CANCER TREATMENT _CHEMOTHERAPY_ALTERNATIVES

Compiled and Edited ByG Vijaya Raghaqvan, CEO, DVS BioLife Ltd, HYDERABAD.

CONTENTS:1. INTRODUCTION2. CHEMOTHERAPY3. COMMON CANCER DRUGS4. ROLE OF UNUSUAL PLANT DERIVATIVES INCLUDING ALKALOIDS FROM POMEGRANATE5. ROLE OF MICROBES LIKE E COLI IN DIAGNOSIS AND IN TREATMENT6. COMPLEMENTARY APPROACHES

1. INTRODUCTION:In the year 2000, malignant tumours were responsible for 12 per cent of the nearly 56 million deaths worldwide from all causes. In many countries, more than a quarter of deaths are attributable to cancer. In 2000, 5.3 million men and 4.7 million women developed a malignant tumour and altogether 6.2 million died from the disease. The report also reveals that cancer has emerged as a major public health problem in developing countries, matching its effect in industrialized nations.

Every year about 85,0000 new cancer cases are diagnosed in India resulting in about 58,0000 cancer related death every year.

Bladder Cancer, Melanoma, Breast Cancer, Non-Hodgkin Lymphoma, Colon and Rectal Cancer, Pancreatic Cancer, Endometrial Cancer, Prostate Cancer, Kidney (Renal Cell) Cancer, Skin Cancer (Nonmelanoma), Leukemia, Thyroid Cancer and Lung Cancer are 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 noninfectious agents, human genes and epigenetic factors. Infectious disease causation has largely been restricted to genes directly responsible for causing cancer after sustaining damage i.e. oncogenes. Lately, evidence has emerged linking infectious agents to a number of chronic diseases. These studies have recognized the influence that acute, atypical, latent and chronic infections may play in tricking the immune system and affecting disease etiology. Similar evidence is emerging in model systems with respect to the role of infectious agents in gastrointestinal, liver and lung cancers. Although viruses have been found in association with breast cancer, skepticism remains about a role for other infectious agents, notably microbes in the disease etiology. Improved experimental designs employed in different cancer studies and a less rigid definition of infectious causation may aid in confirming or refuting a microbe-breast cancer connection. Cancer recurrence could potentially be minimized and treatment options further tailored on a case by case basis if microbes/microbial components/strain variants associated with breast cancer are identified; probiotics are employed to reduce treatment side-effects and 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 cells

Chemotherapy can be given alone or with other treatments. It can help other treatments work 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 Microbial

The following are the common drugs used in Cancer.1. Acarbose 2. Acivicin3. Aclarubicin 4. Acodazole5. Acronine6. Actinomycin D 7. Adenine Phosphate8. Adenosine9. Adozelesin10. Adriamycin 11. Adrucil 12. Alanosine13. Aldesleukin 14. Alemtuzumab 15. Alestramustine16. Alfacalcidol 17. Alitretinoin 18. Alosetron Hcl 19. Alprostadil 20. Altretamine21. Ambamustine22. Ametantrone23. Amifostine 24. Aminoglutethimide 

25. Aminopterin Sodium26. Amonafide27. Amphotericin B28. Amrubicin29. Amsacrine Hcl 30. Amygdalin 31. Anastrozole 32. Anaxirone33. Ancitabine34. Angoroside C 35. Apixaban 36. Arecoline hydrobromide37. Argatroban Anhydrous 38. Arsenic Trioxide 39. Ascomycin 40. Asparaginase41. Atevirdine42. Atosiban 43. Atrimustine44. Axitinib45. 5-Azacytidine46. Azacitidine47. Azasetron Hcl 48. Azatepa49. Azathioprine50. Azetepa51. Batimastat52. Bavituximab53. Benaxibine54. Bendamustine Hcl55. Benexate56. Benzodepa57. Betamerphalan58. Betulinic acid59. Bevacizumab 60. Bicalutamide 61. Bisantrene62. Bisnafide63. Bizelesin64. Bleomycin65. Bofumustine66. Bortezomib 67. Bosutinib68. Brefeldin A 69. Brequinar70. Bromacrylide71. Bromocriptine 72. Bropirimine73. Broxuridine74. Budotitane75. Busulfan 76. Calcifediol 77. Calcipotriol  78. Calcitriol

79. Calcium Folinate80. Calcium Levofolinate 81. Campotothecine82. Canertinib83. Cantharidin 84. Capecitabine 85. Caracemide86. Carbetimer87. Carboplatin 88. Carboquone89. Carmofur 90. carmustine91. Carzelesin92. Catharanthine Tartrate93. Celastrol 94. Cemadotin95. Cephalotaxin96. Cetuximab 97. Chlorambucil 98. Chlorethylaminouracil99. Chlormethine100.Chlornaphazine101.Cisplatin 102.Cladribine103.Cladribine 104.Clanfenur105.Clarithromycin106.Clevudine107.Clodronate Disodium108.Clofarabine109.Clomifene Citrate110.Colchiceinamide111.Colchicine112.Cordycepin 113.Curcumin 114.Cyclocytidine115.Cyclophosphamide116.Cyclosprin117.Cytarabine118.Cytidine119.Dacarbazine120.Dactinomycin121.Daniquidone122.Dapoxetine Hcl123.Dasatinib124.Datelliptium Chloride125.Daunoblastin 126.Daunorubicin127.D-Bicuculline 128.Decarbazine 129.Decitabine130.Deferasirox  131.Deflazacort  132.Defosfamide

133.Demecolcine134.Desonide135.Desoximetasone136.Desoxycorticosterone137.Desoxycortone138.Dexamethasone139.Dexasone 140.Dexniguldipine141.Dexormaplatin142.Dexrazoxane143.Dezaguanine144.Diacetoxysciroenol145.Dianhydrodulcitol146.Dianhydrodulcitolum 147.Diaziquone148.Dibrospidium Chloride149.Dinaline150.Dirithromycin151.Ditercalinium Chloride152.Ditiomustine153.Docetaxel154.Dolasetron mesylate155.Dopan156.Doxifluridine157.Doxorubicin158.D-tetrahydropalmatine159.Ecomustine160.Edatrexate161.Edelfosine162.Eflornithine163.Egenine 164.Elinafide165.Elliptinium Acetate166.Elmustine167.Elsamitrucin168.Emitefur169.Enloplatin170.Enocitabine171.Enpromate172.Entecavir 173.Entricitabine 174.Enzastaurin 175.Epipropidine176.Epirubicin177.Eptaplatin178.Erlotinib 179.Esorubicin180.Estramustine181.Ethoglucid182.7-Ethyl-10-Hydroxycamptothecin Etoglucid183.Etoposide184.Exemestane185.Exenatide Acetate 186.Ezetimibe

187.Fadrozole188.Fazarabine189.Fenclonine190.Fenretinide191.Filgrastim192.Finasteride193.Fingolimod HCL(FTY720)194.Floxuridine 195.Fludarabine196.Flumazenil197.Flumetasone198.Fluoracil199.5-Fluorouracil200.Fluorouracil  201.Fluracil202.Flurocitabine203.Flutamide 204.Folic Acid205.Formestane 206.Formylmerphalan207.Fosquidone208.Fotemustine209.Fotretamine210.Ftorafur211.Fulvestrant 212.Galamustine213.Galanthamine HBr 214.Galocitabine215.Gefitinib216.Gemcitabine217.Gimeracil 218.Giracodazole219.Glyfosfin220.Goserelin 221.Granisetron Hcl 222.Harpagoside  223.Heparin sodium224.Hexarelin 225.Homoharringtonine226.Hydrocamptothecine 227.10-Hydroxycamptothecin228.Hydroxycamptothecin229.Hydroxycarbamide230.Hydroxyurea231.Ibacitabine232.Ibandronate Sodium233.Ibandronic Acid234.Idarubicin Hcl 235.Idoxifene236.Ifosfamide237.Ilmofosine238.Ilomastat239.Imatinib240.Improsulfan

241.Indirubin242.Intoplicine243.Iproplatin244.Irinotecan245.Ivabradine HCL 246.Ixabepilone 247.Ketotrexate248.Lanreotide249.Lapatinib250.Latanoprost  251.L-biopterin 252.Lenalidomide 253.Lentinan 254.Letrozole255.Leucovorin calcium256.Leuprolide Acetate 257.Leuprorelin258.Leurubicin259.Leustatin 260.Levothyroxine 261.Lobaplatin262.Lofexidine HCL  263.Loganin  264.Lometrexol265.Lomustine266.Lonidamine 267.Losoxantrone268.Lupeol 269.Lurtotecan270.Lycopene 271.Lysipressin 272.Mafosfamide273.Mannomustine274.Mannosulfan275.Maraviroc 276.Marimastat277.Masoprocol278.Mechlorethamine279.Mechlorethaminoxide280.Medrysone281.Megastrol acetate 282.Melphalan 283.Menogaril284.Mepitiostane285.Meprednisone286.6-Mercaptopurine 287.Mercaptopurine288.Mesna 289.Metamelfalan290.Methotrexate291.Methoxymerphalan292.Methylprednisolone293.Methylprednisolone Aceponate294.Methylprednisolone Suleptanate

295.Meturedepa296.Metyrosine 297.Miboplatin298.Miltefosine299.Minamestane300.Miproxifene301.Mitindomide302.Mitobronitol303.Mitoclomine304.Mitoflaxone305.Mitoguazone306.Mitolactol307.Mitomycin308.Mitonafide309.Mitopodozide310.Mitoquidone311.Mitosper312.Mitotane 313.Mitotenamine314.Mitoxantrone315.Mitozolomide316.Mizoribine 317.Mofarotene318.Monocrotaline319.Mubritinib 320.Mustine321.Mycophenolate Mofetil  322.Mycophenolic Acid323.Myricetin  324.Myricitrin 325.Nebivolol HCL 326.Nedaplatin327.Nemorubicin328.Neptamustine329.Nigericin330.Nilotinib 331.Nilutamide 332.Nimustine333.Nitrocaphane334.Nocodazole335.Nolatrexed 2HCL  336.Norcantharidin337.Ocaphane338.Octreotide 339.Ondansetron340.Ormaplatin341.Oseltamivir Phosphate 342.Oteracil potassium 343.Oxaliplatin344.Oxipurinol345.Oxylycorium Acetate346.Paclitaxel347.Paliperidone348.palonosetron

349.Pamidronate Disodium350.Pamidronic Acid351.Panitimumab 352.Pazelliptine353.Pegaspargase354.Pemetrexed355.Pentamustine356.Pentetreotide357.Pentostatin358.Perfosfamide359.Phenoxybenzamine HCl360.Pincristine 361.Pipobroman362.Piposulfan363.Pirarubicin364.Pirazofurin365.Piritrexim366.Pirlimycin367.Piroxantrone368.Plomestane369.Plusonermin370.Podophyllotoxin 371.Polymyxin E372.Prednimustine373.Prednisolone374.Procarbazine 375.Procodazole376.Prospidium Chloride377.Psoralen378.Pteropterin379.Pumitepa380.Ractopamine HCL381.Raloxifene Hydrochloride382.Raltegravir383.Raltitrexed384.Ramosetron Hcl 385.Ranimustine 386.Rapamycin  387.Razoxane388.Resveratrol 389.Retigabine390.Riboprine391.Risedronic acid392.Ristocetin A 393.Ritrosulfan394.Rituximab 395.Rocuronium Bromide 396.Roflumilast397.Rogletimide398.Ropinirole HCL  399.Rosuvastatin Calcium 400.Rubitecan 401.Sarcolysin402.Sargramostim

403.Sebriplatin404.Semustine405.Sermorelin 406.Simtrazene407.Sitagliptin Phosphate408.Sizofiran409.Sobuzoxane410.Sodium Borocaptate[10B]411.Solaziquone412.Sonermin413.Sorafenib414.Spiclomazine415.Spirogermanium416.Spiromustine

Following are some excerpts from public domain.

Conventional Alkylating Agents Alkylating agents are one of the earliest and most commonly used chemotherapy agents used for cancer treatments. Their use in cancer treatments started in early 1940s. Majority of alkaline agents are active or dormant nitrogen mustards, which are poisonous compound initially used for certain military purposes. Chlorambucil, Cyclophosphamide, CCNU, Melphalan, Procarbazine, Thiotepa, BCNU, and Busulfan are some of the commonly used alkylating agents.They are more effective in treating slow-growing cancers such as solid tumors and leukemia

Traditional Antimetabolites Structure of antimetabolites (antineoplastic agents) is similar to certain compounds such as vitamins, amino acids, and precursors of DNA or RNA, found naturally in human body. 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 to interfere with the process of division of cancer cells. They are commonly used to treat gastrointestinal tract, breast, and ovary tumors.Methotraxate, which is a commonly used antimetabolites chemotherapy agent, is effective in the S-phase of the cell cycle. It works by inhibiting an enzyme that is essential for DNA synthesis.6-mercaptopurine and 5-fluorouracil (5FU) are two other commonly used antimetabolites. 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 of cancers including breast, esophageal, head, neck, and gastric cancers. 6-mercaptopurine is an analogue of hypoxanthine and is commonly used to treat Acute Lymphoblastic Leukemia (ALL).Other popular antimetabolite chemotherapy drugs are Thioguanine, Cytarabine, Cladribine. Gemcitabine, and Fludarabine.

Anthracyclines Anthracyclines are daunosamine and tetra-hydronaphthacenedione-based chemotherapy agents. These compounds are cell-cycle nonspecific and are used to treat 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, is isolated from mutated strain of Streptomyces. Doxorubicin is more effective in treating solid tumors. Idarubicin, Epirubicin, and Mitoxantrone are few of the other commonly used anthracycline chemotherapy drugs.Anthracyclines work by forming free oxygen radicals that breaks DNA strands thereby inhibiting DNA synthesis and function. These chemotherapeutic agents form a complex with DNA and enzyme to inhibit the topoisomerase enzyme. Topoisomerase is an enzyme class that causes the supercoiling of DNA, allowing DNA repair, transcription, and replication.

Antitumor antibiotics Antitumor antibiotics are also developed from the soil fungus Streptomyces. These drugs are widely used to treat and suppress development of tumors in the body. Similar to anthracyclines, antitumor antibiotics drugs also form free oxygen radicals that result in DNA strand breaks, killing the growth of cancer cells. In most of the cases, these drugs are used in combination with other chemotherapy agents.Bleomycin is one of the commonly used antitumor antibiotic used to treat testicular cancer and hodgkin’s lymphoma.

Monoclonal antibodiesThe treatment is known to be useful in treating colon, lung, head, neck, and breast cancers. Some of the monoclonal drugs are used to treat chronic lymphocytic leukemia, acute myelogenous leukemia, and non-Hodgkin's lymphoma.Monoclonal antibodies work by attaching to certain parts of the tumor-specific antigens and make them easily recognizable by the host’s immune system. They also prevent growth of cancer cells by blocking the cell receptors to which chemicals called ‘growth factors’ attach promoting cell growth.Monoclonal antibodies can be combined with radioactive particles and other powerful anticancer drugs to deliver them directly to cancer cells. Using this method, long term radioactive treatment and anticancer drugs can be given to patients without causing any serious harm to other healthy cells of the body.

Platinum Platinum-based chemotherapy agents work by cross-linking subunits of DNA. These agents act during any part of cell cycle and help in treating cancer by impairing DNA synthesis, transcription, and function.Cisplatin, although found to be useful in treating testicular and lung cancer, is highly toxic 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 has fewer kidney-related side effects. Oxaliplatin, which is third generation platinum-based complex, is found to be helpful in treating colon cancer. Although, oxaliplatin does not cause any toxicity in kidney it can lead to severe neuropathies.  Platinum-based drugs are often used for treatment of mesothelioma.

Role of UNUSUAL Plant Derivatives

They are primarily categorized into four groups: topoisomerase inhibitors, vinca alkaloids, taxanes, and epipodophyllotoxins.

Topoisomerase inhibitors are chemotherapy agents are categorized into Type I and Type 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 (Catharanthus roseus) and are useful in treating leukemias. They are effective in the M phase of the cell cycle and work by inhibiting tubulin assembly in microtubules.Vincristine, Vinblastine, Vinorelbine , and Vindesine are some of the popularly used vinca alkaloid chemotherapy agents used today. Major side effect of vinca alkaloids is that they 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 work in the M-phase of the cell cycle and inhibit the function of microtubules by binding with them. 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 effect of taxanes is that they lower the blood counts in patients.

EpipodophyllotoxinsEpipodophyllotoxins chemotherapy agents are extracted from the American May Apple tree (Podophyllum peltatum). Etoposide and Teniposide are commonly used epipodophyllotoxins chemotherapy agents which are effective in the G1 and S phases of the cell cycle. They prevent DNA replication by stopping the cell from entering the G1 phase and stop DNA replication in the S phase.

Phytoestrogens and Cancer TreatmentPhytoestrogens are polyphenol compounds of plant origin that exhibit a structural similarity to the mammalian steroid hormone 17β-oestradiol. In Asian nations the staple consumption of phyto-oestrogen-rich foodstuffs correlates with a reduced incidence of breast cancer. Human dietary intervention trials have noted a direct relationship between phyto-oestrogen ingestion and a favourable hormonal profile associated with decreased breast cancer risk. However, these studies failed to ascertain the precise effect of dietary phyto-oestrogens on the proliferation of mammary tissue. Epidemiological and rodent studies crucially suggest that breast cancer chemoprevention by dietary phyto-oestrogen compounds is dependent on ingestion before puberty, when the mammary gland is relatively immature. Phyto-oestrogen supplements are commercially marketed for use by postmenopausal women as natural and safe alternatives to hormone replacement therapy. Of current concern is the effect of phyto-oestrogen compounds on the growth of pre-existing breast tumours. Data are contradictory, with cell culture studies reporting

both the oestrogenic stimulation of oestrogen receptor-positive breast cancer cell lines and the antagonism of tamoxifen activity at physiological phyto-oestrogen concentrations. Conversely, phyto-oestrogen ingestion by rodents is associated with the development of less aggressive breast tumours with reduced metastatic potential. Despite the present ambiguity, current data do suggest a potential benefit from use of phyto-oestrogens in breast cancer chemoprevention and therapy.

Phyto-oestrogens may be classified into a number of principal groups [2,7-9]: the isoflavones (genistein, daidzein, biochanin A), the lignans (enterolactone, enterodiol), the coumestans (coumestrol) and the stilbenes (resveratrol). As illustrated in Fig. 1, all are polyphenols sharing structural similarity with the principal mammalian estrogen 17β-oestradiol. Shared features include the presence of a pair of hydroxyl groups and a phenolic ring, which is required for binding to the oestrogen receptor (ER) subtypes α and β. The position of the hydroxyl groups appears to be important in determining ER binding ability and transcriptional activation, with maximal potency achieved at positions four, 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 are present in flaxseed and most fruit and vegetables, and the predominant dietary source of stilbenes is peanuts, grapes and red wine [7,13]. The coumestans are much less frequently consumed within the human diet, but they are more potent activators of ER signalling pathways than are the isoflavones genistein and daidzein [10,14]. By contrast, the stilbene resveratrol is the least potent activator of ER signalling [11].The isoflavones are present in soy as β-glucosides. Metabolism by the gastrointestinal microflora yields a number of metabolites including equol and O-desmethyl-angolensin. Parental compounds and their metabolites are absorbed into the bloodstream, becoming rapidly detectable in the plasma and urine [15-19]. Plasma isoflavone concentrations are considerably elevated in Asian populations as compared with in western ones. A recent comparison of Japanese and UK females revealed an almost 20-fold increase in plasma genistein levels in the Japanese cohort, and daidzein concentrations were similarly elevated by 18-fold [20]. Plasma isoflavone concentrations may accumulate to approximately 100- to 1000-fold higher than endogenous oestradiol levels following the ingestion of soy-rich meal. However, research suggests a decreased ER binding affinity of isoflavone compounds as compared with the mammalian oestrogens [9,10,21,22]. Competition binding assays revealed a 50-fold lower binding affinity of genistein for cytoplasmic ER sites as compared to 17β-oestradiol [23].The complete metabolic activation of soy isoflavones is proposed to occur locally within target tissue. In support of this hypothesis, the analysis of tissue culture supernatants from genistein and biochanin A treated MCF-7 and T47-D cells revealed the presence of hydroxylated and methylated isoflavone metabolites [24]. Current research suggests a role for the CYP family of cytochrome P450 enzymes in the intratumour metabolism of phyto-oestrogen compounds [25,26]. The CYP1B1 enzyme is expressed in a wide range of human tumour types, including breast [27]; however, expression is absent within normal tissue. CYP1B1 is proposed to catalyze the hydroxylation of resveratrol to yield the related stilbene piceatannol [26]. Piceatannol is a tyrosine kinase inhibitor with antileukaemic properties, which differs in structure from resveratrol by the presence of an additional hydroxyl group. A number of plant flavonoids are also putative substrates for the CYP family of enzymes [25]. Maubach and coworkers [28] recently reported the use of high-performance liquid chromatography to quantify isoflavones in normal breast biopsy tissue following consumption of soy for 5 consecutive days. Equol concentrations were approximately fivefold higher in the breast tissue homogenates than in serum,

providing further evidence for the metabolism of phyto-oestrogen compounds within mammary tissue.

Role of phyto-oestrogens in breast cancerSerum concentrations of 17β-estradiol are approximately 40% lower in Asian women than in their Caucasian counterparts [29]. A low lifetime exposure to oestrogen is associated with a reduced risk for breast cancer. Human dietary intervention studies revealed a direct association between the modest consumption of soy products and a reduction in circulating steroid hormone levels. Daily consumption of 154 mg isoflavones for the duration of a single menstrual cycle correlated with substantially decreased plasma concentrations of 17β-oestradiol and progesterone in a cohort of premenopausal women [18]. A longer-term study conducted by Kumar and coworkers [30] similarly reported a moderate decrease in serum oestradiol and oestrone levels following daily ingestion of 40 mg isoflavones for 3 months. Menstrual cycle length was increased by 3.52 days, and the follicular phase of the cycle was extended by 1.46 days. Increased menstrual 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 women failed to reveal a significant effect of 100 mg/day isoflavone consumption either on menstrual cycle length or on serum levels of various steroid hormones, including oestrone, oestradiol and progesterone [19].As a possible explanation for these contradictory data, a study conducted by Duncan and coworkers [31] revealed differential hormonal effects of soy isoflavones depending on the ability to excrete the daidzein metabolite equol. Daily ingestion of 10 mg soy protein by premenopausal equol excretors resulted in a hormonal profile associated with reduced breast cancer risk, characterized by lowered plasma levels of oestrone, oestrone–sulphate and testosterone. Hormone levels however remained unchanged in the equol nonexcretors after soy ingestion.The reduction in steroid hormone levels by phyto-oestrogens is proposed to occur via the direct regulation of 17β-oestradiol biosynthesis and metabolism. Phytochemicals isolated from vegetable extracts effectively suppress the activity of the aromatase enzymes, which are responsible for conversion of androgens to oestrogens [32]. Isoflavone concentrations of 1–10 μmol/l similarly reduced by 50% the activity of the estradiol biosynthetic enzymes 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase [10]. The daily ingestion of 113–202 mg isoflavones by premenopausal women correlated with a 40% increase in the urinary excretion of 2-hydroxyestrone, a putative anticancer metabolite of 17β-oestradiol [33]. The above studies thus suggest a dual chemoprotectant mechanism of soy, in which the isoflavones suppress steroid hormone biosynthesis while promoting the metabolism of oestradiol 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 date have addressed the effects of long-term phyto-oestrogen exposure in humans. Daily dietary supplementation with 45 mg soy isoflavone for 14 days correlated with increased proliferation of normal breast epithelia in a group of 48 premenopausal women [16]. Expression of the ER target protein progesterone receptor was upregulated, suggesting an oestrogenic effect. An identical trial using a larger cohort of 84 premenopausal women conversely found no significant effect of soy consumption on the proliferation of normal breast tissue [17]. A number of recent epidemiological studies similarly failed to correlate soyfood consumption with reduced breast cancer risk. A Japanese prospective study conducted in a cohort of approximately 35,000 women [34] revealed no significant

association between soy consumption during adulthood and breast cancer incidence. The retrospective analysis of soy food intake in a multiethnic cohort of non-Asian breast cancer patients and control individuals residing in the USA similarly failed to correlate soy intake with breast cancer risk [35].Increasing epidemiological evidence suggests that the chemoprotectant effects of phyto-oestrogens are dependent on a lifelong exposure from childhood. A retrospective study revealed decreased soyfood intake during adolescence in a cohort of 1459 Chinese breast cancer patients, as compared with age-matched control individuals [36]. Daily soy consumption between the ages of 13 and 15 was estimated at 6.45 g in the patient group, 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 precise soyfood quantities consumed many years earlier during adolescence. These observations may nonetheless explain the apparent lack of a growth inhibitory effect of soy isoflavones in the adult dietary intervention studies discussed above. Isoflavones are detectable in breast milk following soy consumption [15], implying that the lower breast cancer incidence in Asian countries may be attributable to phyto-oestrogen exposure from birth via breast-feeding. Rodent studies have accordingly revealed the effective transfer of genistein from maternal milk to offspring [37].

Rodent breast cancer modelsMost available information regarding the effects of phyto-oestrogens on tumour initiation and the growth of pre-existing tumours is derived from rodent studies. A number of similarities do exist between mammary gland development in rodents and humans. In both species the differentiation of breast tissue to form lobules and terminal end-bud structures occurs prepubertally. Further maturation does take place throughout adulthood, giving rise to alveolar buds, which become alveoli during pregnancy and lactation [38].Rodent dietary intervention studies using phyto-oestrogens have reported chemopreventive activities when feeding is initiated before puberty, at a time when the mammary gland is undergoing development [3,38-40]. The consumption of a resveratrol-supplemented diet by adolescent rats served to decrease sensitivity to the chemical carcinogens 7,12-dimethylbenz(a)anthracene (DMBA) and N-methyl-N-nitrosaurea [39,40]. DMBA treatment induced mammary tumours in 45% of the resveratrol-treated rodents, increasing to 75% in the group receiving a control diet. An extended tumour latency period in excess of 3 weeks was observed in the resveratrol treatment groups, with the resultant tumours retaining a more differentiated morphology as compared with control animals [18,39]. Resveratrol consumption was also associated with the reduced mammary expression of a number of proteins that are putatively involved in malignant progression, including cyclo-oxygenase-2, matrix metalloprotease-9 and nuclear factor-κB. Similar findings have been noted in prepubertal rats fed an isoflavone-containing diet before tumour initiation using DMBA. Despite having no effect on mammary tumour incidence, soy isoflavone consumption was associated with an increased tumour latency period. The resultant tumours excised from the soy-fed animals were smaller in size and exhibited a more differentiated phenotype compared with control animals [42].It has been proposed that phyto-oestrogens protect against cancer development in adolescent rodents by promoting maturation of the mammary gland. Analysis of breast tissue from prepubertal rats injected with genistein revealed a decrease in the number of immature terminal end-buds, together with an increase in the more differentiated lobules type II [38]. Genistein treatment of human breast cancer cell lines has similarly been found to induce the expression of a number of maturation markers, including casein, lipid droplets and intercellular adhesion molecule-1 [43].

The effect of soy isoflavones on spontaneous tumour development was recently investigated using neu-ErbB2 over-expressing transgenic mice, which characteristically develop multiple mammary tumours during adulthood [40]. Tumour initiation was temporarily delayed following the consumption of an isoflavone mix, but no chemoprotective effects were observed in mice consuming either genistein or daidzein in isolation. 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 [40]. Antimetastatic activities of the isoflavones were similarly revealed in a study in which mice were fed an isoflavone-supplemented diet before injection with the metastatic 4526 mammary carcinoma cell line [44]. The isoflavone diet was continued following surgical excision of the resultant mammary tumour at a size of 1.0 cm diameter. Both the incidence and size of macroscopically detectable lung metastases were significantly reduced in the soy-fed mice, suggesting a potential clinical application of soy isoflavones in the prevention of metastasis.

Phyto-oestrogens and tamoxifenThe selective ER modulator tamoxifen is used clinically in the adjuvant treatment of oestrogen-dependent breast cancer. The drug is also administered as a prophylactic to individuals who are at high risk for developing the disease [45,46]. Side effects associated with tamoxifen therapy include menopause-like symptoms such as hot flushes, joint pain, sleep disorders and depression, which may be reduced by the use of hormone replacement therapy (HRT) [47,48]. Long-term HRT is associated with an increased risk for mammary carcinogenesis, and its use by breast cancer patients is therefore discouraged. As a natural alternative, patients may self-medicate with soy isoflavone supplements to alleviate the tamoxifen-induced menopausal symptoms [49]. Published literature regarding the ingestion of dietary phyto-oestrogens by breast cancer patients and survivors is, however, controversial [50,51].The consumption of genistein by athymic mice antagonized the ability of tamoxifen to inhibit the proliferation of oestrogen-dependent mammary tumours [52]. Tumour suppression by tamoxifen correlated with decreased expression of the ER-inducible genes presenelin-2 (pS2) and cyclin D1. Tumour growth was significantly enhanced in mice simultaneously exposed to tamoxifen and genistein, whereas levels of pS2 and cyclin D1 expression were increased. Physiological concentrations of genistein were similarly found to reverse the antagonistic effects of 4-hydroxytamoxifen on ER signalling pathways [53], promoting the binding of ER-α to the positively acting steroid receptor coactivator (SRC)-1. A recent tissue culture study conversely reported a synergistic antiproliferative effect of tamoxifen and genistein [54]. The proliferation of a panel of dysplastic and cancerous breast cell lines was inhibited by tamoxifen in a dose-dependent manner, and growth was more potently suppressed by combined treatment with tamoxifen and genistein.

Hormone-dependent mechanisms of phyto-oestrogen actionOestrogen signalling typically involves the diffusion of ligand through the cell cytoplasm and subsequent binding to the nuclear receptor subtypes ER-α and ER-β. Ligand-bound receptors dimerise and associate with oestrogen response element (ERE) and activator protein-1 element located in the promoter region of target genes, thereby activating transcription. The association between receptor dimers and DNA response elements is enhanced by the binding of cofactor proteins, such as amplified in breast cancer-1, thyroid hormone receptor-associated protein, SRC-1, glutamate receptor interacting protein-1 and translation initiation factor 2 [55]. Examples of ERE-induced genes include

PR, c-fos, bcl-2 and cathepsin D, whereas pS2 and cyclin D1 are transcribed via the activator protein-1 response element.In breast carcinoma cell lines containing functional ER subtypes the isoflavones exert a biphasic growth effect, stimulating cellular proliferation at concentrations below 5 μmol/l and 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 the G2/M checkpoint [23,54,56,58-60]. Current research suggests a principal signalling role of ER-β in response to isoflavone exposure [61]. Whereas 17β-oestradiol binds to ER-α and ER-β with equal affinity, the soy isoflavones selectively associate with ER-β [62]. Receptor binding assays revealed an eightfold to 16-fold increase in the affinity of genistein, daidzein and biochanin A for ER-β as compared with ER-α [63]. In ER-negative breast cancer cells transfected to express ER-α alone, genistein was only weakly able to stimulate gene transcription through the ERE. By contrast, genistein effectively bound to ER-β and promoted the association of cofactor proteins, thereby regulating downstream ER-β-mediated gene transcription [62]. The preferential binding affinity of genistein for ER-β similarly resulted in a respective 12,000-fold and 33-fold increase in the recruitment of translation initiation factor-2 and SRC-1a to ER-β as compared with ER-α [22]. An enhanced transcriptional activity in response to genistein was, however, noted in cells transfected to express both receptor subtypes as compared with cells solely expressing ER-β [64]. Although ER-α is itself unable to mediate isoflavone signal transduction, it was postulated that the presence of the receptor subtype may enhance ER-β signalling via the formation of ER-α/β heterodimers. These observations imply that the precise tissue-specific effects of the soy isoflavones are dependent on the expression levels and ratios of ER-α and ERβ. The various cofactors are similarly expressed in a tissue-specific manner, therefore further influencing the cellular 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 [39]. Resveratrol exhibits a relatively weak ER-binding affinity as compared with oestradiol [65]; however, unlike the soy 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 for both receptor subtypes, stimulating ERE transcriptional activity through either ER-α or ER-β alone [21]. 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 of the ER target genes pS2 and PR [65]. Recent studies proposed that the cell cycle inhibitor protein p21WAF1 is a potential downstream target of resveratrol-induced ER signalling pathways [66]. The treatment of ER-α-expressing breast cancer cells with resveratrol resulted in a 23-fold increase in p21WAF1 gene expression, as determined by cDNA microarray analysis. The resveratrol-mediated induction of p21WAF1 was blocked by treatment with the pure anti-oestrogen ICI 182,780, confirming p21WAF1 gene regulation as an ER-mediated event.

Hormone-independent mechanismsAt concentrations in excess of 25 μmol/l, the soy isoflavones are capable of inducing apoptosis in human breast cancer cells [23,67-69]. ER-negative cell lines retain sensitivity to the apoptotic effects of soy isoflavones, thereby confirming that apoptosis occurs in a hormone-independent manner. Apoptosis was effectively induced in the ER-α-negative MDA-MB-231 breast cancer cell line by genistein and daidzein concentrations of 50–100 μmol/l [59,60]. In MCF-7 cell cultures the induction of cell

death by treatment with genistein coincided with the increased expression of the proapoptotic proteins Bax and p53 [67]. Breast cancer cell lines expressing mutant p53 also undergo apoptosis in response to phyto-oestrogen treatment, thereby implying apoptosis induction by both p53-dependent and p53-independent mechanisms [67,70]. The polyphenol epigallocatechin (EGC) is principally found in green tea and is proposed to have anticancer properties. Treatment of p53-mutant breast cancer cells with 100 μmol/l EGC induced a 40% increase in apoptosis, correlating with increased Bax expression and reduced levels of the antiapoptotic protein Bcl-2 [70]. EGC-induced apoptosis was abolished following treatment with anti-Fas neutralizing antibodies or caspase inhibitors, suggesting the involvement of Fas signalling pathways. Although phyto-oestrogen compounds are effective inducers of apoptosis in cell culture models, it is unlikely that plasma isoflavone concentrations would accumulate to the required levels for 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 moderate consumption of soy products [15,52], although it is possible that higher levels may be present in target tissues. In a recently reported study, equol concentrations within breast tissue were found to exceed serum levels; however, the reverse was true for genistein and daidzein [28]. A recent in vitro study [68] revealed the flavone baicalein to be a more potent inducer of apoptosis than genistein. Baicalein is isolated from the plant Scutellariae radix and is a common ingredient in herbal tea preparations. A concentration of 10 μmol/l baicalein induced significant cell death in MCF-7 cell cultures, suggesting baicalein as a potentially useful pharmacological agent in breast cancer therapy.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 in the absence of functional ER occurs via the inhibition of tyrosine kinase activity. The protein 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 and epidermal growth factor (EGF). In ER-negative breast cancer cultures 5 μmol/l genistein negated the stimulatory effects of TGF-α, IGF-I and IGF-II, implying the inhibition of tyrosine kinase activity [23]. The human EGF receptor-2 oncogene (Her-2) is constitutively overexpressed in approximately 30% of breast cancers and is associated with a poor patient prognosis [71]. Research using breast cancer cell lines suggests that dietary phyto-oestrogens are capable of repressing EGF receptor activity. The inhibition of tyrosine kinase activity by 5 μmol/l genistein in MCF-7 cells correlated with the repression of EGF receptor tyrosine phosphorylation in response to EGF stimulation [23]. Similar findings were reported in a recent study investigating the chemoprotective effects of the green tea polyphenol epigallocatechin-3 gallate (EGCG). The treatment of Her-2/neu over-expressing mouse mammary cells with 20–80 μg/ml EGCG inhibited proliferation in a dose-dependent manner, correlating with a reduction in Her-2/neu signalling activity [72]. The basal tyrosine phosphorylation of Her-2/neu was decreased by approximately 96% following treatment with 80 μg/ml EGCG. Downstream activities of the signalling proteins phosphoinositide 3-kinase, Akt and nuclear factor-κB were similarly repressed, suggesting a potential clinical application of EGCG in breast cancer therapy.The soy isoflavones have additionally been proposed to regulate the proliferation of breast epithelia via an alternative mechanism involving the modulation of TGF-β synthesis [73]. In normal mammary tissue TGF-β maintains proliferative homeostasis by inhibiting the growth of epithelial cells [74,75]. The incubation of human mammary epithelial cells with 5 μmol/l genistein induced a fivefold increase in the level of TGF-β secretion [76]. The further analysis of media conditioned with human mammary epithelial

cells revealed the presence of the active as opposed to latent form of TGF-β, thus implying 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.

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Effects of Pomegranate Chemical Constituents/Intestinal Microbial Metabolites on CYP1B1 in 22Rv1 Prostate Cancer Cells

Sashi G. Kasimsetty†, Dobroslawa Bialonska† , Muntha K. Reddy†, Cammi Thornton‡, Kristine L. Willett‡§ and Daneel Ferreira*†§ † Department of Pharmacognosy‡ Department of Pharmacology§ National Center for Natural Product Research, Research Institute for Pharmaceutical SciencesSchool of Pharmacy, The University of Mississippi, University, Mississippi 38677

Institute of Environmental Sciences, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, PolandJ. Agric. Food Chem., 2009, 57 (22), pp 10636–10644DOI: 10.1021/jf902716rPublication Date (Web): October 26, 2009Copyright © 2009 American Chemical Society*To whom correspondence should be addressed. Tel: 662-915-7026. Fax: 662-915-6975. E-mail: dferreir@olemiss.edu.

AbstractThe cytochrome P450 enzyme, CYP1B1, is an established target in prostate cancer chemoprevention. Compounds inhibiting CYP1B1 activity are contemplated to exert beneficial effects at three stages of prostate cancer development, that is, initiation, progression, and development of drug resistance. Pomegranate ellagitannins/microbial metabolites were examined for their CYP1B1 inhibitory activity in a recombinant CYP1B1-mediated ethoxyresorufin-O- deethylase (EROD) assay. Urolithin A, a microbial metabolite, was the most potent uncompetitive inhibitor of CYP1B1-mediated EROD activity, exhibiting 2-fold selectivity over CYP1A1, while urolithin B was a noncompetitive inhibitor with 3-fold selectivity. The punicalins and punicalagins exhibited potent CYP1A1 inhibition with 5−10-fold selectivity over CYP1B1. Urolithins, punicalins, and punicalagins were tested for their 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1 inhibitory activity in the 22Rv1 prostate cancer cell line. Urolithins A and B showed a decrease in their CYP1-mediated EROD inhibitory IC50 values upon increasing their treatment 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 upon 24 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 the proximity of their CYP1 inhibitory IC50 values. Urolithins A and B were studied for their cellular uptake and inhibition of TCDD-induced CYP1B1 expression. Cellular uptake experiments demonstrated a 5-fold increase in urolithin uptake by 22Rv1 cells. Western blots of the CYP1B1 protein indicated that the urolithins interfered with the expression of CYP1B1 protein. Thus, urolithins were found to display a dual mode mechanism by decreasing CYP1B1 activity and expression.

o IntroductionDietary intervention to prevent carcinogenesis has been well-established in epidemiological studies. The consumption of fruits and vegetables is considered to be a safeguard against various forms of cancers. Polyphenols are the major constituents of fruit and vegetable diets and are believed to elicit a number of biological properties due to their antioxidant and anticarcinogenic activities (1). A number of flavonoids such as quercetin, chrysin, apigenin, and luteolin have been investigated for their cytochrome P450 1 (CYP1) enzyme inhibition activities, indicating that flavonoid-related anticarcinogenesis is mediated in part by CYP1 inhibition (2). Ellagic acid, the hydrolysis product of ellagitannins, exhibits anticarcinogenic effects by inhibition of CYP1A1-dependent activation of procarcinogens. A number of ellagic acid analogues showed similar inhibitory activities against CYP1-mediated benzo[a]pyrene activation (3). Ellagic acid is a major polyphenol in pomegranate juice. Pomegranate juice polyphenols showed a strong inhibitory activity against estrogen-dependent MCF-7 cell lines. In in vivo studies, pomegranate juice exhibited 47% inhibition of cancerous lesion

formation induced by the carcinogen, 7,12-dimethylbenz[a]anthracene (4), indicating its potential use as an adjuvant therapeutic in human breast cancer treatment. Pomegranate juice components are also believed to exert cancer chemopreventive activity against skin and colon cancer. Importantly, the consumption of pomegranate juice decreased the clinical reemergence of prostate cancer-specific antigen in prostate cancer patients after primary therapy (5, 6). Pomegranate ellagitannins are transformed by human colonic flora into bioavailable organic molecules called urolithins (7). The pomegranate microbial metabolites preferentially accumulate in prostate, colon, and intestinal tissues relative to other organs in a mouse model and exert their beneficial effects to a greater extent in those tissues (5). Pomegranate constituents inhibited prostate cancer cell growth by affecting their proliferation, gene expression, invasion, and apoptosis but had a less profound effect on normal prostate epithelial cells. However, the concentrations at which they exhibited antiproliferation activity against human prostate cancer cell lines were higher than the physiologically available concentrations (18.6 μM when 1 L of juice is consumed for 5 days) (5, 8). These results suggest that there might be some other pathways through which pomegranate constituents exert cancer chemoprevention. To explore the other possible prostate cancer chemopreventive pathways, we studied the effects of pomegranate juice ellagitannins and their microbial metabolites on CYP1B1-induced carcinogenesis. Previously, it was shown that pomegranate juice consumption resulted in lowered total hepatic CYP content and also decreased CYP1A2 and CYP3A. Therefore, the anticarcinogenic effects of pomegranate juice could be partly attributed to their ability to inhibit CYP activity/expression (9). Our work represents the first report concerning the effects of pomegranate ellagitannins on CYP1B1 inhibition as a means for cancer chemoprevention.CYPs are responsible for the bioactivation of endogenous compounds, drugs, dietary chemicals, and xenobiotics. The CYP1 isoforms, CYP1A1, CYP1A2, and CYP1B1, are of major importance because they activate a number of polycyclic aromatic hydrocarbons (PAHs) to genotoxic compounds leading to tumorogenesis (10). CYP1B1 is abundantly expressed extrahepatically in steroidogenic (ovaries, testes, and adrenal glands) and steroid-responsive (breast, uterus, and prostate) tissues. The CYP1B1 enzyme plays an important role not only in the initiation and promotion of cancer but also in the development of drug resistance. The CYP1B1 enzyme alone accounts for activation of 15 PAHs, six heterocyclic amines, and two nitropolycyclic hydrocarbons into mutagenic and carcinogenic compounds, which cause DNA damage and initiate cancer formation. CYP1B1 is also involved in the metabolism of endogenous compounds such as 17β-estradiol to an active metabolite, 4-hydroxyestradiol (4-OH-E2), which has been implicated in breast cancer initiation. In comparison, CYP1A1 converts 17β-estradiol into 2-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. The increased expression of CYP1B1 could generate an excessive number of genotoxic metabolites, which may attack the DNA of normal cells, thus allowing for cancer

promotion. Although the augmented CYP1B1 expression does not cause tumor invasion or metastasis, it leads to deactivation of anticancer drugs such as flutamide in prostate cancer treatment and docetaxel in breast cancer treatment (12). Considering the crucial role played by CYP1B1 in cancer inititation, promotion, and resistance development, it is an attractive molecular target for cancer chemoprevention. The expression of the CYP1 family is regulated by the aryl hydrocarbon receptor (AhR). The ligands of AhR range from environmental contaminants to plant- or diet-derived constituents such as curcumin and carotenoids (13). Because CYP1B1 is a therapeutic target in prostate cancer, we hypothesized that pomegranate constituents/metabolites might exert prostate cancer chemoprevention through CYP1B1 inhibition as one of the plausible mechanisms. Our results indicate a previously unexplored pathway through which pomegranate juice constituents may contribute to prostate cancer chemoprevention.Materials and MethodsIsolation and Identification of Pomegranate Juice Ellagitannins The extraction of ellagitannins was performed by a procedure described previously, by use of a step gradient consisting of an increasing amount of methanol in water. The commercial POMx (100 mL) was diluted to 500 mL with Millipore purified water and successively 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-16 column 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 pressure afforded a tannin fraction (XAD-n-BuOH) (1.3 g). This was further purified on Sephadex LH-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. A follow-up of fractionation and further purification of all of the fractions on Sephadex LH-20 column chromatography using H2O:MeOH gradient, MeOH, and MeOH:Me2CO gradient system afforded the compounds gallic acid, hexahydroxydiphenic acid (HHDP), gallagic acid, punicalins, and punicalagins. The latter compounds (punicalins and punicalagins) exist in solution as the α- and β-anomers as well as acyclic hydroxyaldehyde analogues (14) (Figure 1). The compounds were identified using LC-MS retention time, UV absorption pattern, molecular mass, and 1H NMR spectra. The LC-MS system consisted of Waters Micromass ZMTQ mass spectrophotometer, Waters 2695 Separation Module, and Waters 996 Photodiode Array Detector. Mass spectra were recorded in negative mode, using a capillary voltage of 4000/3500 V and a gas temperature of 300 °C. The column used was a 150 mm × 3.0 mm i.d., 5 μm, Luna C18 100 Å (Phenomenex, Torrance, CA). The analysis was performed using a 2.5% acetic acid in water (solvent A) and 2.5% acetic acid in methanol (solvent B), starting from 100% A for 5 min, 0−60% B for 15 min, and 60−100% B for the next 15 min. The flow rate was 0.3 mL/min with the

pressure set at 900−1500 mmHg. Figure 1. Structures of ellagitannins and urolithins.Synthesis of Urolithins Chemicals Resorcinol, 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 Compounds The high-performance liquid chromatography (HPLC) system consisted of a Waters Delta 600, a Waters 600 controller, a Waters 996 Photodiode Array Detector, and a 3.0 mm × 150 mm column (Phenomenex, ODS 5 μm C18 100 Å). Analyses were performed in the gradient system A, 2.5% aqueous acetic acid, and B, 2.5% acetic acid in methanol, starting from 100% A for 5 min, 0−60% B for 15 min, and 60−100% B for 15 min. The flow rate was 1 mL/min, and the pressure was 600−800 mmHg. The elution of metabolites 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 the condensation of resorcinol or pyrogallol with an appropriately substituted benzoic acid by the modified protocols described by Ito et al. (15). The structures of urolithins were confirmed by their molecular mass and comparison of observed and reported 1H NMR data 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 microbial metabolites on recombinant CYP1A1 and CYP1B1, a 96-well plate EROD assay was used (16). Concentrations of the test compounds ranged from 0.5 to 30 μM. Inhibition kinetics of CYP1B1-mediated EROD activity was determined similarly. Concentrations of 0.5 and 1 μM were used for urolithins A and B in triplicate.22Rv1 Prostate Cell EROD Assay To 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-well plate format (32). The test compounds were studied for their effects on cell-based 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1-mediated EROD activity. The cells were treated with TCDD for 24 h to induce CYP1 expression. The cells were also cotreated with compounds,

for either 30 min or 24 h, at concentrations ranging from 6.75 to 50 μM. The cells were treated with DMSO, ellagitannins, and urolithins alone to evaluate their ability to induce CYP1 expression in the absence of TCDD.Microsome Preparation Cells 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 appropriate amount of the lysis buffer [10 mM Tris, pH 7.5, 10 mM KCl, and 0.5 mM ethylenediaminetetraacetic acid (EDTA)] was added, and the cells were transferred to the glass tube of a Teflon homogenizer and kept on ice for 10 min. This was followed by the addition of an appropriate amount of homogenization buffer [0.25 M KH2PO4, 0.15 M KCl, 10 mM EDTA, and 0.25 mM phenylmethanesulfonyl fluoride (PMSF)]. The cells were then broken by 12 manual strokes on a tight-fitting Teflon homogenizer. After centrifugation at 15000g/20 min/4 °C, the supernatant was again centrifuged at 105000g/90 min/4 °C. Microsomal pellets were resuspended in microsomal dilution buffer [0.1 M KH2PO4, 20% glycerol, 10 mM EDTA, 0.25 mM PMSF, and 0.1 mM dithiothreitol (DTT)] and stored in aliquots at −80 °C.Western Blotting Microsomal protein (5−10 μg) samples were prepared by heating at 95 °C for 5 min in the sample buffer comprising 0.5 μM Tris HCl (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate (SDS), 5% mercaptoethanol, and 0.001% bromophenol blue. The samples were resolved by precast criterion SDS-polyacrylamide gel electrophoresis (PAGE) gel (10%) at 200 V for 45−50 min (Bio-Rad Laboratories, Hercules, CA). The proteins were then transferred to a PVDF membrane (Bio-Rad Laboratories) at 100 V for 90 min. Following transfer, the membranes were blocked in blocking buffer for 1 h followed by incubation in CYP1B1 primary antibody (1:1000, antirat CYP1B1 polyclonal antibody, kindly donated by Dr. Thomas R. Sutter). After they were washed, the membranes were incubated in buffer containing the horseradish peroxidase-conjugated secondary antibody (1:30000, antigoat IgG peroxidase conjugate, Sigma Chemicals) for 2 h. The membrane was washed and developed using LumiGLO Reserve chemiluminescent substrate (KPL, Inc., Gaithersburg, MD). The signals were detected using a CCD camera (VersaDoc Imaging System, Bio-Rad Laboratories). Human CYP1B1 supersomes were used to make standard curves of known protein concentrations (0.25−2 pmol). Standard curves were used to quantitate the CYP1 protein amounts in the samples using the Quantity One quantitation software (Bio-Rad Laboratories). Statistical differences between the control and the treated samples were determined using one-way analysis of variance (ANOVA) followed by Newman−Keuls posthoc (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 of 10000 cells/well and allowed to settle for 30 min at 37 °C. The compounds, diluted appropriately in RPMI-1640 medium, were added to the cells and again incubated for 48 h. The number of viable cells was determined using the neutral red assay procedure (17).Induction of Phase II Conjugating Enzymes Assay The assays were performed according to standard procedures described by Kirlin et al. (18)Cellular Uptake of Urolithins A and B 22Rv1 cells were incubated with 20 μM urolithins A and B in the RPMI-1640 media for 0.5, 6, 12, and 24 h time periods. After incubation, the cells were washed and

harvested by trypsinization. The cells were extracted with acidified MeOH. The samples were analyzed by reverse phase HPLC at 288 nm detection. The experiment was done in triplicate. A standard curve of urolithins A and B was prepared from which the amount of urolithin uptake was determined. The amount of urolithin uptake was adjusted to the amount of protein.Results and DiscussionPomegranate fruit consists of two major classes of polyphenols, flavonoids and ellagitannins. The flavonoids include quercetin, kaempferol, and myricetin (19). These flavonoids have been reported to exhibit CYP1 inhibitory activities (16). Pomegranate juice consumption has been found to decrease the expression/activity of total hepatic CYP content (9). Pomegranate juice obtained by hydrostatic pressing of whole fruit predominantly contains ellagitannins. Ellagitannins, with the exception of ellagic acid, have not been previously studied for their CYP1 inhibitory activities. Ellagic acid has been 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 AhR antagonism or their effects on the downstream products of AhR signal transduction pathways (21). However, ellagic acid decreased CYP1A1-dependent BaP activity independent of the AhR-responsive element (3). The proposed mechanism of inhibition of CYP1A1-dependent BaP activation involved scavenging of the carcinogen by ellagic acid through chemical binding (22). Therefore, our objective was to study the effects of a selection 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 a recombinant CYP1A1- and CYP1B1-dependent EROD assay. The IC50 values for CYP1B1 inhibition ranged from 1.15 ± 0.65 μM for urolithin A to 137 ± 11.08 μM for urolithin D. CYP1A1 IC50 values ranged from 1.5 ± 0.32 μM for punicalins to 2907 ± 168 μM for urolithin D (Table 1, section A). Urolithins exhibited higher selectivity toward CYP1B1 EROD inhibition as compared to CYP1A1, although the selectivity was not significant. 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 CYP1B1 inhibition. Punicalins and punicalgins were 10- and 5-fold more selective toward CYP1A1 inhibition.Table 1. Results of Recombinant CYP1-Mediated EROD Assay and Kinetic Parametersa

Section A  CYP1B1 CYP1A1    IC50 ± SEM

(μM)Ki ± SEM

(μM)IC50 ± SEM

(μM)Ki ± SEM

(μM)Ki(CYP1A1:CYP1B1)

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 0.10

Section A  CYP1B1 CYP1A1    IC50 ± SEM

(μM)Ki ± SEM

(μM)IC50 ± SEM

(μM)Ki ± SEM

(μM)Ki(CYP1A1:CYP1B1)

PG 2.6±0.79 0.58±0.02 2.67±0.48 0.109 ±0.20 0.19Section B

  urolithin A urolithin B  DMSO 0.5 μM 1 μM DMSO 0.5 μM 1 μM

Vmax 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 using recombinant human CYP1B1 and CYP1A1 enzymes. For section B, kinetic parameters, Vmax (pmol/mg/min), and Km (μM) ± standard errors (n = 3) for the inhibition of recombinant human CYP1B1 by urolithin A and B (0.5 and 1 μM), determined by nonlinear regression curve fit using the Michaelis−Menten equation ([S] vs V) plot using GraphPad Prism.Urolithins A and B are the major microbial metabolites of pomegranate chemical constituents detected in human systemic circulation. These metabolites exhibited lower IC50 values in recombinant CYP1 inhibition as compared to all other tested compounds. Therefore, a study was conducted to investigate the mechanism of action of CYP1B1 inhibition by urolithins A and B. The concentrations of inhibitors used were in the vicinity of their calculated IC50 values, that is, 0.5 and 1 μM. EROD activities were determined with substrate concentrations ranging from 0.1 to 2.0 μM. Kinetic parameters, Vmax and Km, were calculated using the Michaelis−Menten equation ([S] vs V curve). Double reciprocal plots were plotted using 1/[S] and 1/V (Figure 2) from which Ki values were calculated (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 using Cheng−Prusoff equations (16). The calculated Vmax and Km for urolithin A changed significantly with an increasing concentration of inhibitor, suggesting an uncompetitive type of inhibition. However, the Vmax and Km of urolithin B did not differ significantly upon increase of inhibitor concentration, suggesting a noncompetitive type

of inhibition. Figure 2. Double reciprocal plots for the inhibition of in vitro EROD activity of CYP1B1 by urolithins 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 inhibitor or DMSO prior to initiation of reaction. Each experiment was done three times in duplicate.In our study, urolithins A and B, structural analogues of ellagic acid, exhibited a significant inhibition of CYP1-dependent EROD activity. The results suggested that a hydroxy 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 in accordance with a previous study by Barch et al. (7). However, in our study, additional hydroxy 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 the hydroxy groups to give 8-O-methylurolithin A and 8,9-di-O-methylurolithin C decreased the activity, suggesting that the phenolic hydroxy groups are important for CYP1 inhibitory activity. To probe the importance of the lactone group for CYP1-dependent EROD activity inhibition, HHDP and gallic acid were tested for their CYP1-dependent EROD activity. The results showed that hydrolysis of the lactone functionality did not result in CYP1 inhibition (data not

shown). The punicalins and punicalagins were potent inhibitors of CYP1-dependent EROD activity with Ki values (Table 1, section A) comparable to the dietary flavonoids. The dietary 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-mediated EROD activities. The apparent Ki values of inhibition of human recombinant CYP1B1 and CYP1A1 were 14 ± 3 and 52 ± 2 nM for kaempferol, whereas they were 23 ± 2 and 77 ± 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 ranging from 2.0 to 9.3 μM with mixed type inhibition (24). In another study, quercetin inhibited recombinant 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 depends upon 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 plasma concentrations ranged from 0.01, 0.05, and 0.1 μM upon consumption of onion, tea, and endive, respectively (27, 28). Bioavailable concentrations of quercetin and kaempferol from 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 Ki values ranging from 60 nM to 0.2 μM. These concentrations are bioavailable (127 ± 81 nM) upon consumption of 2 g of blanched parsley (14). Bioavailability of rutin from tomato puree as detected in plasma was calculated to be 0.1 μM (25), which was around 60-fold lower than the reported Ki of CYP1B1 inhibition. The bioavailability of many other flavonoids is still unclear. However, dietary flavonoids could inhibit CYP1-mediated bioactivation of environmental and dietary carcinogens into genotoxic compounds and prevent cancer initiation in alimentary canal-related cancers because they come into direct contact with the digestive epithelium of the digestive system (29). However, the prostate cancer chemopreventive effects of flavonoids (30) depending on the bioavailability are still debated. It is therefore important to choose an appropriate dietary

supplement that can release adequate amounts of cancer chemopreventive compounds into plasma, whenever it is consumed, with an intended pharmacological activity.Pomegranate juice ellagitannins have been extensively studied for their bioavailability and their biological effects. In one study, it was established that the punicalagins hydrolyze into ellagic acid and other smaller polyphenols that are responsible for the bioactivity of ellagitannins. In a study performed on human subjects, consumption of 180 mL of pomegranate juice (equivalent to 25 mg of ellagic acid and 319 mg of punicalagins) resulted in detection of ellagic acid in plasma with a maximum concentration of 31.9 ng/mL (0.1 μM) (31). Previous studies also indicated that the bioavailability of ellagic acid from pomegranate juice, pomegranate liquid concentrate extract, 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 and CYP1B1 with Ki values 0.618 and 0.58 μM, respectively. Punicalins and punicalagins exhibited approximately a 10- and 5-fold selectivity for CYP1A1 over CYP1B1. Thus, consumption of pomegranate juice could be beneficial in decreasing CYP1-mediated oral, esophageal, and colon cancers. However, these ellagitannins cannot exhibit a systemic CYP1 inhibition activity because they are metabolized in the colon by microflora into smaller organic molecules called urolithins.Bioavailability studies indicate that maximum plasma concentrations of urolithins A and B reach concentrations of 4−18 μM in human subjects (8, 32). Urolithins A and B inhibited human recombinant CYP1A1 and CYP1B1 with Ki values in their bioavailable concentration range. Our particular interest was to explore the beneficial effects of urolithins in prostate cancer. Studies indicate that 15% of prostate cancer patients, who have undergone a radical prostectomy, had a biochemical recurrence of prostate-specific antigen (PSA). Among them, 34% of patients developed distant metastases within 15 years (33). It was evident that consumption of pomegranate juice delayed the doubling time of the PSA by 39 months after primary therapy (7). The effects were ascribed to the antiproliferative, apoptotic, and antioxidant effects of pomegranate constituents, 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 that polymorphisms in CYP1B1 and PSA genes increased the risk and aggressiveness of prostate cancer (35). Any dietary constituent with CYP1B1 inhibitory activity could potentially lead to prostate cancer chemoprevention. There were no previous reports about the ability of pomegranate constituents/metabolites to inhibit CYP1B1-dependent carcinogenesis.Therefore, we studied the capability of pomegranate constituents to inhibit CYP1B1-induced metabolic activation in a prostate cancer cell line, 22Rv1. The cells were treated with TCDD for 24 h to induce CYP1B1 protein expression. Then, the cells were treated with punicalins, punicalagins, or urolithins for 30 min. Following 30 min of incubation, urolithins A and B significantly decreased TCDD-induced EROD activity at the highest concentration used (Figure 3A). The IC50 values calculated for cell-based CYP1 inhibition 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 vitro recombinant CYP1-mediated EROD inhibitory IC50. Punicalins and punicalagins did not inhibit cell-based CYP1-mediated EROD activity at the highest concentration used (50 μM). To ascertain whether a decrease in IC50 occurred upon longer incubation, the cells were 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 inhibited CYP1-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 EROD activity 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 did not 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 A and B (Table 2). The compounds alone did not induce EROD activity after 24 h of incubation. Because the prostate cells were treated with the test compounds for 24 h, it was imperative to investigate if the compounds exhibited any cytotoxicity. A neutral red dye uptake assay was used to measure the cytotoxicity of the test compounds. The data were probit transformed followed by linear regression and IC50 calculation. The cytotoxic IC50 values ranged from 20.6 ± 4.58 μM for 8,9-di-O-methylurolithin C to 108 ± 3.994 μM for urolithin B (Table 3). The results indicate that the cytotoxicities of urolithin A and B had no contribution toward decreased CYP1-mediated EROD activity. The results also indicate that urolithin C, 8,9-di-O-methylurolithin C, and 8-O-methylurolithin A were false positives in the prostate cell EROD assay. The activity was not because of CYP1 inhibition but due to cytotoxicity. This conclusion was verified based on four facts: (1) These compounds inhibited recombinant CYP1-mediated EROD activity at higher concentrations as compared to urolithins A and B; (2) these compounds did not inhibit prostate cell EROD activity upon 30 min of incubation; (3) they inhibited prostate cell EROD activity upon 24 h of treatment, with IC50 values lower than those exhibited in the recombinant CYP1-mediated EROD assay; and (4) they exhibited cytotoxicity in the vicinity 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 were exposed 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 a Student−Newman−Keuls posthoc test was used to determine treatment effects. In the same treatment groups, bars with different letters are statistically different.Table 2. IC50 Values for Pomegranate Chemical

Constituents/Microbial Metabolite Mediated Inhibition of EROD Activity in TCDD-Induced 22Rv1 Prostate Cancer Cells

  IC50 ± SEM (μM)chemical constituent 30 min 24 h

urolithin 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 Cellschemical 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 for urolithins A and B. These experiments were performed to determine if the decrease in IC50 values for CYP1-mediated EROD inhibition in 22Rv1 cells was related to increased uptake over a 24 h time period. The experiments indicated that there was a 4.5-fold increase 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 between 6 and 12 h, beyond which the uptake was constant. The results suggested that increased availability of urolithins (from 30 min to 24 h) could have contributed to the decrease in IC50 of CYP1 inhibition (Table 4). The results also indicated that urolithins were 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 protein expression levels, Western blots were performed (Figure 4). Cell treatments were DMSO, TCDD (50 nM), UA (50 μM), UB (50 μM), TCDD + UA (50 μM), TCDD + UB (50 μM), 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) significantly decreased 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% as compared 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 (25 μM) treatment decreased protein (1.9 ± 0.5 pmol/mg) production by 29% as compared to TCDD-induced levels. Western blot analyses suggest that none of the urolithins induce

CYP1B1 basal levels significantly as compared to DMSO. However, cotreatments with urolithins A and B 50 μM and TCDD decreased CYP1B1 protein expression levels as

compared to TCDD alone (Figure 4). Figure 4. (A) Western blot showing the effect of urolithins A and B and their cotreatment with TCDD on CYP1B1 protein expression. Microsomes were prepared after treating cells for 24 h with different treatments (DMSO, 50 nM TCDD, UA 50 μM, UB 50 μM, TCDD + UA 50 μM, TCDD + UA 25 μM, TCDD + UB 50 μM, and TCDD + UB 25 μM), along with standards, 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 in 22Rv1 prostate cancer cells. Each experiment was repeated three times. Global one-way ANOVA with a Student−Newman−Keuls posthoc test was used to determine treatment effects. Bars with different letters are statistically different.Urolithins A and B inhibited CYP1 EROD activity by inhibiting both the protein expression 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 not clear, they are believed to exert an antiproliferative activity on prostate cancer cells, in accordance with previous studies (32, 34). An ideal anticarcinogenic agent would inhibit phase I enzymes, involved in carcinogen activation while inducing the phase II enzymes, responsible for the deactivation of carcinogens by assisting their excretion via increased water solubility. The pomegranate ellagitannins and urolithins were tested for their capacity to induce glutathione S-transferase and quinone O-reductase enzymes. The basal levels of quinone O-reductase and glutathione S-transferase enzymes in 22Rv1 cells 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 of quinone O-reductase or glutathione S-transferase as compared to normal proliferating cells.In conclusion, our study has asserted a previously unknown mechanism of action of pomegranate juice constituents, which could potentially contribute to prostate cancer chemoprevention. In a study to evaluate the safety of consuming large quantities of pomegranate ellagitannins, it was shown that the consumption of pomegranate ellagitannin-enriched extracts (providing 435 and 870 mg of gallic acid equivalents) was safe in humans (36). The study also demonstrated the ability of pomegranate ellagitannins to release antioxidant principles into plasma. In our study, we proved that systemically available metabolites of pomegranate juice are effective inhibitors of CYP1B1 enzyme activity/expression and could lower the incidence of prostate cancer initiation and sustenance. However, some of the cytotoxic metabolites may exhibit their prostate cancer inhibition activity by exerting an antiproliferative effect. These metabolites may also decrease the incidence of drug resistance mediated by CYP1B1-related drug inactivation, if used as an adjuvant during chemotherapy. It is also well-known that prostate cancer typically possesses long latency periods and develops in older men; therefore, cancer chemoprevention by dietary supplement-based intervention is a desirable form of chemotherapy. Pomegranate juice consumption, thus, may be of considerable advantage in prostate cancer chemoprevention, not only in patients with a

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solution. The bugs lit up like blue fireflies. "They became very colorful and easy to see under a microscope," said Liu.The technique could be adapted to identify a wide array of pathogens by mixing and matching from a library of different sugars and polymers that fluoresce different colors under different frequencies of light. If blue means E. coli, fuchsia could one day mean influenza.With funding from a Small Business Innovation Research grant from the National Institutes of Health, Liu is adapting the technique to combat breast cancer. Instead of mannose, he plans to link the fluorescent polymers to a peptide that homes in on cancer cells.Once introduced to the vascular system, the polymers would travel through the body and stick to tumor cells. Then, illuminated by a type of infrared light that shines through human tissue, the polymers would glow, providing a beacon to pinpoint the location of the malignant cells.The technique would allow surgeons to easily identify and remove malignant cells while minimizing damage to healthy tissue.USEFUL IN TREATMENT:New microbial secondary metabolites under preclinical development for cancer treatment.Umezawa H.Limitless numbers of various genetic structures have been formed in chromosomes and plasmids and numerous bioactive compounds are produced by microorganisms. Therefore, it may be said that compounds useful in treatment of cancer will be found more and more in microbial secondary metabolites and more effective antitumor antibiotics and their derivatives, or more effective products producing immune resistance to cancer, will be discovered. In these studies, as discussed in this paper, the most urgent problem is to establish a rational screening principle or system to select compounds worth clinical examination. This is particularly important in the analog area. Bleomycin is an analog of phleomycin chosen because of lower renal toxicity. It has become an antitumor agent of significant value. Macromycin is a new structure which has been found to bind with animal cells and inhibit growth. Neothramycin is a new benzodiazepine antibiotic which has lower toxicity than other structures studied in this class and is active against L1210, Yoshida sarcoma, and Sarcoma 180. Aclacinomycin A is an analog of adriamycin chosen for clinical study based on its low cardiac toxicity and high distribution in mouse lung and spleen. Coriolins are another new structural class. Diketocoriolin B has activity in L1210 leukemia and has been shown to inhibit Na-K-ATPase. Bestatin is a compound which inhibits aminopeptidase B and leucine aminopeptidase has been shown to increase delayed hypersensitivity. Bestatin also increases the effects of other antitumor agents such as adriamycin, and bleomycin.PMID: 705007 [PubMed - indexed for MEDLINE]E. coli Bacteria Could Become a Cancer TreatmentToxin in E. coli Shows Promise as Cancer TreatmentArticle date: 1999/12/07Most people know E. coli as a bacteria that can cause deadly food poisoning. In the future, though, it may save lives as a treatment for cancer.In research published in the journal Blood (Vol. 94, No. 8), Canadian reseachers have shown that a toxin produced by E. coli holds promise as anBreast cancer cells were used in the E. coli experiments.agent for removing multiple myeloma cells from stem cells used in autologous transplantation. Multiple myeloma, a cancer of the immune system cells that are responsible for producing antibodies, is very resistant to standard doses of chemotherapy.In research published in the journal Blood (Vol. 94, No. 8), Canadian reseachers have shown that a toxin produced by E. coli holds promise as an agent for removing multiple myeloma cells from stem cells used in autologous transplantation. Multiple myeloma, a cancer of the immune system cells that are responsible for producing antibodies, is very resistant to standard doses of chemotherapy.In autologous stem cell transplantation, stem cells are removed from a patient?s blood before the patient is given larger than usual doses of chemotherapy, then the stem cells are returned to the patient after chemotherapy. Generally, patients are not able to tolerate such high doses of cancer drugs because too much

chemotherapy is toxic to bone marrow. In autologous transplantation, large doses can be given because the stem cells help the bone marrow to recover.Before replacement, the stem cell grafts have to be purged to destroy any stray cancer cells. One of the risks of the procedure is the tumor cells might not always be caught and could be returned to the patient with the stem cells.Using the shiga-like toxin-1 (SLT-1) from E. coli, the researchers were able to destroy cancer cells from human stem cell cultures. The cells were exposed to the toxin in a test tube, and neither the toxin nor the intact bacteria were ever given to the patients, thereby avoiding the symptoms of E. coli food poisoning. The cancer cells used in their experiments were multiple myeloma, B-cell lymphoma, and breast cancer.Jean Gariepy, PhD, a senior scientist at the Ontario Cancer Institute and one of the researchers who worked on this study, and his colleagues have been studying toxins and their effects on blood cells for a number of years. Their current study builds on earlier research, which showed toxins could eliminate lymphoma from mice stem cells.The stem cell samples in this study were analyzed in the laboratory to see if stray cancer cells were effectively destroyed but they were not returned to the patients. Dr. Gariepy said the next step is to use the toxin to purge stem cell samples from a small number of patients. A half-dozen multiple myeloma patients at the Cross Cancer Institute, Edmonton, Alberta, Canada, will have their stem cells treated with SLT-1. If the experiment is successful, clinical trials could follow.Ralph Vogler, MD, scientific program director for the American Cancer Society (ACS), said this research is preliminary. "Toxins, other than the one from E. coli, have been of some use," Dr. Vogler said. "This offers another approach, but it needs confirmation."E coli derived L-asparaginase II An intravenous formulation containing E. coli-derived L-asparaginase II conjugated with succinimidyl carbonate monomethoxypolyethylene glycol (SC-PEG), with potential antineoplastic activity. L-asparaginase hydrolyzes L-asparagine to L-aspartic acid and ammonia, thereby depleting cells of asparagine; asparagine depletion blocks protein synthesis and tumor cell proliferation, especially in the G1 phase of the cell cycle and ultimately induces tumor cell death. Asparagine is critical to protein synthesis in acute lymphoblastic leukemia (ALL) cells which, unlike normal cells, cannot synthesize this amino acid due to the absence of the enzyme asparagine synthase. Pegylation decreases enzyme antigenicity and increases its half life. SC is used as a PEG linker to facilitate attachment to asparaginase and enhances the stability of the formulation.(http://www.cancer.gov/drugdictionary/?CdrID=595298)Colorectal cancer and E coliColorectal cancer is one of the most prevalent forms of cancer worldwide. The disease has the highest incidence in well developed countries, affecting thousands of people in the United States each year. Although colorectal cancer predominantly affects people with ages over 50, some forms of the disease can also occur in young adults and even children. Colorectal cancer is a life-threatening disease that occurs on the premises of genetically inherited predispositions and environmental factors.According to the causes that lead to the development of colon cancer, there are two main types of the disease: inherited colorectal cancer and acquired (sporadic) colorectal cancer. Inherited colorectal cancer generally occurs due to the transmission of colonic physiologic abnormalities from one generation to another. The underlying cause of inherited colorectal cancer is the formation of colonic polyps, prominent tissues that can eventually become malignant. Inherited colorectal cancer can occur at any age.Sporadic colorectal cancer generally occurs as a consequence of inappropriate diet, unhealthy lifestyle, obesity and physical inactivity. Acquired colorectal cancer is characterized by the formation of colonic tumors and carcinomas. This type of colorectal cancer is more difficult to diagnose and it predominantly affects older adults. Unlike hereditary colorectal cancer, acquired colorectal cancer can be effectively prevented by timely making

lifestyle improvements and dietary adjustments.Regardless of its actual causes, colorectal cancer needs prompt medical intervention. If the disease is discovered early, the medical treatments available today can control the progression of colorectal cancer, improving patients’ life expectancy. However, in present there is no effective cure for colorectal cancer and medical scientists are trying to find more reliable forms of treatment for this type of malignancy.Doctors inform that a diet rich in calcium can both reduce the risks of colorectal cancer and slow down its progression. In the presence of active Escherichia coli bacteria, calcium seems to be a major inhibitor for colorectal cancer. Recent studies have found that the progression of colorectal cancer is slowed down by a type of bacteria that populates the gastrointestinal tract. It seems that Escherichia coli, the bacteria responsible for causing diarrhea, can actually prevent colon cancer cells from multiplying.The toxins produced by Escherichia coli inside the large bowel trigger a release of calcium, slowing down the multiplication rate of carcinoma cells. Medical scientists are currently trying to minimize the side-effects associated with Escherichia coli bacteria in order to introduce this type of organism in future treatments for colorectal cancer. In present, medical scientists focus on laboratory altering this type of bacteria so that it can act as a safe active agent in stopping the division of malignant cells.Chen-Sheng Yeh from National Cheng Kung University, Tainan, and colleagues have coated infectious E-coli bacteria in a gold nanoshell, making biocompatible nanocomposites in which, they say, the E-coli bacteria are alive, but non-toxic to mammalian cells. Yeh explains that the combination of biological microorganisms with the optical properties of gold nanoparticles 'transforms the microorganisms into a weapon for anti-cancer therapy.' The biocompatible nanocomposites exhibit near infra-red absorption, which means they can be irradiated when in tissue and used for phototherapy, where generated heat is used to kill cells. The scientists have shown that the E-coli bacteria coated in gold can target certain cancer cells. This is done by combining them with the antibodies to anti-epidermal growth factor receptors, which are over-expressed by the cancer

cells.    Yeh plans to do further studies to investigate why the E. coli bacteria lost infectious ability after being coated in gold: 'Even though the bacteria@Au composites in this report showed a good biocompatibility, infectious bacteria still possess pathogenic characteristics intrinsically, and more cytotoxicity studies for designing such nano-bio-composites are

required.' (Rachel Cooper )Complementary approaches that may be used with cancer treatmentAcupuncture: Acupuncture is a technique in which very thin needles are put into the body to treat a number of symptoms. It may help with mild pain and some types of nausea. (See our document Acupuncture.)Aromatherapy: Aromatherapy is the use of fragrant substances, called essential oils, that are distilled from plants to alter mood or improve symptoms such as stress or nausea. (See our document Aromatherapy.)Art therapy: Art therapy is used to help people with physical and emotional problems by using creative activities to express emotions. This is done by mainstream therapists with specialized training. (See our document Art Therapy.)Biofeedback: Biofeedback is a treatment method that uses monitoring devices

to help people gain conscious control over physical processes that are usually controlled automatically, such as heart rate, blood pressure, temperature, sweating, and muscle tension. (See our document Biofeedback.)Labyrinth walking: Involves a meditative walk along a set circular pathway that goes to the center and comes back out. Labyrinths can also be "walked" online or on a grooved board following the curved path with a finger. (See our document Labyrinth Walking.)Massage therapy: Massage involves manipulation, rubbing, and kneading of the body's muscle and soft tissue. Some studies suggest massage can decrease stress, anxiety, depression, and pain and increase alertness. (See our document Massage Therapy.)Meditation: Meditation is a mind-body process in which a person uses concentration or reflection to relax the body and calm the mind. (See our document Meditation.)Music therapy: Music therapy is offered by trained healthcare professionals who use music to promote healing and enhance quality of life. (See our document Music Therapy.)Prayer and spirituality: Spirituality is generally described as an awareness of something greater than the individual self. It is often expressed through religion and/or prayer, although there are many other paths of spiritual pursuit and expression. (See our document Spirituality and Prayer.)Tai chi: Tai chi is an ancient Chinese martial art. It is a mind-body system that uses movement, meditation, and breathing to improve health and well being. It has been shown to improve strength and balance in some people. (See our document Tai Chi.)Yoga: Yoga is a form of non-aerobic exercise that involves a program of precise posture and breathing activities. In ancient Sanskrit, the word yoga means "union." (See our document Yoga.)