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Preclinical and Clinical Evaluation of Sulforaphane for Chemoprevention in the Breast Brian Cornblatt, PhD Manager, Oncology Research Office, SJMC Lifetime Probability of Developing Cancer, by Site, Women, US, 1998-2000 Site Risk All sites 1 in 3 Breast 1 in 7 Lung & bronchus 1 in 17 Colon & rectum 1 in 18 Uterine corpus 1 in 38 Non-Hodgkin lymphoma 1 in 57 Ovary 1 in 59 Pancreas 1 in 83 Melanoma 1 in 82 Urinary bladder 1 in 91 Uterine cervix 1 in 128 Source:DevCan: Probability of Developing or Dying of Cancer Software, Version 5.1 Statistical Research and Applications Branch, NCI, 2003. http://srab.cancer.gov/devcan Cancer Statistics 2004, American Cancer Society a B Ner rain vou & O s S the yste r m New Cases Ute rus Mu ltip le M yelo m rea s kem ia No n-H o Lym dgk ph ins om a Pa nc Leu m ctu Ov Re n& Co lo ary Deaths ast Lu Bre ng 211, 240 Women’s Cancers in the U.S. (Mortality Top 10) ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Source: American Cancer Society, Cancer Statistics 2005 DNA to Enzymes… “Chromosomes” DNA “Gene Expression” “Gene Transcripts” “Protein Expression” “Enzymes” ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ DNA to Enzymes… “Chromosomes” DNA transcription mRNA “Gene Expression” “Gene Transcripts” “Protein Expression” “Enzymes” ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ DNA to Enzymes… “Chromosomes” DNA transcription “Gene Expression” “Gene Transcripts” mRNA translation “Protein Expression” “Enzymes” protein ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ DNA to Enzymes… ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Alkylating Agents Nitrogen mustards Nitrosoureas Topisomerase-Targeted Epipodophyllotoxins Anthracyclines Anthracenes Actinomycins Camptothecins Platinum Compounds Aziridines Cisplatin Carbolatin Oxaliplatin DNA Nucleoside Analogs Cytarabine Fludarabine Gemcitabine replication Antimetabolites transcription Folate antagonists Fluoropyrimidines Purine antagonists Ribonucleoside reductase inhibitors mRNA translation protein Mitotic Spidle Poisons Vinca alkaloids Taxanes Epothilones ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Nelson, WG Alkylating Agents Nitrogen mustards Nitrosoureas Topisomerase-Targeted Epipodophyllotoxins Anthracyclines Anthracenes Actinomycins Camptothecins Platinum Compounds Aziridines Cisplatin Carbolatin Oxaliplatin DNA Nucleoside Analogs Cytarabine Fludarabine Gemcitabine replication Antimetabolites transcription Folate antagonists Fluoropyrimidines Purine antagonists Ribonucleoside reductase inhibitors mRNA Monoclonal Antibodies translation HER2-Neu (trastuzumab) CD20 (rituximab) protein Signal Transduction Inhibitors Mitotic Spidle Poisons EGFR inhibitors Vinca alkaloids Taxanes Epothilones Estrogen Antagonists Tamoxifen Raloxifene Nelson, WG Change in the US Death Rates* by Cause, 1950 & 2001 Rate Per 100,000 600 586.8 1950 500 2001 400 300 245.8 200 193.9 180.7 194.4 100 57.5 48.1 21.8 0 Heart Diseases Cerebrovascular Diseases Pneumonia/ Influenza * Age-adjusted to 2000 US standard population. Sources: 1950 Mortality Data - CDC/NCHS, NVSS, Mortality Revised. 2001 Mortality Data–NVSR-Death Final Data 2001–Volume 52, No. 3. http://www.cdc.gov/nchs/data/nvsr/nvsr52/nvsr52_03.pdf Cancer Statistics 2004, American Cancer Society Cancer ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Some Good News… www.free-picture-graphic.org.uk Vegetable Consumption and Breast Cancer Risk • Epidemiological studies (10 case-control and 7 prospective cohort) provide evidence for a modest inverse relationship between quantities of fruit and vegetables consumed and the risk of developing breast cancer • In the majority of the studies, only limited information on cruciferous intake was available ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ http://www.singlesourcephoto.com Cruciferae (aka Brassicaceae, mustard family) broccoli brussels sprouts cauliflower parsnip radish wasabi Chinese cabbage watercress horseradish white mustard ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Cruciferous Vegetables and Chemoprevention HSO4 Myrosinase D-glucose Glucoraphanin Sulforaphane • In cruciferous vegetables, substances known as isothiocyanates exist as stable sugar conjugates called glucosinolates • Glucoraphanin, the predominant glucosinolate in broccoli sprouts, is converted to sulforaphane by myrosinase, a plant enzyme, during chewing of plants or hydolysis by gut microflora ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Sulforaphane’s Mechanism of Action Inside the Cell ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ www.animalport.com Sulforaphane’s Mechanism of Action Inside the Cell C 273 H 274 ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Nrf2 C 288 Keap1 Zn Sulforaphane Nrf2 F-actin ARE Nucleus Wakabayashi et al. PNAS, 2004 Sulforaphane’s Mechanism of Action Inside the Cell ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ S S Nrf2 S S Sulforaphane ARE Nucleus Wakabayashi et al. PNAS, 2004 Sulforaphane’s Mechanism of Action Inside the Cell ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ www.animalport.com Sulforaphane’s Mechanism of Action Inside the Cell ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ S S Nrf2 S S Sulforaphane ARE Nucleus Wakabayashi et al. PNAS, 2004 Sulforaphane’s Mechanism of Action Inside the Cell DNA S S mRNA protein S S Sulforaphane ARE Nucleus Wakabayashi et al. PNAS, 2004 • chaperones • ubiquitin/ proteasomes • detoxifying enzymes • antioxidative enzymes • glutathione biosynthesis • NADPH generation ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Cruciferous Vegetables and Chemoprevention Heme Biliverdin D N A repair D N A B IN D IN G & DAMAGE N E O P LAS IA P hase 11 Phase enzym es Enzymes R E A C T IV E E LE C TR O P H ILE S (U ltim ate C arcinogens) ROS PPhase hase 22 Enzymes enzym es H O -1, ferritins, HO-1 M nS O D , catalase, LTB 4 D H D E TO XIC A TIO N P R O D U C TS PROC A R C IN O G E N S C ytochrom e P 450s O XY G E N Bilirubin NON E LEC TR O P H ILIC M ET A BO LIT ES G S T s, U G Ts, GSTs, NQO1, SULT1A1 Q R , γ-G C S , A F A R ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ • A major mechanism for protection against carcinogenesis involves induction of cytoprotective enzymes Cruciferous Vegetables and Chemoprevention ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ (T Kensler, 2006 Breast Spore Update) Market Stage Broccoli 3-Day Sprouts (3 grams) (150 grams) Freeze-Dried Sprout Extract (150 mg) All preparations contain the same quantity of enzyme inducer activity ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Anthracenes and Cancer ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ www.cardmine.co.uk Prevention of DMBA-induced Mammary Tumors TUMOR INCIDENCE ** p < 0.01 * p < 0.05 1.5 60 * 40 1.0 * ** 20 0.5 ** TUMOR MULTIPLICITY 2.0 80 0.0 0 0 75 150 0 75 150 SULFORAPHANE DOSE (μmole) • At age 47, 48, 49, 50, and 51 days each female Sprague-Dawley rat received by gavage either vehicle, 75 or 150 μmol sulforaphane • On day 50, all rats received DMBA (8.0 mg) • Overall, the incidence, multiplicity, and weight of mammary tumors ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ were significantly reduced, and their development was delayed Hypothesis Broccoli sprouts, which contain abundant quantities of glucoraphanin, the sulforaphane precursor, are safe, effective, inexpensive and a practical means to achieve chemoprevention against breast cancer ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Preclinical Assay: Specific Aims Determine the pharmacokinetic and pharmacodynamic action of sulforaphane in the mammary epithelium of rodents and develop and validate biomarkers to assess chemopreventive efficacy 1. What is the distribution pattern of a single dose of sulforaphane in the mammary gland over time? 2. How does a single dose of sulforaphane affect cytoprotective gene transcript levels over time (NQO1, HO-1)? 3. How does a single dose of sulforaphane affect cytoprotective enzyme activity over time (NQO1)? 4. In which cells of the mammary gland do we see cytoprotective protein induction (HO-1, SULT1A1)? ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Preclinical Assay Design X 39 = 150 μmol/rat delivered in 400 ul corn oil after overnight fasting Pharmacokinetics of Sulforaphane ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Pharmacokinetics of Sulforaphane HPLC & Photodiode Array Detector Pharmacokinetics of Sulforaphane (Data ± SEM, n = 3 rats/group) Effect of Sulforaphane on NQO1 and HO-1 Transcripts RNA Bioanalyzer & RT Detection System Effect of Sulforaphane on NQO1 and HO-1 Transcripts (Data + SEM, n = 3 rats/group) Effect of Sulforaphane on NQO1 Enzymatic Activity Micro Plate Reader ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Effect of Sulforaphane on NQO1 Enzymatic Activity ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ (Data ± SEM, n = 3 rats/group) Localization of HO-1 and SULT1A1 Induction by Sulforaphane ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Localization of HO-1 and SULT1A1 Induction by Sulforaphane (Milk Glands) ucdavis.edu Localization of HO-1 Induction by Sulforaphane (Original magnification X 100; Scale bar = 50 μm) ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Localization of SULT1A1 Induction by Sulforaphane CONTROL 200X 400X TREATED (48 Hours) ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Preclinical Assay: Specific Aims Determine the pharmacokinetic and pharmacodynamic action of sulforaphane in the mammary epithelium of rodents and develop and validate biomarkers to assess chemopreventive efficacy 1. What is the distribution pattern of a single dose of sulforaphane in the mammary gland over time? 2. How does a single dose of sulforaphane affect cytoprotective gene transcript levels over time (NQO1, HO-1)? 3. How does a single dose of sulforaphane affect cytoprotective enzyme activity over time (NQO1)? 4. In which cells of the mammary gland do we see cytoprotective protein induction (HO-1, SULT1A1)? ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Preclinical Assay Major Aim Determine the pharmacokinetic and pharmacodynamic action of sulforaphane in the mammary epithelium of rodents and develop and validate biomarkers to assess chemopreventive efficacy and translate these findings to a human population at elevated risk for breast cancer Clinical Pilot Study Preop bloods Enroll in study Day -7 Start on cruciferous free diet Collect blood and urine Daily dietary checklist 12 hour urine collection (7PM – 7AM) BSP (200 µmol) given w/in 1 hour prior to surgery Day -3 Day -1 Day 0: Prior to surgery Collect tissue blood and urine Day 0: Intraoperatively • Single-arm non-randomized clinical trial in healthy women undergoing reduction mammoplasty surgery at Johns Hopkins • Primary aim: Determine whether sulforaphane and/or its metabolites directly reach human breast tissue Clinical Pilot Study Eligibility criteria: • Non-smokers • No prior history of cancer • Not on aspirin, NSAIDs, antibiotics (for at least 1 week before surgery) • Scheduled to undergo a reduction mammoplasty • Be 18 yrs or older • Have signed informed consent Clinical Pilot Study Characteristics N= 10 Mean age (yrs) Race Caucasians African Americans Pre-op Weight (kg) Pre-menopausal Current ETOH drinkers 30 (21-50) 1 9 99 (68.3 -158.8) 9 6 ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Clinical Pilot Study - Ten women were recruited from April 05 to March 06 - BSP dose (200 µmol ITC) was given on average 53 min (range 16 – 82 ) before surgery - Side effects : 2 individuals complained of a bitter taste in mouth 2 burning sensation in throat 1 burning in stomach Clinical Pilot Study • Average time between BSP and removal was similar between right and left breast tissue: - left - right 117 min ± 36 103 min ± 26 Mean weight of breast tissue removed - left 846 grams ± 412 - right 916 grams ± 433 Clinical Pilot Study (Pharmacokinetics) Specimens Mean ± SE Pre-dose Post dose (~ 110 minutes) N/A Left 2.00 ± 1.95 Breast Tissue (pmol/mg) Urine (µM) Right 1.45 ± 1.12 4.1 ± 5.2 158.9 ± 93.9 Plasma (µM) 0.01 ± 0.02 0.9 ± 0.7 Clinical Pilot Study (Pharmacodynamics) • Due to short interval between ingestion of broccoli sprouts preparation and breast resection unable to evaluate pharmacodynamic effect • Did detect and measure NQO1 and HO-1 transcripts and NQO1 enzymatic activity in the collected mammary tissue • Developing immunostaining assays to enable future localization of cytoprotective enzyme induction NQO1 HO-1 Randomized Case-Control Clinical Trial Diagram of Study Plan Reduction Mammoplasty/ Prophylactic Mastectomy women begin crucifer free diet Day -17 R A N D O M I Z E D BSP (100 µ mol) beverage Surgery Placebo beverage Blood 12-hr urinary 12-hr Urinary Toxicity check DTC Start taking DTC BSP Day -11 Day -10 Day -1 Toxicity check Acceptability Questionnaire Blood, Urine Tissue acquisition Day 0 ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ http://www.singlesourcephoto.com Since 1994: “Cancer Prevention” & “Diet” “Cancer Prevention” & “Fruits and Vegetables” 3690 556 Trends in Consumption of Five or More Recommended Vegetable and Fruit Servings for Cancer Prevention, Adults 18 and Older, US, 1994-2002 35 30 Prevalence (%) 25 24.2 24.4 24.1 24.4 24.5 1994 1996 1998 2000 2002 20 15 10 5 0 Year Note: Data from participating states and the District of Columbia were aggregated to represent the United States. Source: Behavioral Risk Factor Surveillance System CD-ROM (1984-1995, 1996, 1998) and Public Use Data Tape (2000), National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, 1997, 1999, 2000, 2001. Cancer Statistics 2004, American Cancer Society ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Acknowledgements (Preclinical Studies) KENSLER LAB: Tom Kensler Pat Dolan Melinda Yates TALALAY LAB: Paul Talalay Lingxiang Ye Albena Dinkova-Kostova Kala Visvanathan Dr. Angelo DeMarzo Jessica Hicks Funding: NIEHS Training Grant (#ES 07141) ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ Acknowledgements (Clinical Studies) Kala Visvanathan Jed Fahey Nancy Davidson Food preparation and stability Kitty Stephenson Kristina Wade Clinical team Navin Singh Melanie Erb Jaime Flores Pedram Argani Min Sue Shen Elizabeth Garrett-Mayer Kathy Helzlsouer Study Staff Deborah Santor Anita Mayfield Amy Gross Funding: Hopkins Breast SPORE CA88843, Breast Cancer Research Foundation, NCI KO7 (Ca111948) ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________
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