Inhibited Melanoma Tumor Growth Research Articles

KIF15 plays a role in promoting the tumorigenicity of melanoma

Kinesins are a superfamily of motor proteins and are often dysregulated in many cancers. KIF15, which belongs to the kinesin-12 family, has been shown to function in many different cellular processes, including proliferation, apoptosis, differentiation and development. However, the role of KIF15 in melanoma, remains unknown. In this study, the expression levels of KIF15 in melanoma cells lines and tissues were determined via real-time PCR, immunohistochemical staining and western blot. The effect of KIF15 on tumorigenesis was evaluated by using MTT and colony information. The function of KIF15 on cell survival was detected through flow cytometry assay. Microarray assay and bioinformatics analysis were used to find the potential target of KIF15. We show that KIF15 was significantly upregulated in melanoma cells and tissues. The suppression of KIF15 in tumors significantly reduced tumor growth and increased apoptosis in A375 and OCM1 cells. Findings based on the subcutaneous xenograft model were further consistent with the in vitro results that KIF15 knockdown inhibited melanoma tumor growth in vivo. Microarray assay and bioinformatics indicated that BIRC5, CDK4 and WNT5A were three potential targets of KIF15. Taken together, our results suggest that KIF15 plays a positive role in the tumorigenicity of melanoma and it may serve as a novel diagnostic and therapeutic target for melanoma, especially uveal melanoma.

LY341495, an mGluR2/3 Antagonist, Regulates the Immunosuppressive Function of Myeloid-Derived Suppressor Cells and Inhibits Melanoma Tumor Growth.

Myeloid-derived suppressor cells (MDSCs) are immunosuppressive myeloid cells found in patients with cancer and in mouse tumor models. They suppress anti-tumor immunity, resulting in the promotion of tumor growth. The relationship between nutrition and cancer has recently been reported by several research groups. Tumor cells rely on glutaminolysis, in which glutamine is metabolized into glutamate for energy production, and hence, glutamate levels are elevated in tumor-bearing hosts. However, the mechanism of regulation of tumor progression by glutamate still remains unclear. In this study, we found that the metabotropic glutamate receptor (mGluR) 2/3 was expressed on MDSCs, and an mGluR2/3 antagonist LY341495 attenuated the immunosuppressive activity of MDSCs. Furthermore, we observed that LY341495 treatment inhibited B16-F10 melanoma growth in vivo. Taken together, our data suggest that glutamate signaling promotes tumor growth by increasing the potency of immune suppression.

144 - ROS-independent inhibition of melanoma tumor growth by luteolin involves BRAF and the MAPK pathway

Background Melanoma is a reactive oxygen species (ROS)-driven cancer but developing antioxidants as therapeutic drugs have not been successful. Methods We screened a number of antioxidants for their capacity of inhibiting melanoma cell growth and identified luteolin and honokiol as the best candidates. A series of molecular studies were performed in order to delineate the mechanism of action for luteolin. Results Luteolin unexpectedly induced ROS at its IC50 range of concentrations; co-treatment with antioxidants NAC or mito-TEMPO did not rescue cell growth inhibition, although ROS levels were reduced. Luteolin also inhibited PDE4B and AKT kinase pathways, but over-expression of AKT2 or AKT3 did not show a significant effect on reversing the growth inhibition in vitro. Luteolin showed strong inhibition on BRAF kinase at protein and mRNA levels, accompanied by lower levels of phosphorylated MEK1/2 and ERK1/2 kinases. The BRAF mutation status did not show a difference towards luteolin effect. Over-expression of mutant BRAF (BRAFV600E) in A375 or wild-type BRAF in WM3211 cells reversed the luteolin-induced cell growth inhibition. An in silica pharmacophore mapping revealed that BRAF protein is an excellent target for luteolin. Subsequent protein-ligand docking modeling has revealed that luteolin binds to BRAF through Cys531, a potential redox-sensitive residue that is located in the ATP-binding pocket. Further studies revealed that as a single agent luteolin effectively inhibited melanoma tumor growth in vivo. Conclusion BRAF may be a crucial and direct target for luteolin, which explains the outstanding inhibitory effect of luteolin in multiple cancer types.

Resveratrol analogue, trans-4,4′-dihydroxystilbene (DHS), inhibits melanoma tumor growth and suppresses its metastatic colonization in lungs

The prevalence of melanoma and the lack of effective therapy for metastatic melanoma warrant extensive and systematic evaluations of small molecules in cellular and pre-clinical models. We investigated, herein, the antitumor and anti-metastatic effects of trans-4,4′-dihydroxystilbene (DHS), a natural product present in bark of Yucca periculosa, using in vitro and in vivo melanoma murine models. DHS showed potent melanoma cytotoxicity, as determined by MTT and clonogenic assay. Further, DHS induced cytotoxicity was mediated through apoptosis, which was assessed by annexin V-FITC/PI, sub-G1 and caspase activation assays. In addition, DHS inhibited cell proliferation by inducing robust cell cycle arrest in G1-phase. Imperatively, these inhibitory effects led to a significant reduction of melanoma tumor in pre-clinical murine model. DHS also inhibited cell migration and invasion of melanoma cells, which were examined using wound healing and Transwell migration/invasion assays. Mechanistically, DHS modulated the expressions of several key metastasis regulating proteins e.g., MMP-2/9, N-cadherin, E-cadherin and survivin. We also showed the anti-metastatic effect of DHS in a melanoma mediated lung metastasis model in vivo. DHS significantly reduced large melanoma nodule formation in the parenchyma of lungs. Therefore, DHS may represent a promising natural drug in the repertoire of treatment against melanoma tumor growth and metastasis.

The HU177 Collagen Epitope Controls Melanoma Cell Migration and Experimental Metastasis by a CDK5/YAP-Dependent Mechanism

Stromal components not only help form the structure of neoplasms such as melanomas, but they also functionally contribute to their malignant phenotype. Thus, uncovering signaling pathways that integrate the behavior of both tumor and stromal cells may provide unique opportunities for the development of more effective strategies to control tumor progression. In this regard, extracellular matrix-mediated signaling plays a role in coordinating the behavior of both tumor and stromal cells. Here, evidence is provided that targeting a cryptic region of the extracellular matrix protein collagen (HU177 epitope) inhibits melanoma tumor growth and metastasis and reduces angiogenesis and the accumulation of α-SMA-expressing stromal cell in these tumors. The current study suggests that the ability of the HU177 epitope to control melanoma cell migration and metastasis depends on the transcriptional coactivator Yes-associated protein (YAP). Melanoma cell interactions with the HU177 epitope promoted nuclear accumulation of YAP by a cyclin-dependent kinase-5-associated mechanism. These findings provide new insights into the mechanism by which the anti-HU177 antibody inhibits metastasis, and uncovers an unknown signaling pathway by which the HU177 epitope selectively reprograms melanoma cells by regulating nuclear localization of YAP. This study helps to define a potential new therapeutic strategy to control melanoma tumor growth and metastasis that might be used alone or in combination with other therapeutics.

Synthesis and biological evaluation of indole-based UC-112 analogs as potent and selective survivin inhibitors

The anti-apoptotic protein survivin is highly expressed in cancer cells but has a very low expression in fully differentiated adult cells. Overexpression of survivin is positively correlated with cancer cell resistance to chemotherapy and radiotherapy, cancer cell metastasis, and poor patient prognosis. Therefore, selective targeting survivin represents an attractive strategy for the development of anticancer therapeutics. Herein, we reported the extensive structural modification of our recently discovered selective survivin inhibitor UC-112 and the synthesis of thirty-three new analogs. The structure-activity relationship (SAR) study indicated that replacement of the benzyloxy moeity in UC-112 with an indole moiety was preferred to other moieties. Among these UC-112 analogs, 10f, 10h, 10k, 10n showed the most potent antiproliferative activities. Interestingly, they were more potent against the P-glycoprotein overexpressing cancer cell lines compared with the parental cancer cell lines. Mechanistic studies confirmed that new analogs maintained their unique selectivity against survivin among the IAP family members. In vivo study using 10f in a human A375 melanoma xenograft model revealed that it effectively inhibited melanoma tumor growth without observable acute toxicity. Collectively, this study strongly supports the further preclinical development of selective survivin inhibitors based on the UC-112 scaffold.

An indoleamine 2, 3-dioxygenase siRNA nanoparticle-coated and Trp2-displayed recombinant yeast vaccine inhibits melanoma tumor growth in mice

Therapeutic vaccine is a promising approach in cancer therapy. But tumor-associated antigen peptides have weak immunogenicity and cancer patients are often characterized by immunosuppression and tolerance, leading to less efficiency of immunotherapy. We here successfully developed indoleamine 2, 3-dioxygenase (IDO) siRNA nanoparticle-coated and tyrosinase-related protein 2 (Trp2)-displayed recombinant Saccharomyces cerevisiae (YCP). YCPs had positive charges with a diameter of approximately 5μm, resulting in selective phagocytosis by APC cells. YCP-delivered siRNA and Trp2 successfully escaped from phagosomes, efficiently inhibited IDO expression in DCs, promoted the immune reaction of T cell against Trp2, increased the secretion of proinflammatory cytokines such as IFN-γ,TNF-α, and IL-6, and decreased the generation of regulatory T cells. Moreover, YCPs significantly inhibited melanoma tumor growth by alleviating immune tolerance and promoting Trp2-specific CD8+ T cell immune response. These results suggest that Saccharomyces cerevisiae as a combined immunotherapeutic platform to simultaneously delivery IDO-siRNA and Trp2 epitope peptide is a promising vaccine system for melanoma treatment.

Comparison of fusion protein and DC vaccine in inhibition of mouse B16F10 melanoma tumor

Dendritic cell (DC) vaccine and fusion protein vaccine have been put into clinical use in cancer immunotherapy. This study compared DC vaccine and fusion protein vaccine directly in their capability of inducing specific immune response. We used mouse Granulocyte Macrophage-Colony Stimulating Factor (mGM-CSF) fused with gastrin-releasing peptide (GRP) and Gonadotrophin Releasing Hormone (GnRH) respectively to obtain mGM-CSF/GRP6 (mG6) and mGM-CSF/mGGn (mGGn) fusion proteins. We prepared fusion protein vaccine and DC vaccine including mG6 protein vaccine (6P), mGGn protein vaccine (nP), mG6 DC vaccine (6D) and mGGn DC vaccine (nD), then the two proteins were mixed to prepare combination proteins vaccine (6nP) and DC vaccine (6nD). After that, C57BL/6 mice were injected with B16F10 cell line to build melanoma tumor model, and were immunized with vaccines to produce antibodies to inhibit and destruct melanoma tumor cells. The discoveries showed that anti- mGM-CSF-GRP6 and anti- mGM-CSF-mGGn antibody vaccines were successfully created as expected; this was deduced from significant inhibition of melanoma tumor in vivo and significant reduction of tumor weight and volume. The effects of DC groups were better than that of the protein groups and the combination of vaccines were more effective than vaccine given separately. Our results indicate that using combination vaccine provides a new strategy to inhibit melanoma tumor growth but a complete cure of melanoma needs further investigations.

In vivo knockdown of antisense non‐coding mitochondrial RNAs by a lentiviral‐encoded shRNA inhibits melanoma tumor growth and lung colonization

The family of non-coding mitochondrial RNAs (ncmtRNA) is differentially expressed according to proliferative status. Normal proliferating cells express sense (SncmtRNA) and antisense ncmtRNAs (ASncmtRNAs), whereas tumor cells express SncmtRNA and downregulate ASncmtRNAs. Knockdown of ASncmtRNAs with oligonucleotides induces apoptotic cell death of tumor cells, leaving normal cells unaffected, suggesting a potential application for developing a novel cancer therapy. In this study, we knocked down the ASncmtRNAs in melanoma cell lines with a lentiviral-encoded shRNA approach. Transduction with lentiviral constructs targeted to the ASncmtRNAs induced apoptosis in murine B16F10 and human A375 melanoma cells in vitro and significantly retarded B16F10 primary tumor growth in vivo. Moreover, the treatment drastically reduced the number of lung metastatic foci in a tail vein injection assay, compared to controls. These results provide additional proof of concept to the knockdown of ncmtRNAs for cancer therapy and validate lentiviral-shRNA vectors for gene therapy.

Synthesis and Pharmacological Evaluation of Selective Histone Deacetylase 6 Inhibitors in Melanoma Models.

Only a handful of therapies offer significant improvement in the overall survival in cases of melanoma, a cancer whose incidence has continued to rise in the past 30 years. In our effort to identify potent and isoform-selective histone deacetylase (HDAC) inhibitors as a therapeutic approach to melanoma, a series of new HDAC6 inhibitors based on the nexturastat A scaffold were prepared. The new analogues 4d, 4e, and 7b bearing added hydrophilic substituents, so as to establish additional hydrogen bonding on the rim of the HDAC6 catalytic pocket, exhibit improved potency against HDAC6 and retain selectivity over HDAC1. Compound 4d exhibits antiproliferative effects on several types of melanoma and lymphoma cells. Further studies indicates that 4d selectively increases acetylated tubulin levels in vitro and elicits an immune response through down-regulating cytokine IL-10. A preliminary in vivo efficacy study indicates that 4d possesses improved capability to inhibit melanoma tumor growth and that this effect is based on the regulation of inflammatory and immune responses.

Abstract 4976: Ricolinostat, a selective HDAC6 inhibitor with immunomodulatory properties, has significant antimelanoma activity in vitro and in vivo

Abstract Melanoma is a less common but the most aggressive type of skin cancer and the rates of melanoma have been rising for the last 30 years. Immunotherapy may represent a new treatment paradigm to benefit melanoma patients. Previous studies have identified HDAC6 playing a central role in the regulating melanoma immunogenicity. We have shown that genetic knockdown (KD) of HDAC6 in murine and human melanoma cells resulted in an increased the expression of MHCI and costimulatory molecules as well as melanoma associated antigens. In vivo studies demonstrated that the growth of melanoma cells lacking HDAC6 was significantly delayed as compared to wild-type cells. This growth was further delayed when HDAC6KD melanoma bearing mice were treated with anti-PD-1 antibodies. Recently, we have shown that in vitro treatment of murine melanoma cells with Ricolinostat decreases PD-L1 expression and increases the expression of acetyl tubulin. Surprisingly, in vivo treatment of B16 melanoma bearing animals with increasing concentrations of ricolinostat resulted in a dose-dependent inhibition of melanoma growth (p<0.05). No toxicities were observed at the doses studied; however, the antitumor effect exerted by ricolinostat was not observed when melanoma bearing SCID mice were treated with this compound, indicating that an intact host immune system is required for the observed antitumor activity. In lieu of these previously unknown immunomodulatory properties of ricolinostat, we next assessed the effects of this compound upon T-cells and whether ricolinostat could augment the efficacy of checkpoint blockade in vivo. Murine T-cells were activated with anti-CD3 plus anti-CD28 in the presence or absence of ricolinostat. Then, those T-cells were adoptively transferred into B16 melanoma bearing mice. T-cell subpopulations from the lymph nodes were analyzed ex vivo. We show here that treatment of murine T-cells with ricolinostat resulted in a significant increase in central memory T-cells endowed with a strong anti-melanoma activity in vivo as compared to control group (p<0.05). Finally, the addition of ricolinostat treatment to either anti-CTLA4 or anti-PD1 treatment was associated with an enhanced inhibition of melanoma tumor growth. In summary, our results have identified HDAC6 as a novel target for melanoma immunotherapy and point out ricolinostat as an attractive agent to add to the immuno-oncology armamentarium. Citation Format: Fengdong Cheng, Sodre Andressa, Jie Chen, Alejandro Villagra, David Woods, Jeffrey Weber, Steven Quayle, Jones Simon, Eduardo Sotomayor. Ricolinostat, a selective HDAC6 inhibitor with immunomodulatory properties, has significant antimelanoma activity in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4976. doi:10.1158/1538-7445.AM2017-4976

Disease-associated extracellular loop mutations in the adhesion G protein-coupled receptor G1 (ADGRG1; GPR56) differentially regulate downstream signaling

Mutations to the adhesion G protein-coupled receptor ADGRG1 (G1; also known as GPR56) underlie the neurological disorder bilateral frontoparietal polymicrogyria. Disease-associated mutations in G1 studied to date are believed to induce complete loss of receptor function through disruption of either receptor trafficking or signaling activity. Given that N-terminal truncation of G1 and other adhesion G protein-coupled receptors has been shown to significantly increase the receptors' constitutive signaling, we examined two different bilateral frontoparietal polymicrogyria-inducing extracellular loop mutations (R565W and L640R) in the context of both full-length and N-terminally truncated (ΔNT) G1. Interestingly, we found that these mutations reduced surface expression of full-length G1 but not G1-ΔNT in HEK-293 cells. Moreover, the mutations ablated receptor-mediated activation of serum response factor luciferase, a classic measure of Gα12/13-mediated signaling, but had no effect on G1-mediated signaling to nuclear factor of activated T cells (NFAT) luciferase. Given these differential signaling results, we sought to further elucidate the pathway by which G1 can activate NFAT luciferase. We found no evidence that ΔNT activation of NFAT is dependent on Gαq/11-mediated or β-arrestin-mediated signaling but rather involves liberation of Gβγ subunits and activation of calcium channels. These findings reveal that disease-associated mutations to the extracellular loops of G1 differentially alter receptor trafficking, depending on the presence of the N terminus, and differentially alter signaling to distinct downstream pathways.

Aspirin attenuates melanoma tumor growth via PGF2α‐SOX‐2 dependent pathways

IntroductionExposure to pro‐oxidative stressors including cancer therapies via its ability to generate reactive oxygen species (ROS) produce potent oxidized lipid mediator, platelet‐activating factor (PAF) and PAF‐like agonists. We have shown that these PAF‐agonists significantly: 1) augmented the growth of subcutaneously implanted murine B16F10 melanoma tumors in a PAF‐receptor (PAF‐R) dependent manner; 2) impeded the efficacy of cancer chemotherapeutic agents and radiation therapy in murine melanoma models and; 3) detected in the perfusates and tumors collected from chemotherapy and radiation therapy‐treated melanoma and non‐melanoma cancer patients. As melanoma tumors often acquire resistance to the known chemotherapy, radiation therapy and even novel targeted therapeutic regimens, it is necessary to investigate an alternative approach for its intervention. Aspirin (ASN) has classically been used as an anti‐inflammatory drug and also possesses anti‐cancer activity in‐part via its ability to inhibit enzyme cyclooxygenases. While anti‐carcinogenic activity of ASN has been shown in other cancers, little is known about its effects in melanoma.ObjectivesThe current studies were sought to investigate the mechanism and test the hypothesis if ASN‐mediated anti‐melanoma activity is via inhibition of PAF‐R activation.MethodologyCell proliferation and survival assay, apoptosis by caspase 3/7 luminescence assay, immunoblotting, gene array, quantitative real time PCR and flow cytometry studies were performed. These assays were conducted in stably PAF‐R‐expressing (B16‐PAFR) and deficient melanoma (B16‐MSCV) cells and C57BL/6‐wild‐type (WT) and PAF‐R knockout (PAFR−/−) mice.Results and DiscussionWe demonstrate that ASN inhibits the survival of B16‐PAFR and B16‐MSCV cells at similar rates in a time and dose dependent manner via inducing apoptosis as measured increased caspase 3/7 activity. These ASN‐mediated effects were blocked by PGF2α addition but not by PGE2. Importantly, ASN‐mediated significant suppression of murine B16‐PAFR and B16‐MSCV melanoma tumor growth was independent to both cellular and stromal PAF‐R expression. Moreover, gene array analysis of melanoma tumors revealed an inhibition of SOX‐2 oncogene by ASN. Furthermore, PGF2α add back upregulated SOX‐2 and rescued melanoma cells from ASN‐mediated inhibition of cell survival and induction of apoptosis.ConclusionASN targets PGF2α and downstream SOX‐2 to inhibit melanoma tumor growth in a PAF‐R independent pathway and provide the rationale for its implication as a novel melanoma therapy.Support or Funding InformationNIH K22 ES023850 and WSU‐RIF 190002

Noncalcemic 20-hydroxyvitamin D3 inhibits human melanoma growth in in vitro and in vivo models.

A novel pathway of vitamin D3 (D3) metabolism, initiated by C20-hydroxylation of D3 by CYP11A1, has been confirmed to operate in vivo. Its major product, 20(OH)D3, exhibits antiproliferative activity in vitro comparable to that of 1,25(OH)2D3, but is noncalcemic in mice and rats. To further characterize the antimelanoma activity of 20(OH)D3, we tested its effect on colony formation of human melanoma cells in monolayer culture and anchorage-independent growth in soft agar. The migratory capabilities of the cells and cell-cell and cell-extracellular matrix interactions were also evaluated using transwell cell migration and spheroid toxicity assays. To assess the antimelanoma activity of 20(OH)D3 in vivo, age-matched immunocompromised mice were subcutaneously implanted with luciferase-labelled SKMel-188 cells and were randomly assigned to be treated with either 20(OH)D3 or vehicle (n=10 per group). Tumor size was measured with caliper and live bioimaging methods, and overall health condition expressed as a total body score scale. The following results were observed: (i) 20(OH)D3 inhibited colony formation both in monolayer and soft agar conditions, (ii) 20(OH)D3 inhibited melanoma cells in both transwell migration and spheroid toxicity assays, and (iii) 20(OH)D3 inhibited melanoma tumor growth in immunocompromised mice without visible signs of toxicity. However, although the survival rate was 90% in both groups, the total body score was higher in the treatment group compared to control group (2.8 vs. 2.55). In conclusion, 20(OH)D3, an endogenously produced secosteroid, is an excellent candidate for further preclinical testing as an antimelanoma agent.

IL2 Variant Circumvents ICOS+ Regulatory T-cell Expansion and Promotes NK Cell Activation.

Clinical responses to high-dose IL2 therapy are limited due to selective expansion of CD4+CD25+Foxp3+ T-regulatory cells (Treg), especially ICOS+ Tregs, rather than natural killer (NK) cells and effector T cells. These ICOS+ Tregs are highly suppressive and constitutively express high levels of IL2Rα (CD25) and CD39. Here, we characterized the effect of a mutant form of IL2 (F42K), which preferentially binds to the lower affinity IL2Rβγ with reduced binding to CD25, on Tregs, effector NK cells, and T-cell subsets. Unlike wild-type (WT) IL2, F42K did not efficiently induce the expansion of highly suppressive ICOS+ Tregs in peripheral blood mononuclear cells (PBMC) from healthy controls and melanoma patients. Instead, it promoted the expansion of CD16+CD56+ NK cells and CD56hiCD16- NK cell subsets in both short- and long-term cultures, with enhanced Bcl-2 expression. Stimulation of PBMCs with F42K induced expression of more NK cell activation molecules, such as NKp30, NKp44, DNAM-1, NKG2D, 4-1BB/CD137, and Tim-3, than WT IL2. F42K induced greater upregulation of TRAIL, and NK-mediated cytolytic activity was increased against both autologous and HLA-mismatched melanoma cells compared with WT IL2. Gene expression analysis revealed distinct gene expression profiles stimulated by F42K, WT IL2, and IL15. F42K therapy in vivo also induced a dramatic reduction in the expansion of ICOS+ Tregs, promoted NK cell expansion, and inhibited melanoma tumor growth more efficiently than WT IL2 and more effectively than anti-CTLA-4. Our findings suggest that F42K could be a potential substitute for WT IL2 as a cytokine therapy for cancer. Cancer Immunol Res; 4(11); 983-94. ©2016 AACR.

Peroxisome proliferator-activated receptor alpha deficiency impairs regulatory T cell functions: Possible application in the inhibition of melanoma tumor growth in mice.

Regulatory T (Treg) cells are important to induce and maintain immunological self-tolerance. Although the progress accomplished in understanding the functional mechanism of Treg cells, intracellular molecules that control the mechanisms of their suppressive capacity are still on investigation. The present study showed that peroxisome proliferator-activated receptor-alpha deficiency impaired the suppressive activity of Treg cells on CD4+CD25- and CD8+ T cell proliferation. In Treg cells, PPARα gene deletion also induced a decrease of migratory abilities, and downregulated the expression of chemokine receptors (CCR-4, CCR-8 and CXCR-4) and p27KIP1 mRNA. Treg cells from PPARα-/- mice also lost their anergic property. Since low Treg activity, as observed in PPARα-/- mice, is known to be associated with the inhibition of tumor growth, we inoculated these mice with B16 melanoma cells and assessed tumor proliferation. In PPARα-/- mice, cancer growth was significantly curtailed, and it was correlated with high expression of granzyme B and perforin mRNA in tumor bed. Degranulation of cytolytic molecules by CD8+ T cells, assessed by a perforin-release marker CD107a expression, was higher in PPARα-/- mice than that in wild-type mice. Tumor-infiltrating lymphocytes (TIL) in melanoma tumors in PPARα-/- mice exhibited high pro-inflammatory Th1 phenotype. Consistently, adoptive transfer into lymphopenic RAG2-/- mice of total PPARα-/-splenic T cells inhibited more the growth rate of B16 tumor than the wild type splenic T cells. Our findings suggest that PPARα deficiency, by diminishing Treg cell functions and upregulating pro-inflammatory T cell phenotype, exerts an invivo anti-cancer properties.

668. Platelets Engineered to Express Interleukin-24 Inhibited Melanoma Tumor Growth in Mice

Background: Activated platelets secrete agents (e.g., cytokines) that can promote solid tumor growth & cancer metastasis. Previous studies showed that hematopoietic stem cells (HSC) can be genetically modified to induce platelets to express & secrete proteins to establish hemostasis in animal models of bleeding disorders. Interleukin-24 (IL-24) is a cytokine (normally produced by activated monocytes, macrophages & Thelper cells) with cytotoxic & anti-angiogenic activity preferentially towards cancer cells. Thus, we hypothesized it may be feasible use HSC gene transfer to target platelet synthesis, storage & secretion of IL-24 to inhibit tumor growth. Aims: To investigate if HSC gene transfer targeting IL-24 synthesis, storage & secretion from platelets can inhibit melanoma in mice. Methods: C57BlL/6 mice were transplanted with bone marrow transduced with a lentiviral construct encoding a megakaryocytespecific ITGA2B gene promoter driving synthesis of IL-24 gene. Four weeks after transplant, IL-24 protein was characterized in platelets by flow cytometry. Murine melanoma cells (1×106) were implanted in mice at 5 weeks after transplant. Tumor size was measured in IL24 & control groups using a digital caliper periodically for 4 weeks. Then mice were sacrificed to record tumor mass. Results: Flow cytometry showed that HSC gene transfer of murine bone marrow led to synthesis & storage of IL-24 in platelets. An elisa assay showed that activated platelets could secrete IL-24 in vitro. At 30 days after implant of melanoma cells, platelet IL24 mice displayed significantly smaller (≈50%) tumor size (550 ± 101mm3) & weight (599 ± 120 mg) compared to control mice with larger tumor size (1120 ± 114 mm3) & weight (1391 ± 134 mg) n≈5 mice/group (p<0.05) indicating that platelet IL24 inhibited tumor growth in vivo. Conclusion: HSC gene transfer can be utilized to induce synthesis, storage and secretion of anti-oncogenic agent IL24 in platelets. Melanoma tumor challenge in mice with platelet IL24 showed a significant decrease in tumor growth suggesting that platelets may serve as an ideal therapeutic vehicle to treat cancer.

Antitumor and Antimetastatic Effect of Small Immunostimulatory RNA against B16 Melanoma in Mice

Small interfering RNAs, depending on their structure, delivery system and sequence, can stimulate innate and adaptive immunity. The aim of this study was to investigate the antitumor and antimetastatic effects of immunostimulatory 19-bp dsRNA with 3’- trinucleotide overhangs (isRNA) on melanoma B16 in C57Bl/6 mice. Recently developed novel cationic liposomes 2X3-DOPE were used for the in vivo delivery of isRNA. Administration of isRNA/2X3-DOPE complexes significantly inhibits melanoma tumor growth and metastasis. Histopathological analysis of spleen cross sections showed hyperplasia of the lymphoid white pulp and formation of large germinal centers after isRNA/2X3-DOPE administration, indicating activation of the immune system. The treatment of melanoma-bearing mice with isRNA/2X3-DOPE decreases the destructive changes in the liver parenchyma. Thus, the developed isRNA displays pronounced immunostimulatory, antitumor and antimetastatic properties against melanoma B16 and may be considered a potential agent in the immunotherapy of melanoma.

Epigallocatechin-3-O-gallate up-regulates microRNA-let-7b expression by activating 67-kDa laminin receptor signaling in melanoma cells.

MicroRNAs (miRNAs) are non-coding RNAs involved in various biological processes by regulating their target genes. Green tea polyphenol (−)-epigallocatechin-3-O-gallate (EGCG) inhibits melanoma tumor growth by activating 67-kDa laminin receptor (67LR) signaling. To examine the effect of EGCG on miRNA expression in melanoma cells, we performed miRNA microarray analysis. We showed that EGCG up-regulated miRNA-let-7b expression through 67LR in melanoma cells. The EGCG-induced up-regulation of let-7b led to down-regulation of high mobility group A2 (HMGA2), a target gene related to tumor progression. 67LR-dependent cAMP/protein kinase A (PKA)/protein phosphatase 2A (PP2A) signaling pathway activation was involved in the up-regulation of let-7b expression induced by EGCG. These findings provide a basis for understanding the mechanism of miRNA regulation by EGCG.

7-Hydroxydehydronuciferine induces human melanoma death via triggering autophagy and apoptosis.

Melanoma is the deadliest cancer. We identified 7-hydroxydehydronuciferine (7-HDNF) isolated from the leaves of Nelumbo nucifera Gaertn cv. Rosa-plena to be a bio-active agent that antagonizes melanoma tumor growth in mice xenograft model in vivo. Cell proliferation assay demonstrated strong anticancer effects of 7-HDNF to exhibit a dose-dependent behaviour and displayed minor cytotoxicities on normal human skin cells, including epidermal keratinocytes and melanocytes, and dermal fibroblasts. With acridine orange (AO) staining and flow analysis, we found 7-HDNF induced the formation of intracellular vacuoles and the augmentation of acidic vesicular organelles (AVO). The apoptotic cell death ratio was measured via two-dimensional flow cytometry by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double stained to confirm the cellular membrane asymmetry lost. One-dimensional flow cytometric analysis showed 7-HDNF increased the cellular arrest in cell cycle at the G2/M phase. Through Western blot examinations, protein expressions were discovered to verify autophagy and apoptosis response mechanisms sharing the associated pathways. Finally, 7-HDNF presented a high-quality antimigratory activity in wound-healing assay. Overall, 7-HDNF presented high-quality anticancer bio-functions and inhibited melanoma tumor growth in vivo and in vitro.