NRN1 interacts with Notch to increase oncogenic STAT3 signaling in melanoma.

BACKGROUND: Melanoma is a highly heterogeneous cancer, in which frequent changes in activation of signaling pathways lead to a high adaptability to ever changing tumor microenvironments. The elucidation of cancer specific signaling pathways is of great importance, as demonstrated by the inhibitor of the common BrafV600E mutation PLX4032 in melanoma treatment. We therefore investigated signaling pathways that were influenced by neurotrophin NRN1, which has been shown to be upregulated in melanoma. METHODS: Using a cell culture model system with an NRN1 overexpression, we investigated the influence of NRN1 on melanoma cells' functionality and signaling. We employed real time cell analysis and spheroid formation assays, while for investigation of molecular mechanisms we used a kinase phosphorylation kit as well as promotor activity analysis followed by mRNA and protein analysis. RESULTS: We revealed that NRN1 interacts directly with the cleaved intracellular domain (NICD) of Notch1 and Notch3, causing a potential retention of NICD in the cytoplasm and thereby reducing the expression of its direct downstream target Hes1. This leads to decreased sequestration of JAK and STAT3 in a Hes1-driven phosphorylation complex. Consequently, our data shows less phosphorylation of STAT3 while presenting an accumulation of total protein levels of STAT3 in association with NRN1 overexpression. The potential of the STAT3 signaling pathway to act in both a tumor suppressive and oncogenic manner led us to investigate specific downstream targets - namely Vegf A, Mdr1, cMet - which were found to be upregulated under oncogenic levels of NRN1. CONCLUSIONS: In summary, we were able to show that NRN1 links oncogenic signaling events between Notch and STAT3 in melanoma. We also suggest that in future research more attention should be payed to cellular regulation of signaling molecules outside of the classically known phosphorylation events.

Loss of miR-101-3p in melanoma stabilizes genomic integrity, leading to cell death prevention.

Malignant melanoma remains the most lethal form of skin cancer, exhibiting poor prognosis after forming distant metastasis. Owing to their potential tumor-suppressive properties by regulating oncogenes and tumor suppressor genes, microRNAs are important player in melanoma development and progression. We defined the loss of miR-101-3p expression in melanoma cells compared with melanocytes and melanoblast-related cells as an early event in tumor development and aimed to understand the tumor suppressive role of miR-101-3p and its regulation of important cellular processes. Reexpression of miR-101-3p resulted in inhibition of proliferation, increase in DNA damage, and induction of apoptosis. We further determined the nuclear structure protein Lamin B1, which influences nuclear processes and heterochromatin structure, ATRX, CASP3, and PARP as an important direct target of miR-101-3p. RNA sequencing and differential gene expression analysis after miR-101-3p reexpression supported our findings and the importance of loss of mir-101-3p for melanoma progression. The validated functional effects are related to genomic instability, as recent studies suggest miRNAs plays a key role in mediating this cellular process. Therefore, we concluded that miR-101-3p reexpression increases the genomic instability, leading to irreversible DNA damage, which leads to apoptosis induction. Our findings suggest that the loss of miR-101-3p in melanoma serves as an early event in melanoma progression by influencing the genomic integrity to maintain the increased bioenergetic demand.

Membrane Transporters and Channels in Melanoma.

Recent research has revealed that ion channels and transporters can be important players in tumor development, progression, and therapy resistance in melanoma. For example, members of the ABC family were shown to support cancer stemness-like features in melanoma cells, while several members of the TRP channel family were reported to act as tumor suppressors.Also, many transporter proteins support tumor cell viability and thus suppress apoptosis induction by anticancer therapy. Due to the high number of ion channels and transporters and the resulting high complexity of the field, progress in understanding is often focused on single molecules and is in total rather slow. In this review, we aim at giving an overview about a broad subset of ion transporters, also illustrating some aspects of the field, which have not been addressed in detail in melanoma. In context with the other chapters in this special issue on "Transportome Malfunctions in the Cancer Spectrum," a comparison between melanoma and these tumors will be possible.

Nuclear AREG affects a low-proliferative phenotype and contributes to drug resistance of melanoma.

AMPHIREGULIN (AREG) is a multifaceted molecule, which acts not only as an extracellular ligand for EGF receptor (EGFR), but also as an intracellular signaling molecule. It remains elusive, however, whether AREG has a tumor suppressive or oncogenic role in melanoma. Here, we found that several melanoma cell lines express AREG, but the expression does not correlate with that of EGFR. Recombinant AREG and the neutralizing antibody experiments showed that intracellular AREG plays an important role in melanoma, implying a divergent function of AREG in addition to the role as a ligand for EGFR. Further investigation of this mechanism revealed that particularly nuclear-localized AREG regulates IGF-1R, P21 (Cip1/Waf1), TP53 and JARID1B protein accumulation in the nucleus. Furthermore, manipulation of nuclear AREG levels has influence on heterochromatin condensation (HP1beta, SETDB1) and trimethylation of histones H3K9 and H3K4. As these molecules correspond to previously identified markers for slow-cycling drug resistant cells, we speculate that nuclear AREG predisposes cells to resistance to therapy. According to the hypothesis, we detected the accumulation of AREG in the nucleus of SK-Mel-28-VR, which was cultured under Vemurafenib (VR) selection pressure, and this correlates with JARID1B expression. Here, knockdown of AREG makes the previously resistant cells more sensitive to VR treatment, resulting in inhibited proliferation. Taken together, we suggest that nuclear AREG affects a slow-cycling phenotype and increases resistance to VR, raising a possibility that AREG might be a potential therapeutic target for resistance in melanoma.

Acidified Nitrite Contributes to the Antitumor Effect of Cold Atmospheric Plasma on Melanoma Cells.

Cold atmospheric plasma (CAP) is partially ionized gas near room temperature with previously reported antitumor effects. Despite extensive research and growing interest in this technology, active components and molecular mechanisms of CAP are not fully understood to date. We used Raman spectroscopy and colorimetric assays to determine elevated nitrite and nitrate levels after treatment with a MiniFlatPlaster CAP device. Previously, we demonstrated CAP-induced acidification. Cellular effects of nitrite and strong extracellular acidification were assessed using live-cell imaging of intracellular Ca2+ levels, cell viability analysis as well as quantification of p21 and DNA damage. We further characterized these observations by analyzing established molecular effects of CAP treatment. A synergistic effect of nitrite and acidification was found, leading to strong cytotoxicity in melanoma cells. Interestingly, protein nitration and membrane damage were absent after treatment with acidified nitrite, thereby challenging their contribution to CAP-induced cytotoxicity. Further, phosphorylation of ERK1/2 was increased after treatment with both acidified nitrite and indirect CAP. This study characterizes the impact of acidified nitrite on melanoma cells and supports the importance of RNS during CAP treatment. Further, it defines and evaluates important molecular mechanisms that are involved in the cancer cell response to CAP.

Cold Atmospheric Plasma Changes the Amino Acid Composition of Solutions and Influences the Anti-Tumor Effect on Melanoma Cells.

Cold Atmospheric Plasma (CAP) is an ionized gas near room temperature. Its anti-tumor effect can be transmitted either by direct treatment or mediated by a plasma-treated solution (PTS), such as treated standard cell culture medium, which contains different amino acids, inorganic salts, vitamins and other substances. Despite extensive research, the active components in PTS and its molecular or cellular mechanisms are not yet fully understood. The purpose of this study was the measurement of the reactive species in PTS and their effect on tumor cells using different plasma modes and treatment durations. The PTS analysis yielded mode- and dose-dependent differences in the production of reactive oxygen and nitrogen species (RONS), and in the decomposition and modification of the amino acids Tyrosine (Tyr) and Tryptophan (Trp). The Trp metabolites Formylkynurenine (FKyn) and Kynurenine (Kyn) were produced in PTS with the 4 kHz (oxygen) mode, inducing apoptosis in Mel Im melanoma cells. Nitrated derivatives of Trp and Tyr were formed in the 8 kHz (nitrogen) mode, elevating the p16 mRNA expression and senescence-associated ß-Galactosidase staining. In conclusion, the plasma mode has a strong impact on the composition of the active components in PTS and affects its anti-tumor mechanism. These findings are of decisive importance for the development of plasma devices and the effectiveness of tumor treatment.

miR-488-5p and its role in melanoma.

Due to their ability to regulate dozens to hundreds of target genes simultaneously and, therefore, influence several oncogenic pathways at the same time, microRNAs are a fascinating research object in melanoma. MicroRNAs have been identified as regulators of tumor proliferation, invasion and metastasis in melanoma. More precisely, it has been published that dysregulation of miR-488 contibutes to the progression of several cancer entities. However, the biological functions of miR-488, in special miR-488-5p in melanoma, remain unclear. This study showed the involvement of miR-488-5p in Wnt/ß-catenin pathway and the function as a tumor suppressor. Transfection of miR-488-5p mimic led to inhibition of proliferation, migration, anchorage independent growth and led to induction of apoptosis. These data indicated that miR-488-5p acts as a tumor suppressor and is lost during melanoma development. The loss of miR-488-5p was confirmed in vivo by in situ hybridization on melanoma tissue.

Wild type Kirsten rat sarcoma is a novel microRNA-622-regulated therapeutic target for hepatocellular carcinoma and contributes to sorafenib resistance.

OBJECTIVE: Sorafenib is the only effective therapy for advanced hepatocellular carcinoma (HCC). Combinatory approaches targeting mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)- and phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K)/protein-kinase B(AKT) signalling yield major therapeutic improvements. RAS proteins regulate both RAF/MAPK and PI3K/AKT signalling. However, the most important RAS isoform in carcinogenesis, Kirsten rat sarcoma (KRAS), remains unexplored in HCC. DESIGN: Human HCC tissues and cell lines were used for expression and functional analysis. Sorafenib-resistant HCC cells were newly generated. RNA interference and the novel small molecule deltarasin were used for KRAS inhibition both in vitro and in a murine syngeneic orthotopic HCC model. RESULTS: Expression of wild type KRAS messenger RNA and protein was increased in HCC and correlated with extracellular-signal regulated kinase (ERK) activation, proliferation rate, advanced tumour size and poor patient survival. Bioinformatic analysis and reporter assays revealed that KRAS is a direct target of microRNA-622. This microRNA was downregulated in HCC, and functional analysis demonstrated that KRAS-suppression is the major mediator of its inhibitory effect on HCC proliferation. KRAS inhibition markedly suppressed RAF/ERK and PI3K/AKT signalling and proliferation and enhanced apoptosis of HCC cells in vitro and in vivo. Combinatory KRAS inhibition and sorafenib treatment revealed synergistic antitumorigenic effects in HCC. Sorafenib-resistant HCC cells showed elevated KRAS expression, and KRAS inhibition resensitised sorafenib-resistant cells to suppression of proliferation and induction of apoptosis. CONCLUSIONS: KRAS is dysregulated in HCC by loss of tumour-suppressive microRNA-622, contributing to tumour progression, sorafenib sensitivity and resistance. KRAS inhibition alone or in combination with sorafenib appears as novel promising therapeutic strategy for HCC.

The importance of melanoma inhibitory activity gene family in the tumor progression of oral cancer.

Oral squamous cell carcinoma has a high potential for locoregional invasion and nodal metastasis. Consequently, early detection of such malignancies is of immense importance. The melanoma inhibitory activity (MIA) gene family comprises MIA, MIA2, transport and Golgi organization protein 1 (TANGO), and otoraplin (OTOR). These members of the MIA gene family have a highly conserved Src homology 3 (SH3)-like structure. Although the molecules of this family share 34-45% amino acid homology and 47-59% cDNA sequence homology, those members, excluding OTOR, play different tumor-associated functions. MIA has a pivotal role in the progression and metastasis of melanoma; MIA2 and TANGO have been suggested to possess tumor-suppressive functions; and OTOR is uniquely expressed in cochlea of the inner ear. Therefore, the definite functions of the MIA gene family in cancer cells remain unclear. Since the members of the MIA gene family are secreted proteins, these molecules might be useful tumor markers that can be detected in the body fluids, including serum and saliva. In this review, we described the molecular biological functions of the MIA gene family in oral cancer.

Robo3A and Robo3B expression is regulated via alternative promoters and mRNA stability.

BACKGROUND: The transmembrane receptor family Roundabout (Robo) was described to have an essential role in the developing nervous system. Recent studies demonstrated that Robo3 shows an altered expression in rheumatoid arthritis as well as in melanoma. CONTEXT AND PURPOSE OF THE STUDY: Until today no detailed studies of the two Robo3 isoforms (Robo3A and Robo3B) and their roles in rheumatoid arthritis synovial fibroblasts, respectively malignant melanoma are available. To get a better understanding regarding the role of Robo3A and Robo3B in the molecular process of rheumatoid arthritis and melanoma the exact characterization of expression and regulation is object of this study. RESULTS: mRNA and protein expression of the transcriptional variants were analyzed by quantitative RT-PCR respectively western blotting and revealed particularly enhanced expression of Robo3B in rheumatoid arthritis and melanoma. Promoter assays and inhibitor studies also disclosed that there is apparently a cell- and isoform-specific regulation of the Robo3 expression. Finally, dissimilar mRNA stabilities of Robo3A and Robo3B are identified as decisive posttranscriptional gene expression control. CONCLUSION: In summary, this study supported an isotype specific role of Robo3B in disease hinting to different functional roles of each isoform.

Systematic investigation of CMTM family genes suggests relevance to glioblastoma pathogenesis and CMTM1 and CMTM3 as priority targets.

The novel CKLF-like Marvel Transmembrane Domain-containing gene family (CMTM) consists of 8 members (CMTM1-8). As little is known about the oncogenic impact of these genes, we aimed to systematically investigate the relevance of CMTMs to glioblastoma pathogenesis. We performed mRNA expression analyses and survival correlations in glioblastoma patients. Moreover, we analyzed the impact of RNAi-based silencing and overexpression of CMTM family genes on tumor cell proliferation and invasion in vitro. CMTMs appeared to be widely regulated in the group of glioblastomas relative to non-neoplastic brain (NB) tissue (significant upregulation for CMTM2, 3, and 6 and significant downregulation for CMTM 4 and 8). For CMTM1, 5 and 7, we found aberrant expression levels in individual tumors. Functionally, CMTM1, 3, and 7 promoted tumor cell invasion, while CMTM1 additionally enhanced cell proliferation. In a large clinically annotated dataset, higher CMTM1 and 3 expression was significantly correlated with shorter overall survival. Our data thus suggest CMTM1 and 3 as priority targets in glioblastomas. Using a human phosphokinase protein expression profiling assay, we can provide first insights into signalling of these two genes that might be conveyed by growth factor receptor, Src family kinase and WNT activation.

Enhanced expression of BMP6 inhibits hepatic fibrosis in non-alcoholic fatty liver disease.

OBJECTIVE: Bone morphogenetic protein 6 (BMP6) has been identified as crucial regulator of iron homeostasis. However, its further role in liver pathology including non-alcoholic fatty liver disease (NAFLD) and its advanced form non-alcoholic steatohepatitis (NASH) is elusive. The aim of this study was to investigate the expression and function of BMP6 in chronic liver disease. DESIGN: BMP6 was analysed in hepatic samples from murine models of chronic liver injury and patients with chronic liver diseases. Furthermore, a tissue microarray comprising 110 human liver tissues with different degree of steatosis and inflammation was assessed. BMP6-deficient (BMP6(-/-)) and wild-type mice were compared in two dietary NASH-models, that is, methionine choline-deficient (MCD) and high-fat (HF) diets. RESULTS: BMP6 was solely upregulated in NAFLD but not in other murine liver injury models or diseased human livers. In NAFLD, BMP6 expression correlated with hepatic steatosis but not with inflammation or hepatocellular damage. Also, in vitro cellular lipid accumulation in primary human hepatocytes induced increased BMP6 expression. MCD and HF diets caused more hepatic inflammation and fibrosis in BMP6(-/-) compared with wild-type mice. However, only in the MCD and not in the HF diet model BMP6(-/-) mice developed marked hepatic iron overload, suggesting that further mechanisms are responsible for protective BMP6 effect. In vitro analysis revealed that recombinant BMP6 inhibited the activation of hepatic stellate cells (HSCs) and reduced proinflammatory and profibrogenic gene expression in already activated HSCs. CONCLUSIONS: Steatosis-induced upregulation of BMP6 in NAFLD is hepatoprotective. Induction of BMP6-signalling may be a promising antifibrogenic strategy.

Aberrant expression of c-Jun in glioblastoma by internal ribosome entry site (IRES)-mediated translational activation.

Although the protooncogene c-Jun plays a critical role in cell proliferation, cell death, and malignant transformation, DNA microarray screens have identified only a few human cancer types with aberrant expression of c-Jun. Here, we show that c-Jun accumulation is robustly elevated in human glioblastoma and that this increase contributes to the malignant properties of the cells. Most importantly, the increase in c-Jun protein accumulation occurs with no corresponding increase in c-Jun mRNA or the half-life of the c-Jun protein but, rather, in the translatability of the transcript. The c-Jun 5'UTR harbors a potent internal ribosomal entry site (IRES) with a virus-like IRES domain that directs cap-independent translation in glioblastoma cells. Accumulation of c-Jun is not dependent on MAPK activity but can be stimulated by a cytoskeleton-dependent pathway. Our findings provide evidence that human c-Jun is an IRES-containing cellular transcript that contributes to cancer development through translational activation. This previously undescribed mechanism of c-Jun regulation might also be relevant to other types of human cancer and offers unique potential targets for therapy.

Regulation and function of the atypical cadherin FAT1 in hepatocellular carcinoma.

In human cancers, giant cadherin FAT1 may function both, as an oncogene and a tumor suppressor. Here, we investigated the expression and function of FAT1 in hepatocellular carcinoma (HCC). FAT1 expression was increased in human HCC cell lines and tissues compared with primary human hepatocytes and non-tumorous liver tissue as assessed by quantitative PCR and western blot analysis. Combined immunohistochemical and tissue microarray analysis showed a significant correlation of FAT1 expression with tumor stage and proliferation. Suppression of FAT1 expression by short hairpin RNA impaired proliferation and migration as well as apoptosis resistance of HCC cells in vitro. In nude mice, tumors formed by FAT1-suppressed HCC cells showed a delayed onset and more apoptosis compared with tumors of control cells. Both hepatocyte growth factor and hypoxia-mediated hypoxia-inducible factor 1 alpha activation were identified as strong inducers of FAT1 in HCC. Moreover, demethylating agents induced FAT1 expression in HCC cells. Hypoxia lead to reduced levels of the methyl group donor S-adenosyl-L-methionine (SAM) and hypoxia-induced FAT1 expression was inhibited by SAM supplementation in HCC cells. Together, these findings indicate that FAT1 expression in HCC is regulated via promotor methylation. FAT1 appears as relevant mediator of hypoxia and growth receptor signaling to critical tumorigenic pathways in HCC. This knowledge may facilitate the rational design of novel therapeutics against this highly aggressive malignancy.

Melanoma inhibitory activity in melanoma diagnostics and therapy - a small protein is looming large.

Malignant melanoma is a highly aggressive cancer with a very poor prognosis after the onset of metastasis. We have previously demonstrated that the protein melanoma inhibitory activity (MIA) is involved in the metastasis of and immunosuppression in malignant melanoma. Recently, we further established MIA as a therapeutic target to inhibit metastatic spread in malignant melanoma. We could show that an inhibition of MIA by a synthetic peptide decreased both the number of metastases as well as immunosuppression in a murine model of malignant melanoma. To control recurrence after surgical resection of a primary lesion, it is paramount to have diagnostic tools available that can detect a relapse due to the strong metastatic potential of melanoma. This follow-up is maintained with periodic re-examinations. Due to high cost and the associated radiation exposure, radiology examinations are avoided if possible. The analysis of prognostic markers in patient serum is therefore attractive. In this review, we focus on the quantitative analysis of the MIA protein as a prognostic tool because it has proven to be a useful serum marker for documenting disease progression of malignant melanoma. The MIA quantification assay itself is readily performed using an ELISA kit and common laboratory equipment. Because analysing MIA serum levels in combination with other established markers such as S100B improves their prognostic value, we feel that the quantification of MIA in the serum, among other markers, should be performed as a general standard of care in patients at risk of developing metastatic melanoma.

Alternative Wnt-signaling axis leads to a break of oncogene-induced senescence.

Oncogene-induced senescence (OIS) is an important process that suppresses tumor development, but the molecular mechanisms of OIS are still under investigation. It is known that BRAFV600E-mutated melanocytes can overcome OIS and develop melanoma, but the underlying mechanism is largely unknown. Using an established OIS model of primary melanocytes transduced with BRAFV600E, YAP activity was shown to be induced in OIS as well as in melanoma cells compared to that in normal epidermal melanocytes. This led to the assumption that YAP activation itself is not a factor involved in the disruption of OIS. However, its role and interaction partners potentially change. As Wnt molecules are known to be important in melanoma progression, these molecules were the focus of subsequent studies. Interestingly, activation of Wnt signaling using AMBMP resulted in a disruption of OIS in BRAFV600E-transduced melanocytes. Furthermore, depletion of Wnt6, Wnt10b or ß-catenin expression in melanoma cells resulted in the induction of senescence. Given that melanoma cells do not exhibit canonical Wnt/ß-catenin activity, alternative ß-catenin signaling pathways may disrupt OIS. Here, we discovered that ß-catenin is an interaction partner of YAP on DNA in melanoma cells. Furthermore, the ß-catenin-YAP interaction changed the gene expression pattern from senescence-stabilizing genes to tumor-supportive genes. This switch is caused by transcriptional coactivation via the LEF1/TEAD interaction. The target genes with binding sites for LEF1 and TEAD are involved in rRNA processing and are associated with poor prognosis in melanoma patients. This study revealed that an alternative YAP-Wnt signaling axis is an essential molecular mechanism leading to OIS disruption in melanocytes.

The Role of T-Cadherin (CDH13) in Treatment Options with Garcinol in Melanoma.

Targeted therapies with chemotherapeutic agents and immunotherapy with checkpoint inhibitors are among the systemic therapies recommended in the guidelines for clinicians to treat melanoma. Although there have been constant improvements in the treatment of melanoma, resistance to the established therapies continues to occur. Therefore, the purpose of this study was to explore the function of garcinol with regards to specific cancer properties such as proliferation and apoptosis. Garcinol, a natural compound isolated from the plant also known as mangosteen (Garcinia mangostana), is a newly discovered option for cancer treatment. Numerous pharmaceutical substances are derived from plants. For example, the derivates of camptothecin, extracted from the bark of the Chinese tree of happiness (Camptotheca acuminate), or paclitaxel, extracted from the bark of the Western yew tree (Taxus brevifolia), are used as anti-cancer drugs. Here, we show that garcinol reduced proliferation and induced apoptosis in melanoma cell lines. In addition, we found that those cells that are positive for the expression of the cell-cell adhesion molecule T-cadherin (CDH13) respond more sensitively to treatment with garcinol. After knock-down experiments with an siRNA pool against T-cadherin, the sensitivity to garcinol decreased and proliferation and anti-apoptotic behavior of the cells was restored. We conclude that patients who are T-cadherin-positive could especially benefit from a therapy with garcinol.

Transcription factor activating enhancer-binding protein 2ε (AP2ε) modulates phenotypic plasticity and progression of malignant melanoma.

Malignant melanoma, the most aggressive form of skin cancer, is often incurable once metastatic dissemination of cancer cells to distant organs has occurred. We investigated the role of Transcription Factor Activating Enhancer-Binding Protein 2ε (AP2ε) in the progression of metastatic melanoma. Here, we observed that AP2ε is a potent activator of metastasis and newly revealed AP2ε to be an important player in melanoma plasticity. High levels of AP2ε lead to worsened prognosis of melanoma patients. Using a transgenic melanoma mouse model with a specific loss of AP2ε expression, we confirmed the impact of AP2ε to modulate the dynamic switch from a migratory to a proliferative phenotype. AP2ε deficient melanoma cells show a severely reduced migratory potential in vitro and reduced metastatic behavior in vivo. Consistently, we revealed increased activity of AP2ε in quiescent and migratory cells compared to heterogeneously proliferating cells in bioprinted 3D models. In conclusion, these findings disclose a yet-unknown role of AP2ε in maintaining plasticity and migration in malignant melanoma cells.

Cold atmospheric plasma, a new strategy to induce senescence in melanoma cells.

Over the past few years, the application of cold atmospheric plasma (CAP) in medicine has developed into an innovative field of research of rapidly growing importance. One promising new medical application of CAP is cancer treatment. Different studies revealed that CAP may potentially affect the cell cycle and cause cell apoptosis or necrosis in tumor cells dependent on the CAP device and doses. In this study, we used a novel hand-held and battery-operated CAP device utilizing the surface micro discharge (SMD) technology for plasma production in air and consequently analysed dose-dependent CAP treatment effects on melanoma cells. After 2 min of CAP treatment, we observed irreversible cell inactivation. Phospho-H2AX immunofluorescence staining and Flow cytometric analysis demonstrated that 2 min of CAP treatment induces DNA damage, promotes induction of Sub-G1 phase and strongly increases apoptosis. Further, protein array technology revealed induction of pro-apoptotic events like p53 and Rad17 phosphorylation of Cytochrome c release and activation of Caspase-3. Interestingly, using lower CAP doses with 1 min of treatment, almost no apoptosis was observed but long-term inhibition of proliferation. H3K9 immunofluorescence, SA-ß-Gal staining and p21 expression revealed that especially these low CAP doses induce senescence in melanoma cells. In summary, we observed differences in induction of apoptosis or senescence of tumor cells in respond to different CAP doses using a new CAP device. The mechanism of senescence with regard to plasma therapy was so far not described previously and is of great importance for therapeutic application of CAP.

Cold Atmospheric Plasma Triggers Apoptosis via the Unfolded Protein Response in Melanoma Cells.

Cold atmospheric plasma (CAP) describes a partially ionized gas carrying large amounts of reactive oxygen (ROS) and nitrogen species (RNS). Numerous studies reported strong antitumor activity of CAP, thus rendering it a promising approach for tumor therapy. Although several cellular mechanisms of its cytotoxicity were identified in recent years, the exact molecular effects and contributing signaling pathways are yet to be discovered. We discovered a strong activation of unfolded protein response (UPR) after CAP treatment with increased C/EBP homologous protein (CHOP) expression, which was mainly caused by protein misfolding and calcium loss in the endoplasmic reticulum. In addition, both ceramide level and ceramide metabolism were reduced after CAP treatment, which was then linked to the UPR activation. Pharmacological inhibition of ceramide metabolism resulted in sensitization of melanoma cells for CAP both in vitro and ex vivo. This study identified a novel mechanism of CAP-induced apoptosis in melanoma cells and thereby contributes to its potential application in tumor therapy.