In vitro and in vivo effects of polyhaemoglobin-tyrosinase on murine B16F10 melanoma

Melanoma is an increasingly common fatal skin cancer. Many groups are carrying out research on potential treatments for melanoma. One of these approaches has shown that lowering tyrosine can inhibit the growth of melanoma in cell cultures and of B16BL6 melanoma in mice. However, humans cannot tolerate tyrosine-restricted diets for lowering tyrosine because of nausea, vomiting and weight loss. We report here our preparation and characterization of a novel soluble polyhaemoglobin-tyrosinase complex. This preparation prevents native tyrosinase from having adverse effects and from rapid removal after injection. The preparation inhibited murine B16F10 melanoma cell growth in culture and delayed its growth in a mice model. Intravenous injection of the preparation lowers the systemic tyrosine level without causing adverse effects such as vomiting and weight loss in mice. It is therefore possible that this complex could be useful in the treatment of human melanoma.     

In vitro assay demonstrates similar invasion profiles for B16F1 and B16F10 murine melanoma cells

The variants of the B16 murine melanoma cell line were assayed for their invasive characteristics in the membrane invasion culture system (MICS) and concomitantly tested for their ability to form lung metastases in vivo. Specifically, the B16F1 (low metastatic variant) and the B16F10 (high metastatic variant) murine melanoma cell lines were examined for their ability to invade human amniotic basement membranes (BMs) in vitro and simultaneously examined for lung colony formation in vivo. The B16F1 and B16F10 cell lines both demonstrated similar invasion profiles over 72 h with the total percent invasion through the BMs for both cell lines not exceeding 5.0%. In vivo observations reconfirmed the significant difference in the metastatic capabilities of the 2 variants. These data suggest that tumor cells with differing metastatic propensities can invade an amniotic BM at similar rates, but their survival and metastatic lesion forming capabilities in vivo may vary considerably.     

In vivo characterization of a doxorubicin resistant B16 melanoma cell line

A doxorubicin-resistant line of B16 melanoma (B16VDXR) was obtained in vitro by continuous exposure to increasing concentrations of doxorubicin of an in vitro line (B16V) derived from the in vivo transplanted B16 melanoma. When injected s.c. into mice, B16VDXR exhibited histological features, metastatic behaviour, doubling time and tumourigenic potential similar to those of the parental B16V line. Tumours obtained by implantation of B16VDXR, however, had longer latency and permitted a longer survival time than B16V and had, as in vitro, a higher DNA content. After i.v. inoculation, B16VDXR cells had lower lung colonizing capability compared to B16V. B16V and B16VDXR had significantly lower metastatic potential compared to the B16 melanoma from which they derived. Doxorubicin treatment significantly delayed the growth of B16 and B16V transplanted s.c. and increased the life span of animals bearing B16V. B16VDXR was resistant to doxorubicin treatment when the in vitro resistance index was greater than 100. While the doxorubicin-resistance phenotype was stable in vitro for 50 passages, in vivo the resistance phenotype was lost in 5 passages and tumours grown from s.c. inocula of mixtures of similar percentages of sensitive and resistant cells behaved as sensitive tumours. Cis-diamminedichloroplatinum (II), although marginally active in animals bearing B16V, was highly effective in B16VDXR bearing animals, suggesting a collateral cis-diamminedichloroplatinum (II) sensitivity of the B16VDXR line. After a single i.v. administration, doxorubicin reached initially, in the B16VDXR line, levels similar to those found in the B16 and B16V lines, but its release was faster from the resistant line in comparison with the sensitive ones. Doxorubicin-resistance was not overcome by more frequent treatments with doxorubicin. This doxorubicin-resistant tumour line obtained in vitro and used as a first in vivo transplant, may be a suitable metastaizing model for in vivo study of the mechanisms of resistance and of collateral sensitivity and for screening new drugs.     

Regression of established B16F10 melanoma with a recombinant Listeria monocytogenes vaccine.

We have previously shown that Listeria monocytogenes, a gram-positive, facultative intracellular bacterium, is a potent vector for targeting tumor-specific antigens to the immune system. In the present study, we extend these studies to the highly tumorigenic mouse melanoma B16F10, transduced with a model tumor antigen. We are able to induce the regression of primary tumors and established lung metastases by parenteral immunization with a L. monocytogenes recombinant that expresses the same antigen. Adjunctive therapy with granulocyte macrophage colony-stimulating factor or a vaccinia-based vaccine does not result in an improved cure rate over the L. monocytogenes vaccine alone. Tumor regression is accompanied by the expression of inflammatory cytokines in the tumor.     

Nitric oxide-mediated regulation of hypoxia-induced B16F10 melanoma metastasis

Tumour hypoxia is associated with resistance to therapy and with increased invasion and metastatic potential. Recent studies in our laboratory have shown that the hypoxic up-regulation of tumour cell invasiveness and chemoresistance is in part due to reduced nitric oxide (NO) signaling. Using B16F10 murine melanoma cells, we demonstrate here that the increased metastatic potential associated with exposure to hypoxia is mediated by a reduction in cGMP-dependent NO-signaling. Pre-incubation of B16F10 cells in hypoxia (1% vs. 20% O(2)) for 12 hr increased lung colonization ability by over 4-fold. This effect of hypoxia on metastasis was inhibited by co-incubation with low concentrations of the NO-mimetic drugs glyceryl trinitrate (GTN) and diethylenetriamine NO adduct (DETA/NO). In a manner similar to hypoxia, pharmacological inhibition of NO synthesis resulted in a significant increase in lung nodule formation, an effect that was prevented by co-incubation with GTN. An important NO-signaling pathway involves the activation of soluble guanylyl cyclase and the consequential generation of cGMP. Culture in the presence of a non-hydrolysable cGMP analogue (8-Br-cGMP) abrogated the hypoxia-induced lung nodule formation, suggesting that the effects of NO on metastasis are mediated via a cGMP-dependent pathway. These findings suggest that a novel mechanism whereby hypoxia regulates metastatic potential involves a downstream inhibition of cGMP-dependent NO signaling.     

Effects of N-acetyl-glucosamine-coated glycodendrimers as biological modulators in the B16F10 melanoma model in vivo

Glyco-coat changes on cancer cells due to aberrant glycosylation are potential targets for immune recognition through lectin-like receptors on immune cells. These cells include natural killer (NK), CD8+ and CD4+ lymphocytes, all reported to have, together with cytokines, important functions in antitumor immunity. The aim of this study was to evaluate a possible role of synthetic monodisperse multivalent neo-glycoconjugates, namely glycodendrimers, as a new approach to anticancer immune modulation through carbohydrate-mediated immune recognition. Octavalent polyamidoamine dendrimers functionalized with N-acetyl-glucosamine residues (PAMAM-GlcNAc8), with in vitro high affinity for the recombinant lymphocyte receptor NKR-P1A, were employed. To follow the fate of the compound, a fluorescent marker was conjugated to the tetra-branched semi-component of the dendrimer. Tumor development and immunity were evaluated in C57BL/6 mice. Animals were inoculated with B16F10 melanoma cells and underwent different protocols of PAMAM-GlcNAc8 administration. Advantages on survival and reduction of tumor growth were obtained in dose-dependent manner, by IP route. Increase of CD69+ cells in the spleen and their appearance inside the tumors, early progressive release of IL-1beta, a later production of INFgamma and IL-2 concomitant to an increment of CD4+ cells were observed. Cytotoxicity assays, performed ex vivo, showed an enhanced NK cell activity proportioned to the percentage of activated NK cells. Our data suggest that well-defined multivalent neo-glycoconjugates can stimulate an antitumor immune response engaging both innate and acquired immunity.     

Estrous cycle influences organ-specific metastasis of B16F10 melanoma cells

Some clinical studies suggest that timing of surgery during specific menstrual phases may influence the chances of survival for premenopausal women with breast cancer, whereas other studies failed to find this effect. Because most breast cancer deaths are attributable to metastases, we hypothesized that aspects of the metastatic process might be sensitive to cyclic hormonal fluctuations. Our goal was to develop a mouse model to assess possible mechanisms for the effect of the menstrual cycle on metastatic ability. To separate the effects of the hormonal milieu on the host versus the cancer cells, we began by using melanoma cells. We report here that the estrous phase at the time of entry of B16F10 melanoma cells into the circulation leads to marked differences in organ-specific metastasis, suggesting that this concept merits additional study.     

Tumor regression of B16F10 melanoma in vivo by prevention of neovascularization: study on theophylline

The aim of this study was to determine the effect of theophylline on neovascularization and tumor regression in murine B16F10 melanoma. Theophylline had no direct toxicity to host and significantly reduced (p < 0.001) tumor volume and neovascularization in B16F10 melanoma implanted murine model. The effect of theophylline on neovascularization was observed distinctly in histologic analysis. This effect is mediated, in part by blocking endothelial cell proliferation, thereby preventing neovascularization of the tumor. Further investigations with theophylline can elucidate the exact mechanism of action which characterize neovascularization activity.     

Neoadjuvant immunotherapy with interferon of the spontaneously metastasizing murine B16F10L melanoma.

We have previously demonstrated that administration of interferon a/b (IFN) for 4-5 days after challenge with a transplantable Moloney sarcoma virus-induced tumor completely inhibited tumor development. In the present study, we examined the therapeutic effects of IFN on mortality induced by metastatic dissemination of the B16F10L murine melanoma. IFN was administered at various times in relation to the surgical removal of primary tumor: days -5 to -1 prior to tumor excision (neo-adjuvant protocol), or for 5 days after tumor excision, beginning on days 1, 6 or 11 after excision of the primary tumor (adjuvant protocols). The neo-adjuvant protocol was superior to all other protocols, significantly increasing percentage survival (56% vs. 0%) and median survival time (greater than 84 days vs. 33 days) compared to untreated controls, as well as to all adjuvant protocols. In contrast, IFN treatment on days 1 to 5 after excision of the primary tumor decreased median survival time of cases compared to untreated controls (20 days vs. 33 days). Both IFN-induced inhibition and enhancement of metastatic dissemination were dose-dependent, with higher amounts of IFN producing greater inhibition or enhancement. The superior therapeutic efficacy of the neo-adjuvant IFN treatment was associated with increased spleen and lung-derived natural killer cell cytolytic activity (on days -4, 0 and 2) followed by a later (day 13) increase in lung-associated cytolytic T-cell responses.     

Effect of retinoic acid on integrin receptors of B16F10 melanoma cells

The intriguing problem of metastasis requires the spreading of metastatic cells through the basement membrane barrier. The interaction of the basement membrane with the metastatic cell is a cell surface activity involving the function of integrin receptors. Integrins are a group of alpha,beta heterodimeric proteins responsible for transducing intracellular signals on binding to the extracellular matrix proteins present in the basement membrane. To understand the role of integrin receptors in tumor metastasis, the cell surface receptor functions were modulated by All Trans Retinoic Acid (ATRA) treatment in B16F10 tumor cells. Our experimental results clearly indicate that All Trans Retinoic Acid (ATRA) inhibit metastatic potential of highly metastatic B16F10 melanoma cells by 1) downregulating the cell surface integrin receptors against ECM proteins specially laminin and vitronectin and 2) by inhibiting the 72 kd collagenase activity.     

Alphastatin-C a new inhibitor of endothelial cell activation is a pro-arteriogenic agent in vivo and retards B16-F10 melanoma growth in a preclinical model

Most characterized angiogenic modulators are proteolytic fragments of structural plasma and/or matrix components. Herein, we have identified a novel anti-angiogenic peptide generated by the in vitro hydrolysis of the C-terminal moiety of the fibrinogen alpha chain, produced by the snake venom metalloprotease bothropasin (SVMP), a hemorrhagic proteinase in Bothrops jararaca venom. The 14-amino acids peptide (alphastatin-C) is a potent antagonist of basic fibroblast growth factor, induced endothelial cell (HUVEC-CS) proliferation, migration and capillary tube formation in matrigel. It also inhibits cell adhesion to fibronectin. The basis of the antagonism between bFGF and alphastatin-C is elucidated by the inhibition of various bFGF induced signaling pathways and their molecular components modification, whenever the combination of the stimuli is provided, in comparison to the treatment with bFGF only. To corroborate to the potential therapeutic use of alphastatin-C, we have chosen to perform in vivo assays in two distinct angiogenic settings. In chick model, alphastatin-C inhibits chorioallantoic membrane angiogenesis. In mouse, it efficiently reduces tumor number and volume in a melanoma model, due to the impairment of tumor neovascularization in treated mice. In contrast, we show that the alphastatin-C peptide induces arteriogenesis, increasing pial collateral density in neonate mice. alphastatin-C is an efficient new antiangiogenic FGF-associated agent in vitro, it is an inhibitor of embryonic and tumor vascularization in vivo while, it is an arteriogenic agent. The results also suggest that SVMPs can be used as in vitro biochemical tools to process plasma and/or matrix macromolecular components unraveling new angiostatic peptides.     

Suramin augments the antitumor and antimetastatic activity of pentoxifylline in B16F10 melanoma

Rapid tumor growth and metastasis are 2 major problems associated with treatment of malignant melanoma. Therefore, drugs that can intervene these processes are of clinical importance. Pentoxifylline (PTX), a methyl xanthine derivative, has been shown to inhibit B16F10 melanoma tumor growth and metastasis. We hypothesized that suramin when combined with PTX enhances its antineoplastic effects, which we have examined using the B16F10 mouse melanoma model. Suramin in simultaneous or sequential combination potentiated the cytotoxic effects of PTX on B16F10 cells. PTX arrested cells in the G0-G1 phase and suramin augmented the effects. Both the drugs inhibited F10 adhesion to laminin, matrigel and collagen type IV and showed enhanced inhibition in combination The combination also demonstrated significantly higher inhibition in cell motility (p = 0.002) and invasion through matrigel (p = 0.005) as compared to the single agents. Suramin synergized with PTX in its effects on secretion of MMP-9 gelatinase. DBA2/J mice implanted with intradermal B16F10 tumor were used as a model to study tumor growth. Animals were intratumorally treated with 50 mg/kg of PTX, 10 mg/kg of suramin and their combinations. Simultaneous administration of the drugs inhibited tumor growth by 5- to 6-folds. Tumor growth was completely blocked in sequential regimen with regression in some cases. The number and size of metastatic nodules on lung was also reduced significantly by the combination treatment. In conclusion, the novel combination of PTX and suramin has synergistic antitumor and antimetastatic activity in B16F10 melanoma and may be a promising approach in treatment of patients suffering from malignant melanoma.     

Liposomal encapsulation enhances the antitumour efficacy of the vascular disrupting agent ZD6126 in murine B16.F10 melanoma

Vascular disrupting agents (VDAs) are able to affect selectively tumour endothelial cell morphology resulting in vessel occlusion and widespread tumour cell necrosis. However, single-agent antitumour activity of VDAs is typically limited, as tumour regrowth occurs rapidly following drug treatment. To improve the therapeutic effectiveness of VDAs, we investigated liposomal targeting using ZD6126 as a model VDA. ZD6126 is a phosphate-prodrug of the tubulin-binding vascular disrupting agent ZD6126 phenol. ZD6126 was encapsulated into long circulating PEG-liposomes for passive targeting and PEG-liposomes conjugated with peptide ligands containing the RGD-motif for active targeting to alpha(v)-integrins on tumour endothelial cells. ZD6126 could be stably encapsulated, and liposomes displayed minimal leakage in vitro (<10% in 3 weeks). In vivo, upon intravenous injection, free ZD6126 was rapidly converted into ZD6126 phenol, which was cleared from the circulation within minutes. In contrast, ZD6126 encapsulated into either RGD-targeted or PEG liposomes showed prolonged blood circulation times (t(1/2)=10 h), and ZD6126 phenol exposure was also prolonged (t(1/2)=8 h). Both liposomal formulations displayed tumour accumulation plus hepatosplenic uptake by local macrophages. The altered pharmacokinetics and tissue distribution profiles of both liposomal ZD6126 formulations resulted both in single-dose and multiple-dose regimes, in improved therapeutic efficacy in established murine B16.F10 melanomas compared with free ZD6126. The passively and actively targeted liposomes showed equal antitumour efficacy, indicating that delivery of ZD6126 to the tumour tissue may suffice to disrupt tumour blood vessels without the need for specific targeting to the tumour endothelium.     

Inhibition by analogues of L-tyrosine transport by B16/F10 melanoma cells

The effect of a number of L-tyrosine (L-Tyr) analogues on L-Tyr uptake by B16/F10 malignant melanocytes is reported. This amino acid can be taken up by two of the most ubiquitous transport systems found in animals cells, L and presumably ASC. L-Tyr analogues devoid of the amino group, like p-hydroxyphenyl pyruvic acid and related compounds, and L-Tyr analogues devoid of the carboxyl group, such as tyramine, do not affect L-Tyr uptake. The other aromatic amino acids, L-Phe and L-Trp, and the L-Tyr analogues DL-m-Tyr, L-diiodotyrosine and L-dopa, strongly inhibit the uptake of L-Tyr. This suggests that these chemicals are transported more efficiently than L-Tyr. The ASC system does not show stereospecificity, but the L system has greater affinity for L-Tyr than for D-Tyr. The ASC system also has greater affinity for tyrosine isomers with the hydroxyl group in the ortho and meta positions. The presence of a methyl group at the alpha-carbon of L-Tyr and L-dopa also increases the affinity of the ASC system for these agents. In contrast, alpha-methylation decreases the affinity of the L system in comparison to L-Tyr. Finally, L-Tyr esters do not inhibit, but stimulate the transport of L-Tyr, mainly by the ASC system.     

In vivo and in vitro characteristics of interleukin 6-transfected B16 melanoma cells.

Interleukin 6 (IL-6) is a multifunctional cytokine important in the inflammatory response. Its potential role as an antitumor agent has been suggested by its demonstrated activity in a variety of tumor models. The mechanism of antitumor activity has been proposed to be its enhancement of cytotoxic T-cell function. In the current work we demonstrate clear antitumor activity for this cytokine in a nonimmunogenic tumor system. B16 melanoma cells transfected with the human IL-6 complementary DNA demonstrated slower tumor growth in vivo. Tumors that developed from these cells had a prominent stromal matrix, an easily recognized infiltration of inflammatory cells, fewer mitotic figures, and fewer blood vessels. These in vivo findings corresponded with a greater adhesion of the IL-6-transfected B16 cells to stromal matrix proteins (laminin, fibronectin, and vitronectin) and a less prominent vascular response in an intradermal angiogenesis assay. Therefore, we propose that with weakly antigenic tumors, such as B16 melanoma, IL-6 may mediate important antitumor responses by nonspecific proinflammatory mechanisms.     

小鼠黑色素瘤B16F10-Red细胞株的建立与生物学特性分析

目的:构建表达香菇珊瑚红色荧光蛋白(discosomasp red fluorescent protein,DsRed)的小鼠黑色素瘤B16F10-Red细胞株,并检测其生物学特性.方法:用GenEscortTMⅡ转染试剂将pDsRed质粒导入小鼠黑色素瘤B16F10细胞,G418加压培养联合极限稀释法建立稳定、高水平表达DsRed的单克隆细胞系.FCM检测B16F10和B16F10-Red细胞的细胞周期.比较B16F10-Red和B16F10细胞的克隆球形成能力和小鼠体内致瘤能力.结果:稳定表达DsRed的小鼠黑色素瘤B16F10-Red细胞株基本保持了其亲代细胞的特征,能在C57BL/6小鼠腹部皮下形成肿瘤并继续生长和转移.结论:B16F10-Red细胞株构建成功,其移植瘤模型成瘤率和转移情况同B16F10肿瘤相比无明显差别.     

Decreased B16F10 melanoma growth and impaired vascularization in telomerase-deficient mice with critically short telomeres.

Endothelial cell function and angiogenesis are modulated by aging. However, the underlying molecular mechanisms are largely unknown. Here we show that in telomerase-deficient mice Terc(-/-), short telomeres result in a sharp decrease in angiogenesis in both Matrigel implants and murine melanoma grafts. In the latter model, decreased microvessel counts in late generation Terc(-/-) mice led to diminished tumor cell proliferation and increased tumor cell apoptosis, resulting in a lower tumor growth rate. Our results indicate that telomere length is a key molecular determinant of angiogenic potential in vivo and that telomere length modifiers and telomerase inhibitors could be useful antiangiogenic agents.