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Cancer has become a consistent concern globally and increasingly fatal. Malignant melanoma is a rising concern, with its increased mortality. Kaempferia parviflora Wall. ex Baker (K. parviflora (KP)), commonly known as black ginger, is well known for its medicinal contributions. For the first time, in the following study we investigated the antimelanoma potential of Laos KP extracts in human cell lines. KP extracts (KPE) in methanol, DCM, and ethyl acetate showed strong cell inhibition in both melanomas, with KPE-DCM being particularly effective in inhibiting melanoma cell migration, invasion, and proliferation by inducing cell cycle arrest and apoptosis, while KPE-Hexane exhibited a low cell inhibition rate and a more limited effect. KPE affected the increased expression of caspase-3, PARP andBax and the decreased expression of the BcL-2, Mu-2-related death-inducing gene (MUDENG, MuD) protein. Furthermore, KPE enhanced apoptotic cells in the absence and presence of the pancaspase inhibitor Z-VAD-FMK. Interestingly, these apoptotic cells were significantly suppressed by the caspase inhibitor. Moreover, elevated mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) levels, suggestive of KPE's mitochondrial-mediated apoptosis in melanoma cells, were also confirmed. KPE treatment increased MMP levels, and upregulated the generation of ROS in A375 cells but not in A2058 cells. However, pretreatment with an ROS scavenger (NAC) suppressed KPE-induced cell death and ROS generation. These results clearly pointed out KPE-induced mitochondrial-mediated apoptotic cell death as the mechanism behind the inhibition of the human melanoma cells. Future studies exploring the role of specific ROS sources and their interaction with mitochondrial dynamics could deepen the existing understanding on KPE-induced apoptosis. [ABSTRACT FROM AUTHOR]

Ewelina Waglewska,1 Julita Kulbacka,2,3 Urszula Bazylinska1 1Department of Physical and Quantum Chemistry, Faculty of Chemistry, Wroclaw University of Science and Technology, Wroclaw, Poland; 2Department of Molecular and Cellular Biology, Faculty of Pharmacy, Wroclaw Medical University, Wroclaw, Poland; 3State Research Institute Centre for Innovative Medicine, Department of Immunology and Bioelectrochemistry, Vilnius, LithuaniaCorrespondence: Urszula Bazylinska, Department of Physical and Quantum Chemistry, Faculty of Chemistry, Wroclaw University of Science and Technology, Wybrzeze Wyspianskiego 27, Wroclaw, 50-370, Poland, Tel +48 713202183, Email urszula.bazylinska@pwr.edu.plPurpose: Numerous failures in melanoma treatment as a highly aggressive form of skin cancer with an unfavorable prognosis and excessive resistance to conventional therapies are prompting an urgent search for more effective therapeutic tools. Consequently, to increase the treatment efficiency and to reduce the side effects of traditional administration ways, herein, it has become crucial to combine photodynamic therapy as a promising therapeutic approach with the selectivity and biocompatibility of a novel colloidal transdermal nanoplatform for effective delivery of hybrid cargo with synergistic effects on melanoma cells.Methods: The self-assembled bilosomes, co-stabilized with L-α-phosphatidylcholine, sodium cholate, Pluronic® P123, and cholesterol, were designated, and the stability of colloidal vesicles was studied using dynamic and electrophoretic light scattering, also provided in cell culture medium (Dulbecco’s Modified Eagle’s Medium). The hybrid compounds – a classical photosensitizer (Methylene Blue) along with a complementary natural polyphenolic agent (curcumin), were successfully co-loaded, as confirmed by UV-Vis, ATR-FTIR, and fluorescent spectroscopies. The biocompatibility and usefulness of the polymer functionalized bilosome with loaded double cargo were demonstrated in vitro cyto- and phototoxicity experiments using normal keratinocytes and melanoma cancer cells.Results: The in vitro bioimaging and immunofluorescence study upon human skin epithelial (A375) and malignant (Me45) melanoma cell lines established the protective effect of the PEGylated bilosome surface. This effect was confirmed in cytotoxicity experiments, also determined on human cutaneous (HaCaT) keratinocytes. The flow cytometry experiments indicated the enhanced uptake of the encapsulated hybrid cargo compared to the non-loaded MB and CUR molecules, as well as a selectivity of the obtained nanocarriers upon tumor cell lines. The phyto-photodynamic action provided 24h-post irradiation revealed a more significant influence of the nanoplatform on Me45 cells in contrast to the A375 cell line, causing the cell viability rate below 20% of the control.Conclusion: As a result, we established an innovative and effective strategy for potential metastatic melanoma treatment through the synergism of phyto-photodynamic therapy and novel bilosomal-origin nanophotosensitizers. Keywords: nanovesicular carriers, human melanoma, A375 cells, Me45 cells, antitumor activity, phyto-photodynamic therapy

Purpose: To investigate the effect of dose-rate on nitric oxide (NO)-induced apoptosis in A375 human melanoma cells. Methods: The A375 cells were exposed to NO at a steady-state concentration of 7 µM, similar to the level estimated to occur in vivo in inflamed tissues, and delivered by diffusion through silastic tubing. Trypan blue dye exclusion assay was used to assess cell proliferation and viability. Cell cycle was studied by propidium iodide (PI) staining while Annexin V-FITC and PI assays were used to evaluate apoptosis. Western blotting assay was carried out to determine protein levels of p53, Bax, DR4, DR5, Fas (CD95), Procaspase 8, Procaspase 9 and Procaspase 3. Results: Viability of A375 cells was reduced by 22 % after 24 h leading to extensive apoptosis and cell cycle arrest following exposure to a cumulative dose of 3360 µM/min NO. Treatment with NO stimulated p53 and triggered mitochondrial apoptotic events by inducing conformational changes in Bax. Also, activation of caspase 9 and 3, DR4, DR5, Fas (CD95) and caspase 8 appeared to be mediated concurrently by death receptor processing and downstream caspases. Conclusion: Nitric oxide (NO) induces programmed cell death, thus indicating that it could serve as an effective inhibitor to halt the progression of melanoma or as an enhancer to improve therapeutic strategies for treatment. [ABSTRACT FROM AUTHOR]

Metastatic melanoma has a very poor prognosis. Statins, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) inhibitors, are cholesterol-lowering agents with a potential for cancer treatment. The inhibition of HMGCR by statins, however, induces feedback, which paradoxically upregulates HMGCR expression via sterol regulatory element-binding protein-2 (SREBP2). Dipyridamole, an antiplatelet agent, is known to inhibit SREBP2 upregulation. We aimed to demonstrate the efficacy of statin–dipyridamole combination treatment in both human and spontaneously occurring canine melanoma cell lines. The half maximal inhibitory concentration (IC50) of atorvastatin showed a 68–92% reduction when combined with dipyridamole, compared with that of atorvastatin alone. In some melanoma cell lines, cell proliferation was suppressed to almost zero by the combination treatment (≥3 μM atorvastatin). Finally, the BRAF inhibitor, vemurafenib, further potentiated the effects of the combined statin–dipyridamole treatment in BRAF V600E mutation-bearing human melanoma cell lines. In conclusion, the inexpensive and frequently prescribed statin–dipyridamole combination therapy may lead to new developments in the treatment of melanoma and may potentiate the effects of vemurafenib for the targeted therapy of BRAF V600E-mutation bearing melanoma patients. The concordance between the data from canine and human melanoma cell lines reinforces this possibility. [ABSTRACT FROM AUTHOR]

Cancer has become a consistent concern globally and increasingly fatal. Malignant melanoma is a rising concern, with its increased mortality. Kaempferia parviflora Wall. ex Baker (K. parviflora (KP)), commonly known as black ginger, is well known for its medicinal contributions. For the first time, in the following study we investigated the antimelanoma potential of Laos KP extracts in human cell lines. KP extracts (KPE) in methanol, DCM, and ethyl acetate showed strong cell inhibition in both melanomas, with KPE-DCM being particularly effective in inhibiting melanoma cell migration, invasion, and proliferation by inducing cell cycle arrest and apoptosis, while KPE-Hexane exhibited a low cell inhibition rate and a more limited effect. KPE affected the increased expression of caspase-3, PARP andBax and the decreased expression of the BcL-2, Mu-2-related death-inducing gene (MUDENG, MuD) protein. Furthermore, KPE enhanced apoptotic cells in the absence and presence of the pancaspase inhibitor Z-VAD-FMK. Interestingly, these apoptotic cells were significantly suppressed by the caspase inhibitor. Moreover, elevated mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) levels, suggestive of KPE’s mitochondrial-mediated apoptosis in melanoma cells, were also confirmed. KPE treatment increased MMP levels, and upregulated the generation of ROS in A375 cells but not in A2058 cells. However, pretreatment with an ROS scavenger (NAC) suppressed KPE-induced cell death and ROS generation. These results clearly pointed out KPE-induced mitochondrial-mediated apoptotic cell death as the mechanism behind the inhibition of the human melanoma cells. Future studies exploring the role of specific ROS sources and their interaction with mitochondrial dynamics could deepen the existing understanding on KPE-induced apoptosis.

Abstract Background Melanoma is one of the most aggressive and deadliest skin tumor. Cholesterol content in melanoma cells is elevated, and a portion of it accumulates into lipid rafts. Therefore, the plasma membrane cholesterol and its lateral organization might be directly linked with tumor development. ATP Binding Cassette A1 (ABCA1) transporter modulates physico-chemical properties of the plasma membrane by modifying cholesterol distribution. Several studies linked the activity of the transporter with a different outcome of tumor progression depending on which type. However, no direct link between human melanoma progression and ABCA1 activity has been reported yet. Methods An immunohistochemical study on the ABCA1 level in 110 patients-derived melanoma tumors was performed to investigate the potential association of the transporter with melanoma stage of progression and prognosis. Furthermore, proliferation, migration and invasion assays, extracellular-matrix degradation assay, immunochemistry on proteins involved in migration processes and a combination of biophysical microscopy analysis of the plasma membrane organization of Hs294T human melanoma wild type, control (scrambled), ABCA1 Knockout (ABCA1 KO) and ABCA1 chemically inactivated cells were used to study the impact of ABCA1 activity on human melanoma metastasis processes. Results The immunohistochemical analysis of clinical samples showed that high level of ABCA1 transporter in human melanoma is associated with a poor prognosis. Depletion or inhibition of ABCA1 impacts invasion capacities of aggressive melanoma cells. Loss of ABCA1 activity partially prevented cellular motility by affecting active focal adhesions formation via blocking clustering of phosphorylated focal adhesion kinases and active integrin β3. Moreover, ABCA1 activity regulated the lateral organization of the plasma membrane in melanoma cells. Disrupting this organization, by increasing the content of cholesterol, also blocked active focal adhesion formation. Conclusion Human melanoma cells reorganize their plasma membrane cholesterol content and organization via ABCA1 activity to promote motility processes and aggressiveness potential. Therefore, ABCA1 may contribute to tumor progression and poor prognosis, suggesting ABCA1 to be a potential metastatic marker in melanoma.

Melatonin (N-acetyl-5-methoxytryptamine, MEL), its kynurenic (N1-acetyl-N2-formyl-5-methoxykynurenine, AFMK) and indolic derivatives (6-hydroxymelatonin, 6(OH)MEL and 5-methoxytryptamine, 5-MT) are endogenously produced in human epidermis. Melatonin, produced by the pineal gland, brain and peripheral organs, displays a diversity of physiological functions including anti-inflammatory, immunomodulatory, and anti-tumor capacities. Herein, we assessed their regulatory effect on melanogenesis using amelanotic (A375, Sk-Mel-28) and highly pigmented (MNT-1, melanotic) human melanoma cell lines. We discovered that subjected compounds decrease the downstream pathway of melanin synthesis by causing a significant drop of cyclic adenosine monophosphate (cAMP) level, the microphthalmia-associated transcription factor (MITF) and resultant collapse of tyrosinase (TYR) activity, and melanin content comparatively to N-phenylthiourea (PTU, a positive control). We observed a reduction in pigment in melanosomes visualized by the transmission electron microscopy. Finally, we assessed the role of G-protein-coupled seven-transmembrane-domain receptors. Obtained results revealed that nonselective MT1 and MT2 receptor antagonist (luzindole) or selective MT2 receptor antagonist (4-P-PDOT) did not affect dysregulation of the melanin pathway indicating a receptor-independent mechanism. Our findings, together with the current state of the art, provide a convenient experimental model to study the complex relationship between metabolites of melatonin and the control of pigmentation serving as a future and rationale strategy for targeted therapies of melanoma-affected patients. [ABSTRACT FROM AUTHOR]

Background: Melanoma is one of the most aggressive and deadliest skin tumor. Cholesterol content in melanoma cells is elevated, and a portion of it accumulates into lipid rafts. Therefore, the plasma membrane cholesterol and its lateral organization might be directly linked with tumor development. ATP Binding Cassette A1 (ABCA1) transporter modulates physico-chemical properties of the plasma membrane by modifying cholesterol distribution. Several studies linked the activity of the transporter with a different outcome of tumor progression depending on which type. However, no direct link between human melanoma progression and ABCA1 activity has been reported yet. Methods: An immunohistochemical study on the ABCA1 level in 110 patients-derived melanoma tumors was performed to investigate the potential association of the transporter with melanoma stage of progression and prognosis. Furthermore, proliferation, migration and invasion assays, extracellular-matrix degradation assay, immunochemistry on proteins involved in migration processes and a combination of biophysical microscopy analysis of the plasma membrane organization of Hs294T human melanoma wild type, control (scrambled), ABCA1 Knockout (ABCA1 KO) and ABCA1 chemically inactivated cells were used to study the impact of ABCA1 activity on human melanoma metastasis processes. Results: The immunohistochemical analysis of clinical samples showed that high level of ABCA1 transporter in human melanoma is associated with a poor prognosis. Depletion or inhibition of ABCA1 impacts invasion capacities of aggressive melanoma cells. Loss of ABCA1 activity partially prevented cellular motility by affecting active focal adhesions formation via blocking clustering of phosphorylated focal adhesion kinases and active integrin β3. Moreover, ABCA1 activity regulated the lateral organization of the plasma membrane in melanoma cells. Disrupting this organization, by increasing the content of cholesterol, also blocked active focal adhesion formation. Conclusion: Human melanoma cells reorganize their plasma membrane cholesterol content and organization via ABCA1 activity to promote motility processes and aggressiveness potential. Therefore, ABCA1 may contribute to tumor progression and poor prognosis, suggesting ABCA1 to be a potential metastatic marker in melanoma. [ABSTRACT FROM AUTHOR]

This study aimed to synthesize four new semisynthetic derivatives of natural oleanolic acid (OA) and, based on an analysis of their cytotoxic and anti-proliferative effects against human MeWo and A375 melanoma cell lines, select those with anti-cancer potential. We also screened the treatment time with the concentration of all four derivatives. We synthesized oxime 2 and performed its acylation with carboxylic acids into new derivatives 3a, 3b, 3c and 3d according to the methods previously described. Colorimetric MTT and SRB assays were used to measure the anti-proliferative and cytotoxic activity of OA and its derivatives 3a, 3b, 3c and 3d against melanoma cells. Selected concentrations of OA, the derivatives, and different time periods of incubation were used in the study. The data were analyzed statistically. The present results revealed the possible anti-proliferative and cytotoxic potential of two selected OA derivatives 3a and 3b, on A375 and MeWo melanoma cells, especially at concentrations of 50 μM and 100 μM at 48 h of incubation (p < 0.05). Further studies will be necessary to analyze the proapoptotic and anti-cancer activities of 3a and 3b against skin and other cancer cells. The bromoacetoxyimine derivative (3b) of OA morpholide turned out to be the most effective against the tested cancer cells. [ABSTRACT FROM AUTHOR]

Given the known pro-oxidant status of tumour cells, the development of anti-proliferative strategies focuses on products with both anti- and pro-oxidant properties that can enhance antitumour drug cytotoxicity. We used a C. zeylanicum essential oil (CINN-EO) and assessed its effect on a human metastatic melanoma cell line (M14). Human PBMCs and MDMs from healthy donors were used as normal control cells. CINN-EO induced cell growth inhibition, cell cycle perturbation, ROS and Fe(II) increases, and mitochondrial membrane depolarization. To assess whether CINN-EO could affect the stress response, we analysed iron metabolism and stress response gene expression. CINN-EO increased HMOX1, FTH1, SLC7A11, DGKK, and GSR expression but repressed OXR1, SOD3, Tf, and TfR1 expression. HMOX1, Fe(II), and ROS increases are associated with ferroptosis, which can be reversed by SnPPIX, an HMOX1 inhibitor. Indeed, our data demonstrated that SnPPIX significantly attenuated the inhibition of cell proliferation, suggesting that the inhibition of cell proliferation induced by CINN-EO could be related to ferroptosis. Concurrent treatment with CINN-EO enhanced the anti-melanoma effect of two conventional antineoplastic drugs: the mitochondria-targeting tamoxifen and the anti-BRAF dabrafenib. We demonstrate that CINN-EO-mediated induction of an incomplete stress response specifically in cancer cells affects the proliferation of melanoma cells and can enhance drug cytotoxicity. [ABSTRACT FROM AUTHOR]

Copyright of Farmacia is the property of Societatea de Stiinte Farmaceutice Romania and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Clinical outcomes of melanoma patients pointed out a gender disparity that supports a correlation between sex hormone activity on estrogen receptors (ER) and melanoma development and progression. Here, we found that the epithelial-to-mesenchymal transition (EMT) of melanoma cells induced by extracellular acidosis, which is a crucial hallmark of solid cancers, correlates with the expression of ERβ, the most representative ER on melanoma cells. Extracellular acidosis induces an enhanced expression of ERβ in female cells and EMT markers remain unchanged, while extracellular acidosis did not induce the expression of ERβ in male cells and EMT was strongly promoted. An inverse relationship between ERβ expression and EMT markers in melanoma cells of different sex exposed to extracellular acidosis was revealed by two different technical approaches: florescence-activated cell sorting of high ERβ expressing cell subpopulations and ERβ receptor silencing. Finally, we found that ERβ regulates EMT through NF-κB activation. These results demonstrate that extracellular acidosis drives a differential ERβ regulation in male and female melanoma cells and that this gender disparity might open new perspectives for personalized therapeutic approaches. [ABSTRACT FROM AUTHOR]

Abstract Background Malignant melanomas frequently metastasize to the brain, but metastases in the cerebellum are underrepresented compared with metastases in the cerebrum. Methods We established animal models by injecting intracardially in athymic nude fox1nu mice two human melanoma cell lines, originating from a cerebral metastasis (HM19) and a cerebellar metastasis (HM86). Results Using magnetic resonance imaging (MRI), metastases were first detected after a mean of 34.5 days. Mean survival time was 59.6 days for the mice in the HM86 group and significantly shorter (43.7 days) for HM19‐injected animals (p

Human malignant melanoma exhibits imbalances in redox status, leading to activation of many redox-sensitive signaling pathways. APE/Ref-1 is a multifunctional protein that serves as a redox chaperone that regulates many nuclear transcription factors and is an important mechanism in cancer cell survival of oxidative stress. Previous studies showed that APE/Ref-1 is a potential druggable target for melanoma therapy. In this study, we synthesized a novel APE/Ref-1 inhibitor, bis-cinnamoyl-1,12-dodecamethylenediamine (2). In a xenograft mouse model, compound 2 treatment (5 mg/kg) significantly inhibited tumor growth compared to the control group, with no significant systemic toxicity observed. We further synthesized compound 2 analogs to determine the structure-activity relationship based on their anti-melanoma activities. Among those, 4-hydroxyphenyl derivative (11) exhibited potent anti-melanoma activities and improved water solubility compared to its parental compound 2. The IC50 of compound 11 was found to be less than 0.1 μM. Compared to other known APE/Ref-1 inhibitors, compound 11 exhibited increased potency in inhibiting melanoma proliferation. As determined by luciferase reporter analyses, compound 2 was shown to effectively inhibit H2O2-activated AP-1 transcription activities. Targeting APE/Ref-1-mediated signaling using pharmaceutical inhibitors is a novel and effective strategy for melanoma treatment with potentially high impact. [ABSTRACT FROM AUTHOR]

Background: Malignant melanomas frequently metastasize to the brain, but metastases in the cerebellum are underrepresented compared with metastases in the cerebrum. Methods: We established animal models by injecting intracardially in athymic nude fox1nu mice two human melanoma cell lines, originating from a cerebral metastasis (HM19) and a cerebellar metastasis (HM86). Results: Using magnetic resonance imaging (MRI), metastases were first detected after a mean of 34.5 days. Mean survival time was 59.6 days for the mice in the HM86 group and significantly shorter (43.7 days) for HM19‐injected animals (p < 0.001). In the HM86 group, the first detectable metastasis was located in the cerebellum in 15/55 (29%) mice compared with none in the HM19 group (p < 0.001). At sacrifice, cerebellar metastases were found in 34/55 (63%) HM86‐injected mice compared with 1/53 (2%) in the HM19‐injected (p < 0.001) mice. At that time, all mice in both groups had detectable metastases in the cerebrum. Comparing macroscopic and histologic appearances of the brain metastases with their clinical counterparts, the cell line‐based tumors had kept their original morphologic characteristics. Conclusions: The present work demonstrates that human brain‐metastatic melanoma cells injected intracardially in mice had retained inherent characteristics also in reproducing interaction with subtle microenvironmental brain tissue compartment‐specific features. The models offer new possibilities for investigating tumor‐ and host‐associated factors involved in determining tissue specificity of brain metastasis. [ABSTRACT FROM AUTHOR]

Onconase (ONC) is a monomeric amphibian "pancreatic-type" RNase endowed with remarkable anticancer activity. ONC spontaneously forms traces of a dimer (ONC-D) in solution, while larger amounts can be formed when ONC is lyophilized from mildly acidic solutions. Here, we report the crystal structure of ONC-D and analyze its catalytic and antitumor activities in comparison to ONC. ONC-D forms via the three-dimensional swapping of the N-terminal α-helix between two monomers, but it displays a significantly different quaternary structure from that previously modeled [Fagagnini A et al., 2017, Biochem J 474, 3767-81], and based on the crystal structure of the RNase A N-terminal swapped dimer. ONC-D presents a variable quaternary assembly deriving from a variable open interface, while it retains a catalytic activity that is similar to that of ONC. Notably, ONC-D displays antitumor activity against two human melanoma cell lines, although it exerts a slightly lower cytostatic effect than the monomer. The inhibition of melanoma cell proliferation by ONC or ONC-D is associated with the reduction of the expression of the anti-apoptotic B cell lymphoma 2 (Bcl2), as well as of the total expression and phosphorylation of the Signal Transducer and Activator of Transcription (STAT)-3. Phosphorylation is inhibited in both STAT3 Tyr705 and Ser727 key-residues, as well as in its upstream tyrosine-kinase Src. Consequently, both ONC species should exert their anti-cancer action by inhibiting the pro-tumor pleiotropic STAT3 effects deriving either by its phospho-tyrosine activation or by its non-canonical signaling pathways. Both ONC species, indeed, increase the portion of A375 cells undergoing apoptotic cell death. This study expands the variety of RNase domain-swapped dimeric structures, underlining the unpredictability of the open interface arrangement upon domain swapping. Structural data also offer valuable insights to analyze the differences in the measured ONC or ONC-D biological activities. [ABSTRACT FROM AUTHOR]

This article represents the synthesis and characterizations of Au NPs immobilized and carboxymethyl lignin (CML) modified Fe 3 O 4 nanoparticles (Fe 3 O 4 @CML/Au NPs) following a bio-inspired protocol without the participation of any toxic and harmful reductant or stabilizers. Following various physicochemical methodologies, such as FT-IR, FE-SEM, TEM, EDX, XRD, VSM, and ICP-OES, the textural characteristics and different structural aspects were evaluated. The Fe 3 O 4 @CML/Au NPs nanocomposite was subsequently explored towards the catalytic reduction of diverse aromatic nitro functions using green conditions. An excellent yield were achieved within very short reaction time. Nine recycling runs of the nanocatalyst were completed without a discernible loss of catalytic activity, thanks to its easy magnetic recovery. The DPPH assay was carried out to examine the antioxidant effectiveness. The Fe 3 O 4 @CML/Au NPs nanocomposite inhibited half of the DPPH in a 250 μg/mL solution. To measure the anti-human melanoma efficacy of Fe 3 O 4 @CML/Au NPs nanocomposite, MTT assay was applied on HT144, MUM2C, IPC-298 and SKMEL24 cell lines. Fe 3 O 4 @CML/Au NPs nanocomposite had high anti-human melanoma efficacy on above tumor cells. The best finding of anti-human melanoma properties of Fe 3 O 4 @CML/Au NPs nanocomposite was seen in the case of the SKMEL24 cell line. The IC 50 of Fe 3 O 4 @CML/Au NPs nanocomposite was 137, 145, 185, and 125 μg/mL against HT144, MUM2C, IPC-298 and SKMEL24 cells, respectively. This research exhibited remarkable anti-human melanoma and antioxidant efficacies of Fe 3 O 4 @CML/Au NPs nanocomposite in the in vitro condition. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: The role of tumor-associated B cells in human cancer is only starting to emerge. B cells typically undergo a series of developmental changes in phenotype and function, however, data on the composition of the B cell population in human melanoma are largely absent including changes during tumor progression and their potential clinical significance.Methods: In this study, we compared the number and distribution of six major B cell and antibody secreting cell subpopulations outside tertiary lymphoid structures in whole tumor sections of 154 human cutaneous melanoma samples (53 primary tumors without subsequent metastasis, 44 primary tumors with metastasis, 57 metastatic samples) obtained by seven color multiplex immunohistochemistry and automated tissue imaging and analysis.Results: In primary melanomas, we observed the highest numbers for plasmablast-like, memory-like, and activated B cell subtypes. These cells showed a patchy, predominant paratumoral distribution at the invasive tumor-stroma margin. Plasma cell-like cells were hardly detected, germinal center- and transitional/regulatory-like B cells not at all. Of the major clinicopathologic prognostic factors for primary melanomas, metastasis was associated with decreased memory-like B cell numbers and a higher age associated with higher plasmablast-like cell numbers. When we compared the composition of B cell subpopulations in primary melanomas and metastatic samples, we found a significantly higher proportion of plasma cell-like cells at distant metastatic sites and a higher proportion of memory-like B cells at locoregional than distant metastatic sites. Both cell types were detected mainly in the para- and intratumoral stroma.Conclusion: These data provide a first comprehensive and comparative spatiotemporal analysis of major B cell and antibody secreting cell subpopulations in human melanoma and describe metastasis-, tumor stage-, and age-associated dynamics, an important premise for B cell-related biomarker and therapy studies.

There is increasing evidence that tertiary lymphoid structures (TLS) control not only local adaptive B cell responses at melanoma tumor sites but also the cellular composition and function of other immune cells. In human melanoma, however, a comprehensive analysis of TLS phenotypes, density and spatial distribution at different disease stages is lacking. Here we used 7-color multiplex immunostaining of whole tissue sections from 103 human melanoma samples to characterize TLS phenotypes along the expression of established TLS-defining molecular and cellular components. TLS density and spatial distribution were determined by referring TLS counts to the tissue area within defined intra- and extratumoral perimeters around the invasive tumor front. We show that only a subgroup of primary human melanomas contains TLS. These TLS rarely formed germinal centers and mostly located intratumorally within 1 mm distance to the invasive tumor front. In contrast, melanoma metastases had a significantly increased density of secondary follicular TLS. They appeared preferentially in stromal areas within an extratumoral 1 mm distance to the invasive tumor front and their density varied over time and site of metastasis. Interestingly, secondary follicular TLS in melanoma often lacked BCL6+ lymphatic cells and canonical germinal center polarity with the formation of dark and light zone areas. Our work provides an integrated qualitative, quantitative and spatial analysis of TLS in human melanoma and shows disease progression- and site-associated changes in TLS phenotypes, density and spatial distribution. The frequent absence of canonical germinal center polarity in melanoma TLS highlights the induction of TLS maturation as a potential additive to future immunotherapy studies. Given the variable evaluation strategies used in previous TLS studies of human tumors, an important asset of this study is the standardized quantitative evaluation approach that provides a high degree of reproducibility. [ABSTRACT FROM AUTHOR]

Malignant melanoma is an aggressive type of skin cancer, rarely detected in the early stages. Various sets of methods and techniques, including dermatoscopical inspection of the "ABCDE" signs of the lesion, imaging techniques or microscopical, immunohistochemical and serological biomarkers are available and used nowadays to diagnose malignant melanoma. To date, different biomarkers were proposed for melanoma, but only a few, including circulating proteins, such as lactate dehydrogenase, molecular and metabolite biomarkers, have reached clinical applications. Gangliosides represent an emerging class, being used as tumor markers and targets of antibody therapy in melanomas, based on their elevated abundance in melanoma, especially of GM3 and GD3, when compared with the corresponding normal tissues. The conjunction of mass spectrometry (MS) with ion mobility separation (IMS) demonstrated an elevated potential in detection and identification of low abundant components, with biomarker role, in extremely complex biological mixtures. Therefore, here, a native ganglioside extract originating from human melanoma was investigated for the first time by IMS MS to provide the first profiling of gangliosides in this type of cancer. The present approach revealed the high incidence of species belonging to GD3 and GM3 classes, as well as of de- N -acetyl GM3 (d-GM3) and de- N -acetyl GD3 (d-GD3), characteristic for human melanoma. Additionally, the structure of two molecules characterized by shorter glycan chains associated to melanoma, were investigated in detail. The present approach brings valuable data related to this type of cancer, completing the existing inventory of melanoma-associated biomarkers and opens new directions for further research in this field. • IMS MS was applied to a native human melanoma ganglioside (GG) mixture. • IMS MS generated the first inventory of malignant human melanoma GGs. • IMS MS revealed the elevated incidence of GM3 and GD3 bearing different ceramides. • De- N -acetyl GM3, de- N -acetyl GD3and N -glycolyl GM3 were detected in melanoma. • IMS CID MS/MS confirmed the structure of GD3 (d18:1/16:0) and GD3 (d18:1/24:1). [ABSTRACT FROM AUTHOR]

Copyright of Journal of China Medical University is the property of Journal of China Medical University Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Accumulating evidence suggests that abnormal expression and dysfunction of microRNA is involved in development of cancers. However, the function of miR-520f especially in human melanoma remains elusive. In the current study, the underlying function of miR-520f in human melanoma was investigated. Our study demonstrated that the miR-520f level in human melanoma cell lines and clinical tissues was increased. Overexpression of miR-520f promoted cell proliferation by using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation, anchorage-independent growth assay, and 5-bromo-2-deoxyuridine assays. Furthermore, we revealed that miR-520f could interact with circular RNA Itchy E3 ubiquitin protein ligase (ITCH) 3′-untranslated region and suppress ITCH expression in human melanoma cells. The inhibitory effect of miR-520f-in could be partially restored by knockdown of ITCH in human melanoma cells. In summary, this study provides novel insights into miR-520f act as a crucial role in the regulation of human melanoma cell growth via regulating ITCH, which might be a potential biomarker and therapeutic target of human melanoma. [ABSTRACT FROM AUTHOR]

Despite the success of immunotherapy using checkpoint blockade, many patients with solid tumors remain refractory to these treatments. In human cancer, the experimental options to investigate the specific effects of antibodies blocking inhibitory receptors are limited and it is still unclear which cell types are involved. We addressed the question whether the direct interaction between T cells and tumor cells can be enforced through blocking a set of inhibitory receptors including PD-1, TIM-3, BTLA and LAG-3, blocked either individually or in dual combinations with the anti-PD-1 antibody, and to determine the condition that induces maximal T cell function preventing tumor cell proliferation. Using short-term Melan-A-specific or autologous re-stimulations, checkpoint blockade did not consistently increase cytokine production by tumor-derived expanded T cells. We next set up a 5-day co-culture assay with autologous melanoma cell lines and expanded tumor infiltrating T cells, originating from tumor specimens obtained from 6 different patients. Amongst all combos tested, we observed that blockade of LAG-3 alone, and more strongly when combined with PD-1 blockade, enforced T cell responses and tumor cell growth control. The combination of anti-LAG-3 plus anti-PD-1 acted through CD8 T cells and led to increased IFNγ production and cytotoxic capacity. Our results show that LAG-3 and PD-1 are regulating the direct interaction between tumor cells and autologous T cells, suggesting that therapy effects may be promoted by enhanced access of the corresponding blocking reagents to the tumor microenvironment.

Activated antigen-experienced B cells play an unexpected complex role in anti-tumor immunity in human melanoma patients. However, correlative studies between B cell infiltration and tumor progression are limited by the lack of distinction between functional B cell subtypes. In this study, we examined a series of 59 primary and metastatic human cutaneous melanoma specimens with B cell infiltration. Using seven-color multiplex immunohistochemistry and automated tissue imaging and analysis, we analyzed the spatiotemporal dynamics of three major antigen-experienced B cell subpopulations expressing lymphotoxin alpha (LTA/TNFSF1) or interleukin-10 (IL-10) outside tertiary lymphoid structures. The expression of both LTA and IL-10 was not restricted to a particular B cell subtype. In primary melanomas, these cells were predominantly found at the invasive tumor-stroma front and, in metastatic melanomas, they were also found in the intratumoral stroma. In primary melanomas, decreased densities of LTA+ memory-like and, to a lesser extent, activated B cells were associated with metastasis. Compared with metastatic primary tumors, B cell infiltrates in melanoma metastases were enriched in both LTA+ memory-like and LTA+ activated B cells, but not in any of the IL-10+ B cell subpopulations. Melanoma disease progression shows distinct dynamics of functional B cell subpopulations, with the regulation of LTA+ B cell numbers being more significant than IL-10+ B cell subpopulations.

Despite the success of immunotherapy using checkpoint blockade, many patients with solid tumors remain refractory to these treatments. In human cancer, the experimental options to investigate the specific effects of antibodies blocking inhibitory receptors are limited and it is still unclear which cell types are involved. We addressed the question whether the direct interaction between T cells and tumor cells can be enforced through blocking a set of inhibitory receptors including PD-1, TIM-3, BTLA and LAG-3, blocked either individually or in dual combinations with the anti-PD-1 antibody, and to determine the condition that induces maximal T cell function preventing tumor cell proliferation. Using short-term Melan-A-specific or autologous re-stimulations, checkpoint blockade did not consistently increase cytokine production by tumor-derived expanded T cells. We next set up a 5-day co-culture assay with autologous melanoma cell lines and expanded tumor infiltrating T cells, originating from tumor specimens obtained from 6 different patients. Amongst all combos tested, we observed that blockade of LAG-3 alone, and more strongly when combined with PD-1 blockade, enforced T cell responses and tumor cell growth control. The combination of anti-LAG-3 plus anti-PD-1 acted through CD8 T cells and led to increased IFNγ production and cytotoxic capacity. Our results show that LAG-3 and PD-1 are regulating the direct interaction between tumor cells and autologous T cells, suggesting that therapy effects may be promoted by enhanced access of the corresponding blocking reagents to the tumor microenvironment. [ABSTRACT FROM AUTHOR]

Kalantuboside B (KB), a natural bufadienolide derivative extracted from the succulent plant Kalanchoe tubiflora , is well-known for its cardiotonic, immunomodulatory, and anti-inflammatory properties. In this study, we tested in vitro and in vivo anti-cancer efficacy with low concentrations of KB (5–30 ng/mL; 8.7–52.2 nM) on A2058 melanoma cells; and for the molecular mechanisms that underlie them. KB significantly inhibited the cell viability and colony formation via arresting the cell cycle at G2/M phase. There was an association with a decrease in Cyclin A/B1, Cdc25C, and Cdc2 expressions. Further, this treatment indicated the induction of apoptosis, DNA fragmentation, cytochrome c release, and caspase-3, -8, -9, and -12 activation, and PARP cleavage, which shows that mitochondrial, death-receptor, and ER-stress signaling pathways are involved. KB-induced autophagy was apparent from enhanced LC3-II accumulation, GFP-LC3 puncta, and AVO formation. Surprisingly, KB-mediated cell death was potentiated by 3-MA and CQ to suggest the role of autophagy as a cytoprotective mechanism. Moreover, KB-treated A2058 cells enhanced intracellular ROS generation and antioxidant NAC prevented apoptosis and reversed cytoprotective autophagy. Interestingly, KB-induced apoptosis (PARP cleavage) and cytoprotective autophagy (LC3-II accumulation) were mediated by the up-regulation of the ERK signaling pathway. It was also shown that KB promoted cytoprotective autophagy by a calcium dependent-p53 downregulation pathway. In vivo data showed that KB suppressed tumor growth significantly in A2058-xenografted nude mice. A Western blot indicated cell-cycle inhibition (cyclin A reduction), apoptosis induction (PARP cleavage and Bcl-2 inhibition), and cytoprotective autophagy (LC3-II upregulation and p53 downregulation) in KB-treated A2058-xenografted mice. Our findings suggested that KB-induced ROS pathway plays a role in mediating the apoptosis and cytoprotective autophagy in human melanoma cells. Thus, KB is considered to be a putative anti-tumor agent. Image 1 • Kalantuboside B (KB, 8.7–52.2 nM) shows anti-tumor activity in human melanoma cells. • KB inhibited melanoma cell proliferation via G 2 /M cell-cycle arrest and apoptosis. • KB induced autophagy in A2058 cells as a cell-survival mechanism. • ROS pathway plays a role in KB-mediated apoptosis and cytoprotective autophagy. • KB mediated the suppression of A2058 xenograft tumor growth in athymic nude mice. [ABSTRACT FROM AUTHOR]

Introduction: The human melanoma is a type of invasive tumor the treatment of which is challenging. To better understand the proton irradiation mechanisms as one of the widely applied therapy for this type of cancer, bioinformatics analysis of proteomics outcome could be beneficial. Methods: Protein-protein interaction network analysis of the differentially expressed proteins (DEPs) of melanoma BLM (BRO lung metastasis) cells in the treatment of 3 Gy dosage proton therapy was performed in this study via Cytoscape V.3.7.2. and its integrated plug-ins. Results: Eighteen DEPs were searched for network constructions and limited numbers of query +neighbor proteins were found central. The hub-bottlenecks (i.e. central nodes) were GAPDH, ACTB, ALB, AKT1, TP53, and EGFR. The fist mentioned proteins were from DEPs. The enrichment analysis of these elements identified nitric-oxide synthase regulator activity and the positive regulation of the norepinephrine uptake that may be the key to the mechanisms of proton therapy. Conclusion: In conclusion, the identified central nodes (EGFR, TP53, ALB, AKT1, GAPDH, and ACTB) and the related biological terms are the critical affected genes and biological terms in the irradiated melanoma cells. [ABSTRACT FROM AUTHOR]

Human malignant melanoma is one of the most aggressive cancers, accompanied with poor prognosis, metastatic evolution and high mortality. Many strategies have been developed using BRAF and MEK inhibitors in spite of the classic therapy with alkylating agents, but failure related to the ability of the tumor to activate alternative proliferation pathways occurred after promising initial successes. Poly(ADP-ribose) polymerase (PARP) enzymes are well known to be crucial for DNA damage response, and PARP inhibition results in the accumulation of DNA strand breaks that induce cell injury. For this reason, PARP-inhibitors (PARPi) have become promising tools to counteract many cancer types. One of the most used by clinicians is olaparib, that, however, showed again cancer resistance in patients. Thus, new generation molecules have been designed mainly to counteract this problem. Among them, we chose to test AZD2461 on the particularly aggressive human melanoma A375 cell line. This drug is a PARPi significantly less prone than olaparib to undergo the P-glycoprotein-mediated efflux mechanism, one of those responsible for resistance, that in turn is the main adversity in melanoma therapy. Then, we analysed AZD2461 also together with the enzyme onconase (ONC) on the same A375 cells, to investigate if the combination of drugs could possibly increase the in vitro antitumor activity. ONC is a small amphibian "pancreatic-type" ribonuclease that is able to exert a remarkable antitumor activity against many cancers, either in vitro or in vivo , principally because it can evade the ubiquitous ribonuclease cytosolic inhibitor thanks to its structural determinants. Hence, ONC became relevant in the use of protein-drug strategies against incurable cancers. The studies performed in this work showed that both drugs definitely affect A375 cells viability by inducing cytostatic and pro-apoptotic effects in a time- and dose-dependent manner, either if administered alone or in combination. Although we registered low synergistic effects with the combination of the two drugs, we found that AZD2461 did not induce resistance in A375 after two months treatment with high concentration of this molecule. Moreover, we underline that A375 cells treated for a prolonged time with AZD2461 were definitely more susceptible than parental A375 cells to the pro-apoptotic action of ONC. Considering also the different inhibitory effects of the two drugs on TNF-α gene expression and NF-κB DNA-binding, the tuning of their combined delivery to the A375 tumor cell line might open a promising scenario for future therapeutic applications devoted to defeat human melanoma. [ABSTRACT FROM AUTHOR]

Introduction: The main purpose of the present research was to study the anticancer effects of methylwogonin in A375 human malignant melanoma cells by evaluating its effects on apoptosis, DNA fragmentation, cancer cell invasion and the mTOR/PI3K/AKT signalling pathway.Material and Methods: Effects on cell cytotoxicity were evaluated by MTT assay while a clonogenic assay determined the effects of methylwogonin on colony formation. Fluorescence microscopy evaluated apoptotic effects of methylwogonin in these cells using acridine orange/propidium iodide and Hoechst 33342 staining dyes. Gel electrophoresis evaluated the effects of methylwogonin on DNA fragmentation while the Matrigel invasion assay evaluated the effects of the drug on cancer cell invasion. Effects of methylwogonin on the mTOR/PI3K/AKT signalling pathway were evaluated by western blot assay.Results: Methylwogonin induces concentration-dependent as well as time-dependent growth inhibitory effects inducing significant cytotoxicity in these cancer cells. Methylwogonin led to dose-dependent inhibition of colony formation in A375 human malignant melanoma cells. The number of cell colonies decreased significantly as the methylwogonin dose increased from 0, 50, 150, to 300 μM. Methylwogonin treatment of cells at lower doses led to yellow fluorescence (early apoptosis), which changed to red/orange fluorescence, indicating late apoptosis at higher doses. Similar results were obtained using Hoechst 33342 staining, revealing that 50, 150 and 300 μM doses of methylwogonin led to significant morphological changes including chromatin condensation, fragmented nuclei and cellular shrinkage. DNA ladder formation was also observed, and this effect increased with increasing doses of methylwogonin. Methylwogonin also inhibited cancer cell invasion in a dose-dependent manner.Conclusions: Different doses of methylwogonin led to concentration-dependent downregulation of phosphorylated PI3K, AKT and mTOR. [ABSTRACT FROM AUTHOR]

Abstract In this work was evaluated the cytotoxic activity of dehydrodieugenol B (1) and methyldehydrodieugenol B (2) isolated from Nectandra leucantha (Lauraceae) through cytokinesis-block micronucleus (CBMN) and Comet assay. Compounds 1 and 2 displayed in vitro toxicity against human melanoma cells (SKMEL-147) with IC 50 values of 4.4 and 43.6 μg/mL, respectively. The interaction of these compounds with resistant human melanoma cell line SKMEL-29 was also investigated. Obtained results showed a concentration-response relationship for DNA damage (DI and D%) in SKMEL-29 cells for compounds 1 and 2 causing an increase in DNA damage on their lower concentrations. Concerning the cytokinesis-block micronucleus cytome (CBMNCyt) assay, all treatments demonstrated an increase in cytostatic and cytotoxic indexes. In micronucleus quantification, compound 1 displayed higher index in comparison to control. Regarding necrotic and apoptotic cells, treatments with SKMEL-29 cells demonstrated 100% of cell death induced by compounds 1 and 2 at 25 and 88 μg/mL, respectively. Additionally, it was observed that apoptosis is prevalent in SKMEL-147 cells treated with compound 1 , while necrotic cells were observed in SKMEL-29 cells treated with both compounds. In conclusion, compounds 1 and 2 are suggested as promising cytotoxic agents against human melanoma resistant cells, emphasizing the potential use of these neolignans for the treatment of melanoma. Highlights • Neolignans 1–2 were isolated from twigs of Nectandra leucantha (Lauraceae) • Neolignans 1–2 displayed toxicity against human melanoma cells SKMEL-147. • Neolignans 1–2 are suggested as promising genotoxic and cytotoxic agents against human melanoma resistant cells. [ABSTRACT FROM AUTHOR]

We have recently described that DNA-damage inducing drug DTIC, administered before peptide (Melan-A and gp100)-vaccination, improves anti-tumor CD8+ Melan-A-specific T-cell functionality, enlarges the Melan-A+ TCR repertoire and impacts the overall survival of melanoma patients. To identify whether the two Ags employed in the vaccination differently shape the anti-tumor response, herein we have carried out a detailed analysis of phenotype, anti-tumor functionality and TCR repertoire in treatment-driven gp100-specific CD8+ T cells, in the same patients previously analyzed for Melan-A. We found that T-cell clones isolated from patients treated with vaccination alone possessed an Early/intermediate differentiated phenotype, whereas T cells isolated after DTIC plus vaccination were late-differentiated. Sequencing analysis of the TCRBV chains of 29 treatment-driven gp100-specific CD8+ T-cell clones revealed an oligoclonal TCR repertoire irrespective of the treatment schedule. The high anti-tumor activity observed in T cells isolated after chemo-immunotherapy was associated with low PD-1 expression. Differently, T-cell clones isolated after peptide-vaccination alone expressed a high level of PD-1, along with LAG-3 and TIM-3, and were neither tumor-reactive nor polyfunctional. Blockade of PD-1 reversed gp100-specific CD8+ T-cell dysfunctionality, confirming the direct role of this co-inhibitory molecule in suppressing anti-tumor activity, differently from what we have previously observed for Melan-A+CD8+ T cells, expressing PD-1 but highly functional. These findings indicate that the functional advantage induced by combined chemo-immunotherapy is determined by the tumor antigen nature, T-cell immune-checkpoints phenotype, TCR repertoire diversity and anti-tumor T-cell quality and highlights the importance of integrating these parameters to develop effective immunotherapeutic strategies.

The focus of the present review is to investigate the role of melanin in the radioprotection of melanoma and attempts to sensitize tumors to radiation by inhibiting melanogenesis. Early studies showed radical scavenging, oxygen consumption and adsorption as mechanisms of melanin radioprotection. Experimental models of melanoma in hamsters and in gerbils are described as well as their use in biochemical and radiobiological studies, including a spontaneously metastasizing ocular model. Some results from in vitro studies on the inhibition of melanogenesis are presented as well as radio-chelation therapy in experimental and clinical settings. In contrast to cutaneous melanoma, uveal melanoma is very successfully treated with radiation, both using photon and proton beams. We point out that the presence or lack of melanin pigmentation should be considered, when choosing therapeutic options, and that both the experimental and clinical data suggest that melanin could be a target for radiosensitizing melanoma cells to increase efficacy of radiotherapy against melanoma. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) have emerged as critical regulators for cancer development and progression of human melanoma. However, the potential molecular mechanism of miR-767 in human melanoma has not been intensively investigated. In this present study, we confirmed that miR-767 was frequently up-regulated in human melanoma tissues and cell lines. Ectopic expression of miR-767 promoted cell proliferation in human melanoma cell lines A375 and WM35, whereas miR-767-in reversed the function. Bioinformatics analysis revealed that cylindromatosis (CYLD) was hypothesized to be a possible target gene of miR-767, and this was confirmed by luciferase activity assay. Knockdown of CYLD counteracted the proliferation arrest by miR-767-in in melanoma cells A375 and WM35. In conclusion, our study indicated that miR-767 acted as a role of tumor promoter by targeting CYLD in human melanoma, and might serve as a prognostic or therapeutic target for human melanoma. [ABSTRACT FROM AUTHOR]

Accumulating evidence indicated that aberrantly expressed microRNAs play critical roles in the initiation and progression of human cancers. However, the underlying functions of miR-493 in human melanoma remains unknown. Here, our study found that miR-493 expression was downregulated in human melanoma tissues and cells. Overexpression of miR-493 suppressed cell proliferation and cell cycle in human melanoma cell line A375. IRS4 was defined as a target for downregulation by miR-493 and was confirmed by luciferase assay. We also found that knockdown of IRS4 counteracted the proliferation promotion by miR-493 inhibitor. In summary, these results demonstrated that miR-493 acts as a tumor suppressor and inhibits cell proliferation and cell cycle in human melanoma by directly targeting IRS4. [ABSTRACT FROM AUTHOR]