Growth Of B16F10 Melanoma Cells Research Articles

Paulownin elicits anti-tumor effects by enhancing NK cell cytotoxicity through JNK pathway activation.

Paulownin, a natural compound derived from Paulownia tomentosa wood, exhibits various physiological functions, including anti-bacterial and anti-fungal effects. However, the impact of paulownin on natural killer (NK) cell immune activity remains largely unknown. In this study, we investigated the effect of paulownin on NK cell activity both in vitro and in vivo, and explored its potential mechanisms. NK-92 cells were used for in vitro experiments and a BALB/c mouse model with B16F10 cells injected subcutaneously were used for in vivo anti-tumor analysis. We found that paulownin enhanced the cytolytic activity of NK-92 cells against leukemia, human colon, and human lung cancer cell lines. Paulownin treatment increased the expression of the degranulation marker protein CD107a and cytolytic granules, including granzyme B and perforin in NK-92 cells. Moreover, these enhancements of cytotoxicity and the expression of cytolytic granules induced by paulownin were also observed in human primary NK cells. Signaling studies showed that paulownin promoted the phosphorylation of JNK. The increased perforin expression and elevated cytotoxic activity induced by paulownin were effectively inhibited by pre-treatment with a JNK inhibitor. In vivo studies demonstrated that the administration of paulownin suppressed the growth of B16F10 melanoma cells allografted into mice. Paulownin administration promoted the activation of NK cells in the spleen of mice, resulting in enhanced cytotoxicity against YAC-1 cells. Moreover, the anti-tumor effects of paulownin were reduced upon the depletion of NK cells. Therefore, these results suggest that paulownin enhances NK cell cytotoxicity by activating the JNK signaling pathway and provide significant implications for developing new strategies for cancer immunotherapy.

Continuous topical application of microencapsulated recombinant human epidermal growth factor does not promote the progression of established melanoma in animals

Microencapsulated epidermal growth factor (EGF) device has been applied topically for the management of several types of wounds to accelerate wound healing and prevent scar formation. However, it remains unclear whether such EGF device induced neoplastic transformation in the skin. In this study, we exploited a well-established murine B16-F10 melanoma model, coupled with MTT viability and colony formation assays, to investigate the influence of microencapsulated recombinant EGF (Me-EGF; brand name NewEpi®) and its ingredients on the tumorigenicity of skin cancer cells in vitro and in vivo. The results indicated that Me-EGF did not stimulate the viability nor the anchorage-dependent growth of B16-F10 melanoma cells. Western blot analysis showed that Me-EGF treatment increased the total and phosphorylated EGFR expression without affecting the HER2 expression in B16-F10 melanoma cells. In mice bearing established B16-F10 melanoma, continuous application of Me-EGF for 14 days did not enhance the melanoma tumor burden compared with control groups. Immunohistochemical analysis also revealed the similar expression of proliferative index Ki-67 between Me-EGF-treated melanoma and other groups. Altogether, these results suggest that the application of Me-EGF device did not promote the oncogenic potential of B16-F10 melanoma in vitro and in vivo.

DB3 from Antarctic lichen inhibits the growth of B16F10 melanoma cells in vitro and in vivo

Malignant melanoma is a fatal disease with an increasing global incidence. Despite numerous studies focused on anti-cancer drugs, a variety of side effects of cancer treatment remain challenging. Thus, there is a pressing need to identify novel anti-cancer agents with minimal cytotoxicity and side effects. DB3 (1,3,7,9-tetrahydroxy-2,8-dimethyl-4,6-di[ethanoyl]dibenzofuran) is a member of the dibenzofuran family and is extracted from Ramalina terebrata (Antarctic lichen). We investigated if DB3 exerted an antitumor effect on B16F10 melanoma cells. The results revealed that DB3 exerted time- and dose-dependent reduction of cell viability by inducing apoptosis and significantly suppressing cell proliferation through cell cycle arrest in the G0/G1 phase in B16F10 melanoma cells. Additionally, DB3 impeded the migration and invasiveness of B16F10 cells. Subsequently, we observed that DB3 decreased the expression levels of Cdk4/Cyclin D1 and the phosphorylation of p38, JNK, ERK, and AKT. Furthermore, DB3 decreased melanoma tumor growth in a mouse tumor syngraft model. Based on these findings, we propose that DB3 possesses potential for use as an anti-cancer agent for melanoma treatment.

Shikonin Causes Non-apoptotic Cell Death in B16F10 Melanoma.

Melanoma treatment is highly resistant to current chemotherapeutic agents. Due to its resistance towards apoptotic cell death, non-apoptotic cell death pathways are sought after. We investigated a Chinese herbal medicine, shikonin, and its effect on B16F10 melanoma cells in vitro. Cell growth of B16F10 melanoma cells treated with shikonin was analyzed using an MTT assay. Shikonin was combined with necrostatin, an inhibitor of necroptosis; caspase inhibitor; 3-methyladenine, an inhibitor of autophagy; or N-acetyl cysteine, an inhibitor of reactive oxygen species. Flow cytometry was used to assess types of cell death resulting from treatment with shikonin. Cell proliferation was also analyzed utilizing a BrdU labeling assay. Monodansylcadaverine staining was performed on live cells to gauge levels of autophagy. Western blot analysis was conducted to identify specific protein markers of necroptosis including CHOP, RIP1, and pRIP1. MitoTracker staining was utilized to identify differences in mitochondrial density in cells treated with shikonin. Analysis of MTT assays revealed a large decrease in cellular growth with increasing shikonin concentrations. The MTT assays with necrostatin, 3-methyladenine, and N-acetyl cysteine involvement, suggested that necroptosis, autophagy, and reactive oxygen species are a part of shikonin's mechanism of action. Cellular proliferation with shikonin treatment was also decreased. Western blotting confirmed that shikonin-treated melanoma cells increase levels of stress-related proteins, e.g., CHOP, RIP, pRIP. Our findings suggest that mainly necroptosis is induced by the shikonin treatment of B16F10 melanoma cells. Induction of ROS production and autophagy are also involved.

Direct anti-tumoral effects of sulfated fucans isolated from echinoderms: a possible role of neuropilin-1/β1 integrin endocytosis and focal adhesion kinase (FAK) degradation.

Hypercoagulability, a major complication of metastatic cancers, has usually been treated with heparins from natural sources, or with their synthetic derivatives, which are under intense investigation in clinical oncology. However, the use of heparin has been challenging for patients with risk of severe bleeding. While the systemic administration of heparins, in pre-clinical models, has shown primarily attenuating effects on metastasis, their direct effect on established solid tumors has generated contradictory outcomes. We investigated the direct antitumoral properties of two sulfated fucans isolated from marine echinoderms, FucSulf1 and FucSulf2, which exhibit anticoagulant activity with mild hemorrhagic potential. Unlike heparin, sulfated fucans significantly inhibited tumor cell proliferation (by ~30-50 percent), and inhibited tumor migration and invasion in vitro. We found that FucSulf1 and FucSulf2 interacted with fibronectin (FN) as efficiently as heparin, leading to loss of prostate cancer and melanoma cell spreading. The sulfated fucans increased the endocytosis of β1 integrin and neuropilin-1 (NRP-1) chains, two cell receptors implicated in FN-dependent adhesion. The treatment of cancer cells with both sulfated fucans, but not with heparin, also triggered intracellular focal adhesion kinase (FAK) degradation, with a consequent overall decrease in activated FAK levels. Finally, only sulfated fucans inhibited the growth of B16-F10 melanoma cells implanted in the dermis of syngeneic C57/BL6 mice. FucSulf1 and FucSulf2 arise from this study as candidates for the design of possible alternatives to long-term treatments of cancer patients with heparins, with the advantage of also controlling local growth and invasion of malignant cells.

Combination Therapy of Curcumin and Disulfiram Synergistically Inhibits the Growth of B16-F10 Melanoma Cells by Inducing Oxidative Stress

Oxidative stress plays a central role in the pathophysiology of melanoma. Curcumin (CUR) is a polyphenolic phytochemical that stimulates reactive oxygen species (ROS) production, while disulfiram (DSS) is a US FDA-approved drug for the treatment of alcoholism that can act by inhibiting the intracellular antioxidant system. Therefore, we hypothesized that they act synergistically against melanoma cells. Herein, we aimed to study the antitumor potential of the combination of CUR with DSS in B16-F10 melanoma cells using in vitro and in vivo models. The cytotoxic effects of different combination ratios of CUR and DSS were evaluated using the Alamar Blue method, allowing the production of isobolograms. Apoptosis detection, DNA fragmentation, cell cycle distribution, and mitochondrial superoxide levels were quantified by flow cytometry. Tumor development in vivo was evaluated using C57BL/6 mice bearing B16-F10 cells. The combinations ratios of 1:2, 1:3, and 2:3 showed synergic effects. B16-F10 cells treated with these combinations showed improved apoptotic cell death and DNA fragmentation. Enhanced mitochondrial superoxide levels were observed at combination ratios of 1:2 and 1:3, indicating increased oxidative stress. In vivo tumor growth inhibition for CUR (20 mg/kg), DSS (60 mg/kg), and their combination were 17.0%, 19.8%, and 28.8%, respectively. This study provided data on the potential cytotoxic activity of the combination of CUR with DSS and may provide a useful tool for the development of a therapeutic combination against melanoma.

In vivo and in vitro evidence for growth hormone-like bioactivity of Rhizoma Anemarrhenae extract

Certain herbs used in traditional Chinese medicine may produce a growth-enhancing effect by promoting the secretion of growth hormone (GH) by the pituitary gland or mimicking the function of GH. In this study, we aimed to identify herbs that could serve as GH alternatives. A reporter gene assay for GH was developed, and 100 different herbal extracts were assayed. We found that Rhizoma Anemarrhenae (RA) water extracts exhibited transactivation activities that stimulate the activation of signal transducer and activator of transcription 5 (STAT5). The growth-promoting effect of RA in NB2–11 cells was inhibited by co-treatment with GH receptor (GHR)-Fc fusion protein. Unlike GH, RA extracts did not enhance the growth of B16F10 melanoma cells. The activation of the Janus kinase 2-STAT5 signaling pathway was confirmed in both NB2–11 cells and WI-38 human normal lung fibroblasts; the activation was inhibited by co-treatment with GHR-Fc fusion protein. Docking analysis of the active ingredients of RA, including mangiferin, neomangiferin, isomangiferin, anemarsaponin E, 7-O-methylmangiferin, officinalisinin I, timosaponin BII, timosaponin AI, and timosaponin AIII, using SWISSDOCK indicated a direct interaction of these compounds with GHR. The growth-promoting effects and activation of STAT5 were also confirmed. Moreover, we found that RA extract significantly increased the height of the tibial growth plate and stimulated the production of insulin-like growth factor 1 in the serum, liver, and muscle tissues. Our findings provide evidence that herbal extracts, particularly, RA extracts, can promote growth by mimicking GH bioactivity.

Metabolic installation of macrophage-recruiting glycan ligand on tumor cell surface for in vivo tumor suppression

Synthetic probes that could direct immune cells against tumors are potential immunotherapeutics. We herein report in vivo tumor suppression via an intravenously injected abiotic sialic acid (TCCSia) that could be metabolically incorporated into tumor cell surface to yield of a high affinity ligand (TCCSiaα2,3-Gal) of Siglec-1 specifically expressed on macrophages. We observed marked suppression of pulmonary metastasis and subcutaneous tumor growth of B16F10 melanoma cells in mice with TCCSia, suggesting the utility of abiotic sialic acid to modulate tumor immunity via recruiting Siglec+ immune cells.

Dual Photothermal/Chemotherapy of Melanoma Cells with Albumin Nanoparticles Carrying Indocyanine Green and Doxorubicin Leads to Immunogenic Cell Death.

Recent focus on cancer immunotherapies has led to significant interest in the development of therapeutic strategies that can lead to immunogenic cell death (ICD), which can cause activation of an immune response against tumor cells and improve immunotherapy outcomes by enhancing the immunogenicity of the tumor microenvironment. In this work, a nanomedicine-mediated combination therapy is used to deliver the ICD inducers doxorubicin (Dox), a chemotherapeutic agent, and indocyanine green (ICG), a photothermal agent. These agents are loaded into nanoparticles (NPs) of bovine serum albumin (BSA) that are prepared through a desolvation process. The formulation of BSA NPs is optimized to achieve NPs of 102.6nm in size and loadings of 8.55% and 5.69% (w/w) for ICG and Dox, respectively. The controlled release of these agents from the BSA NPs is confirmed. Upon laser irradiation for 2.5min, NPs at a dose of 62.5μg mL-1 are able to increase the temperature of the cells by 7 °C and thereby inhibit the growth of B16F10 melanoma cells in vitro. Surface presentation of heat shock proteins and calreticulin from the cells after treatment confirmed the ability of the Dox/ICG loaded BSA NPs to induce ICD in the melanoma cells.

Integrase-Defective Lentiviral Vector Is an Efficient Vaccine Platform for Cancer Immunotherapy

Integrase-defective lentiviral vectors (IDLVs) have been used as a safe and efficient delivery system in several immunization protocols in murine and non-human primate preclinical models as well as in recent clinical trials. In this work, we validated in preclinical murine models our vaccine platform based on IDLVs as delivery system for cancer immunotherapy. To evaluate the anti-tumor activity of our vaccine strategy we generated IDLV delivering ovalbumin (OVA) as a non-self-model antigen and TRP2 as a self-tumor associated antigen (TAA) of melanoma. Results demonstrated the ability of IDLVs to eradicate and/or controlling tumor growth after a single immunization in preventive and therapeutic approaches, using lymphoma and melanoma expressing OVA. Importantly, LV-TRP2 but not IDLV-TRP2 was able to break tolerance efficiently and prevent tumor growth of B16F10 melanoma cells. In order to improve the IDLV efficacy, the human homologue of murine TRP2 was used, showing the ability to break tolerance and control the tumor growth. These results validate the use of IDLV for cancer therapy.

Nintedanib promotes antitumour immunity and shows antitumour activity in combination with PD-1 blockade in mice: potential role of cancer-associated fibroblasts

BackgroundCancer-associated fibroblasts (CAFs) in the tumour microenvironment (TME) suppress antitumour immunity, and the tyrosine kinase inhibitor nintedanib has antifibrotic effects.MethodsWe performed a preclinical study to evaluate whether nintedanib might enhance antitumour immunity by targeting CAFs and thereby improve the response to immune checkpoint blockade (ICB).ResultsWhereas nintedanib did not suppress the growth of B16-F10 melanoma cells in vitro, it prolonged survival in a syngeneic mouse model of tumour formation by these cells, suggestive of an effect on the TME without direct cytotoxicity. Gene expression profiling indeed showed that nintedanib influenced antitumour immunity and fibrosis. Tumoural infiltration of CD8+ T cells and granzyme B production were increased by nintedanib, and its antitumour activity was attenuated by antibody-mediated depletion of these cells, indicating that nintedanib suppressed tumour growth in a CD8+ T cell-dependent manner. Moreover, nintedanib inhibited the proliferation and activation of fibroblasts. Finally, the combination of nintedanib with ICB showed enhanced antitumour efficacy in B16-F10 tumour-bearing mice.ConclusionsOur results suggest that nintedanib targeted CAFs and thereby attenuated the immunosuppressive nature of the TME and promoted the intratumoural accumulation and activation of CD8+ T cells, with these effects contributing to enhanced antitumour activity in combination with ICB.

Study on the Synthesis and Biological Activities of N-Alkylated Deoxynojirimycin Derivatives with a Terminal Tertiary Amine

A series of N-alkylated deoxynojirimycin (DNJ) derivatives connected to a terminal tertiary amine at the alkyl chains of various lengths were prepared. These novel synthetic compounds were assessed for preliminary glucosidase inhibition and anticancer activities in vitro. Potent and selective inhibition was observed among them. Compound 7d (IC50 = 0.052 mM) showed improved and selective inhibitory activity against ?-glucosidase compared to DNJ (IC50 = 0.65 mM). In addition, analysis of the kinetics of enzyme inhibition by using Lineweaver-Burk plots indicated that 7d inhibited ?-glu-cosidase in a competitive manner, suggesting that 7d was expected to bind to the active site of ?-glucosidase. Compounds 8b and 8c were found to be moderate and selective inhibitors of ?-glucosidase. Nevertheless, none of compounds inhib-ited the growth of B16F10 melanoma cells.

Abstract A79: Inhibition of p53-MDM2 protein interaction reduces tumor growth in a mouse melanoma model

Abstract Tumor suppressor protein p53 is mutated in close to 50% of human tumors. Under steady state conditions, the two E3 ligases MDM2 and MDM4 interact with and inhibit the transcriptional activity of p53. Inhibition of p53-MDM2/4 interaction to reactivate p53 has therefore been considered a therapeutic strategy. In this study, we tested p53-MDM2 protein-protein interaction inhibition using the small molecule AMG 232, which is currently being tested in phase 1b/2a clinical trials. In vitro, AMG 232 induced a significant, p53-dependent growth arrest in the mouse melanoma cell line B16-F10. Using mass spectrometry-based proteomics, we identified differential protein expression patterns following AMG 232 treatment of B16-F10 melanoma cells. In vivo, the growth of B16-F10 melanoma cells implanted in WT C57BL/6 mice was significantly reduced by AMG 232 treatment. Our data demonstrate that AMG 232 induces a p53-dependent tumor growth arrest in an immunocompetent mouse model, and we are currently testing whether AMG232 can synergize with checkpoint immunotherapy. Citation Format: Katrine Ingelshed, Danai Lianoudaki, Dilraj Lama, Silke Sohn, Nicolas Fritz, Long Jiang, Fredrik Wermeling, Mikael Karlsson, David P. Lane, Saikiran K. Sedimbi. Inhibition of p53-MDM2 protein interaction reduces tumor growth in a mouse melanoma model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A79.

Development of Vaccines with Self-assembled Carriers That Deliver Drugs to Target Organs

I have developed novel ternary complexes of various vaccines with cationic materials and anionic polymers. Plasmid DNA (pDNA) encoding firefly luciferase was used as a model drug to form adequate ternary complexes. Cationic binary complexes were constructed using pDNA and polyethylenimine, and these binary complexes were coated with various anionic polymers to form ternary complexes. These ternary complexes significantly improved cytotoxicity and aggregation with erythrocytes in comparison to the binary complexes. On the other hand, most of those ternary complexes showed little in vitro transgene efficiency because of their anionic surface charge. γ-Polyglutamic acid (γ-PGA)-ternary complexes, however, demonstrated high in vitro transgene efficiency. After the intravenous administration of γ-PGA-ternary complexes to mice, extremely high gene expression was detected in the marginal zone of the spleen, which is rich in antigen-presenting cells. This spleen-specific phenomenon of γ-PGA-ternary complexes appeared to be suited to DNA vaccines against cancer. I therefore examined the preventive effect of γ-PGA-ternary complexes containing pUb-M, a pDNA encoding melanoma surface antigen, against melanoma-bearing mice. Vaccinations of γ-PGA-ternary complexes into mice significantly suppressed the tumor growth of B16-F10 melanoma cells subcutaneously injected into the mice. In the same manner, vaccinations of γ-PGA-ternary complexes containing ovalbumin (OVA) completely suppressed the growth of E.G7-OVA cells expressing OVA. These results strongly suggest that γ-PGA-ternary complexes are useful in the manufacture of specific tumor vaccines.

349P - Flavonoids from ethanol extracts of euphorbia hirta inhibit melanoma growth and metastatic potential

BackgroundEuphorbia hirta has many health benefits for enhancing human health. It exhibits various antioxidant properties. Our aim was to investigate the effects of several polyphenols from Euphorbia hirta ethanolic extract on the growth and metastatic potential of B16F10 melanoma cells in vitro and to measure the observable changes of cell morphology upon exposure to the extract. MethodsThe cytotoxic activity of the Euphorbia hirta extract on B16F10 melanoma cell line was investigated in vitro using 3‐ (4, 5‐dimethyl thiazol‐2yl) ‐2, 5‐diphenyl tetrazolium bromide (MTT). B16F10 (human Melanoma) cell lines were cultured in DMEM supplemented with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/ml), streptomycin (100 μg/ml) and amphotericin B (5 μg/ml) in an humidified atmosphere of 5% CO2 at 37°C until confluent. The cells were dissociated with Trypsin solution (0.2% trypsin, 0.02% EDTA in PBS). The stock cultures were grown in 25 cm2 culture flasks and all experiments were carried out in 96 micro-titer plates. After culturing and proliferation of B16F10 cells, these cells were exposed to various ethanolic extract of E hirta. The potency of plant extract concentration was calculated in terms of a percent decrease in viable B16F10 cells as compared to the control value. ResultsThe in vitro growth of B16F10 melanoma cells in the presence of a series of polyphenolic compounds (10–9 to 10–5 M) was evaluated. Quercetin, apigenin, EGCG, resveratrol, curcumin, and tamoxifen inhibited the growth of B16F10 cells in a concentration‐dependent manner. The concentrations needed to inhibit cell growth by 50% (IC50) are reported. The results of MTT assay test showed that the ethanol extract of Euphorbia hirta was tested for the anticancer activity and it showed the most effective inhibition of B16F10 cells proliferation. ConclusionsWe assessed the cytotoxic effect of Euphorbia hirta extract in cell culture. In defiance of astonishing advances in modern medicine, such as surgery, radiotherapy, chemotherapy, and immunotherapy, melanoma cancer disease remains a worldwide health problem, thus endeavoring the search for a new alternate approach. Legal entity responsible for the studyThe author. FundingHas not received any funding. DisclosureThe author has declared no conflicts of interest.

Ellagic acid, extracted from Sanguisorba officinalis, induces G1 arrest by modulating PTEN activity in B16F10 melanoma cells.

In Chinese medicine, herbal medicine is commonly used to treat individuals suffering from many types of diseases. We thus expected that some herbal medicines would contain promising compounds for cancer chemotherapy. Indeed, we found that Sanguisorba officinalis extracts strongly inhibit the growth of B16F10 melanoma cells, and we identified ellagic acid (EA) as the responsible ingredient. B16F10 cells treated with EA exhibited strong G1 arrest accompanied by accumulation of p53, followed by inactivation of AKT. Addition of a PTEN inhibitor, but not a p53 inhibitor, abrogated the EA-induced AKT inactivation and G1 arrest. The PTEN inhibitor also diminished EA-induced p53 accumulation. Furthermore, EA apparently increased the protein phosphatase activity of PTEN, as demonstrated by the reduced phosphorylation level of FAK, a protein substrate of PTEN. Furthermore, an in vitro PTEN phosphatase assay on PIP3 showed the direct modulation of PTEN activity by EA. These results suggest that EA functions as an allosteric modulator of PTEN, enhancing its protein phosphatase activity while inhibiting its lipid phosphatase activity. It is notable that a combination of EA and cisplatin, a widely used chemotherapy agent, dramatically enhanced cell death in B16F10 cells, suggesting a promising strategy in chemotherapy.

Broad and systemic immune-modulating capacity of plant-derived dsRNA.

Double-stranded RNA (dsRNA) is well characterized as an inducer of anti-viral interferon responses. We previously reported that dsRNA extracted from a specific edible plant possesses an immune-modulating capacity to confer, in mice, resistance against respiratory viruses, including the H1N1 strain of the influenza A virus (IAV). We report here that the systemic immune-activating capacity of the plant-derived dsRNA protected mice from infection by a highly virulent H5N1 strain of the IAV. In addition, subcutaneous inoculation of the dsRNA together with the inactivated virion of the H5N1 strain of the IAV suppressed the lethality of the viral infection as compared with individual inoculation of either dsRNA or HA protein, suggesting its potential usage as a vaccination adjuvant. Moreover, intra-peritoneal inoculation of the dsRNA limited the growth of B16-F10 melanoma cells through the activation of NK cells in murine models. Taken together, this study demonstrated the systemic immune-modulating capacity of a plant-derived dsRNA and its potential for nucleic acid-based clinical applications.

RETRACTED: The therapeutic potential of human adipose-derived mesenchymal stem cells producing CXCL10 in a mouse melanoma lung metastasis model

Interferon γ-induced protein 10 kDa (IP-10) is a potent chemoattractant and has been suggested to enhance antitumor activity and mediate tumor regression through multiple mechanisms of action. Multiple lines of evidence have indicated that genetically-modified adult stem cells represent a potential source for cell-based cancer therapy. In the current study, we assessed therapeutic potential of human adipose derived mesenchymal stem cells (hADSC) genetically-modified to express IP-10 for the treatment of lung metastasis in an immunocompetent mouse model of metastatic melanoma. A Piggybac vector encoding IP-10 was employed to transfect hADSC exvivo. Expression and bioactivity of the transgenic protein from hADSCs expressing IP-10 were confirmed prior to invivo studies. Our results indicated that hADSCs expressing IP-10 could inhibit the growth of B16F10 melanoma cells and significantly prolonged survival. Immunohistochemistry analysis, TUNEL assay and western blot analysis indicated that hADSCs expressing IP-10 inhibited tumor cell growth, hindered tumor infiltration of Tregs, restricted angiogenesis and significantly prolonged survival. In conclusion, our results demonstrated that targeting metastatic tumor sites by hADSC expressing IP-10 could reduce melanoma tumor growth and lung metastasis.

Activated Natural Killer Cells Mediate the Suppressive Effect of Interleukin-4 on Tumor Development via STAT6 Activation in an Atopic Condition Melanoma Model

A protective effect of allergy for cancer has been suggested, but the results are somewhat conflicting, and the mechanism remains elusive. Interleukin-4 (IL-4) signaling has been identified as a potentially important pathway in the development of allergies and the suppression of cancer development. To evaluate the allergy responses in IL-4–mediated tumor development, we compared the growth of B16F10 melanoma cells in 4% phthalic anhydride (PA)-treated IL-4/Luc/CNS-1 transgenic mice (IL-4 mice) and acetone-olive oil (AOO)–treated IL-4 mice as a control for 3 weeks. Much higher allergic responses and natural killer (NK) and STAT6 activation were found in PA-treated IL-4 mice compared with AOO-treated IL-4 control mice. Tumor volume and weight showed an inverse association with the higher allergic response and were significantly reduced in the PA-treated IL-4 mice when compared with those of AOO-treated IL-4 control mice. Significantly higher activation of STAT6, as well as IL-4 and NK cell activation, was found in the tumor tissues of PA-treated IL-4 mice. Infiltration of immune cells and cytokine levels were also higher in the tumor tissues of PA-treated IL-4 mice. We further found that IL-4–activated NK-92MI cells showed increased anticancer effects in human melanoma cells. Overall, these results showed that allergy responses further accelerated the IL-4–induced inhibition of tumor development through the activation of STAT6 pathways.

1,2,3-Triazolyl esterization of PAK1-blocking propolis ingredients, artepillin C (ARC) and caffeic acid (CA), for boosting their anti-cancer/anti-PAK1 activities along with cell-permeability.

Artepillin C (ARC) and caffeic acid (CA) are among the major anti-cancer ingredients of propolis, and block the oncogenic/melanogenic/ageing kinase PAK1. However, mainly due to their COOH moiety, cell-permeability of these herbal compounds is rather limited. Thus, in this study, in an attempt to increase their cell-permeability without any significant loss of their water-solubility, we have esterized both ARC and CA with the water-soluble 1,2,3-triazolyl alcohol through Click Chemistry. We found that this esterization boosts the anti-cancer activity of ARC and CA by 100 and over 400 folds, respectively, against the PAK-dependent growth of A549 lung cells, but show no effect on the PAK1-independent growth of B16F10 melanoma cells. Confirming this "selective" toxicity, these esters are still capable of blocking the kinase PAK1 strongly in cell culture (with IC50 around 5 µM), and the anti-PAK1 activity of 15A (ARC ester) and 15C (CA ester) appears to be 30-fold and 140-fold higher than ARC and CA, respectively. The 15A and 15C are 8-fold and 70-fold more cell-permeable (through the multi-drug resistant cell line EMT6) than ARC and CA, respectively. These data altogether suggest that both 15A and 15C would be far more useful than propolis for the treatment of a wide variety of PAK1-dependent diseases/disorders such as cancers, Alzheimer's diseases (AD), hypertension, diabetes (type 2), and hyper-pigmentation.