A comparative study of sphingolipids in transplanted melanomas with high and low metastatic activity

The content of sphingolipids in M3 and B16/F10 melanomas with a high metastatic potential and in Claudman's and B16/F1 melanomas with a low metastatic potential was studied. It was shown that the content of total lipid-bound sialic acids and ganglioside GM3 in melanomas with a high metastatic potential is considerably higher than that in melanomas with a low metastatic potential. On the other hand, the ceramide to glucosylceramide molar ratio is higher in melanomas with a low metastatic potential. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.     

Down-regulation of glutathione and Bcl-2 synthesis in mouse B16 melanoma cells avoids their survival during interaction with the vascular endothelium

B16 melanoma (B16M) cells with high GSH content show high metastatic activity. However, the molecular mechanisms linking GSH to metastatic cell survival are unclear. The possible relationship between GSH and the ability of Bcl-2 to prevent cell death was studied in B16M cells with high (F10) and low (F1) metastatic potential. Analysis of a Bcl-2 family of genes revealed that B16M-F10 cells, as compared with B16M-F1 cells, overexpressed preferentially Bcl-2 (approximately 5.7-fold). Hepatic sinusoidal endothelium-induced B16M-F10 cytotoxicity in vitro increased from approximately 19% (controls) to approximately 97% in GSH-depleted B16M-F10 cells treated with an antisense Bcl-2 oligodeoxynucleotide (Bcl-2-AS). l-Buthionine (S,R)-sulfoximine-induced GSH depletion or Bcl-2-AS decreased the metastatic growth of B16M-F10 cells in the liver. However, the combination of l-buthionine (S,R)-sulfoximine and Bcl-2-AS abolished metastatic invasion. Bcl-2-overexpressing B16M-F1/Tet-Bcl-2 and B16M-F10/Tet-Bcl-2 cells, as compared with controls, showed an increase in GSH content, no change in the rate of GSH synthesis, and a decrease in GSH efflux. Thus, Bcl-2 overexpression may increase metastatic cell resistance against oxidative/nitrosative stress by inhibiting release of GSH. In addition, Bcl-2 availability regulates the mitochondrial GSH (mtGSH)-dependent opening of the permeability transition pore complex. Death in B16M-F10 cells was sharply activated at mtGSH levels below 30% of controls values. However, this critical threshold increased to approximately 60% of control values in Bcl-2-AS-treated B16M-F10 cells. GSH ester-induced replenishment of mtGSH levels (even under conditions of cytosolic GSH depletion) prevented cell death. Our results indicate that survival of B16M cells with high metastatic potential can be challenged by inhibiting their GSH and Bcl-2 synthesis.     

Membrane-associated cathepsin L: a role in metastasis of melanomas

Subcellular distribution of cathepsin L, the major protein released by transformed or ras transfected fibroblasts, was examined in murine liver, murine B16 amelanotic melanoma and human A2058 melanoma after sequential differential and Percoll density gradient centrifugation. In both murine and human melanomas, cathepsin L activity was found to be enriched in plasma membrane fractions; cathepsin L in these fractions was in both native and acid activatable forms. Plasma membrane fractions from B16 melanoma subpopulations of "low" and "high" metastatic potential were assayed for activity of cathepsin L and of heat stable endogenous inhibitors. The relative specific activity of cathepsin L was 7-fold greater in the subpopulation of "high" metastatic potential, whereas cysteine proteinase inhibitory activity was 5-fold less. Since cathepsin L can degrade intact basement membrane, this membrane-associated cathepsin L may well contribute to metastatic spread of melanomas.     

Investigation of tumor metastatic potential with N-[18F]fluoroacetyl-d-glucosamine

In order to investigate the metastatic potential of tumors in vivo by measuring hyaluronic acid metabolism, C57BL/6 mice with B16 melanoma variants and C3H/He mice with FM3A tumor variants were evaluated using N-[¹⁸F]fluoroacetyl-d-glucosamine (¹⁸F-GlcNFAc). The uptake of ¹⁸F-GlcNFAc was slightly higher (P < 0.05) in B16-F10 tumors (high metastatic potential) than in B16-F1 (low metastatic potential). Analysis of metabolites showed that acid-insoluble fraction was the largest one in the liver by 60 min, whereas in the tumors, phosphates fraction was the major metabolite. Slower metabolism in tumors was suggested, and it may be one of the reasons for the difficulty of detecting the characteristics of their hyaluronic acid synthesis. ¹⁸F-GlcNFAc uptake by FM3A variants showed no significant correlation with their metastatic potential. In addition, N-acetyl-d-[l-¹⁴C]glucosamine, 2-deoxy-d-[l-¹⁴C]glucose and [6-³H]thymidine failed to demonstrate any difference between tumors' metastatic variants in vivo.     

Histone Deacetylase-3 Mediates Positive Feedback Relationship between Anaphylaxis and Tumor Metastasis

Allergic inflammation has been known to enhance the metastatic potential of tumor cells. The role of histone deacetylase-3 (HDAC3) in allergic skin inflammation was reported. We investigated HDAC3 involvement in the allergic inflammation-promotion of metastatic potential of tumor cells. Passive systemic anaphylaxis (PSA) induced HDAC3 expression and FcϵRI signaling in BALB/c mice. PSA enhanced the tumorigenic and metastatic potential of mouse melanoma cells in HDAC3- and monocyte chemoattractant protein 1-(MCP1)-dependent manner. The PSA-mediated enhancement of metastatic potential involved the induction of HDAC3, MCP1, and CD11b (a macrophage marker) expression in the lung tumor tissues. We examined an interaction between anaphylaxis and tumor growth and metastasis at the molecular level. Conditioned medium from antigen-stimulated bone marrow-derived mouse mast cell cultures induced the expression of HDAC3, MCP1, and CCR2, a receptor for MCP1, in B16F1 mouse melanoma cells and enhanced migration and invasion potential of B16F1 cells. The conditioned medium from B16F10 cultures induced the activation of FcϵRI signaling in lung mast cells in an HDAC3-dependent manner. FcϵRI signaling was observed in lung tumors derived from B16F10 cells. Target scan analysis predicted HDAC3 to be as a target of miR-384, and miR-384 and HDAC3 were found to form a feedback regulatory loop. miR-384, which is decreased by PSA, negatively regulated HDAC3 expression, allergic inflammation, and the positive feedback regulatory loop between anaphylaxis and tumor metastasis. We show the miR-384/HDAC3 feedback loop to be a novel regulator of the positive feedback relationship between anaphylaxis and tumor metastasis.     

E‐cadherin determines Caveolin‐1 tumor suppression or metastasis enhancing function in melanoma cells

The role of caveolin‐1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E‐cadherin in CAV1‐dependent tumor suppression. Here, we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E‐cadherin, to unravel how CAV1 affects tumor growth and metastasis and to assess how co‐expression of E‐cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10 (cav‐1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E‐cadherin expression in B16F10 (E‐cad) cells reduces subcutaneous tumor formation and lung metastasis when intravenously injected. Importantly, co‐expression of CAV1 and E‐cadherin in B16F10 (cav‐1/E‐cad) cells abolishes tumor formation, lung metastasis, increased Rac‐1 activity, and cell migration observed with B16F10 (cav‐1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac‐1 activation in these cells.     

HPLC analysis of supernatant soluble factors from tumor cell variants with defined metastatic status

In the present study, we report a simple and sensitive procedure to study supernatant soluble factors of short time cultured cells by high performance gel permeation chromatography (HPGPC). This procedure enables a direct analysis with no preparatory procedures prior to application in the chromatographic column and eliminates tedious and often low reproducible techniques, required during conventional assay of culture medium. In addition, we show that HPGPC analysis is suitable for discriminating between two variants of a rhabdomyosarcoma with different metastatic potential, while two distinct cell lines (3LL carcinoma and B16F10 melanoma), which do not differ in their metastatic potential, do not present noticeable qualitative differences in their chromatographic secretion pattern.     

Reconstitution of the Gs protein from B16 melanoma clones of high and low experimental metastatic potential into S49 cyc-membranes

The ability of a series of B16 melanoma clones to form experimental lung metastases in syngeneic mice has been shown to correlate positively with adenylate cyclase activity. (Sheppard et al, Int. J. Cancer 37 (1986) 713-722). To begin to identify the components of the adenylate cyclase complex that account for enhanced enzyme activity in highly metastatic tumor populations, cholate extracts containing the GTP-binding protein GS from B16 melanoma clones of different metastatic capacities were reconstituted with membranes prepared from S49 cyc-, a variant lymphoma cell line that lacks GS function. The results revealed that extracts from a highly metastatic B16 clone (F10-C23) reconstituted significantly greater adenylate cyclase activities in S49 cyc- membranes than parallel preparations from a B16 clone (F1-C29) of low metastatic capacity. The data suggest that aberrations in GS function may contribute to the heightened responsiveness of adenylate cyclase observed in B16 melanoma clones of increased metastatic potential.     

Intertissue flow of glutathione (GSH) as a tumor growth-promoting mechanism: interleukin 6 induces GSH release from hepatocytes in metastatic B16 melanoma-bearing mice

B16 melanoma F10 (B16-F10) cells with high glutathione (GSH) content show high metastatic activity in vivo. An intertissue flow of GSH, where the liver is the main reservoir, can increase GSH content in metastatic cells and promote their growth. We have studied here possible tumor-derived molecular signals that could activate GSH release from hepatocytes. GSH efflux increases in hepatocytes isolated from mice bearing liver or lung metastases, thus suggesting a systemic mechanism. Fractionation of serum-free conditioned medium from cultured B16-F10 cells and monoclonal antibody-induced neutralization techniques facilitated identification of interleukin (IL)-6 as a tumor-derived molecule promoting GSH efflux in hepatocytes. IL-6 activates GSH release through a methionine-sensitive/organic anion transporter polypeptide 1- and multidrug resistance protein 1-independent channel located on the sinusoidal site of hepatocytes. Specific siRNAs were used to knock down key factors in the main signaling pathways activated by IL-6, which revealed a STAT3-dependent mechanism. Our results show that IL-6 (mainly of tumor origin in B16-F10-bearing mice) may facilitate GSH release from hepatocytes and its interorgan transport to metastatic growing foci.     

Silencing of Diphthamide Synthesis 3 (Dph3) Reduces Metastasis of Murine Melanoma

Melanoma is the most dangerous skin cancer due to its highly metastatic potential and resistance to chemotherapy. Currently, there is no effective treatment for melanoma once it is progressed to metastatic stage. Therefore, further study to elucidate the molecular mechanism underlying the metastasis of melanoma cells is urgently required for the improvement of melanoma treatment. In the present study, we found that diphthamide synthesis 3 (Dph3) is involved in the metastasis of B16F10 murine melanoma cells by insertional mutagenesis. We demonstrated that Dph3 disruption impairs the migration of B16F10 murine melanoma cells. The requirement of Dph3 in the migration of melanoma cells was further confirmed by gene silencing with siRNA in vitro. In corresponding to this result, overexpression of Dph3 significantly promoted the migratory ability of B16F10 and B16F0 melanoma cells. Moreover, down regulation of Dph3 expression in B16F10 melanoma cells strikingly inhibits their cellular invasion and metastasis in vivo. Finally, we found that Dph3 promotes melanoma migration and invasion through the AKT signaling pathway. To conclude, our findings suggest a novel mechanism underlying the metastasis of melanoma cells which might serve as a new intervention target for the treatment of melanoma.     

LC/MS-Based Quantitative Proteomic Analysis of Paraffin-Embedded Archival Melanomas Reveals Potential Proteomic Biomarkers Associated with Metastasis

Background

Melanoma metastasis status is highly associated with the overall survival of patients; yet, little is known about proteomic changes during melanoma tumor progression. To better understand the changes in protein expression involved in melanoma progression and metastasis, and to identify potential biomarkers, we conducted a global quantitative proteomic analysis on archival metastatic and primary melanomas.

Methodology and Findings

A total of 16 metastatic and 8 primary cutaneous melanomas were assessed. Proteins were extracted from laser captured microdissected formalin fixed paraffin-embedded archival tissues by liquefying tissue cells. These preparations were analyzed by a LC/MS-based label-free protein quantification method. More than 1500 proteins were identified in the tissue lysates with a peptide ID confidence level of >75%. This approach identified 120 significant changes in protein levels. These proteins were identified from multiple peptides with high confidence identification and were expressed at significantly different levels in metastases as compared with primary melanomas (q-Value<0.05).

Conclusions and Significance

The differentially expressed proteins were classified by biological process or mapped into biological system networks, and several proteins were implicated by these analyses as cancer- or metastasis-related. These proteins represent potential biomarkers for tumor progression. The study successfully identified proteins that are differentially expressed in formalin fixed paraffin-embedded specimens of metastatic and primary melanoma.     

The effects of patchouli alcohol and combination with cisplatin on proliferation,apoptosis and migration in B16F10 melanoma cells

Melanoma is a highly metastatic cancer with a low incidence rate, but a high mortality rate. Patchouli alcohol (PA), a tricyclic sesquiterpene, is considered the main active component in Pogostemon cablin Benth, which improves wound healing and has anti-tumorigenic activity. However, the pharmacological action of PA on anti-melanoma remains unclear. Thus, the present study aimed to investigate the role of PA in the proliferation, cell cycle, apoptosis and migration of melanoma cells. These results indicated that PA selectively inhibited the proliferation of B16F10 cells in a dose- and time-dependent manner. It induced cell cycle arrest at the G₀/G₁ phase and typical morphological changes in apoptosis, such as chromatin condensation, DNA fragmentation and apoptotic bodies. In addition, PA reduced the migratory ability of B16F10 cells by upregulating E-cadherin and downregulating p-Smad2/3, vimentin, MMP-2 and MMP-9 expression. PA was also found to strongly suppress tumour growth in vivo. Furthermore, PA combined with cisplatin synergistically inhibited colony formation and migration of B16F10 cells and attenuated the development of resistance to treatment. Therefore, the results of this study indicate that PA may play a pivotal role in inducing apoptosis and reducing the migration of melanoma cells, and may thus be a potential candidate for melanoma treatment.     

Selective Expression of PNA-Binding Glycoconjugates by Invasive Human Melanomas: A New Marker of Metastatic Potential

Alterations of cell-surface glycoconjugates have been associated with invasiveness and metastatic capacity in a number of experimental and human tumors (bladder and colon cancer). We have recently shown that human melanoma cells from variants selected for high metastatic potential in an animal model bind the lectin peanut agglutinin (PNA), and that human melanoma cell populations enriched for PNA binding cells generate a higher frequency of metastases when xenografted into immune suppressed neonatal rats. We have therefore sought cells binding PNA in biopsied human melanocytic tumors and compared frequencies of PNA binding by cells from benign nevi, early and late primary melanomas, and metastatic melanomas. Sections of conventionally processed tissues were deparaffinised and exposed to biotinylated PNA; PNA fixation was revealed by the avidine/peroxidase/AEC technique. In 51 specimens tested, PNA appears to react electively with invasive tumors, since only one of the 7 early primary melanomas (Clark III) reacted while 13/23 late primary melanomas (Clark III-V), and 4/21 melanoma metastases were reactive. In addition, only 1/17 benign nevi bound PNA. In primary tumors, the reactive cells were exclusively invasive tumors cells in the dermis. PNA reactive material was observed in the cytoplasm and plasma membrane of reactive cells. Hence, alterations in composition and cellular localisation of glycoconjugates detectable by lectin histochemistry in melanoma cells may be markers of metastatic potential that may be applicable on an individual patient basis.     

Induction of 103-kDa gelatinase/type IV collagenase by acidic culture conditions in mouse metastatic melanoma cell lines.

Gelatinases/type IV collagenases have been shown to be involved in tumor invasion and metastasis. In this study, we examined the effect of culture medium pH on the secretion of the gelatinases from mouse B16 melanoma cell lines and human tumor cell lines using zymography analysis. The highly metastatic clone F10 of B16 melanoma did not secrete any gelatinase in neutral culture media (pH 7.1-7.3), whereas it secreted a high level of a 103-kDa gelatinase in an initial pH range of 5.4-6.1. The addition of an excess amount of glucose into a neutral culture medium also induced the gelatinase secretion from the cells by decreasing the medium pH during incubation. The extent of the acid-induced gelatinase secretion by the B16 melanoma cell lines was in the order of BL6 greater than F10 greater than F1 much greater than the parent B16 line, in good agreement with the order of their metastatic potentials. Two human cell lines (A549 and HT1080) secreted a higher level of a 90-kDa gelatinase at pH 6.8 compared with pH 7.3. The acid-induced gelatinase secretion from B16-F10 cells was blocked by cycloheximide, indicating that the enzyme induction was due to de novo synthesis. When in vitro tumor cell invasion was assayed in Boyden chambers, B16-F10 cells incubated in an acidic medium exerted a more active migration through type IV collagen gel than those in a neutral medium. These results suggest that the acidic environment formed around tumor tissues may be an important factor in invasion and metastasis of some types of tumors.     

Metastatic melanoma imaging using a novel Tc-99m-labeled lactam-cyclized alpha-MSH peptide

The purpose of this study was to determine the metastatic melanoma imaging property of ^99mTc(EDDA)-HYNIC-Aoc-Nle-CycMSH_hex {hydrazinonicotinamide-8-aminooctanoic acid-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH₂}. HYNIC-Aoc-Nle-CycMSH_hex was synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The IC₅₀ value of HYNIC-Aoc-Nle-CycMSH_hex was 0.78?±?0.13?nM for B16/F10 melanoma cells. ^99mTc(EDDA)-HYNIC-Aoc-Nle-CycMSH_hex displayed significantly higher uptake (14.26?±?2.74 and 10.45?±?2.31%?ID/g) in B16/F10 metastatic melanoma-bearing lung than that in normal lung (0.90?±?0.15 and 0.53?±?0.14%?ID/g) at 2 and 4?h post-injection, respectively. B16/F10 pulmonary metastatic melanoma lesions were clearly visualized by SPECT/CT using ^99mTc(EDDA)-HYNIC-Aoc-Nle-CycMSH_hex as an imaging probe at 2?h post-injection, underscoring its potential as an imaging probe for metastatic melanoma detection.     

DNA repair and repair fidelity in metastatic variants of the B16 murine melanoma

We have examined the concept of genomic instability in relation to the metastatic progression of low (F1) and high metastasis (BL6, ML8) clones of the B16 mouse melanoma, by using a mutation assay, and DNA strand break repair and repair fidelity assays. The frequency of induced ouabain resistant colonies between the variant cell lines was consistent with the difference between their metastatic properties. Survival data for X-irradiation and bleomycin were similar among the 3 cell lines. When X-rays or bleomycin were used to induce strand breakage, no difference was detectable in either the rate or extent of DNA repair using the techniques of alkaline unwinding and alkaline elution for total strand breaks, and neutral elution for double strand breaks. DNA repair fidelity was measured using the PMH16 plasmid. A Kpn I restriction site was used to introduce a break within the gpt gene of the plasmid, prior to transfection. We found that ～ 100% and ～ 65% of the highly metastatic ML8 and BL6 clones, respectively, religated the gene with the required fidelity, compared with only ～ 25% of the low metastasis F1 clones. In summary, the metastatic variants show similar sensitivities to X-irradiation and bleomycin, but a differential response to EMS. This difference is not reflected in any subsequent DNA strand break religation, but the variants do differ in their fidelity of repair. However, although the fidelity of DNA religation is related to metastatic potential, it is not consistent with the mutation frequency data. © 1993 Wiley-Liss, Inc.     

Preparation,In Vivo Administration,Dose-Limiting Toxicities,and Antineoplastic Activity of Cytochalasin B

An effective and inexpensive protocol for producing cytochalasins A and B is being disclosed to propose a viable method by which to examine the in vivo antineoplastic activity of these congeners in preclinical tumor-bearing mammalian models. In addition, we determine the maximum tolerated doses of cytochalasin B using multiple routes and formulations, characterize the tissue distribution of intravenous bolus cytochalasin B, and assess the in vivo antineoplastic activity of cytochalasin B in comparison in doxorubicin in Balb/c mice challenged intradermally with M109 murine lung carcinoma. We also examine the effects of cytochalasin B against several other murine neoplastic cell lines (Lewis lung, LA4, B16F10, and M5076). Finally, we examine a potential mechanism of the antimetastatic activity of cytochalasin B by observing the effects of the agent on the secretion of N-acetylglucosaminidase (GlcNACase) by B16BL6 and B16F10 murine melanomas in vitro. The results of the study can be summarized as follows: 1) Cytochalasin B can be safely administered intravenously, intraperitoneally, and subcutaneously in murine models, with the maximum tolerated dose of all routes of administration being increased by liposome encapsulation. 2) Cytochalasin B can significantly inhibit the growth of tumors in mice challenged with M109, Lewis lung, LA4, B16F10, or M5076, producing long-term survival against lung carcinomas and adenocarcinomas (M109, Lewis lung, and LA4) and B16F10 melanoma, but not M5076 sarcoma. These effects were comparable to intraperitoneally administered doxorubicin. 4) Low concentrations of cytochalasin B inhibit the secretion of GlcNACase, indicating that cytochalasin B may inhibit metastatic progression by mechanisms not directly associated with its influence on cell adhesion and motility.