Carla Giordano

Gli effetti dei fitoestrogeni nel trattamento delle malattie mitocondriali

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t0 vehicle 17bE2 ici+17bE2

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0123456

1 2 3 4 5PGC-1α PGC-1β NRF1 NRF2 TfamC

to ICI E2 ICI+E2

0200400600800

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to 20' 30' 1h 3h 24h

mtD

NA

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y/ce

ll

Gl+E2

Glu

Gal+E2

Gal

A B0

200400600800

100012001400

t0 vehicle 17bE2 ici+17bE2

mtD

NA

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y/ce

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B

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0123456

1 2 3 4 5PGC-1α PGC-1β NRF1 NRF2 Tfam0123456

1 2 3 4 5PGC-1α PGC-1β NRF1 NRF2 TfamC

to ICI E2 ICI+E2

0200400600800

100012001400

to 20' 30' 1h 3h 24h

mtD

NA

cop

y/ce

ll

Gl+E2

Glu

Gal+E2

Gal

A B

   *   toE2ICI+E2

toE2ICI+E2

toE2ICI+E2

toE2ICI+E2WT  11778  

0,0

0,2

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1,0

1,2

1,4

control 11778/ND4 3460/ND1 14484/ND6

*   **   *   *  

   control                      11778                  3460                  14484  

ROS

glu glu+E2 gal gal+E2 gal+E2+I0

50

100

150control11778346014484

°°° †

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°

*MFI

mit negative control merge ERβ negative control

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May the selective target of ERβ prevent or delay the development of LHON in unaffected

mutation carriers?

ERER

N Engl J Med 1999; 340:1801-1811 Journal of Endocrinology 1999; 163: 379-383

Erα,  ERβ  

ERα,    ERβ    

ESTROGEN  RECEPTORS  

ERα,    ERβ    

ERβ  

ER

   C2  

 C1 ER    Estrogen Responsive

ER

TRANSCRIPTION  Elements

5’AGGTCAnnnTGACCT 3’

citosol

17β-E

GENOMIC  ACTION  OF  ESTROGENS      

ER P

GFR

ESTROGEN RECEPTORS AS A TARGET FOR BREAST CANCER THERAPY

In 1896, Dr George Beatson noted that removal of the ovaries caused tumor regression for some women with metastatic breast cancer Selective estrogen receptor modulators (i.e. Tamoxifen) are well established in the treatment of estrogen receptor-positive breast tumors.

Estrogens had been used for the treatment of postmenopausal symptoms and osteoporosis, but were found to cause serious side-effects such as breast cancer, endometrial cancer, and thromboembolism. In the early 1990 SERMs have been developed which offer the benefit of dissociating the favorable estrogenic effects on bone and the cardiovascular system from unfavorable stimulatory effects associated with breast and endometrial cancer. Raloxifen is currently the only SERM marketed for the prevention and treatment of osteoporosis. Potential side-effects of this drug are increased risk of blood clots, vasomotor symptoms, and hot flushes

ESTROGEN REPLACEMENT THERAPY AFTER MENOPAUSE

Erβ localize to membranes and mitochondria,

 

ERβ  

 

Studies in ERα, ERβ, and Erα/β knockout mice shed light on the distinctive biology of ERs •  ERα has a more profound effect on the development and

function of the mammary gland and uterus and on the maintenance of metabolic and skeletal homeostasis

•  Erβ has more pronounced effects on the central nervous system.

•  Selective activation of ERβ avoids/limits ERα-mediated sexual modulation and proliferative responses.

Endocrinology  150:  770–783,  2009  

Phytoestrogens

 These  findings  have  recently  led  to  a  clinical  trial  of  the  phyto-­‐SERM  formulaMon  designed  to  evaluate  the  dosage/safety,  pharmacokineMcs,  and  proof-­‐of-­‐concept  efficacy  to  miMgate  hot  flash  frequency  and  memory  deficits  in  menopausal  women  (ClinicalTrials.gov  idenMfier:  NCT01723917).  

May the selective target of ERβ prevent or delay the development of LHON in unaffected

mutation carriers?

Methods

•  We treated 11778/ND4-LHON and control cybrids either with 100 nM genistein (G), daidzein (D) and equol (Eq) or with a combination of these compounds (G+D+Eq).

•  To highlight the pathological phenotype, cybrids were grown in glucose-free medium supplemented with galactose, a condition forcing cells to rely on oxidative phosphorylation for ATP synthesis.

###  p<  0.001,  for  11778  vehicle  vs  WT  vehicle.  *p<0.05,  ***p<  0.001  for  11778  treated  vs  11778  vehicle.  

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       WT                                                  11778    

 Vehicle      

*  ####  

***  ***  

             E2          G                D            Eq          GDEq  100nM  

*   *  

% o

f via

ble

cells

in g

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tose

The selected combination of phytoestrogens increases cell

viability of LHON cybrids in galactose medium

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11778  

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300nM  

*  

% o

f via

ble

cells

in g

alac

tose

The increase of LHON cybrids viability after incubation with phytoestrogens is associated with

decreased apoptosis    WT                                        11778  

 Vehicle      

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               E2                      GDEq  100nM  

Phytoestrogens action on cell viability is mediated by ERβ

   CTRL- ESRα

ERα

Effect of phytoestrogens on cell viability are evident also in LHON

patient’s derived fibroblasts bearing the 11778/ND4 mutation

Untreated   G+D+Eq  

       WT                                                                      11778  

0  

0,02  

0,04  

0,06  

0,08  

0,1  

0,12  

0,14  

1   2   3   4   5   6   7              V              E2        GDEq            V            E2        GDEq  

%  of  raM

o  of  m

itochon

drial  

area/sarcoplasmic  area  

****  ****  ****  

****  

The selected combination of phytoestrogens increase mitochondrial density

***p<0.001for    treated  cells  vs    vehicle.  

       WT                                                                      11778  

0  

0,02  

0,04  

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0,08  

0,1  

0,12  

0,14  

1   2   3   4   5   6   7              V              E2        GDEq            V            E2        GDEq  

%  of  raM

o  of  m

itochon

drial  

area/sarcoplasmic  area  

****  ****  ****  

****  

Untreated   G+D+Eq  

The selected combination of phytoestrogens increase mitochondrial density

 

***p<0.001  for  treated  cells    vs    vehicle.  

The selected combination of phytoestrogens is associated with up-regulation of the master modulator

of mitochondrial biogenesis  

*p<0.05,**p<  0.01,  ***p<0.001  for  treated  cells  vs  vehicle.    

Core2

NDUFA9

COX IV

SDHA

COX I

WT 11778 G+D+Eq G+D+Eq

The selected combination of phytoestrogens increase the expression of mitochondrial and nuclear encoded

respiratory chain subunits  

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fmol

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l/min

*  **  

           WT                                                                                                          11778  

V                  E2          GDEq        V                  E2            GDEq                            V                    E2        GDEq          V              E2            GDEq        

Supplementation of medium with the selected combination of phytoestrogens increases the rate of

oxygen consumption, especially in galactose medium

The selected combination of phytoestrogens decrease

superoxide levels in 11778 cells

Vehicle                                            G+D+Eq                                      Vehicle                                      G+D+Eq  

Glucose                                                                                                                Galactose  

11778  

WT  

Vehicle      E2        G+D+Eq      

WT  SOD2  

AcMn  11778  

SOD2  

AcMn  

Vehicle      E2        G+D+Eq      

The selected combination of phytoestrogens increases levels and activity of SOD2 and

reduce levels of H202  

#  

*  

Future directions  

•  To dissect the intracellular signaling involved in ERβ activation

•  To screen the effect of additional estrogen-like molecules on LHON metabolism and on other mitochondrial disorders (α-zearalanol, epigallocatechin gallate, epicatechin and baicalin, etc)

•  To evaluate the effect on animal models •  Clinical trials

Anna  Ghelli,  Valerio  Carelli  

Annalinda  Pisano,  Elena  Perli,  Maurizia  Orlandi,  Carmela  Preziuso,    Giulia  d’AmaM      Antonio  Campese,  Paola  Grazioli