Theophylline administration markedly reduces hepatic and pulmonary implantation of B16-F10 melanoma cells in mice

Theophylline-treated B16-F10 melanoma cells show a lower experimental metastatic potential in vivo. To identify the possible mechanism(s) involved and on the basis of previous reports, we tested the induction of apoptosis in B16-F10 cells. Fluorescence activated cell sorter (FACS) analysis and p53 overexpression in theophylline-treated B16-F10 melanoma cells appeared to suggest enhanced cell death by apoptosis. The in vivo effects of orally administered theophylline in mice were investigated using different treatment schedules in mice that had undergone hepatic or pulmonary colonization with tumour cells. Mice received theophylline in their drinking water according to different protocols: (i) from 3 days before tumour cell inoculation until animal sacrifice ('early treatment'); (ii) from 3 days before until 3 days after tumour cell inoculation ('short treatment'); or (iii) from 3 days after tumour cell inoculation until animal sacrifice ('late treatment'). In the 'early treatment' group, the number of melanoma foci was reduced by 92.3% in the liver and 81.4% in the lung compared with control animals (P < 0.001). In the 'short treatment' group, there was an 80.2% and 72.2% reduction in liver and lung metastases, respectively (P < 0.001). In the 'late treatment' group, the inhibition of metastasis was 59.7% for liver and 45.3% for lung (P < 0.005). Survival studies showed that 50% of the 'early' theophylline-treated animals died 33.2 +/- 2.0 days after intrasplenic injection (control group: 23.1 1.8 days; P < 0.001) and 33.9 +/- 2.5 days after tail vein injection (control group: 24.1 +/- 1.4 days; P < 0.001). Taken together, these observations provide useful information for the potential clinical application of theophylline as a chemotherapeutic agent against malignant melanoma.     

Differences in the post-translational modification of proteins by polyamines between weakly and highly metastatic B16 melanoma cells

The identification of (γ-glutamyl)polyamines in proteolytic digest of proteins from the cytosolic and particulate fractions of B16-F10 and B 16-F1O^Lr6 cell lines, originating from a spontaneous tumor in C57BL/6 mice, indicates that polyamines are incorporated into melanoma cell proteins by transglutaminases (TGases-EC 2.3.2.13). The levels of spermidine-derived protein cross-links were found to be inversely related with the meta-static potential of the 2 melanoma lines. Characterization of TGase activity in the 2 tumor cell lines showed 3 types of enzyme. The soluble cellular TGase activity (TGase C) was higher, and increased more, during the growth of the least metastasizing clone B16-F10^Lr6 than in the B16-F10 line, which is the most metastasizing. Consistently, NN⁸-bis(γ-glutamyl) spermidine, which is responsible for protein cross-link formation, was present in greater amount in B16-F10Lr6 cells. The enhancement by theophylline of soluble-TGase activity and spermidine-dependent protein cross-links of B16-F10 cells reduced, with linear dose dependence, the ability of these cells to penetrate through human fibronectin-coated membrane in an in vitro assay of invasiveness. Our data confirm and extend earlier observations indicating that the propensity of a tumor to metastasize can be indirectly related to intracellular levels of TGase activity, and provide the basis for some speculation concerning the role of polyamines as modifiers of murine melanoma cell proteins in metastasis.     

Comparison of the in vitro and in vivo effects of retinoids either alone or in combination with cisplatin and 5-fluorouracil on tumor development and metastasis of melanoma

Purpose  Retinoids have previously been reported to inhibit proliferation of melanoma cell lines in vitro. However, the relative antimetastatic efficacy of various retinoids on melanoma in vivo is unknown. Therefore, we investigated the effects of different retinoids on the invasion and metastasis of murine melanoma B16-F10 cells in vitro and in vivo. Based on the findings, the antitumor effects of a selected retinoid either alone or in combination with cisplatin were also investigated in a preclinical mouse melanoma model. Methods  Cell proliferation and invasion analyses of murine melanoma B16-F10 cells were assessed in the presence of different retinoids, either alone or in combination with cisplatin (CDDP) or 5-fluorouracil (5-FU). Experimental lung metastasis assay was performed in this study to investigate the antimetastatic efficacy of retinoids. Additionally, a mouse melanoma model was used to assess the antitumor efficacy of a selected retinoid in combination with cisplatin. Results  Retinoids showed significant antiproliferation and anti-invasion effects on murine melanoma B16-F10 cells. Pretreatment with retinoids increased the sensitivity to CDDP but not to 5-FU in in-vitro. Moreover, the number of metastatic colonies formed in the lungs of mice injected intravenously with B16-F10 cells was significantly reduced by injecting the respective retinoid once a day for 10 days. Treatment with a combination of cisplatin and 13-cis-retinoic acid resulted in a significant reduction in primary tumor size and the number of lung metastatic nodules in melanoma-bearing mice. Conclusion  These results suggest that retinoids not only exhibit antimetastatic effect, but also enhance the antitumor activity of cisplatin in vivo.     

Decrease of polyamine levels and enhancement of transglutaminase activity in selective reduction of B16-F10 melanoma cell proliferation induced by atrial natriuretic peptide

The atrial natriuretic peptide (ANP) at physiological levels reduced the proliferation of highly metastatic murine (B16-F10) and human (SK-MEL 110) melanoma cell lines whereas rat aortic smooth muscle (RASM) cells were unaffected. In RASM cells, the levels of proliferation markers (putrescine, spermidine and spermine) increase after 24 h of epidermal growth factor (EGF) stimulation (RASM-EGF), but strongly decrease after 24 h of exposition to ANP. The B16-F10 cell line, which received no EGF stimulation, showed a similar decrease in polyamine content after ANP treatment. Furthermore, the enzymatic activity of a differentiation marker (transglutaminase) was increased for both RASM-EGF and B16-F10 cells after 24 h of treatment with 10(-10) mol/l ANP, concomitantly with the observed inhibition of polyamine biosynthesis and cell growth. Data obtained on B16-F10 cells treated with 8Br-GMPc or with an ANP analogue (cANF) support the involvement of the type C ANP receptor (NRP-C) in hormone effects. From the overall results, it appears that ANP may play a role in the inhibition of cellular growth under hyperproliferative conditions, as shown for RASM-EGF cells. The B16-F10 melanoma cell line showed similar results, but in the absence of mitogen stimulation. This observation suggests that the constitutive hyperproliferative state of tumor cells may be a sufficient condition to favor the ANP inhibitory effects on cell growth. This finding is particularly interesting in the light of a possible use of ANP as a potential selective antineoplastic agent.     

Correlation of gangliosides GM2 and GM3 with metastatic potential to lungs of mouse B16 melanoma

Ten B16 mouse melanoma cell lines with increasing metastatic potential to lungs (B16LuF1 to B16LuF10) were generated by in-vitro & in-vivo selection technique starting with B16F1 melanoma cell line. The number of metastatic tumor nodules in lungs rose with increasing metastatic potential. Tumor cell gangliosides of B16LuF1 to B16LuF10 cell lines, analysed and compared with TLC, showed eight major ganglioside bands. Band1 to band6 corresponded with standard gangliosides GT1b, GD1b, GD1a, GM1, GM2 and GM3 respectively. Band7 and Band8 could not be identified. The concentration of total as well as individual ganglioside bands of B16LuF1 to B16LuF10 cells appeared to rise with increasing metastatic potential. Gangliosides from the plasma of these cell lines (B16LuF1 to B16LuF10) maintained in-vivo in C57BL/6 mice on TLC analysis gave eight major ganglioside bands, similar to those of cells. Plasma gangliosides appeared to rise with increasing metastatic potential. However, it was interesting to see that only band5 and band6 gangliosides in plasma increased almost linearly with increasing metastatic potential. The remaining six ganglioside bands in the plasma did not show such correlation. Band5 and Band6 gangliosides corresponded with standard gangliosides GM2 and GM3 respectively. Gangliosides of the spent culture media, secreted by these cell lines in-vitro in tissue culture also gave eight major ganglioside bands, similar to that of cells. Spent culture media gangliosides appeared to increase with increasing metastatic potential. However, concentration of only band5 and band6 gangliosides of spent culture media increased almost linearly with increasing metastatic potential, thus further confirming the role of band5(GM2) and Band6(GM3) gangliosides in regulating metastatic potential of B16-melanoma cells to lung.     

Lu-ECAM-1-mediated adhesion of melanoma cells to endothelium under conditions of flow

Lu-ECAM-1 is a lung-derived, venular endothelial cell adhesion molecule. It promotes the selective adhesion of lung-metastatic B16-F10 melanoma cells to endothelium under static conditions and mediates colonization of the lungs by the same tumor cells. To test whether Lu-ECAM-1 by itself is sufficient to cause vascular arrest of B16-F10 cells, we measured here under conditions of flow tumor cell adhesion to endothelia that express different amounts of Lu-ECAM-1 on their surfaces. At physiological shear stresses, adhesion of B16-F10 melanoma cells to endothelia correlates positively with the amount of Lu-ECAM-1 expression on the endothelial cell surface and inversely with the level of the applied shear stress. Tumor cell trajectories are biphasic; i.e., B16-F10 melanoma cells initially move along the endothelial surface with a velocity similar to the theoretical velocity, then arrest within a fraction of a second. Arrest is permanent for most B16-F10 melanoma cells at all shear stresses tested. Tumor cells never engaged in a rolling motion prior to arrest. Masking of the Lu-ECAM-1 ligand on the surface of B16-F10 melanoma cells with soluble Lu-ECAM-1 impedes arrest of tumor cells on the surface of the test endothelium. Purified Lu-ECAM-1 also mediates B16-F10 arrest, but arrest is mostly transient at shear stresses of 0.59 dynes/cm² and higher, implying adhesion by single receptor/ligand bonds. Our data suggest that Lu-ECAM-1 plays a critical role in the recognition and initial arrest of murine melanoma cells in lung venules. © 1996 Wiley-Liss, Inc.     

Reduced tumorigenicity of B16-F10 mouse melanoma cells transfected with mycobacterial antigen 85A

Immunotherapy based on the administration of the mycobacterium bacillus Calmette-Guerin has been successfully used in the treatment of in situ transitional cell bladder cancer, and may be applicable to the treatment of cutaneous malignant melanoma. Antigen 85A (Ag85A) and heat shock protein 65 kDa (hsp65) are major secreted proteins of Mycobacterium species and potent stimulators of cell-mediated immunity. This study evaluated the ability of Ag85A and hsp65 gene transfection to limit tumor growth by B16-F10 mouse melanoma cells. Immunoblotting confirmed protein expression and secretion by B16-F10 cells that were transiently transfected with plasmid DNA containing the Ag85A or hsp65 gene. Groups of syngeneic C57BL/6 mice were injected subcutaneously with 1x10(5) untransfected B16-F10 cells or B16-F10 cells transiently transfected with either empty vector or vector containing the Ag85A or hsp65 gene. Ag85A-expressing B16-F10 cells exhibited a dramatic 76% reduction (p<0.05, Mann-Whitney U test) in tumor weight in comparison to empty vector controls at 14 days post-inoculation. In contrast, hsp65-transfected B16-F10 cells did not show any change in tumorigenicity. Decreased tumorigenicity by Ag85A-transfected B16-F10 cells was not due to a reduced ability of Ag85A-transfected B16-F10 cells to proliferate since both mock- and Ag85A-transfected B16-F10 cells showed increased in vitro proliferation in comparison to untransfected cells. Hematoxylin and eosin staining revealed that Ag85A-transfected B16-F10 tumors contained an inflammatory leukocyte infiltrate that was not present in hsp65-transfected tumors. Reduced tumor progression by Ag85A-transfected B16-F10 melanoma cells suggests that immunotherapy based on the transient induction of Ag85A expression may be an effective approach for the treatment of cutaneous malignant melanoma.     

Anti-tumoural activity of peripheral blood mononuclear cells against melanoma cells: discrepant in-vitro and in-vivo effects

As tumour cells use multiple mechanisms to escape from chemotherapeutic drugs, the anti-tumoural activity of naive mouse peripheral blood mononuclear cells was examined in this study, using a mouse melanoma cell subline resistant to doxorubicin (B16R). Multicellular spheroids are known to be the most adapted in-vitro model to mimic solid tumours in vivo and are used to investigate many aspects of tumour biology. For in-vitro studies, murine peripheral blood mononuclear cells recovered by Ficoll gradient centrifugation after caudal puncture were co-cultured with multicellular tumour spheroids of B16R cells. Morphological investigations show that peripheral blood mononuclear cells were gathered and focused around the spheroids after 14 h of co-culture and contacts were established within 32 h. Between 38 and 62 h of co-culture, the size of the spheroids decreased significantly. The peripheral blood mononuclear cells exerted cytolytic effects that correlated with the induction of cell death in spheroids of B16R melanoma cells. Immunological investigations to localize and identify peripheral blood mononuclear cells that exerted anti-tumoural effects have shown that spheroids were deeply infiltrated by monocytes/macrophages at a stage in which a significant cytolytic activity and a strong cell death rate were observed. For in-vivo studies, intratumoural injections of syngeneic naive peripheral blood mononuclear cells were administered. A weak potential in-vivo anti-tumoural effect of these cells was observed (inhibition of B16R melanoma growth by 20-25%) but the median survival time of mice treated with peripheral blood mononuclear cells did not increase compared with untreated control mice. Thus, despite anti-tumoural activities of peripheral blood mononuclear cells against the poorly immunogenic and highly metastatic chemoresistant B16 melanoma cells in vitro, a potential anti-melanoma effect in vivo, if present, did not increase the life span of B16R melanoma-bearing mice.     

Membrane lipids and enzymes of cultured high- and low-metastatic B16 melanoma variants

B16 melanoma cell variants were used to determine if the metastatic properties of these cells could be correlated to distinct plasma membrane, microsome, and mitochondrial membrane lipid compositions and membrane-bound enzyme activities in high- and low-metastatic cell variants, respectively. The high-metastatic B16-F10 melanoma cell membranes had lower cholesterol/phospholipid ratios, lower arachidonic acid content, lower polyunsaturated fatty acid content, higher phosphatidylcholine/phosphatidylethanolamine ratios, and higher succinate cytochrome c reductase activity than those of B16-F1 melanoma cell membranes. No differences in cholesterol/phospholipid ratio were noted in the mitochondria. Na+-K+-adenosinetriphosphatase activity and solubility of 5'-nucleotidase activity were also similar. The data indicate that the membrane lipid composition of B16-F10 melanoma cells is distinct from that of B16-F1 melanoma cells and may help to elucidate the molecular basis for the different metastatic properties of these cell lines in vivo.     

4—乙酰胺苯基维甲酸酯抑制肿瘤细胞侵袭重组基底膜及其作用机理

目的研究新维甲类化合物４－乙酰胺苯基维甲酸酯（４－ＡＰＲ）对Ｂ１６－Ｆ１０小鼠黑色素瘤细胞侵袭能力的抑制作用,探讨其作用机理。方法癌细胞侵袭能力用重组基底膜侵袭试验衡量;ＰＡＧＥ底物酶谱方法检测Ⅳ型胶原酶活性;斑点杂交检测ＣＮＥ－２Ｚ细胞ＴＩＭＰ－１ｍＲＮＡ表达;以细胞生长曲线评价药物对Ｂ１６－Ｆ１０细胞的生长抑制作用。结果在１０－５ｍｏｌ／Ｌ和１０－６ｍｏｌ／Ｌ时,４－ＡＰＲ对Ｂ１６－Ｆ１０小鼠黑色素瘤细胞侵袭重组基底膜的抑制率分别为５４．２％和４１．９％,对ＣＮＥ－２Ｚ细胞培养上清中的Ⅳ型胶原酶活性有降低作用。此外,４－ＡＰＲ可抑制Ｂ１６－Ｆ１０细胞与ＬＮ、ＦＮ和Ｍａｔｒｉｇｅｌ的粘附,诱导ＣＮＥ－２Ｚ细胞ＴＩＭＰ－１ｍＲＮＡ表达。结论４－ＡＰＲ抑制Ｂ１６－Ｆ１０细胞侵袭重组基底膜。４－ＡＰＲ的抗侵袭活性与抑制肿瘤细胞的粘附,降低肿瘤细胞培养上清中Ⅳ型胶原酶的活性和（或）诱导肿瘤细胞ＴＩＭＰ－１ｍＲＮＡ表达等有关。     

Laminin gamma 1 chain peptide, C-16 (KAFDITYVRLKF), promotes migration, MMP-9 secretion, and pulmonary metastasis of B16-F10 mouse melanoma cells

Laminin-1, a heterotrimer of alpha 1, beta 1, and gamma 1 chains specific to basement membrane, promotes cell adhesion and migration, proteinase secretion and metastases of tumour cells. Several active sites on the alpha 1 chain have been found to promote B16-F10 melanoma lung colonisation and here we have determined whether additional tumour promoting sites exist on the beta 1 and gamma 1 chains. Recently, we have identified novel cell adhesive peptides derived from laminin beta 1 and gamma 1 chains by systematic screening of synthetic peptides. Nine beta 1 peptides and seven gamma 1 peptides active for cell adhesion were tested for their effects on experimental pulmonary metastases of B16-F10 mouse melanoma cells in vivo. The most active adhesive peptide derived from the gamma 1 chain globular domain, C-16 (KAFDITYVRLKF), significantly enhanced pulmonary metastases of B16-F10 cells, whereas no other peptides showed enhancement. C-16 also stimulated migration of B16-F10 cells in the Boyden chamber assay in vitro. Furthermore, C-16 significantly induced the production of MMP-9 from B16-F10 cells. These results suggest that this specific laminin gamma 1 chain peptide has a metastasis-promoting activity and might be a new molecular target of anti-cancer treatment.     

Effects of DL-alpha-difluoromethylornithine on the growth and metastasis of B16 melanoma in vivo

The effects of DL-alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase, on the growth of experimental mouse B16-F10 melanoma cells were investigated. DFMO (3%) in drinking water was administered to B16-F10 melanoma-bearing mice. At 24 days, B16-F10 melanomas in DFMO-fed mice weighed 75% less than those in control mice (p less than 0.001). DFMO reduced putrescine and spermidine levels in B16-F10 melanoma by 98% and 84%, respectively, and prolonged the mean survival time from 25.9 +/- 1.2 to 35.7 +/- 2.2 days (p less than 0.001). The effects of DFMO on experimental metastasis were also investigated. DFMO treatment resulted in a significant decrease in pulmonary metastasis induced by i.v. injection of B16-F10 melanoma cells.     

Pentoxifylline modulates cell surface integrin expression and integrin mediated adhesion of B16F10 cells to extracellular matrix components

Our previous studies demonstrated that Pentoxifylline (PTX), a phosphodiesterase inhibitor, could inhibit the lung homing of B16-F10 melanoma cells in C57BL/6 mice. In this study we have looked at the effect of PTX on cell surface integrin expression and integrin mediated adhesion of B16-F10 melanoma cells. B16-F10 cells treated with PTX when injected through the tail vein of mice showed a 75% reduction in pulmonary nodules as compared to control untreated cells. PTX brought about a significant reduction in the integrin mediated adhesion of F10 cells to Fibronectin and Vitronectin (58.75% +/- 3.4 S.E and 60% +/- 1.7 S.E respectively if control was considered as 100%). This inhibition in adhesion was evident up to four hours only and treatment for 24 hours brought about an increase in adhesion (135.5% +/- 0.5 S.E). Flow cytometric analysis showed higher surface expressions of alphav, alpha5 and alphaIIb integrin subunits in B16-F10 as compared to the low metastatic cell line B16-F1 suggesting a role for these integrins in determining the metastatic potential. PTX brought about a significant decrease in the cell surface expression of alpha5, alphaIIb and beta1 integrin subunits but not that of the alphav subunit on B16-F10 cells. PTX also brought about a reduction in the total cellular protein levels of beta1 and alphav integrin subunits. Various isoforms of Protein Kinase C (PKC) has been shown to regulate integrin expression, localization and activity. Hence we looked at the effect of PTX on total cellular PKC activity. PTX brought about a significant reduction in total cellular PKC activity (82.66 +/- 0.593). Collectively our results indicate that the antimetastatic action of PTX is mediated, at least in part through its effects on adhesion and the surface expression of specific integrin receptors.     

Comparative analysis of immunocritical melanoma markers in the mouse melanoma cell lines B16, K1735 and S91-M3

The mouse melanoma cell lines B16, K1735 and Cloudman S91-M3 (and various sublines) are frequently used as melanoma models. Extensive comparative data of their immunological features are not available. In order to define the immunological profiles of these cell lines, relevant tumour markers were studied. S91-M3 melanoma cells constitutively expressed high levels of major histocompatibility complex (MHC) I, in contrast to K1735-M2 and B16-F1 cells. MHC II expression was restricted to B16-F1 cells following interferon-gamma treatment. Tyrosinase, tyrosinase-related protein-2 and gp100 were detected in B16-F1 and S91-M3 cells, but not in K1735-M2 cells. Constitutive surface expression and secretion of intercellular adhesion molecule-1 was found on S91-M3 cells. No substantial secretion of interleukin-10 could be detected. In contrast, low levels of latent transforming growth factor-beta were found in the cell supernatants of B16-F1 and K1735-M2 cells. The expression pattern of Fas, FasL and FLICE inhibitory protein was comparable in all three cell lines. Thus our findings indicate that each cell line presents a characteristic immunological profile, confirming that B16-F1 is an appropriate murine tumour model for tumours with low levels of MHC I but expressing melanoma-associated antigens. S91-M3 represents a complementary, more immunogenic model. In contrast, K1735-M2 does not seem to be an appropriate model for melanoma.     

Fatty acid synthase inhibition with Orlistat promotes apoptosis and reduces cell growth and lymph node metastasis in a mouse melanoma model

Fatty acid synthase (FASN) is the enzyme responsible for the endogenous synthesis of the saturated fatty acid palmitate. In contrast to most normal cells, malignant cells depend on FASN activity for growth and survival. In fact, FASN is overexpressed in a variety of human cancers including cutaneous melanoma, in which its levels of expression are associated with a poor prognosis and depth of invasion. Here, we show that the specific inhibition of FASN activity by the antiobesity drug Orlistat or siRNA is able to significantly reduce proliferation and promote apoptosis in the mouse metastatic melanoma cell line B16-F10. These results prompted us to verify the effect of FASN inhibition on the metastatic process in a model of spontaneous melanoma metastasis, in which B16-F10 cells injected in the peritoneal cavity of C57BL/6 mice metastasize to the mediastinal lymph nodes. We observed that mice treated with Orlistat 48 hr after the inoculation of B16-F10 cells exhibited a 52% reduction in the number of mediastinal lymph node metastases, in comparison with the control animals. These results suggest that FASN activity is essential for B16-F10 melanoma cell proliferation and survival while its inactivation by Orlistat significantly reduces their metastatic spread. The chemical inhibition of FASN activity could have a potential benefit in association with the current chemotherapy for melanoma.     

The fatty acid synthase inhibitor orlistat reduces experimental metastases and angiogenesis in B16-F10 melanomas

Background:Fatty acid synthase (FASN) is overexpressed and associated with poor prognosis in several human cancers. Here, we investigate the effect of FASN inhibitors on the metastatic spread and angiogenesis in experimental melanomas and cultured melanoma cells.Methods:The lung colonisation assay and cutaneous melanomas were performed by the inoculation of mouse melanoma B16-F10 cells in C57BL6 mice. Blood vessel endothelial cells (RAEC and HUVEC) were applied to determine cell proliferation, apoptosis, and the formation of capillary-like structures. Vascular endothelial growth factor A (VEGFA) expression was evaluated by quantitative RT-PCR and ELISA in B16-F10, human melanoma (SK-MEL-25), and human oral squamous carcinoma (SCC-9) cells. Conditioned media from these cancer cell lines were used to study the effects of FASN inhibitors on endothelial cells.Results:B16-F10 melanoma-induced metastases and angiogenesis were significantly reduced in orlistat-treated mice. Fatty acid synthase inhibitors reduced the viability, proliferation, and the formation of capillary-like structures by RAEC cells, as well as the tumour cell-mediated formation of HUVEC capillary-like structures. Cerulenin and orlistat stimulated the production of total VEGFA in B16-F10, SK-MEL-25, and SCC-9 cells. Both drugs also enhanced VEGFA(121), (165), (189,) and (165b) in SK-MEL-25 and SCC-9 cells.Conclusion:FASN inhibitors reduce metastasis and tumour-induced angiogenesis in experimental melanomas, and differentially modulate VEGFA expression in B16-F10 cells.     

三肽化合物酪丝缬肽对小鼠黑色素瘤细胞B16-F10侵袭和转移的抑制作用

背景与目的:三肽化合物酪丝缬肽(tyroservaltide,YSV)对实验性肝癌具有明显抑制作用,本研究观察YSV对小鼠黑色素瘤细胞B16-F10侵袭和转移的抑制作用。方法:MTT法检测YSV对B16-F10的细胞毒作用;利用基质胶Matrigel研究YSV对细胞粘附能力的影响;用Transwell小室侵袭模型研究YSV对肿瘤细胞侵袭能力的改变;经C57/BL6小鼠尾静脉注射B16-F10细胞,建立人工肺转移模型,观察YSV对B16-F10肺转移的影响;免疫组化方法观察YSV对细胞间粘附分子(intercellularadhesionmolecule-1,ICAM-1)在肺组织中表达水平的影响。结果:100μg/mlYSV作用48h对B16-F10细胞的增殖抑制率为24.36%;作用24h对B16-F10细胞在Matrigel胶上的粘附抑制率为36.51%;10μg/mlYSV作用48h对B16-F10细胞的侵袭抑制率为36.53%;640μg·(kg·d)-1YSV抑制B16-F10的肺转移,抑制率为62.21%;YSV组ICAM-1的组织量明显少于生理盐水组。结论:YSV具有抑制B16-F10生长和侵袭转移的作用。     

Control of melanogenesis in mouse melanoma cells of varying metastatic potential.

The control of melanin production, tyrosinase activity, and cell replication by melanocyte-stimulating hormone (MSH) and cyclic AMP (cAMP) was examined in differentially metastasizing B16 mouse melanoma variants. In B16-F1 cells (low metastatic potential), MSH or cAMP greatly elevated tyrosinase activity and melanin content while inhibiting cell replication. The same parameters in B16-F5 cells (intermediate metastatic potential) were altered to a much lesser degree, whereas B16-F10 cells (high metastatic potential) were not significantly affected by MSH or cAMP. Therefore, a correlation exists between loss of hormonal regulation and increased metastatic potential.     

Loss of metastatic responsiveness to cell shape modulation in a newly characterized B16 melanoma adhesive cell variant

Repeated selection of an adherent subpopulation from B16-F1 melanoma cells growing in suspension culture on poly(hydroxyethylmethacrylate) [poly(HEMA)] coated plates resulted in the isolation of an adherent variant designated B16-A10. B16-A10 cells are more adherent to poly(hydroxyethylmethacrylate) coated plates than are B16-F1 cells and express an organized actin structure characteristic of highly adherent low metastatic cells as opposed to the poor cytoskeletal organization of B16-F1 cells. Upon growth in suspension, B16-A10 cells do not acquire the enhanced metastatic capability characteristic of B16-F1 cells and they express similar lung colonizing ability irrespective of the culture conditions. The increased metastatic ability of B16-F1 cells in suspension culture has previously been associated with the decreased accessibility of surface proteins to lactoperoxidase catalyzed iodination and with the increased expression of sialylated peanut agglutinin-binding oligosaccharides on these proteins. B16-A10 cells which show no cell shape induced increase in metastatic ability do not undergo alteration in either of these two properties in suspension culture. The absence of these two phenomena on B16-A10 cells grown in suspension indicates that they are interrelated and involved in the increased metastatic ability of B16-F1 cells grown in suspension.