florenz imhof 2006
DESCRIPTION
TRANSCRIPT
Clinical Action and Safety of Red Clover Isoflavones
Martin Imhof
General Public Teaching Hospital Korneuburg/Vienna
Medical University - Vienna
• Million Women Study Collaborators. Breast cancer and hormone- replacement therapy in the Million Women Study. Lancet 2003; 362:419-27
• Women´s Health Initiative (WHI) Study – Interrupted due to severe side effects
Herbal Alternatives
to
Hormone Replacement Therapy
How Safe and Effective are Phytohormones?
Are Red Clover Isoflavones Effective ?
A Double Blind, Randomized,
Placebo-controlledCross-Over Study
Week 12Week 4 Week 8
-25
-20
-15
-10
-5
0
Per
cen
tag
e R
edu
ctio
n in
Ho
t F
lush
es
Placebo
Active
Baber RJ, et al. Climacteric 1999
Effect of 40 mg red clover extracts over No. Daily hot flashes and Greene Scores (n=51 postmenopausal)*
*RDBPC study
Tice JA, et al. JAMA 2003
Effect of 80 mg vs. 57 mg red clover extracts vs placebo over No. hot flashes freq and Greene Scores (n=252 postmenopausal)*
*ICE study (RDBPC)
After 12 weeks:After 12 weeks: 80 mg RCE (n=84): 41% reduction80 mg RCE (n=84): 41% reduction 57 mg RCE (n=83): 34%.57 mg RCE (n=83): 34%. Placebo (n=85): 36% (p > 0.05).Placebo (n=85): 36% (p > 0.05).
Van Weijer PH, et al. Maturitas 2002
Effect of 80 mg red clover extracts vs placebo over hot flashes frequenz and Greene Scores (n=30 postmenopausal)*
4 weeks placebo run in phase 4 weeks placebo run in phase (16% reduction).(16% reduction).
After 12 weeks:After 12 weeks: Red clover reduced hot flashes Red clover reduced hot flashes
freq 44%.freq 44%. Placebo no further reduction.Placebo no further reduction.
*RDBPC study
• Placebo-controlled, cross-over, double-blind, randomized • 113 menopausal women, 109 finished this study
• 25 weeks • 80 mg isoflavones daily (2 capsules of menoflavon)
• Within the 25 weeks 4 examinations
• Start: November 2002 - End: February 2005
Menoflavon Study 80mg isoflavones /d of red clover – clinical trial Austria
Examinations and questionnaires
Kupperman scoreKupperman score (index before start >15) (index before start >15) Changes in Changes in blood serum levelsblood serum levels (T, E2, FSH, LH, (T, E2, FSH, LH,
SHBG, HDL, LDL, Triglycerides, Lpa, Cholesterol)SHBG, HDL, LDL, Triglycerides, Lpa, Cholesterol) Effects on the Effects on the endometriumendometrium Vaginal smearVaginal smear
(cytodiagnosis)(cytodiagnosis) Urine-analysisUrine-analysis (urinary excretion of isoflavones) (urinary excretion of isoflavones) Depressions scaleDepressions scale after Zerssen after Zerssen Hospital Anxiety and Depression Scale (Hospital Anxiety and Depression Scale (HADS-DHADS-D) )
Observation studyObservation study: effects of menoflavon on skin, hair, : effects of menoflavon on skin, hair, nails and libido, vaginal atrophy…. nails and libido, vaginal atrophy….
Demographic and anamnestic data
Group A(n=50)
Group B(n=59)
Overall(n=109)
Mean age (yrs.) 54.5 ± 6.2 53.7 ± 7.8 53.5 ± 7.1
Mean BMI 24.5 ± 3.9 24.9 ± 3.9 24.7 ± 3.9
Hysterectomy (%) 18.0 13.6 15.6
Kupperman Index
0
5
10
15
20
25
30
35
40
45
MF11RCE Placebo
gestiegen
gefallen
unverändert
Wilcoxon Test, PVerum < 0,001, PPlacebo < 0,001
Reduction of hot flushes
0
5
10
15
20
25
30
35
40
45
MF11RCE Placebo
zugenommen
abgenommen
unverändert
Wilcoxon Test, PVerum < 0,001, PPlacebo = 0,001
Hot flushes, Boxplots-comparison
Hot flushes were reduced significantly with MF11RCE (p<0,001)
No effect under Placebo
before after
MF11RCE
before afterPlacebo
Reduction sleep disorders
0
10
20
30
40
50
MF11RCE Placebo
Zunahme
Abnahme
keine Veränderung
Wilcoxon Test, PVerum < 0,001, PPlacebo = 0,018
Sleeping behavior - Boxplots-comparison
before afterMF11RCE Placebo
afterbefore
Sleeping behavior improved high significantly with MF11RCE (p<0,001) No effect under Placebo
Mood improvement
0
5
10
15
20
25
30
35
MF11RCE Placebo
verschlechtert
verbessert
unverändert
Wilcoxon Test, PVerum < 0,001, PPlacebo = 0,001
Baseline measures
Group A(n=50)
Group B(n=59)
Overall(n=109)
E2 (pg/ml) 36.30 38.31 37.39
T (ng/ml) 0.43 0.63 0.54
LH (mIU/ml) 30.64 29.92 30.25
FSH (mlU/ml) 60.31 60.45 60.38
SHBG (nmol/l) 61.44 57.69 59.41
Group A(n=41)
Group B(n=51)
Overall(n=92)
Endometrium (mm)
4.3 3.4 3.8
Data are presented as mean values.
Wilcoxon-test MF11RCE vs. placebo
N Negative Ranks
Positive Ranks
p
E2 109 40 43 0,8610,861
T 109 61 36 0.003
LH 109 64 44 0.861
FSH 109 62 47 0.069
SHBG 109 58 51 0.824
ET 92 15 45 0.001
Changes with MF11RCE
Before MF11RCE
After MF11RCE
Change (%)
T (ng/ml) 0.54 ± 0.26 0.66 ± 0.29 22.12
ET (mm) 3.8 ± 1.9 3.2 ± 1.5 -14.69
Data are presented as mean ± SD.
Changes in Endometrium after 90 days of treatment with MF11RCE
Changes in Testosteron serum levels after 90 days of treatment
Maturitas. 2006 Aug 20;55(1):76-81.
Effects of a red clover extract Effects of a red clover extract (MF11RCE(MF11RCE) ) onon endometrium endometrium and sex hormones in and sex hormones in
postmenopausal women. postmenopausal women.
Martin Imhof, Anca Gocan, Franz Reithmayr, Markus Lipovac, Martin Imhof, Anca Gocan, Franz Reithmayr, Markus Lipovac, Claudia Schimitzek4, Johannes HuberClaudia Schimitzek4, Johannes Huber
Results clinical study - 80 mg isoflavones – 113 women Austria
Testosterone levels increasedTestosterone levels increased significantly (p=0,021) Wilcoxon test with MF11RCE, no significantly (p=0,021) Wilcoxon test with MF11RCE, no significant change with placebosignificant change with placebo
Endometrium thicknessEndometrium thickness was was reduced reduced withwith MF11RCE (p<0.001) MF11RCE (p<0.001) Climacteric symptomsClimacteric symptoms as evaluated with as evaluated with Kupperman index was reducedKupperman index was reduced in general in general
(p<0.001) with MF11RCE treatment.(p<0.001) with MF11RCE treatment.o Hot flushes and sweating were reducedHot flushes and sweating were reduced statistically significantly (p<0.001) statistically significantly (p<0.001) o Sleeping behavior improvedSleeping behavior improved highly significanthighly significant (p<0.001) (p<0.001) o DepressionDepression and and nervousnessnervousness were significantly reduced (p<0.001) were significantly reduced (p<0.001) o Lack of concentration Lack of concentration was significantly reduced (p<0.001) was significantly reduced (p<0.001)
EstradiolEstradiol, , FSHFSH and and LHLH plasmaplasma levels did not change significantly comparing both groups levels did not change significantly comparing both groups
Design clinical study - 80 mg isoflavones – 60 women Ecuador
Double-blind, placebo controlled, randomized, cross-overDouble-blind, placebo controlled, randomized, cross-over
90 days 2 capsules menoflavon/day - wash out - 90 days Placebo resp. 90 days 2 capsules menoflavon/day - wash out - 90 days Placebo resp. vice versavice versa
60 women, average age: 51,3 years60 women, average age: 51,3 years
Inclusion criteria: Kupperman-Index ≥15, no HRT,..Inclusion criteria: Kupperman-Index ≥15, no HRT,..
3 examinations (at the beginning, after 90 days, end)3 examinations (at the beginning, after 90 days, end)
Kupperman-Index, lipids, hormone levels (E2, FSH, LH, Testosterone, Kupperman-Index, lipids, hormone levels (E2, FSH, LH, Testosterone, SHBG, Lipoprotein a), vaginal cytologySHBG, Lipoprotein a), vaginal cytology
Effect of 80 mg red clover extracts over Kupperman scoring after 3 months (n=53
postmenopausal)
Hidalgo L, Chedraui P, et al. Gynecol Endocrinol, 2005, In press
0
5
10
15
20
25
30
baseline red clover placebo
baseline red clover placebo
The effect of red clover supplementation over lipid profile
a vs. baseline; b vs. red clover phase; * p < 0.05;
ParameterBaseline
n=53After isoflavone
supplementation n=53After placebo
n=53
Total cholesterol (mg/dL)
223.9 37.6
214 32.2 a * 220.4 34.1 b
HDL-C (mg/dL)39.7 11.5
40 9.6 a 41.1 10 b
LDL-C (mg/dL)146.8 29.9
129.7 39.4 a * 140 35.2 b
Triglycerides (mg/dL)
199.6 77.8
181.1 72.3 a * 242.7 166.9 b *
Lipoprotein A (mg/dL)
41.2 36.9
22.8 26.9 a * 20.5 25.8 b
Hidalgo L, Chedraui P, et al. Gynecol Endocrinol, 2005, In press
The effect of red clover supplementation over hormonal profile
ParameterBaseline
n=53After isoflavone
supplementation n=53After placebo
n=53
SHBG (nmol/L) 50.1 23.7
46.9 21.9 a 49 22 b
FSH (mIU/mL)63.7 22.6
63.3 26.1 a 62.6 22.1 b
LH (mIU/mL) 27.7 13.8
30.8 13.4 a 28.7 14 b
17 β estradiol (pg/mL)
23.8 8.8
22.4 7.2 a 20.9 4.6 b
Testosterone (ng/dL)
24.8 11.2
21.4 5 a 23.2 11.3 b
Hidalgo L, Chedraui P, et al. Gynecol Endocrinol, 2005, In press
Results clinical study - 80 mg isoflavones – 60 women Ecuador
No significant effect on BMI, weight and blood pressure in both groupsNo significant effect on BMI, weight and blood pressure in both groups
significant effect on Kupperman-Index, lipidssignificant effect on Kupperman-Index, lipids
Compared to placebo menoflavon significantly decreased:Compared to placebo menoflavon significantly decreased:
• menopausal symptoms, like:menopausal symptoms, like:
• hot flushes, night sweats, sleeping disorders, nervousness, hot flushes, night sweats, sleeping disorders, nervousness, depression, dizziness, ....depression, dizziness, ....
• Triglyceride levelTriglyceride level
menoflavon had a positive effect over the vaginamenoflavon had a positive effect over the vagina
CONCLUSIONS
80 mg (MF11RCE) Red Clover does 80 mg (MF11RCE) Red Clover does significantly decrease menopausal complaints significantly decrease menopausal complaints
80 mg (MF11RCE) Red Clover significantly 80 mg (MF11RCE) Red Clover significantly influences Testosterone level and influences Testosterone level and Endometrium thickness Endometrium thickness
The action of MF11RCE on MCF 7 and Human The action of MF11RCE on MCF 7 and Human Endothelial Breast Cells does not show a risk Endothelial Breast Cells does not show a risk of tumour induction in analysis of gene of tumour induction in analysis of gene activation activation
Phytohormones can stimulate MCF 7 cell proliferation
0,00
10,00
20,00
30,00
40,00
50,00
60,00
70,00
80,00
ASR (W)
Finland Greece China Northern Europe Southern Europe East Asia
Prostate
Breast
Colon/ Rectum Man
Colon/ Rectum Woman
Cancer Mondial/ Globocan 2000
Epidemiological Data CancerEpidemiological Data Cancer
Cell proliferation is not
sufficient to describe
the risk of tumour development
Experiment 1
Cells: - MCF-7 mamma carcinoma cell line
Phytohormones: - Red clover - Trifolium pratense, (MF11RCE) - “synthetic” Genistein and Daidzein (Indofine).
Technology:- 20K Chip arrays (Affymetrix)
Experimental design:
MCF-7 were examined after 12 and 24 hours.
Experimental groups:- untreated cells- combination of genistein plus daidzein (2,5 µg/ml each)- red clover extract (5 µg/ml)
Flowchart of experimental analysis
c-DNA
Scan Hybridize
(16 hours)
mRNA
IVT
(Biotin-UTPBiotin-CTP)
Labeled fragments
L L
L
L
Cells
Comparison of the mRNA expression pattern in MC-F7 cells stimulated with red-clover extract with that of
daidzin and genestin stimulated cells.
22.000 genes were screened by DNA array and for each gene 11 oligos for perfect match and 11 oligos for mismatch are analyzed. Therefore a total of
500.000 spots were quantitatively analyzed.
Results
A significant increase (>2times) in mRNA expression
induced by phytohormones was seen for 117 genes.
The functional repertoire of these genes cover
-Growth arrest genes
-DNA repair genes
(GADD34)
-Growth factors
(insulin-like growth factor binding protein 4)
Selected Gene Tree: Affymetrix Experiment (Default Interpretation)Colored by: Affymetrix Experiment, Default Interpretation
203720_s_at (ERCC1)207348_s_at (LIG3)218685_s_at (SMUG1)201236_s_at (BTG2)204461_x_at (RAD1)219510_at (POLQ)201746_at (TP53)203565_s_at (MNAT1)202239_at (ADPRTL1)204766_s_at (NUDT1)31861_at (SMBP2)204093_at (CCNH)207405_s_at (RAD17)205811_at (POLG2)207598_x_at (XRCC2)219317_at (POLI)202330_s_at (UNG)205091_x_at (RECQL)34063_at (RecQ5)203719_at (ERCC1)204828_at (RAD9)202332_at (CSNK1E)219502_at (FLJ10858)202726_at (LIG1)205733_at (BLM)203725_at (GADD45A)218428_s_at (REV1L)218961_s_at (PNKP)201459_at (RUVBL2)203210_s_at (RFC5)201529_s_at (RPA1)205024_s_at (RAD51)206066_s_at (RAD51C)208393_s_at (RAD50)205189_s_at (FANCC)203564_at (FANCG)203806_s_at (FANCA)205909_at (POLE2)204603_at (EXO1)203577_at (GTF2H4)207891_s_at (TREX2)202453_s_at (GTF2H1)204408_at (APEX2)204884_s_at (HUS1)203422_at (POLD1)203616_at (POLB)207347_at (ERCC6)208386_x_at (DMC1)202466_at (POLS)203409_at (DDB2)207945_s_at (CSNK1D)
CLUSTERED DNA REPAIR GENES
Red CloverSynthetic Phytohormones
unstimulated
Y-axis: Affymetrix Experiment, Default InterpretationColored by: Control_12Gene List: Tumor Suppressor (376), 204159_at selected
Control_12 Control_24 G+D_12h G+D_24h redc_12h redc_24h
Sample0.01
0.1
1
10
100
Control_12 Control_24 G+D_12h G+D_24h redc_12h redc_24h
Sample0.01
0.1
1
10
100
CDKN2C
Cyclin dependent kinase inhibitor
CLUSTERED TUMOR SUPRESSOR GENES
Results
THE GENES DIFFERENTIALLLY UP-REGULATED BY
SYNTHETIC PHYTOESTROGENS ARE
“METALLOTHIONEINS” (MT)
AND ARE BELIVED TO PLAY AN ROLE IN
TUMORGENESIS.
Y-axis: Affymetrix Experiment, Default InterpretationColored by: G+D_12hGene List: like 217165_x_at (MT1F) (0.95) (70)
Control_12 Control_24 G+D_12h G+D_24h redc_12h redc_24h
Sample0.01
0.1
1
10
100
Control_12 Control_24 G+D_12h G+D_24h redc_12h redc_24h
Sample0.01
0.1
1
10
100
12h 24hRed Clover
12h 24hSynthetic
Phytohormones
12h 24hunstimulated
Dec
reas
e I I
ncre
ase
EXTRACT OF GENES THAT ARE DIFFERENTLY REGULATED
Conclusion
Application of red clover and synthetic phytohormones causes a transcriptional regulation of 117 genes in MCF-7.
The effect of synthetic Daidzein/Genistein differs significantly from that of red clover by upregulation of transcription of 10 genes (Metallothionins).
This first results do not implicate a major influence of red clover on the proliferation or differentiation of MCF-7 cells.
Experiment 2
Cells:
- Cell culture from primary mammae epithelial
cells (healthy cells)
Technology:- 20K Chip arrays (Affymetrix)
Experiment 2
Pooled sera of 5 menopausal women . Before and after intake of MF11RCE (menoflavon) for 3 month
• Control group = before intake of MF11RCE
• Treatment group = after intake of MF11RCE
Regulation of Transcription
0100200300400500600700800900
1000
TC
F4
TC
F7L2
MAFB
MXI1
ID4
HO
P
BTF3
BAZ1A
Gene
Estim
at
control
treatment
Regulation of Transcription
• MF11RCE inhibits the expression of transcriptional genes (TCF4,TCF7L2,MAFB,MXI1,ID4,HOP,BTF3,BAZ1A) and genes in relation to ribosomal subunit joining and the initiation of translation (EIF3S5, EIF4EBP, RPL29,RPS16)
= MF11RCE can influence cell proliferation and differentiation by down regulation of cellular transcription and translation
Genes involved in regulation of DNA
0
100
200
300
400
500
600
Gene
Estim
at
control
treatment
Genes involved in regulation of DNA
• MF11RCE down regulates genes involved with DNA replication.
(GMNN,H2AFV,HIST1H2,HMGB2,MC5,PCNA,PRKDC,RM1)
= MF11RCE has an anti-proliferative effect through inhibition of DNA replication
Apoptosis
0200400600800
100012001400160018002000
AFURS1 ENC1 PORIMIN
genes
sig
nal e
stim
ate
s
control
treatment
Apoptosis • MF11RCE induces Expression of
(AFURS1, ENC1/PIG10 p53 induced gene 10) and inhibits Porimin
= MF11RCE can rise the p53 level (ENCI/PIG10) and activate apoptosis.
= MF11RCE can induce senescence (AFURS1)
Regulation of cell proliferation
0
200
400
600
800
1000
1200
genes
signal
est
imat
es
control
treatment
Regulation of cell proliferation
• MF11RCE inhibits JAG1,RARRES, VEGF,MET. (cell proliferation) and cell differentation (Keratin 10, Keratin 19).
= MF11RCE inhibits genes of cell-proliferation and cell-differentation
In a culture of primary mammae epithelial cells MF11RCE down regulates genes for
– Transcription and translation– Protein synthesis– DNA replication– Cell proliferation and up regulates genes for
– Apoptosis
Conclusion
MF11RCE seems not to be inductive for tumor and/or tumor growth in a healthy mamma epithelial cells culture.
Further study are necessary to improve if Isoflavones can protect against tumor development an/or tumor growth
• Georg Steiner • Michael Loeffler• Anka Gocan • Marianne Imhof • Kristian Hrachowitz• Sylvia Molzer• Markus Lipovac
• Johannes Huber