BACKGROUND AND PURPOSEDespite new therapeutic approaches, metastatic melanomas still have a poor prognosis. Statins reduce low-density lipoprotein cholesterol and exert anti-inflammatory and anti-proliferative actions. We have recently shown that simvastatin triggers an apoptotic burst in human metastatic melanoma cells by the synthesis of an autocrine factor. EXPERIMENTAL APPROACHThe current in vitro study was performed in human metastatic melanoma cell lines (A375, 518a2) and primary human melanocytes and melanoma cells. The secretome of simvastatin-stressed cells was analysed with two-dimensional difference gel electrophoresis and MS. The signalling pathways involved were analysed at the protein and mRNA level using pharmacological approaches and siRNA technology. KEY RESULTSSimvastatin was shown to activate a stress cascade, leading to the synthesis of 15-deoxy-12,14-PGJ2 (15d-PGJ2), in a p38-and COX-2-dependent manner. Significant concentrations of 15d-PGJ2 were reached in the medium of melanoma cells, which were sufficient to activate caspase 8 and the mitochondrial pathway of apoptosis. Inhibition of lipocalin-type PGD synthase, a key enzyme for 15d-PGJ2 synthesis, abolished the apoptotic effect of simvastatin. Moreover, 15d-PGJ2 was shown to bind to the fatty acid-binding protein 5 (FABP5), which was up-regulated and predominantly detected in the secretome of simvastatin-stressed cells. Knockdown of FABP5 abolished simvastatin-induced activation of PPAR-γ and amplified the apoptotic response. CONCLUSIONS AND IMPLICATIONSWe characterized simvastatin-induced activation of the 15d-PGJ2/FABP5 signalling cascades, which triggered an apoptotic burst in melanoma cells but did not affect primary human melanocytes. These data support the rationale for the pharmacological targeting of 15d-PGJ2 in metastatic melanoma. Abbreviations15d-PGJ2, 15-deoxy-12,14-PGJ2; FABP5, fatty acid-binding protein
The interleukin (IL)-6 inhibits the growth of early-stage melanoma cells, but not metastatic cells. Metastatic melanoma cells are susceptible to statin-induced apoptosis, but this is not clear for early-stage melanoma cells. This study aimed to investigate the IL-6 susceptibility of melanoma cells from different stages in the presence of simvastatin to overcome loss of growth arrest. ELISA was used to detect secreted IL-6 in human melanoma cells. The effects of IL-6 were measured by western blots for STAT3 and Bcl-2 family proteins. Apoptosis and proliferation were measured by caspase 3 activity, Annexin V staining, cell cycle analysis, and a wound-healing assay. Human metastatic melanoma cells A375 and 518A2 secrete high amounts of IL-6, in contrast to early-stage WM35 cells. Canonical IL-6 signaling is intact in these cells, documented by transient phosphorylation of STAT3. Although WM35 cells are highly resistant to simvastatin-induced apoptosis, coadministration with IL-6 enhanced the susceptibility to undergo apoptosis. This proapoptotic effect of IL-6 might be explained by a downregulation of Bcl-XL, observed only in WM35 cells. Furthermore, the IL-6 receptor blocking antibody tocilizumab was coadministered and unmasked an IL-6-sensitive proportion in the simvastatin-induced caspase 3 activity of metastatic melanoma cells. These results confirm that simvastatin facilitates apoptosis in combination with IL-6. Although endogenous IL-6 secretion is sufficient in metastatic melanoma cells, exogenously added IL-6 is needed for WM35 cells. This effect may explain the failure of simvastatin to reduce melanoma incidence in clinical trials and meta-analyses.
Cancer cells rewire metabolism to sustain high proliferation rates. Beside glycolysis and glutaminolysis, amino acids substitute as energy source, feed fatty acid biosynthesis and represent part of the secretome of transformed cells, including melanoma. We have therefore investigated acetate, pyruvate and the amino acid composition of the secretome of human melanoma cells representing the early slow (WM35, WM278, WM793b and VM21) and metastatic fast (A375, 518a2, 6F and WM8) growth phase in order to identify possible signalling components within these profiles. Proliferation assays and a principle component analysis revealed a stringent difference between the fast and slow growing melanoma cells. Moreover, upon inhibition of the mevalonate pathway, glutamic acid and alanine were identified as the central difference in the conditional media. A supplementation of the media with glutamic acid and the combination with alanine significantly accelerated the proliferation, migration and invasion of early stage melanoma cells, but not metastatic cells. Finally, the inhibition of the mevalonate pathway abolished the growth advantage of the melanoma cells in a time dependent manner. Taken together, these data corroborate a stage specific response in growth and aggressiveness to extracellular glutamic acid and alanine, indicative for microenvironmental signalling of individual amino acids.
Background: Statins may trigger apoptosis in tumour cells in vitro and, particularly, melanoma cells are susceptible to statin induced apoptosis. Lipophilic statins (e.g. Atorvastatin, Simvastatin) induce strong caspase 3 activation after 48h of treatment, whereas hydrophilic statins (e.g. Pravastatin, Rosuvastatin) do not trigger caspase activation. Recently, we demonstrated the presence of an autocrine amplification loop which increases apoptosis by a caspase 8 dependent pathway after treatment with lipophilic statins (Minichsdorfer and Hohenegger, Br J Ph 2009). It is known that activation of caspase 2 may prime melanoma cells for death receptor induced apoptosis, however, although an association between caspase 2 activation and RhoB upregulation was shown, the significance of this interaction is still not clear. Methods: We investigated the activity of caspase 2, 3 and 8 in A375 and 518A2 human metastatic melanoma cells after statin treatment in the presence and absence of a caspase 2 inhibitor (Z-VDVAD-FMK) or cycloheximide by cleavage of specific fluorescent caspase substrates. Upregulation of RhoB after statin treatment as well as the influence of the co-treatment with the isoprenoids farnesylpyrophosphate (FPP) or geranylgeranylpyrophosphate (GGPP) was studied by Western blot. Results: Exposure of these cells to simvastatin led to increased caspase 2 activation which is abrogated by co-application of the caspase 2 inhibitor Z-VDVAD-FMK. Moreover, treatment with Z-VDVAD-FMK also prevented caspase 8 and caspase 3 activation. We could show that production of an autocrine factor needs an intact translation machinery. Inhibition of translation by co-administration of cycloheximide prevented apoptotic morphological changes as well as caspase 3 and caspase 8 activation in A375 and 518A2 cells. Since caspase 2 may interact with RhoB, we also investigated the effect of statin treatment on RhoB expression and observed an upregulation of RhoB which was inhibited by the addition of cycloheximide in both cell lines. However, isoprenoids were not able to reverse RhoB upregulation or PARP cleavage in 518A2 cells. Conclusion: Overall, our data provide evidence for an important role of caspase 2 in statin induced apoptosis of 2 human metastatic melanoma cell lines. These findings may help to understand the function of caspase 2 in the induction of apoptosis. Moreover, we observed upregulation of RhoB following statin treatment in these cell lines. However, additional experiments are necessary to fully understand the role of RhoB in statin induced apoptosis. Citation Format: Christoph Minichsdorfer, Christine Wasinger, Evelyn Sieczkowski, Atil Bihter, Gerwin Heller, Sabine Zöchbauer-Müller, Martin Hohenegger. Statin induced apoptosis in human melanoma cells is prevented by inhibition of caspase 2 and translational activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2940. doi:10.1158/1538-7445.AM2013-2940
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