Hyperforin Elicits Cytostatic/Cytotoxic Activity in Human Melanoma Cell Lines, Inhibiting Pro-Survival NF-κB, STAT3, AP1 Transcription Factors and the Expression of Functional Proteins Involved in Mitochondrial and Cytosolic Metabolism

Abstract

Hyperforin (HPF), the main component responsible for the antidepressant action of Hypericum perforatum, displays additional beneficial properties including anti-inflammatory, antimicrobic, and antitumor activities. Among its antitumor effects, HPF activity on melanoma is poorly documented. Melanoma, especially BRAF-mutated melanoma, is still a high-mortality tumor type and the currently available therapies do not provide solutions. We investigated HPF’s antimelanoma effectiveness in A375, FO1 and SK-Mel-28 human BRAF-mutated cell lines. Cell viability assays documented that all melanoma cells were affected by low HPF concentrations (EC50% 2–4 µM) in a time-dependent manner. A Br-deoxy-uridine incorporation assay attested a significant reduction of cell proliferation accompanied by decreased expression of cyclin D1 and A2, CDK4 and of the Rb protein phosphorylation, as assessed by immunoblots. In addition, the expression of P21/waf1 and the activated form of P53 were increased in A375 and SK-Mel-28 cells. Furthermore, HPF exerts cytotoxic effects. Apoptosis is induced 24 h after HPF administration, documented by an increase of cleaved-PARP1 and a decrease of both Bcl2 and Bcl-xL expression levels. Autophagy is induced, attested by an augmented LC3B expression and augmentation of the activated form of AMPK. Moreover, HPF lowers GPX4 enzyme expression, suggesting ferroptosis induction. HPF has been reported to activate the TRPC6 Ca++ channel and/or Ca++ and Zn++ release from mitochondria stores, increasing cytosolic Ca++ and Zn++ concentrations. Our data highlighted that HPF affects many cell-signaling pathways, including signaling induced by Ca++, such as FRA1, pcJun and pCREB, the expression or activity of which are increased shortly after treatment. However, the blockage of the TRPC6 Ca++ channel or the use of Ca++ and Zn++ chelators do not hinder HPF cytostatic/cytotoxic activity, suggesting that damages induced in melanoma cells may pass through other pathways. Remarkably, 24 h after HPF treatment, the expression of activated forms of the transcription factors NF-κB P65 subunit and STAT3 are significantly lowered. Several cytosolic (PGM2, LDHA and pPKM2) and mitochondrial (UQCRC1, COX4 and ATP5B) enzymes are downregulated by HPF treatment, suggesting a generalized reduction of vital functions in melanoma cells. In line with these results is the recognized ability of HPF to affect mitochondrial membrane potential by acting as a protonophore. Finally, HPF can hinder both melanoma cell migration and colony formation in soft agar. In conclusion, we provide evidence of the pleiotropic antitumor effects induced by HPF in melanoma cells.

Lamivudine, Doravirine, and Cabotegravir Downregulate the Expression of Human Endogenous Retroviruses (HERVs), Inhibit Cell Growth, and Reduce Invasive Capability in Melanoma Cell Lines

“…The anchorage-independent growth of A375, FO-1, SK-Mel-28, and MeWo melanoma cells was assessed through colony formation in soft agar, as previously described [73]. Initially, the bottom layer of 6-well plates was filled with 1% low gelling temperature agarose (Sigma-Merck, Milan, Italy) dissolved in 2X DMEM, 20% FBS, and 2% antibioticantimycotic solution.…”

Ferroptosis as a promising therapeutic strategy for melanoma

“…Gambogenic acid significantly inhibits migration, invasion, and EMT in MM cells through inducing ferroptosis via the p53/SLC7A11/GPX4 signaling pathway ( Wang et al, 2020 ). Hyperforin inhibits cell growth by inducing apoptosis, autophagy, and ferroptosis through a reduction in GPX4 expression ( Cardile et al, 2023 ). RSL3-induced cell death is fully reversed by ferrostatin-1 in A375 melanoma cells, indicating their high susceptibility to ferroptosis ( Tyurina et al, 2023 ).…”

Harnessing the Potential of Non-Apoptotic Cell Death Processes in the Treatment of Drug-Resistant Melanoma

“…In some cases, suppressing autophagy in melanoma cells can enhance pyroptosis [ 71 ]. On the other hand, autophagy can promote pyroptosis by facilitating the formation of inflammasomes or by aiding in the release of inflammatory cytokines [ 72 , 73 ].…”

“…Other compounds such as gallic acid and hyperforin, well-known antioxidants, have demonstrated the ability to inhibit cell viability and induce ferroptosis in melanoma cells by decreasing GPX4 activity [ 72 , 88 ]. Additionally, engineered ultra-small silica nanoparticles developed by Kim and colleagues effectively induced ferroptosis in melanoma cells and reduced tumor size in melanoma-bearing mice [ 89 ].…”