Prostate Cancer A Primary Care Perspective

Screening for Anti-Cancer Agents in Herbal Medicines: “ Prostate: what’s in it for the tomato ? “ Emma Guns, Ph.D. Natural Health Products: Pharmacological Research Program Screening for Anti-Cancer Agents in Herbal Medicines: “ Prostate: what’s in it for the tomato ? “ Emma Guns, Ph.D. Natural Health Products: Pharmacological Research Program Anti-Cancer Activity: should we focus on lycopene ? • Both epidemiologic and case-control studies have associated increased consumption of tomato products and greater blood concentrations of lycopene with a reduced risk of mortality from several cancers, including prostate cancer. • In prostate cancer a reduction in risk of 33% was found among men who consumed > 10 servings of tomato products per week (30-50mg per day) (Giovanucci et al., 1995, JNCI 87:1767). • Levels of serum PSA were found to decline in patients who received lycopene tomato extract (Kucuk et al., 2001, Cancer Epidemiol Biomarkers Prev 10:861). • Lycopene has now been implicated in epidemiologic studies to be preventive against a broad range of epithelial cancers (prostate, lung) Lycopene • Lycopene is abundant in several fruits including guava, watermelon, pink grapefruit, rosehips and tomatoes. • Tomato products (such as tomato sauce, tomato paste and ketchup) are the major dietary source of lycopene. • • Lycopene is a carotenoid in the same family as ß-carotene. It is a powerful antioxidant that has shown beneficial activity in several diseases (cardiovascular, cancer, Alzheimer`s disease, multiple sclerosis). Anti-Cancer Activity • In vitro data conclusively supports an anti-proliferative effect of lycopene and other tomato derived carotenoids on prostate and other cancer cells (data from Guns lab to follow). • Preclinical data in animal models suggests a preventive role for lycopene/tomato extracts (most evident for prostate and lung). • Preventive effects have recently been shown to be enhanced in animals treated with whole tomato extracts vs lycopene (prostate cancer). • Lycopene serum levels have recently been associated with more rapid clearance of an oncogenic HPV infection…… prevention of cervical cancer. Lycopene or carotenoids ? • Results supporting therapeutic effects of lycopene in animal models are inconclusive. Lycopene vs whole tomato extract ? Lycopene Quantitation in Lycomato™ Lycopene Standard Curve 12000000 Area under curve 10000000 8000000 6000000 4000000 2000000 0 0 20 40 60 80 100 120 [Lycopene), uM Lycomato™ : 3% lycopene plus other carotenoids Standard curve from HPLC for pure lycopene obtained from LKT laboratories. HPLC chromatogram for Lycomato™ shown below (lycopene RT = 12.04 min) Absorbance Units 0.30 0.20 0.10 0.00 -0.10 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 Elution Time, Minutes Lycomato on Cytotoxicity LNCaP Cells LYCOPENE CYTOTOXICITY AT 24 HOURS (Crystal Violet Assay) ABSORBANCE AT 592 nm 120.0 100.0 80.0 60.0 40.0 20.0 0.0 0 1.00E10 LYCOPENE CONCENTRATION (M) The ability of lycopene to kill LNCaP cells in culture was confirmed using a crystal violet assay. Lycomato™ is cytotoxic to LNCaP cells at concentrations of 1μM and above. The arrow marks the hypothetical pharmacologically achievable concentration. 1.00E09 1.00E08 1.00E07 1.00E06 1.00E05 Lycomato on proliferation in LNCaP Cells L y co p en e effect o n M ito tic In d ex o f L N C aP cells in 5 % csfb s 6 M arch 2 0 0 3 0 .1 0 M ito tic In d ex (% B rd U P o sitiv e C ells) 0 .0 8 1µM 0 .0 6 Nikita data 3.3µM 5µM 0 .0 4 0 .0 2 0 .0 0 co n tro l 1 0 -6 1 0 -5 d ilu ted 3 x 1 0 -5 d ilu ted 2 x L yco p en e (n M ) Lycomato on Protein Kinase Phosphorylation Phosphosite Screen, Kinexus 2002 12000 10000 8000 6000 4000 2000 0 d d d d un cla ss ifie 9 k1 8 ss ifie ss ifie pY 52 pY 41 ss ifie cd Control Lycopene Intensity cla cla src src un un Protein LNCaP cells were treated for 10 minutes with 10 -8 M lycopene and protein was screened using Western blot by Kinexus (www.kinexus.ca) for changes in the phosphorylation states of an array of proteins. un cla Rb Lycomato on Protein Kinase Phosphorylation Phosphosite Screen, Kinexus 2002 12000 10000 8000 6000 4000 2000 0 d d d d un cla ss ifie 9 k1 8 ss ifie ss ifie pY 52 pY 41 ss ifie cd Control Lycopene Intensity cla cla src src un un Protein LNCaP cells were treated for 10 minutes with 10 -8 M lycopene in lycomato and protein was screened using Western blot by Kinexus (www.kinexus.ca) for changes in the phosphorylation states of an array of proteins. un cla Rb Cell signaling made simple: Soluble growth factors Extracellular matrix Surface receptor Integrin Ras ERK Rho Cytoskeletal tension Cyclin D1 CDK 4/6 P21/p27 G0 G1 pRb E2F Cyclin E CDK 2 M G2 S Cell signaling made simple: Soluble growth factors Extracellular matrix Surface receptor Integrin Ras ERK Rho Cytoskeletal tension Cyclin D1 CDK 4/6 P21/p27 G0 G1 pRb E2F Cyclin E CDK 2 M G2 S Lycomatoon Retinoblastoma Phosphorylation The Kinexus data indicated that lycomato altered the phosphorylation state of the tumour suppressor gene Retinoblastoma. This finding was validated by using Western blot with lycomato treated LNCaP cells 1 Hour Co 10-10 10-8 10-6 4 Hours Co 10-10 10-8 10-6 P780 RB P795 RB P807/811 RB Total RB LNCaP cells were treated with lycopene in lycomato and and a Western blot of cell lysates was probed for changes in the phosphorylation state of Retinoblastoma at several sites. Phosphorylation at tyrosine 780 was markedly decreased by lycomato treatment. Cell signaling made simple: Soluble growth factors Extracellular matrix Surface receptor Integrin Ras ERK Rho Cytoskeletal tension Cyclin D1 CDK 4/6 P21/p27 G0 G1 pRb E2F Cyclin E CDK 2 M G2 S Lycomato on cdk or p27 protein levels 10 minutes [Lycopene] uM 30 minutes 8 hours 0 0.001 0.01 0.1 24 hours 0.001 0.01 0.1 1.0 10 0 0.001 0.01 0.1 1.0 10 0.001 0.01 0.1 1.0 10 Cdk 1 Cdk 4 Cdk 5 p27 1.0 10 Cell signaling made simple: IGF-1 Extracellular matrix IGF-1 receptor Integrin Ras ERK Rho Cytoskeletal tension Cyclin D1 CDK 4/6 p21/p27 G0 G1 pRb E2F Cyclin E CDK 2 M G2 S Insulin-Like Growth Factor Axis Components Ligands: Insulin-like Growth Factors I (IGF-I) Binding Proteins: IGFBP 1 – 6 BP-2 & 5 are upregulated in tumours and promote the IGF response, BP 3 is generally antagonistic to IGFmediated signaling YP- IGF-I IGFBPs IGF-I -ss- -ss- -ss-YP Extracellular Intracellular Receptors: IGF-IR Tyrosine kinase YP- T YPYP- K T -YP -YP K -YP -YP -YP -YP YPYPYP- Differentiation Proliferation Survival IGF-I and Cancer • Serum IGF-I levels are correlated with increased risk for developing prostate cancer (Chan et al., 1998, Sci. 279:563). • Human population studies strongly indicate high circulating levels of IGF-1 and low IGF-BP3 with lung cancer (Wakai et al., Cancer Res. 2002). • Serum lycopene levels inversely correlate with IGF-I levels (Mucci et al., 2001, BJU Int. 87:814). • Lycopene treatment in MCF 7 cells (breast cancer cells) has been shown to downregulate IGF-IR signaling (Levy et al 1995, Nutr. Cancer 24:257). • Upregulation of IGF-BP3 has been inversely correlated with lung cancer incidence in ferrets exposed to smoke carcinogens (Lui et al., Cancer Res 2003). IGF-I Receptor Expression IGF-IR PY99 - - - + + + - + + + + + - - + + - + - + Lyc-o-mato 48h R1881 10-9 M 48h IGF-I 100 ng/ml 10 min > IGF-IR ß subunit > P-IGF-IR ß subunit >Loading control IGF-I Receptor (IGF-IR) was immunoprecipitated from cells treated with 1M lycopene in lycomato, R1881, and/or IGF-I. Lycomato produced a decrease in the expression of IGF-IR in the LNCaP cells but had no effect upon the phosphorylation state of the receptor. Effects of Lycomato on IGF Signalling The impacts of lycomato treatment upon components of the IGF signalling pathway were investigated in LNCaP cells using Western blot. IGFBP2 Expression - - - + + + - + + + + + Lyc-o-mato 48h - - + + R1881 10-9 M 48h - + - + IGF-I 100 ng/ml 10 min > IGFBP2 > Loading control Protein was prepared from LNCaP cells treated with 1M lycopene in lycomato, the synthetic androgen R1881, and/or IGF-I. Western blot shows that lycomato induces increased expression of IGF Binding Protein 2 (IGFBP2) Lycomato Interaction with the Androgen Receptor To determine whether lycomato was acting through the androgen receptor a reporter gene assay was used. Effects of Lycomato on Androgenic Response in LNCaP Cells 1000000 100000 Lycomato plus 0.05nM DHT, hAR Lycomato plus 0.5nM DHT, hAR Lycomato only, hAR RLU 10000 1000 100 0.0001 0.01 1 [Lycopene], uM 100 LNCaP cells transfected with AR and an androgen responsive luciferase reporter were treated with lycopene. At concentrations >1M an agonist effect was seen in the absence of DHT. Lycomato behaves as a weak antagonist against the action of DHT. Summary…. what’s in it for the tomato ? • • Lycomato does not alter cdk 1, 4, 5 or p27 expression. Lycomato inhibits phosphorylation of the tumour suppressor, Rb, thus preserving growth suppression properties. • Lycomato treatment opposes androgen`s effect on IGF-I signaling by decreasing IGF-I receptor expression. • Analogous to androgen suppression, IGF-BP2 levels increase upon treatment with lycomato. • In the presence of androgen, lycomato acts a weak antagonist of androgenic response. At higher lycomato concentrations stimulation of the androgenic pathway occurs in the absence of androgen. And…. is lycopene all that’s in it for the tomato ? • -carotene, -carotene, -carotene, phytoene, phytofluene – all shown to accumulate in prostate tissue upon dietary inclusion of tomato. • In Acycloretinoic acid has been shown to be an oxidative product of lycopene in pig and human liver fractions. • Bioleau et al. have recently shown whole tomato extract had greater efficacy than lycopene treatment alone on lethal prostate cancer development in a rat model: PC induced by N-methyl-N-nitrosourea and testosterone treatment. And…. is lycopene all that’s in it for the tomato ? NO • -carotene, -carotene, -carotene, phytoene, phytofluene – all shown to accumulate in prostate tissue upon dietary inclusion of tomato. Lycopene accumulation is most pronounced. • In Acycloretinoic acid has been shown to be an oxidative product of lycopene in pig and human liver fractions. • Bioleau et al. have recently shown whole tomato extract had greater efficacy than lycopene treatment alone on lethal prostate cancer development in a rat model: PC induced by N-methyl-N-nitrosourea and testosterone treatment. ………….likelihood is that lycopene has some anti-proliferative activities which are enhanced in the context of tomato extract either in an additive or synergistic manner. • Many Thanks to: • Dr. Simon Cowell • Nikita Ivanov • Dr. Michael Cox • Dr. Paul Rennie • Catherine Wood • Venket Rao, U. of Toronto • Dr. Vasu Venketswaran, Princess Margaret Hospital, University of TO • Prostate Cancer Research Foundation of Canada (research grant) • Canadian Prostate Cancer Research Initiative (Bio-NET grant)