Ocoxin Increases the Antitumor Effect of BRAF Inhibition and Reduces Cancer Associated Fibroblast-Mediated Chemoresistance and Protumoral Activity in Metastatic Melanoma

Whereas the prevalence of several cancer types is decreasing, skin malignancies are growing more common every year. Malignant melanoma is the most aggressive form of skin cancer with high metastatic capacity. In most cases, malignant melanoma shows acquired therapy resistance. We evaluated the ability of Ocoxin, a natural compound-based antioxidant and anti-inflammatory nutritional complement, to exert an antitumor effect in melanoma. To do so, the cytotoxicity of Ocoxin in a panel of BRAF-mutated murine and human melanoma cell lines was tested alone and in combination with BRAF inhibitor Vemurafenib. Our results revealed a potent cytotoxic effect of Ocoxin against melanoma cells and a synergic effect when combined with Vemurafenib, reducing viability and increasing apoptosis. Besides, Ocoxin interferes with the cell cycle, impairs the inherent and fibroblast-mediated melanoma cell migration, and reduces resistance to BRAF inhibition. Proteomic analysis revealed reduced tumor secretion of inflammatory factors Galectin-1, Osteopontin, CCL5, and CCL9 upon treatment with Ocoxin. Moreover, RNASeq showed that Ocoxin downregulated the cell cycle and proliferation-related genes. In vivo, Ocoxin reduced the number of lung metastasis of YUMM-1.7 melanoma cells. Therefore, Ocoxin arises as a good candidate for clinical trials analyzing the beneficial effects in patients suffering from this cutaneous malignancy.

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Luteolin inhibits proliferation and induces apoptosis of human melanoma cells in vivo and in vitro by suppressing MMP-2 and MMP-9 through the PI3K/AKT pathway

Food & Function ◽

10.1039/c8fo02013b ◽

2019 ◽

Vol 10 (2) ◽

pp. 703-712 ◽

Cited By ~ 19

Author(s):

Xin Yao ◽

Wei Jiang ◽

Danhong Yu ◽

Zhaowei Yan

Keyword(s):

Malignant Melanoma ◽

Incidence Rate ◽

Melanoma Cell ◽

Melanoma Cells ◽

Human Melanoma ◽

Akt Pathway ◽

Human Melanoma Cells ◽

Cell Metastasis

Since the incidence rate of malignant melanoma is increasing annually, development of drugs against melanoma cell metastasis has become more urgent.

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Nuclear Localization of BRAFV600E Is Associated with HMOX-1 Upregulation and Aggressive Behavior of Melanoma Cells

Cancers ◽

10.3390/cancers14020311 ◽

2022 ◽

Vol 14 (2) ◽

pp. 311

Author(s):

Mourad Zerfaoui ◽

Eman Toraih ◽

Emmanuelle Ruiz ◽

Youssef Errami ◽

Abdallah S. Attia ◽

...

Keyword(s):

Cell Proliferation ◽

Nuclear Localization ◽

Melanoma Cell ◽

Braf Inhibitor ◽

Melanoma Cells ◽

Heme Oxygenase 1 ◽

Biological Targets ◽

Melanoma Cell Proliferation

Background: Previously, we have demonstrated that nuclear BRAFV600E is associated with melanoma aggressiveness and vemurafenib resistance. However, the underlying mechanisms of how nuclear localization of BRAFV600E promotes cell aggressiveness have not yet been investigated. Despite therapeutic advancements targeting cutaneous melanoma, unknown cellular processes prevent effective treatment for this malignancy, prompting an urgent need to identify new biological targets. This study aims to explore the association of inducible heme oxygenase 1 (HMOX-1) with nuclear BRAFV600E in promoting melanoma aggressiveness. Methods: Proteomics analysis was performed to identify the interacting partner(s) of nuclear BRAFV600E. Immunohistochemistry was applied to evaluate the levels of HMOX-1 and nuclear BRAFV600E expression in melanoma and adjacent healthy tissues. Immunofluorescence assessed the nuclear localization of BRAFV600E in vemurafenib-resistant A375R melanoma cells. Further study of HMOX-1 knockdown or BRAFV600E overexpression in melanoma cells suggested a role for HMOX-1 in the regulation of cell proliferation in vivo and in vitro. Finally, Western blot analysis was performed to confirm the pathway by which HMOX-1 mediates Akt signaling. Results: Proteomics results showed that HMOX-1 protein expression was 10-fold higher in resistant A375R cells compared to parental counterpart cells. In vitro and in vivo results illustrate that nuclear BRAFV600E promotes HMOX-1 overexpression, whereas HMOX-1 reduction represses melanoma cell proliferation and tumor growth. Mechanistic studies revealed that HMOX-1 was associated with nuclear BRAFV600E localization, thus promoting melanoma proliferation via a persistent activation of the AKT pathway. Conclusions: Our results highlight a previously unknown mechanism in which the nuclear BRAFV600E/HMOX-1/AKT axis plays an essential role in melanoma cell proliferation. Targeting HMOX-1 could be a novel method for treating melanoma patients who develop BRAF inhibitor resistance.

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P2X7 promotes metastatic spreading and triggers release of miRNA-containing exosomes and microvesicles from melanoma cells

Cell Death and Disease ◽

10.1038/s41419-021-04378-0 ◽

2021 ◽

Vol 12 (12) ◽

Author(s):

Anna Pegoraro ◽

Elena De Marchi ◽

Manuela Ferracin ◽

Elisa Orioli ◽

Michele Zanoni ◽

...

Keyword(s):

Malignant Melanoma ◽

Melanoma Cell ◽

P2x7 Receptor ◽

Metastatic Malignant Melanoma ◽

Melanoma Cells ◽

Vesicular Release ◽

And Migration ◽

Metastatic Spreading

AbstractTumor growth and metastatic spreading are heavily affected by the P2X7 receptor as well as microvesicles and exosomes release into the tumor microenvironment. P2X7 receptor stimulation is known to trigger vesicular release from immune and central nervous system cells. However, P2X7 role in microvesicles and exosomes delivery from tumor cells was never analyzed in depth. Here we show that P2X7 is overexpressed in patients affected by metastatic malignant melanoma and that its expression closely correlates with reduced overall survival. Antagonism of melanoma cell-expressed P2X7 receptor inhibited in vitro anchorage-independent growth and migration and in vivo dissemination and lung metastasis formation. P2X7 stimulation triggered the release of miRNA-containing microvesicles and exosomes from melanoma cells, profoundly altering the nature of their miRNA content, as well as their dimensions and quantity. Among the more than 200 miRNAs that we found up-or-down-modulated for each vesicular fraction tested, we identified three miRNAs, miR-495-3p, miR-376c-3p, and miR-6730-3p, that were enriched in both the exosome and microvesicle fraction in a P2X7-dependent fashion. Interestingly, upon transfection, these miRNAs promoted melanoma cell growth or migration, and their vesicular release was minimized by P2X7 antagonism. Our data unveil an exosome/microvesicle and miRNA-dependent mechanism for the pro-metastatic activity of the P2X7 receptor and highlight this receptor as a suitable prognostic biomarker and therapeutic target in malignant melanoma.

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Inhibitory Effects of Trehalose on Malignant Melanoma Cell Growth: Implications for a Novel Topical Anticancer Agent on the Ocular Surface

ISRN Ophthalmology ◽

10.5402/2012/968493 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 6

Author(s):

Takashi Kudo ◽

Kimio Takeuchi ◽

Yu-ichi Ebina ◽

Mitsuru Nakazawa

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Cell Growth ◽

Malignant Melanoma ◽

Melanoma Cell ◽

Ocular Surface ◽

Melanoma Cells ◽

Apoptotic Cells ◽

Inhibitory Effects ◽

Malignant Melanoma Cell

Purpose. To investigate the inhibitory effects of trehalose on malignant melanoma cell growth. Methods. We cultured human malignant melanoma cells in a medium containing trehalose (control/2.5%/5.0%/7.5%/10.0%) and used the MTT assay to evaluate the growth activities. Subsequently, trehalose was topically instilled on subconjunctivally inoculated melanoma cells in F334/NJcl-rmu/rmu rats, followed by a histopathological evaluation of tumor growth. Using flow cytometry, we compared the distribution of the cell cycle, rate of apoptotic cells, and intracellular factors related to the cell cycle in cultured melanoma cells after trehalose treatment. Results. The MTT study showed that proliferation of melanoma cells was significantly inhibited by ≧ 5% of trehalose concentrations in the culture media. Subconjunctivally inoculated melanoma cell masses were significantly smaller in eyes administered trehalose as compared to controls. Flow cytometry analyses demonstrated that the trehalose groups had increased rates of G2/M phase cells and apoptotic cells in the cell culture. These cells also exhibited increased expressions of cell-cycle inhibitory factors. Conclusions. The current results show trehalose inhibits malignant melanoma cell growth by inducing G2/M cell cycle arrest and apoptosis, suggesting trehalose as a potential candidate for a topical agent to inhibit proliferation of malignant tumor cells of the ocular surface.

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Targeting BET proteins in melanoma: A novel treatment approach.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.9091 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 9091-9091

Author(s):

Luca Paoluzzi ◽

Miguel F. Segura ◽

Barbara Fontanals-Cirera ◽

Avital Gaziel-Sovran ◽

Maria V Guijarro ◽

...

Keyword(s):

Cell Cycle ◽

Melanoma Cell ◽

Statistical Significance ◽

Melanoma Cells ◽

Western Blots ◽

In Vivo Models ◽

Mutational Status ◽

Bet Inhibitors

9091 Background: Manipulation of key epigenetic regulators in melanoma proliferation is emerging as a new therapeutic strategy. Bromodomain-containing proteins such as the extraterminal domain (BET) family are components of transcription factor complexes and determinants of epigenetic memory. We investigated the expression of BRD4, a BET family member in melanoma cell lines and tissues, and the effects of its inhibition with the small molecule compounds MS436 and MS417 in in vitro and in vivo models of melanoma. Methods: BRD2 and BRD4 expression were analyzed by immunohistochemistry. We tested the effects of pharmacological or RNAi-mediated inhibition of BRD4 in melanoma cells using crystal violet-based assays for proliferation/colony formation and flow-cytometry for cell cycle analysis. The molecular effects of BRD4 suppression were examined using RNA sequencing, Real-Time quantitative PCR and western blots for p27, p21, MYC, ERK1 and SKP2. In the in vivo xenograft experiments NOD/SCID/IL2γR-/-mice were injected with melanoma cells and treated with MS417. Statistical significance was determined by unpaired t-test (GraphPad). Results: BRD4 was found significantly upregulated in primary and metastatic melanoma tissues compared to melanocytes and nevi (p<0.001). Treatment with BET inhibitors impaired melanoma cell proliferation in vitro and tumor growth and metastatic behavior in vivo, effects that were mostly recapitulated by individual silencing of BRD4. Rapidly after BET displacement, key cell cycle genes (SKP2, ERK1 and c-MYC) were downregulated concomitantly with the accumulation of CDK inhibitors (p21, p27), followed by melanoma cell cycle arrest. BET inhibitor efficacy was not influenced by BRAF or NRAS mutational status. Conclusions: Our results demonstrate for the first time a role for BRD4 in melanoma maintenance and support the role of BET proteins as novel targets in melanoma. Further investigation in the clinical setting is warranted.

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Cell-cycle-controlled radiation therapy was effective for treating a murine malignant melanoma cell line in vitro and in vivo

Scientific Reports ◽

10.1038/srep30689 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 14

Author(s):

Keisuke Otani ◽

Yoko Naito ◽

Yukako Sakaguchi ◽

Yuji Seo ◽

Yutaka Takahashi ◽

...

Keyword(s):

Cell Cycle ◽

Radiation Therapy ◽

Malignant Melanoma ◽

Cell Line ◽

Melanoma Cell ◽

Melanoma Cell Line ◽

Malignant Melanoma Cell

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BRAFV600 inhibition alters the microRNA cargo in the vesicular secretome of malignant melanoma cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1705206114 ◽

2017 ◽

Vol 114 (29) ◽

pp. E5930-E5939 ◽

Cited By ~ 45

Author(s):

Taral R. Lunavat ◽

Lesley Cheng ◽

Berglind O. Einarsdottir ◽

Roger Olofsson Bagge ◽

Somsundar Veppil Muralidharan ◽

...

Keyword(s):

Malignant Melanoma ◽

Melanoma Cells ◽

Resistant Cell ◽

Resistant Cell Line ◽

Braf Inhibitors ◽

Braf Inhibition ◽

Survival Pathway ◽

Tumor Tissues

The BRAF inhibitors vemurafenib and dabrafenib can be used to treat patients with metastatic melanomas harboring BRAFV600 mutations. Initial antitumoral responses are often seen, but drug-resistant clones with reactivation of the MEK–ERK pathway soon appear. Recently, the secretome of tumor-derived extracellular vesicles (EVs) has been ascribed important functions in cancers. To elucidate the possible functions of EVs in BRAF-mutant melanoma, we determined the RNA content of the EVs, including apoptotic bodies, microvesicles, and exosomes, released from such cancer cells after vemurafenib treatment. We found that vemurafenib significantly increased the total RNA and protein content of the released EVs and caused significant changes in the RNA profiles. RNA sequencing and quantitative PCR show that cells and EVs from vemurafenib-treated cell cultures and tumor tissues harvested from cell-derived and patient-derived xenografts harbor unique miRNAs, especially increased expression of miR-211–5p. Mechanistically, the expression of miR-211–5p as a result of BRAF inhibition was induced by increased expression of MITF that regulates the TRPM1 gene resulting in activation of the survival pathway. In addition, transfection of miR-211 in melanoma cells reduced the sensitivity to vemurafenib treatment, whereas miR-211–5p inhibition in a vemurafenib resistant cell line affected the proliferation negatively. Taken together, our results show that vemurafenib treatment induces miR-211–5p up-regulation in melanoma cells both in vitro and in vivo, as well as in subsets of EVs, suggesting that EVs may provide a tool to understand malignant melanoma progression.

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The Induction of Apoptosis in A375 Malignant Melanoma Cells bySutherlandia frutescens

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2016/4921067 ◽

2016 ◽

Vol 2016 ◽

pp. 1-14 ◽

Cited By ~ 5

Author(s):

Nicola B. van der Walt ◽

Zahra Zakeri ◽

Marianne J. Cronjé

Keyword(s):

Malignant Melanoma ◽

Basic Mechanism ◽

Melanoma Cells ◽

Caspase Activation ◽

Nuclear Condensation ◽

Induced Apoptosis ◽

Apoptosis Inducing Factor ◽

A375 Melanoma Cell Line ◽

A375 Cells

Sutherlandia frutescensis a medicinal plant indigenous to Southern Africa and is commonly known as the “cancer bush.” This plant has traditionally been used for the treatment of various ailments, although it is best known for its claims of activity against “internal” cancers. Here we report on its effect on melanoma cells. The aim of this study was to investigate whether an extract ofS. frutescenscould induce apoptosis in the A375 melanoma cell line and to outline the basic mechanism of action.S. frutescensextract induced apoptosis in A375 cells as evidenced by morphological features of apoptosis, phosphatidylserine exposure, nuclear condensation, caspase activation, and the release of cytochromecfrom the mitochondria. Studies in the presence of a pan-caspase inhibitor allude to caspase-independent cell death, which appeared to be mediated by the apoptosis inducing factor. Taken together, the results of this study show thatS. frutescensextract is effective in inducing apoptosis in malignant melanoma cells and indicates that furtherin vivomechanistic studies may be warranted.

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Beta3-Tubulin Is Critical for Microtubule Dynamics, Cell Cycle Regulation, and Spontaneous Release of Microvesicles in Human Malignant Melanoma Cells (A375)

International Journal of Molecular Sciences ◽

10.3390/ijms21051656 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1656

Author(s):

Mohammed O. Altonsy ◽

Anutosh Ganguly ◽

Matthias Amrein ◽

Philip Surmanowicz ◽

Shu Shun Li ◽

...

Keyword(s):

Cell Cycle ◽

Malignant Melanoma ◽

Structural Changes ◽

Melanoma Cells ◽

Neoplastic Cell ◽

Primary Component ◽

Cellular Growth ◽

Class Iii ◽

M Cell ◽

Human Malignant Melanoma

Microtubules (MTs), microfilaments, and intermediate filaments, the main constituents of the cytoskeleton, undergo continuous structural changes (metamorphosis), which are central to cellular growth, division, and release of microvesicles (MVs). Altered MTs dynamics, uncontrolled proliferation, and increased production of MVs are hallmarks of carcinogenesis. Class III beta-tubulin (β3-tubulin), one of seven β-tubulin isotypes, is a primary component of MT, which correlates with enhanced neoplastic cell survival, metastasis and resistance to chemotherapy. We studied the effects of β3-tubulin gene silencing on MTs dynamics, cell cycle, and MVs release in human malignant melanoma cells (A375). The knockdown of β3-tubulin induced G2/M cell cycle arrest, impaired MTs dynamics, and reduced spontaneous MVs release. Additional studies are therefore required to elucidate the pathophysiologic and therapeutic role of β3-tubulin in melanoma.

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Antitumor Effect of a Novel Spiro-Acridine Compound is Associated with Up-Regulation of Th1-Type Responses and Antiangiogenic Action

Molecules ◽

10.3390/molecules25010029 ◽

2019 ◽

Vol 25 (1) ◽

pp. 29 ◽

Cited By ~ 1

Author(s):

Daiana K. Frade Silva ◽

Sâmia S. Duarte ◽

Thaís M. H. Lisboa ◽

Rafael C. Ferreira ◽

Ana Luíza de O. Lopes ◽

...

Keyword(s):

Cell Cycle ◽

Antitumor Activity ◽

Tumor Cells ◽

Antitumor Effect ◽

Inflammatory Responses ◽

Lethal Dose ◽

Ehrlich Ascites Carcinoma ◽

Antitumor Effects ◽

Th2 Immune Response

Tumor cells have specific features, including angiogenesis induction, cell cycle dysregulation, and immune destruction evasion. By inducing a T helper type 2 (Th2) immune response, tumor cells may favor immune tolerance within the tumor, which allows progression of cancer growth. Drugs with potential antitumor activity are the spiro-acridines, which is a promising new class of acridine compounds. Herein, the novel spiro-acridine (E)-5′-oxo-1′-((3,4,5-trimethoxybenzylidene)amino)-1′,5′-dihydro-10H-spiro[acridine-9,2′-pyrrole]-4′-carbonitrile (AMTAC-17) was synthesized and tested for antitumor effects. Toxicity evaluation was performed in mice after acute treatment (2000 mg/kg, intraperitoneally, i.p.). The Ehrlich ascites carcinoma model was used to investigate the antitumor activity of AMTAC-17 (12.5, 25, or 50 mg/kg, i.p.) after seven days of treatment. Effects on the cell cycle, angiogenesis, and inflammatory responses were investigated. LD50 (lethal dose 50%) was estimated to be higher than 5000 mg/kg. AMTAC-17 reduced the Ehrlich tumor’s total viable cancer cells count and peritumoral micro-vessels density, and induced an increase in the sub-G1 peak. Additionally, there was an increase of Th1 cytokine profile levels (IL-1β, TNF-α, and IL-12). In conclusion, the spiro-acridine compound AMTAC-17 presents low toxicity, and its in vivo antitumor effect involves modulation of the immune system to a cytotoxic Th1 profile and a reduction of tumor angiogenesis.

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