Abstract
Melanoma remains incurable skin cancer, and targeting heat shock protein 90 (HSP90) is a promising therapeutic approach. In this study, we investigate the effect of 17-aminogeldanamycin, a potent HSP90 inhibitor, on nuclear factor-kappa B (NF-κB) activity in BRAFV600E and NRASQ61R patient-derived melanoma cell lines. We performed time-lapse microscopy and flow cytometry to monitor changes in cell confluence and viability. The NF-κB activity was determined by immunodetection of phospho-p65 and assessment of expression of NF-κB-dependent genes by quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Constitutive activity of p65/NF-κB was evident in all melanoma cell lines. Differences in its level might be associated with genetic alterations in CHUK, IL1B, MAP3K14, NFKBIE, RIPK1, and TLR4, while differences in transcript levels of NF-κB-inducible genes revealed by PCR array might result from the contribution of other regulatory mechanisms. 17-Aminogeldanamycin markedly diminished the level of phospho-p65, but the total p65 protein level was unaltered, indicating that 17-aminogeldanamycin inhibited activation of p65/NF-κB. This conclusion was supported by significantly reduced expression of selected NF-κB-dependent genes: cyclin D1 (CCND1), C-X-C motif chemokine ligand 8 (CXCL8), and vascular endothelial growth factor (VEGF), as shown at transcript and protein levels, as well as secretion of IL-8 and VEGF. Our study indicates that 17-aminogeldanamycin can be used for efficient inhibition of NF-κB activity and the simultaneous diminution of IL-8 and VEGF levels in the extracellular milieu of melanoma.
Highlights
Melanoma is one of the most aggressive human cancers, posing a serious clinical problem worldwide due to an increasing incidence and limited therapeutic regimens for the advanced stage of the disease [1,2]
Patient-derived melanoma cell lines used in the present study exhibited a constitutive activity of NF-κB that is in agreement with our previous studies reporting substantial levels of phosphorylated p65 in cells harboring mutations in either BRAF or RAS [22,23], and indicating that NF-κB activity is independent of extracellular growth factor stimuli [56]
We have found a large diversity of transcript levels between three representative melanoma cell lines, and this was observed when transcript levels of three selected genes were assessed in six cell lines of either the BRAF or NRAS subtype
Summary
Melanoma is one of the most aggressive human cancers, posing a serious clinical problem worldwide due to an increasing incidence and limited therapeutic regimens for the advanced stage of the disease [1,2]. The majority of melanomas harbor mutations in either BRAF or RAS or NF1 that result in hyperactivation of the RAS/RAF/MEK/ERK (hereafter MAPK) signaling pathway [3]. This has propelled the development of targeted therapeutics, the treatment with BRAFV600 inhibitors such as vemurafenib [4,5] or dabrafenib [6,7] almost inevitably results in drug-resistant disease despite an initially potent response [8,9]. 41–81% melanoma patients do not respond to immunotherapy, which is another treatment option currently used in the clinics [14] This indicates that alternative or complementary drug targets are needed
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