WM35 Cells Research Articles

CD44 and CD271 expression and chemoresistance in BRAF V600E/K mutation–positive melanoma.

e21542 Background: Melanoma stem cells (MSC) are thought to play a role in cellular proliferation, migration, and chemotherapeutic resistance. We investigated native expression of several putative MSC biomarkers in human BRAF V600E/K mutation positive melanoma cell lines of different phases—radial growth phase (WM35), vertical growth phase (WM278)—and metastatic (LM-Mel-45, LM-Mel-71) cell lines. We examined cell viability of select cell subpopulations during in vitro treatment with BRAF and MEK inhibitor monotherapy and combination therapy. Methods: Using flow cytometry, we assessed surface expression of putative MSC biomarkers: CD44, CD271, CD133, CD186, and CD20. We sorted each cell line by CD44 and CD271 expression with a predetermined hierarchical gating strategy and compared 4 chemotherapy conditions: control, BRAF inhibition with 1.25 µM dabrafenib, MEK inhibition with 0.125 µM trametinib, and combination therapy. Cells (25,000) were collected directly into chemotherapy treated media and incubated for 72 hours. Cell viability was measured by MTT assay. Statistical analysis used mixed-effects ANOVA and Tukey HSD. Results: No significant expression of CD133, CD186, or CD20 was found in any cell line. Over 99% of live cells were CD44+ in all 4 cell lines. Only WM35 and LM-Mel-71 had both CD44+/CD271+ and CD44+/CD271- subpopulations. CD271 expression was restricted to CD44+ cells. Among live cells, the CD44+/CD271+ subpopulation was 31.2% for WM35 and 14.4% for LM-Mel-71. For WM35, cell viability in CD44+/CD271+ cells was greater than CD44+/CD271- cells for all treatments (p < 0.001). For LM-Mel-71 cells, CD271 expression did not affect cell viability within any treatment (p = 0.64). For chemotherapeutic treatments, cell viability in CD44+/CD271- subpopulation was higher in LM-Mel-45 cells than WM278 (p < 0.03) or LM-Mel-71 (p < 0.002), but not WM35 cells (p > 0.05). Trametinib monotherapy and combination therapy had similar cell viability in all 4 cell lines (p > 0.05). Conclusions: To our knowledge, our study is the first to show differences in phenotypic expression of CD44 and CD271 across nonmetastatic and metastatic melanoma cell lines and suggests co-expression of CD44 and CD271 confers increased chemotherapeutic resistance to in vitro BRAF and MEK inhibitor treatment in radial growth phase but not metastatic melanoma. More research is needed to better understand the role of CD44 and CD271 co-expression in melanoma initiation, progression, and chemotherapeutic resistance. CD271 remains a potential immunotherapeutic drug target in the treatment of BRAF V600E/K mutation positive melanoma.

Cell viability and chemotherapeutic resistance across melanoma lines from various stages of progression

Background: The melanoma stem cell (MSC) hypothesis holds that a subpopulation of cells with stem-cell like features are important for tumor progression and chemoresistance. We examined four V600E BRAF-mutated melanoma lines at various stages of melanoma progression. Within each cell line, we assessed the expression of putative MSC markers: CD20, CD44, CD133, CD186, and CD271. Subsequently, we assessed cell viability in the four lines during mono- or combination chemotherapy relative to surface marker expression. Methods: We examined two primary melanoma lines: WM35 (radial growth phase) and WM278 (vertical growth phase). Additionally, we analyzed two metastatic melanoma lines from brain metastases: LM-Mel-71 and LM-Mel-45, exhibiting epithelial and fibroblastic morphologies, respectively. MSC surface marker expression was determined using a Biorad S3e cell sorter to collect 25,000 cells each into one of 4 chemotherapeutic treatments on a 96-well plate. The treatments included: control, dabrafenib (1.25 μM, BRAF inhibitor), trametinib (0.125 μM, MEK inhibitor), and a combination of dabrafenib (1.25 μM) and trametinib (0.125 μM). Following 72 hours of incubation, we analyzed cell viability using an MTT assay. Statistical analysis was performed using ANOVA and Tukey posthoc testing. Results: All cell lines exhibited over 99% CD44+ cells. The only surface markers detected were CD44 and CD271; the remaining markers were undetectable across the four cell lines. LM-Mel-71 and WM35 cells presented both CD44+/CD271+ (14% and 31%, respectively) population and a larger CD44+/CD271- population. LM-Mel-45 and WM278 cells displayed a CD44+/CD271- population. LM-Mel-45 consistently exhibited higher viability than LM-Mel-71 (p<0.002) or WM271 (p<0.03) under both control and the three chemotherapeutic regimens. For LM-Mel-71, cell viability did not differ with CD271 expression in any treatment condition (p=0.64). For WM35 cells, viability was significantly higher in CD271+ compared to CD271- cells under all treatment conditions. Based on cell viability, monotherapy with trametinib or combination therapy appeared more effective than monotherapy with dabrafenib alone across all melanoma lines. Conclusions: The results suggest that there is not a simple relationship between CD271 expression and the origin of the cell line. While CD44+CD271+ expression may correlate with increased cell viability — as observed in WM35 radial growth phase melanoma — such an association is not universal, as CD44+/CD271+ and CD44+/CD271- cells demonstrated similar viability under each chemotherapeutic condition. Additional factors beyond the examined surface markers likely modulate melanoma progression and BRAF/MEK chemotherapy effcacy. Supported by A T Still Univeristy Student Research Fund Grant 560-950 to EH and MR. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Blocking of Drug Resistance Channels by Selected Hydrophobic Statins in Chemoresistance Human Melanoma.

Despite the development of modern drugs, drug resistance in oncology remains the main factor limiting the curability of patients. This paper shows the use of a group of hydrophobic statins to inhibit drug resistance (Pgp protein). In a chemoresistance melanoma cell model, viability, necroptosis with DNA damage, the absorption of the applied pharmaceuticals, and the functional activity of the ABCB1 drug transporter after administration of docetaxel or docetaxel with a selected hydrophobic statin were studied. Taxol-resistant human melanoma cells from three stages of development were used as a model: both A375P and WM239A metastatic lines and radial growth phase WM35 cells. An animal model (Mus musculus SCID) was developed for the A375P cell line. The results show that hydrophobic statins administered with docetaxel increase the accumulation of the drug in the tumor cell a.o. by blocking the ABCB1 channel. They reduce taxol-induced drug resistance. The tumor size reduction was observed after the drug combination was administrated. It was shown that the structural similarity of statins is of secondary importance, e.g., pravastatin and simvastatin. Using cytostatics in the presence of hydrophobic statins increases their effectiveness while reducing their overall toxicity.

Discovery of novel CDK2 inhibitors using multistage virtual screening and in vitro melanoma cell lines.

Cyclin-dependent kinases 2 (CDK2) is a serine/threonine-protein kinase, which plays a key role in the regulation of cell cycle and is related to the occurrence and development of melanoma. In this study, we identified potent inhibitors for CDK2 by combining a multistage virtual screening strategy with bioassay validations. The biochemical activity of compounds was validated with ADP-Glo™ Kinase assay in vitro, and the results indicated that the biochemical activity of compound 1 (C1) was better than other selected compounds. Cell viability assay showed that the minimum inhibition concentration of C1 for CDK2 was lower than 4μM. Further functional test results showed that C1 exerted significant antiproliferative, pro-apoptosis, and anti-migration activity in melanoma cell lines (A375 cells, WM35 cells, and A875 cells). Our findings suggested that the C1, virtually screened from compound libraries, as the novel inhibitor of CDK2, may be further developed as an effective therapeutic agent in the treatment of melanoma lines.

Capsaicin Regulates Mitochondrial Fission to Promote Melanoma Cell Apoptosis

Abstract Objective: Capsaicin (CPS) is a major component of the red pepper, and its anti-tumor property has been confirmed. However, the underlying mechanism of this anti-tumor effect has not been fully clarified, so we conducted this study to evaluate the role of mitochondrial fission and subsequent mitochondrial dysfunction in CPS-induced apoptosis of melanoma cells. Methods: Two melanoma cell lines and melanocytes were treated with CPS alone or in combination with ruthenium red (a transient receptor potential vanilloid 1 [TRPV] antagonist), Z-VAD-FMK (a pan-caspase inhibitor), or N-acetyl-L-cysteine (an antioxidant). Cell vitality was tested using a cell counting kit-8 assay. The expression levels of related proteins were examined by Western blotting. Apoptosis, intracellular reactive oxygen species, mitochondrial membrane potential, adenosine triphosphate levels, and mitochondrial dynamics were analyzed by flow cytometry, luminometry, and confocal laser microscopy, respectively, and compared between groups. Results: CPS treatment significantly inhibited the vitality of melanoma cells (For A2058 cells: 0 vs. 120 μmol/L: [100.00% ± 0%] vs. [51.02% ± 6.40%], P < 0.05; For WM35 cells: 0 vs. 120 μmol/L: [100.00% ± 0%] vs. [51.80% ± 3.45%], P < 0.05) but exerted less impact on normal melanocytes. CPS promoted melanoma cell apoptosis through TRPV channels and the caspase cascade. CPS treatment then led to TRPV channel-dependent mitochondrial dysfunction with an increase in reactive oxygen species generation (For A2058 cells: CPS vs. CPS+RR: [2.34 ± 0.30] vs. [1.34 ± 0.12], P < 0.05; For WM35 cells: CPS vs. CPS+RR: [2.25 ± 0.25] vs. [1.65 ± 0.13], P < 0.05), dissipation of the mitochondrial membrane potential (Control vs. CPS: [1.00 ± 0] vs. [0.61 ± 0.08], P < 0.05), and adenosine triphosphate reduction (P < 0.05). In addition, reactive oxygen species generation contributed to CPS-induced melanoma cell apoptosis. Mitochondrial fission was subsequently proved to connect CPS treatment to mitochondrial dysfunction, which was also TRPV channel-dependent, thereby inducing melanoma cell apoptosis. Conclusion: Our study highlights the role of mitochondrial fission and its related mitochondrial dysfunction in mediating the pro-apoptotic effect of CPS in melanoma. These findings deepen our understanding of the mechanisms underlying the anti-tumor activity of CPS and indicate the clinical relevancy of extending the use of this agent for cancer therapy.

Effect of miR-19b-3p targeting coiled-coil domain containing 6 on melanoma cell proliferation, invasion and tumor volume in mice

Objective To investigate the mechanism of microRNA (miRNA, miR) -19b-3p targeted regulation of coiled-coil domain containing 6 (CCDC6) in melanoma cell proliferation, invasion and mouse tumor volume. Methods Fifty-six cases of melanoma tissue and fifty-six healthy people were collected. The relative expression of miR-19b-3p and CCDC6 was detected by real-time quantitative polymerase chain reaction (Real-time PCR). The proliferation of WM35 cells was detected by cell counting kit-8 (CCK-8). The invasion ability of WM35 cells was detected by cell invasion assay. The levels of CCDC6 and p-protein kinase B (Akt) protein were detected by Western blotting. The relationship between miR-19b-3p and CCDC6 was verified by luciferase report analysis. The nude mouse tumor model was established to explore the effect of miR-19b-3p on tumor volume. Results Compared with benign sputum tissue (1.43±0.38), miR-19b-3p levels in melanoma tissues (2.89±0.46) were significantly increased (t=6.823, P<0.05), and miR-19b-3p was significantly associated with stage Ⅲ/Ⅳ (χ2=12.087, P<0.05) and distant metastasis (χ2=12.600, P<0.05). Compared with the blank control group [miR-19b-3p: 1.16±0.28; Cell invasion: (26.35±4.73)/HF] and the miR control group [miR-19b-3p: 1.22±0.30; Cell invasion: (28.16±4.80)/HF], the miR-19b-3p expression level and cell invasion ability of miR-19b-3p mimetic group [miR-19b-3p: 4.06±0.28, t=3.977, t=4.003, P<0.05; Cell invasion: (49.64±5.11)/HF, t=3.981, t=4.500, P<0.05] were significantly increased (P<0.05), while the miR-19b-3p expression level and cell invasion ability were significantly decreased in the miR-19b-3p inhibitor group [miR-19b-3p: 0.41±0.08, t=3.771, t=3.579, P<0.05; Cell invasion: (14.47±0.96)/HF, t=3.669, t=4.004, P<0.05], P<0.05. The results of CCK-8 assay showed that the proliferation of miR-19b-3p mimetic cells was significantly increased, while the proliferation was significantly decreased in miR-19b-3p inhibitor group at 24, 48 and 72 h (F=4.026, 4.609, 7.446, P<0.05). Compared with the miR control group [volume: (467.34±36.75) mm3; CCDC6: 1.00±0.12; miR-19b-3p: 2.17±0.42], the volume of melanoma tissue in the miR-19b-3p mimic group [(1 058.66±66.17) mm3] was significantly increased, the expression of CCDC6 was significantly decreased (0.42±0.08), and the level of miR-19b-3p was significantly increased (4.01±0.66), t=22.836, 8.129, 10.663, P<0.05. Conclusion MiR-19b-3p can promote the invasion and proliferation of melanoma cells, and its mechanism is closely related to the regulation of downstream CCDC6 and p-Akt. Key words: Melanoma; MicroRNA; Coiled-coil domain containing 6; Invasion; Proliferation

Abstract 1543: Upregulation of mannose 6-phosphate receptor (MPR) has a potential to increase the efficacy of combined therapy for melanoma patients

Abstract BRAF -targeting therapy demonstrates impressive clinical response in patients with metastatic melanoma. However, patients quickly develop resistance associated with rapid tumor progression. Therefore, additional therapeutic approaches are necessary. Our group has previously shown that transient induction of cation-independent mannose 6-phosphate receptor (MPR) is important for the antitumor effect of chemo-immunotherapy. In this study, we aimed to decipher the role of MPR during possible combination therapy of BRAF inhibitor with adoptive T-cell therapy of BRAF inhibitor-resistant melanoma. For this aim, we have used PLX4720 as a BRAFV600E inhibitor and observed that in vitro PLX4720 treatment of human V600E-positive melanoma cells, including WM-35, SK-Mel-624 and WM983B, resulted in a dose-dependent upregulation of MPR on the cell surface. Importantly, increased levels of MPR on the cell surface induced the uptake of recombinant Granzyme B, which is a major mechanism of cell killing by cytotoxic T cells (CTL). We found that PLX4720 treatment rendered WM35 cells more prone to lysis by HLA-matching tumor-infiltrating lymphocytes (TILs) and increased cell killing is not due to PLX4720 treatment itself but increased levels of MPR on the cell surface. In order to prove this, we generated MPR-overexpressing WM35 cell line and showed that upregulation of MPR, independently of PLX4720 treatment, sensitizes melanoma cells for T cell killing in vitro. We further showed that PLX4720 treatment leads to upregulation of MPR in vivo by a mouse model bearing xenograft WM35-derived subcutaneous tumors as well. According to our data, 3 days of PLX4720 treatment is enough to induce MPR in tumors and this effect continues up to 6 days after 5 days treatment ends. Unexpectedly, we found that PLX4720 caused upregulation of MPR in resistant cell lines and that those cell lines became more sensitive to cytotoxic effect of TILs. Thus, our data suggest that adoptive T-cell therapy can be potentially effective in melanoma resistant to BRAF inhibitor. (This study is supported by NIH grant P50 CA168536.) Citation Format: Cigdem Atay, Sergio Lavilla-Alonso, Dmitry Gabrilovich. Upregulation of mannose 6-phosphate receptor (MPR) has a potential to increase the efficacy of combined therapy for melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1543.

Differentially expressed circRNAs in melanocytes and melanoma cells and their effect on cell proliferation and invasion.

Circular RNAs (circRNAs) play critical roles in the occurrence of human diseases, including cancer. However, the detailed functions of circRNAs in melanoma have not been fully elucidated. In the present study, a circRNA microarray was performed to analyze the variability of circRNAs in the low-metastatic melanoma WM35 cell line and in the high-metastatic melanoma WM451 cell line in comparison to control human melanocytes. The results revealed that five circRNAs were upregulated and four circRNAs were downregulated in both the WM35 and WM451 cells. qRT-PCR revealed an upregulated expression of circ0000082 and circ0016418 and a downregulation of circ0023988, circ0008157 and circ0030388 in the cells which was consistent with the results of the microarray assay. Functional tests revealed that knockdown of circ0023988, circ0008157 or circ0030388 significantly promoted the proliferation and invasion of the WM35 cells. Following the silencing of circ0000082 or circ0016418 in WM451 cells, the proliferation and invasion of the WM451 cells were inhibited. Bioinformatic analysis predicted that the circ0000082-, circ0023988- and circ0008157-circRNA-miRNA-mRNA network may participate in the occurrence, development, invasion and metastasis of malignant tumors. The present study revealed several differentially expressed circRNAs, indicating that the newly identified circRNAs may provide new therapeutic targets for melanoma.

Targeting Insulin-Like Growth Factor-I and Extracellular Matrix Interactions in Melanoma Progression

Insulin-like growth factor (IGF)-I binds to the ECM protein vitronectin (VN) through IGF binding proteins (IGFBPs) to enhance proliferation and migration of skin keratinocytes and fibroblasts. Although evidence exists for the role of individual components of the complex (IGF-I, IGFBP-3 and VN), the cellular functions stimulated by these proteins together as a complex remains un-investigated in melanoma cells. We report here that the IGF-I:IGFBP-3:VN trimeric complex stimulates a dose-dependent increase in the proliferation and migration of WM35 and Sk-MEL28 melanoma cells. In 3D Matrigel™ and hydrogel cultures, both cell lines formed primary tumor-like spheroids, which increased in size in a dose-dependent manner in response to the trimeric complex. Furthermore, we reveal IGFBP-3:VN protein complexes in malignant melanoma and squamous cell carcinoma patient tissues, where the IGFBP-3:VN complex was seen to be predominantly tumor cell-associated. Peptide antagonists designed to target the binding of IGF-I:IGFBP-3 to VN were demonstrated to inhibit IGF-I:IGFBP-3:VN-stimulated cell migration, invasion and 3D tumor cell growth of melanoma cells. Overall, this study provides new data on IGF:ECM interactions in skin malignancies and demonstrates the potential usefulness of a growth factor:ECM-disrupting strategy for abrogating tumor progression.

Effects of tumor suppressor gene Dickkopf-3 on proliferation and apoptosis of cutaneous malignant melanoma cells

Objective To investigate the expression of Dickkopf-3 (DKK3) in human malignant melanoma cell lines and tissues, and to evaluate effects of DKK3 on the proliferation and apoptosis of malignant melanoma cell line A375. Methods Reverse transcription PCR (RT-PCR) and real-time fluores-cence-based quantitative PCR (qRT-PCR) were performed to measure the mRNA expression of DKK3 in human malignant melanoma cell lines HM, A375, WM451, WM35, SK-MEL-1, Hs-695T and MDA-MB-435s, as well as in 38 primary melanoma tissues, 4 metastatic melanoma tissues and 20 pigmented nevus tissues. Cultured malignant melanoma A375 cells were divided into 2 groups to be transfected with pcDNA3.1 (+) -Flag-DKK3 (experiment group) and pcDNA3.1 (+) -Flag-Vector (control group) respectively. The overexpression of DKK3 was verified by RT-PCR. Cell counting kit-8 (CCK8) assay and plate colony formation assay were performed to evaluate the proliferative activity of A375 cells, flow cytometry was conducted to detect apoptosis of A375 cells, and Western blot analysis was performed to determine the expression of cell cycle- and cell apoptosis-related proteins. Results The mRNA expression of DKK3 was downregulated in WM35 cells, absent in HM cells, A375 cells, WM451 cells, SK-MEL-1 cells and Hs-695T cells, but upregulated in MDA-MB-435s cells. Compared with pigmented nevus tissues, the mRNA expression of DKK3 was significantly decreased in malignant melanoma tissues (P < 0.001) . Compared with the control group (100%) , cell colony formation was markedly suppressed in the experiment group (23.22% ± 3.55%) , and the proliferative activity of A375 cells was also significantly inhibited in the experiment group 24, 48, 72 hours after the transfection (all P < 0.05) . Flow cytometry showed that compared with the control group, A375 cells were significantly arrested in G1 phase (48.68% ± 3.92% vs. 25.38% ± 2.92%, P < 0.001) , and the apoptosis rate of A375 cells was significantly increased in the experiment group (P < 0.001) . Compared with the control group, the experiment group showed significantly higher expression of p21, Bax, cleaved-parp and cleaved-casp3, but significantly lower expression of cyclin D1 and Bcl2 (all P < 0.001) . Conclusion DKK3 expression is downregulated in human malignant melanoma tissues, so it may serve as a potential tumor suppressor gene involved in the development of cutaneous malignant melanoma. Key words: Melanoma; Cell proliferation; Apoptosis; DKK3; Tumor suppressor genes

MicroRNA-33a-5p increases radiosensitivity by inhibiting glycolysis in melanoma.

Glycolysis was reported to have a positive correlation with radioresistance. Our previous study found that the miR-33a functioned as a tumor suppressor in malignant melanoma by targeting hypoxia-inducible factor1-alpha (HIF-1α), a gene known to promote glycolysis. However, the role of miR-33a-5p in radiosensitivity remains to be elucidated. We found that miR-33a-5p was downregulated in melanoma tissues and cells. Cell proliferation was downregulated after overexpression of miR-33a-5p in WM451 cells, accompanied by a decreased level of glycolysis. In contrast, cell proliferation was upregulated after inhibition of miR-33a-5p in WM35 cells, accompanied by increased glycolysis. Overexpression of miR-33a-5p enhanced the sensitivity of melanoma cells to X-radiation by MTT assay, while downregulation of miR-33a-5p had the opposite effects. Finally, in vivo experiments with xenografts in nude mice confirmed that high expression of miR-33a-5p in tumor cells increased radiosensitivity via inhibiting glycolysis. In conclusions, miR-33a-5p promotes radiosensitivity by negatively regulating glycolysis in melanoma.

Solamargine triggers cellular necrosis selectively in different types of human melanoma cancer cells through extrinsic lysosomal mitochondrial death pathway.

BackgroundPrevious reports showed that the Steroidal Glycoalkaloid Solamargine inhibited proliferation of non-melanoma skin cancer cells. However, Solamargine was not tested systematically on different types of melanoma cells and was not simultaneously tested on normal cells either. In this study we aimed to investigate the effect of Solamargine and the mechanism involved in inhibiting the growth of different types of melanoma cells.MethodsSolamargine effect was tested on normal cells and on another three melanoma cell lines. Vertical growth phase metastatic and primary melanoma cell lines WM239 and WM115, respectively and the radial growth phase benign melanoma cells WM35 were used. The half inhibitory concentration IC50 of Solamargine was determined using Alamarblue assay. The cellular and subcellular changes were assessed using light and Transmission Electron Microscope, respectively. The percentage of cells undergoing apoptosis and necrosis were measured using Flow cytometry. The different protein expression was detected and measured using western blotting. The efficacy of Solamargine was determined by performing the clonogenic assay. The data collected was analyzed statistically on the means of the triplicate of at least three independent repeated experiments using one-way ANOVA test for parametric data and Kruskal–Wallis for non-parametric data. Differences were considered significant when the P values were less than 0.05.ResultsHereby, we demonstrate that Solamargine rapidly, selectively and effectively inhibited the growth of metastatic and primary melanoma cells WM239 and WM115 respectively, with minimum effect on normal and benign WM35 cells. Solamargine caused cellular necrosis to the two malignant melanoma cell lines (WM115, WM239), by rapid induction of lysosomal membrane permeabilization as confirmed by cathepsin B upregulation which triggered the extrinsic mitochondrial death pathway represented by the release of cytochrome c and upregulation of TNFR1. Solamargine disrupted the intrinsic apoptosis pathway as revealed by the down regulation of hILP/XIAP, resulting in caspase-3 cleavage, upregulation of Bcl-xL, and Bcl2, and down regulation of Apaf-1 and Bax in WM115 and WM239 cells only. Solamargine showed high efficacy in vitro particularly against the vertical growth phase melanoma cells.ConclusionOur findings suggest that Solamargine is a promising anti-malignant melanoma drug which warrants further attention.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0287-4) contains supplementary material, which is available to authorized users.

Tocilizumab unmasks a stage-dependent interleukin-6 component in statin-induced apoptosis of metastatic melanoma cells.

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.

Silencing the KCNK9 potassium channel (TASK-3) gene disturbs mitochondrial function, causes mitochondrial depolarization, and induces apoptosis of human melanoma cells.

TASK-3 (KCNK9 or K2P9.1) channels are thought to promote proliferation and/or survival of malignantly transformed cells, most likely by increasing their hypoxia tolerance. Based on our previous results that suggested mitochondrial expression of TASK-3 channels, we hypothesized that TASK-3 channels have roles in maintaining mitochondrial activity. In the present work we studied the effect of reduced TASK-3 expression on the mitochondrial function and survival of WM35 and A2058 melanoma cells. TASK-3 knockdown cells had depolarized mitochondrial membrane potential and contained a reduced amount of mitochondrial DNA. Compared to their scrambled shRNA-transfected counterparts, they demonstrated diminished responsiveness to the application of the mitochondrial uncoupler [(3-chlorophenyl)hydrazono]malononitrile (CCCP). These observations indicate impaired mitochondrial function. Further, TASK-3 knockdown cells presented reduced viability, decreased total DNA content, altered cell morphology, and reduced surface area. In contrast to non- and scrambled shRNA-transfected melanoma cell lines, which did not present noteworthy apoptotic activity, almost 50% of the TASK-3 knockdown cells exhibited strong Annexin-V-specific immunofluorescence signal. Sequestration of cytochrome c from the mitochondria to the cytosol, increased caspase 3 activity, and translocation of the apoptosis-inducing factor from mitochondria to cell nuclei were also demonstrated in TASK-3 knockdown cells. Interference with TASK-3 channel expression, therefore, induces caspase-dependent and -independent apoptosis of melanoma cells, most likely via causing mitochondrial depolarization. Consequently, TASK-3 channels may be legitimate targets of future melanoma therapies.

Abstract 336: IL-6 primes melanoma cells from early stages to statin induced apoptosis

Abstract Introduction: Statins trigger apoptosis in tumour cells in vitro and, particularly, melanoma cells are susceptible to statin induced apoptosis. Growth of normal melanocytes and early stage melanoma can be inhibited by IL-6, whereas metastatic melanoma cells are mostly resistant to the anti-proliferative effects of IL-6. It has been shown that nearly 50% of metastatic melanoma secrete IL-6 into the supernatant. Nevertheless until now the stage dependant effects of IL-6 on melanoma cells are not fully understood. Methods: The secretion of IL-6 by A375, 518A2 human metastatic melanoma cells was compared with WM 35 cells which derive from an early lesion (radial growth phase) and investigated with ELISA. The sensitivity toward statin induced caspase 3 activation was investigated in these cells in the absence and presence of IL-6 or an anti IL-6 receptor antibody (tocilizumab). Apoptosis was also confirmed by FACS analyses for AnnexinV/PI staining and proliferation by cell cycle analysis using PI staining in such treated cells. Phosphorylation of Stat3 and the regulation of pro- (Bax, Bak) and anti-apoptotic proteins (Bcl-2, Bxl-XL) were studied by Western blot. Results: Melanoma cells derived from late stage lesions (A375, 518A2) secrete high amounts of IL-6 in contrast to WM35 cells. IL-6 signalling is intact in A375, 518A2 and WM 35 cells indicated by IL-6 triggered phosphorylation of Stat-3. Most interestingly, the A375 and 518A2 cells show a high sensitivity to simvastatin induced apoptosis with EC50 of 0,52 μM (518A2) and 1,7 μM (A375) in contrast to WM35 cells which are more resistant (EC50 16 μM). Co-treatment with IL-6 leads to an augmentation of the statin induced apoptosis in WM35 cells. Statin induced apoptosis can be mediated by the intrinsic apoptotic pathway which requires the loss of mitochondrial membrane potential. This is often alleviated by a downregulation of antiapoptotic Bcl-2 family members. Interestingly IL-6 treatment led to a marked decrease in the amount of Bcl-2 and Bcl-XL in WM35 cells which may explain the increased activation of apoptosis. Since A375 and 518A2 secrete high amounts of IL-6 we investigated the effects of the anti IL-6 receptor antibody tocilizumab. Conversely interruption of the IL-6 signalling with tocilizumab had no effect on the proliferation of 518A2 and A375 cells. Moreover the simvastatin induced caspase 3 activation was not decreased by blockage of the IL-6 signalling. Conclusion: Taken together our data prove different sensitivities of melanoma cells from various stages to statin induced apoptosis. Moreover, IL-6 facilitates apoptosis in early stage melanoma cells by the down-regulation of anti-apoptotic proteins. Nevertheless, these pro-apoptotic effects of IL-6 are not observed in metastatic melanoma cells indicating a stage specific role of IL-6 in the development and tumorigenesis of melanoma. Citation Format: Christoph Minichsdorfer, Christine Wasinger, Martin Hohenegger. IL-6 primes melanoma cells from early stages to statin induced apoptosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 336. doi:10.1158/1538-7445.AM2014-336

Modulation of matrix elasticity with PEG hydrogels to study melanoma drug responsiveness

Metastatic melanoma is highly resistant to drug treatment, and the underlying mechanisms of this resistance remain unclear. Increased tissue stiffness is correlated with tumor progression, but whether increased tissue stiffness contributes to treatment resistance in melanoma is not known. To investigate the effect of substrate stiffness on melanoma cell treatment responsiveness, PEG hydrogels were utilized as a cell culture system to precisely vary matrix elasticity and investigate melanoma cell responses to a commercially available pharmacological inhibitor (PLX4032). The tensile moduli were varied between 0.6 and 13.1 kPa (E) and the effects of PLX4032 on metabolic activity, apoptosis, and proliferation were evaluated on human cell lines derived from radial growth phase (WM35) and metastatic melanoma (A375). The A375 cells were found to be stiffness-independent; matrix elasticity did not alter cell morphology or apoptosis with PLX4032 treatment. The WM35 cells, however, were more dependent on substrate modulus, displaying increased apoptosis and smaller focal adhesions on compliant substrates. Culturing melanoma cells on PEG hydrogels revealed stage-dependent responses to PLX4032 that would have otherwise been masked if cultured strictly on TCPS. These findings demonstrate the utility of PEG hydrogels as a versatile in vitro culture platform with which to investigate the molecular mechanisms of melanoma biology and treatment responsiveness.

Abstract 3251: Blockade of HIF-1 with a small molecule inhibitor WP1066 in melanoma.

Abstract WP1066 was shown to exert its potent inhibitory activity against several types of human cancer including melanoma in vitro and in vivo via suppression of JAK2/STAT3/STAT5 activation. However, if there is any other additional molecular mechanism(s) by which WP1066 generates its anti-tumor effects remain largely unknown. Here, we report a novel function for WP1066 in inducing the degradation of both HIF-1α and HIF-1β subunits in human melanoma cells. We found constitutive activation of HIF-1α and an increased expression of hexokinase II, a down target of HIF-1α, in normoxia in 6/6 of melanoma cell lines, but not in other tumor types. Both hypoxia and cobalt chloride, a transition metal that mimics hypoxia, further increased HIF-1α protein accumulation in a time-dependent manner in WM35 and SKMEL-1 melanoma cells. WP1066 selectively decreased the constitutive and hypoxia and cobalt chloride induced increase in the level of HIF-1α in a dose-dependent manner with a half-life (T1/2) of ∼13, ∼10, and ∼10 min in WM35 cells. Moreover, WP1066 decreased the T1/2 of HIF-1α following cycloheximide treatment from ∼13 to ∼7 min suggesting that WP1066 induced HIF-1α instability is not mediated via inhibition of its translation. Further, inhibition of HIF-1α protein accumulation by WP1066 was not abolished in the presence of MG132 and proteasome inhibitor I, a potent inhibitor of the 26S proteasome, suggesting that the 26S proteasome-system is not responsible for WP1066-induced HIF-1α instability. Immunoprecipitation, confocal microscopy, and cellular fractionation analyses revealed that WP1066 induced ubiquitinylation, aggregation and degradation of HIF-1α in WM35 cells. Finally, WP1066 directly inhibits activity of rhUSP5 and rUCH-L1. Altogether, our study identified a novel function for WP1066 in inducing HIF-1 instability that may also contribute to the anti-cancer effects of WP1066 in human melanoma. Citation Format: Arumugam Jayakumar, Jana Rauvolfova, Hanying Bao, Izabela Fokt, Stanislaw Skora, Amy Heimberger, Waldemar Priebe. Blockade of HIF-1 with a small molecule inhibitor WP1066 in melanoma. [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 3251. doi:10.1158/1538-7445.AM2013-3251

Temporally designed treatment of melanoma cells by ATRA and polyI

In the last three decades, the incidence of melanoma has increased worldwide and no effective treatment modalities have been developed yet. All-trans retinoic acid (ATRA) and polyinosinic:polycytidylic acid (polyI:C) are strong inducers of toll-like receptor 3 (TLR3) and MDA5 expression, and polyI:C-induced TLR3 and MDA5 signaling specifically causes cell death in melanoma cells in vitro. We addressed the question of whether ATRA pretreatment could enhance the efficacy of polyI:C and, if so, would ATRA have any additional effects on this process. We found that the combined treatment of human melanoma cells with ATRA and polyI:C strongly increased the expression of TLR3 and MDA5 in both WM35 and WM983A cells associated with significantly higher mRNA and secreted levels of interferon β (IFNβ), CXCL1, CXCL8/IL-8, CXCL9, and CXCL10 than cells treated with either ATRA or polyI:C. Silencing of MDA5 by siRNA moderately affected IFNβ secretion, whereas TLR3 knockdown interfered with both CXCL chemokine and IFNβ production. Furthermore, the supernatants of ATRA+polyI:C-activated cultures increased the migration of both human monocyte-derived macrophages and CD1a dendritic cells significantly as compared with the supernatants of cells treated with either ATRA or polyI:C, and this effect occurred in a TLR3-dependent manner. In conclusion, consecutive treatment with ATRA and polyI:C results in strong, TLR3/MDA5-mediated chemokine and IFN responses in cultured human melanoma cells, which triggers a functional migratory response in professional antigen-presenting cells. This novel mode of concomitant activation may represent a more efficient treatment option for future melanoma therapy.

165 - Effects of IL6 on Statin Induced Apoptosis in Human Melanoma Cells

ABSTRACT Statins trigger apoptosis in primary cells and tumour cells. In particular, melanoma cells have been found to be susceptible to statin-induced apoptosis, although only after longer incubation times. Cells derived from metastatic melanoma cell lines (A375, 518A2) secrete high amounts of IL-6 in contrast to WM 35 cells which derive from an early lesion. However, IL-6 did not ameliorate the statin induced apoptosis in A375 and 518A2 cells. Conversely, in WM35 cells IL-6 led to a marked decrease in Cyclin D1 levels and enhanced the simvastatin induced apoptosis. Melanoma cells from early growth stage are more resistant to simvastatin induced apoptosis than metastatic melanoma cells. IL-6 plays a major role in the progression of melanoma. Interestingly, IL-6 has no cytostatic effect on metastatic melanoma cells but primes WM 35 cells for statin induced apoptosis. These pro-apoptotic stimuli confirm possible therapeutic potentials and may guide feasibility for more potent statins in anti-cancer strategies and help to understand the dualistic effect of IL-6 during melanoma tumorigenesis. This work was supported by Herzfeldeŕsche Familienstiftung and FWF (grant P22385). Disclosure All authors have declared no conflicts of interest.

Proline dehydrogenase is essential for proline protection against hydrogen peroxide-induced cell death

Proline metabolism has an underlying role in apoptotic signaling that influences tumorigenesis. Proline is oxidized to glutamate in the mitochondria, with the rate-limiting step catalyzed by proline dehydrogenase (PRODH). PRODH expression is inducible by p53, leading to increased proline oxidation, reactive oxygen species formation, and induction of apoptosis. Paradoxical to its role in apoptosis, proline also protects cells against oxidative stress. Here we explore the mechanism of proline protection against hydrogen peroxide stress in melanoma WM35 cells. Treatment of WM35 cells with proline significantly increased cell viability, diminished oxidative damage of cellular lipids and proteins, and maintained ATP and NADPH levels after exposure to hydrogen peroxide. Inhibition or siRNA-mediated knockdown of PRODH abolished proline protection against oxidative stress, whereas knockdown of Δ1-pyrroline-5-carboxylate reductase, a key enzyme in proline biosynthesis, had no impact on proline protection. Potential linkages between proline metabolism and signaling pathways were explored. The combined inhibition of the mammalian target of rapamycin complex 1 (mTORC1) and mTORC2 eliminated proline protection. A significant increase in Akt activation was observed in proline-treated cells after hydrogen peroxide stress along with a corresponding increase in the phosphorylation of the forkhead transcription factor class O3a (FoxO3a). The role of PRODH in proline-mediated protection was validated in the prostate carcinoma cell line PC3. Knockdown of PRODH in PC3 cells attenuated phosphorylated levels of Akt and FoxO3a and decreased cell survival during hydrogen peroxide stress. The results provide evidence that PRODH is essential in proline protection against hydrogen peroxide-mediated cell death and that proline/PRODH helps activate Akt in cancer cells.