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.     

Effects of alpha-difluoromethylornithine on the growth of experimental Wilms' tumor and renal adenocarcinoma

Because polyamines are essential for cellular growth and differentiation, and because human renal carcinomas have spermidine levels that are higher than those in normal renal tissue, effects of 2-difluoromethylornithine (DFMO) on the growth of experimental renal tumors were investigated. DFMO is a specific enzyme-activated irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme controlling polyamine biosynthesis. DFMO (2%) in drinking water was administered to BALB/c mice with intrarenal transplants of a renal adenocarcinoma cell suspension and to Wistar/Furth rats with s.c. transplants of a Wilms' tumor. At 28 days, renal carcinomas in DFMO-fed mice weighed 72% less than those in control animals (p less than 0.001). Wilms' tumor weight was not affected by DFMO feeding. DFMO caused 72 to 75% inactivation of ornithine decarboxylase activity and reduced putrescine levels in renal carcinoma and Wilms' tumor, reduced spermidine levels in Wilms' tumor, and apparently raised spermine levels in the latter as a consequence. DNA content was not affected by DFMO feeding. The mean number of lung metastases in DFMO-fed, renal carcinoma-bearing mice was 0.1 and in controls was 1.4 (p less than 0.001). DFMO feeding increased survival of mice bearing renal carcinomas by 3.0 +/- 0.8 (S.E.) days (p less than 0.05), i.e., from 30.5 +/- 0.8 days to 33.5 +/- 1.2 days. DFMO did not affect the growth of Wilms' tumor; however, in renal adenocarcinoma, it reduced growth, prevented lung metastases, and increased survival.     

4-1BBL联合Hsp70-肿瘤抗原肽抑制小鼠黑色素瘤肺转移

背景与目的正调节性共刺激分子表达偏低是肿瘤逃避机体免疫系统攻击的重要原因之一,增加这些分子的表达是促进抗肿瘤免疫的研究热点。小鼠黑色素瘤B16-F1细胞株是弱免疫原性癌细胞株。本研究旨在观察4-1BBL联合Hsp70-抗原肽对小鼠黑色素瘤肺转移的抑制作用,并探讨其机制。方法建立小鼠黑色素瘤肺转移模型,Hsp70-B16抗原肽小鼠皮下免疫,同时从尾静脉注射4-1BBL表达质粒p4-1BBL。于接种后第17天,解剖小鼠取肺组织,解剖显微镜下计数黑色素瘤肺转移结节的数目,并检测小鼠部分免疫学指标及肝、肾功能。结果4-1BBL联合Hsp70-B16抗原肽治疗组小鼠黑色素瘤肺转移结节数降至50±8,明显少于二者单独治疗组的500±80和450±40;联合治疗组小鼠血清IL-2和IFN-γ的分泌水平分别增加了4倍和3倍,与对照组相比均存在显著性差异(P<0.01);单独应用4-1BBL基因或与Hsp70-B16抗原肽联合应用对小鼠肝、肾的功能均无明显影响。结论表达4-1BBL联合应用Hsp70-B16抗原肽主要通过增强外周T淋巴细胞功能活性而有效抑制小鼠黑色素瘤肺转移。     

Effect of polyamine depletion in vivo by DL-alpha-difluoromethylornithine on functionally distinct populations of tumoricidal effector cells in normal and tumor-bearing mice

The objective of the present investigation was to examine the effect of in vivo polyamine depletion by DL-alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase, on cell-mediated tumoricidal activity in normal and tumor-bearing (B16 melanoma) mice. DFMO treatment in vivo for 6 days reduced splenic leukocyte polyamine levels and the induction of cytotoxic T-lymphocytes (greater than 50%) in both normal and tumor-bearing mice. However, substantially less inhibition was observed in the ability to generate cytotoxic T-lymphocytes following 18 days of DFMO treatment. In contrast, DFMO treatment for 6 or 18 days did not impair splenic natural cell-mediated cytotoxicity, assessed against natural killer sensitive YAC-1 target cells and natural cytotoxic sensitive WEHI-164 target cells, in normal or tumor-bearing mice. Natural cell-mediated cytotoxicity was not observed against fresh B16 melanoma cells. However, macrophage-mediated tumoricidal activity directed against B16 melanoma cells was augmented 79% following 6 but not 18 days of DFMO treatment. These results demonstrate that DFMO can exert very selective effects on functionally distinct populations of antitumor effector cells in vivo depending upon the schedule of DFMO administration.     

Genetic disruption of host nitric oxide synthase II gene impairs melanoma-induced angiogenesis and suppresses pleural effusion

Our previous study showed that genetic disruption of nitric oxide (NO) synthase II (NOS II) expression inhibits the metastatic ability of non-immunogenic B16 melanoma cells in syngeneic mice. In the present study, the mechanisms for this metastasis suppression were determined. B16-BL6 and B16-F10 murine melanoma cells were injected i.v. into syngeneic wild-type (NOS II(+/+)) and NOS II-null (NOS II(-/-)) C57BL/6 mice. Both melanoma cells produced less and smaller experimental pulmonary metastases in NOS II(-/-) mice than in NOS II(+/+) mice. Moreover, less metastatic pleural effusion was observed in NOS II(-/-) mice than in NOS II(+/+) mice. Immunohistochemical analyses indicated that absence of NOS II expression was correlated with decreased vascular endothelial growth factor expression and tumor-associated vascular formation. After activation with lipopolysaccharide and IFN-gamma, neither melanoma cell line produced detectable levels of NO. Our data demonstrate that tumor-induced expression of host NOS II enhances melanoma metastasis and pleural effusion, at least in part, through regulation of vascular formation and vascular permeability.     

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。     

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.     

Role of asparagine-linked carbohydrates in pulmonary metastasis of B16-F10 murine melanoma cells: implication through glycosylation inhibition by nojirimycin.

N-linked oligosaccharide moieties of cell surface glycoconjugates may be involved in the metastatic potential of tumour cells. Many studies have examined the anti-metastatic properties of inhibitors of carbohydrate synthesis or processing in vitro. However, there has so far been little evidence of such inhibitory factors on metastasis in vivo because of the general high toxicity of the inhibitors. In this study, we found nojirimycin (NM) to be a substantially non-toxic carbohydrate synthesis inhibitor that inhibited the experimental metastasis of B16-F10 melanoma cells not only in vitro but also in vivo. When NM was administered intraperitoneally to syngeneic C57BL/6 mice, the inhibition was dose-dependent, with 40-75% suppression of pulmonary colonization observed after 5 days exposure to NM (at 0.4 or 4 mg/day). An in vitro lectin sensitivity assay showed that NM-treated B16-F10 cells were less susceptible to the lectin concanavalin-A, suggesting alteration or decrease of high mannose-type carbohydrate moieties on the cell surface. Further in vitro analysis of adhesiveness between B16-F10 cells and endothelial cells demonstrated that NM treatment causes reduced binding of B16-F10 cells to endothelial cells. We have also studied the effect of NM on natural killer (NK) cells in mice. NM treatment elicited no substantial increase in the cytotoxic activity of splenic NK cells against YAC-1 target cells. These results indicate that NM acts on the process of adhesion and that specific structures of the cell surface carbohydrate moieties may be involved in the colonization phase of metastasis in vivo.     

Effect of inhibition of polyamine biosynthesis by DL-alpha-difluoromethylornithine on the growth and melanogenesis of B16 melanoma in vitro and in vivo

The objective of the present study was to investigate the effect of polyamine depletion by alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase, on the growth and differentiation of B16 melanoma cells grown in culture and also as solid tumors in mice. Polyamine depletion by DFMO (2.5 mM) resulted in a complete inhibition of cell growth in culture and a 90% inhibition of viability of melanoma cells as determined by clonogenic assay at the end of 7 days after DFMO treatment. These results indicate that polyamine depletion induced by DFMO is cytotoxic to B16 melanoma cells in culture. Furthermore a 2- to 5-fold increase in tyrosinase activity and 10-fold accumulation of melanine were observed in polyamine depleted cells compared to control cultures. These effects of DFMO could easily be reversed by the addition of putrescine simultaneously with DFMO. Administration of different doses of DFMO in drinking water to B16 melanoma tumor bearing mice also resulted in an increase in tyrosinase activity and a dose dependent inhibition (86-90%) of tumor growth. Although one cannot rule out the possibility of induction of differentiated phenotype as a result of antiproliferative activity of DFMO, the data presented indicate that the unique sensitivity of melanoma to DFMO may be due to a combination of cell growth inhibition and concomitant induction of differentiation upon polyamine depletion. The results of the present study indicate that polyamines play an important role in growth and differentiation of melanoma and also provide an example of inhibition of tumor cell growth by induction of cellular differentiation.     

Inhibition of intravascular mouse melanoma dissemination by recombinant human interferon alpha A/D

The effects of pure recombinant human interferon alpha A/D (IFN alpha A/D) on natural killer (NK) activity and the experimental lung metastasis of B16-F10 melanoma were studied. Treatment of C57BL/6 mice with IFN alpha A/D augmented splenic NK activity and also inhibited the experimental lung metastasis of B16-F10 melanoma in a dose-dependent manner. The augmentation of NK activity and the inhibition of experimental lung metastasis by IFN alpha A/D were completely abolished in anti-asialo GM1-pretreated mice. These results suggested that the effector cells which inhibited melanoma metastasis in the present system were mainly NK cells, and that it was by activating NK cells that IFN alpha A/D had its effect. We next studied the timing of IFN alpha A/D administration for the most effective prevention of melanoma metastasis. The inhibitory effect of IFN alpha A/D was most pronounced when it was given 12 hr before or at the same time as melanoma inoculation. This suggested that melanoma cells were susceptible to NK cells only for a short period of time after intravascular invasion.     

B16 murine melanoma and aging: slower growth and longer survival in old mice

The growth characteristics and colonization potential of a transplantable melanoma administered to young (3 mo) and old (24 mo) C57BL/6 mice were investigated. After sc injection of B16-F10 melanoma cells, tumor growth was slower, and final tumor volume was less in the older mice. Furthermore, after iv injection of B16-F1 melanoma cells, the number of pulmonary colonies was also less, and the survival was greater in the older mice. These findings indicate an age advantage in this experimental tumor model that may be attributed to either physical or immunologic factors.     

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.     

Inhibitory effect of murine recombinant interferon (beta) on the pulmonary metastasis of B-16 melanoma

The antitumor effect of murine recombinant interferon (beta) [Mu-rIFN(beta)] was examined on artificial metastasis of B-16 melanoma in C57BL/6 mice. The numbers of pulmonary nodules were significantly decreased to 16.7 +/- 4.7 (P less than 0.01), 9.5 +/- 4.2 (P less than 0.01), 7.1 +/- 5.6 (P less than 0.01) in mice given 20,000 units of Mu-rIFN(beta) intraperitoneally (ip) 24, 6, and 3 hr before intravenous tumor inoculation, respectively, compared with the control group of mice (57.1 +/- 1.4), if B-16 melanoma cells (5 X 10(5] were intravenously injected 28 days before the experiment. In mice given 20,000 units of Mu-rIFN(beta) ip 24, 6, and 3 hr before the experiment, the natural killer (NK) activities of spleen cells against YAC-1 cells were elevated to 45.5 +/- 6.1%, 53.7 +/- 3.4%, 43.2 +/- 6.5%, respectively, compared with NK activities in control mice (20.3 +/- 3.1%). Similarly, NK activities against B-16 melanoma cells were also elevated in mice given Mu-rIFN(beta). Pretreatment with anti-asialo GM1 antibody and carrageenan reduced the inhibitory effect of Mu-rIFN(beta) on the pulmonary metastasis. In vitro colony inhibition of more than 50% was not observed even if B-16 melanoma cells were incubated with 100,000 units/ml of Mu-rIFN(beta). From these results, it can be concluded that the inhibition of pulmonary metastasis by Mu-rIFN(beta) is mediated via host defense mechanisms and that NK cells and macrophages are both important for the inhibition.     

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.     

Inhibition of murine melanoma experimental metastasis by recombinant desulfatohirudin, a highly specific thrombin inhibitor

Recombinant desulfatohirudin (r-hirudin), a highly specific inhibitor of thrombin, was examined to determine whether it would inhibit production of experimental lung metastasis by B16-F10 melanoma cells. In in vitro assays using mouse plasma, the high level of procoagulant activity in B16-F10 cells was significantly inhibited by r-hirudin in a dose-dependent manner. From 15 to 120 min after s.c. administration into C57BL/6 mice, r-hirudin (10 mg/kg) markedly prolonged clotting time in a time course pattern that directly correlated with that of blood distribution of 125I-labeled r-hirudin. The production of experimental lung metastasis by B16-F10 cells was significantly inhibited by r-hirudin administered s.c. at time points ranging from 120 min before to 60 min after tumor cell inoculation with the most significant effects found in mice given r-hirudin 15 or 2 min before the i.v. injection of tumor cells. The organ distribution of [125I]IdUrd-labeled tumor cells demonstrated a clear difference in the lungs of mice treated with r-hirudin and the lungs of control mice, and these differences directly correlated with the number of lung tumor colonies found 3 weeks later. The inhibition of lung metastasis was not due to direct antitumor effects of r-hirudin. These results suggest that inhibition of coagulation events by r-hirudin significantly inhibit experimental lung metastasis during a critical time of 60 min after the entry of tumor cells into the circulation.     

Modulation of the lung colonization of B16-F1 melanoma cells by citrus pectin.

CONTEXT: Studies have shown that the galactoside-containing simple sugars and anti-galactoside-binding lectin antibodies may affect experimental tumor cell metastasis. However, the limited number of reagents used thus far necessitate further observations. PURPOSE: Natural citrus pectin (CP) and pH-modified CP (MCP), rich in galactose residues, were used to study the involvement of carbohydrates containing galactoside residues in cellular interaction in vitro and in lung colonization in vivo of B16-F1 melanoma cells. METHODS: B16-F1 melanoma cells were incubated with various concentrations of CP and MCP. Their ability to form homotypic aggregation in vitro and tumor lung colonization in vivo in 8-week-old female C57BL/6 mice was then analyzed. RESULTS: The CP binds to the surface of B16-F1 melanoma cells; this binding can be inhibited by lactose at a concentration of 0.15 M. Intravenous injection of the murine B16-F1 melanoma cells with the natural CP resulted in a significant increase (up to threefold) in the appearance of tumor colonies in the lung and in increased homotypic aggregation properties of the cells, while injection of MCP significantly decreased B16-F1 experimental metastasis (greater than 90%). CONCLUSIONS: Tumor galactoside-binding proteins mediate cellular recognition by linking oligosaccharides with terminal D-galactoside residues on adjacent cells. Successful interference with such a process with MCP may lead to a reduced ability to form tumor cell emboli and metastasis. IMPLICATIONS: These findings imply that the galactose-containing carbohydrate side chains of CP might mimic or compete with the natural ligand(s) of the tumor galactoside-binding protein (gal-lectin) and thus affect cellular interactions relevant for metastasis.     

Suppression of growth and hepatic metastasis of murine B16FO melanoma cells by a novel nutrient mixture

Highly metastatic melanoma is resistant to existing therapies. Our main objective was to investigate the effect of a nutrient mixture (NM) on B16FO tumor growth and hepatic metastasis. Tumor growth was studied in athymic nude male mice, 5-6 weeks old, inoculated with 10(6) B16FO melanoma cells subcutaneously and fed either a regular diet or one supplemented with 0.5% NM. Four weeks later, the mice were sacrificed and their tumors excised, weighed and processed for histology. Metastasis was studied in C57BL/6 mice, which received 10(6) B16FO melanoma cells by intrasplenic injection, as well as a regular or 0.5% NM-supplemented diet for 2 weeks. Survival was studied in C57BL/6 mice receiving 10(6) B16FO melanoma cells intraperitoneally (i.p.) followed by the regular, NM-supplemented, or regular diet in addition to being administered with 2 mg NM injection 3 times per week. NM inhibited the growth of B16FO melanoma cells by 50%. Lesions in the two groups were consistent with malignant melanoma. Mice were injected with B16FO cells in the spleen. Those fed the regular diet developed large black spleens and livers indicating growth in the spleen and metastasis to the liver. In contrast, mice supplemented with NM showed less growth in spleen, but also reduced metastasis to the liver. The survival time of mice receiving NM supplementation and B16FO cells i.p. was greater than in mice which were fed the regular diet. To confirm effects in vivo, we investigated the effect of NM on murine B16FO melanoma cells in vitro, including cell proliferation by MTT assay, morphology by hematoxylin and eosin (H&E) staining and apoptosis using live green caspase detection kit. In vitro, NM was not toxic at 100 microg/ml concentration, but exhibited 44% toxicity over the control at 500 and 1000 microg/ml. H&E did not indicate any changes up to 100 microg/ml. NM induced slight apoptosis at 100 microg/ml, moderate at 500 and extensive at 1000 microg/ml concentration.     

Inhibition of experimental metastasis by castanospermine in mice: blockage of two distinct stages of tumor colonization by oligosaccharide processing inhibitors

The extent of maturation of the oligosaccharide subunits of tumor cell glycoproteins appears to correlate with malignant potential, suggesting that modification of oligosaccharide structures may alter metastatic capacity. Castanospermine, a recently discovered inhibitor of glucosidase I, was tested for its effect on experimental metastasis of B16-F10 murine melanoma cells and was compared to treatment with swainsonine and tunicamycin. All three drugs block different steps in the pathway of glycoprotein processing yet each was a potent inhibitor of pulmonary colonization after i.v. injection of treated cells into C57BL/6 mice (greater than or equal to 80% inhibition). This result indicates a generality of inhibition of experimental metastasis by blockage of protein glycosylation or oligosaccharide processing and strongly implicates carbohydrate residues in at least one critical step of the metastatic cascade. Cytotoxic side effects could not account for the inhibitory activity. In order to identify a possible mechanism of inhibition of colonization, the adhesive behavior and pulmonary retention properties of B16-F10 cells treated with the above inhibitors were examined. Tunicamycin-treated B16-F10 cells exhibited poor adhesion to substrate-adsorbed fibronectin and laminin, whereas both castanospermine- and swainsonine-treated cells possessed near normal adhesive capacity; furthermore, the initial rate of loss of tunicamycin-treated cells from the lungs of mice was substantially greater than either control, castanospermine- or swainsonine-treated cells. These data suggest that these processing inhibitors can block experimental metastasis by at least two different mechanisms. The antimetastatic effect of tunicamycin may be related to interference in tumor cell-extracellular matrix interactions, whereas treatment with castanospermine or swainsonine appears to block at a stage distal to initial tumor cell arrest.     

Effects of alpha-difluoromethylornithine and 5-fluorouracil on the proliferation of a human colon adenocarcinoma cell line

Because alpha-difluoromethylornithine (DFMO) reduces the incidence of experimental colon cancers, inhibits the growth of human lung cancer cells and human leukemia cells in culture, and in combination with methylglyoxal (bis)guanylhydrazone induces remission in children with leukemia, its effectiveness against a human colon adenocarcinoma cell line (Colo 205) was tested alone and in combination with 5-fluorouracil (5-FU). Both DFMO (2 X 10(-4) M) and 5-FU (10(-6) M) inhibited Colo 205 cell proliferation. Above 5 X 10(-4) M DFMO (p less than 0.001) and at 10(-4) M 5-FU (p less than 0.001), Colo 205 growth was completely inhibited. Although DFMO did not sensitize Colo 205 cells to a noninhibitory concentration of 5-FU, the effectiveness of inhibitory concentrations of 5-FU and DFMO in reducing Colo 205 cell growth was additive. DFMO (2 X 10(-4) M) caused 89 to 93% inhibition of ornithine decarboxylase activity (p less than 0.001) and reduced levels of putrescine (93%; p less than 0.01) and spermidine (57%; p less than 0.02). Growth rate and the intracellular putrescine and spermidine contents were restored by 10(-6) M putrescine. DFMO could be an effective chemotherapeutic agent against human colonic cancer because of its effects at such unusually low concentrations in vitro.     

Lack of correlation between interferon levels induced by polyribonucleotides and their antimetastatic effect.

The inhibitory effect of poly(A)poly(U) on the pulmonary metastasis of B16-F10 melanoma was examined in comparison with that of poly(I,C)-L,C and poly(I)poly(C). The correlation between interferon (IFN) level and antimetastatic effect was also investigated. Intraperitoneal injection of poly(A)poly(U) (50 mg/kg) into C57BL/6 mice 24 h before intravenous inoculation of B16-F10 melanoma (1 X 10(5] caused a significant decrease (p less than 0.01) in the number of pulmonary nodules 14 days after tumor challenge. But poly(I,C)-L,C (1 or 0.2 mg/kg) and poly(I)poly(C) (5 mg/kg or 1 mg/kg) did not. From the kinetic study of IFN levels induced by polyribonucleotides, poly(I,C)-L,C showed the most potent IFN-inducing activity, followed by poly(I)poly(C) and poly(A)poly(U), in this order. Plasma IFN reached a peak at 6 h and still continued to be detected at 24 h after intraperitoneal injection of the polyribonucleotides. Against B16-F10 melanoma, the cytotoxicity of spleen cells stimulated by poly(A)poly(U) (50 mg/kg) was significantly (p less than 0.05) higher than that of spleen cells stimulated by poly(I)poly(C) (5 mg/kg) both at 12 and 24 h after intraperitoneal injection of those agents. The above results that there is no correlation between the IFN levels induced by polyribonucleotides and their antimetastatic effect. More extensive study of poly(A)poly(U) might give more fruitful results, which will give valuable information for future clinical trials of this lowly toxic promising agent.