Hyperforin Enhances Heme Oxygenase-1 Expression Triggering Lipid Peroxidation in BRAF-Mutated Melanoma Cells and Hampers the Expression of Pro-Metastatic Markers

Abstract

Hyperforin (HPF) is an acylphloroglucinol compound found abundantly in Hypericum perforatum extract which exhibits antidepressant, anti-inflammatory, antimicrobial, and antitumor activities. Our recent study revealed a potent antimelanoma effect of HPF, which hinders melanoma cell proliferation, motility, colony formation, and induces apoptosis. Furthermore, we have identified glutathione peroxidase-4 (GPX-4), a key enzyme involved in cellular protection against iron-induced lipid peroxidation, as one of the molecular targets of HPF. Thus, in three BRAF-mutated melanoma cell lines, we investigated whether iron unbalance and lipid peroxidation may be a part of the molecular mechanisms underlying the antimelanoma activity of HPF. Initially, we focused on heme oxygenase-1 (HO-1), which catalyzes the heme group into CO, biliverdin, and free iron, and observed that HPF treatment triggered the expression of this inducible enzyme. In order to investigate the mechanism involved in HO-1 induction, we verified that HPF downregulates the BTB and CNC homology 1 (BACH-1) transcription factor, an inhibitor of the heme oxygenase 1 (HMOX-1) gene transcription. Remarkably, we observed a partial recovery of cell viability and an increase in the expression of the phosphorylated and active form of retinoblastoma protein when we suppressed the HMOX-1 gene using HMOX-1 siRNA while HPF was present. This suggests that the HO-1 pathway is involved in the cytostatic effect of HPF in melanoma cells. To explore whether lipid peroxidation is induced, we conducted cytofluorimetric analysis and observed a significant increase in the fluorescence of the BODIPY C-11 probe 48 h after HPF administration in all tested melanoma cell lines. To discover the mechanism by which HPF triggers lipid peroxidation, along with the induction of HO-1, we examined the expression of additional proteins associated with iron homeostasis and lipid peroxidation. After HPF administration, we confirmed the downregulation of GPX-4 and observed low expression levels of SLC7A11, a cystine transporter crucial for the glutathione production, and ferritin, able to sequester free iron. A decreased expression level of these proteins can sensitize cells to lipid peroxidation. On the other hand, HPF treatment resulted in increased expression levels of transferrin, which facilitates iron uptake, and LC3B proteins, a molecular marker of autophagy induction. Indeed, ferritin and GPX-4 have been reported to be digested during autophagy. Altogether, these findings suggest that HPF induced lipid peroxidation likely through iron overloading and decreasing the expression of proteins that protect cells from lipid peroxidation. Finally, we examined the expression levels of proteins associated with melanoma cell invasion and metastatic potential. We observed the decreased expression of CD133, octamer-4, tyrosine-kinase receptor AXL, urokinase plasminogen activator receptor, and metalloproteinase-2 following HPF treatment. These findings provide further support for our previous observations, demonstrating the inhibitory effects of HPF on cell motility and colony formation in soft agar, which are both metastasis-related processes in tumor cells.

Keywords: BACH-1; CD133; FSP1; LC3B; MMP-2; NRF-2; ferritin; ferroptosis; heme oxigenase-1; uPAR.

Figure 1

Hyperforin induces heme oxygenase-1 (HO-1) expression in melanoma cell lines. A375, SK-Mel-28, and FO-1 melanoma cells were treated with 2 or 3 µM of hyperforin (HPF) for 24 h. (Top) Representative immunoblots show the expression levels of HO-1, phosphorylated form of nuclear factor E2-related factor-2 (pNRF-2), heme-binding transcription factor BTB and CNC homology 1 (BACH-1), and glyceraldehyde-3-P dehydrogenase (GAPDH) after 24 h of treatment with 2 or 3 µM of HPF. (Bottom) Histograms represent the mean values ± S.D. of protein expression levels, as measured via densitometry in three independent experiments and normalized with GAPDH expression. Statistical analysis was performed using Student’s t-test for treated samples compared to the respective untreated samples after data normalization. Significance levels are denoted as follows: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 2

Hyperforin affects cell viability and the expression of cell-cycle-regulating proteins in cells silenced using HMOX-1 siRNA. A375, SK-Mel-28, and FO-1 melanoma cells were transfected with either HMOX-1 siRNA or a scrambled siRNA. After 24 h of transfection, cells were treated with or without 3 µM of HPF. (A) Representative immunoblots show the expression levels of HO-1, pNRF-2, BACH-1, phosphorylated form of retinoblastoma protein (pRB), Cyclin D1, and GAPDH after 24 h of treatment with 3 µM of HPF. The histograms display the mean values ± S.D. of protein expression levels, measured via densitometry from three independent experiments and normalized to GAPDH expression. All comparisons were performed against each respective untreated sample after data normalization. (B) The SRB cell viability assay was performed on A375 (green), SK-Mel-28 (red), and FO-1 (blue) cells. Before treatment with 3 µM of HPF for 48 h, cells were transfected with either HMOX-1 siRNA or a scrambled siRNA. Statistical comparisons were conducted using Student’s t-test for unpaired samples for immunoblots and paired samples for SRB cell viability assay. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

Hyperforin induces lipid peroxidation and affects the expression of proteins associated with iron homeostasis. (A) A375, SK-Mel-28, and FO-1 melanoma cells were treated with or without HPF for 48 h, followed by labeling with BODIPY C-11 probe. Fluorescence was measured using cytofluorimetric analysis. (B) Cells were transfected with either HMOX-1 siRNA or scrambled siRNA. After 24 h of transfection, cells were treated with or without 3 µM of HPF. Representative immunoblots showed the expression levels of glutathione peroxidase-4 (GPX-4), solute carrier family 7 member 11 (SLC7A11), ferroptosis suppressor protein 1 (FSP1), transferrin, ferritin heavy chain (FTH), light chain 3 B (LC3B), and GAPDH. Histograms represent the mean values ± S.D. of protein expression levels measured via densitometry from three independent experiments and normalized to GAPDH expression. Student’s t-test was used for unpaired samples to each untreated sample after data normalization. * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001.

Figure 4

Hyperforin affects the expression of proteins involved in tumor invasion and metastatic potential. Cell were transfected with either HMOX-1 siRNA or a scrambled siRNA. After 24 h of transfection, the cells were treated with or without 3 µM of HPF. Representative immunoblots show the expression level of CD133 antigen, octamer-4 (OCT-4), tyrosine-protein kinase receptor UFO (AXL), urokinase plasminogen activator receptor (uPAR), metalloproteinase-2 (MMP-2), and GAPDH as a loading control. The histograms represent the mean values ± S.D. of protein expression levels, which were measured via densitometry and derived from three independent experiments. Statistical comparisons were conducted using Student’s t-test for unpaired samples after data normalization with GAPDH expression. * p < 0.05; ** p < 0.01; *** p < 0.001.