Alternatives to chemotherapy

1. CANCER TREATMENT
_CHEMOTHERAPY_ALTERNATIVES
Compiled and Edited By
G Vijaya Raghaqvan, CEO, DVS BioLife Ltd, HYDERABAD.
CONTENTS:
1. INTRODUCTION
2. CHEMOTHERAPY
3. COMMON CANCER DRUGS
4. ROLE OF UNUSUAL PLANT DERIVATIVES INCLUDING ALKALOIDS FROM POMEGRANATE
5. ROLE OF MICROBES LIKE E COLI IN DIAGNOSIS AND IN TREATMENT
6. COMPLEMENTARY APPROACHES
1. INTRODUCTION:
In the year 2000, malignant tumours were responsible for 12 per cent of the nearly 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

2. breast cancer are identified; probiotics are employed to reduce treatment side-effects
and if microbes could effectively be harnessed in immunotherapy.
2. Chemotherapy
Chemotherapy 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. Acivicin
3. Aclarubicin
4. Acodazole
5. Acronine
6. Actinomycin D
7. Adenine Phosphate
8. Adenosine
9. Adozelesin
10. Adriamycin
11. Adrucil
12. Alanosine
13. Aldesleukin
14. Alemtuzumab
15. Alestramustine
16. Alfacalcidol
17. Alitretinoin

3. 18. Alosetron Hcl
19. Alprostadil
20. Altretamine
21. Ambamustine
22. Ametantrone
23. Amifostine
24. Aminoglutethimide
25. Aminopterin Sodium
26. Amonafide
27. Amphotericin B
28. Amrubicin
29. Amsacrine Hcl
30. Amygdalin
31. Anastrozole
32. Anaxirone
33. Ancitabine
34. Angoroside C
35. Apixaban
36. Arecoline hydrobromide
37. Argatroban Anhydrous
38. Arsenic Trioxide
39. Ascomycin
40. Asparaginase
41. Atevirdine
42. Atosiban
43. Atrimustine
44. Axitinib
45. 5-Azacytidine
46. Azacitidine
47. Azasetron Hcl
48. Azatepa
49. Azathioprine
50. Azetepa
51. Batimastat
52. Bavituximab
53. Benaxibine
54. Bendamustine Hcl
55. Benexate
56. Benzodepa
57. Betamerphalan
58. Betulinic acid
59. Bevacizumab
60. Bicalutamide
61. Bisantrene
62. Bisnafide
63. Bizelesin

4. 64. Bleomycin
65. Bofumustine
66. Bortezomib
67. Bosutinib
68. Brefeldin A
69. Brequinar
70. Bromacrylide
71. Bromocriptine
72. Bropirimine
73. Broxuridine
74. Budotitane
75. Busulfan
76. Calcifediol
77. Calcipotriol
78. Calcitriol
79. Calcium Folinate
80. Calcium Levofolinate
81. Campotothecine
82. Canertinib
83. Cantharidin
84. Capecitabine
85. Caracemide
86. Carbetimer
87. Carboplatin
88. Carboquone
89. Carmofur
90. carmustine
91. Carzelesin
92. Catharanthine Tartrate
93. Celastrol
94. Cemadotin
95. Cephalotaxin
96. Cetuximab
97. Chlorambucil
98. Chlorethylaminouracil
99. Chlormethine
100. Chlornaphazine
101. Cisplatin
102. Cladribine
103. Cladribine
104. Clanfenur
105. Clarithromycin
106. Clevudine
107. Clodronate Disodium
108. Clofarabine
109. Clomifene Citrate

5. 110. Colchiceinamide
111. Colchicine
112. Cordycepin
113. Curcumin
114. Cyclocytidine
115. Cyclophosphamide
116. Cyclosprin
117. Cytarabine
118. Cytidine
119. Dacarbazine
120. Dactinomycin
121. Daniquidone
122. Dapoxetine Hcl
123. Dasatinib
124. Datelliptium Chloride
125. Daunoblastin
126. Daunorubicin
127. D-Bicuculline
128. Decarbazine
129. Decitabine
130. Deferasirox
131. Deflazacort
132. Defosfamide
133. Demecolcine
134. Desonide
135. Desoximetasone
136. Desoxycorticosterone
137. Desoxycortone
138. Dexamethasone
139. Dexasone
140. Dexniguldipine
141. Dexormaplatin
142. Dexrazoxane
143. Dezaguanine
144. Diacetoxysciroenol
145. Dianhydrodulcitol
146. Dianhydrodulcitolum
147. Diaziquone
148. Dibrospidium Chloride
149. Dinaline
150. Dirithromycin
151. Ditercalinium Chloride
152. Ditiomustine
153. Docetaxel
154. Dolasetron mesylate
155. Dopan

6. 156. Doxifluridine
157. Doxorubicin
158. D-tetrahydropalmatine
159. Ecomustine
160. Edatrexate
161. Edelfosine
162. Eflornithine
163. Egenine
164. Elinafide
165. Elliptinium Acetate
166. Elmustine
167. Elsamitrucin
168. Emitefur
169. Enloplatin
170. Enocitabine
171. Enpromate
172. Entecavir
173. Entricitabine
174. Enzastaurin
175. Epipropidine
176. Epirubicin
177. Eptaplatin
178. Erlotinib
179. Esorubicin
180. Estramustine
181. Ethoglucid
182. 7-Ethyl-10-Hydroxycamptothecin Etoglucid
183. Etoposide
184. Exemestane
185. Exenatide Acetate
186. Ezetimibe
187. Fadrozole
188. Fazarabine
189. Fenclonine
190. Fenretinide
191. Filgrastim
192. Finasteride
193. Fingolimod HCL(FTY720)
194. Floxuridine
195. Fludarabine
196. Flumazenil
197. Flumetasone
198. Fluoracil
199. 5-Fluorouracil
200. Fluorouracil
201. Fluracil

7. 202. Flurocitabine
203. Flutamide
204. Folic Acid
205. Formestane
206. Formylmerphalan
207. Fosquidone
208. Fotemustine
209. Fotretamine
210. Ftorafur
211. Fulvestrant
212. Galamustine
213. Galanthamine HBr
214. Galocitabine
215. Gefitinib
216. Gemcitabine
217. Gimeracil
218. Giracodazole
219. Glyfosfin
220. Goserelin
221. Granisetron Hcl
222. Harpagoside
223. Heparin sodium
224. Hexarelin
225. Homoharringtonine
226. Hydrocamptothecine
227. 10-Hydroxycamptothecin
228. Hydroxycamptothecin
229. Hydroxycarbamide
230. Hydroxyurea
231. Ibacitabine
232. Ibandronate Sodium
233. Ibandronic Acid
234. Idarubicin Hcl
235. Idoxifene
236. Ifosfamide
237. Ilmofosine
238. Ilomastat
239. Imatinib
240. Improsulfan
241. Indirubin
242. Intoplicine
243. Iproplatin
244. Irinotecan
245. Ivabradine HCL
246. Ixabepilone
247. Ketotrexate

8. 248. Lanreotide
249. Lapatinib
250. Latanoprost
251. L-biopterin
252. Lenalidomide
253. Lentinan
254. Letrozole
255. Leucovorin calcium
256. Leuprolide Acetate
257. Leuprorelin
258. Leurubicin
259. Leustatin
260. Levothyroxine
261. Lobaplatin
262. Lofexidine HCL
263. Loganin
264. Lometrexol
265. Lomustine
266. Lonidamine
267. Losoxantrone
268. Lupeol
269. Lurtotecan
270. Lycopene
271. Lysipressin
272. Mafosfamide
273. Mannomustine
274. Mannosulfan
275. Maraviroc
276. Marimastat
277. Masoprocol
278. Mechlorethamine
279. Mechlorethaminoxide
280. Medrysone
281. Megastrol acetate
282. Melphalan
283. Menogaril
284. Mepitiostane
285. Meprednisone
286. 6-Mercaptopurine
287. Mercaptopurine
288. Mesna
289. Metamelfalan
290. Methotrexate
291. Methoxymerphalan
292. Methylprednisolone
293. Methylprednisolone Aceponate

9. 294. Methylprednisolone Suleptanate
295. Meturedepa
296. Metyrosine
297. Miboplatin
298. Miltefosine
299. Minamestane
300. Miproxifene
301. Mitindomide
302. Mitobronitol
303. Mitoclomine
304. Mitoflaxone
305. Mitoguazone
306. Mitolactol
307. Mitomycin
308. Mitonafide
309. Mitopodozide
310. Mitoquidone
311. Mitosper
312. Mitotane
313. Mitotenamine
314. Mitoxantrone
315. Mitozolomide
316. Mizoribine
317. Mofarotene
318. Monocrotaline
319. Mubritinib
320. Mustine
321. Mycophenolate Mofetil
322. Mycophenolic Acid
323. Myricetin
324. Myricitrin
325. Nebivolol HCL
326. Nedaplatin
327. Nemorubicin
328. Neptamustine
329. Nigericin
330. Nilotinib
331. Nilutamide
332. Nimustine
333. Nitrocaphane
334. Nocodazole
335. Nolatrexed 2HCL
336. Norcantharidin
337. Ocaphane
338. Octreotide
339. Ondansetron

10. 340. Ormaplatin
341. Oseltamivir Phosphate
342. Oteracil potassium
343. Oxaliplatin
344. Oxipurinol
345. Oxylycorium Acetate
346. Paclitaxel
347. Paliperidone
348. palonosetron
349. Pamidronate Disodium
350. Pamidronic Acid
351. Panitimumab
352. Pazelliptine
353. Pegaspargase
354. Pemetrexed
355. Pentamustine
356. Pentetreotide
357. Pentostatin
358. Perfosfamide
359. Phenoxybenzamine HCl
360. Pincristine
361. Pipobroman
362. Piposulfan
363. Pirarubicin
364. Pirazofurin
365. Piritrexim
366. Pirlimycin
367. Piroxantrone
368. Plomestane
369. Plusonermin
370. Podophyllotoxin
371. Polymyxin E
372. Prednimustine
373. Prednisolone
374. Procarbazine
375. Procodazole
376. Prospidium Chloride
377. Psoralen
378. Pteropterin
379. Pumitepa
380. Ractopamine HCL
381. Raloxifene Hydrochloride
382. Raltegravir
383. Raltitrexed
384. Ramosetron Hcl
385. Ranimustine

11. 386. Rapamycin
387. Razoxane
388. Resveratrol
389. Retigabine
390. Riboprine
391. Risedronic acid
392. Ristocetin A
393. Ritrosulfan
394. Rituximab
395. Rocuronium Bromide
396. Roflumilast
397. Rogletimide
398. Ropinirole HCL
399. Rosuvastatin Calcium
400. Rubitecan
401. Sarcolysin
402. Sargramostim
403. Sebriplatin
404. Semustine
405. Sermorelin
406. Simtrazene
407. Sitagliptin Phosphate
408. Sizofiran
409. Sobuzoxane
410. Sodium Borocaptate[10B]
411. Solaziquone
412. Sonermin
413. Sorafenib
414. Spiclomazine
415. Spirogermanium
416. 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

12. 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 antibodies
The 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.

13. 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 alkaloids
Vinca 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.
Taxanes
Taxanes 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.

14. Epipodophyllotoxins
Epipodophyllotoxins 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 Treatment
Phytoestrogens 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

15. 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 cancer
Serum 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

16. 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 models
Most 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-

17. 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 tamoxifen
The 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

18. 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 action
Oestrogen 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

19. 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 mechanisms
At 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

20. 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.
Abbreviations
DMBA = 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
Sciences
School of Pharmacy, The University of Mississippi, University, Mississippi 38677
Institute of Environmental Sciences, Jagiellonian University, Gronostajowa 7, 30-387
Krakow, Poland
J. Agric. Food Chem., 2009, 57 (22), pp 10636–10644
DOI: 10.1021/jf902716r
Publication Date (Web): October 26, 2009
Copyright © 2009 American Chemical Society
*To whom correspondence should be addressed. Tel: 662-915-7026. Fax: 662-915-
6975. E-mail: dferreir@olemiss.edu.
Abstract
The 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

26. 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.
Introduction
Dietary 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

27. 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 Methods
Isolation 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-

28. 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

29. 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

30. 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 Discussion
Pomegranate 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

31. Section A
CYP1B1 CYP1A1
IC50 ± SEM Ki ± SEM IC50 ± SEM Ki ± SEM Ki(CYP1A1:CYP1B1)
(μM) (μM) (μM) (μM)
UA 1.15 ± 0.65 0.25 ± 0.14 12.4 ± 4.7 0.51 ± 0.18 2.05
UB 1.55 ± 0.49 0.34 ± 0.17 26.8 ± 12.9 1.11 ± 0.53 3.28
UC 39.9 ± 29 8.74 ± 0.65 790 ± 75 32.7 ± 3.11 3.74
UD 137 ± 11.08 30.10 ± 25 2907 ± 168 120.3 ± 69.6 3.99
MUA 1.49 ± 0.39 0.327 ± 0.08 59.8 ± 8.7 2.47 ± 0.36 7.57
DMUC 89.6 ± 9.7 19.6 ± 2.13 657 ± 74.3 27.2 ± 3.00 1.39
PL 2.82 ± 0.33 0.618 ± 0.07 1.5 ± 0.32 0.062 ± 0.00 0.10
PG 2.6 ± 0.79 0.58 ± 0.02 2.67 ± 0.48 0.109 ± 0.20 0.19
Section B
urolithin A urolithin B
DMSO 0.5 μM 1 μM DMSO 0.5 μM 1 μM
Vmax 339 ± 148 97.3 ± 14.6 66 ± 6.02 355 ± 103 150.9 ± 14.9 99.5 ± 8.53
Km 3.414 ± 2 2 ± 0.47 1.6 ± 0.25 4.78 ± 1.81 2.10 ± 0.33 1.9 ± 0.26
IC50 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.

32. 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).

33. 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

34. 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

35. 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.

36. 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 ± 1.3
urolithin B 38 ± 3.9 17.9 ± 1.8
urolithin C NA 26.89 ± 2.5
8-O-methylurolithin A NA 14.8 ± 2.2
8,9-di-O-methylurolithin C NA 11.5 ± 2.8
punicalins NA NA
punicalagins NA NA
Table 3. IC50 Values for Urolithin-Mediated Cytotoxicity of 22Rv1 Prostate Cells
chemical constituents IC50 ± SEM (μM)
urolithin A 98.7 ± 4.2 (a)
urolithin B 108 ± 3.9 (a)
urolithin C 36 ± 3.5 (b)
8-O-methylurolithin A 23.3 ± 3.9 (c)
8,9-di-O-methylurolithin C 20.6 ± 4.6 (c)
Following cell-based EROD assays, cellular uptake experiments were performed 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 protein
time (h) urolithin A urolithin B
0.5 5.2 ± 0.4 5.7 ± 0.8
6 9.6 ± 0.4 8.0 ± 1.1
12 20.1 ± 0.5 21.9 ± 1.1
24 23.9 ± 0.6 25.2 ± 1.6
The 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

37. 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.