Characterization in vitro and in vivo of progressively adriamycin-resistant B16-BL6 mouse melanoma cells

Adriamycin (ADR)-resistant sublines of B16-BL6 mouse melanoma selected by exposure to increasing concentrations of ADR were characterized in vitro for growth properties and in vivo for tumorigenicity and pulmonary metastases. The progressively resistant sublines adapted to grow in the presence of 0.025, 0.05, 0.1, and 0.25 microgram/ml ADR in monolayer culture were found to be 5-, 10-, 20-, and 40-fold ADR-resistant, respectively, compared to the parental sensitive cells, using a soft-agar colony assay and continuous ADR treatment for 7 days. The doubling time in monolayer culture of the parent sensitive and progressively ADR-resistant sublines of B16-BL6 melanoma cells was approximately 16-18 h. Although the colony-forming efficiency in soft agar of parental sensitive cells was only 0.5-4%, the 5-, 10-, 20-, and 40-fold ADR-resistant sublines had colony-forming efficiencies of 15, 20, 30, and 77%, respectively. Tumorigenicity in C57BL/6 mice of progressively ADR-resistant sublines was similar to parental sensitive cells following s.c. and i.p. implantation of 10(5)-10(6) tumor cells. Experimental pulmonary metastases were significantly lower in ADR-resistant sublines with progressive resistance. Additionally, unlike the parental sensitive and 5-fold ADR-resistant B16-BL6 cells, the 10-, 20-, and 40-fold ADR-resistant sublines were spontaneously nonmetastatic. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical detection of P-glycoprotein revealed the presence of a Mr 170,000 plasma membrane glycoprotein in the 40-fold ADR-resistant subline and its counterpart maintained for 1 year in ADR-free medium. Results from this study suggest that progressively ADR-resistant B16-BL6 mouse melanoma cells selected in vitro demonstrate a marked increase in colony formation in soft agar and a decrease in the ability to produce pulmonary metastases, without alterations in tumorigenicity.     

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.     

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.     

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.     

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.     

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).     

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.     

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     

A novel role for autologous tumour cell vaccination in the immunotherapy of the poorly immunogenic B16-BL6 melanoma.

The growth of immunogenic tumours stimulates the generation of tumour-sensitized, but not functional, pre-effector T cells in the draining lymph nodes. These pre-effector cells can mature into effector cells upon in-vitro stimulation with anti-CD3 and IL-2. In the current study, using a defined, poorly immunogenic tumour, B16-BL6 melanoma, the pre-effector cell response was not evident during progressive tumour growth but was elicited by vaccination with irradiated tumour cells admixed with Corynebacterium parvum. After anti-CD3/IL-2 activation, these cells were capable of mediating the regression of established pulmonary metastases. The efficacy of the vaccine depended on the doses of both tumour cells and the adjuvant. While higher numbers of tumour cells were more effective, an optimal dose (12.5 micrograms) of C. parvum was required. The dose of irradiation was not a critical factor. After vaccination, kinetic studies revealed that the pre-effector cell response was evident 4 days later and declined after 14 days. These observations illustrate the potential role of active immunization in the cellular therapy of cancer.     

The production of acidic polysaccharides by 5-bromodeoxyuridine-treated B16 mouse melanoma cells.

B16 melanoma cells were treated in culture with 5-bromo-2-deoxyuridine. The cell-associated and released proteoglycans and sialoglycopeptides were compared to those of control cultures treated with thymidine. The 5-bromo-2-deoxyuridine-treated cultures showed a marked reduction in the proportion of cell-associated proteoglycans and sialoglycopeptides, an increase in the synthesis of hyaluronic acid, the absence of high-molecular-weight chondroitin sulfate, and the presence of increased amounts of heparan sulfate in the media. In addition, the 5-bromo-2-deoxyuridine-treated cells had a higher DNA content and were larger than controls.     

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.     

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.     

Multiple differences between wild-type B16 melanoma cells and a wheat germ agglutinin resistant clone.

A low-metastatic, glycosylation-defective variant of the B16 murine melanoma was obtained by Tao and Burger (1977) through selection with wheat germ agglutinin. We found that variant and parental (wild-type) cell lines were equally invasive when confronted with precultured embryonic chick heart fragments in vitro. Also, a short-term in vivo arrest assay showed no significant differences. After intravenous injection, wild-type cells killed the recipient mice faster than did the variant cells. We were able to confirm the changes in glycosylation at the enzyme level. In addition, we showed that the pattern of endogenous lectins was strikingly different, at least at the quantitative level. We also looked at another set of receptor proteins, namely receptors for neurotransmitters coupled to adenylate cyclase. The response to the vasoactive intestinal peptide and prostaglandins was lower in the variant cells, which also had a delayed response to cholera toxin. Although most of the data can be explained by altered glycosylation in the variant cells, the large number of differences between variant and parent cells makes it difficult to identify the biochemical basis of altered metastatic behaviour. This might also be the case with other pairs of cells differing in metastatic activity.     

Evaluation of toxicity following electrically mediated interleukin-12 gene delivery in a B16 mouse melanoma model.

PURPOSE: Interleukin-12 (IL-12) has potential as an immunotherapeutic agent for the treatment of cancer but is unfortunately associated with toxicity. Delivery of a plasmid encoding IL-12 with electroporation induces an antitumor effect in the B16 mouse melanoma model without serious side effects. To translate this observation to the clinic, an evaluation of toxicity was done in the mouse model. EXPERIMENTAL DESIGN: Weight change, tumor response, blood chemistry and hematology values, and serum IL-12 levels were evaluated. Multiple tissues were analyzed histopathologically. RESULTS: A pronounced reduction in tumor volume, including a large percentage of complete regressions, was observed after electrically mediated gene therapy. No significant increases in serum IL-12 levels were detected. Tumor-bearing mice showed an increased number of atypical hematology values when compared with normal naive controls. Statistically significant differences in chemistry and hematology values were observed sporadically in most of the standard chemistry and hematology categories in all groups. The only histopathologic abnormality specific to the animals receiving both plasmid and electroporation was inflammation associated with the kidney at the last time point. CONCLUSIONS: In general, mice that received both plasmid and electroporation showed the least abnormal histopathologic findings and were found to be in the best health, reflecting the reduced burden of disease. No significant toxic effects due to the IL-12 gene therapy were observed.     

Characterization of B16 melanoma cells resistant to the CC-1065 analogue U-71,184

U-71,184 is a CC-1065 analogue which is highly cytotoxic in vitro and has a broad spectrum of antitumor activity in vivo. Against B16 cells, U-71,184 was 8-fold and 253-fold more potent than Actinomycin D and Adriamycin, respectively. U-71,184 killed 90% of B16 cells at 0.01 ng/ml levels of drug in the medium, which was equivalent to an intracellular concentration of about 8 pg/10(6) cell (= 2 x 10(-8) pmol/cell). A B16 cell line resistant to U-71,184 developed after 3 months of in vitro exposure to gradually increasing concentrations of the drug. The sensitive and resistant cell lines were cloned and a B16/R clone was selected which was 60 to 100 times more resistant to U-71,184 than the cloned sensitive parent (B16/S). Cells grown in the absence of U-71,184 for 2 months retained resistance to the drug. B16/R was slightly cross-resistant only to Adriamycin but not to Actinomycin D, vinblastine, or colchicine. Among alkylating agents, it was slightly cross-resistant to Melphalan but not to 1,3-bis(2-chloroethyl)-1-nitrosourea or cisplatin. B16/R did not overexpress mdr mRNA. Therefore, this cell line does not exhibit the multidrug-resistant phenotype. Most karyotypes of B16/R had a marker chromosome which carried an aberrantly staining region apparently containing repetitive replication of the same segment. Resistance can be partly accounted for by the approximately 10-fold lesser uptake of [3H]-U-71,184 in B16/R, as compared to B16/S. B16/R was cross-resistant in varying degrees to several other CC-1065 analogues. The ratio of the 50% lethal dose of U-71,184 for B16/R, as compared to B16/S, was about 60 (i.e., R/S = 60). In comparison, the following compounds had an R/S ratio of less than 20 (i.e., modest level of cross-resistance to U-71,184): U-68,819, U-73,975, U-75,500, U-75,559, and CC-1065. In contrast, the following compounds had an R/S ratio greater than 20 (i.e., highly cross-resistant to U-71,184): U-71,184 analogues U-71,185, U-73,903, and U-75,012; U-73,975 analogues U-75,613, U-75,032, and U-73,896; and CC-1065 enantiomer U-76,915. We cannot yet explain the difference in the level of cross-resistance between these compounds in vitro. B16/S and B16/R cells were tumorigenic in mice and B16/R was resistant to U-71,184 in vivo. There was no clear indication of cross-resistance of B16/R in vivo to Adriamycin, Actinomycin D, cisplatin, or Melphalan. However, U-73,975, a compound with modest cross-resistance in vitro, was significantly cross-resistant in vivo.     

表达单纯疱疹病毒受体HVEM的黑色素瘤B16-ova-HVEM细胞系的构建及成瘤后oHSV2治疗效果初探

目的：构建可被单纯疱疹病毒感染的新型细胞系B16-ova-HVEM,体外验证目的基因的表达并初步探究其成瘤后,应用Ⅱ型溶瘤单纯疱疹病毒（oHSV2）的治疗效果。方法构建表达单纯疱疹病毒受体（HVEM）的质粒载体,脂质体转染B16-ova细胞,嘌呤霉素筛选表达HVEM的阳性克隆。体外验证后进行体内检测,B16-ova-HVEM荷瘤C57BL/6小鼠,成瘤后分为两组,每组5只小鼠,给予oHSV2治疗为oHSV2治疗组,荷瘤未治疗的小鼠为对照组。治疗后测量肿瘤大小,观察小鼠生存期;流式细胞术检测两组小鼠外周血中CD4+、CD8+T细胞和髓系来源的抑制性细胞（MDSC）比例变化。结果质粒DNA测序和酶切鉴定结果均显示成功构建含HVEM的质粒;荧光显微镜下可观察到筛选后细胞发绿色荧光;流式细胞术检测筛选后细胞GFP阳性率为98.3%;RT-PCR验证结果显示筛选后细胞含有目的基因HVEM;oHSV2在感染复数（MOI）为0.1、0.5和1的情况下均能明显感染筛选出的细胞;动物实验显示,oHSV2体内治疗后抑瘤效果显著（P﹤0.001）;流式检测结果显示oHSV2治疗后小鼠外周血中CD4+、CD8+T细胞比例明显高于对照组（P﹤0.01）,而MDSC比例明显低于对照组（P﹤0.01）。结论成功构建B16-ova-HVEM细胞系,其既可被OT-1小鼠T细胞特异性识别又可被单纯疱疹病毒感染,为肿瘤特异性免疫治疗和溶瘤病毒联合治疗的研究奠定基础。     

Generation and partial characterization of melanoma sublines resistant to lymphokine activated killer (LAK) cells. Relevance to doxorubicin resistance

To see whether a tumor cell population may contain cells resistant to lymphokine-activated killer (LAK) lymphocytes, cells from a LAK-sensitive melanoma line (Me 665/2) were co-cultured with LAKs. Three sublines were obtained after 1, 2 or 3 immunoselection cycles. Immunoselected (IS) sublines show reduced proliferation, decreased reactivity to the monoclonal antibody (MAb) R24 and appeared morphologically more differentiated in comparison with the parental Me 665/2 line. A progressively reduced sensitivity to LAKs was observed in IS sublines with a more than 8-fold reduction in LAK susceptibility. A reduced complement (C)-mediated lysis was also observed in IS sublines. Since we have previously shown that LAK sensitivity of melanoma cells may be associated with Doxorubicin (Dx) resistance, the sensitivity to Dx was tested in these lines. An augmented sensitivity to Dx was noted in IS sublines as compared with Me 665/2. The differences in LAK susceptibility between the IS sublines and the parental Me 665/2 line remained stable for 2 weeks but declined and disappeared thereafter. These results indicate that (1) a LAK-sensitive tumor line may contain a subpopulation of cells which are significantly less lysed by LAKs; (2) a correlation between LAK sensitivity and susceptibility to C-mediated lysis is also present; and (3) increased sensitivity to Dx is evident in the IS sublines.