Abnormally banded chromosomal regions in doxorubicin-resistant B16-BL6 murine melanoma cells

B16-BL6 murine melanoma cells were selected for cytogenetic evaluation during the stepwise development of increasing resistance in vitro to the antitumor antibiotic, doxorubicin (DOX). Karyotypic studies demonstrated extensive heteroploidy with both numerical and structural abnormalities which were not present in the parental DOX-sensitive B16-BL6 cells. Trypsin-Giemsa banding revealed the presence of several marker chromosomes containing abnormally banding regions (ABRs) in the 44-fold B16-BL6 DOX-resistant subline. These ABRs appeared to be more homogeneously staining at the higher DOX concentrations. Length measurements (ABR index) in seven banded metaphases indicated a direct correlation with increasing DOX concentration. When the DOX-resistant cells were grown in drug-free medium for 1 yr, the drug-resistant phenotype gradually declined in parallel with the level of resistance and the ABR index. DOX-induced cytogenetic damage examined by sister chromatid exchange methodology in parental B16-BL6 cells indicated a linear sister chromatid exchange:DOX dose-response relationship. However, after continuous treatment of parental B16-BL6 cells with DOX (0.01 microgram/ml) for 30 days, sister chromatid exchange scores were found to return to base-line values. The B16-BL6 resistant cells demonstrated a cross-resistant phenotype with N-trifluoroacetyladriamycin-14-valerate, actinomycin D, and the Vinca alkaloids but not with 1-beta-D-arabinofuranosylcytosine. The results suggest that ABR-containing chromosomes in DOX-resistant sublines may represent cytogenetic alterations of specific amplified genes involved in the expression of DOX resistance. Further studies are required to identify and define the possible gene products and to correlate their relationship to the cytotoxic action of doxorubicin.     

Combined effect of cisplatin and caffeine on murine B16-BL6 melanoma cells

Combined effect of cisplatin and caffeine on murine B16-BL6 melanoma cells was studied. Synergistic inhibition of the cell growth was observed when caffeine (2 mM) was added continuously after one hour exposure of cisplatin. On the other hand, when caffeine was added before one hour exposure of cisplatin or one hour simultaneous exposure with cisplatin, synergistic effect was not shown. In the analysis of DNA histogram obtained from flow cytometry, S and G2/M accumulation was observed by the treatment of cisplatin and that accumulation was reduced by the combination of cisplatin and caffeine. From this findings, it was suggested that caffeine would inhibit DNA repair process. Furthermore, according to morphological studies with hematoxylin-eosin stain and Fontana-Masson stain, the addition of caffeine alone resulted in mild swelling of melanoma cells and the decrease of nuclear-cytoplasmic ratio. The combination of cisplatin and caffeine caused marked swelling of melanoma cells and remarkable increase of dendrite-like processes. Melanogenesis was also enhanced by the addition of these two drugs. Many matured melanosomes, increases of mitochondria, Golgi's apparatus and endoplasmic reticula were observed by the use of electron microscope. These findings implied that the combination of cisplatin and caffeine induced a differentiation of murine melanoma cells.     

Therapeutic efficacy of interleukin-2 activated killer cells against adriamycin resistant mouse B16-BL6 melanoma.

Development of multidrug-resistance (MDR) remains a major cause of failure in the treatment of cancer with chemotherapeutic agents. In our efforts to explore alternative treatment regimens for multidrug-resistant tumors we have examined the sensitivity of MDR tumor cell lines to lymphokine activated killer (LAK) cells. Adriamycin (ADM) resistant B16-BL6 melanoma, L1210 and P388 leukemic cell lines were tested for sensitivity to lysis by LAK cells in vitro. While ADM-resistant B16-BL6 and L1210 sublines were found to exhibit at least 2-fold greater susceptibility to lysis by LAK cells, sensitivity of ADM-resistant P388 cell was similar to that of parental cells. Since ADM-resistant B16-BL6 cells were efficiently lysed by LAK cells in vitro, the efficacy of therapy with LAK cells against the ADM-resistant B16-BL6 subline in vivo was evaluated. Compared to mice bearing parental B16-BL6 tumor cells, the adoptive transfer of LAK cells and rIL2 significantly reduced formation of experimental metastases (P less than 0.009) and extended median survival time (P less than 0.001) of mice bearing ADM-resistant B16-BL6 tumor cells. Results suggest that immunotherapy with LAK cells and rIL2 may be a useful modality in the treatment of cancers with the MDR phenotype.     

Inhibition of B16-BL6 melanoma growth in mice by methionine-enkephalin

The antitumor effect of methionine-enkephalin [( Met]enkephalin) was demonstrated in C57BL/6J mice inoculated with B16-BL6 melanoma cells. Local subcutaneous tumor growth was inhibited with a 50-micrograms dose daily for 7 or 14 days. The antitumor effect of [Met]enkephalin was inhibited by the administration of the opioid receptor antagonist naloxone. Naloxone alone had no significant effect on tumor growth.     

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.     

Adhesion-induce protein tyrosine phosphory-lation is associated with invasive and metastatic potentials in B16-BL6 melanoma cells

Objective: The interaction of cancer cell with extracellular matrix (ECM) happens as an earlier and specific event in the invasive and metastatic cascade. To explore the key element(s) in cancer metastasis and observe the cell-ECM interaction and its role. Methods: To interrupt the cell-ECM interaction by suppression of adhesion-induced protein tyrosine phosphorylation with protein tyrosine kinase inhibitor genistein in B16-B16 mouse melanoma cells. Results: When B16-BL6 cells attached to Matrigel, a solubilized basement membrane preparation from EHS sarcoma, a 125 kDa protein increased its phosphotyrosine content dramatically. In contrast, when the cells were pretreated with 20μM or 30 μM genistein for 3 days, it was revealed a less increase in the phosphotyrosine content of this 125 kDa protein in response to cell attachment to ECM was revealed with immunoblot analysis. Accompanied by the lower level of adhesion-induced protein tyrosine phosphorylation the genistein-treated cells exhibited a decrease in their capabilities of adhesion to Matrigel and invasion through reconstituted basement membrane. The potentials of and forming lung metastatic nodules were also shown to be decreased dramatically in these genistein-treated cells. Conclusion: It was suggested that protein tyrosine phosphorylation in cell-ECM interaction might be associated with invasive and metastatic potentials in cancer cells.     

Growth inhibition and differentiation of murine melanoma B16-BL6 cells caused by the combination of cisplatin and caffeine

We preliminarily investigated the combined effects of cisplatin and caffeine on murine melanoma B16-BL6 cells in vitro. When caffeine was added before or simultaneously with cisplatin, there was little growth inhibition. The addition of 2.0 mM caffeine after 1 h of exposure to cisplatin inhibited growth and induced cell differentiation. This treatment resulted in fewer cells, and the numbers of melanosomes and mitochondria and the amount of Golgi's complex and endoplasmic reticulum were increased. DNA histograms obtained by flow cytometry showed that cells treated with cisplatin alone accumulated in the G2/M phase, with a partial G2 block. The addition of 2.0 mM caffeine after 1 h of treatment with cisplatin reduced this block. Caffeine caused murine melanoma B16-BL6 cells treated with cisplatin to differentiate, and this inhibited growth.     

In vivo immunologic selection of class I major histocompatibility complex gene deletion variants from the B16-BL6 melanoma

The mechanism by which tumor allografts escape host immunologic attack was investigated. B16-BL6 cells (the bladder 6 subline of the B16 melanoma) (H-2b) were transfected with a gene (Dd) encoding an allogeneic class I major histocompatibility complex antigen. Clones that expressed Dd antigen were injected into the footpads of nonimmune syngeneic mice, syngeneic immune mice, and nude mice. Under conditions of immunologic selection a clone that contained multiple copies of the transfected gene formed variants that lacked the transfected gene. Primary tumors and pulmonary metastases of immunized mice and pulmonary metastases of nonimmunized mice had lost the Dd gene and, in most cases, all of the associated plasmid. In contrast, in immunodeficient nude mice, primary tumors and pulmonary metastases retained the Dd gene and the associated plasmid. Deletion of genes encoding cell surface antigens may be one of the mechanisms by which allogeneic tumors escape immunologic attack.     

Induction of autocrine factor inhibiting cell motility from murine B16-BL6 melanoma cells by alpha-melanocyte stimulating hormone

We have previously reported that neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) successfully inhibited Matrigel invasion and haptotactic migration of B16-BL6 melanoma cells towards both fibronectin and laminin without affecting their growth. In the present study, we investigated the inhibitory mechanism of tumor cell motility by alpha-MSH. Alpha-MSH significantly blocked the autocrine motility factor (AMF)-enhanced cell motility. However, alpha-MSH did neither prevent the secretion of AMF from B16-BL6 cells nor alter the expression level of AMF receptor (gp78). On the other hand, alpha-MSH induced the secretion of the motility inhibitory factor(s) from B16-BL6 cells in a concentration- and time-dependent manner. The induction of the motility inhibitor(s) was proportional to increasing levels of intracellular cAMP induced by alpha-MSH as well as forskolin, and the activity was abolished by an adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA). The motility-inhibiting activity in conditioned medium (CM) from alpha-MSH-treated B16-BL6 cells was found to have a m.w. below 3 kDa after fractionation. This activity was abolished by boiling but insensitive to trypsin. The treatment of tumor cells with cycloheximide reduced the activity in alpha-MSH-stimulated CM. Our results suggest that alpha-MSH inhibited the motility of B16-BL6 cells through induction of autocrine factor(s).     

Hyperthermic enhancement of rhodamine 123 cytotoxicity in B16 mouse melanoma cells in vitro

The effect of elevated temperature on cytotoxicity of rhodamine 123 (R123) was tested in vitro on B16 mouse melanoma cells. Simultaneous 1-h exposure to R123 and hyperthermia (43 degrees C for 1 h) resulted in marked enhancement of R123 cytotoxicity. Thermal enhancement of R123 cytotoxicity occurred at temperatures as low as 38 degrees C. Heat treatment (43 degrees C for 1 h) given immediately before or after R123 exposure (37 degrees C for 1 h) yielded no significant increase in cytotoxicity over that expected for strict additivity. The effects of heat on two mechanisms reported to be associated with R123 cytotoxicity were evaluated: (a) target inactivation by R123; and (b) R123 intracellular accumulation. Hyperthermia caused an increased rate of target inactivation by R123 and also caused an increased net intracellular accumulation of R123. This indicates that at least two mechanisms are responsible for the synergistic cytotoxicity of R123 and hyperthermia.     

Genistein inhibits growth of B16 melanoma cells in vivo and in vitro and promotes differentiation in vitro

Consumption of soy products has been linked to a reduced mortality and morbidity from a number of cancers. Genistein, one of the principal soy isoflavones, has been shown to inhibit the growth of a number of tumour cell lines in vitro; however, a role of genistein in retarding tumour growth in vivo is less well documented. In this study, in addition to examining the effects of genistein on the growth of murine B16 melanoma cells in vitro, we have examined the effects of feeding a genistein-rich diet on s.c. growth of these tumour cells in mice. In vitro, the melanoma cells showed an increase in sensitivity to genistein with increasing time of exposure, culminating in a 50% growth inhibition (IC₅₀) at 12.5 μM after 7 days. Genistein at 25 μM induced micronucleus formation after 24 hr and at concentrations as low as 2.5 μM induced morphological changes indicative of differentiation. Growth of solid tumours implanted into female C57BL/6J mice was inhibited by 50% when mice were fed genistein for 1 week before and for 1 week after inoculation with B16 melanoma cells. Plasma genistein concentrations at the time of tumour removal were 1.1 μM, which is similar to levels reported in humans consuming diets high in soybeans or soybean products, while control animals had no detectable genistein in plasma. Our results provide additional in vivo evidence suggesting that genistein retards the growth of implanted tumours, adding further to studies suggesting that this isoflavonoid is a biologically active component of soy foods. Int. J. Cancer 72:860–864, 1997. © 1997 Wiley-Liss, Inc.     

Quercetin inhibits the invasion of murine melanoma B16-BL6 cells by decreasing pro-MMP-9 via the PKC pathway

Purpose: On the basis of the inhibitory effect of quercetin on the invasion of melanoma B16-BL6 cells previously reported by us, the mechanisms of quercetin-mediated inhibition of invasion were further investigated in the present study.Methods: The ability of B16-BL6 cells to invade and migrate was evaluated in terms of the numbers of cells penetrating a reconstituted basement membrane in the Transwell coculture system. The relative levels and activities of matrix metalloproteinase-9 (MMP-9) and MMP-2 were determined by gelatin zymography and quantified using LabWorks 4.0 software.Results: The quercetin-mediated inhibition of invasion was partially blocked by phorbol-12,13-dibutyrate (PDB), a PKC (protein kinase C) activator, and by doxorubicin, a PKC inhibitor. Only the proforms of MMP-9 (92 kDa) and MMP-2 (72 kDa) were detected by gelatin zymography. Quercetin dose-dependently decreased the gelatinolytic activity of pro-MMP-9. Doxorubicin also markedly reversed the quercetin-induced decrease. Quercetin showed a dose-dependent antagonism of increases in gelatinolytic activity of pro-MMP-9 induced by PDB and free fatty acid (another PKC activator).Conclusions: Together with the report that quercetin directly reduces PKC activity, the results reported here suggest that quercetin may inhibit the invasion of B16-BL6 cells by decreasing pro-MMP-9 via the PKC pathway. Published online: 30 October 2003     

木鳖子醇提物抑制小鼠黑素瘤B16细胞体内外侵袭转移的实验研究

目的:观察木鳖子醇提物(ethanol extract from cochinchina momordica seed,CMSEE)对小鼠黑素瘤细胞B16及其荷瘤小鼠移植瘤侵袭转移的影响,初步探讨其作用机制.方法:通过划痕实验和体外迁移实验检测不同质量浓度CMSEE(20、25、30μg/mL)作用24和48 h后,B16细胞侵袭和转移能力的改变;分别用RT-PCR和Western印迹法检测不同质量浓度CMSEE(20、25、30μg/mL)作用24 h以及25μg/mL CMSEE作用不同时间(0、6、12、24、36、48 h)对B16细胞中基质金属蛋白酶(matrix metalloproteinase,MMP)-2、MMP-9 mRNA及其蛋白以及基质金属蛋白酶组织抑制因子(tissue inhibitor of metalloproteinase,TIMP)-1、TIMP-2蛋白表达的影响.以B16细胞建立小鼠荷瘤模型,用10 mg/kg CMSEE灌胃治疗荷瘤小鼠,HE染色观察各组小鼠的肿瘤、肝、脾组织的病理变化.结果:CMSEE(20～30 μg/mL)可明显抑制B16细胞的侵袭和转移能力,可使B16细胞划痕的直径明显变宽(P＜0.05),穿过Transwell小室膜的细胞数显著减少(P＜0.05),并呈浓度依赖性;CMSEE(20～30μg/mL)可明显下调B16细胞中MMP-2、MMP-9 mRNA及蛋白的表达(P＜0.05),并有浓度依赖性;而CMSEE对B16细胞中TIMP-1和TIMP-2蛋白的表达无明显影响(P＞0.05).CMSEE可有效抑制小鼠体内黑素瘤的侵袭和转移.结论:CMSEE在体内外均能明显抑制小鼠B16细胞的侵袭和转移能力,其作用机制可能与其抑制MMP-2和MMP-9的表达有关.     

Verapamil potentiation of doxorubicin resistance development in B16 melanoma cells both in vitro and in vivo

The effect of the combined administration of doxorubicin (DX) and verapamil (VRP) on the induction of DX resistance of B16 melanoma cells, was investigated both in vitro and in vivo. Cells grown in the presence of increasing concentrations of DX and of 1 microM VRP, tested at several passages, were more resistant than cells grown with DX alone. The treatment of B16 melanoma bearing mice with the maximal tolerated dose of DX (12 mg kg-1 i.v.) and of VRP (25 mg kg-1 i.p.) selected a line (B16-DX. VRP) completely resistant to DX after 17 transplants, while treatment with DX alone selected a DX resistant line after 27 transplants. Lung metastases were significantly lower in the B16-DX. VRP line compared to the original B16 melanoma. The results suggest that the association of VRP with DX increases the rate of resistance development to DX.     

Receptor-mediated uptake of low-density lipoprotein by B16 melanoma cells in vitro and in vivo in mice.

Selective delivery of cytotoxic anti-neoplastic drugs can diminish the severe side-effects associated with these drugs. Many malignant tumours express high levels of low-density lipoprotein (LDL) receptors on their membranes. Therefore, LDL may be used as a carrier to obtain selective delivery of anti-neoplastic drugs to tumours. The present study was performed to investigate the feasibility of the murine B16 tumour/mouse model for the evaluation of LDL-mediated tumour therapy. LDL binds with high affinity to LDL receptors on cultured B16 cells (Kd, 5.9 +/- 2.3 micrograms ml-1; Bmax 206 +/- 23 ng LDL mg-1 cell protein). After binding and internalisation, LDL was very efficiently degraded: 724 +/- 19 ng LDL mg-1 cell protein h-1. Chloroquine and ammonium chloride completely inhibited the degradation of LDL by the B16 cells, indicating involvement of lysosomes. LDL receptors were down-regulated by 70% after preincubation of B16 cells with 300 micrograms ml-1 LDL, indicating that their expression is regulated by intracellular cholesterol. To evaluate the uptake of LDL by the B16 tumour in vivo, tissue distribution studies were performed in C57/B1 mice inoculated with B16 tumours. For these experiments, LDL was radiolabelled with tyramine cellobiose, a non-degradable label, which is retained in cells after uptake. At 24 h after injection of LDL, the liver, adrenals and the spleen were found to be the major organs involved in LDL uptake, with tissue-serum (T/S) ratios of 0.82 +/- 0.08, 1.17 +/- 0.20 and 0.69 +/- 0.08 respectively. Of all the other tissues, the tumour showed the highest uptake of LDL (T/S ratio of 0.40 +/- 0.07). A large part of the LDL uptake was receptor mediated, as the uptake of methylated LDL was much lower. Although the LDL uptake by the liver, spleen and adrenals is higher than that by the tumour, the LDL receptor-mediated uptake by these organs may be selectively down-regulated by methods that do not affect the expression of LDL receptors on tumour cells. It is concluded that the B16 tumour-bearing mouse constitutes a good model to evaluate the effectiveness of LDL-mediated delivery of cytotoxic (pro)drugs to tumours in vivo.     

Inhibition of the metastatic spread and growth of B16-BL6 murine melanoma by a synthetic matrix metalloproteinase inhibitor

The synthetic matrix metalloproteinase inhibitor batimastat was tested for its ability to inhibit growth and metastatic spread of the B16-BL6 murine melanoma in syngeneic C57BL/6N mice. Intraperitoneal administration of batimastat resulted in a significant inhibition in the number of lung colonies produced by B16-BL6 cells injected i.v. The effect of batimastat on spontaneous metastases was examined in mice inoculated in the hind footpad with B16-BL6 melanoma. The primary tumor was removed surgically after 26-28 days. Batimastat was administered twice a day from day 14 to day 28 (pre-surgery) or from day 26 to day 44 (post-surgery). With both protocols, the median number of lung metastases was not significantly affected, but there was a significant reduction in the weight of the metastases. Finally, the effect of batimastat was examined on s.c. growth of B16-BL6 melanoma. Batimastat administered daily, starting at day of tumor transplantation, resulted in a significant growth delay, whereas treatment starting at advanced stage tumor only reduced tumor growth marginally. Our results indicate that a matrix metalloproteinase inhibitor can not only prevent the colonization of secondary organs by B16-BL6 cells but also limit the growth of solid tumors.     

Preventive effect of oral administration of 6-(methylsulfinyl)hexyl isothiocyanate derived from wasabi (Wasabia japonica Matsum) against pulmonary metastasis of B16-BL6 mouse melanoma cells

AIM: Effect of oral administration of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) or a 6-MITC-containing T-wasabi fraction from wasabi root (Wasabia japonica Matsum) to inhibit the macroscopic pulmonary metastasis was studied with a murine B16-BL6 melanoma model. METHOD: Two administration routes, subcutaneous or intravenous, and two administration times, prior to or concomitant with tumor inoculation, of 6-MITC or T-wasabi against the metastatic foci formation in C57BL/6J mouse lungs were compared. RESULTS: The number of metastasized foci per lung in either subcutaneous or intravenous injection was significantly reduced by intake of 6-MITC or a T-wasabi fraction. The maximum reduction by a T-wasabi fraction reached to 82%. Fifty-six percent of foci formation was inhibited by a 2 week-prior administration of 6-MITC (200 microM), whereas only 27% inhibition was obtained by a concomitant administration with tumor inoculation. Neither 6-MITC nor T-wasabi at tested concentrations showed any toxic effects. DISCUSSION: Together with our previous results, a component of the Japanese pungent spice, wasabi appears to inhibit not only tumor cell growth but also tumor metastasis. Therefore, 6-MITC from wasabi is apparently a useful dietary candidate for controlling tumor progression.     

Effects of theophylline treatment on mouse B-16 melanoma cells in vitro

The effects of theophylline treatment on mouse B-16 melanoma cell growth, metabolism, and membrane antigen expression in vitro were studied. Theophylline treatment inhibited DNA synthesis and the cell growth rate, and caused an elevation of intracellular cAMP levels. Cells treated with theophylline became elongated and assumed a normal fibroblast-like morphology. Theophylline treatment of B-16 cells also reduced the levels of tumor specific antigen and H-2 antigen detectable on the cell membrane.     

Ex vivo and in vivo delivery of anti-tissue factor short interfering RNA inhibits mouse pulmonary metastasis of B16 melanoma cells.

PURPOSE: The coagulation trigger tissue factor has been implicated in tumor growth, angiogenesis, and metastasis. In this study, we explore the effects of ex vivo and in vivo delivery of short interfering RNA (siRNA) targeting tissue factor on B16 melanoma colonization of the lung in a murine model for metastasis. The purposes of this work are to establish a noncytotoxic in vivo model for investigation of tissue factor function and provide preclinical assessment of the therapeutic potential of tissue factor siRNA for prevention of metastasis. EXPERIMENTAL DESIGN AND RESULTS: C57BL/6 mice were evaluated for pulmonary metastases following tail vein injection of B16 cells transfected with either active or inactive siRNA. Mice receiving cells transfected with active siRNA had significantly lower numbers of pulmonary tumors compared with mice injected with control cells (transfected with inactive siRNA). The average time point at which the mice started to exhibit tumor-associated stress was also increased significantly from 22 days for the control group to 27 days for the experimental group (P = 0.01). In a therapeutically more relevant model, where the siRNA was delivered i.p. and the cells (untransfected) by tail vein injection, an inhibitory effect on metastasis was observed when the siRNA treatment was initiated either before or at the time of cell injection. CONCLUSIONS: The results suggest that tissue factor has a crucial function in promoting lung tumor metastasis of blood-borne tumor cells in the early stages of the tumor take process and further suggest that treatment with tissue factor siRNA may become a viable clinical strategy for prevention of tumor metastasis.