B16 Mouse Melanoma Cell Line Research Articles

Abstract 5371: Repurposing erbb2 (HER2/neu) as a neoantigen target for neu-negative tumors using a novel erbb2-encoding lentivirus

Abstract INTRODUCTION: HER2/neu is a potent biomarker for which anti-HER2/neu antibodies offer significant responses. However, many cancers, such as melanoma, have less than 5% expression of HER2/neu. The purpose of this study was to determine whether induced expression of erbb2 (mouse HER2/neu analogue) could function as a neoantigen target for anti-erbb2 antibody. METHODS: RT-PCR was used to construct a rat erbb2-encoding vector using the live, non-replicating lentivirus pLenti6.3. erbb2-encoding pLenti6.3 was transfected in vitro into the B16 mouse melanoma cell line, which has less than 5% endogenous expression of erbb2. The generated erbb2+ B16 cell line was designated B16/neu. Anti-erbb2 antibody (7.16.4) was used to treat C57Bl/6 male and female mice bearing B16/neu (200 ug intraperitoneally three times per week for 3 weeks). To confirm activity of 7.16.4, this antibody (100 ug per T75 flask) was co-cultured with B16/neu in vitro, and cell growth/confluence was measured. RESULTS: The novel B16/neu cell line expressed approximately 50% erbb2 in vitro on flow cytometry analysis, compared to 2% errb2 expression in naïve B16. Erbb2/neu expression was confirmed on fluorescent Western blot. Anti-erbb2 antibody (7.16.4) slowed the growth of B16/neu in vitro, confirming the suppressive effects of 7.16.4. In B16/neu-bearing mice, 7.16.4 resulted in effective control of tumor growth and a complete tumor response rate of 35.7%, (5/14 mice) compared to placebo controls (p < 0.01). Interestingly, B16/neu tumors demonstrated slower growth kinetics compared to naïve B16 tumors (p = 0.013), suggesting immune recognition of erbb2 in naïve C57Bl/6 mice as a neoantigen target. No adverse reactions were observed with 7.16.4. In vivo inoculation of erbb2-encoding pLenti6.3 in naïve B16 tumors and treatment of in vivo transfected B16/neu tumors with 7.16.4 comprise ongoing experiments. CONCLUSIONS: erbb2 expression on B16 melanoma was successfully achieved using a novel erbb2-encoding lentivirus. Induced erbb2 served as a neoantigen target for anti-erbb2 antibody. This preclinical study provides translational evidence that induced HER2/neu expression may be utilized to repurpose anti-HER2 antibodies for human malignancies that do not normally express high levels of HER2/neu. Citation Format: Emmanuel M. Gabriel, Brian Necela, Deborah Bahr, Jamie Kaplan, Kristopher Attwood, Keith Knutson. Repurposing erbb2 (HER2/neu) as a neoantigen target for neu-negative tumors using a novel erbb2-encoding lentivirus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5371.

Tumor-Selective Cascade-Amplified Dual-Prodrugs Activation for Synergistic Oxidation-Chemotherapy

Tumor-Selective Cascade-Amplified Dual-Prodrugs Activation for Synergistic Oxidation-Chemotherapy

Mitofusin-2 Negatively Regulates Melanogenesis by Modulating Mitochondrial ROS Generation.

Inter-organellar communication is emerging as one of the most crucial regulators of cellular physiology. One of the key regulators of inter-organellar communication is Mitofusin-2 (MFN2). MFN2 is also involved in mediating mitochondrial fusion–fission dynamics. Further, it facilitates mitochondrial crosstalk with the endoplasmic reticulum, lysosomes and melanosomes, which are lysosome-related organelles specialized in melanin synthesis within melanocytes. However, the role of MFN2 in regulating melanocyte-specific cellular function, i.e., melanogenesis, remains poorly understood. Here, using a B16 mouse melanoma cell line and primary human melanocytes, we report that MFN2 negatively regulates melanogenesis. Both the transient and stable knockdown of MFN2 leads to enhanced melanogenesis, which is associated with an increase in the number of mature (stage III and IV) melanosomes and the augmented expression of key melanogenic enzymes. Further, the ectopic expression of MFN2 in MFN2-silenced cells leads to the complete rescue of the phenotype at the cellular and molecular levels. Mechanistically, MFN2-silencing elevates mitochondrial reactive-oxygen-species (ROS) levels which in turn increases melanogenesis. ROS quenching with the antioxidant N-acetyl cysteine (NAC) reverses the MFN2-knockdown-mediated increase in melanogenesis. Moreover, MFN2 expression is significantly lower in the darkly pigmented primary human melanocytes in comparison to lightly pigmented melanocytes, highlighting a potential contribution of lower MFN2 levels to higher physiological pigmentation. Taken together, our work establishes MFN2 as a novel negative regulator of melanogenesis.

Bone morphogenetic protein induces bone invasion of melanoma by epithelial–mesenchymal transition via the Smad1/5 signaling pathway

Oral malignant melanoma, which frequently invades the hard palate or maxillary bone, is extremely rare and has a poor prognosis. Bone morphogenetic protein (BMP) is abundantly expressed in bone matrix and is highly expressed in malignant melanoma, inducing an aggressive phenotype. We examined the role of BMP signaling in the acquisition of an aggressive phenotype in melanoma cells in vitro and in vivo. In five cases, immunohistochemistry indicated the phosphorylation of Smad1/5 (p-Smad1/5) in the nuclei of melanoma cells. In the B16 mouse and A2058 human melanoma cell lines, BMP2, BMP4, or BMP7 induces morphological changes accompanied by the downregulation of E-cadherin, and the upregulation of N-cadherin and Snail, markers of epithelial–mesenchymal transition (EMT). BMP2 also stimulates cell invasion by increasing matrix metalloproteinase activity in B16 cells. These effects were canceled by the addition of LDN193189, a specific inhibitor of Smad1/5 signaling. In vivo, the injection of B16 cells expressing constitutively activated ALK3 enhanced zygoma destruction in comparison to empty B16 cells by increasing osteoclast numbers. These results suggest that the activation of BMP signaling induces EMT, thus driving the acquisition of an aggressive phenotype in malignant melanoma.Bone morphogenetic protein (BMP) induces epithelial–mesenchymal transition (EMT) both B16 mouse and A2058 human melanoma cell lines. The injection of B16 cells expressing constitutively activated ALK3 enhanced zygoma destruction compared with empty B16 cells, suggesting that the activation of BMP signaling enhances bone invasion by inducing EMT in melanoma cells.

Abstract 1845: Enhancing the efficacy of antitumor antibodies with immune checkpoint inhibitors and targeted therapy in melanoma

Abstract The therapeutic outcomes of current strategies in melanoma treatment have greatly improved within the last decade. However, the persistent relatively low success rate of current standard-of-care strategies underscores the urgent need for improvement of these therapeutic approaches. The use of specific antitumor antibodies recently transformed the cancer therapeutics landscape with successes such as anti-HER2 in breast cancer, and anti-EGFR in HNSCC and colorectal cancer. However, antitumor antibodies have not been developed for the treatment of melanoma. Here, using mouse preclinical models of melanoma (both solid tumor and lung metastasis), we found that the therapeutic efficacy of the specific antitumor antibody TA99 (anti-TYRP1) is significantly enhanced by immune checkpoint blockade. This was associated with a greater CD8+, CD8+/Foxp3+, NK1.1+ and dendritic cell infiltrate, suggestive of an increased anti-tumor immune response. Further, MEK inhibition (MEKi) increased the expression of melanosomal antigens in B16 and YUMM mouse melanoma cell lines in vitro. We next combined TA99 and MEKi to treat B16 melanoma in vivo and observed a synergistic reduction in tumor growth. Treatment of mice bearing YUMM melanoma (BRAF mutant) with TA99/MEKi resulted in the complete elimination of tumors in the majority of animals, and produced durable responses. Moreover, we found a synergistic effect when B16 tumor-bearing mice were treated with TA99 combined with MEKi and immune checkpoint blockade (anti-PD1 and anti-CTLA4). Our findings suggest that MEKi induced an increased expression of tumor-associated antigens, which, in combination with antitumor antibodies, generated a robust adaptive antitumor response that was sustained by immune checkpoint inhibition therapy. These improved therapeutic effects of specific antitumor antibodies by current standard-of-care therapeutics have potential near-term translation to the clinic, in light of fully humanized anti-TYRP1 antibodies currently in clinical development. Citation Format: Rolando Pérez-Lorenzo, Stephanie O. Erjavec, Raphael Clynes, Angela M. Christiano. Enhancing the efficacy of antitumor antibodies with immune checkpoint inhibitors and targeted therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1845.

Biological studies of an ICG-tagged aptamer as drug delivery system for malignant melanoma

Malignant melanoma accounts for about 1% of all skin malignant tumors and represents the most aggressive and lethal form of skin cancer. Clinically, there exist different therapeutic options for melanoma treatment, such as surgery, chemotherapy, radiotherapy, photodynamic therapy and immunotherapy. However, serious adverse effects usually arise, and survival rates are still low because a high number of patients present relapses within 6–9 months after therapy.AS1411 is a G-quadruplex (G4) aptamer capable of tumor-specific recognition, since it binds to nucleolin, a multi-functional protein expressed in many different types of cancer cells. In this work, we present a novel drug delivery system composed of AS1411 and indocyanine green (ICG) to track its accumulation in tumoral cells in a melanoma mouse model. Using a simple supramolecular strategy, we conjugated the complex AS1411-ICG with C8 ligand, an acridine orange derivative with potential anticancer ligand. Then, we performed in vitro cytotoxicity experiments using the B16 mouse melanoma cell line, and in vivo experiments using a B16 mouse melanoma model to study biodistribution and histological changes. The circular dichroism (CD) data suggest that C8 does not affect the parallel G4 topology of AS1411-ICG, whereas it increases its thermal stability. Incubation of B16 melanoma cells with the AS1411-ICG complex associated with C8 increases the cytotoxicity compared with AS1411-ICG alone.From the in vivo studies, we conclude that both AS1411-ICG and AS1411-ICG-C8 presented the potential to accumulate preferentially in tumor tissues. Moreover, these compounds seem to be efficiently removed from the mice's bodies through kidney clearance.In summary, these results suggest that these complexes derived from AS1411 aptamer could act as a delivery system of ligands with antitumoral activity for in vivo melanoma therapy.

A milbemycin compound isolated from Streptomyces Sp. FJS31-2 with cytotoxicity and reversal of cisplatin resistance activity in A549/DDP cells

Streptomyces Sp FJS31-2 is a strain isolated from special habitat soils in the early stage of our laboratory for producing a new type of halogenated type II polyketide antibiotic with good anti-MRSA activity. In this experiment, a variety of chromatographic and spectroscopic methods was used to isolate and identify a milbemycin compound VM48130 from the ethyl acetate extract of the fermentation products. To investigate its bioactivity, Cell Counting Kit-8 (CCK-8) assay was used to test the cytotoxic activity of the compound against a variety of cancer cells (human liver cancer cell line MHCC97H and SK-Hep1, human nasopharyngeal carcinoma cell line CNE1, mouse melanoma cell line B16, human colon cancer cell line LOVO, human lung adenocarcinoma cell line A549) and normal cells (human bronchial epithelial cell line 16HBE, human normal liver cell line L02, human nasopharyngeal epithelial cell line NP69). The results showed that the compound had significant cytotoxic activity against the above cancer cells, and the IC50 values were 21.96 ± 1.45, 22.18 ± 0.55, 19.42 ± 0.71, 18.61 ± 1.68, 18.62 ± 0.67, 18.52 ± 0.64 μM, respectively. Furthermore, the CCK-8 method was used to evaluate the compound's reversal of cisplatin resistance in multidrug resistant cisplatin-resistant human lung adenocarcinoma (A549/DDP) cells. The results indicated that when the compound concentration was 0.5 μM, the reversal fold (RF) reached 6.25 and showed a dose-dependent effect. At 5 μM, the RF reached 8.35, which was approximately equivalent to the reversal effect of the positive drug verapamil at the same concentration. The expression of MDR1, MRP1, LRP, MAST1 resistance genes and the corresponding proteins were analyzed by quantitative RT-PCR and Western blot assay, and found that the compound could significantly down-regulate the expression of these genes and proteins. These results indicated that VM48130 had the potential of being a lead compound for the treatment or adjuvant treatment of cancer.

Regulation of eIF2α by RNF4 Promotes Melanoma Tumorigenesis and Therapy Resistance

Among the hallmarks of melanoma are impaired proteostasis and rapid development of resistance to targeted therapy that represent a major clinical challenge. However, the molecular machinery that links these processes is unknown. Here we describe that by stabilizing key melanoma oncoproteins, the ubiquitin ligase RNF4 promotes tumorigenesis and confers resistance to targeted therapy in melanoma cells, xenograft mouse models, and patient samples. In patients, RNF4 protein and mRNA levels correlate with poor prognosis and with resistance to MAPK inhibitors. Remarkably, RNF4 tumorigenic properties, including therapy resistance, require the translation initiation factor initiation elongation factor alpha (eIF2α). RNF4 binds, ubiquitinates, and stabilizes the phosphorylated eIF2α (p-eIF2α) but not activating transcription factor 4 or C/EBP homologous protein that mediates the eIF2α-dependent integrated stress response. In accordance, p-eIF2α levels were significantly elevated in high-RNF4 patient-derived melanomas. Thus, RNF4 and p-eIF2α establish a positive feed-forward loop connecting oncogenic translation and ubiquitin-dependent protein stabilization in melanoma.

Alkylation enhances biocompatibility and siRNA delivery efficiency of cationic curdlan nanoparticles

Cationic curdlan derivatives are a class of promising carriers for nucleic acid delivery including short interfering RNA (siRNA). While our previous studies demonstrated the siRNA delivery efficiency of aminated curdlan derivatives, the associated cytotoxicity issue remained unsolved. To investigate the effects of alkylation on the toxicity as well as the transfection efficiency, we conjugated short alkyl chains to 6-amino-6-deoxy-curdlan (6AC-100). The cytotoxicity of alkylated 6AC-100 derivatives (denote CuVa polymers) decreased with the increase of the degree of substitution (DS). CuVa3, with the highest DS, showed a 50% decreased cytotoxicity compared to 6AC-100 to 6AC-100 at a concentration of 140 μg/mL. The CuVa polymers readily complexed with siRNA to form nanoparticles, and induced significant knockdown of a disease related gene (STAT3) in mouse melanoma cell line B16. However, B16 cells transfected with siSTAT3 complexed to CuVa3 showed the highest phenotypic changes. These findings suggest that CuVa polymers have significantly enhanced biocompatibility and may be a promising delivery system for delivery of therapeutic siRNAs.

Abstract 4520: Lymphatic vessels regulate exosome trafficking from tumors

Abstract Prior to metastasis, tumor-draining lymph nodes and distant organs are altered by a number of tumor-derived factors that help to both suppress host immunity as well as form an environment that supports metastatic tumor cells, i.e., a pre-metastatic niche. Recent studies suggest that pre-metastatic niches are induced by vesicles, including exosomes, secreted by tumor cells and cells of the tumor microenvironment. Here, we investigated the contribution of lymphatic vessels, particularly the lymphatic endothelial cells (LECs), in the active regulation of tumor exosome production and trafficking. To track exosomes, we purified and fluorescently labeled exosomes from in vitro cultures of the mouse melanoma cell line B16 F10 and of the human melanoma cell line Me260LN. Upon injection into the mouse ear dermis, fluorescent exosomes were observed within minutes in draining lymphatics, but not in surrounding blood vessels. Using the B16 F10 melanoma model overexpressing the lymphangiogenic growth factor VEGF-C, we found that upon intratumoral injection of exosomes, LECs within the tumor microenvironment abundantly take up exosomes, and at higher levels than blood endothelial cells, delivering exosomes to draining lymph nodes and eventually to the blood. In transgenic mice lacking dermal lymphatics, exosomes injected intratumorally were absent in the lymph nodes and present in far lower amounts in the blood compared to those injected into wild type mice. Additionally, while the density of endogenous blood exosomes increased upon tumor growth in wild type mice, no significant change in blood exosome density was observed in transgenic mice. Using an in vitro model where LECs are seeded onto porous inserts, we found that LEC transport of tumor exosomes occurs preferentially from their basal to apical side, and that some of these exosomes were taken up and stored intracellularly. Together, these findings suggest that lymphatic vessels constitute a major route of tumor exosome distribution to the systemic circulation and that LECs actively regulate exosome transport by active uptake. Citation Format: Lea Maillat, Lambert Potin, Witold W. Kilarski, Melody A. Swartz. Lymphatic vessels regulate exosome trafficking from tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4520.

Teraphtal decreased the sensitivity tumor cells to doxorubicine in vitro but does not affect its antitumor effect in vivo .

Introduction . Anthracycline antibiotic doxorubicin (DOX) is widely used in clinical oncology. It is known that hemin, endogenious compound, has the ability to modulate DOX cytotoxicity. We found that DOX toxicity against mammalian cancer cells can be decreased in vitro in the presence of teraftal (ТF), the component anticancer binaric catalytic system (TF + ascorbic acid).Purpose . To study the influence of TF on anticancer effect of DOX.Materials and methods . The mouse melanoma cell line B16 / F10 and mouse transplanted tumor B16 were used. The TF ability to protect from DOX-induced cell death were measured by MTT-assay, flow сytometry, light microscopy, cytochemical determination of ß-galactosidase expression, radiometric assay and tumor growth inhibition assay in vivo.Results. The sensitivity of mouse melanoma cell line B16 / F10 to DOX decreased in the presence TF (10–20 mkM) in the mean by 4–6 fold. The same mechanism takes part into the decrease of DOX cytotoxicity at the presence of TF / hemin khown which connects with the cell ability to accumulate of drug. TF protect the mouse melanoma cells B16 / F10 from apoptosis, induced by DOX throwing switching on cell premature senescence programme. The antitumor effect of DOX against mouse transplanted melanoma B16 at presence of TF was the same as DOX alone.Conclusions. The TF potency to decrease the sensitivity of cancer cells to DOX in vitro does not correlate with its ability to modulate аnthracycline antibiotics anticancer effect in vivo.

Abstract 3795: Novel mechanism underlying tumor immune evasion through tumor endothelial cells

Abstract Background: There has been a recent interest in how tumor cells escape from immune surveillance while establishing tumor microenvironments. Tumor endothelial cells (TECs), which constitute the lining of the tumor vessels, develop in the tumor microenvironment and come in contact with circulating immune cells. As such, TECs may control the trafficking or the anti-tumor reaction of an immune cell. We hypothesized that TECs may have an immune-suppressive potential in tumor microenvironments; thus, TECs may contribute to tumor immune-evasion. Objective: We aimed to investigate the immune-suppressive functions of TECs. Materials and Methods: We established a mouse cancer-model using the B16 mouse melanoma cell line and isolated TECs from the tumors. The immunological character of TECs was assessed by performing flow cytometric phenotyping of TECs and in vitro antigen endocytosis assay using exogenous antigen ovalbumin (OVA). Next, to identify the antigen presentation and immune-suppressive functions of TECs, B16 cells transfected with ovalbumin (B16-OVA) were inoculated in mice and the TECs isolated from the B16-OVA tumor were analyzed. An antibody reacting specifically to complex of OVA-derived antigen presented with mouse MHC class I was used to test antigen presentation. Further, to verify antigen-specific suppressive functions, TECs isolated from B16-OVA tumors were co-cultured with OVA-specific CD8+ T cells (OT-I cells) stimulated by OVA-pulsed bone marrow derived dendritic cells (BMDCs) in vitro. In this suppression assay, we performed a blocking assay using an anti-programmed death-ligand 1 (PD-L1) antibody. We also performed an antigen-specific cytotoxicity assay using B16-OVA cells as targets and OVA-specific CD8+ T cells which had been co-cultured with TECs as effectors. Results: TEC expressed MHC class I/II and co-stimulating molecules and PD-L1, suggesting that TECs had antigen-presenting and immune-suppressive properties. We found that TECs endocytosed B16-OVA derived protein and presented tumor-derived OVA peptides with their MHC class I molecule. TECs from B16-OVA tumor showed significantly greater suppression of OT-I CD8+ T cell proliferation compared to those from non-transfected B16 tumor. Similarly, in a cytotoxicity assay, TECs from B16-OVA tumor suppressed cytotoxicity of CD8+ T cells in an antigen specific manner. Strikingly, this suppressive function of TECs for the proliferation and cytotoxicity of OVA-specific T cells was abrogated by blocking PD-L1 using an anti-PD-L1 antibody, suggesting that the PD-1/PD-L1 axis contributes towards the immune-evasion mechanism. Conclusions: TECs have the characteristics of antigen presenting cells and regulate the immune response of tumor antigen specific T cells via the PD-1/PD-L1 pathway. Our data may suggest the novel mechanism of tumor immune-evasion through TECs. Citation Format: Kazuhiro Taguchi, Takashi Onoe, Hideki Ohdan. Novel mechanism underlying tumor immune evasion through tumor endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3795.

Targeted Delivery of Auristatin-Modified Toxins to Pancreatic Cancer Using Aptamers.

Pancreatic cancer is one of the most lethal malignancies. Treatment with the first-line agent, gemcitabine, is often unsuccessful because it, like other traditional chemotherapeutic agents, is non-specific, resulting in off-target effects that necessitate administration of subcurative doses. Alternatively, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) are highly toxic small molecules that require ligand-targeted delivery. MMAE has already received FDA approval as a component of an anti-CD30 antibody-drug conjugate, brentuximab vedotin. However, in contrast to antibodies, aptamers have distinct advantages. They are chemicals, which allows them to be produced synthetically and facilitates the rapid development of diagnostics and therapeutics with clinical applicability. In addition, their small size allows for enhanced tissue distribution and rapid systemic clearance. Here, we assayed the toxicity of MMAE and MMAF conjugated to an anti-transferrin receptor aptamer, Waz, and an anti-epidermal growth factor receptor aptamer, E07, on the pancreatic cancer cell lines Panc-1, MIA PaCa-2, and BxPC3. In vitro, our results indicate that these aptamers are a viable option for the targeted delivery of toxic payloads to pancreatic cancer cells.

Investigations on the molecular mode of action of the novel immunostimulator ZelNate: Activation of the cGAS-STING pathway in mammalian cells

Bovine respiratory disease (BRD) is usually prevented or treated with vaccines and/or antibiotics. The use of antibiotics is, however, of concern due to the potential promotion of microbial resistance and the occurrence of residues. Recently an alternative aid in the treatment of BRD, the cationic lipid/bacterial plasmid DNA liposome-based immunomodulator ZelNate, has entered the veterinary market. In the present study, we provide data on the molecular mode of action of ZelNate. Despite the presence of numerous non-methylated CpG motifs in its plasmid DNA, ZelNate proved to be inactive on human and mouse toll-like receptor 9 (TLR9) in cell culture, in both recombinant and natural cellular receptor settings. However, in the human monocyte cell line THP1 and in the mouse melanoma cell line B16, ZelNate activates strongly the stimulator of interferon genes (STING) pathway, which is known to lead predominantly to interferon response factor 3 (IRF3) activation. Further analysis in THP1 cells suggests that the ZelNate plasmid DNA activates STING via interaction with cyclic guanylate adenylate synthase (cGAS), but not via interferon induced gene 16 (IFI16). Our in vitro observations suggest that ZelNate may act predominantly via the cGAS/STING/IRF3 pathway.

Radiation Therapy and Talimogene Laherparepvec (TVEC) Synergize in the Treatment of Melanoma

Abstract Talimogene laherparepvec (TVEC), an oncolytic virus, is an FDA-approved therapy that increases the overall survival of locally advanced and metastatic melanoma patients. Radiation therapy (RT) in combination with immunotherapies improves response rates (compared to either modality alone). Therefore, we hypothesized that combination RT and TVEC results in a synergistic therapeutic effect on melanoma. We treated 8 human melanoma cell lines with radiation (0, 4, or 8 Gy) and/or T-VEC (0, 0.01, 0.1, or 1 MOI) and determined its therapeutic effects using the AlamarBlue cell viability assay. Treatment of UACC257 melanoma cells with combination RT (8 Gy) and TVEC (1 MOI) resulted in a significant decrease in tumor cell viability (a decrease of 44.9%) compared to no treatment, RT alone (33.2%, P=0.0004), or T-VEC alone (24.2%, P=0.0088), with similar findings in other cell lines. We then injected a B16 mouse melanoma cell line, permissive to T-VEC infection, into B6 mice to assess in vivo synergy. Mice were treated when tumors were ~7mm × 7mm. RT resulted in an early 20% decrease in tumor size within 4 days, followed by an increase thereafter. Intra-tumoral TVEC resulted in no clear benefit until 9 days post-treatment, when it showed a slight decrease in size sustained for ~10 days. Combination treatment, however, led to an early response that was sustained until 18 days after the initial treatment. When tumors were assessed 18 days after initial treatment, a 3-fold or more increase in tumor size was lowest in the combination T-VEC + RT group (17%) compared to the control (100%), RT (60%), or TVEC (50%) groups. These data propose that combination T-VEC + RT may be beneficial for cancer patients, and studies establishing the mechanism of synergy are ongoing.

Characterization and Potential Antitumor Activity of Polysaccharide from Gracilariopsis lemaneiformis.

Substances with valuable antitumor properties have been identified in many marine algae, including an edible polysaccharide from the marine alga Gracilariopsis lemaneiformis (PGL). We previously reported transcriptome profiling data showing that PGL induced transcriptional alterations generate anti-lung cancer activity. To identify how PGL is detrimental to tumors, we purified PGL to characterize its chemical composition, molecular weight, and sugar and protein content and investigated its antitumor activity. We demonstrated that PGL exerted its antitumor activities by modulating cell viability, morphology, apoptosis, and the apoptosis-related Fas/FasL signaling pathway in the human lung cancer cell line A549, the gastric cancer cell line MKN28, and the mouse melanoma cell line B16. Our data provide the first evidence that PGL inhibits cell proliferation by inducing apoptosis, which is largely mediated by Fas/FasL in cancer cells, suggesting that PGL might be a novel therapeutic agent against cancer.

RACK1 cooperates with NRASQ61K to promote melanoma in vivo

Melanoma is the deadliest skin cancer. RACK1 (Receptor for activated protein kinase C) protein was proposed as a biological marker of melanoma in human and domestic animal species harboring spontaneous melanomas. As a scaffold protein, RACK1 is able to coordinate the interaction of key signaling molecules implicated in both physiological cellular functions and tumorigenesis. A role for RACK1 in rewiring ERK and JNK signaling pathways in melanoma cell lines had been proposed. Here, we used a genetic approach to test this hypothesis in vivo in the mouse. We show that Rack1 knock-down in the mouse melanoma cell line B16 reduces invasiveness and induces cell differentiation. We have developed the first mouse model for RACK1 gain of function, Tyr::Rack1-HA transgenic mice, targeting RACK1 to melanocytes in vivo. RACK1 overexpression was not sufficient to initiate melanomas despite activated ERK and AKT. However, in a context of melanoma predisposition, RACK1 overexpression reduced latency and increased incidence and metastatic rate. In primary melanoma cells from Tyr::Rack1-HA, Tyr::NRasQ61K mice, activated JNK (c-Jun N-terminal kinase) and activated STAT3 (signal transducer and activator of transcription 3) acted as RACK1 oncogenic partners in tumoral progression. A sequential and coordinated activation of ERK, JNK and STAT3 with RACK1 is shown to accelerate aggressive melanoma development in vivo.

Cell stress response to two different types of polymer coated cobalt ferrite nanoparticles

Potential nanoparticle (NP) toxicity is one of crucial problems that limit the applicability of NPs. When designing NPs for biomedical and biotechnological applications it is thus important to understand the mechanisms of their toxicity. In this study, we analysed the stress responses of previously designed polyacrylic acid (PAA) and polyethylenimine (PEI) coated NPs on primary human myoblasts (MYO) and B16 mouse melanoma cell line. Negatively charged PAA did not induce cell toxicity, reactive oxygen species (ROS) or activate the transcription factor NF-κB in either cell line even at high concentrations (100μg/ml). On the other hand, positively charged PEI NPs induced a concentration dependent necrotic cell death and an increase in ROS following 24h incubation already at low concentrations (>4μg/ml). Moreover, PEI NPs induced NF-κB activation 15–30min after incubation in MYO cells, most probably through activation of TLR4 receptor. Interestingly, there was no NF-κB response to PEI NPs in B16 cells.

Radiation Changes the Metabolic Profiling of Melanoma Cell Line B16.

Radiation therapy can be an effective way to kill cancer cells using ionizing radiation, but some tumors are resistant to radiation therapy and the underlying mechanism still remains elusive. It is therefore necessary to establish an appropriate working model to study and monitor radiation-mediated cancer therapy. In response to cellular stress, the metabolome is the integrated profiling of changes in all metabolites in cells, which can be used to investigate radiation tolerance mechanisms and identify targets for cancer radiation sensibilization. In this study, using 1H nuclear magnetic resonance for untargeted metabolic profiling in radiation-tolerant mouse melanoma cell line B16, we comprehensively investigated changes in metabolites and metabolic network in B16 cells in response to radiation. Principal component analysis and partial least squares discriminant analysis indicated the difference in cellular metabolites between the untreated cells and X-ray radiated cells. In radiated cells, the content of alanine, glutamate, glycine and choline was increased, while the content of leucine, lactate, creatine and creatine phosphate was decreased. Enrichment analysis of metabolic pathway showed that the changes in metabolites were related to multiple metabolic pathways including the metabolism of glycine, arginine, taurine, glycolysis, and gluconeogenesis. Taken together, with cellular metabolome study followed by bioinformatic analysis to profile specific metabolic pathways in response to radiation, we deepened our understanding of radiation-resistant mechanisms and radiation sensibilization in cancer, which may further provide a theoretical and practical basis for personalized cancer therapy.

Triterpenoids from the fruit of Schisandra glaucescens

Five new triterpenoids, named schiglausins P-T (1–5), together with twelve known analogues (6–17), were isolated from the fruit of Schisandra glaucescens Diels. Their structures were determined by various spectroscopic methods, including HRESIMS, 1D and 2D NMR spectra and CD experiment. Additionally, all these compounds were tested for their cytotoxicities against B16 mouse melanoma cell line. Compounds 8, 11, 14, and 15 exhibited moderate anti-proliferative effects against B16 cells with IC50 values ranging from 3.64 to 27.00μM.