberty, when the mammary gland isrelatively immature. Phyto-oestrogen supplements are commercially marketed for use bypostmenopausal women as natural and safe alternatives to hormone replacementtherapy. Of current concern is the effect of phyto-oestrogen compounds on the growth ofpre-existing breast tumours. Data are contradictory, with cell culture studies reportingboth the oestrogenic stimulation of oestrogen receptor-positive breast cancer cell linesand the antagonism of tamoxifen activity at physiological phyto-oestrogenconcentrations. Conversely, phyto-oestrogen ingestion by rodents is associated with thedevelopment of less aggressive breast tumours with reduced metastatic potential.Despite the present ambiguity, current data do suggest a potential benefit from use ofphyto-oestrogens in breast cancer chemoprevention and therapy.Phyto-oestrogens may be classified into a number of principal groups [2,7-9]: theisoflavones (genistein, daidzein, biochanin A), the lignans (enterolactone, enterodiol),the coumestans (coumestrol) and the stilbenes (resveratrol). As illustrated in Fig. 1, allare polyphenols sharing structural similarity with the principal mammalian estrogen 17β-oestradiol. Shared features include the presence of a pair of hydroxyl groups and aphenolic ring, which is required for binding to the oestrogen receptor (ER) subtypes αand β. The position of the hydroxyl groups appears to be important in determining ERbinding ability and transcriptional activation, with maximal potency achieved at positionsfour, six and seven [10-12]. The isoflavones are naturally found in soybeans and soy-based food products, including tofu, soy milk, textured soy protein and miso. Lignans arepresent in flaxseed and most fruit and vegetables, and the predominant dietary source ofstilbenes is peanuts, grapes and red wine [7,13]. The coumestans are much lessfrequently consumed within the human diet, but they are more potent activators of ERsignalling pathways than are the isoflavones genistein and daidzein [10,14]. By contrast,the stilbene resveratrol is the least potent activator of ER signalling .The isoflavones are present in soy as β-glucosides. Metabolism by the gastrointestinalmicroflora yields a number of metabolites including equol and O-desmethyl-angolensin.Parental compounds and their metabolites are absorbed into the bloodstream, becomingrapidly detectable in the plasma and urine [15-19]. Plasma isoflavone concentrations areconsiderably elevated in Asian populations as compared with in western ones. A recentcomparison of Japanese and UK females revealed an almost 20-fold increase in plasmagenistein levels in the Japanese cohort, and daidzein concentrations were similarly
elevated by 18-fold . Plasma isoflavone concentrations may accumulate toapproximately 100- to 1000-fold higher than endogenous oestradiol levels following theingestion of soy-rich meal. However, research suggests a decreased ER binding affinityof isoflavone compounds as compared with the mammalian oestrogens [9,10,21,22].Competition binding assays revealed a 50-fold lower binding affinity of genistein forcytoplasmic ER sites as compared to 17β-oestradiol .The complete metabolic activation of soy isoflavones is proposed to occur locally withintarget tissue. In support of this hypothesis, the analysis of tissue culture supernatantsfrom genistein and biochanin A treated MCF-7 and T47-D cells revealed the presence ofhydroxylated and methylated isoflavone metabolites . Current research suggests arole for the CYP family of cytochrome P450 enzymes in the intratumour metabolism ofphyto-oestrogen compounds [25,26]. The CYP1B1 enzyme is expressed in a wide rangeof human tumour types, including breast ; however, expression is absent withinnormal tissue. CYP1B1 is proposed to catalyze the hydroxylation of resveratrol to yieldthe related stilbene piceatannol . Piceatannol is a tyrosine kinase inhibitor withantileukaemic properties, which differs in structure from resveratrol by the presence ofan additional hydroxyl group. A number of plant flavonoids are also putative substratesfor the CYP family of enzymes . Maubach and coworkers  recently reported theuse of high-performance liquid chromatography to quantify isoflavones in normal breastbiopsy tissue following consumption of soy for 5 consecutive days. Equol concentrationswere approximately fivefold higher in the breast tissue homogenates than in serum,providing further evidence for the metabolism of phyto-oestrogen compounds withinmammary tissue.Role of phyto-oestrogens in breast cancerSerum concentrations of 17β-estradiol are approximately 40% lower in Asian womenthan in their Caucasian counterparts . A low lifetime exposure to oestrogen isassociated with a reduced risk for breast cancer. Human dietary intervention studiesrevealed a direct association between the modest consumption of soy products and areduction in circulating steroid hormone levels. Daily consumption of 154 mg isoflavonesfor the duration of a single menstrual cycle correlated with substantially decreasedplasma concentrations of 17β-oestradiol and progesterone in a cohort of premenopausalwomen . A longer-term study conducted by Kumar and coworkers  similarlyreported a moderate decrease in serum oestradiol and oestrone levels following dailyingestion of 40 mg isoflavones for 3 months. Menstrual cycle length was increased by3.52 days, and the follicular phase of the cycle was extended by 1.46 days. Increasedmenstrual cycle length may serve to reduce the total number of cycles per lifetime,therefore decreasing the total exposure of breast epithelia to endogenous oestrogens.Conversely, a year-long dietary intervention trial involving 34 premenopausal womenfailed to reveal a significant effect of 100 mg/day isoflavone consumption either onmenstrual cycle length or on serum levels of various steroid hormones, includingoestrone, oestradiol and progesterone .As a possible explanation for these contradictory data, a study conducted by Duncanand coworkers  revealed differential hormonal effects of soy isoflavones dependingon the ability to excrete the daidzein metabolite equol. Daily ingestion of 10 mg soyprotein by premenopausal equol excretors resulted in a hormonal profile associated withreduced breast cancer risk, characterized by lowered plasma levels of oestrone,oestrone–sulphate and testosterone. Hormone levels however remained unchanged inthe equol nonexcretors after soy ingestion.The reduction in steroid hormone levels by phyto-oestrogens is proposed to occur viathe direct regulation of 17β-oestradiol biosynthesis and metabolism. Phytochemicals
isolated from vegetable extracts effectively suppress the activity of the aromataseenzymes, which are responsible for conversion of androgens to oestrogens .Isoflavone concentrations of 1–10 μmol/l similarly reduced by 50% the activity of theestradiol biosynthetic enzymes 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase . The daily ingestion of 113–202 mg isoflavones bypremenopausal women correlated with a 40% increase in the urinary excretion of 2-hydroxyestrone, a putative anticancer metabolite of 17β-oestradiol . The abovestudies thus suggest a dual chemoprotectant mechanism of soy, in which theisoflavones suppress steroid hormone biosynthesis while promoting the metabolism ofoestradiol to the protective 2-hydroxylated metabolites.Despite their apparent effect on endogenous hormone levels, the role of phyto-oestrogens in breast cancer initiation and development is unclear. Few studies to datehave addressed the effects of long-term phyto-oestrogen exposure in humans. Dailydietary supplementation with 45 mg soy isoflavone for 14 days correlated with increasedproliferation of normal breast epithelia in a group of 48 premenopausal women .Expression of the ER target protein progesterone receptor was upregulated, suggestingan oestrogenic effect. An identical trial using a larger cohort of 84 premenopausalwomen conversely found no significant effect of soy consumption on the proliferation ofnormal breast tissue . A number of recent epidemiological studies similarly failed tocorrelate soyfood consumption with reduced breast cancer risk. A Japanese prospectivestudy conducted in a cohort of approximately 35,000 women  revealed no significantassociation between soy consumption during adulthood and breast cancer incidence.The retrospective analysis of soy food intake in a multiethnic cohort of non-Asian breastcancer patients and control individuals residing in the USA similarly failed to correlatesoy intake with breast cancer risk .Increasing epidemiological evidence suggests that the chemoprotectant effects of phyto-oestrogens are dependent on a lifelong exposure from childhood. A retrospective studyrevealed decreased soyfood intake during adolescence in a cohort of 1459 Chinesebreast cancer patients, as compared with age-matched control individuals . Daily soyconsumption between the ages of 13 and 15 was estimated at 6.45 g in the patientgroup, increasing to 7.23 g in the control cohort. A potential flaw in the study, however,concerns the ability of women up to the age of 64 years to recall accurately the precisesoyfood quantities consumed many years earlier during adolescence. Theseobservations may nonetheless explain the apparent lack of a growth inhibitory effect ofsoy isoflavones in the adult dietary intervention studies discussed above. Isoflavones aredetectable in breast milk following soy consumption , implying that the lower breastcancer incidence in Asian countries may be attributable to phyto-oestrogen exposurefrom birth via breast-feeding. Rodent studies have accordingly revealed the effectivetransfer of genistein from maternal milk to offspring .Rodent breast cancer modelsMost available information regarding the effects of phyto-oestrogens on tumour initiationand the growth of pre-existing tumours is derived from rodent studies. A number ofsimilarities do exist between mammary gland development in rodents and humans. Inboth species the differentiation of breast tissue to form lobules and terminal end-budstructures occurs prepubertally. Further maturation does take place throughoutadulthood, giving rise to alveolar buds, which become alveoli during pregnancy andlactation .Rodent dietary intervention studies using phyto-oestrogens have reportedchemopreventive activities when feeding is initiated before puberty, at a time when themammary gland is undergoing development [3,38-40]. The consumption of a resveratrol-
supplemented diet by adolescent rats served to decrease sensitivity to the chemicalcarcinogens 7,12-dimethylbenz(a)anthracene (DMBA) and N-methyl-N-nitrosaurea[39,40]. DMBA treatment induced mammary tumours in 45% of the resveratrol-treatedrodents, increasing to 75% in the group receiving a control diet. An extended tumourlatency period in excess of 3 weeks was observed in the resveratrol treatment groups,with the resultant tumours retaining a more differentiated morphology as compared withcontrol animals [18,39]. Resveratrol consumption was also associated with the reducedmammary expression of a number of proteins that are putatively involved in malignantprogression, including cyclo-oxygenase-2, matrix metalloprotease-9 and nuclear factor-κB. Similar findings have been noted in prepubertal rats fed an isoflavone-containing dietbefore tumour initiation using DMBA. Despite having no effect on mammary tumourincidence, soy isoflavone consumption was associated with an increased tumour latencyperiod. The resultant tumours excised from the soy-fed animals were smaller in size andexhibited a more differentiated phenotype compared with control animals .It has been proposed that phyto-oestrogens protect against cancer development inadolescent rodents by promoting maturation of the mammary gland. Analysis of breasttissue from prepubertal rats injected with genistein revealed a decrease in the number ofimmature terminal end-buds, together with an increase in the more differentiated lobulestype II . Genistein treatment of human breast cancer cell lines has similarly beenfound to induce the expression of a number of maturation markers, including casein, lipiddroplets and intercellular adhesion molecule-1 .The effect of soy isoflavones on spontaneous tumour development was recentlyinvestigated using neu-ErbB2 over-expressing transgenic mice, which characteristicallydevelop multiple mammary tumours during adulthood . Tumour initiation wastemporarily delayed following the consumption of an isoflavone mix, but nochemoprotective effects were observed in mice consuming either genistein or daidzein inisolation. An equal rate of tumour growth was noted in the control and treatment groups,although the isoflavone group exhibited a lower incidence of lung metastases .Antimetastatic activities of the isoflavones were similarly revealed in a study in whichmice were fed an isoflavone-supplemented diet before injection with the metastatic 4526mammary carcinoma cell line . The isoflavone diet was continued following surgicalexcision of the resultant mammary tumour at a size of 1.0 cm diameter. Both theincidence and size of macroscopically detectable lung metastases were significantlyreduced in the soy-fed mice, suggesting a potential clinical application of soy isoflavonesin the prevention of metastasis.Phyto-oestrogens and tamoxifenThe selective ER modulator tamoxifen is used clinically in the adjuvant treatment ofoestrogen-dependent breast cancer. The drug is also administered as a prophylactic toindividuals who are at high risk for developing the disease [45,46]. Side effectsassociated with tamoxifen therapy include menopause-like symptoms such as hotflushes, joint pain, sleep disorders and depression, which may be reduced by the use ofhormone replacement therapy (HRT) [47,48]. Long-term HRT is associated with anincreased risk for mammary carcinogenesis, and its use by breast cancer patients istherefore discouraged. As a natural alternative, patients may self-medicate with soyisoflavone supplements to alleviate the tamoxifen-induced menopausal symptoms .Published literature regarding the ingestion of dietary phyto-oestrogens by breast cancerpatients and survivors is, however, controversial [50,51].The consumption of genistein by athymic mice antagonized the ability of tamoxifen toinhibit the proliferation of oestrogen-dependent mammary tumours . Tumoursuppression by tamoxifen correlated with decreased expression of the ER-inducible
genes presenelin-2 (pS2) and cyclin D1. Tumour growth was significantly enhanced inmice simultaneously exposed to tamoxifen and genistein, whereas levels of pS2 andcyclin D1 expression were increased. Physiological concentrations of genistein weresimilarly found to reverse the antagonistic effects of 4-hydroxytamoxifen on ER signallingpathways , promoting the binding of ER-α to the positively acting steroid receptorcoactivator (SRC)-1. A recent tissue culture study conversely reported a synergisticantiproliferative effect of tamoxifen and genistein . The proliferation of a panel ofdysplastic and cancerous breast cell lines was inhibited by tamoxifen in a dose-dependent manner, and growth was more potently suppressed by combined treatmentwith tamoxifen and genistein.Hormone-dependent mechanisms of phyto-oestrogen actionOestrogen signalling typically involves the diffusion of ligand through the cell cytoplasmand subsequent binding to the nuclear receptor subtypes ER-α and ER-β. Ligand-boundreceptors dimerise and associate with oestrogen response element (ERE) and activatorprotein-1 element located in the promoter region of target genes, thereby activatingtranscription. The association between receptor dimers and DNA response elements isenhanced by the binding of cofactor proteins, such as amplified in breast cancer-1,thyroid hormone receptor-associated protein, SRC-1, glutamate receptor interactingprotein-1 and translation initiation factor 2 . Examples of ERE-induced genes includePR, c-fos, bcl-2 and cathepsin D, whereas pS2 and cyclin D1 are transcribed via theactivator protein-1 response element.In breast carcinoma cell lines containing functional ER subtypes the isoflavones exert abiphasic growth effect, stimulating cellular proliferation at concentrations below 5 μmol/land inhibiting growth in a dose-dependent manner at elevated doses [23,43,56,57].Growth inhibition correlates with decreased DNA synthesis and cell cycle arrest at theG2/M checkpoint [23,54,56,58-60]. Current research suggests a principal signalling roleof ER-β in response to isoflavone exposure . Whereas 17β-oestradiol binds to ER-αand ER-β with equal affinity, the soy isoflavones selectively associate with ER-β .Receptor binding assays revealed an eightfold to 16-fold increase in the affinity ofgenistein, daidzein and biochanin A for ER-β as compared with ER-α . In ER-negative breast cancer cells transfected to express ER-α alone, genistein was onlyweakly able to stimulate gene transcription through the ERE. By contrast, genisteineffectively bound to ER-β and promoted the association of cofactor proteins, therebyregulating downstream ER-β-mediated gene transcription . The preferential bindingaffinity of genistein for ER-β similarly resulted in a respective 12,000-fold and 33-foldincrease in the recruitment of translation initiation factor-2 and SRC-1a to ER-β ascompared with ER-α . An enhanced transcriptional activity in response to genisteinwas, however, noted in cells transfected to express both receptor subtypes as comparedwith cells solely expressing ER-β . Although ER-α is itself unable to mediateisoflavone signal transduction, it was postulated that the presence of the receptorsubtype may enhance ER-β signalling via the formation of ER-α/β heterodimers. Theseobservations imply that the precise tissue-specific effects of the soy isoflavones aredependent on the expression levels and ratios of ER-α and ERβ. The various cofactorsare similarly expressed in a tissue-specific manner, therefore further influencing thecellular response to dietary phyto-oestrogens.The stilbene resveratrol is structurally similar to the synthetic oestrogen diethylstilbestrol.Treatment of breast cancer cell lines with resveratrol represses proliferation in a dose-dependent manner, inducing G2/M phase cell cycle arrest . Resveratrol exhibits arelatively weak ER-binding affinity as compared with oestradiol ; however, unlike thesoy isoflavones, it is able to bind to both ER-α and ER-β with equal affinity. In cells
transfected to express either ER-α or ER-β resveratrol was found to act as an agonist forboth receptor subtypes, stimulating ERE transcriptional activity through either ER-α orER-β alone . Similar agonist activity was observed in MCF-7 breast cancer cells,which predominantly express the ER-α isoform. Resveratrol induced the dose-dependent activation of ERE-mediated transcription, also upregulating the expression ofthe ER target genes pS2 and PR . Recent studies proposed that the cell cycleinhibitor protein p21WAF1 is a potential downstream target of resveratrol-induced ERsignalling pathways . The treatment of ER-α-expressing breast cancer cells withresveratrol resulted in a 23-fold increase in p21WAF1 gene expression, as determined bycDNA microarray analysis. The resveratrol-mediated induction of p21WAF1 was blocked bytreatment with the pure anti-oestrogen ICI 182,780, confirming p21WAF1 gene regulationas an ER-mediated event.Hormone-independent mechanismsAt concentrations in excess of 25 μmol/l, the soy isoflavones are capable of inducingapoptosis in human breast cancer cells [23,67-69]. ER-negative cell lines retainsensitivity to the apoptotic effects of soy isoflavones, thereby confirming that apoptosisoccurs in a hormone-independent manner. Apoptosis was effectively induced in the ER-α-negative MDA-MB-231 breast cancer cell line by genistein and daidzeinconcentrations of 50–100 μmol/l [59,60]. In MCF-7 cell cultures the induction of celldeath by treatment with genistein coincided with the increased expression of theproapoptotic proteins Bax and p53 . Breast cancer cell lines expressing mutant p53also undergo apoptosis in response to phyto-oestrogen treatment, thereby implyingapoptosis induction by both p53-dependent and p53-independent mechanisms [67,70].The polyphenol epigallocatechin (EGC) is principally found in green tea and is proposedto have anticancer properties. Treatment of p53-mutant breast cancer cells with 100μmol/l EGC induced a 40% increase in apoptosis, correlating with increased Baxexpression and reduced levels of the antiapoptotic protein Bcl-2 . EGC-inducedapoptosis was abolished following treatment with anti-Fas neutralizing antibodies orcaspase inhibitors, suggesting the involvement of Fas signalling pathways. Althoughphyto-oestrogen compounds are effective inducers of apoptosis in cell culture models, itis unlikely that plasma isoflavone concentrations would accumulate to the required levelsfor the activation of apoptotic pathways in vivo. It is estimated that plasma phyto-oestrogen concentrations may reach a maximum of 2–4 μmol/l following the moderateconsumption of soy products [15,52], although it is possible that higher levels may bepresent in target tissues. In a recently reported study, equol concentrations within breasttissue were found to exceed serum levels; however, the reverse was true for genisteinand daidzein . A recent in vitro study  revealed the flavone baicalein to be a morepotent inducer of apoptosis than genistein. Baicalein is isolated from the plantScutellariae radix and is a common ingredient in herbal tea preparations. Aconcentration of 10 μmol/l baicalein induced significant cell death in MCF-7 cell cultures,suggesting baicalein as a potentially useful pharmacological agent in breast cancertherapy.Dietary phyto-oestrogens are capable of inhibiting the proliferation of hormone-independent breast cell lines [43,54,58,69]. It has been proposed that growth inhibition inthe absence of functional ER occurs via the inhibition of tyrosine kinase activity. Theprotein tyrosine kinases are involved in a number of growth factor signalling pathways,including transforming growth factor (TGF)-α, insulin-like growth factor (IGF)-I, IGF-II andepidermal growth factor (EGF). In ER-negative breast cancer cultures 5 μmol/l genisteinnegated the stimulatory effects of TGF-α, IGF-I and IGF-II, implying the inhibition oftyrosine kinase activity . The human EGF receptor-2 oncogene (Her-2) is
constitutively overexpressed in approximately 30% of breast cancers and is associatedwith a poor patient prognosis . Research using breast cancer cell lines suggests thatdietary phyto-oestrogens are capable of repressing EGF receptor activity. The inhibitionof tyrosine kinase activity by 5 μmol/l genistein in MCF-7 cells correlated with therepression of EGF receptor tyrosine phosphorylation in response to EGF stimulation. Similar findings were reported in a recent study investigating the chemoprotectiveeffects of the green tea polyphenol epigallocatechin-3 gallate (EGCG). The treatment ofHer-2/neu over-expressing mouse mammary cells with 20–80 μg/ml EGCG inhibitedproliferation in a dose-dependent manner, correlating with a reduction in Her-2/neusignalling activity . The basal tyrosine phosphorylation of Her-2/neu was decreasedby approximately 96% following treatment with 80 μg/ml EGCG. Downstream activitiesof the signalling proteins phosphoinositide 3-kinase, Akt and nuclear factor-κB weresimilarly repressed, suggesting a potential clinical application of EGCG in breast cancertherapy.The soy isoflavones have additionally been proposed to regulate the proliferation ofbreast epithelia via an alternative mechanism involving the modulation of TGF-βsynthesis . In normal mammary tissue TGF-β maintains proliferative homeostasis byinhibiting the growth of epithelial cells [74,75]. The incubation of human mammaryepithelial cells with 5 μmol/l genistein induced a fivefold increase in the level of TGF-βsecretion . The further analysis of media conditioned with human mammary epithelialcells revealed the presence of the active as opposed to latent form of TGF-β, thusimplying a direct link between soy isoflavones and the TGF-β signalling pathway.AbbreviationsDMBA = 7,12-dimethylbenz(a)anthracene;EGC = epigallocatechin;EGF = epidermal growth factor;EGCG = epigallocatechin-3 gallate;ER = oestrogen receptor;ERE = oestrogen response element;HRT = hormone replacement therapy;IGF = insulin-like growth factor;SRC = steroid receptor coactivator;TGF = transforming growth factor.References: Messina MJ: Legumes and soybeans: overview of their nutritional profiles and health effects. Am J Clin Nutr 1999, Suppl:439S-450S. Barnes S: Phytoestrogens and cancer. Baillières Clin Endocrinol Metab 1998, 12:559-579. Lamartiniere CA: Protection against breast cancer with genistein: a component of soy. Am J Clin Nutr 2000, Suppl:1705S-1707S. Dai Q, Shu X-O, Jin F, Potter JD, Kushi LH, Teas J, Gao Y-T, Zheng W: Population-based case- control study of soyfood intake and breast cancer risk in Shanghai. Br J Cancer 2001, 85:372-387. Wu AH, Ziegler RG, Nomura AMY, West DW, Kolonel LN, Horn-Ross PL, Hoover RN, Pike MC: Soy intake and risk of breast cancer in Asians and Asian Americans. Am J Clin Nutr 1998, Suppl:1437S-1443S. Adlercreutz H: Phyto-oestrogens and cancer. Lancet Oncol 2002, 3:364-373.
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inhibitory activity in the 22Rv1 prostate cancer cell line. Urolithins A and B showed adecrease in their CYP1-mediated EROD inhibitory IC50 values upon increasing theirtreatment times from 30 min to 24 h. Urolithin C, 8-O-methylurolithin A, and 8,9-di-O-methylurolithin C caused a potent CYP1-mediated EROD inhibition in 22Rv1 cells upon24 h of incubation. Neutral red uptake assay results indicated that urolithin C, 8-O-methylurolithin A, and 8,9-di-O-methylurolithin C induced profound cytotoxicity in theproximity of their CYP1 inhibitory IC50 values. Urolithins A and B were studied for theircellular uptake and inhibition of TCDD-induced CYP1B1 expression. Cellular uptakeexperiments demonstrated a 5-fold increase in urolithin uptake by 22Rv1 cells. Westernblots of the CYP1B1 protein indicated that the urolithins interfered with the expression ofCYP1B1 protein. Thus, urolithins were found to display a dual mode mechanism bydecreasing CYP1B1 activity and expression.IntroductionDietary intervention to prevent carcinogenesis has been well-established inepidemiological studies. The consumption of fruits and vegetables is considered to be asafeguard against various forms of cancers. Polyphenols are the major constituents offruit and vegetable diets and are believed to elicit a number of biological properties dueto their antioxidant and anticarcinogenic activities (1). A number of flavonoids such asquercetin, chrysin, apigenin, and luteolin have been investigated for their cytochromeP450 1 (CYP1) enzyme inhibition activities, indicating that flavonoid-relatedanticarcinogenesis is mediated in part by CYP1 inhibition (2). Ellagic acid, the hydrolysisproduct of ellagitannins, exhibits anticarcinogenic effects by inhibition of CYP1A1-dependent activation of procarcinogens. A number of ellagic acid analogues showedsimilar inhibitory activities against CYP1-mediated benzo[a]pyrene activation (3). Ellagicacid is a major polyphenol in pomegranate juice. Pomegranate juice polyphenolsshowed a strong inhibitory activity against estrogen-dependent MCF-7 cell lines. In invivo studies, pomegranate juice exhibited 47% inhibition of cancerous lesion formationinduced by the carcinogen, 7,12-dimethylbenz[a]anthracene (4), indicating its potentialuse as an adjuvant therapeutic in human breast cancer treatment. Pomegranate juicecomponents are also believed to exert cancer chemopreventive activity against skin andcolon cancer. Importantly, the consumption of pomegranate juice decreased the clinicalreemergence of prostate cancer-specific antigen in prostate cancer patients afterprimary therapy (5, 6). Pomegranate ellagitannins are transformed by human colonicflora into bioavailable organic molecules called urolithins (7). The pomegranate microbialmetabolites preferentially accumulate in prostate, colon, and intestinal tissues relative toother organs in a mouse model and exert their beneficial effects to a greater extent inthose tissues (5). Pomegranate constituents inhibited prostate cancer cell growth byaffecting their proliferation, gene expression, invasion, and apoptosis but had a lessprofound effect on normal prostate epithelial cells. However, the concentrations at whichthey exhibited antiproliferation activity against human prostate cancer cell lines werehigher than the physiologically available concentrations (18.6 μM when 1 L of juice isconsumed for 5 days) (5, 8). These results suggest that there might be some otherpathways through which pomegranate constituents exert cancer chemoprevention. Toexplore the other possible prostate cancer chemopreventive pathways, we studied theeffects of pomegranate juice ellagitannins and their microbial metabolites on CYP1B1-induced carcinogenesis. Previously, it was shown that pomegranate juice consumptionresulted in lowered total hepatic CYP content and also decreased CYP1A2 and CYP3A.Therefore, the anticarcinogenic effects of pomegranate juice could be partly attributed totheir ability to inhibit CYP activity/expression (9). Our work represents the first report
concerning the effects of pomegranate ellagitannins on CYP1B1 inhibition as a meansfor cancer chemoprevention.CYPs are responsible for the bioactivation of endogenous compounds, drugs, dietarychemicals, and xenobiotics. The CYP1 isoforms, CYP1A1, CYP1A2, and CYP1B1, areof major importance because they activate a number of polycyclic aromatichydrocarbons (PAHs) to genotoxic compounds leading to tumorogenesis (10). CYP1B1is abundantly expressed extrahepatically in steroidogenic (ovaries, testes, and adrenalglands) and steroid-responsive (breast, uterus, and prostate) tissues. The CYP1B1enzyme plays an important role not only in the initiation and promotion of cancer but alsoin the development of drug resistance. The CYP1B1 enzyme alone accounts foractivation of 15 PAHs, six heterocyclic amines, and two nitropolycyclic hydrocarbons intomutagenic and carcinogenic compounds, which cause DNA damage and initiate cancerformation. CYP1B1 is also involved in the metabolism of endogenous compounds suchas 17β-estradiol to an active metabolite, 4-hydroxyestradiol (4-OH-E2), which has beenimplicated in breast cancer initiation. In comparison, CYP1A1 converts 17β-estradiol into2-hydroxyestradiol (2-OH-E2), which is relatively noncarcinogenic as compared to 4-OH-E2 and plays no role in cancer (11). CYP1B1 levels are overexpressed in prostate, lung,esophageal, oral, and colon cancers but not in the corresponding normal tissues. Theincreased expression of CYP1B1 could generate an excessive number of genotoxicmetabolites, which may attack the DNA of normal cells, thus allowing for cancerpromotion. Although the augmented CYP1B1 expression does not cause tumor invasionor metastasis, it leads to deactivation of anticancer drugs such as flutamide in prostatecancer treatment and docetaxel in breast cancer treatment (12). Considering the crucialrole played by CYP1B1 in cancer inititation, promotion, and resistance development, it isan attractive molecular target for cancer chemoprevention. The expression of the CYP1family is regulated by the aryl hydrocarbon receptor (AhR). The ligands of AhR rangefrom environmental contaminants to plant- or diet-derived constituents such as curcuminand carotenoids (13). Because CYP1B1 is a therapeutic target in prostate cancer, wehypothesized that pomegranate constituents/metabolites might exert prostate cancerchemoprevention through CYP1B1 inhibition as one of the plausible mechanisms. Ourresults indicate a previously unexplored pathway through which pomegranate juiceconstituents may contribute to prostate cancer chemoprevention.Materials and MethodsIsolation and Identification of Pomegranate Juice EllagitanninsThe extraction of ellagitannins was performed by a procedure described previously, byuse of a step gradient consisting of an increasing amount of methanol in water. Thecommercial POMx (100 mL) was diluted to 500 mL with Millipore purified water andsuccessively partitioned with EtOAc (3 × 200 mL) and n-BuOH (3 × 200 mL).The n-BuOH extract (2.0 g) was concentrated and subjected to Amberlite XAD-16column chromatography (500 g, 6 cm × 35 cm) and eluted with H2O (2.0 L) and MeOH(2.0 L) successively. The MeOH fraction on removal of solvent under reduced pressureafforded a tannin fraction (XAD-n-BuOH) (1.3 g). This was further purified on SephadexLH-20 CC (6 cm × 55 cm) and eluted with H2O:MeOH (2:8, 350 mL), H2O:MeOH (1:9,500 mL), MeOH (450 mL), and MeOH:Me2CO (1:1, 600 mL) to give nine fractions. Afollow-up of fractionation and further purification of all of the fractions on Sephadex LH-20 column chromatography using H2O:MeOH gradient, MeOH, and MeOH:Me2COgradient system afforded the compounds gallic acid, hexahydroxydiphenic acid (HHDP),gallagic acid, punicalins, and punicalagins. The latter compounds (punicalins andpunicalagins) exist in solution as the α- and β-anomers as well as acyclichydroxyaldehyde analogues (14) (Figure 1). The compounds were identified using LC-
MS retention time, UV absorption pattern, molecular mass, and 1H NMR spectra. TheLC-MS system consisted of Waters Micromass ZMTQ mass spectrophotometer, Waters2695 Separation Module, and Waters 996 Photodiode Array Detector. Mass spectrawere recorded in negative mode, using a capillary voltage of 4000/3500 V and a gastemperature of 300 °C. The column used was a 150 mm × 3.0 mm i.d., 5 μm, Luna C18100 Å (Phenomenex, Torrance, CA). The analysis was performed using a 2.5% aceticacid in water (solvent A) and 2.5% acetic acid in methanol (solvent B), starting from100% A for 5 min, 0−60% B for 15 min, and 60−100% B for the next 15 min. The flowrate was 0.3 mL/min with the pressure set at 900−1500 mmHg.Figure 1. Structures of ellagitannins and urolithins.Synthesis of UrolithinsChemicalsResorcinol, ReagentPlus (99%), 2-bromobenzoic acid (97%), 2-bromo-4,5-dimethoxybenzoic acid (98%), and chlorobenzene were purchased from Sigma Aldrich(St. Louis, MO). 2-Bromo-5-methoxybenzoic acid (98%) was purchased from Alfa Aesar(Ward Hill, MA). Pyrogallol (ACS grade) was purchased from Acros Organics. CuSO4,NaOH, and AlCl3 were purchased from Fisher Scientific (Pittsburgh, PA).Purification of CompoundsThe high-performance liquid chromatography (HPLC) system consisted of a WatersDelta 600, a Waters 600 controller, a Waters 996 Photodiode Array Detector, and a 3.0mm × 150 mm column (Phenomenex, ODS 5 μm C18 100 Å). Analyses were performedin the gradient system A, 2.5% aqueous acetic acid, and B, 2.5% acetic acid inmethanol, starting from 100% A for 5 min, 0−60% B for 15 min, and 60−100% B for 15min. The flow rate was 1 mL/min, and the pressure was 600−800 mmHg. The elution ofmetabolites was monitored at 254 nm.Urolithins (urolithin B, 8-O-methylurolithin A, urolithin A, 8,9-di-O-methylurolithin C,urolithin C, 8,9-di-O-methylurolithin D, and urolithin D) were synthesized by thecondensation of resorcinol or pyrogallol with an appropriately substituted benzoic acid bythe modified protocols described by Ito et al. (15). The structures of urolithins wereconfirmed by their molecular mass and comparison of observed and reported 1H NMRdata with reported data (Figure 1).Recombinant CYP1 Ethoxyresorufin-O-deethylase (EROD) Assay and Inhibition Kinetics
To study the effects of pomegranate chemical constituents and their microbialmetabolites on recombinant CYP1A1 and CYP1B1, a 96-well plate EROD assay wasused (16). Concentrations of the test compounds ranged from 0.5 to 30 μM. Inhibitionkinetics of CYP1B1-mediated EROD activity was determined similarly. Concentrations of0.5 and 1 μM were used for urolithins A and B in triplicate.22Rv1 Prostate Cell EROD AssayTo evaluate the effects of pomegranate constituents and microbial metabolites in a cell-based CYP1 activity, an EROD assay was conducted using 22Rv1 cells in a 48-wellplate format (32). The test compounds were studied for their effects on cell-based2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1-mediated EROD activity. Thecells were treated with TCDD for 24 h to induce CYP1 expression. The cells were alsocotreated with compounds, for either 30 min or 24 h, at concentrations ranging from 6.75to 50 μM. The cells were treated with DMSO, ellagitannins, and urolithins alone toevaluate their ability to induce CYP1 expression in the absence of TCDD.Microsome PreparationCells were seeded in 150 cm2 culture plates and exposed to various treatments for 24 h.The cells were harvested, washed, and spun down (250g/5 min/22 °C). An appropriateamount of the lysis buffer [10 mM Tris, pH 7.5, 10 mM KCl, and 0.5 mMethylenediaminetetraacetic acid (EDTA)] was added, and the cells were transferred tothe glass tube of a Teflon homogenizer and kept on ice for 10 min. This was followed bythe addition of an appropriate amount of homogenization buffer [0.25 M KH2PO4, 0.15 MKCl, 10 mM EDTA, and 0.25 mM phenylmethanesulfonyl fluoride (PMSF)]. The cellswere then broken by 12 manual strokes on a tight-fitting Teflon homogenizer. Aftercentrifugation at 15000g/20 min/4 °C, the supernatant was again centrifuged at105000g/90 min/4 °C. Microsomal pellets were resuspended in microsomal dilutionbuffer [0.1 M KH2PO4, 20% glycerol, 10 mM EDTA, 0.25 mM PMSF, and 0.1 mMdithiothreitol (DTT)] and stored in aliquots at −80 °C.Western BlottingMicrosomal protein (5−10 μg) samples were prepared by heating at 95 °C for 5 min inthe sample buffer comprising 0.5 μM Tris HCl (pH 6.8), 10% glycerol, 2% sodiumdodecyl sulfate (SDS), 5% mercaptoethanol, and 0.001% bromophenol blue. Thesamples were resolved by precast criterion SDS-polyacrylamide gel electrophoresis(PAGE) gel (10%) at 200 V for 45−50 min (Bio-Rad Laboratories, Hercules, CA). Theproteins were then transferred to a PVDF membrane (Bio-Rad Laboratories) at 100 V for90 min. Following transfer, the membranes were blocked in blocking buffer for 1 hfollowed by incubation in CYP1B1 primary antibody (1:1000, antirat CYP1B1 polyclonalantibody, kindly donated by Dr. Thomas R. Sutter). After they were washed, themembranes were incubated in buffer containing the horseradish peroxidase-conjugatedsecondary antibody (1:30000, antigoat IgG peroxidase conjugate, Sigma Chemicals) for2 h. The membrane was washed and developed using LumiGLO Reservechemiluminescent substrate (KPL, Inc., Gaithersburg, MD). The signals were detectedusing a CCD camera (VersaDoc Imaging System, Bio-Rad Laboratories). HumanCYP1B1 supersomes were used to make standard curves of known proteinconcentrations (0.25−2 pmol). Standard curves were used to quantitate the CYP1protein amounts in the samples using the Quantity One quantitation software (Bio-RadLaboratories). Statistical differences between the control and the treated samples weredetermined using one-way analysis of variance (ANOVA) followed by Newman−Keulsposthoc (p < 0.05) using GraphPad Prism software.Neutral Red Cytotoxicity Assay
The assay was performed in 96-well microplates. Cells were seeded at a density of10000 cells/well and allowed to settle for 30 min at 37 °C. The compounds, dilutedappropriately in RPMI-1640 medium, were added to the cells and again incubated for 48h. The number of viable cells was determined using the neutral red assay procedure(17).Induction of Phase II Conjugating Enzymes AssayThe assays were performed according to standard procedures described by Kirlin et al.(18)Cellular Uptake of Urolithins A and B22Rv1 cells were incubated with 20 μM urolithins A and B in the RPMI-1640 media for0.5, 6, 12, and 24 h time periods. After incubation, the cells were washed and harvestedby trypsinization. The cells were extracted with acidified MeOH. The samples wereanalyzed by reverse phase HPLC at 288 nm detection. The experiment was done intriplicate. A standard curve of urolithins A and B was prepared from which the amount ofurolithin uptake was determined. The amount of urolithin uptake was adjusted to theamount of protein.Results and DiscussionPomegranate fruit consists of two major classes of polyphenols, flavonoids andellagitannins. The flavonoids include quercetin, kaempferol, and myricetin (19). Theseflavonoids have been reported to exhibit CYP1 inhibitory activities (16). Pomegranatejuice consumption has been found to decrease the expression/activity of total hepaticCYP content (9). Pomegranate juice obtained by hydrostatic pressing of whole fruitpredominantly contains ellagitannins. Ellagitannins, with the exception of ellagic acid,have not been previously studied for their CYP1 inhibitory activities. Ellagic acid hasbeen shown to inhibit CYP2A2, 3A1, 2C11, 2B1, 2B2, and 2C6 in rat liver microsomes(20) and also inhibited the CYP1A1-dependent activation of benzo[a]pyrene (BaP) (3).Inhibition of CYP1 protein expression and activity by some flavonoids, such as diosmin,diosmetin, quercetin, kaempferol, and myricetin, was believed to be through AhRantagonism or their effects on the downstream products of AhR signal transductionpathways (21). However, ellagic acid decreased CYP1A1-dependent BaP activityindependent of the AhR-responsive element (3). The proposed mechanism of inhibitionof CYP1A1-dependent BaP activation involved scavenging of the carcinogen by ellagicacid through chemical binding (22). Therefore, our objective was to study the effects of aselection of pomegranate ellagitannins and urolithins on the inhibition of CYP1-dependent carcinogen activation.The ability of ellagitannins and urolithins to inhibit CYP1 activity was tested in arecombinant CYP1A1- and CYP1B1-dependent EROD assay. The IC50 values forCYP1B1 inhibition ranged from 1.15 ± 0.65 μM for urolithin A to 137 ± 11.08 μM forurolithin D. CYP1A1 IC50 values ranged from 1.5 ± 0.32 μM for punicalins to 2907 ± 168μM for urolithin D (Table 1, section A). Urolithins exhibited higher selectivity towardCYP1B1 EROD inhibition as compared to CYP1A1, although the selectivity was notsignificant. The Ki values of CYP1B1 and CYP1A1 depicted in Table 1, section A,indicate that 8-O-methylurolithin A exhibited a 7.5-fold selectivity toward CYP1B1inhibition. Punicalins and punicalgins were 10- and 5-fold more selective towardCYP1A1 inhibition.Table 1. Results of Recombinant CYP1-Mediated EROD Assay and Kinetic Parametersa
Section A CYP1B1 CYP1A1 IC50 ± SEM Ki ± SEM IC50 ± SEM Ki ± SEM Ki(CYP1A1:CYP1B1) (μM) (μM) (μM) (μM)UA 1.15 ± 0.65 0.25 ± 0.14 12.4 ± 4.7 0.51 ± 0.18 2.05UB 1.55 ± 0.49 0.34 ± 0.17 26.8 ± 12.9 1.11 ± 0.53 3.28UC 39.9 ± 29 8.74 ± 0.65 790 ± 75 32.7 ± 3.11 3.74UD 137 ± 11.08 30.10 ± 25 2907 ± 168 120.3 ± 69.6 3.99MUA 1.49 ± 0.39 0.327 ± 0.08 59.8 ± 8.7 2.47 ± 0.36 7.57DMUC 89.6 ± 9.7 19.6 ± 2.13 657 ± 74.3 27.2 ± 3.00 1.39PL 2.82 ± 0.33 0.618 ± 0.07 1.5 ± 0.32 0.062 ± 0.00 0.10PG 2.6 ± 0.79 0.58 ± 0.02 2.67 ± 0.48 0.109 ± 0.20 0.19 Section B urolithin A urolithin B DMSO 0.5 μM 1 μM DMSO 0.5 μM 1 μMVmax 339 ± 148 97.3 ± 14.6 66 ± 6.02 355 ± 103 150.9 ± 14.9 99.5 ± 8.53Km 3.414 ± 2 2 ± 0.47 1.6 ± 0.25 4.78 ± 1.81 2.10 ± 0.33 1.9 ± 0.26IC50 and Ki values (±SEM) and the ratio of CYP1A1 to CYP1B1 Ki for urolithins A (UA), B(UB), C (UC), D (UD), 8-O-methylurolithin A (MUA), 8,9-di-O-methylurolithin C (DMUC),punicalins (PL), and punicalagins (PG) mediated inhibition of EROD acitivity usingrecombinant human CYP1B1 and CYP1A1 enzymes. For section B, kinetic parameters,Vmax (pmol/mg/min), and Km (μM) ± standard errors (n = 3) for the inhibition ofrecombinant human CYP1B1 by urolithin A and B (0.5 and 1 μM), determined bynonlinear regression curve fit using the Michaelis−Menten equation ([S] vs V) plot usingGraphPad Prism.Urolithins A and B are the major microbial metabolites of pomegranate chemicalconstituents detected in human systemic circulation. These metabolites exhibited lowerIC50 values in recombinant CYP1 inhibition as compared to all other tested compounds.Therefore, a study was conducted to investigate the mechanism of action of CYP1B1inhibition by urolithins A and B. The concentrations of inhibitors used were in the vicinityof their calculated IC50 values, that is, 0.5 and 1 μM. EROD activities were determinedwith substrate concentrations ranging from 0.1 to 2.0 μM. Kinetic parameters, Vmax andKm, were calculated using the Michaelis−Menten equation ([S] vs V curve). Doublereciprocal plots were plotted using 1/[S] and 1/V (Figure 2) from which Ki values werecalculated (Table 1, section B). The calculated Ki values for urolihins A and B (1.51 ±0.91 and 1.33 ± 0.08 μM) were not statistically different from those calculated by usingCheng−Prusoff equations (16). The calculated Vmax and Km for urolithin A changedsignificantly with an increasing concentration of inhibitor, suggesting an uncompetitivetype of inhibition. However, the Vmax and Km of urolithin B did not differ significantly uponincrease of inhibitor concentration, suggesting a noncompetitive type of inhibition.
Figure 2. Double reciprocal plots for the inhibition of in vitro EROD activity of CYP1B1 byurolithins A and B at 0.5 and 1 μM. EROD substrate concentrations used were 0.1, 0.2,0.3, 0.4, 0.6, 1.0, and 2.0 μM. Recombinant CYP1B1 was preincubated with the inhibitoror DMSO prior to initiation of reaction. Each experiment was done three times induplicate.In our study, urolithins A and B, structural analogues of ellagic acid, exhibited asignificant inhibition of CYP1-dependent EROD activity. The results suggested that ahydroxy group at C-8 and C-3 (urolithins A and B), corresponding to the C-4 and C-4′position of ellagic acid, were required for full CYP1-dependent EROD activity inhibition inaccordance with a previous study by Barch et al. (7). However, in our study, additionalhydroxy groups at C-4 and C-9 of the urolithin pharmacophore (urolithins C and D)resulted in decreased CYP1-dependent EROD inhibitory activity. Methylation of thehydroxy groups to give 8-O-methylurolithin A and 8,9-di-O-methylurolithin C decreasedthe activity, suggesting that the phenolic hydroxy groups are important for CYP1inhibitory activity. To probe the importance of the lactone group for CYP1-dependentEROD activity inhibition, HHDP and gallic acid were tested for their CYP1-dependentEROD activity. The results showed that hydrolysis of the lactone functionality did notresult in CYP1 inhibition (data not shown).
The punicalins and punicalagins were potent inhibitors of CYP1-dependent ERODactivity with Ki values (Table 1, section A) comparable to the dietary flavonoids. Thedietary flavonoids, inhibiting CYP1-dependent metabolic activation of procarcinogens,include quercetin, kaempferol, apigenin, myricetin, and rutin. Quercetin and kaempferol,which are the predominant flavonoids in the human diet, inhibited CYP1-mediatedEROD activities. The apparent Ki values of inhibition of human recombinant CYP1B1and CYP1A1 were 14 ± 3 and 52 ± 2 nM for kaempferol, whereas they were 23 ± 2 and77 ± 5 nM for quercetin (23). In a different study, quercetin inhibited epoxidation of 7,8-dihydro-7,8-benzo[a]pyrene-7,8-diol by CYP1A1 allelic variants with Ki values rangingfrom 2.0 to 9.3 μM with mixed type inhibition (24). In another study, quercetin inhibitedrecombinant CYP1A1 and CYP1B1 activities with Ki values of 0.25 ± 0.04 and 0.12 ±0.02 μM with a mixed type inhibition (16). The bioavailability of the flavonoids dependsupon the source of food; for example, quercetin absorption from tomato puree, apples,and onions was 0.082, 0.34, and 0.74 μM, respectively (25, 26). Kaempferol plasmaconcentrations ranged from 0.01, 0.05, and 0.1 μM upon consumption of onion, tea, andendive, respectively (27, 28). Bioavailable concentrations of quercetin and kaempferolfrom some food sources were less than their reported Ki values of CYP1 inhibition.Apigenin, a flavone present in parsley, exhibits CYP1B1 and CYP1A1 inhibition with Kivalues ranging from 60 nM to 0.2 μM. These concentrations are bioavailable (127 ± 81nM) upon consumption of 2 g of blanched parsley (14). Bioavailability of rutin fromtomato puree as detected in plasma was calculated to be 0.1 μM (25), which was around60-fold lower than the reported Ki of CYP1B1 inhibition. The bioavailability of many otherflavonoids is still unclear. However, dietary flavonoids could inhibit CYP1-mediatedbioactivation of environmental and dietary carcinogens into genotoxic compounds andprevent cancer initiation in alimentary canal-related cancers because they come intodirect contact with the digestive epithelium of the digestive system (29). However, theprostate cancer chemopreventive effects of flavonoids (30) depending on thebioavailability are still debated. It is therefore important to choose an appropriate dietary
supplement that can release adequate amounts of cancer chemopreventive compoundsinto plasma, whenever it is consumed, with an intended pharmacological activity.Pomegranate juice ellagitannins have been extensively studied for their bioavailabilityand their biological effects. In one study, it was established that the punicalaginshydrolyze into ellagic acid and other smaller polyphenols that are responsible for thebioactivity of ellagitannins. In a study performed on human subjects, consumption of 180mL of pomegranate juice (equivalent to 25 mg of ellagic acid and 319 mg ofpunicalagins) resulted in detection of ellagic acid in plasma with a maximumconcentration of 31.9 ng/mL (0.1 μM) (31). Previous studies also indicated that thebioavailability of ellagic acid from pomegranate juice, pomegranate liquid concentrateextract, and pomegranate powder extract was not statistically different (32). In our study,punicalins and punicalagins inhibited CYP1A1 with Ki values of 0.062−0.109 μM andCYP1B1 with Ki values 0.618 and 0.58 μM, respectively. Punicalins and punicalaginsexhibited approximately a 10- and 5-fold selectivity for CYP1A1 over CYP1B1. Thus,consumption of pomegranate juice could be beneficial in decreasing CYP1-mediatedoral, esophageal, and colon cancers. However, these ellagitannins cannot exhibit asystemic CYP1 inhibition activity because they are metabolized in the colon bymicroflora into smaller organic molecules called urolithins.Bioavailability studies indicate that maximum plasma concentrations of urolithins A andB reach concentrations of 4−18 μM in human subjects (8, 32). Urolithins A and Binhibited human recombinant CYP1A1 and CYP1B1 with Ki values in their bioavailableconcentration range. Our particular interest was to explore the beneficial effects ofurolithins in prostate cancer. Studies indicate that 15% of prostate cancer patients, whohave undergone a radical prostectomy, had a biochemical recurrence of prostate-specific antigen (PSA). Among them, 34% of patients developed distant metastaseswithin 15 years (33). It was evident that consumption of pomegranate juice delayed thedoubling time of the PSA by 39 months after primary therapy (7). The effects wereascribed to the antiproliferative, apoptotic, and antioxidant effects of pomegranateconstituents, observed in LNCaP, PC-3, 22Rv1, and DU 145 prostate cancer cell lines(8, 34). A study about gene polymorphisms and risk of prostate cancer showed thatpolymorphisms in CYP1B1 and PSA genes increased the risk and aggressiveness ofprostate cancer (35). Any dietary constituent with CYP1B1 inhibitory activity couldpotentially lead to prostate cancer chemoprevention. There were no previous reportsabout the ability of pomegranate constituents/metabolites to inhibit CYP1B1-dependentcarcinogenesis.Therefore, we studied the capability of pomegranate constituents to inhibit CYP1B1-induced metabolic activation in a prostate cancer cell line, 22Rv1. The cells were treatedwith TCDD for 24 h to induce CYP1B1 protein expression. Then, the cells were treatedwith punicalins, punicalagins, or urolithins for 30 min. Following 30 min of incubation,urolithins A and B significantly decreased TCDD-induced EROD activity at the highestconcentration used (Figure 3A). The IC50 values calculated for cell-based CYP1inhibition by urolithins A and B were 32 ± 8.9 and 38.2 ± 3.94 μM, respectively (Table 2).The results indicate a 28- and 26-fold increase in IC50 values for cell-based CYP1-mediated EROD activity of urolithins A and B, respectively, as compared to their in vitrorecombinant CYP1-mediated EROD inhibitory IC50. Punicalins and punicalagins did notinhibit cell-based CYP1-mediated EROD activity at the highest concentration used (50μM). To ascertain whether a decrease in IC50 occurred upon longer incubation, the cellswere allowed to grow in the presence of compounds and TCDD for 24 h (Figure 3B).The EROD activity results indicated that the compounds more effectively inhibitedCYP1-mediated EROD activity and had IC50 values lower than those following 30 min of
incubation. After 24 h of cotreatment, urolithins A and B inhibited TCDD-induced ERODactivity in prostate cells with IC50 values of 13.3 ± 1.32 and 17.9 ± 1.8 μM, respectively,which were in the vicinity of their bioavailability (8, 32). Punicalins and punicalagins didnot exhibit EROD inhibition even upon 24 h of incubation. Urolithin C, 8-O-methylurolithin A, and 8,9-di-O-methyl urolithin C demonstrated IC50 values of 26.8 ± 2.5,14.8 ± 2.24, and 11.5 ± 2.8 μM, respectively, which were lower than those of urolithins Aand B (Table 2). The compounds alone did not induce EROD activity after 24 h ofincubation. Because the prostate cells were treated with the test compounds for 24 h, itwas imperative to investigate if the compounds exhibited any cytotoxicity. A neutral reddye uptake assay was used to measure the cytotoxicity of the test compounds. The datawere probit transformed followed by linear regression and IC50 calculation. The cytotoxicIC50 values ranged from 20.6 ± 4.58 μM for 8,9-di-O-methylurolithin C to 108 ± 3.994 μMfor urolithin B (Table 3). The results indicate that the cytotoxicities of urolithin A and Bhad no contribution toward decreased CYP1-mediated EROD activity. The results alsoindicate that urolithin C, 8,9-di-O-methylurolithin C, and 8-O-methylurolithin A were falsepositives in the prostate cell EROD assay. The activity was not because of CYP1inhibition but due to cytotoxicity. This conclusion was verified based on four facts: (1)These compounds inhibited recombinant CYP1-mediated EROD activity at higherconcentrations as compared to urolithins A and B; (2) these compounds did not inhibitprostate cell EROD activity upon 30 min of incubation; (3) they inhibited prostate cellEROD activity upon 24 h of treatment, with IC50 values lower than those exhibited in therecombinant CYP1-mediated EROD assay; and (4) they exhibited cytotoxicity in thevicinity of their prostate cell EROD inhibition IC50 values.Figure 3. Effects of increasing concentration (6.75, 12.5, 25, and 50 μM) of urolithin A(UA), urolithin B (UB), urolithin C (UC), 8-O-methylurolithin A (MUA), 8,9-di-O-methylurolithin C (DMUC), punicalins (PL), and punicalagins (PG) on 50 nM TCDD-induced EROD activity in intact 22Rv1 human prostate cancer cells. The cells wereexposed to the compounds for (A) 30 min or (B) 24 h prior to the EROD measurement.
Each experiment was done three times in triplicate. Global one-way (ANOVA) with aStudent−Newman−Keuls posthoc test was used to determine treatment effects. In thesame treatment groups, bars with different letters are statistically different.Table 2. IC50 Values for Pomegranate Chemical Constituents/Microbial MetaboliteMediated Inhibition of EROD Activity in TCDD-Induced 22Rv1 Prostate Cancer Cells IC50 ± SEM (μM) chemical constituent 30 min 24 hurolithin A 32 ± 8.9 13.3 ± 1.3urolithin B 38 ± 3.9 17.9 ± 1.8urolithin C NA 26.89 ± 2.58-O-methylurolithin A NA 14.8 ± 2.28,9-di-O-methylurolithin C NA 11.5 ± 2.8punicalins NA NApunicalagins NA NATable 3. IC50 Values for Urolithin-Mediated Cytotoxicity of 22Rv1 Prostate Cells chemical constituents IC50 ± SEM (μM)urolithin A 98.7 ± 4.2 (a)urolithin B 108 ± 3.9 (a)urolithin C 36 ± 3.5 (b)8-O-methylurolithin A 23.3 ± 3.9 (c)8,9-di-O-methylurolithin C 20.6 ± 4.6 (c)Following cell-based EROD assays, cellular uptake experiments were performed forurolithins A and B. These experiments were performed to determine if the decrease inIC50 values for CYP1-mediated EROD inhibition in 22Rv1 cells was related to increaseduptake over a 24 h time period. The experiments indicated that there was a 4.5-foldincrease in urolithin uptake upon 24 h of incubation as compared to 30 min of incubation.The results also indicated that there was a dramatic increase in urolithin uptake between6 and 12 h, beyond which the uptake was constant. The results suggested thatincreased availability of urolithins (from 30 min to 24 h) could have contributed to thedecrease in IC50 of CYP1 inhibition (Table 4). The results also indicated that urolithinswere metabolically stable in 22Rv1 cells for up to 24 h.Table 4. Uptake of Urolithins A and B by 22Rv1 Cells over a Period of 0.5−24 h uptake in μmol/mg proteintime (h) urolithin A urolithin B0.5 5.2 ± 0.4 5.7 ± 0.86 9.6 ± 0.4 8.0 ± 1.112 20.1 ± 0.5 21.9 ± 1.124 23.9 ± 0.6 25.2 ± 1.6The decrease in IC50 values could also be attributed to the decrease in the TCDD-induced CYP1B1 protein expression. To examine if the treatment affected CYP1 proteinexpression levels, Western blots were performed (Figure 4). Cell treatments wereDMSO, TCDD (50 nM), UA (50 μM), UB (50 μM), TCDD + UA (50 μM), TCDD + UB (50μM), TCDD + UA (25 μM), and TCDD + UB (25 μM). CYP1B1 protein levels were
significantly increased ( 4.5-fold) by TCDD (2.8 ± 0.6 pmol/mg) as compared to DMSO(0.62 ± 0.28 pmol/mg). Cotreatment of TCDD with urolithin A (50 μM) significantlydecreased CYP1B1 protein (1.26 ± 0.38 pmol/mg) production by 54%, while urolithin A(25 μM) decreased CYP1B1 protein (2.00 ± 0.5 pmol/mg) production by 28% ascompared to TCDD-induced cells. Cotreatment of TCDD with urolithin B (50 μM)decreased CYP1B1 protein (0.96 ± 0.4 pmol/mg) production by 65%, and urolithin B (25μM) treatment decreased protein (1.9 ± 0.5 pmol/mg) production by 29% as compared toTCDD-induced levels. Western blot analyses suggest that none of the urolithins induceCYP1B1 basal levels significantly as compared to DMSO. However, cotreatments withurolithins A and B 50 μM and TCDD decreased CYP1B1 protein expression levels ascompared to TCDD alone (Figure 4).Figure 4.(A) Western blot showing the effect of urolithins A and B and their cotreatment withTCDD on CYP1B1 protein expression. Microsomes were prepared after treating cells for24 h with different treatments (DMSO, 50 nM TCDD, UA 50 μM, UB 50 μM, TCDD + UA50 μM, TCDD + UA 25 μM, TCDD + UB 50 μM, and TCDD + UB 25 μM), along withstandards, were loaded on 10% SDS-PAGE. Protein levels were expressed in pmol/mg.(B) Effect of urolithin A and B on the TCDD-induced CYP1B1 protein expression in22Rv1 prostate cancer cells. Each experiment was repeated three times. Global one-way ANOVA with a Student−Newman−Keuls posthoc test was used to determinetreatment effects. Bars with different letters are statistically different.Urolithins A and B inhibited CYP1 EROD activity by inhibiting both the proteinexpression and the activity of CYP1B1. While the influence of urolithin C, 8,9-di-O-methylurolithin C, and 8-O-methylurolithin A on CYP1B1 activity/expression was notclear, they are believed to exert an antiproliferative activity on prostate cancer cells, inaccordance with previous studies (32, 34). An ideal anticarcinogenic agent would inhibitphase I enzymes, involved in carcinogen activation while inducing the phase II enzymes,responsible for the deactivation of carcinogens by assisting their excretion via increasedwater solubility. The pomegranate ellagitannins and urolithins were tested for theircapacity to induce glutathione S-transferase and quinone O-reductase enzymes. Thebasal levels of quinone O-reductase and glutathione S-transferase enzymes in 22Rv1cells were determined to be 1.2 ± 0.32 and 0.51 ± 0.17 μmol min−1 mg protein−1,respectively. However, none of the compounds exhibited (data not shown) induction ofquinone O-reductase or glutathione S-transferase as compared to normal proliferatingcells.