Wogonin suppresses melanoma cell B16-F10 invasion and migration by inhibiting Ras-medicated pathways.
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ABSTRACT:
SUBMITTER: Zhao K
PROVIDER: S-EPMC4159230 | biostudies-literature | 2014
REPOSITORIES: biostudies-literature
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Project description:Melanoma is a common skin tumor that causes a high rate of mortality, especially in Europe, North America and Oceania. Immunosuppressants such as anti-PD-1 have been used in the treatment of malignant melanoma, however, nearly 60% of patients do not respond to these treatments. Sema4D, also called CD100, is expressed in T cells and tumor tissues. Sema4D and its receptor, Plexin-B1, play crucial roles in the process of immune regulation, angiogenesis, and tumor progression. The role of Sema4D in melanoma with anti-PD-1 resistance is poorly understood. Through a combination of molecular biology techniques and in silico analysis, the role of Sema4D in improving anti-PD-L1 sensitivity in melanoma was explored. The results showed that the expression of Sema4D, Plexin-B1 and PD-L1 was significantly increased in B16-F10R cells. Sema4D knockdown synergizes with anti-PD-1 treatment, cell viability, cell invasion and migration were significantly decreased, while the apoptosis was increased, the growth of tumors on the mice was also inhibited. Mechanistically, bioinformatics analysis revealed that Sema4D is involved in the PI3K/AKT signaling pathway; the downregulation of p-PI3K/PI3K and p-AKT/AKT expression were observed in Sema4D knockdown, therefore, nivolumab resistance is related to Sema4D and Sema4D silencing can improve sensitivity to nivolumab via inhibition of the PI3K/AKT signaling pathway.
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Project description:We developed and characterized a 3D collagen hydrogel model for B16.F10 melanoma tumors. Cells in this 3D environment exhibited lower proliferation than cells in the conventional 2D culture environment. Interestingly, the basal expression levels of several genes varied when compared to conventionally grown cells. In each growth environment, a significant number of melanoma cells were transfected by plasmid electroporation (electrotransfer), although expression could only be ascertained on the surface of the 3D constructs. Cellular responses to plasmid entry as demonstrated by pro-inflammatory cytokine and chemokine upregulation varied based on the growth environment, as did the mRNA levels of several putative DNA-specific pattern recognition receptors (DNA sensors). Unexpectedly, when plasmid DNA was delivered while cells where attached in the 2D or 3D environments, the mRNAs of the DNA sensor p204 and the inflammatory mediator TNFα were regulated in cells receiving pulses only. However, we were unable to confirm coordinate upregulation of TNFα and p204 proteins. This study confirms that cell responses differ significantly based on their environment, and demonstrates the difficulty of extending experimental observations between cell environments.
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Project description:In several preclinical tumor models, antitumor effects occur after intratumoral electroporation, also known as electrotransfer, of plasmid DNA devoid of a therapeutic gene. In mouse melanomas, these effects are preceded by significant elevation of several proinflammatory cytokines. These observations implicate the binding and activation of intracellular DNA-specific pattern recognition receptors or DNA sensors in response to DNA electrotransfer. In tumors, IFNβ mRNA and protein levels significantly increased. The mRNAs of several DNA sensors were detected, and DAI, DDX60, and p204 tended to be upregulated. These effects were accompanied with reduced tumor growth and increased tumor necrosis. In B16.F10 cells in culture, IFNβ mRNA and protein levels were significantly upregulated. The mRNAs for several DNA sensors were present in these cells; DNA-dependent activator of interferon regulatory factor (DAI), DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 (DDX60), and p204 were significantly upregulated while DDX60 protein levels were coordinately upregulated. Upregulation of DNA sensors in tumors could be masked by the lower transfection efficiency compared to in vitro or to dilution by other tumor cell types. Mirroring the observation of tumor necrosis, cells underwent a significant DNA concentration-dependent decrease in proliferation and survival. Taken together, these results indicate that DNA electrotransfer may cause the upregulation of several intracellular DNA sensors in B16.F10 cells, inducing effects in vitro and potentially in vivo.
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Project description:The hypoxia responsive protein BNIP3, plays an important role in promoting cell death and/or autophagy, ultimately resulting in a cancer type-dependent, tumour-enhancer or tumour-suppressor activity. We previously reported that in melanoma cells, BNIP3 regulates cellular morphology, mitochondrial clearance, cellular viability and maintains protein expression of CD47, a pro-cancerous, immunosuppressive 'don't eat me' signal. Surface exposed CD47 is often up-regulated by cancer cells to avoid clearance by phagocytes and to suppress immunogenic cell death (ICD) elicited by anticancer therapies. However, whether melanoma-associated BNIP3 modulates CD47-associated immunological effects or ICD has not been explored properly. To this end, we evaluated the impact of the genetic ablation of BNIP3 (i.e. BNIP3KD) in melanoma cells, on macrophage-based phagocytosis, polarization and chemotaxis. Additionally, we tested its effects on crucial determinants of chemotherapy-induced ICD (i.e. danger signals), as well as in vivo anticancer vaccination effect. Interestingly, loss of BNIP3 reduced the expression of CD47 both in normoxic and hypoxic conditions while macrophage phagocytosis and chemotaxis were accentuated only when BNIP3KD melanoma cells were exposed to hypoxia. Moreover, when exposed to the ICD inducer mitoxantrone, the loss of melanoma cell-associated BNIP3 did not alter apoptosis induction, but significantly prevented ATP secretion and reduced phagocytic clearance of dying cells. In line with this, prophylactic vaccination experiments showed that the loss of BNIP3 tends to increase the intrinsic resistance of B16-F10 melanoma cells to ICD-associated anticancer vaccination effect in vivo. Thus, normoxic vs. hypoxic and live vs. dying cell contexts influence the ultimate immunomodulatory roles of melanoma cell-associated BNIP3.
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Project description:PURPOSE:Prenyl ammonium iodides (Amino-Prenols, APs), semi-synthetic polyprenol derivatives were studied as prospective novel gene transfer agents. METHODS:AP-7, -8, -11 and -15 (aminoprenols composed of 7, 8, 11 or 15 isoprene units, respectively) were examined for their capacity to form complexes with pDNA, for cytotoxicity and ability to transfect genes to cells. RESULTS:All the carriers were able to complex DNA. The highest, comparable to commercial reagents, transfection efficiency was observed for AP-15. Simultaneously, AP-15 exhibited the lowest negative impact on cell viability and proliferation--considerably lower than that of commercial agents. AP-15/DOPE complexes were also efficient to introduce pDNA to cells, without much effect on cell viability. Transfection with AP-15/DOPE complexes influenced the expression of a very few among 44 tested genes involved in cellular lipid metabolism. Furthermore, complexes containing AP-15 and therapeutic plasmid, encoding the TIMP metallopeptidase inhibitor 2 (TIMP2), introduced the TIMP2 gene with high efficiency to B16-F10 melanoma cells but not to B16-F10 melanoma tumors in C57BL/6 mice, as confirmed by TIMP2 protein level determination. CONCLUSION:Obtained results indicate that APs have a potential as non-viral vectors for cell transfection.
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Project description:Roots of Glycyrrhiza uralensis have been used as herbal medicine and natural sweetener. By activity-guided phytochemical investigation of the extracts from G.uralensis root, ten flavonoids, namely GF-1?GF-10, of which five were prenylated flavonoids, were found to show antiproliferative effects in melanoma B16-F10 cells. Three of the prenylated flavonoids, namely GF-1, GF-4 and GF-9, significantly induced the differentiation of B16-F10 cells; the inductions included increase of tyrosinase activity, tyrosinase protein, and melanin content. In GF-1 and GF-9 induced melanoma differentiation, the phosphorylation of p38 MAPK (mitogen activated potein kinase) was identified; while GF-4 could trigger the phosphorylation of PI3K/AKT (phosphatidylinositol 3-kinase/Protein Kinase B) signaling. However, application of GF-6 to the melanoma cells did not induce differentiation; but which promoted cell apoptotic signaling, i.e., increase levels of cleaved-PRAP, cleaved-caspase 3, and cleaved-caspase 9. These results suggested that different types of prenylated flavonoids from G.uralensis might have potential anticancer effects against melanoma cells by acting through different signaling pathways.
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Project description:BackgroundMacrophage migration inhibitory factor (MIF) has been proposed as a link between inflammation and tumorigenesis. Despite its potentially broad influence in tumour biology and prevalent expression, the value of MIF as a therapeutic target in cancer remains unclear. We sought to validate MIF in tumour models by achieving a complete inhibition of its expression in tumour cells and in the tumour stroma.MethodsWe used MIF shRNA-transduced B16-F10 melanoma cells implanted in wild-type and MIF-/- C57Bl6 mice to investigate the effect of loss of MIF on tumour growth. Cytokine detection and immunohistochemistry (IHC) were used to evaluate tumours ex vivo.ResultsMacrophage migration inhibitory factor shRNA inhibited expression of MIF protein by B16-F10 melanoma cells in vitro and in vivo. In vitro, the loss of MIF in this cell line resulted in a decreased response to hypoxia as indicated by reduced expression of VEGF. In vivo the growth of B16-F10 tumours was inhibited by an average of 47% in the MIF-/- mice compared with wild-type but was unaffected by loss of MIF expression by the tumour cells. Immunohistochemistry analysis revealed that microvessel density was decreased in tumours implanted in the MIF-/- mice. Profiling of serum cytokines showed a decrease in pro-angiogenic cytokines in MIF-/- mice.ConclusionWe report that the absence of MIF in the host resulted in slower tumour growth, which was associated with reduced vascularity. While the major contribution of MIF appeared to be in the regulation of angiogenesis, tumour cell-derived MIF played a negligible role in this process.
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Project description:As one of the major active ingredients in Radix Scutellariae, wogonin has been shown to be associated with various pharmacological activities on cancer cell growth, apoptosis, and cell invasion and migration. Here, we demonstrated that wogonin may harbor potential anti-metastatic activities in hepatocarcinoma (HCC). The anti-metastasis potential of wogonin and its underlying mechanisms were evaluated by ligand-protein docking approach, surface plasmon resonance assay, and in vitro gelatin zymography studies. Our results showed that wogonin (100 μM, 50 μM) suppressed MHCC97L and PLC/PRF/5 cells migration and invasion in vitro. The docking approach and surface plasmon resonance assay indicated that the potential binding affinity between wogonin and matrix metalloproteinase-9 (MMP-9) may lead to inhibition of MMP-9 activity and further leads to suppression of tumor metastasis. This conclusion was further verified by Western blot results and gelatin zymography analysis. Wogonin might be a potent treatment option for disrupting the tumor metastasis that favors HCC development. The potential active targets from computational screening integrated with biomedical study may help us to explore the molecular mechanism of herbal medicines.
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Project description:PurposeMistletoe extracts are often used in complementary cancer therapy although the efficacy of that therapy is controversially discussed. Approved mistletoe extracts contain mainly water soluble compounds of the mistletoe plant, i.e. mistletoe lectins. However, mistletoe also contains water-insoluble triterpenoids (mainly oleanolic acid) that have anti-tumorigenic effects. To overcome their loss in watery extracts we have solubilized mistletoe triterpenoids with cyclodextrins, thus making them available for in vivo cancer experiments.Experimental designB16.F10 subcutaneous melanoma bearing C57BL/6 mice were treated with new mistletoe extracts containing both water soluble compounds and solubilized triterpenoids. Tumor growth and survival was monitored. In addition, histological examinations of the tumor material and tumor surrounding tissue were performed.ResultsAddition of solubilized triterpenoids increased the anti-tumor effects of the mistletoe extracts, resulting in reduced tumor growth and prolonged survival of the mice. Histological examination of the treated tumors showed mainly tumor necrosis and some apoptotic cells with active caspase-3 and TUNEL staining. A significant decrease of CD31-positive tumor blood vessels was observed after treatment with solubilized triterpenoids and different mistletoe extracts.ConclusionWe conclude that the addition of solubilized mistletoe triterpenoids to conventional mistletoe extracts improves the efficacy of mistletoe treatment and may represent a novel treatment option for malignant melanoma.
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