Carla Giordano, Convegno Mitocon 2014

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Carla Giordano, Convegno Mitocon 2014

  1. 1. Carla Giordano Gli effetti dei fitoestrogeni nel trattamento delle malattie mitocondriali
  2. 2. 0 200 400 600 800 1000 1200 1400 t0 vehicle 17bE2 ici+17bE2 mtDNAcopy/cel B * 4 5RF2 Tfam to ICI E2 ICI+E2 Gl+E2 Glu Gal+E2 Gal B 0 200 400 600 800 1000 1200 1400 t0 vehicle 17bE2 ici+17bE2 mtDNAcopy/cel B * 4 5RF2 Tfam4 5RF2 Tfam to ICI E2 ICI+E2 Gl+E2 Glu Gal+E2 Gal B    *   to E2 ICI+E2 to E2 ICI+E2 to E2 ICI+E2 to E2 ICI+E2 WT   11778   0,0 0,2 0,4 0,6 0,8 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+I 0 50 100 150 control 11778 3460 14484 °°° † *** ++ °°° °°° °° ° * M F I mit negative controlmergeERβ negative control fmol/cell/min
  3. 3. May the selective target of ERβ prevent or delay the development of LHON in unaffected mutation carriers?
  4. 4. 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  
  5. 5. ER    C2    C1 ER     Estrogen Responsive ER TRANSCRIPTION  Elements 5’AGGTCAnnnTGACCT 3’ citosol 17β-E GENOMIC  ACTION  OF  ESTROGENS       ER P GFR
  6. 6. 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.
  7. 7. 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
  8. 8. Erβ localize to membranes and mitochondria,   ERβ  
  9. 9.   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.
  10. 10. Endocrinology  150:  770–783,  2009   Phytoestrogens
  11. 11.  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).  
  12. 12. May the selective target of ERβ prevent or delay the development of LHON in unaffected mutation carriers?
  13. 13. 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.
  14. 14. ###  p<  0.001,  for  11778  vehicle  vs  WT  vehicle.  *p<0.05,   ***p<  0.001  for  11778  treated  vs  11778  vehicle.   0   5   10   15   20   25   30   35   40   1   2   3   4   5   6   7          WT                                                  11778      Vehicle       *   ####   ***  ***                E2          G                D            Eq          GDEq   100nM   *   *   %ofviablecellsingalactose The selected combination of phytoestrogens increases cell viability of LHON cybrids in galactose medium 0   5   10   15   20   25   30   1   2   3   4   5   11778    Vehicle                          E2                      G                  D                  Eq                   300nM   *   %ofviablecellsingalactose
  15. 15. The increase of LHON cybrids viability after incubation with phytoestrogens is associated with decreased apoptosis    WT                                        11778    Vehicle       ##   **   **   0   0,5   1   1,5   2   2,5   1   2   3   4   %ofapoptoticcellsingalactose                E2                      GDEq   100nM  
  16. 16. Phytoestrogens action on cell viability is mediated by ERβ    CTRL- ESRα ERα
  17. 17. Effect of phytoestrogens on cell viability are evident also in LHON patient’s derived fibroblasts bearing the 11778/ND4 mutation
  18. 18. 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  raMo  of  mitochondrial   area/sarcoplasmic  area   ****   ****  ****   ****   The selected combination of phytoestrogens increase mitochondrial density ***p<0.001for    treated   cells  vs    vehicle.  
  19. 19.        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  raMo  of  mitochondrial   area/sarcoplasmic  area   ****   ****  ****   ****   Untreated   G+D+Eq   The selected combination of phytoestrogens increase mitochondrial density   ***p<0.001  for  treated   cells    vs    vehicle.  
  20. 20. 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.    
  21. 21. 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  
  22. 22. 0   0,5   1   1,5   2   2,5   3   3,5   4   4,5   1   2   3   4   5   6   7   8   9   10   11   12   13   fmol/cell/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
  23. 23. The selected combination of phytoestrogens decrease superoxide levels in 11778 cells Vehicle                                            G+D+Eq                                      Vehicle                                      G+D+Eq   Glucose                                                                                                                Galactose   11778   WT  
  24. 24. 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   #   *  
  25. 25. 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
  26. 26. Anna  Ghelli,  Valerio  Carelli   Annalinda  Pisano,   Elena  Perli,  Maurizia   Orlandi,  Carmela   Preziuso,    Giulia   d’AmaM       Antonio  Campese,   Paola  Grazioli  

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