Histone deacetylase inhibitor valproic acid sensitizes B16F10 melanoma cells to cucurbitacin B treatment

Cucurbitacin B (CuB) is reported to have anti-proliferation effects on a variety of tumors including melanoma, and more effective regimens by combination of this agent with others are under investigation. In this study, the anti-melanoma effect of CuB as a single agent and in combination with valproic acid (VPA), an inhibitor of histone deacetylase (HDAC), was evaluated in B16F10, a mouse melanoma cell line. The results demonstrated that CuB inhibited the proliferation of the cell line in a dose-dependent manner. However, it was likely that a pro-survival compensatory response, involving the induction of autophagy and upregulation of anti-apoptotic Bcl-2 protein, was induced by CuB treatment, which might greatly decrease the cytotoxicity of this agent. Supporting this, the melanoma cells were found to be more sensitive to the combination of CuB with chloroquine, a well-known autophagy inhibitor. And CuB-induced autophagy was associated with c-Jun N-terminal kinase (JNK) activation, at least partly, since inhibition of JNK activity by SP600125 could alleviate the autophagy. When CuB was combined with VPA, the two drugs showed synergistic cytotoxicity by induction of cell apoptosis. Moreover, the multiploidization effect of CuB was also suppressed in the presence of VPA. In contrast to the transient activation of JNKs by CuB, the combination of CuB and VPA resulted in prolonged JNK activation, although at low level after 4 h. Our results demonstrated that HDAC inhibitor VPA can sensitize B16F10 cells to CuB treatment through induction of apoptotic pathway.     

Synthesis and biological evaluation of novel N, N'-disubstituted urea and thiourea derivatives as potential anti-melanoma agents

Two series of urea and thiourea derivatives (1a-11a, 1b-11b) have been synthesized; all the 22 compounds were reported for the first time. Their anti-proliferative activities against the melanoma cell line B16-F10 were evaluated. Among the compounds tested, compound 6b exhibited the most potent activity in melanoma cells growth inhibition (IC(50) = 0.33 μM). The bioassay tests showed that anti-proliferative activities of these novel compounds were possibly caused by inhibition of ERK1/2 phosphorylation level. Therefore, compound 6b can be a potential anti-melanoma agent and an inhibitor of ERK1/2 phosphorylation deserving further research.     

Paclitaxel treatment enhances lymphatic metastasis of B16F10 melanoma cells via CCL21/CCR7 axis

Chemotherapeutic drugs have been successfully used to treat several cancers, including melanoma. However, metastasis occasionally occurs after chemotherapy. Here, we reported that paclitaxel (PTX) treatment for B16F10 tumour in mice led to an enhanced lymphatic metastasis of the melanoma cells, although a significant inhibition of tumour growth at the injection site was observed. Further study demonstrated that PTX upregulated the expression of C-C chemokine receptor type 7 (CCR7) in B16F10 cells, enhancing their migration through the activation of JNK and p38 signalling pathways. Loss of CCR7 or blockade of C-C motif chemokine ligand 21 (CCL21)/CCR7 axis abolished the pro-migration effect of PTX on B16F10 melanoma cells. Importantly, combination of PTX and CCR7 mAb could simultaneously delay the tumour growth and reduce the lymphatic metastasis in B16F10 melanoma. The blockade of CCL21/CCR7 axis may collectively serve as a strategy for lymphatic metastasis in some melanoma after chemotherapy.     

Further characterization of a clinically relevant model of melanoma metastasis and an effective vaccine

A major problem in evaluating the effectiveness of tumor cell vaccination and other biological therapies is the variability of experimental models. In this study we have further developed and characterized a model for metastatic melanoma that approximates the major clinical stages of metastatic dissemination: stage I-growth of the primary (local) tumor, stage II-dissemination to regional lymph nodes, and stage III-metastasis to distant organs (lungs). C57BL/6 mice were challenged subcutaneously with B16 F10 murine melanoma cells in the midtail, and within 3 weeks 100% of the mice had local tumors growing in their tails. By 5–7 weeks after challenge, most of the mice had developed metastases to the inguinal lymph nodes and subsequently had metastatic colonies in the lungs and in the bone marrow. Preimmunization of mice with a formalinized extracellular antigen vaccine, derived from B16F10 melanoma cells, provided partial inhibition of the growth of the primary melanoma tumors, as well as reducing the number of metastases to the regional (inguinal) lymph nodes and lungs along with concomitantly increasing survival time. This model for melanoma metastasis provides a reasonable and reproducible test system for the study of anti-melanoma immunity and the different cellular and humoral mechanisms involved.This work was supported in part by National Institutes of Health grants R37 CA45148 and R30 CA13943     

Differentiation-inducing activity of lupane triterpenes on a mouse melanoma cell line

Lupane triterpenes were found to promote melanogenesis, a hallmark of B16 2F2 mouse melanoma cell differentiation. Studies of the structure-activity relationships demonstrated that the keto function at C-3 of the lupane skeleton played important roles in the melanogenic activities of lupane triterpenes on melanoma cells. The carbonyl group at C-17 of lupane triterpenes was essential against their apoptosis-inducing activity against human cancer cells via the inhibition of topoisomerase I. We investigated whether signaling mechanisms were involved in the stimulative effects of lupane triterpenes on the melanogenesis of B16 2F2 cells. In experiments using selective inhibitors against various signal transduction molecules and Western blotting analysis, it was suggested that p38 MAPK was involved in melanoma cell differentiation as a downstream effector of PKA. Lupeol (compound 1), a lupane triterpene, induced dendrite formations, a morphological hallmark of B16 2F2 cell differentiation by rearrangement of the actin cytoskeleton. The activation of cofilin, an actin depolymerizing factor, by compound 1 caused actin fiber disassembly in B16 2F2 cells. Furthermore, compound 1 was shown to inhibit the cell motilities of human melanoma and neuroblastoma in vitro.     

Melanoma Cells Treated with GGTI and IFN-γ Allow Murine Vaccination and Enhance Cytotoxic Response against Human Melanoma Cells

Background

Suboptimal activation of T lymphocytes by melanoma cells is often due to the defective expression of class I major histocompatibility antigens (MHC-I) and costimulatory molecules. We have previously shown that geranylgeranyl transferase inhibition (done with GGTI-298) stimulates anti-melanoma immune response through MHC-I and costimulatory molecule expression in the B16F10 murine model [1].

Methodology/Principal Findings

In this study, it is shown that vaccination with mIFN-gand GGTI-298 pretreated B16F10 cells induces a protection against untreated tumor growth and pulmonary metastases implantation. Furthermore, using a human melanoma model (LB1319-MEL), we demonstrated that in vitro treatment with hIFN-γ and GGTI-298 led to the up regulation of MHC-I and a costimulatory molecule CD86 and down regulation of an inhibitory molecule PD-1L. Co-culture experiments with peripheral blood mononuclear cells (PBMC) revealed that modifications induced by hIFN-γ and GGTI-298 on the selected melanoma cells, enables the stimulation of lymphocytes from HLA compatible healthy donors. Indeed, as compared with untreated melanoma cells, pretreatment with hIFN-γ and GGTI-298 together rendered the melanoma cells more efficient at inducing the: i) activation of CD8 T lymphocytes (CD8+/CD69+); ii) proliferation of tumor-specific CD8 T cells (MelanA-MART1/TCR+); iii) secretion of hIFN-γ; and iv) anti-melanoma specific cytotoxic cells.

Conclusions/Significance

These data indicate that pharmacological treatment of melanoma cell lines with IFN-γ and GGTI-298 stimulates their immunogenicity and could be a novel approach to produce tumor cells suitable for vaccination and for stimulation of anti-melanoma effector cells.     

Interaction between integrin alpha(5) and fibronectin is required for metastasis of B16F10 melanoma cells

In this study, we report the role of integrin alpha(5) in promoting melanoma metastasis. The alpha(5) expression was remarkably elevated in highly metastatic B16F10 melanoma cells compared to lowly metastatic B16F1 cells, whereas no significant changes were detected in those of integrin alpha(4), alpha(v), and beta(1) subunits. Neutralization of alpha(5) with anti-alpha(5) antibody significantly suppressed the potential of B16F10 cells for pulmonary metastasis in mice and inhibited cell adhesion or spreading to fibronectin in vitro. Furthermore, loss of the interaction between alpha(5) and fibronectin diminished cell survival and induced apoptosis in B16F10 cells. Above results provide clear evidence that integrin alpha(5) is positively correlated with melanoma metastasis and might be an anti-melanoma target.     

Antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells

Reactive oxygen species (ROS) generation is linked to dynamic actin cytoskeleton reorganization, which is involved in tumor cell motility and metastasis. Thus, inhibition of ROS generation and actin polymerization in tumor cells may represent an effective anticancer strategy. However, the molecular basis of this signaling pathway is currently unknown. Here, we show that the Ecklonia cava-derived antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Steady-state intracellular ROS levels were higher in malignant B16F10 cells than in parental, nonmetastatic B16F0 cells. Elevation of ROS by H₂O₂ treatment increased migration and invasion ability of B16F0 cells to level similar to that of B16F10 cells, suggesting that intracellular ROS signaling mediates the prometastatic properties of B16 mouse melanoma cells. ROS levels and the cell migration and invasion ability of B16 melanoma cells correlated with Rac1 activation and WAVE2 expression. Overexpression of dominant negative Rac1 and depletion of WAVE2 by siRNA suppressed H₂O₂-induced cell invasion of B16F0 and B16F10 cells. Similarly, dieckol attenuates the ROS-mediated Rac1 activation and WAVE2 expression, resulting in decreased migration and invasion of B16 melanoma cells. In addition, we found that dieckol decreases association between WAVE2 and NADPH oxidase subunit p47^phox. Therefore, this finding suggests that WAVE2 acts to couple intracellular Rac1/ROS signaling to the invasive migration of B16 melanoma cells, which is inhibited by dieckol.     

Role of p38 MAPK in lupeol-induced B16 2F2 mouse melanoma cell differentiation

We examined the signaling mechanisms involved in the differentiation-inducing activity of lupeol toward B16 2F2 melanoma cells. alpha-Melanocyte stimulating hormone (alpha-MSH), forskolin and dibutyryl cAMP, which are believed to be cAMP-elevating agents and analogues, enhanced lupeol-induced B16 2F2 cell differentiation. However, H89, an inhibitor of protein kinase A, completely abolished B16-2F2 cell differentiation induced by lupeol. Furthermore, we studied the role of mitogen-activated protein kinases (MAPKs) in lupeol-induced B16 2F2 cell differentiation. U0126, an inhibitor of MAPK kinases, induced B16 2F2 cell differentiation and enhanced the cell differentiation induced by lupeol. However, SB203580, a selective inhibitor of p38 MAPK, completely blocked lupeol-induced B16 2F2 cell differentiation. Western blot analysis revealed that 10 microM lupeol transiently elevated the level of phosphorylation of p38 MAPK. The phosphorylation of p38 MAPK was detected on the addition of 1 microM lupeone, another lupane triterpene, but was not induced by 1 microM lupeol. These results suggested that lupeol induced B16 2F2 cell differentiation through activation of p38 MAPK, and that the structural differences at C-3 of lupane triterpenes played an important role in the activation of p38 MAPK.     

1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) facilitates curcumin-induced melanoma cell apoptosis by enhancing ceramide accumulation, JNK activation, and inhibiting PI3K/AKT activation

The majority of metastatic melanomas are resistant to different chemotherapeutic agents, consequently, the search for novel anti-melanoma agents and adjuvant is urgent. Here, we found that 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of glycosphingolipid biosynthesis, enhanced curcumin-induced cell growth inhibition and apoptosis in two melanoma cell lines (WM-115 and B16). PDMP facilitated curcumin-induced ceramide accumulation; the latter contributed to melanoma cell apoptosis. PDMP also dramatically enhanced curcumin-induced c-Jun N-terminal kinase activation, which was important to melanoma cell apoptosis. Meanwhile, curcumin plus PDMP treatment largely inhibited the activation of pro-survival PI3K/AKT signal pathway. In conclusion, PDMP-sensitized curcumin-induced melanoma cell growth inhibition and apoptosis in vitro due to changes of multiple signal events. Combining PDMP with curcumin may represent a new therapeutic intervention against melanoma.     

Galangin induces B16F10 melanoma cell apoptosis via mitochondrial pathway and sustained activation of p38 MAPK

Galangin, an active flavonoid present at high concentration in Alpinia officinarum Hance and propolis, shows cytotoxicity towards several cancer cell lines, including melanoma. However, the specific cellular targets of galangin-induced cytotoxicity in melanoma are still unknown. Here, we investigated the effects of galangin in B16F10 melanoma cells and explored the possible molecular mechanisms. Galangin significantly decreased cell viability of B16F10 cells, and also induced cell apoptosis shown by Hoechst 33342 staining and Annexin V-PI double staining flow cytometric assay. Furthermore, upon galangin treatment, disruption of mitochondrial membrane potential was observed by JC-1 staining. Western blotting analysis indicated that galangin activated apoptosis signaling cascades by cleavage of procaspase-9, procaspase-3 and PARP in B16F10 cells. Moreover, galangin significantly induced activation of phosphor-p38 MAPK in a time and dose dependent manner. SB203580, an inhibitor of p38, partially attenuated galangin-induced apoptosis in B16F10 cells. Taken together, this work suggests that galangin has the potential to be a promising agent for melanoma treatment and may be further evaluated as a chemotherapeutic agent.     

The molecular mechanisms of vitamin C on cell cycle regulation in B16F10 murine melanoma

Vitamin C has inconsistent effects on malignant tumor cells, which vary from growth stimulation to apoptosis induction. It is well known that melanoma cells are more susceptible to vitamin C than any other tumor cells, but the precise mechanism remains to be elucidated. In the present study, the proliferation of B16F10 melanoma cells was suppressed by vitamin C, which induced growth arrest in a dose-dependent manner without cytotoxic effects. Therefore, we investigated the changes in cell cycle distribution of B16F10 melanoma cells by staining DNAs with propidium iodide (PI). The growth inhibition of B16F10 melanoma by vitamin C was associated with an arrest of cell cycle distribution at G1 stage. In addition, the levels of p53-p21Waf1/Cip1 increased during G1 arrest, which were essential for vitamin C-induced cell cycle arrest. The increased p21Waf1/Cip1 inhibited CDK2. Moreover, the activity of p53-p21Waf1/Cip1 pathway was closely related with the activation of checkpoint kinase 2 (Chk2). Inhibitor of the PI3K-family, LY294002 and the ATM/ATR inhibitor, caffeine, blocked vitamin C-induced growth arrest in B16F10 melanoma cells. These results suggest that vitamin C might be a potent agent to inhibit proliferative activity of melanoma cells via the regulation of Chk2-p53-p21Waf1/Cip1 pathway.     

Increasing ceramides sensitizes genistein-induced melanoma cell apoptosis and growth inhibition

The aim of the current study is to investigate the effect of ceramides on genistein-induced anti-melanoma effects in vitro. We found that exogenously added cell-permeable short-chain ceramides (C6) dramatically enhanced genistein-induced growth inhibition and apoptosis in cultured melanoma cells. Genistein treatment only induced a moderate intracellular ceramides accumulation in B16 melanoma cells. Two different agents including 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a ceramide glucosylation inhibitor, and the sphingosine kinase-1 (SphK1) inhibitor II (SKI-II), a sphingosine (ceramides precursor) phosphorylation inhibitor, both facilitated genistein-induced ceramides accumulation and melanoma cell apoptosis. Co-administration of ceramide (C6) and genistein induced a significant Akt inhibition and c-jun-NH(2)-kinase (JNK) activation, caspase-3 cleavage and cytochrome c release. Caspase-3 inhibitor z-DVED-fmk, JNK inhibitor SP 600125, or to restore Akt activation by introducing a constitutively active form of Akt (CA-Akt) diminished ceramide (C6) and genistein co-administration-induced in vitro anti-melanoma effect. Our study suggests that increasing cellular level of ceramides may sensitize genistein-induced anti-melanoma effects.     

Involvement of extracellular signal-regulated kinase in nobiletin-induced melanogenesis in murine B16/F10 melanoma cells

Nobiletin contributes to pharmacological activities such as anti-cancer and anti-inflammatory effects, but little is known about its effect on melanogenesis. In this study, we found that nobiletin increased melanin content and tyrosinase activity in murine B16/F10 melanoma cells. Furthermore, inhibition of the extracellular signal-regulated kinase (ERK) pathway with U0216 resulted in inhibition of nobiletin-induced melanin synthesis and tyrosinase expression.     

CCN2 expression and localization in melanoma cells

The matricellular protein connective tissue growth factor (CTGF, CCN2) is overexpressed in several forms of cancer and may represent a novel target in anti-cancer therapy. However, whether CCN2 is expressed in melanoma cells is unknown. The highly metastatic murine melanoma cell line B16(F10) was used for our studies. Real time polymerase chain reaction analysis was used to detect mRNA expression of CCN1, CCN2, CCN3 and CCN4 in Western blot and immunofluorescence analyses were used to detect CCN2 protein. Inhibitors of signal transduction cascades were used to probe the mechanism underlying CCN2 expression in B16(F10) cells. CCN2 was expressed in B16(F10) cells, and was reduced by the FAK/src inhibitor PP2 and the MEK/ERK inhibitor U0126 indicating that CCN2 acts downstream of these pathways in B16(F10) murine melanoma cells. Expression of CCN1, CCN3 and CCN4 was not reduced by PP2 or U0126; in fact, expression of CCN4 mRNA was elevated by PP2 or U0126 treatment. To our surprise, CCN2 protein was detected in the nuclei of B16(F10) cells, and was undetectable in the cytoplasm. CCN2 was expressed in B16(F10) melanoma cells, adding to the list of cancer cells in which CCN2 is expressed. Of the CCN family members tested, only CCN2 is downstream of the highly oncogenic MEK/ERK pathway. CCN2 should be further evaluated for a possible role in melanoma growth and progression.