Background. Malignant melanoma (MM) is the most fatal skin cancer whose incidence is critically increasing in the last decades. Recent molecular therapies are giving excellent results in the remission of MM but most of them develops an acquired drug resistance, limiting the therapeutic efficacy. The search for new compounds able to overcome drug resistance is therefore essential. Vanadium is a trace element present in the diet and essential in small quantities for normal metabolism and growth of mammals. Antitumor effects of vanadium compounds are described against several tumors, but only few data are reported regarding vanadium effect against MM. We already demonstrated the anticancer activity against MM cell lines of four different Vanadium species (Rozzo et al,. 2017) being A375 the most sensitive cell line. The inorganic anion vanadate(V) indicated with VN and the oxidovanadium(IV) complex [VO(dhp)2] where dhp is 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonato (indicated with VS2) showed an IC50 of 4,7 and 2,6 uM respectively against A375 causing apoptosis and cell cycle arrest in two different cell cycle phases. In order to better understand the molecular pathways involved in the antitumoral activity of these two species we investigated if the apoptosis was caused by ROS production and evaluated different cell cycle check points. Material and Methods. Detection of Reactive Oxigen Species (ROS) generated inside the cells following treatment of A375 cell line with VN and VS2 species was carried out using the ROS-ID Total detection kit (ENZO) through a flow cytometry assay. To explore the differential action on cell cycle arrest caused by VN and VS2 we carried out a western blot analysis of several proteins expression at four different time points (3 ,6 ,16 and 24 hours). In particular the phosphorylation of: retinoblastoma tumor suppressor protein (Ser 780-phosphorylated Rb), Endoplasmic Reticulum kinases ERK1 and ERK2, Cell division control protein 25 (Cdc25) phosphatase, and the expression of p21 cell cycle inhibitor, following treatments, were investigated. Results. First, we confirmed the pro-apoptotic activity by VN and VS2 on A375 cells (Rozzo et al, 2017). Then, we demonstrated that VN and VS2 treatments were able to generate ROS production inside the cells in a time and dose dependent manner. Moreover, using NAC (N-acetyl cysteine), a general ROS scavenger, before Vanadium treatments, we were able to abate ROS levels induced by VN and VS2. Inhibition of ROS production was concomitant with significant abrogation of VN and VS2 induced apoptosis, demonstrating it was caused by ROS. The two V species analyzed seemed to induce cell cycle arrest at different phases (G2/M checkpoint for VN and G1/S for VS2). To clarify whether and how VN and VS2 exert differential action on cell cycle progression of A375 cells, we analyzed the level of key proteins, implicated in regulating G1/S and G2/M transitions, through western blot analysis of VN and VS2- treated cells. Levels of phosphorylated Rb (Ser780), considered a hallmark of the G1/S transition, were observed to decrease, in a time and dose dependent manner, for both VN and VS2-treated samples, confirming a block in G1 phase for V treated cells. Unexpectedly, Cdc25C phosphatase, which regulate the G2/M transition, was observed to be dephosphorylated at Ser216 in the V-treated samples, showing not to be involved in the G2 cell cycle block apparently induced by VN. In addition, cdc25C expression was also increased by VN treatments, not being degraded by any of the two V compounds, as it was previously reported in literature. Both VN and VS2 treatments induced a strong increase of p21 expression, thus demonstrating the involvement of this cyclin dependent kinase inhibitor in the cell cycle arrest induced by these compounds. ERK hyper-phosphorylation, due to MAPK pathway constitutive activation in A375 cells owning BRAF V660E oncogenic variant, was strongly reduced by both VN and VS2 treatments, showing inactivation of MAPK pathway. Conclusions. Our results confirm the antitumor properties of vanadium against melanoma cells highlighting its ability to induce apoptosis through generation of ROS and cell cycle arrest by contrasting MAPK pathway activation. Such inactivation is probably involved in a block of ciclin D transcription taking to a consequent arrest in G1 phase of cell cycle. Moreover, the strong induction of p21 expression observed, with consequent inhibition of Cdk4/2 and hypo-phosphorylation of Rb, is an additional evidence of cell cycle arrest in G1 phase. The G2 arrest observed after VN treatments has to be better investigated.
DISSECTING MOLECULAR PATHWAYS INVOLVED IN THE ANTIPROLIFERATIVE EFFECTS OF VANADIUM COMPOUNDS IN A375 MALIGNANT MELANOMA CELL LINE.
Carla Maria Rozzo
2018
Abstract
Background. Malignant melanoma (MM) is the most fatal skin cancer whose incidence is critically increasing in the last decades. Recent molecular therapies are giving excellent results in the remission of MM but most of them develops an acquired drug resistance, limiting the therapeutic efficacy. The search for new compounds able to overcome drug resistance is therefore essential. Vanadium is a trace element present in the diet and essential in small quantities for normal metabolism and growth of mammals. Antitumor effects of vanadium compounds are described against several tumors, but only few data are reported regarding vanadium effect against MM. We already demonstrated the anticancer activity against MM cell lines of four different Vanadium species (Rozzo et al,. 2017) being A375 the most sensitive cell line. The inorganic anion vanadate(V) indicated with VN and the oxidovanadium(IV) complex [VO(dhp)2] where dhp is 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonato (indicated with VS2) showed an IC50 of 4,7 and 2,6 uM respectively against A375 causing apoptosis and cell cycle arrest in two different cell cycle phases. In order to better understand the molecular pathways involved in the antitumoral activity of these two species we investigated if the apoptosis was caused by ROS production and evaluated different cell cycle check points. Material and Methods. Detection of Reactive Oxigen Species (ROS) generated inside the cells following treatment of A375 cell line with VN and VS2 species was carried out using the ROS-ID Total detection kit (ENZO) through a flow cytometry assay. To explore the differential action on cell cycle arrest caused by VN and VS2 we carried out a western blot analysis of several proteins expression at four different time points (3 ,6 ,16 and 24 hours). In particular the phosphorylation of: retinoblastoma tumor suppressor protein (Ser 780-phosphorylated Rb), Endoplasmic Reticulum kinases ERK1 and ERK2, Cell division control protein 25 (Cdc25) phosphatase, and the expression of p21 cell cycle inhibitor, following treatments, were investigated. Results. First, we confirmed the pro-apoptotic activity by VN and VS2 on A375 cells (Rozzo et al, 2017). Then, we demonstrated that VN and VS2 treatments were able to generate ROS production inside the cells in a time and dose dependent manner. Moreover, using NAC (N-acetyl cysteine), a general ROS scavenger, before Vanadium treatments, we were able to abate ROS levels induced by VN and VS2. Inhibition of ROS production was concomitant with significant abrogation of VN and VS2 induced apoptosis, demonstrating it was caused by ROS. The two V species analyzed seemed to induce cell cycle arrest at different phases (G2/M checkpoint for VN and G1/S for VS2). To clarify whether and how VN and VS2 exert differential action on cell cycle progression of A375 cells, we analyzed the level of key proteins, implicated in regulating G1/S and G2/M transitions, through western blot analysis of VN and VS2- treated cells. Levels of phosphorylated Rb (Ser780), considered a hallmark of the G1/S transition, were observed to decrease, in a time and dose dependent manner, for both VN and VS2-treated samples, confirming a block in G1 phase for V treated cells. Unexpectedly, Cdc25C phosphatase, which regulate the G2/M transition, was observed to be dephosphorylated at Ser216 in the V-treated samples, showing not to be involved in the G2 cell cycle block apparently induced by VN. In addition, cdc25C expression was also increased by VN treatments, not being degraded by any of the two V compounds, as it was previously reported in literature. Both VN and VS2 treatments induced a strong increase of p21 expression, thus demonstrating the involvement of this cyclin dependent kinase inhibitor in the cell cycle arrest induced by these compounds. ERK hyper-phosphorylation, due to MAPK pathway constitutive activation in A375 cells owning BRAF V660E oncogenic variant, was strongly reduced by both VN and VS2 treatments, showing inactivation of MAPK pathway. Conclusions. Our results confirm the antitumor properties of vanadium against melanoma cells highlighting its ability to induce apoptosis through generation of ROS and cell cycle arrest by contrasting MAPK pathway activation. Such inactivation is probably involved in a block of ciclin D transcription taking to a consequent arrest in G1 phase of cell cycle. Moreover, the strong induction of p21 expression observed, with consequent inhibition of Cdk4/2 and hypo-phosphorylation of Rb, is an additional evidence of cell cycle arrest in G1 phase. The G2 arrest observed after VN treatments has to be better investigated.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
