Tissue-protective activity of selenomethionine and D-panthetine in B16 melanoma-bearing mice under doxorubicin treatment is not connected with their ROS scavenging potential.

AIM: To evaluate molecular mechanisms of tissue-protective effects of antioxidants selenomethionine (SeMet) and D-pantethine (D-Pt) applied in combination with doxorubicin (Dx) in B16 melanoma-bearing-mice. METHODS: Impact of the chemotherapy scheme on a survival of tumor-bearing animals, general nephro- and hepatotoxicity, blood cell profile in vivo, and ROS content in B16 melanoma cells in vitro was compared with the action of Dx applied alone. Nephrotoxicity of the drugs was evaluated by measuring creatinine indicator assay, hepatotoxicity was studied by measuring the activity of ALT/AST enzymes, and myelotoxicity was assessed by light microscopic analysis of blood smears. Changes in ROS content in B16 melanoma cells under Dx, SeMet, and D-Pt action in vitro were measured by incubation with fluorescent dyes dihydrodichlorofluoresceindiacetate (DCFDA, H2O2-specific) and dihydroethidium (DHE, O2--specific), and further analysis at FL1 (DCFDA) or FL2 channels (DHE) of FACScan flow cytometer. The impact of aforementioned compounds on functional status of mitochondria was measured by Rhodamine 123 assay and further analysis at FL1 channel of FACScan flow cytometer. RESULTS: Selenomethionine (1200 µg/kg) and D-pantethine (500 mg/kg) in combination with Dx (10 mg/kg) significantly reduced tumor-induced neutrophilia, lymphocytopenia, and leukocytosis in comparison to Dx treatment alone. Moreover, SeMet and D-Pt decreased several side effects of Dx, namely an elevated creatinine level in blood and monocytosis, thus normalizing health conditions of B16 melanoma-bearing animals. CONCLUSIONS: Our results showed that antioxidants selenomethionine and D-pantethine possess significant nephroprotective and myeloprotective activity toward Dx action on murine B16 melanoma in vivo, but fail to boost a survival of B16 melanoma-bearing animals. The observed cytoprotective effects of studied antioxidants are not directly connected with their ROS scavenging.

Butein inhibits metastatic behavior in mouse melanoma cells through VEGF expression and translation-dependent signaling pathway regulation.

BACKGROUND: Melanoma is an aggressive skin cancer and a predominant cause of skin cancer-related deaths. A previous study has demonstrated the ability of butein to inhibit tumor proliferation and invasion. However, the anti-metastatic mechanisms and in vivo effects of butein have not been fully elucidated. METHODS: MTT cell viability assays were used to evaluate the antitumor effects of butein in vitro. Cytotoxic effects of butein were measured by lactate dehydrogenase assay. Anti-migratory effects of butein were evaluated by two-dimensional scratch and transwell migration assays. Signaling transduction and VEGF-releasing assays were measured by Western blotting and ELISA. We also conducted an experimental analysis of the metastatic potential of tumor cells injected into the tail vein of C57BL/6 mice. RESULTS: We first demonstrated the effect of butein on cell viability at non-cytotoxic concentrations (1, 3, and 10 µM). In vitro, butein was found to inhibit the migration of B16F10 cells in a concentration-dependent manner using transwell and scratch assays. Butein had a dose-dependent effect on focal adhesion kinase, Akt, and ERK phosphorylation in B16F10 cells. Butein efficiently inhibited the mTOR/p70S6K translational inhibition machinery and decreased the production of VEGF in B16F10 cells. Furthermore, the in vivo antitumor effects of butein were demonstrated using a pulmonary metastasis model. CONCLUSION: The results of the present study indicate the potential utility of butein in the treatment of melanoma.

Shifting wavelengths of ultraweak photon emissions from dying melanoma cells: their chemical enhancement and blocking are predicted by Cosic's theory of resonant recognition model for macromolecules.

During the first 24 h after removal from incubation, melanoma cells in culture displayed reliable increases in emissions of photons of specific wavelengths during discrete portions of this interval. Applications of specific filters revealed marked and protracted increases in infrared (950 nm) photons about 7 h after removal followed 3 h later by marked and protracted increases in near ultraviolet (370 nm) photon emissions. Specific wavelengths within the visible (400 to 800 nm) peaked 12 to 24 h later. Specific activators or inhibitors for specific wavelengths based upon Cosic's resonant recognition model elicited either enhancement or diminishment of photons at the specific wavelength as predicted. Inhibitors or activators predicted for other wavelengths, even within 10 nm, were less or not effective. There is now evidence for quantitative coupling between the wavelength of photon emissions and intrinsic cellular chemistry. The results are consistent with initial activation of signaling molecules associated with infrared followed about 3 h later by growth and protein-structural factors associated with ultraviolet. The greater-than-expected photon counts compared with raw measures through the various filters, which also function as reflective material to other photons, suggest that photons of different wavelengths might be self-stimulatory and could play a significant role in cell-to-cell communication.

Pathways of tumor development and progression in drug-induced nonmelanoma skin cancer: a new hope or the next great confusion?

The factors that lead to the clinical manifestation of the nonmelanocytic skin tumors are different. Ultraviolet radiation, infections with human papillomaviruses, and inherited or iatrogenic-induced immunosuppression (in cases of autoimmune diseases and organ transplant recipients) are considered to be some of the most important generators and/or costimulating factors supporting the appearance of "de-novo" mutations and obstruct, in one or another way, the cell cycle arrest, the programmed cell death (apoptosis), and the immunosurveillance. Preconditions are thus created for the initial persistence and subsequent proliferation of the malignant cell branch in the genome, with the simultaneous increase of the risk of nonmelanocytic skin tumor manifestation.A number of medical drugs that possess a currently well-known selective, targeting, and immunomodulating effect, like the TNF-alpha inhibitors for example, most probably possess an additional blocking action on the death receptors within the framework of the extrinsic apoptotic pathway. In this way, they seem to be one of the major factors for the clinical manifestation not only of nonmelanocytic skin but also of a number of other type of tumors with a dependency on the genetic predisposition of each separate patient.This article focuses the attention on the basic exogenic and endogenic factors that affect the regulatory processes of the cellular cycle, apoptosis, immunosurveillance, and the human inflammasome in patients with nonmelanocytic skin tumors. These processes are interwoven in a complex network and are controlled by (1) the genome regulator p53, (2) its interaction with the proapoptotic acting proteins Bak and Bax, (3) as well as the interaction with the key regulatory protein of the inflammasome-ASC/TMS1.As a process, the malignant transformation is exceptionally dynamic, plastic, and adaptive. The exterior "interferences", on the part of the clinician, in the form of a planned therapy should be targeted at the simultaneous impact on the various pathogenetic chains with the objective of bringing the tumor cells to their total collapse. This can be made possible only after the careful and simultaneous-or parallel-examination of a much greater number of markers that serve to characterize the process of the malignant transformation-a fact, which is currently being disregarded by many researchers.

B7-H1-dependent sex-related differences in tumor immunity and immunotherapy responses.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are immunopathogenic in cancers by impeding tumor-specific immunity. B7-homologue 1 (B7-H1) (CD274) is a cosignaling molecule with pleiotropic effects, including hindering antitumor immunity. In this study, we demonstrate sex-dependent, B7-H1-dependent differences in tumor immunity and response to immunotherapy in a hormone-independent cancer, murine B16 melanoma. Antitumor immunity was better in B7-H1(-/-) females versus males as a result of reduced regulatory T cell function in the B7-H1(-/-) females, and clinical response following B7-H1 blockade as tumor immunotherapy was significantly better in wild-type females than in males, owing to greater B7-H1 blockade-mediated reduction of Treg function in females. Wild-type female Tregs expressed significantly lower B7-H1 versus males but were insensitive to estrogen in vitro. Female B7-H1(-/-) Tregs were exquisitely sensitive to estrogen-mediated functional reduction in vitro, suggesting that B7-H1 effects occur before terminal Treg differentiation. Immune differences were independent of known B7-H1 ligands. Sex-dependent immune differences are seldom considered in designing immune therapy or interpreting immunotherapy treatment results. Our data demonstrate that sex is an important variable in tumor immunopathogenesis and immunotherapy responses through differential Treg function and B7-H1 signaling.

Stat3 mediates myeloid cell-dependent tumor angiogenesis in mice.

The underlying molecular mechanisms that cause immune cells, mediators of our defense system, to promote tumor invasion and angiogenesis remain incompletely understood. Constitutively activated Stat3 in tumor cells has been shown to promote tumor invasion and angiogenesis. Therefore, we sought to determine whether Stat3 activation in tumor-associated inflammatory cells has a similar function. We found that Stat3 signaling mediates multidirectional crosstalk among tumor cells, myeloid cells in the tumor stroma, and ECs that contributes to tumor angiogenesis in mice. Myeloid-derived suppressor cells and macrophages isolated from mouse tumors displayed activated Stat3 and induced angiogenesis in an in vitro tube formation assay via Stat3 induction of angiogenic factors, including VEGF and bFGF. Stat3-regulated factors produced by both tumor cells and tumor-derived myeloid cells also induced constitutive activation of Stat3 in tumor endothelium, and inhibiting Stat3 in ECs substantially reduced in vitro tumor factor-induced endothelial migration and tube formation. In vivo assays demonstrated the requirement for Stat3 signaling in tumor-associated myeloid cells for tumor angiogenesis. Our results indicate that, by virtue of the ability of Stat3 in tumor cells and tumor-derived myeloid cells to upregulate expression of factors that activate Stat3 in ECs, Stat3 mediates multidirectional crosstalk among tumor cells, tumor-associated myeloid cells, and ECs that contributes to tumor angiogenesis.

Bone marrow stem and progenitor cell contribution to neovasculogenesis is dependent on model system with SDF-1 as a permissive trigger.

Adult bone marrow (BM) contributes to neovascularization in some but not all settings, and reasons for these discordant results have remained unexplored. We conducted novel comparative studies in which multiple neovascularization models were established in single mice to reduce variations in experimental methodology. In different combinations, BM contribution was detected in ischemic retinas and, to a lesser extent, Lewis lung carcinoma cells, whereas B16 melanomas showed little to no BM contribution. Using this spectrum of BM contribution, we demonstrate the necessity for site-specific expression of stromal-derived factor-1alpha (SDF-1alpha) and its mobilizing effects on BM. Blocking SDF-1alpha activity with neutralizing antibodies abrogated BM-derived neovascularization in lung cancer and retinopathy. Furthermore, secondary transplantation of single hematopoietic stem cells (HSCs) showed that HSCs are a long-term source of neovasculogenesis and that CD133(+)CXCR4(+) myeloid progenitor cells directly participate in new blood vessel formation in response to SDF-1alpha. The varied BM contribution seen in different model systems is suggestive of redundant mechanisms governing postnatal neovasculogenesis and provides an explanation for contradictory results observed in the field.

Inhibitory effect of rose hip (Rosa canina L.) on melanogenesis in mouse melanoma cells and on pigmentation in brown guinea pigs.

The compounds present in rose hips exerting an inhibitory action against melanogenesis in B16 mouse melanoma cells were investigated by dividing an aqueous extract of rose hips (RE) into four fractions. The 50% ethanol eluate from a DIAION HP-20 column significantly reduced the production of melanin and was mainly composed of procyanidin glycosides. We also found that this 50% ethanol eluate reduced the intracellular tyrosinase activity and also had a direct inhibitory effect on tyrosinase obtained as a protein mixture from the melanoma cell lysate. We also investigated the effect of orally administering RE on skin pigmentation in brown guinea pigs, and found that the pigmentation was inhibited together with the tyrosinase activity in the skin. These data collectively suggest that proanthocyanidins from RE inhibited melanogenesis in mouse melanoma cells and guinea pig skin, and could be useful as a skin-whitening agent when taken orally.

Temporal analysis of type 1 interferon activation in tumor cells following external beam radiotherapy or targeted radionuclide therapy.

Rationale: Clinical interest in combining targeted radionuclide therapies (TRT) with immunotherapies is growing. External beam radiation therapy (EBRT) activates a type 1 interferon (IFN1) response mediated via stimulator of interferon genes (STING), and this is critical to its therapeutic interaction with immune checkpoint blockade. However, little is known about the time course of IFN1 activation after EBRT or whether this may be induced by decay of a TRT source. Methods: We examined the IFN1 response and expression of immune susceptibility markers in B78 and B16 melanomas and MOC2 head and neck cancer murine models using qPCR and western blot. For TRT, we used 90Y chelated to NM600, an alkylphosphocholine analog that exhibits selective uptake and retention in tumor cells including B78 and MOC2. Results: We observed significant IFN1 activation in all cell lines, with peak activation in B78, B16, and MOC2 cell lines occurring 7, 7, and 1 days, respectively, following RT for all doses. This effect was STING-dependent. Select IFN response genes remained upregulated at 14 days following RT. IFN1 activation following STING agonist treatment in vitro was identical to RT suggesting time course differences between cell lines were mediated by STING pathway kinetics and not DNA damage susceptibility. In vivo delivery of EBRT and TRT to B78 and MOC2 tumors resulted in a comparable time course and magnitude of IFN1 activation. In the MOC2 model, the combination of 90Y-NM600 and dual checkpoint blockade therapy reduced tumor growth and prolonged survival compared to single agent therapy and cumulative dose equivalent combination EBRT and dual checkpoint blockade therapy. Conclusions: We report the time course of the STING-dependent IFN1 response following radiation in multiple murine tumor models. We show the potential of TRT to stimulate IFN1 activation that is comparable to that observed with EBRT and this may be critical to the therapeutic integration of TRT with immunotherapies.

The urokinase plasminogen activator receptor promotes efferocytosis of apoptotic cells.

The urokinase receptor (uPAR), expressed on the surface of many cell types, coordinates plasmin-mediated cell surface proteolysis for matrix remodeling and promotes cell adhesion by acting as a binding protein for vitronectin. There is great clinical interest in uPAR in the cancer field as numerous reports have demonstrated that up-regulation of the uPA system is correlated with malignancy of various carcinomas. Using both stable cell lines overexpressing uPAR and transient gene transfer, here we provide evidence for a non-reported role of uPAR in the phagocytosis of apoptotic cells, a process that has recently been termed efferocytosis. When uPAR was expressed in human embryonic kidney cells, hamster melanoma cells, or breast cancer cells (BCCs), there was a robust enhancement in the efferocytosis of apoptotic cells. uPAR-expressing cells failed to stimulate engulfment of viable cells, suggesting that uPAR enhances recognition of one or more determinant on the surface of the apoptotic cell. uPAR-mediated engulfment was not inhibited by expression of mutant beta5 integrin, nor was alphavbeta5 integrin-mediated engulfment modulated by cleavage of uPAR by phosphatidylinositol-specific phospholipase C. Further, we found that the more aggressive BCCs had a higher phagocytic capacity that correlated with uPAR expression and cleavage of membrane-associated uPAR in MDA-MB231 BCCs significantly impaired phagocytic activity. Because efferocytosis is critical for the resolution of inflammation and production of anti-inflammatory cytokines, overexpression of uPAR in tumor cells may promote a tolerogenic microenvironment that favors tumor progression.

Involvement of peripheral adenosine 5'-triphosphate and P2X purinoceptor in pain-related behavior produced by orthotopic melanoma inoculation in mice.

Adenosine 5'-triphosphate (ATP) plays an important role in nociceptive processing. We used a mouse model of skin cancer pain to investigate the role of ATP in cancer pain. Orthotopic inoculation of B16-BL6 melanoma cells into the hind paw produced spontaneous licking of the tumor-bearing paw. Intraperitoneal injection of the P2 purinoceptor antagonist suramin suppressed spontaneous licking dose-dependently. Two P2X purinoceptor antagonists also suppressed spontaneous licking. An intraplantar injection of ATP, which did not induce licking in the healthy paw, increased licking of the tumor-bearing paw. Spontaneous firing of the tibial nerve was significantly increased in tumor-bearing mice and was inhibited by suramin. Extracellular concentration of ATP was significantly increased in the tumor-bearing paw than in the normal paw. ATP is concentrated in the culture medium of melanoma, lung cancer and breast cancer cells, but not fibroblasts. The P2X(3) receptor was expressed in about 40% of peripherin-positive small and medium-sized neurons in the dorsal root ganglia. P2X(3)-positive neurons were significantly increased in melanoma-bearing mice. These results suggest that ATP and P2X, especially P2X(3), receptors are involved in skin cancer pain, due to the increased release of ATP and increased expression of P2X(3) receptors in the sensory neurons.

Host deficiency in Vav2/3 guanine nucleotide exchange factors impairs tumor growth, survival, and angiogenesis in vivo.

Vav guanine nucleotide exchange factors modulate changes in cytoskeletal organization through activation of Rho, Rac, and Cdc42 small GTPases. Although Vav1 expression is restricted to the immune system, Vav2 and Vav3 are expressed in several tissues, including highly vascularized organs. Here, we provide the first evidence that Vav2 and Vav3 function within the tumor microenvironment to promote tumor growth, survival, and neovascularization. Host Vav2/3 deficiency reduced microvascular density, as well as tumor growth and/or survival, in transplanted B16 melanoma and Lewis lung carcinoma models in vivo. These defects were due in part to Vav2/3 deficiency in endothelial cells. Vav2/3-deficient endothelial cells displayed reduced migration in response to tumor cells in coculture migration assays, and failed to incorporate into tumor vessels and enhance tumor volume in tumor-endothelial cotransplantation experiments. These data suggest that Vav2/3 guanine nucleotide exchange factors play a critical role in host-mediated tumor progression and angiogenesis, particularly in tumor endothelium.

Chronic alcohol consumption enhances myeloid-derived suppressor cells in B16BL6 melanoma-bearing mice.

We previously found that chronic alcohol consumption decreases the survival of mice bearing subcutaneous B16BL6 melanoma. The underlying mechanism is still not completely understood. Antitumor T cell immune responses are important to inhibiting tumor progression and extending survival. Therefore, we examined the effects of chronic alcohol consumption on the functionality and regulation of these cells in C57BL/6 mice that chronically consumed 20% (w/v) alcohol and subsequently were inoculated subcutaneously with B16BL6 melanoma cells. Chronic alcohol consumption inhibited melanoma-induced memory T cell expansion and accelerated the decay of interferon (IFN)-gamma producing T cells in the tumor-bearing mice. Foxp3(+)CD4(+)CD25(+) regulatory T cells were not affected; however, the percentage of myeloid-derived suppressor cells (MDSC) was significantly increased in the peripheral blood and spleen. T cell proliferation as determined by carboxyfluorescein succinimidyl ester labeling experiments in vitro was inhibited by alcohol consumption relative to control water-drinking melanoma-bearing mice. Collectively, these data show that chronic alcohol consumption inhibits proliferation of memory T cells, accelerates the decay of IFN-gamma producing CD8(+) T cells, and increases MDSC, all of which could be associated with melanoma progression and reduced survival.

Hematopoietic- and neurologic-expressed sequence 1 (Hn1) depletion in B16.F10 melanoma cells promotes a differentiated phenotype that includes increased melanogenesis and cell cycle arrest.

The Hematopoietic- and neurologic-expressed sequence 1 (Hn1) gene encodes a small protein that is highly conserved among species. Hn1 expression is upregulated in regenerating neural tissues, including the axotomized adult rodent facial motor nerve and dedifferentiating retinal pigment epithelial cells of the Japanese newt. It is also expressed in numerous tissues during embryonic development as well as in regions of the adult brain that exhibit high plasticity. Hn1 has also been reported as a marker for human ovarian carcinoma and it is expressed in high-grade human gliomas. This study was directed toward understanding the function of Hn1 in a murine melanoma cell line. Hn1 mRNA and protein were identified in B16.F10 cells and in tumors formed from these cells. Inhibition of Hn1 protein expression with siRNA increased melanogenesis. Hn1-depleted cells expressed higher levels of the melanogenic proteins tyrosinase and Trp2 and an increased interaction between actin and Rab27a. The in vitro cell growth rate of Hn1-depleted cells was significantly reduced due to G1/S cell cycle arrest. This was consistent with a reduction in the phosphorylation of retinoblastoma protein as well as lower levels of p27 and increased expression of p21. Decreased expression of c-Met, the receptor for hepatocyte growth factor, was also detected in the Hn1-depleted cells, however HGF-dependent stimulation of phosphorylated-ERK was unaffected. Hn1 depletion also led to increased basal levels of phosphorylated p38 MAPK, while basal ERK phosphorylation was reduced. Moreover, Hn1-depleted cells had reduced expression of transcription factors MITF and USF-1, and increased expression of TFE3. These data, coupled with reports on Hn1 expression in regeneration and development, suggest that Hn1 functions as a suppressor of differentiation in cells undergoing repair or proliferation.

Hypoxia influences linearly patterned programmed cell necrosis and tumor blood supply patterns formation in melanoma.

To investigate the possibility that tumor cells undergoing linearly patterned programmed cell necrosis (LPPCN) establish a spatial foundation for vasculogenic mimicry (VM) and to reveal that hypoxia influences LPPCN formation as well as Endo G and DNase 1 expression, 78 C57 mice were divided evenly into two groups and engrafted with B16 melanoma. Starting 9 days after inoculation, subgroups of mice were killed every 2 days. LPPCN and the tumor blood supply vessel types were counted and Endo G and DNase 1 mRNA expression were measured. Additionally, 124 cases of human melanoma samples were collected to assess the clinical significance of LPPCN and VM. The data revealed that regions of LPPCN were positive for caspase-3, caspase-9 and Bax, and negative for TUNEL staining. Electron microscopy images indicated that these cells took on the morphologic changes of necrosis. There was more DNase I mRNA expression in the hypoxic group than in the control group (P<0.05) in vitro, and the expression of Endo G mRNA in the hypoxic groups was significantly higher than that in the control groups both in vitro and in vivo (P<0.05). VM and LPPCN cell numbers in the ischemic group were higher than those in the control group in the early stage of tumor growth. Finally, the survival time for patients whose samples showed LPPCN and VM was significantly shorter than that of patients with one or neither of those factors. We speculated that under hypoxic conditions, some melanoma cells might undergo LPPCN, thus providing a spatial foundation for VM channel formation.

YIGSR, a synthetic laminin pentapeptide, inhibits experimental metastasis formation.

The invasion of tumor cells through basement membranes is a critical step in the formation of metastases. The binding of the malignant cells to laminin in the basement membranes allows their attachment and activates their invasiveness. Recently a synthetic nonapeptide from the B1 chain sequence of laminin was identified as a major site for cell binding. A pentapeptide within the nonapeptide sequence was found to reduce the formation of lung colonies in mice injected with melanoma cells and also to inhibit the invasiveness of the cells in vitro.

Cell investigations simultaneously with exposure to 2.45 GHz microwaves.

The paper presents two microwave (MW) exposure systems (MWESs) that permit observations and measurements on cell cultures during their exposure to MW of 2.45 GHz: MWES-1 and MWES-2. MWES-1 is designed for the measurement of the cell membrane fluorescence anisotropies (MFA) simultaneously with MW exposure. MWES-2 is designed for the cells culture exploration under an inverted microscope before, during and after MW exposure. MWES-1 consists mainly of a 2.45 GHz microwave generator (MWG-2.45 GHz-SAIREM) of 0-25 W, equipped with forward power and reflected power displaying, and an adjustable coaxial antenna immersed directly into the cuvette with the cells-suspension of a Spex type spectrofluorometer. The MW effect on membrane fluidity of B16F10 malignant melanoma (B16F10-MM) cells in suspension were investigated with MWES-1, by MFA measurements. We observed a MW induced transition temperature (ITT) rising strongly during the MW exposure as compared with ITT obtained by classical heating (CH). The MWES-2 consists of the MWG-2.45 GHz-SAIREM generator and a rectangular waveguide applicator with traveling wave placed between the condenser and the objective of a Zeiss Axiovert 200 microscope, equipped with a fluorescence device and image acquisition. The MW effects on shape and apoptosis of the B16F10-MM cells were investigate with MWES-2. The B16F10-MM cells exhibited visible shape changes during MW exposure up to 37 degrees C. The MW exposure induced cells apoptosis/necrosis in several seconds after that MW are applied, beginning with SAR = 1.5 W/sample, compared to CH controls exposed at the same temperature dynamics.

Factors Affecting Growth Kinetics and Spontaneous Metastasis in the B16F10 Syngeneic Murine Melanoma Model.

Melanoma is an immunogenic tumor that can metastasize quickly to proximal and distal sites, thus complicating the application of therapeutic modalities. Numerous mouse model systems have been used to gain understanding of the immunobiology and metastatic potential of melanoma. Here, we report the optimization of a syngeneic mouse melanoma model protocol using the mouse B16-derived melanoma cell line B16F10 that ensures the production of tumors on mice pinnae that are similar in size between animals and that enlarge in a time-dependent manner. In this model, B16F10 cells are first allowed to develop tumors after injection in the interscapular area or flank of C57BL/6J mice. Subsequently, the tumors are harvested, cells dissociated and injected into mouse pinnae. Dose-dependent studies revealed that injection of 2 × 105 cells allowed for slow tumor enlargement, producing tumors averaging 100 mm³ within 2 to 3 wk with a metastatic frequency of 100%. This experimental protocol will be useful in dissecting the immunobiology of melanoma tumor development and metastasis and the evaluation of immunotherapeutic antimelanoma therapies.

Flower extracts from Paeonia decomposita and Paeonia ostii inhibit melanin synthesis via cAMP-CREB-associated melanogenesis signaling pathways in murine B16 melanoma cells.

This investigation seeks to identify the effects of the EtOAc fractions of different flower parts of Paeonia decomposita (Pd) and Paeonia ostii (Po) on melanogenesis and their mechanisms of action in B16 melanoma cells. Cell viability assay showed that Pd-1, Po-1 (the petals of Pd or Po), Pd-3 and Po-3 (the stamens of Pd or Po) at 25 µg/ml produced lower toxic activities in B16 cells. Pd-1 and Po-1 extracts considerably reduced the melanin content and inhibited tyrosinase and DOPA oxidase activity. Moreover, Pd-1 and Po-1 down-regulated the expressions of MC1R, MITF, TRP-1, TRP-2, and tyrosinase. These extracts also reduce cAMP levels and inhibited the phosphorylation of CREB, which might be due to the presence of high concentrations of phenolic compounds and flavonoids. Our results suggested that Pd-1 and Po-1 are able to modulate the cAMP-CREB signaling pathway and down-regulate the melanogenesis-related proteins resulting in the observed anti-melanogenic effects. PRACTICAL APPLICATIONS: In China, the flower of Paeonia is often consumed as a dietary supplement and as an additive in skin whitening products. In November 2013, the National Health and Family Planning Commission of the People's Republic of China has approved the flower of Paeonia ostii as a novel food resource. The current study firstly demonstrated that the effects of EtOAc fractions of the petals of Paeonia decomposita (Pd) and Paeonia ostii (Po) considerably reduced the melanin content in B16 cells, which is due to the modulation of the cAMP-CREB signaling pathway followed by the down-regulation of melanogenesis-related proteins. Pd and Po extracts, as natural tyrosinase inhibitors, may serve as good candidates in food additives, cosmetic materials, or even in treating hyperpigmentation diseases.

Extracellular Vesicles Shedding Promotes Melanoma Growth in Response to Chemotherapy.

Extracellular vesicles (EVs) are emerging as key players in intercellular communication. EVs can transfer biological macromolecules to recipient cells, modulating various physiological and pathological processes. It has been shown that tumor cells secrete large amounts of EVs that can be taken up by malignant and stromal cells, dictating tumor progression. In this study, we investigated whether EVs secreted by melanoma cells in response to chemotherapy modulate tumor response to alkylating drugs. Our findings showed that human and murine melanoma cells secrete more EVs after treatment with temozolomide and cisplatin. We observed that EVs shed by melanoma cells after temozolomide treatment modify macrophage phenotype by skewing macrophage activation towards the M2 phenotype through upregulation of M2-marker genes. Moreover, these EVs were able to favor melanoma re-growth in vivo, which was accompanied by an increase in Arginase 1 and IL10 gene expression levels by stromal cells and an increase in genes related to DNA repair, cell survival and stemness in tumor cells. Taken together, this study suggests that EVs shed by tumor cells in response to chemotherapy promote tumor repopulation and treatment failure through cellular reprogramming in melanoma cells.