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Indice Figure
Figura 1. Struttura dei recettori umani cannabinoidi CB1 e CB2… 7 Figura 2. Signalling pathway individuati fino ad oggi innescati dal GPR55...……….10 Figura 3. Rappresentazione schematica dei signalling pathway associati all’attivazione dei recettori cannabinoidi indotta da CB agonisti………..……… 12 Figura 4. Principali signalling pathway coinvolti nell’azione anticancro esercitata da agonisti cannabinoidi……….. 14 Figura 5. Struttura della cute. In dettaglio: strato basale
dell’epidermide e disposizione dei melanociti rispetto alle cellule che lo compongono……….. 15
Figura 6. Istotipi di melanoma (classificazione della World Health Organization, WHO)………..17 Figura 7. Struttura chimica AM251………..23 Figura 8. Strutture dei composti saggiati utilizzati nello studio……27 Figura 9. Cellule A375 a basso (sinistra) e alto (destra) tasso di crescita………....28 Figura 10. Cellule HDFa a basso (sinistra), medio (centro) e alto (destra) tasso di crescita………. 29 Figura 11. a) Struttura chimica del colorante DAPI b) Spettro di assorbimento ed emissione del colorante DAPI……….33 Figura 12. Bioconversione del substrato WST-1 in sale di formazano solubile………...37 Figura 13. Schema della procedura per l’estrazione dell’RNA
(RNeasy Mini kit)……….. 41
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Figura 14. Meccanismo d’azione della sonda EvaGreen Sso7d….. 46
Figura 15. Grafico del segnale di amplificazione ottenuto dopo real time PCR………48 Figura 16. Retta che indica la relazione tra la quantità di campione e il CT………... 48 Figura 17. Valutazione della vitalità in cellule A375, dopo
trattamento con AM251 (0,1-50 µM) per 24, 48 e 72 ore; 1% FBS..54 Figura 18. Valutazione della vitalità in cellule HDFa dopo
trattamento con AM251 (0,1-50 µM) per 48 e 72 ore; 1% FBS……55 Figura 19. Valutazione della vitalità in cellule A375 dopo trattamento con indometacina (0,1-100 µM), rofecoxib (0,1-100 µM) e celecoxib (0,1-100 µM) per 48 ore; 1% FBS……….56 Figura 20. Valutazione della vitalità in cellule A375dopo trattamento per 48 ore con a) LPI (0,1-10 µM) b) O1602 (0,1-10 µM) c)
CID16020046 (0,1-10 µM )………...57 Figura 21. Valutazione della vitalità in cellule A375 dopo
trattamento per 48 ore con AM251 (0,1-50 µM) in presenza di
A967079 1µM (a) e HC030031 10µM (b)……….59
Figura 22. Valutazione dei livelli di calcio intracellulare con sonda
Fluo 4 AM basale a) e dopo perfusione con AM251 (0,1-10 µM) (b) e
con ionomicina 5µM (c)……….60
Figura 23. Valutazione della percentuale di cellule nelle fasi G1/G0,
S e G2/M del ciclo cellulare di cellule A375 trattate con AM251 5
µM………..62
Figura 24. Valutazione dei livelli di espressione di Survivina, Bcl-2 e
Bax dopo trattamento di cellule A375 con AM251 5 µM per 48 ore,
normalizzato rispetto alla GAPDH (a) e alla β actina (b)…………..64
Figura 25. Valutazione della morfologia nucleare di cellule A375
trattate con AM251 5 µM per 48 ore (b) e controllo (a.)…………...65
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