Recent advances in melanoma therapy have significantly improved the prognosis of metastasized melanoma. However, large therapeutic gaps remain that need to be closed by new strategies. Antiapoptotic Bcl-2 proteins critically contribute to apoptosis deficiency and therapy resistance. They can be targeted by BH3 mimetics, small molecule antagonists that mimic the Bcl-2 homology domain 3 (BH3) of proapoptotic BH3-only proteins. By applying in vitro experiments, we aimed to obtain an overview of the possible suitability of BH3 mimetics for future melanoma therapy. Thus, we investigated the effects of ABT-737 and ABT-263, which target Bcl-2, Bcl-xL and Bcl-w as well as the Bcl-2-selective ABT-199 and the Mcl-1-selective S63845, in a panel of four BRAF-mutated and BRAF-WT melanoma cell lines. None of the inhibitors showed significant effectiveness when used alone; however, combination of S63845 with each one of the three ABTs almost completely abolished melanoma cell survival and induced apoptosis in up to 50–90% of the cells. Special emphasis was placed here on the understanding of the downstream pathways involved, which may allow improved applications of these strategies. Thus, cell death induction was correlated with caspase activation, loss of mitochondrial membrane potential, phosphorylation of histone H2AX, and ROS production. Caspase dependency was demonstrated by a caspase inhibitor, which blocked all effects. Upregulation of Mcl-1, induced by S63845 itself, as reported previously, was blocked by the combinations. Indeed, Mcl-1, as well as XIAP (X-linked inhibitor of apoptosis), were strongly downregulated by combination treatments. These findings demonstrate that melanoma cells can be efficiently targeted by BH3 mimetics, but the right combinations have to be selected. The observed pronounced activation of apoptosis pathways demonstrates the decisive role of apoptosis in the loss of cell viability by BH3 mimetics.

BACKGROUND:A disruption of sebocyte differentiation and lipogenesis has fatal consequences and can cause a wide spectrum of skin diseases, from acne vulgaris to sebaceous carcinoma, however, the relevant molecular mechanisms have not been fully clarified.OBJECTIVES:The induction of autophagy and apoptosis in human sebocytes in response to biologically relevant fatty acids was investigated.METHODS:Free fatty acids (arachidonic acid, linoleic acid, palmitic acid, and palmitoleic acid) and the pan-caspase inhibitor QVD-Oph were added to the supernatant of cultured human SZ95 sebocytes. Individual relevant proteins were analyzed by Western blotting. Apoptosis and cell viability were determined, and typical autophagy structures were detected through electron microscopy. To obtain cell growth curves, cell confluence was continuously monitored by real-time cell analysis.RESULTS:Fatty acids induced the development of intracellular lipid droplets with subsequent apoptosis, whereas arachidonic acid caused the most rapid effect. Cleavage products of caspase-3 were only detected in arachidonic acid-induced apoptosis. The high basal apoptotic rate of cultured SZ95 sebocytes was strongly suppressed by QVD-Oph. Fatty acid-induced apoptosis was also markedly inhibited by QVD-Oph, whereas intracellular lipid droplets further accumulated. While cell viability after incubation with linoleic acid, palmitic acid, or palmitoleic acid and QVD-Oph was comparable with that of non-treated controls, arachidonic acid significantly reduced cell viability and cell density despite the concomitant pan-caspase inhibitor treatment. Using electron microscopy, typical autophagy structures were detected, such as autophagosomes and autolysosomes, at the basal level, which became more pronounced after treatment with fatty acids.CONCLUSIONS:Our findings contribute to a better understanding of the inflammation-associated mechanisms of lipogenesis and cell death induction in human sebocytes and may help to unveil the effects of fatty acid-rich human nutrition.

Targeting of MAP kinase pathways by BRAF inhibitors has evolved as a key therapy for BRAF-mutated melanoma. However, it cannot be applied for BRAF-WT melanoma, and also, in BRAF-mutated melanoma, tumor relapse often follows after an initial phase of tumor regression. Inhibition of MAP kinase pathways downstream at ERK1/2, or inhibitors of antiapoptotic Bcl-2 proteins, such as Mcl-1, may serve as alternative strategies. As shown here, the BRAF inhibitor vemurafenib and the ERK inhibitor SCH772984 showed only limited efficacy in melanoma cell lines, when applied alone. However, in combination with the Mcl-1 inhibitor S63845, the effects of vemurafenib were strongly enhanced in BRAF-mutated cell lines, and the effects of SCH772984 were enhanced in both BRAF-mutated and BRAF-WT cells. This resulted in up to 90% loss of cell viability and cell proliferation, as well as in induction of apoptosis in up to 60% of cells. The combination of SCH772984/S63845 resulted in caspase activation, processing of poly (ADP-ribose) polymerase (PARP), phosphorylation of histone H2AX, loss of mitochondrial membrane potential, and cytochrome c release. Proving the critical role of caspases, a pan-caspase inhibitor suppressed apoptosis induction, as well as loss of cell viability. As concerning Bcl-2 family proteins, SCH772984 enhanced expression of the proapoptotic Bim and Puma, as well as decreased phosphorylation of Bad. The combination finally resulted in downregulation of antiapoptotic Bcl-2 and enhanced expression of the proapoptotic Noxa. In conclusion, combined inhibition of ERK and Mcl-1 revealed an impressive efficacy both in BRAF-mutated and WT melanoma cells, and may thus represent a new strategy for overcoming drug resistance.

Background: Cannabinoids are mainly used for recreational purposes, but also made their way into oncology, since these substances can be taken to increase appetite in tumour cachexia. Since there are some hints in the literature that cannabinoids might have some anti-cancerous effects, the aim of this study was to study if and how cannabinoids mediate pro-apoptotic effects in metastatic melanoma in vivo and in vitro and its value besides conventional targeted therapy in vivo. Methods: Several melanoma cell lines were treated with different concentrations of cannabinoids, and anti-cancerous efficacy was assessed by proliferation and apoptosis assays. Subsequent pathway analysis was performed using apoptosis, proliferation, flow cytometry and confocal microscopy data. The efficacy of cannabinoids in combination with trametinib was studied in NSG mice in vivo. Results: Cannabinoids reduced cell viability in multiple melanoma cell lines in a dose-dependent way. The effect was mediated by CB1, TRPV1 and PPARα receptors, whereby pharmacological blockade of all three receptors protected from cannabinoid-induced apoptosis. Cannabinoids initiated apoptosis by mitochondrial cytochrome c release with consecutive activation of different caspases. Essentially, cannabinoids significantly decreased tumour growth in vivo and were as potent as the MEK inhibitor trametinib. Conclusions: We could demonstrate that cannabinoids reduce cell viability in several melanoma cell lines, initiate apoptosis via the intrinsic apoptotic pathway by cytochrome c release and caspase activation and do not interfere with commonly used targeted therapy.

Fungal infections have increased considerably over the last decades, becoming progressively resistant to common drugs. UVC light has shown microbiological eradication effects, whereby the wavelength of 254 nm is strongly carcino‐ and mutagenic. Therefore, 222 and 233 nm, which do not significantly harm skin cells, were tested for their antifungal effects. Microbicidal doses were reached at 40 mJ/cm2 for both wavelengths, resulting in only minor superficial skin damage (<20 μm). UVC irradiation with far‐UVC <240 nm represents a new opportunity to effectively eradicate even larger pathogens on tissue causing no or strongly reduced DNA and tissue damage.

New therapeutic strategies are needed for cutaneous T-cell lymphoma (CTCL), and the plant extract ingenol mebutate (PEP005) may be considered. PEP005 has been approved for actinic keratosis, and proapoptotic activities were described in different cancer cells. Here, we aimed to investigate its efficacy in four CTCL cell lines and its mode of action. While HuT-78 and HH responded with induced apoptosis as well as with loss of cell viability and cell proliferation, MyLa and SeAx remained resistant. Interestingly, both sensitive and resistant cells showed caspase-8 activation and enhanced levels of reactive oxygen species (ROS), while final caspase-3 activation was restricted to sensitive cells. Apoptosis induction was prevented by the caspase inhibitor QVD-Oph as well as by the antioxidant vitamin E. Caspase activation by PEP005 may be explained to some extent by the downregulation of the caspase antagonistic proteins c-FLIP and XIAP in sensitive cells, whereas both proteins were strongly expressed in resistant cells. Finally, PEP005 resulted in the activation of proapoptotic PKCδ, and the PKC inhibitor bisindolylmaleimide I reduced apoptosis, caspase-3 processing and ROS production, as well as restored cell viability. In conclusion, PKCδ appeared as a central player in apoptosis regulation in CTCL cells, also suggesting its therapeutic targeting.

The term sinecatechins designates an extract containing a high percentage of catechins obtained from green tea, which is commercially registered as Veregen or Polyphenon E (PE) and may be considered for treatment of cutaneous squamous cell carcinoma (cSCC) and actinic keratosis (AK). As shown here, treatment of four cSCC cell lines with 200 µg/mL of PE resulted in strong, dose-dependent decrease in cell proliferation (20–30%) as well as strongly decreased cell viability (4–21% of controls, 48 h). Effects correlated with loss of mitochondrial membrane potential, whereas early apoptosis was less pronounced. At the protein level, some activation of caspase-3 and enhanced expression of the CDK inhibitor p21 were found. Loss of MMP and induced cell death were, however, largely independent of caspases and of the proapoptotic Bcl-2 proteins Bax and Bak, suggesting that sinecatechins induce also non-apoptotic, alternative cell death pathways, in addition to apoptosis. Reactive oxygen species (ROS) were downregulated in response to PE at 4 h, followed by an increase at 24 h. The contributory role of initially reduced ROS was supported by the antioxidant N-acetyl cysteine, which in combination with PE further enhanced the negative effects on cell viability. Thus, sinecatechins inhibited cell proliferation and viability of cSCC cells, which could suggest the use of PE for AK treatment. The mechanisms appear as linked to an imbalance of ROS levels.

Long-term, curative treatment of cutaneous T-cell lymphomas (CTCL) remains a major challenge. Therapy resistance is often based on apoptosis deficiency, and may depend on antiapoptotic Bcl-2 proteins, such as Bcl-2, Bcl-xL, Bcl-w and Mcl-1. For their targeting, several antagonists have been generated, which mimic the Bcl-2 homology domain 3 (BH3 mimetics). As dysregulation and overexpression of Mcl-1 has been reported in CTCL, the use of Mcl-1 inhibitors appears as an attractive strategy. Here, we investigated the effects of the selective Mcl-1 inhibitor S63845 in a series of four CTCL cell lines, in comparison to ABT-263 and ABT-737 (inhibitors of Bcl-2, Bcl-xL and Bcl-w). In two cell lines (HH, HuT-78), S63845 resulted in significant apoptosis induction, decrease in cell viability, loss of mitochondrial membrane potential and caspase activation, while two other cell lines (MyLa, SeAx) remained completely resistant. An inverse correlation was found, as S63845-resistant cells were highly sensitive to ABT-263/-737, and S63845-sensitive cells showed only moderate sensitivity to ABTs. Combinations of S63845 and ABT-263 partially yielded synergistic effects. As concerning Bcl-2 protein expression, weaker Mcl-1 expression was found in S63845-resistant MyLa and SeAx, while for Bcl-2 and Bcl-xL, the lowest expression was found in the highly sensitive cell line HH. The most striking difference between S63845-resistant and -sensitive cells was identified for Bcl-w, which was exclusively expressed in S63845-resistant cells. Thus, CTCL may be efficiently targeted by BH3 mimetics, providing the right target is preselected, and Bcl-w expression may serve as a suitable marker.

INTRODUCTION:Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine that may paradoxically induce either apoptosis or cell survival. It mediates its activity through binding of TNF-receptor (TNFR) 1 or 2. TNFR1 is mainly responsible for transmitting apoptotic signals. The activation of apoptotic mechanisms can either be intrinsic (mitochondrial) or extrinsic (death receptors). Death ligands such as TNF-related apoptosis-inducing ligand (TRAIL) specifically induce extrinsic apoptosis, while cytostatic drugs such as 5-fluorouracil (5FU) induce intrinsic apoptosis.OBJECTIVES:To investigate the effects of TNFα on apoptosis in malignant and normal human keratinocytes.METHODS:Human cutaneous squamous cell carcinoma (SCC) cell line SCC-13 and immortalized human keratinocytes HaCaT as well as primary normal human keratinocytes (PNHK) were stimulated with TNFα and then treated either with TRAIL or 5FU. Cell viability and cell proliferation, DNA fragmentation, apoptosis, and cytotoxicity were determined by WST-1 proliferation assay, ELISA, flow cytometry, and colorimetric analysis of lactate dehydrogenase, respectively. In addition, Western blotting was performed for analysis of caspase-3.RESULTS:TNFα affected viability of SCC-13 and HaCaT cells in combination with 5FU or TRAIL. In contrast, TNFα did not influence cell viability of PNHK. It enhanced the apoptotic effects of both extrinsic and intrinsic stimuli in SCC-13 and HaCaT. In clear contrast, TNFα protected PNHK against TRAIL- and 5FU-induced apoptosis. The effects were dose-dependent and TNFα-specific; furthermore, the apoptosis pathway was caspase-dependent.CONCLUSIONS:In summary, opposing effects of TNFα in malignant versus normal human keratinocytes were observed with possibly relevant clinical implications, when patients are treated with TNFα inhibitors.