Human Melanoma Growth Research Articles

Retargeting NK‐92 for anti‐melanoma activity by a TCR‐like single‐domain antibody

The efficacy of immunotherapy based on natural killer (NK) cells is hampered by intrinsic non-specific cytotoxicity and insufficient activation of NK cells. Here, we confer the T-cell receptor-like (TCR-like) specificity on NK cells, taking advantage of both the innate and adaptive immune arms of the immune response to generate enhanced anti-melanoma activity. The TCR-like antibody (Ab) GPA7 was selected against melanoma-associated gp100/human leukocyte antigen (HLA)-A2 complex and then fused to intracellular domain of CD3-ζ chain. This fusion construct was incorporated into NK-92MI cell line and expressed as a chimeric antigen receptor on the surface of the cell. The anti-tumour activity of the transgenic NK-92MI-GPA7-ζ cell line was assessed against melanoma in vitro and in vivo. The engineered NK-92MI-GPA7-ζ cells could recognize melanoma cells in the context of HLA-A2 and showed enhanced killing of both melanoma cell lines and primary melanoma. Furthermore, adoptively transferred NK-92MI-GPA7-ζ cells significantly suppressed the growth of human melanoma in a xenograft model in mice. Collectively, these results demonstrate that the TCR-like Ab, GPA7, could redirect NK cells to target the intracellular antigen gp100 and enhance anti-melanoma activity, providing a promising immunotherapeutic strategy to prevent and treat melanoma.

Selective Inhibition of p300 HAT Blocks Cell Cycle Progression, Induces Cellular Senescence, and Inhibits the DNA Damage Response in Melanoma Cells

Epigenetic events, including covalent post-translational modifications of histones, have been demonstrated to play critical roles in tumor development and progression. The transcriptional coactivator p300/CBP possesses both histone acetyltransferase (HAT) activity as well as scaffolding properties that directly influence the transcriptional activation of targeted genes. We have used a potent and specific inhibitor of p300/CBP HAT activity, C646, in order to evaluate the functional contributions of p300/CBP HAT to tumor development and progression. Here we report that C646 inhibits the growth of human melanoma and other tumor cells and promotes cellular senescence. Global assessment of the p300 HAT transcriptome in human melanoma identified functional roles in promoting cell cycle progression, chromatin assembly and activation of DNA repair pathways through direct transcriptional regulatory mechanisms. Additionally, C646 is shown to promote sensitivity to DNA damaging agents, leading to the enhanced apoptosis of melanoma cells following combination treatment with cisplatin. Together, our data suggest that p300 HAT activity mediates critical growth regulatory pathways in tumor cells and may serve as a potential therapeutic target for melanoma and other malignancies by promoting cellular responses to DNA damaging agents that are currently ineffective against specific cancers.

Variant G6PD levels promote tumor cell proliferation or apoptosis via the STAT3/5 pathway in the human melanoma xenograft mouse model

BackgroundGlucose-6-phosphate dehydrogenase (G6PD), elevated in tumor cells, catalyzes the first reaction in the pentose-phosphate pathway. The regulation mechanism of G6PD and pathological change in human melanoma growth remains unknown.MethodsHEM (human epidermal melanocyte) cells and human melanoma cells with the wild-type G6PD gene (A375-WT), G6PD deficiency (A375-G6PD∆), G6PD cDNA overexpression (A375-G6PD∆-G6PD-WT), and mutant G6PD cDNA (A375-G6PD∆-G6PD-G487A) were subcutaneously injected into 5 groups of nude mice. Expressions of G6PD, STAT3, STAT5, cell cycle-related proteins, and apoptotic proteins as well as mechanistic exploration of STAT3/STAT5 were determined by quantitative real-time PCR (qRT-PCR), immunohistochemistry and western blot.ResultsDelayed formation and slowed growth were apparent in A375-G6PD∆ cells, compared to A375-WT cells. Significantly decreased G6PD expression and activity were observed in tumor tissues induced by A375-G6PD∆, along with down-regulated cell cycle proteins cyclin D1, cyclin E, p53, and S100A4. Apoptosis-inhibited factors Bcl-2 and Bcl-xl were up-regulated; however, apoptosis factor Fas was down-regulated, compared to A375-WT cells. Moderate protein expressions were observed in A375-G6PD∆-G6PD-WT and A375-G6PD∆-G6PD-G487A cells.ConclusionsG6PD may regulate apoptosis and expression of cell cycle-related proteins through phosphorylation of transcription factors STAT3 and STAT5, thus mediating formation and growth of human melanoma cells. Further study will, however, be required to determine potential clinical applications.

Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth

Here, we show that forced expression of a B-cell lymphoma 2 (bcl-2) protein lacking residues 1 to 36 at the N-terminal, including the entire Bcl-2 homology 4 (BH4) domain, determines reduction of in vitro and in vivo human melanoma growth. Noteworthy, melanoma cells in vivo exhibit markedly increased autophagy, as response to expression of bcl-2 protein deleted of its BH4 domain. This observation led to the identification of a novel gain of function for bcl-2 protein lacking the BH4 domain. In particular, upon different autophagic stimuli in vitro, overexpression of bcl-2 protein deleted of BH4 domain induces autophagosome accumulation, conversion of microtubule-associated protein 1 light chain 3B-II, reduced expression of p62/SQSTM1 protein, and thereby enhanced autophagic flux. The relevance of Beclin-1 is evidenced by the fact that 1) the autophagy-promoting and growth-inhibiting properties are partially rescued by Beclin-1 knockdown in cells expressing bcl-2 protein lacking the BH4 domain, 2) Beclin-1 only interacts with wild-type but not with deleted bcl-2, and 3) BH4 domain removal from bcl-2 protein does not influence in vitro and in vivo growth of tumor cells expressing low levels of endogenous Beclin-1. These results provide new insight into molecular mechanism of bcl-2 functions and represent a rationale for the development of agents interfering with the BH4 domain of bcl-2 protein.

The critical role of mast cell‐derived hypoxia‐inducible factor‐1α in human and mice melanoma growth

Mast cells play an important role in tumorigenesis. Histamine released from mast cells stimulates new vessel formation by acting through the histamine1 (H1) receptor. Despite the evidence of the role of mast cells in tumor growth and angiogenesis, the potential mechanism remains to be elucidated. Therefore, we investigated the role of mast cell-derived HIF-1α in melanoma growth. Here, we identify that the most positive cells for HIF-1α staining are seen in mast cells of human and animal melanoma tissue. The number of the stromal cell types (fibroblasts, macrophages and endothelial cells) was also increased in melanoma tissues. In activated bone marrow-derived mast cells (BMMCs), expressions of HIF-1α and VEGF were increased. Histamine also induced the expressions of HIF-1α and VEGF in BMMCs. H1 receptor antagonists significantly improved overall survival rates and substantially suppressed tumor growth as well as the infiltration of mast cells and levels of VEGF through the inhibition of HIF-1α expression in B16F10 melanoma-bearing mice. Furthermore, the injection of HIF-1α depleted BMMCs markedly inhibited the growth of tumors and migration of mast cells and increased the survival rate of the mice. These findings emphasize that the growth of melanoma can actually be exacerbated by mast cell-derived HIF-1α. In aggregate, our results reveal a novel role for mast cell-derived HIF-1α in the melanoma microenvironment and have important implications for the design of therapeutic strategies.

Blocking glutamate‐mediated signalling inhibits human melanoma growth and migration

Glutamate is an excitatory neurotransmitter that has been shown to regulate the proliferation, migration and survival of neuronal progenitors in the central nervous system through its action on metabotropic and ionotropic glutamate receptors (GluRs). Antagonists of ionotropic GluRs have been shown to cause a rapid and reversible change in melanocyte dendritic morphology, which is associated with the disorganization of actin and tubulin microfilaments in the cytoskeleton. Intracellular expression of microtubule-associated protein (MAP) 2a affects the assembly, stabilization and bundling of microtubules in melanoma cells; stimulates the development of dendrites; and suppresses melanoma cell migration and invasion. In this study, we investigated the relationship between glutamate-mediated signalling and microtubules, cell dendritic morphology and melanoma cell motility. We found that metabotropic GluR1 and N-methyl-d-aspartate receptor antagonists increased dendritic branching and inhibited the motility, migration and proliferation of melanoma cells. We also demonstrated that the invasion and motility of melanoma cells are significantly inhibited by the combination of increased expression of MAP2a and either metabotropic GluR1 or N-methyl-d-aspartate receptor antagonists. Moreover, the blockade of glutamate receptors inhibited melanoma growth in vivo. Collectively, these results demonstrate the importance of glutamate signalling in human melanoma and suggest that the blockade of glutamate receptors is a promising novel therapy for treating melanoma.

Merlin Is a Negative Regulator of Human Melanoma Growth

Merlin is encoded by the neurofibromatosis type 2 (NF2) gene and is a member of the Band 4.1 protein family. This protein acts as a linker that connects cell surface proteins to the actin cytoskeleton. Defects caused by mutations of the NF2 gene give rise to NF2 disease, which is generally characterized by the formation of bilateral vestibular schwannomas and, to a lesser extent, meningiomas and ependymomas. In addition to these tumor types, NF2 is mutated and/or merlin expression is reduced or lost in numerous non-NF2 associated tumors, including melanoma. However, the role of merlin in human melanoma growth and the mechanism underlying its effect are currently unknown. In the present study, we show that merlin knockdown enhances melanoma cell proliferation, migration, and invasion in vitro and that decreased merlin expression promotes subcutaneous melanoma growth in immunocompromised mice. Concordantly, we find that increased expression of merlin in a metastatic melanoma cell line reduced their in vitro migration and proliferation, and diminished their ability to grow in an anchorage independent manner. Increased merlin expression also inhibits in vivo growth of these melanoma cells. Lastly, we demonstrate that higher merlin levels in human melanoma cells promote the H2O2-induced activation of MST1/2 Ser/Thr kinases, which are known tumor suppressors in the Hippo signaling pathway. Taken together, these results provide for the first time evidence that merlin negatively regulates human melanoma growth, and that loss of merlin, or impaired merlin function, results in an opposite effect. In addition, we show that increased merlin expression leads to enhanced activation of the MTS1/2 kinases, implying the potential roles of MST1/2 in mediating the anti-melanoma effects of merlin.

A Coreceptor-Independent Transgenic Human TCR Mediates Anti-Tumor and Anti-Self Immunity in Mice

Recent advancements in T cell immunotherapy suggest that T cells engineered with high-affinity TCR can offer better tumor regression. However, whether a high-affinity TCR alone is sufficient to control tumor growth, or the T cell subset bearing the TCR is also important remains unclear. Using the human tyrosinase epitope-reactive, CD8-independent, high-affinity TCR isolated from MHC class I-restricted CD4(+) T cells obtained from tumor-infiltrating lymphocytes (TIL) of a metastatic melanoma patient, we developed a novel TCR transgenic mouse with a C57BL/6 background. This HLA-A2-restricted TCR was positively selected on both CD4(+) and CD8(+) single-positive cells. However, when the TCR transgenic mouse was developed with a HLA-A2 background, the transgenic TCR was primarily expressed by CD3(+)CD4(-)CD8(-) double-negative T cells. TIL 1383I TCR transgenic CD4(+), CD8(+), and CD4(-)CD8(-) T cells were functional and retained the ability to control tumor growth without the need for vaccination or cytokine support in vivo. Furthermore, the HLA-A2(+)/human tyrosinase TCR double-transgenic mice developed spontaneous hair depigmentation and had visual defects that progressed with age. Our data show that the expression of the high-affinity TIL 1383I TCR alone in CD3(+) T cells is sufficient to control the growth of murine and human melanoma, and the presence or absence of CD4 and CD8 coreceptors had little effect on its functional capacity.

Abstract 4730: Inhibition of the p53-HDMX interaction sensitizes melanoma to chemotherapy

Abstract Melanoma is the leading cause of death from skin disease worldwide. It is an aggressive cancer that is often resistant to conventional chemotherapy and radiation-based therapy. The combination of a low survival rate in metastatic disease and lack of successful treatment options underscores the need to elucidate novel methods to target melanoma. More than 95% of human melanomas retain wild type p53, the protein responsible for maintaining genomic integrity; and HDMX, a negative regulator of p53 that neutralizes p53, has recently been found to be overexpressed in 65% of human cutaneous melanoma. These features suggest that targeting the p53-HDMX interaction in melanoma could not only restore the tumor suppressor function of p53, but also sensitize melanoma cells to treatment with conventional chemotherapeutic agents which rely on an intact p53 response. We have previously demonstrated that a hydrocarbon-stapled α-helix based on the transactivation domain of p53 (SAH-p53-8) efficiently disrupts p53-HDM2 and p53-HDMX complexes allowing for p53 to induce apoptotic cell death. Here, we show that SAH-p53-8 treatment is cytotoxic to metastatic melanoma cells by a p53-dependent mechanism. Additionally, SAH-p53-8 sensitizes melanoma to treatment with either cisplatin or the HDM2-specific inhibitor Nutlin-3. Importantly, the inhibition of the p53-HDMX complex arrests the growth of human metastatic melanoma in vivo. The restoration of functional p53 activity provides a basis for a previously unrecognized targeted therapy for metastatic melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4730. doi:1538-7445.AM2012-4730

Abstract LB-310: Changes in the gene expression profile of A375 human melanoma cells induced by over-expression of multifunctional pigment epithelium-derived factor

Abstract Pigment epithelium-derived factor (PEDF) is a broad-spectrum angiogenesis inhibitor that displays potent anti-metastatic activity in multiple tumor types. We have previously shown that PEDF prevents primary tumor growth and metastatic spread of human melanoma in mouse experimental models. Consistent with these observations, PEDF expression is lost at the late stages of melanoma progression, allowing melanoma cells to become angiogenic, migratory and invasive. PEDF's ability to modify the interplay between the host and tumor tissues strongly supports its use as a therapeutic agent for the treatment of metastatic melanoma. However, transition to the clinic requires a more detailed knowledge of the molecular mechanisms underpinning PEDF's activity. In this study we describe changes in the gene expression profile of A375 human melanoma cells induced by PEDF over-expression. PEDF modulated diverse categories of genes known to be involved in angiogenesis and migration. It downregulated cytokines like interleukin 8 and extracellular matrix proteins like collagen IV, while it upregulated fibronectin. Multiple transcripts previously described as contributing to the acquisition of malignant phenotype by melanoma were also diminished by PEDF over-expression, among which we validated galectin 3 and jagged 1. Additionally, PEDF downregulated S100β and melanoma inhibitory activity (MIA), which are widely used in the pathological diagnosis of melanoma. Interestingly, PEDF increased the expression of melanophilin and decreased rab27A, which are relevant targets for melanosome transport; suggesting that PEDF could directly impinge on melanocytic lineage-specific processes. Our study identifies new molecular targets and signaling pathways that may potentially contribute to determine PEDF's ability to restrict the aggressiveness of A375 human melanoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-310. doi:1538-7445.AM2012-LB-310

Abstract 3864: Biological basis of pan-ErbB receptor targeting for malignant melanoma treatment

Abstract Dysregulated activation of the epidermal growth factor receptor (ErbB) family (EGFR, ErBB2, ErbB3, and ErbB4) is involved in promoting the cellular growth of human melanoma. EGFR, ErbB3, and ErbB4 are promising targets of malignant melanoma for new molecular drug development. However, the biology of ErbB family signaling pathways indicates that functional redundancy and heterodimerization among ErbB family members can greatly compromise the therapeutic efficacy of single ErbB targeting tyrosine kinase inhibitors (TKIs). In fact, Phase II clinical trials have already shown that EGFR (ErbB1) targeting TKIs provide limited benefits to melanoma patients. Here, this study has compared single vs. multiple ErbB targeting TKIs for inhibiting the growth of melanoma cells with different ErbB family protein expression profiles. We examined the expression of ErbB family members in 14 different human melanoma cell lines (MEL-526, M14, UPCI-MEL 527.1 and 11 SK-MEL cells). EGFR was expressed in 5 of the cell lines examined (35.7 %). ErbB2 was present in 13 lines (92.9 %); ErbB3 was found in 12 lines (85.7 %); and ErbB4 was found in all the cell lines. Furthermore, individual cell lines had a particular dominant form of ErbB protein. This differential expression pattern of ErbB family proteins was also seen in melanoma tissues from patients. Out of 14 melanoma cases examined by immunohistochemistry, 8 samples (61.5 %) were positive for ErbB1 and ErbB4 expression while ErbB2 was detected in 11 samples (84.6 %). We also found that Neuregulin 3 (which ranged from 500 to 76500 pg/ml) and Neuregulin 4 (which ranged from 26-300 pg/ml), which activate ErbB3 and/or ErbB4, are the major ErbB ligands released by the human melanoma cell lines. TGFβ and Amphiregulin, which mainly bind to EGFR, were released in relatively lower levels by the majority of the cell lines, but high levels of these ligands were distinctly found in individual cell lines. Among all the cell lines examined, HB-EGF was barely undetectable. In addition, different sensitivities towards single ErbB targeting tyrosine kinase inhibitors (TKIs) and pan-ErbB targeting TKIs were seen among the melanoma cells. For example, the TKI sensitivities of MEL526 which expressed no EGFR but high ErbB2, 3, and 4 proteins, was found to be: lapatinib (IC50: 6.7 μM) > canertinib (IC50: 12.4 μM) = erlotinib (IC50: 12.9 μM) > gefitinib (IC50: 23 μM). Similarly, M14 and UPCI-MEL527.1, which also have no EGFR expression but express ErbB 2, 3, 4, also responded better to lapatinib and canertinib respectively than to gefitinib or erlotinib. Our study is the first to suggest that the pan-ErbB targeting approach may be more effective in treatment malignant melanoma. Our findings also suggest that future screening of melanoma patients for their dominantly expressed ErbB protein types can potentially serve as a guide for the choice of effective ErbB TKI in treating malignant melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3864. doi:1538-7445.AM2012-3864

Changes in the gene expression profile of A375 human melanoma cells induced by overexpression of multifunctional pigment epithelium-derived factor

Pigment epithelium-derived factor (PEDF) is a broad-spectrum angiogenesis inhibitor that displays potent antimetastatic activity in multiple tumor types. We have previously shown that PEDF prevents primary tumor growth and metastatic spread of human melanoma in mouse experimental models. Consistent with these observations, PEDF expression is lost at the late stages of melanoma progression, allowing melanoma cells to become angiogenic, migratory, and invasive. PEDF's ability to modify the interplay between the host and tumor tissues strongly supports its use as a therapeutic agent for the treatment of metastatic melanoma. However, transition to the clinic requires a more detailed knowledge of the molecular mechanisms underpinning PEDF's activity. In this study, we describe changes in the gene expression profile of A375 human melanoma cells induced by PEDF overexpression. PEDF modulated diverse categories of genes known to be involved in angiogenesis and migration. It downregulated cytokines such as interleukin-8 and extracellular matrix proteins such as collagen IV, while it upregulated fibronectin. Multiple transcripts previously described as contributing to the acquisition of malignant phenotype by melanoma were also diminished by PEDF overexpression, among which we validated galectin 3 and jagged 1. In addition, PEDF downregulated S100β and melanoma inhibitory activity, which are widely used in the pathological diagnosis of melanoma. Interestingly, PEDF increased the expression of melanophilin and decreased rab27A, which are relevant targets for melanosome transport; suggesting that PEDF could directly impinge on melanocytic lineage-specific processes. Our study identifies new molecular targets and signaling pathways that may potentially contribute to determine PEDF's ability to restrict the aggressiveness of A375 human melanoma cells.

AMP kinase-related kinase NUAK2 affects tumor growth, migration, and clinical outcome of human melanoma

The identification of genes that participate in melanomagenesis should suggest strategies for developing therapeutic modalities. We used a public array comparative genomic hybridization (CGH) database and real-time quantitative PCR (qPCR) analyses to identify the AMP kinase (AMPK)-related kinase NUAK2 as a candidate gene for melanomagenesis, and we analyzed its functions in melanoma cells. Our analyses had identified a locus at 1q32 where genomic gain is strongly associated with tumor thickness, and we used real-time qPCR analyses and regression analyses to identify NUAK2 as a candidate gene at that locus. Associations of relapse-free survival and overall survival of 92 primary melanoma patients with NUAK2 expression measured using immunohistochemistry were investigated using Kaplan-Meier curves, log rank tests, and Cox regression models. Knockdown of NUAK2 induces senescence and reduces S-phase, decreases migration, and down-regulates expression of mammalian target of rapamycin (mTOR). In vivo analysis demonstrated that knockdown of NUAK2 suppresses melanoma tumor growth in mice. Survival analysis showed that the risk of relapse is greater in acral melanoma patients with high levels of NUAK2 expression than in acral melanoma patients with low levels of NUAK2 expression (hazard ratio = 3.88; 95% confidence interval = 1.44-10.50; P = 0.0075). These data demonstrate that NUAK2 expression is significantly associated with the oncogenic features of melanoma cells and with the survival of acral melanoma patients. NUAK2 may provide a drug target to suppress melanoma progression. This study further supports the importance of NUAK2 in cancer development and tumor progression, while AMPK has antioncogenic properties.

Inhibitory effect of dendritic cells combined with cytokine-induced killer cells on the growth and liver metastasis of human primary malignant melanoma of the small intestine

Objective To investigate the inhibitory effect of dendritic cells (DCs) and homologous cytokine-induced killer cells (CIKs) on the growth and liver metastasis of human primary malignant melanoma of the small intestine. Methods A histologically intact liver metastasis fragment derived from a surgical specimen of patients with primary malignant melanoma of the small intestine was implanted in the mucous layer of small intestine in nude mice to construct the liver metastasis model of the malignant melanoma. Peripheral blood mononuclear cells from healthy donors and patients with malignant melanoma of the small intestine were isolated. DCs and CIKs were separately produced according to corresponding culture method, and the DC-CIKs were harvested after coculorthotopically implanted in 56 nude mice, and 72 hours later, all the mice were equally divided into normal saline group (0.3 ml), mitomycin treatment group (400 μg/0.3 ml), healthy donor CIK group (6 × 107/0.3 ml),healthy donor DC-CIK group (3 × 107/0.3 ml), autologous CIK treatment group (6 × 107/0.3 ml), autologous DC-CIK group (6 × 107/0. 3 ml) and DC-CIK (6 × 107/0.3 ml) + mitomycin (400 μg/0. 3 ml) treatment group. Mice in the experimental groups were administered the agents once daily by vena caudalis injection for 28 days. Mice were sacrificed 72 hours after treatment, and the orthotopic xenografts were removed for weighing,and the liver metastases were simultaneously evaluated by macroscopy and microscopy. All data were analyzed using the analysis of variance, LSD test and chi-square test. Results A liver metastatic model of human primary malignant melanoma of the small intestine ( HSIM-0603 ) was established. Of the control group, healthy donor CIK group, healthy donor DC-CIK group, autologous CIK treatment group, autologous DC-CIK group and DC-CIK +mitomycin group, the tumor weights were ( 1.18 ± 0. 17), (0.71 ± 0.06), (0.68 ± 0. 15 ), (0. 43 ± 0.08 ),(0.67 ± 0.07 ), (0.37 ± 0.08 ) and (0.20 ± 0.05 ) g, respectively; the tumor inhibition rates were 0, 39.82%,42.37%, 65.56%, 43.22%, 68.64% and 83.05%, respectively; liver metastases were detected in 8, 8, 5,4, 4, 3, 2 rats, respectively, in the above mentioned groups. There were significant differences in the tumor weight, tumor inhibition rate and liver metastasis rate among the seven groups (F = 152. 300, x2= 200. 538,94. 325, P ＜ 0.05 ). Conclusions The liver metastatic model could be applied to the studies on the pathogenesis,invasion, metastasis and treatment of human primary malignant melanoma of the small intestine. DC-CIK has the potential in inhibiting the growth and liver metastasis of the malignant melanoma of the small intestine. Key words: Neoplasms of small intestine; Malignant melanoma; Liver metastasis; Animal model; Dendritic cells; Killer cells; Immunotherapy

The promotion of human malignant melanoma growth by mesoporous silica nanoparticles through decreased reactive oxygen species

The concept that mesoporous silica nanoparticles (MSNs) are regarded as ideal novel drug delivery carriers in tumor therapy has been introduced extensively, but the effects of MSNs on tumor growth have received little attention. Here a model of nude mice xenografted with human malignant melanoma cells (A375) was used to investigate the effect of MSNs on tumor growth. Surprisingly, we found that MSNs have no toxicity to human malignant melanoma but increasing tumor growth in vivo. It was also confirmed that MSNs significantly promoted A375 cell proliferation and accelerated cell cycle progression in vitro. Cellular uptake mechanism showed that MSNs may affect molecular behavior of A375 cells when they entered into cytoplasm. Then, a detailed mechanism indicated that the promotion effect induced by MSNs was due to the decreasing of endogenous reactive oxygen species (ROS) in cells. Further results demonstrated that the upregulation of anti-apoptotic molecules Bcl-2 expression and the inhibition of NF-κB activation by MSNs may promote cell proliferation in a redox-sensitive signal pathway. These results show that tumor growth can be regulated by nanocarriers themselves in a ROS-dependent manner and imply that nanocarriers are not necessarily suitable for all kinds of tumor therapy in development drug delivery system.

Resveratrol and Resveratrol Analogues—Structure—Activity Relationship

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a compound found in wine and is held responsible for a number of beneficial effects of red wine. Besides the prevention of heart disease and significant anti-inflammatory effects, resveratrol might inhibit tumor cell growth and even play a role in the aging process. We here describe the structure-activity relationship of resveratrol and analogues of resveratrol regarding the free radical scavenging and antitumor effects of this exciting natural compound. In addition, we have synthesized a number of analogues of resveratrol with the aim to further improve the beneficial effects of resveratrol. Our studies were based on the analysis of structural properties, which were responsible for the most important effects of this compound. Striking in vivo effects can be observed with hexahydroxystilbene (M8), the most effective synthetic analogue of resveratrol. We could show that M8 inhibits tumor as well as metastasis growth of human melanoma in two different animal models, alone and in combination with dacarbacine.

Targeted inhibition of inducible nitric oxide synthase inhibits growth of human melanoma in vivo and synergizes with chemotherapy.

Aberrant expression of inflammatory molecules, such as inducible nitric oxide (NO) synthase (iNOS), has been linked to cancer, suggesting that their inhibition is a rational therapeutic approach. Whereas iNOS expression in melanoma and other cancers is associated with poor clinical prognosis, in vitro and in vivo studies suggest that iNOS and NO can have both protumor and antitumor effects. We tested the hypothesis that targeted iNOS inhibition would interfere with human melanoma growth and survival in vivo in a preclinical model. We used an immunodeficient non-obese diabetic/severe combined immunodeficient xenograft model to test the susceptibility of two different human melanoma lines to the orally-given iNOS-selective small molecule antagonist N(6)-(1-iminoethyl)-l-lysine-dihydrochloride (L-nil) with and without cytotoxic cisplatin chemotherapy. L-nil significantly inhibited melanoma growth and extended the survival of tumor-bearing mice. L-nil treatment decreased the density of CD31+ microvessels and increased the number of apoptotic cells in tumor xenografts. Proteomic analysis of melanoma xenografts with reverse-phase protein array identified alterations in the expression of multiple cell signaling and survival genes after L-nil treatment. The canonical antiapoptotic protein Bcl-2 was downregulated in vivo and in vitro after L-nil treatment, which was associated with increased susceptibility to cisplatin-mediated tumor death. Consistent with this observation, combination therapy with L-nil plus cisplatin in vivo was more effective than either drug alone, without increased toxicity. These data support the hypothesis that iNOS and iNOS-derived NO support tumor growth in vivo and provide convincing preclinical validation of targeted iNOS inhibition as therapy for solid tumors.

Cathepsin B inhibition interferes with metastatic potential of human melanoma: an in vitro and in vivo study

BackgroundCathepsins represent a group of proteases involved in determining the metastatic potential of cancer cells. Among these are cysteinyl- (e.g. cathepsin B and cathepsin L) and aspartyl-proteases (e.g. cathepsin D), normally present inside the lysosomes as inactive proenzymes. Once released in the extracellular space, cathepsins contribute to metastatic potential by facilitating cell migration and invasiveness.ResultsIn the present work we first evaluated, by in vitro procedures, the role of cathepsins B, L and D, in the remodeling, spreading and invasiveness of eight different cell lines: four primary and four metastatic melanoma cell lines. Among these, we considered two cell lines derived from a primary cutaneous melanoma and from a supraclavicular lymph node metastasis of the same patient. To this purpose, the effects of specific chemical inhibitors of these proteases, i.e. CA-074 and CA-074Me for cathepsin B, Cathepsin inhibitor II for cathepsin L, and Pepstatin A for cathepsin D, were evaluated. In addition, we also analyzed the effects of the biological inhibitors of these cathepsins, i.e. specific antibodies, on cell invasiveness. We found that i) cathepsin B, but not cathepsins L and D, was highly expressed at the surface of metastatic but not of primary melanoma cell lines and that ii) CA-074, or specific antibodies to cathepsin B, hindered metastatic cell spreading and dissemination, whereas neither chemical nor biological inhibitors of cathepsins D and L had significant effects. Accordingly, in vivo studies, i.e. in murine xenografts, demonstrated that CA-074 significantly reduced human melanoma growth and the number of artificial lung metastases.ConclusionsThese results suggest a reappraisal of the use of cathepsin B inhibitors (either chemical or biological) as innovative strategy in the management of metastatic melanoma disease.

CXCR1 and CXCR2 enhances human melanoma tumourigenesis, growth and invasion

The aggressiveness of malignant melanoma is associated with differential expression of CXCL-8 and its receptors, CXCR1 and CXCR2. However, the precise functional role of these receptors in melanoma progression remains unclear. In this study, we investigate the precise functional role of CXCR1 and CXCR2 in melanoma progression. CXCR1 or CXCR2 were stably overexpressed in human melanoma cell lines, SBC-2 (non-tumourigenic) and A375P (low-tumourigenic) exhibiting low endogenous expression of receptors. Functional assays were performed to study the resulting changes in cell proliferation, motility and invasion, and in vivo tumour growth using a mouse xenograft model. Our data demonstrated that CXCR1- or CXCR2-overexpressing SBC-2 and A375P melanoma cells had enhanced proliferation, chemotaxis and invasiveness in vitro. Interestingly, CXCR1 or CXCR2 overexpression in SBC-2 cells induced tumourigenicity, and A375P cells significantly enhanced tumour growth as examined in vivo. Immunohistochemical analyses showed significantly increased tumour cell proliferation and microvessel density and reduced apoptosis in tumours generated from CXCR1- or CXCR2-overexpressing melanoma cells. CXCR1- or CXCR2-induced modulation of melanoma cell proliferation and migration was observed to be mediated through the activation of ERK1/2 phosphorylation. Together, these studies demonstrate that CXCR1 and CXCR2 play essential role in growth, survival, motility and invasion of human melanoma.

Small-Molecule Antagonists for CXCR2 and CXCR1 Inhibit Human Melanoma Growth by Decreasing Tumor Cell Proliferation, Survival, and Angiogenesis

Melanoma, the most aggressive form of skin cancer, accounts for 75% of all skin cancer-related deaths and current therapeutic strategies are not effective in advanced disease. In the current study, we have investigated the efficacy of orally active small-molecule antagonist targeting CXCR2/CXCR1. Human A375SM melanoma cells were treated with SCH-479833 or SCH-527123, and their effect on proliferation, motility, and invasion was evaluated in vitro. We examined the downstream signaling events in the cells following treatment with antagonists. For in vivo studies, A375SM cells were implanted subcutaneously into athymic nude mice followed by administration of SCH-479833, SCH-527123, or hydroxypropyl-beta-cyclodextrin (20%) orally for 21 days and their effect on tumor growth and angiogenesis was evaluated. Our data show that SCH-479833 or SCH-527123 inhibited the melanoma cell proliferation, chemotaxis, and invasive potential in vitro. Treatment of melanoma cells with SCH-479833 or SCH-527123 also inhibited tumor growth. Histologic and histochemical analyses showed significant (P < 0.05) decreases in tumor cell proliferation and microvessel density in tumors. Moreover, we observed a significant increase in melanoma cell apoptosis in SCH-479833- or SCH-527123-treated animals compared with controls. Together, these studies show that selectively targeting CXCR2/CXCR1 with orally active small-molecule inhibitors is a promising therapeutic approach for inhibiting melanoma growth and angiogenesis.