The antitumour effects of hyperforin compared to its more traditional use as an antidepressant has barely been explored in melanoma cells. The existing medicines in the treatment of melanoma are poorly effective, particularly of BRAF-mutated melanoma, which continues to be a malignant type with a high fatality rate. In this study, we examined the antitumour activity of hyperforin in human melanoma cell lines A375, FO1, and SKMEL-28 harbouring BRAF mutation. Melanoma cell viability was shown to be adversely affected by hyperforin in a time-dependent manner (EC50% 2-4 µM, after 72 hours). The Br-deoxy-Uridine incorporation experiment demonstrated a significant decrease in cell proliferation rates, which was accompanied by lower production of cyclin D1 and A2, CDK4, and Rb protein phosphorylation, as determined by immunoblots. A375 and SKMEL-28 cells also showed enhanced expression of p21/waf1 and the activated form of p53. After 24 hours of hyperforin treatment, cleaved-PARP1 levels rose while Bcl-2 and Bcl-xL expression levels fell, indicating that apoptosis had been activated. An increased expression of LC3B and the activated form of AMPK both indicated that autophagy was initiated. Hyperforin lowers GPX4 enzyme expression, suggesting ferroptosis induction, as well. Finally, hyperforin treatment resulted in the downregulation of several cytosolic (PGM2, LDHA, and pPKM2) and mitochondrial (UQCRC1, COX4, and ATP5B) enzyme expression, suggesting a widespread loss of critical metabolic functions in melanoma cells. These findings could be supported by hyperforin’s known capability to lower mitochondrial membrane potential by functioning as a protonophore. It was also demonstrated that hyperforin could hinder both melanoma cell migration and colony formation in soft agar. It can be concluded that hyperforin is able to counteract melanoma progression by pleiotropic antitumour effects.
Gli effetti antitumorali dell'iperforina rispetto al suo uso più tradizionale come antidepressivo sono stati appena esplorati nelle cellule di melanoma. I farmaci esistenti nel trattamento del melanoma sono scarsamente efficaci, in particolare del melanoma BRAF-mutato, che continua ad essere un tipo maligno con un alto tasso di mortalità. In questo studio, abbiamo esaminato l'attività antitumorale dell'iperforina nelle linee cellulari di melanoma umano A375, FO1 e SKMEL-28 che presentano la mutazione BRAF. È stato dimostrato che la vitalità cellulare del melanoma è influenzata negativamente dall'iperforina in modo tempo dipendente (EC50% 2-4 µM, dopo 72 ore). L'esperimento di incorporazione di Br-deossi-Uridina ha dimostrato una significativa diminuzione dei tassi di proliferazione cellulare, che è stata accompagnata da una minore produzione di ciclina D1 e A2, CDK4 e la proteina Rb fosforilata, come determinato dagli immunoblot. Le cellule A375 e SKMEL-28 hanno anche mostrato una maggiore espressione di p21/waf1 e la forma attivata di p53. Dopo 24 ore di trattamento con iperforina, i livelli di PARP1 tagliata sono aumentati mentre i livelli di espressione di Bcl-2 e Bcl-xL sono diminuiti, indicando che l'apoptosi era stata attivata. Un'aumentata espressione di LC3B e la forma attivata di AMPK indicavano entrambi che l'autofagia era stata avviata. L'iperforina abbassa l'espressione dell'enzima GPX4, suggerendo anche l'induzione della ferroptosi. Infine, il trattamento con iperforina ha portato alla downregulation di diversi enzimi citosolici (PGM2, LDHA e pPKM2) e mitocondriali (UQCRC1, COX4 e ATP5B), suggerendo una perdita diffusa delle funzioni metaboliche critiche nelle cellule di melanoma. Questi risultati potrebbero essere supportati dalla nota capacità dell'iperforina di abbassare il potenziale della membrana mitocondriale funzionando come un protonoforo. È stato inoltre dimostrato che l'iperforina potrebbe ostacolare sia la migrazione delle cellule di melanoma che la formazione di colonie nell'agar. Si può concludere che l'iperforina è in grado di contrastare la progressione del melanoma mediante effetti antitumorali pleiotropici.
In vitro antitumor activity of Hyperforin in A375, FO1 and SKMEL-28 human melanoma cell lines
ADDO, SOLOMON SAFORO
2021/2022
Abstract
The antitumour effects of hyperforin compared to its more traditional use as an antidepressant has barely been explored in melanoma cells. The existing medicines in the treatment of melanoma are poorly effective, particularly of BRAF-mutated melanoma, which continues to be a malignant type with a high fatality rate. In this study, we examined the antitumour activity of hyperforin in human melanoma cell lines A375, FO1, and SKMEL-28 harbouring BRAF mutation. Melanoma cell viability was shown to be adversely affected by hyperforin in a time-dependent manner (EC50% 2-4 µM, after 72 hours). The Br-deoxy-Uridine incorporation experiment demonstrated a significant decrease in cell proliferation rates, which was accompanied by lower production of cyclin D1 and A2, CDK4, and Rb protein phosphorylation, as determined by immunoblots. A375 and SKMEL-28 cells also showed enhanced expression of p21/waf1 and the activated form of p53. After 24 hours of hyperforin treatment, cleaved-PARP1 levels rose while Bcl-2 and Bcl-xL expression levels fell, indicating that apoptosis had been activated. An increased expression of LC3B and the activated form of AMPK both indicated that autophagy was initiated. Hyperforin lowers GPX4 enzyme expression, suggesting ferroptosis induction, as well. Finally, hyperforin treatment resulted in the downregulation of several cytosolic (PGM2, LDHA, and pPKM2) and mitochondrial (UQCRC1, COX4, and ATP5B) enzyme expression, suggesting a widespread loss of critical metabolic functions in melanoma cells. These findings could be supported by hyperforin’s known capability to lower mitochondrial membrane potential by functioning as a protonophore. It was also demonstrated that hyperforin could hinder both melanoma cell migration and colony formation in soft agar. It can be concluded that hyperforin is able to counteract melanoma progression by pleiotropic antitumour effects.The text of this website © Università degli studi di Padova. Full Text are published under a non-exclusive license. Metadata are under a CC0 License
https://hdl.handle.net/20.500.12608/42391