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Role of B-Estrogen receptors stimulation in
the prevention and treatment
of colorectal polyps
Mariabeatrice Principi
Gastroenterology Section
D.E.T.O.
University of Bari
Estrogens regulate growth, differentiation, and
functioning of various target tissues
both within and outside the reproductive system.
The biological actions of estrogens are mediated by
estrogen binding
to one of two specific
Estrogen Receptors (ER)
ER-α and ER-β
Messa C, Di Leo A. Scand J Gastroenterol 2000
ER-α and ER-β exhibit
tissue-specific expression.
Variations in the phenotypes
of knock-out mice lacking ER-α or ER-β suggest
that these proteins have different biological activities.
In vitro and invivo studies in ER-β knock-out mice indicate
that ER-β is a modulator of ER-α activity.
ER-β is able to reverse the effects of ER-α and
to inhibit Estradiol-dependent proliferation.
Couse JF. J Steroid Biochem Mol Biol 2000
Hall JM Endocrinology 1999
Liu MM J Biol Chem 2002
Normal cells
ERα↑
α↑ ERβ↑↑↑
β↑↑↑
ERβ
β
Balance ERα
α
Growth
inhibition
Proliferation
Tumor cells
ERα↑↑↑
α↑↑↑ ERβ↓
β↓
Bardin, Endocrinol related cancer 2004
ERs and CRC
Incidence and mortality rates for CRC: F < M
(Potter,1993)
Risk and benefit of estrogen plus progestin in healthy
postmenopausal women: principal results from the Women’s
Health Initiative randomized controlled trial.
Rossouw J.E. et al. JAMA 2002
Reciprocal expression of ERα
α and ERβ
β is associated with estrogenmediated modulation of intestinal tumorigenesis.
Weyant M.J et al Cancer Research 2001
ER-β expression in large bowel adenomas: Implications in colon
carcinogenesis.
A. Di Leo et al Digestive and liver disease, 40 (2008) 260–266
The sharp decline of estrogen receptors
expression
in long-lasting ulcerative-associated carcinoma
M. Principi et al. (under review)
Silymarin
•Extract from the seeds of milk thistle
(Silybum marianum).
•is a mixture of four isomeric flavonoids (silybinin,
isosilybinin, silydianin and silychristin).
It is already used for the treatment of alcoholic
liver disease and as an anti-fibrotic agent.
•It is a selective ER-β agonist.
Wuttke-Seidlova D. J of Steroid Biochem & Mol Biol 2003
dietary administration of silymarin significantly
suppressed the development of
azoxymethane(AOM)-induced rat colonic carcinoma,
in conjunction with modulation
of cell growth in colonic adenocarcinoma.
Phytoestrogens, compounds derived
from plants, are structurally and functionally
related to estrogens
with an increased binding affinity vs ERβ.
• Isoflavones
: contained
many foods
Biologically
activein forms
are: such as
soy, legumes, tofu.
• •Lignans
: contained
in fruits,
vegetables,
Isoflavones:
Genistein
and
Daidzein grains
(corn, wheat, barley, rice, bran).
• Lignans: Enterolactone and Enterodiol
• Coumestans: contained in cereals
• Coumestans: Coumestrol
Diet
SYL
LIG
SYL+LIG
Reduced number and size of polyps
Lowest number of mice bearing HGD polyps
Barone M. et al Carcinogenesis 2010
Cancer
in DSS colitis
Modified Diet
Standard Diet
number and size of polyps
Histological score
C57BL/J6 male mice
aged 4 weeks (weight 22gr)
ER-alpha and beta
expression
M. Principi et al. (under review)
100th day
“Color-view Miniendoscopic System”
(Karl Storz, Tuttlingen, Germany).
Number of polyps
Volume of polyps
Number of
polyps <
3mm
Number of
polyps
≥ 3mm
Total polyp
number
Modified Diet
2.43±1.5
0.93±1.00
3.36±1.78
Modified Diet
2.17 ± 2.55 12.99 ± 19.66 15.16 ± 20.12
Standard Diet
5.68±3.26
3.52±2.1
9.20±3.26
Standard Diet
7.93 ± 5.71
Polyp volume Polyp volume
<3mm
≥ 3mm
Total volume
134.34 ±
113.58
ERβ LI (%)
Histological score
SCORE 0 SCORE 1 SCORE 2 SCORE 3
Modified
Diet
Standard
Diet
126.41 ±
113.89
72.73%
10.39%
9.09%
7.79%
46.78%
12.28%
10.53%
30.41%
Modified
Diet
Standard
Diet
Polyp
Proximal
Mucosa
Distal
Mucosa
Healthy
Mucosa
57,66±7,25
43,78±2,55
63,85±3,43
_
24,32±3,74
52,98±5,53
46,59±2,15
_
Could there be a possible chemopreventive
effect also in humans?
“A randomised, double-blind placebo-controlled study
to assess the effect of supplements containing a
selected mixture
of specific estrogen receptor beta agonists
on proliferative (Ki.67)
and apoptotic biomarkers (Tunel and Caspase-3)
of intestinal mucosa.”
n= 600 patients interviewed potential participants
Sixty patients, naïve for previous or
concomitant CRC chemoprevention, were randomised to:
Placebo or Active Dietary Intervention (ADI)
175 mg Silymarin +
20 mg flaxseed extract +
750 mg oat fiber extract
For 60 days before surveillance
screening colonoscopy.
Assessment of cell proliferation, apoptosis and levels of ERβ
in the non-adenomatous mucosa.
Urinary lignans-assessed phytoestrogens intake in the diet
showing active dietary intervention (ADI) compliance.
n=60 patients enrolled and randomised 1:1
n=30 ADI
T0
n=30 PLACEBO
TWICE/DAY for 60 days
n=10 drop-outs (7 Placebo, 3 ADI)
T60
n=50 final patients population
Phytoestrogen intake and compliance were assessed by
urinary measurements i.e., enterolignans enterodiol
(ED) and enterolactone (EL), at T0, T30 and T60
Endoscopy and Histology
Pancolonoscopy with polypectomy and 8 biopsies
Microbiological and Biochemical Analysis
ERβ and ERα mRNA (RT-PCR)
ERβ and ERα proteins (ELISA)
Immunohistochemistry, immunofluorescence
ER-β and ER-α expression,
Apoptosis (Caspase-3),
Proliferation (Ki-67)
Endoscopy and Histology
21/50 patients with recurrent polyps
Immunohistochemistry,
immunofluorescence
Microbiological and
Biochemical Analysis
ERβ protein
ERβ mRNA
ERα protein
ERα mRNA
ERβ/ERα protein
-α
NONRECURRENT
ERβ
protein
ERβ
mRNA
ERβ
LI
ERα
protein
ERα
mRNA
ERα
LI
TUNEL
LI
Caspase-3
LI
Ki-67
LI
0.805±0.13
2.278±1.1
9
47,533±15.4
7
0.423±0.0
6
0.295±0.2
15.333±9.7
6
39.444±15.
6
55.33±20.41
6
43.777±17.2
4
1.105±1.0
7
34,875±16.6
7
0.532±0.1
1
0.159±0.3
2
16.750±8.8
6
31.375±12.9
3
29.53±9.13
45.31±11.29
ADI
PL
0.773±0.13
In patients without polyp recurrence, there was a significant increase of the
ER-β pattern (mRNA, protein and LI) in the ADI vs the placebo group.
Chemopreventive dietary manipulation,
targeted to the selective upregulation of ER-β,
of intestinal carcinogenesis ????
Scopo della tesi
È stato condotto uno studio randomizzato, in doppio cieco, controllato
con placebo, allo scopo di valutare se una supplementazione dietetica
di breve durata (60 gg) con una miscela brevettata di fitoestrogeni e
fibre insolubili (EVIENDEP®), fosse in grado di determinare una
significativa variazione dell’espressione degli Ers nella mucosa colica
sana di pazienti sottoposti a sorveglianza endoscopica per una
pregressa polipectomia.
È stata valutata, inoltre, l’espressione di biomarkers di proliferazione
epiteliale (Ki-67) e di apoptosi (TUNEL e Caspasi-3)
L’analisi dei risultati è stata condotta in due step:
1. EVIENDEP Vs PLACEBO
2.
Suddivisione in 4 sottogruppi:
ADI con polipi recidivanti
ADI senza polipi
recidivanti
PL con polipi recidivanti
PL senza polipi recidivanti
Materiali e metodi
1. DISEGNO DELLO STUDIO E POPOLAZIONE
IN ESAME600 pazienti potenzialmente arruolabili
Maschi e femmine in post-menopausa
Età ≥ 50 anni
Criteri
preliminari
Pregressa colonscopia con polipectomia
Colonscopia di sorveglianza programmata a 3-5
anni
SCREENI
NG
CRITERI DI INCLUSIONE
CRITERI DI ESCLUSIONE
Parametri biochimici ematici (Hb ≥
MICI, neoplasie maligne, malattia
12 g/dl, PLT ≥ 120.000/mm3,
renale, BMI ≥ 30, anemia o
Azotemia ≤ 40 mg/dl,M)
coagulopatie
P.A. normale o ipertensione
Chemioterapia o Tx
controllata farmacologicamente
corticosteroidea entro 6 mesi
dall’arruolamento
Tx antiaggregante o
anticoagulante entro 30 gg
dall’arruolamento
HRT, SERMS, entro 6 mesi
dall’arruolamento
ASA e altri FANS entro 6 mesi
2. ENDOSCOPIA E ISTOLOGIA
Sono stati registrati numero,
localizzazione, dimensioni dei polipi
visualizzati durante l’esame endoscopico
È stato seguito un protocollo standard
per la rimozione:
Polipi < 0.5 cm elettrocoagulazione
Polipi > 0.5 cm rimozione con pinza
bioptica ed esame istologico
Sono state eseguite 8 biopsie su mucosa sana del sigma per ogni
partecipante:
7 in azoto liquido per analisi biochimiche e microbiologiche
1 fissata in formalina al 4% per l’immunoistochimica.
3. ANALISI BIOCHIMICHE E
MICROBIOLOGICHE
mRNA di ERβ e di ERα attraverso real time polymerase chain reaction (RTPCR);
Proteine di ERβ e di ERα attraverso dosaggio ELISA (Enzyme-Linked
ImmunoSorbant Assay).
4. IMMUNOISTOCHIMICA E
IMMUNOFLUORESCENZA
Espressione dei ERs;
Proliferazione cellulare attraverso il Ki-67;
Apoptosi attraverso la Caspasi-3.
Il dosaggio della Caspasi 3 è stato condotto in modo da permettere la
valutazione sia della sua espressione esclusiva che della co-espressione con
ERβ.
Risultati
1. RISULTATI ENDOSCOPICI ED
ISTOLOGICI
n=50 hanno eseguito la colonscopia al
T60
n=21 (42%) riscontro di
polipi recidivanti
58% elettrocoagulati in situ
42% esame istologico
9% iperplastici
14% tubulari
14% tubulari con displasia
di basso grado
5% tubulo-villosi con
displasia di basso grado
2. ANALISI MOLECOLARE DEI RECETTORI DEGLI
ESTROGENI
Placebo
ADI (N=27)
(N=23)
mean±
±SD
P value
mean±
±SD
ERβ protein
0,768±0,10
0,822±0,08
0.04
ERβ mRNA
0,994±0,99
1,266±1,24
0.1
ERα protein
0,51±0,11
0,49±0,12
0.5
ERα mRNA
0,139±0,28
0,230±0,24
0.2
1,571±0,42
1,734±0,20
0.07
ERβ/ERα
protein
3. VALUTAZIONE DEI MARKERS
IMMUNOISTOCHIMICI
LI
Placebo (N=23)
ADI (N=27)
mean±
±SD
mean±
±SD
P value
ERβ
37,708±5,31
39,222±2.69
0.06
ERα
20,416±10,71
16,481±10,67
0.2
ERβ/ERα
2,437±1,53
6,564±10,04
0.06
TUNEL
31,541±11,54
35,592±14,97
0.07
Caspase-3
29,717±7,98
32,937±13,54
0.1
Ki-67
44,833±10,38
53,923±20,91
0.07
Il test di correlazione di Spearman ha mostrato che, nel gruppo ADI, l’ ERβ LI è
direttamente collegato al TUNEL e alla Caspasi-3
ERβ E APOPTOSI
A.
Espressione del recettore β degli estrogeni nucleare (segnale rosso);
B.
Espressione della Caspasi-3 nucleare (segnale verde);
C.
Contrasto nucleare con TOPRO-3 (segnale blu);
D.
Fusione dei tre segnali. Entrambe le molecole sono localizzate nel terzo superiore delle cripte.
4. ESPRESSIONE DEI BIOMARKERS NEI PAZIENTI CON E SENZA
RECIDIVA DI POLIPI
RECURRENT
ADI
PL
NONRECURRENT
ERβ
protein
ERβ
mRNA
ERβ
LI
ERα
protein
ERα
mRNA
ERα
LI
TUNEL
LI
Caspase-3
LI
Ki-67
LI
0.822±0.0
8
0.730±0.9
35,142±18.8
0.518±0.1
0.209±0.2
18.785±10.0
0
31.93±13.3
29.09±12.7
53.14±21.73
9
0
3
6
3
9
0.763±0.0
1.474±0.6
38,833±12.8
4
0.485±0.1
0.09±0.11
32.00±10.5
31.000±3.8
5
2
ERβ
protein
ERβ
mRNA
ERβ
LI
ERα
protein
ERα
mRNA
ERα
LI
TUNEL
LI
Caspase-3
LI
Ki-67
LI
0.805±0.13
2.278±1.1
9
47,533±15.4
7
0.423±0.0
6
0.295±0.2
15.333±9.7
6
39.444±15.
6
55.33±20.41
6
43.777±17.2
4
1.105±1.0
7
34,875±16.6
7
0.532±0.1
1
0.159±0.3
2
16.750±8.8
6
31.375±12.9
3
29.53±9.13
45.31±11.29
29.50±10.23
1
43.66±10.31
4
ADI
PL
0.773±0.13
Riassumendo…
Questo studio è stato progettato per verificare se un supplemento dietetico
costituito da silimarina e lignani potesse modificare i livelli di Ers nella mucosa
colica umana.
L’EVIENDEP ha determinato un aumento significativo del contenuto medio delle
proteine di ERβ, come pure una tendenza verso l’aumento del rapporto ERβ/ ERα.
I livelli di mRNA di ERβ, invece, pur risultando più elevati rispetto al gruppo
Placebo non hanno raggiunto differenze significative.
L’espressione di Erα (proteine ed mRNA) si è rivelata simile tra i due gruppi.
È stata mostrata una proporzionalità diretta tra ERβ e l’apoptosi, confermando
quanto è stato dimostrato in precedenza nel modello murino ApcMin/+ .
La somministrazione di EVIENDEP è stata incapace di influenzare le recidive dei
polipi, in quanto il breve periodo di somministrazione (60gg) non è stato
progettato con lo scopo di dimostrare l’effetto chemiopreventivo dell’ADI sulla
crescita dei polipi intestinali.
Conclusioni e sviluppi futuri
In conclusione, questo studio ha chiaramente confermato il
ruolo di ERβ sul controllo dell’apoptosi e la possibilità che la
sua espressione possa essere modificata da un supplemento
dietetico a base di fitoestrogeni.
Questi risultati potrebbero stimolare ulteriori studi, anche più
estesi, volti a intraprendere l’obiettivo di dimostrare le
potenzialità dell’EVIENDEP nell’ambito della
chemioprevenzione secondaria del CCR.
GRAZIE PER L’ATTENZIONE
Tab.1: Study design and flow
N=600 patients contacted by phone for study presentation and invitation to participate.
•20% refused to participate or to undergo the preliminary interview
•20% had the scheduled screening colonoscopy already performed
•40% were prescribed cardioaspirin
•5% developed an active tumor (breast, liver, stomach), or other invalidating pathology
•5% were deceased
n=60 enrolled and 1:1 randomized
n= 60 urinary lignan assessment at baseline (T0)
n=30 allocated to Placebo
n=30 allocated to ADI
n=26 completed the 60 days oral supplementation
n=4 withdrew consent in advance of T60 colonoscopy
n=3 lost to T60 colonoscopy:
n=1 protocol violation because of NSAID
prescription for flu symptoms
n=1 protocol violation because not inscribed
in surveillance program by screening
colonoscopy
n=1 dropped-out because of surgery
n=23 underwent T60 colonoscopy
and assessed for study end-points
n=27 completed the 60 days oral supplementation
n=3 withdrew consent in advance of T60 colonoscopy
n= 50 urinary lignan assessment at 30 day
of supplementation (T30)
n= 50 urinary lignan at
60 day of supplementation (T60)
n=27 underwent T60 colonoscopy
and assessed for study end-points
n=50 Per Protocol Population (PPP)
Tab. 2: Baseline Demographics (PPP) and comparison between the study groups
DEMOGRAPHIC DATA
Sex
Age (years)
Weight (kg)
Height (cm)
Season of
Enrolment
PL
(n=23)
ADI
(n=27)
PPP
(n=50)
Male
n (%)
17 (73,9%)
19 (70,4%)
36 (72,0%)
Female
n (%)
6 (26,1%)
8 (29,6%)
14 (28,0%)
61,6±5,2
62,6±6,5
62,1±5,9
60,0
63,0
61,0
75,5±13,8
78,8±12,6
77,1±13,1
80,0
80,0
80,0
169,5±9,0
170,6±9,5
170,1±9,2
172,0
172,0
172,0
mean±sd
median
mean±sd
median
mean±sd
median
Spring
n (%)
2 ( 8,7%)
5 (18,5%)
7 (14,0%)
Summer
n (%)
2 ( 8,7%)
6 (22,2%)
8 (16,0%)
Autumn
n (%)
15 (65,2%)
11 (40,8%)
26 (52,0%)
Winter
n (%)
4 (17,4%)
5 (18,5%)
9 (18,0%)
P*
1.00
0.56
0.32
0.67
0.29
Tab. 3: Urinary lignan (ng/ml) at different timepoints
Placebo
(n=23)
ADI
(n=27)
P
ED+ELT0
697.8±963.3
410.3±336.9
0.27
ED+ELT30
472.4±475.5
3300.1±1535.5
<.001
ED+ELT60
116.0±235.2
327.5±313.1
<.001
Δ ED+ELT30-T0
-251.2±1085.9
2889.8±1445.0
<.001
Δ ED+ELT60-T0
-393.9±415.0
-82.8±380.7
<.02
EDT0
117.3±135.6
51.8±55.0
0.06
EDT30
65.6±63.1
1084.6±1066.9
<.001
EDT60
17.2±48.5
77.2±75.1
<.001
Δ EDT30-T0
-57.1±156.4
1032.8±1041.6
<.001
Δ EDT60-T0
-89.9±105.6
25.4±96.7
<.001
ELT0
580.5±853.3
358.5±335.0
0.35
ELT30
391.3±440.8
2215.5±1366.2
<.001
ELT60
81.6±177.4
250.3±275.2
<.001
Δ ELT30-T0
-189.2±931.1
1857.0±1199.1
<.001
Δ ELT60-T0
-498.8±861.9
-108.2±320.1
<.01
Tab 4: ERs protein content and patients distribution around the ERs cut-off value
ERβ
β
mean±
±sd
Median (cutoff value)
≤ 0.82
ERα
α
PL
(n=23)
ADI
(n=27)
PPP
(n=50)
0.77±
±0.
1
0.82±
±0.1
0.8±
±0.1
0.76
0.84
15
(65.22%)
P
0.04
10
(37.04%)
0.82
25
(50.0%)
> 0.82
8
(34.78%)
17
(62.96%)
25
(50.0%)
mean±
±sd
0.51±
±0.1
0.49±
±0.1
0.50±
±0.1
Median (cutoff value)
0.50
0.45
0.47
≤ 0.47
8 (34.8%)
18
(66.7%)
26
(52.0%)
0.05
0.44
0.02
> 0.47
15
(65.2%)
9
(33.3%)
24
(48.0%)
Tab.5:Biomarkers in the non adenomatous mucosa of non-recurrent and recurrent patients
BIOMARKER
IN
NORMAL
MUCOSA
NON-RECURRENT
N (%)
RECURRENT
N (%)
PPP
N=25
PLACEBO
N=7/23 (30.4)
In situ n=3
(42.9)
P+H n=4 (57.1)
ADI
N=18/27 (66.7)(a)
(P=0.03)
In situ n=10
(55.6)
P+H n=8
(44.4)
0.161±
±0.2
mean±
±sd
(%:IHC)
PPP
N=25
PLACEBO
N=16/23 (69.6)
ERα mRNA
0.202±
±0.3
0.149±
±0.3
0.296±
±0.3
0.132±
±0.2
0.057±
±0.1
ERα (%)
16.240±
±9.0
16.750±
±8.9
15.333±
±9.8
19.840±
±12.0
27.000±
±11.4(e)
(P= 0.04)
ERα (Elisa)
0.496±
±0.1
0.532±
±0.1
0.431±
±0.1(c)
(P= 0.02)
0.506±
±0.1
0.473±
±0.1
0.518±
±0.1 (f)
(P= 0.05)
Ki-67 (%)
48.920±
±15.6
45.313±
±11.3
55.333±
±20.4
50.583±
±19.2
44.286±
±9.6
53.176±
±21.8
ERβ mRNA
1.483±
±1.3
1.036±
±1.1
2.278±
±1.2(c)
(P= 0.03)
0.838±
±0.9(b)
(P= 0.03)
1.041±
±0.8
0.759±
±1.0(f)
(P= 0.004)
ADI
N=9/27 (33.3)
17.056±
±11.3
ERβ (%)
39.440±
±17.1
34.875±
±16.7
47.556±
±15.4
36.560±
±15.4
40.429±
±12.5
35.056±
±16.5
ERβ (Elisa)
0.785±
±0.1
0.773±
±0.1
0.806±
±0.1(a)
(P= 0.04)
0.807±
±0.1
0.767±
±0.0
0.822±
±0.1(d)
(P= 0.04)
Caspase-3 (%)
33.120±
±12.5
29.563±
±9.2
39.444±
±15.6
30.125±
±10.1
31.500±
±3.8
29.667±
±11.5
TUNEL (%)
35.840±
±15.5
31.375±
±12.9
43.778±
±17.2(c)
(P= 0.04)
31.60±
±11.4
31.857±
±9.6
31.500±
±12.2(f)
(P= 0.03)
Fig 1. Colocalization of ERβ and Caspase-3 immunostained cells in cryptae-villi
from normal mucosa. From upper left to right panel: ERβ (red), Caspase-3
(green), TOPRO-3 (blue) counterstained nuclei in cryptae-villi, colocalization of
the three stainings at x400.From lower left to right panel: ERβ; Caspase-3;
TOPRO-3 (blue); co-localization at x1200.
The presence of estrogen receptor
(cytosolic and nuclear)
in colorectal tissue has been demonstrated
in several studies
TC. Alford et al. Cancer- 1979
NY. Zhu et al. Clin Med J -1981
A. Di Leo et al. Dis Colon Rectum 1992
Polyamine levels are higher
in ER negative colon cancer
than ER positive
•The estrogen receptors are poorly expressed in the tumor
mucosa compared to healthy mucosa surrounding the tumor.
•Cancers of the colon ER - have a worse prognosis of ER +.
The subtypes of the estrogen receptor
ER-α and ER-β have been demonstrated
in both colorectal and gastric tissue
with a different expression of
ER-β in tumor tissue than normal
N. Takano et al. Cancer Letters 2002;176:129-35
A. Cavallini, A. Di Leo. Dig Dis Sci 2002; 47:2720-8
ER-β
mRNA
–
+
c-myc and cyclins ( A;D1)
p21(Cip1) and p27(Kip1)
cell cycle arrest
(phaseG2)
(Paruthiyl, Cancer Res 2004)
Estrogens regulate
growth, differentiation, and functioning
of various target tissues
both within and outside the
reproductive system.
Fisher B Cancer Res 1984
Couse JF Endocr Rev 1999
Pettersson K Annu Rev Physiol 2001
Francavilla A, Di Leo A. Hepatology 1989
Messa C, Di Leo A. Scand J Gastroenterol 2000
Barone M, Di Leo A. Dig Dis Sci 2006
ESTROGENS
• Steroid hormones
• Major production site: ovary
• Estradiol: the most powerful body estrogen
History of Estrogen Receptor
Identification of estrogen receptor
Jensen E.W. , DeSombre E.R.:
"Mechanisms of action of female sex hormones"
Annu Rev Biochem. 1972
Discovery of receptor β (485AA, pm 54.2 Kda)
S. Mosselman, J. Polman, R. Dijkema :
“ER β: identification and characterization of a novel oestrogen receptor“
FEBS LETT- 1996
Estrogen Receptors contain the evolutionarily
conserved structural and functional domains
DNA binding
AF-1
ERα
ERβ
NH-
NH-
Ligand binding
Dimerization
AF-2
-COOH
-COOH
ER-α and ER-β are the products of separates genes present
on distinct chromosomes (locus 6q25.1 and locus 14q23-24.1,
respectively)
Menasce LP. Genomics 1993
Estrogen receptors genomic pathway.
Estrogens (E2) enter the cell by diffusion and bind to specific receptor
proteins (ERs). Upon estrogen binding, ERs either bind effectively to
DNA elements of their target genes or activate other genes through
the interaction with AP-1 site.
In both cases, ERs activate transcription, protein synthesis and finally
several biological effects.
Estrogen receptors non-genomic pathways
Palmitoylation of cytosolic ERs allows them to localize at the plasma membrane
where they associate with caveolin-1 (CAV).
Upon estradiol (E2) stimulation, ER-a is de-palmitoylated and dissociated from
caveolin-1, stimulating signals of cell proliferation.
On the contrary, after binding to ER-b, E2 increases the association of the
receptorial complex with caveolin-1 and p38 (a member of the MAPK family), in
order to promote apoptosis
ER-α
α
Cell Growth
ER-β
Inhibition
Cell Growth
Estrogen
and
colorectal cancer
Inverse association between the risk of
large adenomas (> 1 cm) and current use
of hormone replacement therapy.
(Grodstein F. Ann Intern Med. 1998)
Normal
N=4
UC
N=8
UC-Dysplasia
N=4)
P value°
BETA ER
35.0±3.3
26.4±15.3
31.2±1.7
0.17
ALPHA ER
21.5±1.9
23.4±6.2
25.5±1.0
0.22
BETA/ALPHA
1.6±0.2
1.2±0.7
1.2±0.1
0.15
Tunel
16.7±1.7
17.0±4.4
22.7±0.9
0.05
Ki-67
24.5±4.8
39.6±10.7
65.2±8.8
0.004
Tunel/Ki-67
0.7±0.1
0.4±0.2
0.5±0.3
0.14
The fall of estrogen receptors expression in long-lasting ulcerative-associated
carcinoma
M. Principi et al.
Normal
(8)
UC
(14)
LGD
(10)
HGD
(12)
CRC
(12)
p value
Subanalisys
ER α
24.2 ± 5.2
23.3 ± 4.3
25.4 ± 1.2
42.2 ± 6.2
59.8 ± 5.1
< 0.0001
CRC > HGD > LGD
= UC= Normal
ER β
34.3 ± 3.1
26.8 ± 7.8
29.4 ± 3.7
19.6 ± 5.1
18.4 ± 8.1
< 0.0001
LGD = Normal > UC
> HGD = CRC
Beta/alpha
1.42
1.15
1.16
0.46
0.31
Ki 67
26.02 ± 3.3
37.9 ± 6.4
45.7 ± 6.2
60.6 ± 5.2
71.1 ± 5.1
< 0.0001
CRC > HGD > LGD
> UC > Normal
TUNEL
17.9 ± 3.5
20.1 ± 2.5
30.7 ± 5.2
15.3 ± 3.1
10.1 ± 1.9
< 0.0001
LGD > UC = Normal
> HGD > CRC
TUNEL/Ki 67
0.68
0.53
0.67
0.25
0.14
ANOVA più correzione secondo Bonferroni
Preliminary data
Etiopathogenesis of Colorectal Cancer
Environmental factors (diet, exercise, obesity, smoking and
alcohol intake )
Genetic factor:
-Approximately 25% of colorectal cancers occur in
individuals with a family history of the disease, including 5%
caused by the genetic syndromes familial adenomatous polyposis
(FAP) or hereditary non-polyposis colorectal cancer (HNPCC).
- Risk is also higher in individuals with inflammatory bowel
disease.
Prognosis.
The overall 5-year survival rate for colorectal cancer in England
and Wales is approximately 50% but varies according to the
stage of disease at diagnosis.
Colorectal Tumorigenesis
Several lines of epidemiologic, clinical
and experimental evidences have been
reported showing that estrogen hormones
may be involved in malignant
colorectal tumors.
Epidemiological
Data
Biological
Data
The presence of estrogen receptor
(cytosolic and nuclear)
in colorectal tissue has been demonstrated
in several studies
TC. Alford et al. Cancer- 1979
NY. Zhu et al. Clin Med J -1981
A. Di Leo et al. Dis Colon Rectum 1992
Polyamine levels are higher
in ER negative colon cancer
than ER positive
•The estrogen receptors are poorly expressed in the tumor
mucosa compared to healthy mucosa surrounding the tumor.
•Cancers of the colon ER - have a worse prognosis of ER +.
The subtypes of the estrogen receptor
ER-α and ER-β have been demonstrated
in both colorectal and gastric tissue
with a different expression of
ER-β in tumor tissue than normal
N. Takano et al. Cancer Letters 2002;176:129-35
A. Cavallini, A. Di Leo. Dig Dis Sci 2002; 47:2720-8
Experimental
Data
Dimethylhydrazine
risk of CRC in rats (M>>F)
Administration of 17β
β estradiol
CRC-dimethylhydrazine induced in male rats
and female
(Singh S. Gut 1995)
Protective effect of estrogen on the
development of colorectal cancer has
been studied in an animal model
APCMin / + mice
C57BL/6J-Min/+mice:
After ovariectomy
intestinal adenomas (77%)
Administration of 17betaestradiol
intestinal adenomas
ER-β
β
ER-α
α
(Weyant M.J. Cancer Research 2001)
ER beta and
Colorectal Cancer
Today
We have recently demonstrated a significant
reduction in the expression of ER-β
already in the precancerous
stage of colorectal cancer.
Di Leo et al Digestive Liver Disease 2008
In this study adenomatous samples were
obtained from 25 patients
(M/F: 13/12, aged 55-75 years, mean 65 years)
found to be affected
by colonic polyps and treated with
endoscopic polypectomy.
Results
Normal colonic
mucosa
Adenomatous
tissue
Fig 1 Immunohistochemical evaluation of ER-β expression in normal colonic
mucosa and adenomas. (A) Normal colonic mucosa in which most enterocytes,
with the exception of some (black arrows), show consistent immunoreactivity
for ER-β (B) Adenomatous tissue in which only cells clusters (black arrow)
show nuclear positivity for ER-β.
Results
Fig 2 Evaluation of ER-α and ER-β
labelling index in normal colonic mucosa
and in adenomatous polyps.
Fig 3 Evaluation of PCNA and PARP
labelling indexes in normal colonic
mucosa and adenomas.
Results
Fig 4. Correlation between ER-β expression and PCNA
labelling index in adenomas.
Normal mucosa
ER-β
Results
Adenomatous tissue
ER-β
PCNA
PCNA
ER-β and PCNA
ER-β and PCNA
Fig 5 Evaluation by confocal microscopy of ER-β and PCNA immunolabelling. Inverse relationship
exists between ER-β and PCNA expression. Colonocytes showed elevated ER-β (5A) and low PCNA
immunolabelling (5B) in normal mucosa as compared to adenomatous polyps (5D and E, respectively).
Our data not only confirm the involvement
of ER-β in colorectal cancer but suggest a
possible explanation for the protective
effect of oestrogens on
cancer development.
In order to verify the key role
of ER-β the pathogenesis of CRC, our
research group has carried out an
experimental study of 6 patients with
“Familial Adenomatous Polyposis” (FAP)
(4 ♂ and 2 ♀)) aged between
29 and 52 years who undergoing colonoscopy
and subsequent colectomy.
Barone M, Di Leo A. Scand J of Gastroent 2010
Results
Normal mucosa
Adenomas with
low-grade dysplasia
High-grade
dysplasia
Carcinomatous
tissue
Fig 1. ER-β expression in normal, dysplastic and carcinomatous colon tissues from
patients with FAP.
Results
Normal mucosa
Adenomas with
low-grade dysplasia
High-grade
dysplasia
Carcinomatous
tissue
Fig 2. ERa expression in normal, dysplastic and carcinomatous colon tissues from
patients with FAP.
Results
Normal mucosa
Adenomas with
low-grade dysplasia
High-grade
dysplasia
Carcinomatous
tissue
Fig 3. Ki-67 expression in normal, dysplastic and carcinomatous colon tissues
from patients with FAP.
Results
Normal mucosa
High-grade
dysplasia
Adenomas
with low-grade
dysplasia
Carcinomatous
tissue
Fig 4. Cell apoptosis evaluated by TUNEL in normal, dysplastic and carcinomatous
colon tissues from patients with FAP.
Results
Fig.5 Correlation of ER-β expression with Ki-67 or TUNEL. The reported values were
obtained in normal, mild dysplastic and high dysplastic tissues of six patients.
This study demonstrates a precise
correlation between reduced ER-β expression and
progression toward neoplasia concomitant with
decreased apoptosis, in FAP patients.
Our results support the evaluation
of ER-β levels as a surrogate biomarker prognostic for
tumor progression, and suggest the use
of ER-β as possible therapeutic target
for chemoprevention
in patients at high risk of colonic neoplasia.
Confirmation of the role of ER-β
in
intestinal carcinogenesis
ESTROGENS:
possible chemopreventive
but many side effects,
mediated primarily by
ERα!!!
Future Prospects
Phytoestrogens
Epidemiological studies have shown a low
incidence of CRC among asian populations whose
diet is rich in soy, a source of
Phytoestrogens (isoflavones).
Dietary Factor
easily modified!!!
Phytoestrogens
Phytoestrogens are compounds derived
from plants, are structurally and functionally
related to estrogens with increased binding
affinity vs ERβ.
Classification of Phytoestrogens
Phytoestrogens are divided into
3 main groups:
• Isoflavones: content in many foods such as
soy, legumes, tofu.
• Lignans: content in fruits, vegetables, grains
(corn, wheat, barley, rice, bran).
• Coumestans: content in the forage.
Phytoestrogens
Biologically active forms are:
• Isoflavones: Genistein and Daidzein
• Lignans: Enterolactone and Enterodiol
• Coumestans: Coumestrol
Silymarin
•Extracted from the seeds of milk thistle
[Silybum marianum].
•Is a mixture of four isomeric flavonoids (silibinin,
isosilibinin, silydianin and silychristin).
•It is already used as for the treatment of alcoholic
liver disease and as an anti fibrotic agent.
•Is a selective ER-β agonist.
Wuttke-Seidlova D. J of Steroid Biochem & Mol Biol 2003
Kohno et al. have shown that dietary administration
of silymarin significantly suppressed the development
of azoxymethane(AOM)-induced rat colonic carcinoma,
in conjunction with modulation of cell growth in the
colonic adenocarcinoma.
120 rats aged 5 weeks were randomly divided into 7 groups. Groups 1–5 received 3 weekly s.c. injections of AOM
(15 mg/kg body weight). Rats in groups 2 and 3 were fed diets containing 100 or 500 ppm silymarin for 4 weeks,
respectively, commencing 1 week before the first dose of AOM.Groups 4 and 5 were fed diets mixed with 100 and
500 ppm silymarin, respectively, for 30 weeks of the postinitiation phase, starting 1 week after the last
administration of AOM. Group 6 was fed the diet mixed with 500 ppm silymarin for the entire study period (34
weeks). Group 7 served as an untreated control.
We recently conducted an experimental study to
evaluate the association between phytoestrogensdietary factors and the development of colorectal
cancer in male mice genetically predisposed to
develop intestinal polyps (pre-cancerous) as carriers
of the somatic mutation of tumor suppressor gene
Adenomatous Polyposis Coli (APC).
Barone M, Di Leo A. Carcinogenesis 2010
Forty-five intact male Apc
Min/+
mice:
- 15 diet fed carcinogenic (control)
- 15 fed with control diet with the addition of
0.02% silymarin
- 9 mice fed with control diet + 6.24% lignin
- 6 mice fed with control diet with the addition of
0,005% silymarin + 6,24% lignin
Results
Results
Results
24 h
Fig 1. ERβ:ERα mRNA ratio and
ERβ and ERα protein expression
in WT and ApcMin/+ mice.
48 h
72 h
Fig 2. Enterocyte migration along the
crypt/villus axis at different times after
BrdU labeling in Apc Min/+ mice.
Results
Non-adenomatous
tissue in ApcMin/+
mice
Fig 3. Epithelial cell proliferation in WT
animals and in normal and adenomatous
tissues in ApcMin/+ mice.
Adenomatous tissue
in ApcMin/+ mice
Fig 4. Cell apoptosis evaluated by TUNEL
and cleaved caspase-3 (CC-3) protein
expression in WT and ApcMin/+ mice.
Our data support appropriate dietary
management as a feasible approach to
counteract polyp development on the
Apc mutated mucosa, targeted to the
selective upregulation of ERβ.
In our experimental conditions,
ERβ increased with no significant change
in ERa expression, thus supporting ERβ
as an inhibitory mediator of aberrant
intestinal proliferative phenomena,
favoring the maintenance of proper
pro-apoptotic activity.
Our dietary-managed ApcMin/+ mice
produced a reduced tumor multiplicity
and size, an increased epithelial cell
migration, a reduced cell proliferation
and an increased apoptosis and a lowgrade dysplasia.
Animal models: C57BL/6J-APC
min/+
and wild-type male mice
APC min/+ mice were divided into two groups of 18 animals each
and given:
0.2 mL vehicle (0.5% w/v carboxy methyl cellulose and 0.025%
Tween 20 in distilled water)
or 750 mg silibinin/kg body weight by p.o. gavage in 0.2 mL
vehicle for 5 d/wk for 13 wk.
Negative controls (n = 9 per group) of wild-type mice were given
vehicle or same silibinin treatment as for APC min/+ mice.
Results
Fig 1. Silibinin treatment of APCmin/+ mice resulted in a strong inhibition in
intestinal tumorigenesis in terms of decreased polyp number, size, and appearance
In small intestine
Results
Fig 2. Silibinin feeding inhibits proliferation and induces apoptosis selectively
in small intestinal polyps of APCmin/+ mice.
This study clearly show:
In vivo antiproliferative and proapoptotic effects of
silibinin in polyps, which collectively contribute to its
strong chemopreventive efficacy against spontaneous
intestinal tumorigenesis in APCmin/+ mice.
Present findings underscore the possibility that
silibinin would be an effective agent in CRC prevention
trials for patients with FAP.
Further demonstration of in vivo efficacy
chemopreventive dietary manipulation of
intestinal carcinogenesis !!!
Possible chemopreventive effect also
in humans?
“We can conclude that estrogens are important in
protecting against CRC initiation and progression,
and that the protective effect most likely is
mediated by ERβ.
Several clinical trials of ERβ-specific ligands are
ongoing for different indications such as the
prevention of menopausal symptoms. To our
knowledge, however, no clinical trials of ERβspecific ligands are being or have been performed
for the prevention or treatment of CRC. Such trials
are needed to finally establish the significance of
ERβ as a target of CRC prevention or therapy.”
NOTE
La Sicurezza è stata valutata attraverso la determinazione
dei segni vitali ed esami ematochimici al basale (T0), dopo
30 (T30) e 60 (T60) giorni di supplementazione
La Compliance dei pazienti all’assunzione degli integratori
è stata valutata mediante la misurazione dei livelli di
fitoestrogeni urinari (ED ed EL) a T0, T30 e T60
Normal mucosa
ER-β
Adenomatous tissue
ER-β
PCNA
PCNA
ER-β and PCNA
ER-β and PCNA
Fig 5 Evaluation by confocal microscopy of ER-β and PCNA immunolabelling. Inverse relationship
between ER-β and PCNA expression. Colonocytes showed elevated ER-β (5A) and low PCNA
immunolabelling (5B) in normal mucosa as compared to adenomatous polyps (5D and E, respectively).
We recently conducted an experimental study to
evaluate the association between phytoestrogensdietary factors and the development of colorectal
cancer in male mice genetically predisposed to
develop intestinal polyps (pre-cancerous) as carriers
of the somatic mutation of tumor suppressor gene
Adenomatous Polyposis Coli (APC).
Barone M, Di Leo A. Carcinogenesis 2010
Our data support appropriate dietary
management as a feasible approach to
counteract polyp development on the
Apc mutated mucosa, targeted to the
selective upregulation of ERβ.
In our experimental conditions,
ERβ increased with no significant change
in ERa expression, thus supporting ERβ
as an inhibitory mediator of aberrant
intestinal proliferative phenomena,
favoring the maintenance of proper
pro-apoptotic activity.
Our dietary-managed ApcMin/+ mice
produced a reduced tumor multiplicity
and size, an increased epithelial cell
migration, a reduced cell proliferation
and an increased apoptosis and a lowgrade dysplasia.
Our group recently conducted a
randomized double blind and
placebo controlled study
to evaluate the impact of
ERβ-targeted dietary supplementation
on top of the common
diet on balance between proliferation and
apoptosis in colon epithelial.

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