Vernonia cinerea Less. inhibits tumor cell invasion and pulmonary metastasis in C57BL/6 mice

The effect of Vernonia cinerea Less. extract on the inhibition of lung metastasis induced by B16F-10 melanoma cells was studied in C57BL/6 mice. V cinerea extract significantly (P < .001) inhibited lung tumor formation (78.8%) and significantly increased the life span (72.5%). Moreover, lung collagen hydroxyproline, uronic acid, and hexosamine and also serum sialic acid, γ-glutamyltransferase (GGT), and vascular endothelial growth factor (VEGF) levels were found to be significantly (P < .001) lower in treated animals compared with untreated controls. Histopathological analysis of the lung tissues also correlated with these findings. V cinerea treatment significantly inhibited the invasion of B16F-10 melanoma cells across the collagen matrix of the Boyden chamber. V cinerea also inhibited the migration of B16F-10 melanoma cells across a polycarbonate filter in vitro. It downregulated the production and expression of proinflammatory cytokines such as TNF (tumor necrosis factor)-α, IL (interleukin)-1β, IL-6, and GM-CSF (granulocyte monocyte colony-stimulating factor). V cinerea extract administration could suppress or downregulate the expression of matrix metalloproteinase (MMP)-2, MMP-9, lysyl oxidase, prolyl hydroxylase, K-ras, extracellular signal-regulated kinase (ERK)-1, ERK-2, and VEGF and also upregulate the expression of nm-23, tissue inhibitor of metalloproteinase (TIMP-1), and TIMP-2 in the lung tissue of metastasis-induced animals. It also inhibited the protein expression of MMP-2 and MMP-9 in gelatin zymographic analysis of B16F-10 cells. These results indicate that V cinerea could inhibit the metastatic progression of B16F-10 melanoma cells in C57BL/6 mice by regulating MMPs, VEGF, prolyl hydroxylase, lysyl oxidase, ERK-1, ERK-2, TIMPs, nm23, and proinflammatory cytokine gene expression in metastatic lung tissue.     

C57BL/6小鼠的血循环DNA的定量研究

目的探讨循环DNA的定量检测在荷瘤C57BL/6小鼠中的应用价值.方法以异位接种法在C57BL/6小鼠中建立B16黑色素瘤肝转移模型,以"微量基因组抽提试剂盒"抽提血清DNA,用SYBR Green I斑点荧光染色法对DNA进行定量.结果在正常小鼠中,血循环DNA为(62.4±25.52)ng/mL、荷瘤伴有肝转移小鼠为(241±73.7)ng/mL(P＜0.01);小鼠的血循环DNA水平与小鼠肝转移的分期相关,Ⅰ期、Ⅱ期肝转移小鼠分别为(131.11±54.32)ng/mL和(170±10.96)ng/mL,Ⅲ期、Ⅳ期小鼠为(235±85.54)ng/mL和(271±65.76)ng/mL.用健脾理气中药抑制肿瘤生长和转移的同时,荷瘤小鼠血循环DNA也同步下降.结论循环DNA定量检测能客观反应小鼠荷瘤程度,并能动态反应药物的疗效,是一具有应用前景的肿瘤标志物.     

Possible two-stage transplacental liver carcinogenesis in C57BL/6 mice.

A single SC injection of 2-acetylaminofluorene (AAF) was given to pregnant C57BL/6 mice on day 15 of gestation, and the offspring subsequently given twice-weekly injections of phorbol for 25 weeks. Control groups included: (1) untreated; (2) AAF-treated mothers (kept under observation for 18 months, as with all the other groups); (3) untreated offspring of untreated mothers; (4) untreated offspring of AAF-treated mothers, and (5) phorbol-treated offspring of untreated mothers. The incidence of hepatomas in the phorbol-treated offspring of AAF-injected mothers was 8/74 (11%), as compared with 2/80 (2.5%) in the untreated offspring of AAF-injected mothers. The AAF-injected mothers themselves developed 3/36 (8%) hepatomas; while all othe other control groups were free from liver tumours. The development of reticulum cell sarcomas, and of a few cases of lung adenomas, in the various groups, was presumably spontaneous. The results seem sufficiently encouraging, as a model for the study of systemic carcinogenesis, to warrant further attempts at two-stage transplacental carcinogenesis, using other potential initiators and promoters.     

Cytofluorometric analysis of thymic interdigitating cells from C57BL/6 mice prior and after leukemogenic X-irradiation

The thymus is populated by various Ia+ cell populations, including epithelial cells, macrophages and dendritic cells. Thymic cell suspensions were stained with an anti-Ia antibody and shown by cytofluorometry to contain a small number of strongly Ia+ cells characterized by a large diameter. The cell population was separated with the aid of the fluorescence-activated cell sorter (FACS) and characterized. They were shown to express high levels of membranal Ia antigens; they demonstrated ATPase activity and displayed the ultrastructural features characteristic of the previously described thymic interdigitating cells. C57BL/6 mice were submitted to various regimens of X-irradiation. Whereas exposure to a single dose of X-irradiation was followed by an increase in the percentage of strongly Ia+ cells, exposure to a leukemogenic regimen of fractionated X-irradiation led to a decrease in the percentage and absolute numbers of these cells in the thymus. Of the C57BL/6 mice that were irradiated with fractionated X-irradiation, 77% developed leukemia. Intravenous injection of syngeneic bone marrow one day following the last irradiation or protection of the femur during irradiation prevented both the appearance of leukemia and the disappearance of interdigitating cells. Therefore an inverse correlation between the presence of thymic dendritic cells and the incidence of leukemia in C57BL/6 mice could be demonstrated. These findings are discussed in relation to the putative role of dendritic cells in the thymus.     

Neonatal UVB exposure accelerates melanoma growth and enhances distant metastases in Hgf-Cdk4(R24C) C57BL/6 mice

Genetically engineered mouse models offer new opportunities to experimentally investigate the impact of UV on melanoma pathogenesis. Here we irradiated a cohort of newborn 15 Hgf-Cdk4(R24C) mice on the pigmented C57BL/6 background with one erythemogenic dose of 6 kJ/m(2) UVB and compared the development of nevi and melanoma with a cohort of 30 untreated Hgf-Cdk4(R24C) mice. Neonatal UVB exposure decreased the latency and accelerated the growth of primary melanomas resulting in a significantly decreased time from melanoma onset to melanoma-related death (61 days vs. 96 days). Interestingly, we did not observe differences in the development of melanocytic nevi. Histopathological investigations revealed that UVB irradiation shifted the spectrum of melanomas toward a more aggressive phenotype with increased tumor cell proliferation, invasive growth and enhanced angiogenesis. Accordingly, we observed distal melanoma metastases in the lungs more frequently in the UV-irradiated than in the untreated cohort of Hgf-Cdk4(R24C) mice (73% vs. 47%). UVB-induced melanomas only contained very few infiltrating immune cells and expressed very low levels of proinflammatory chemokines. Taken together, our results demonstrate that neonatal UVB exposure promoted the early appearance of rapidly enlarging primary melanomas in Hgf-Cdk4(R24C) C57BL/6 mice which showed enhanced invasive and metastatic behaviour without a persistent tumor-associated inflammatory response. The preferential impact of UVB irradiation on the progression of melanoma without an effect on the development of nevi supports the hypothesis that the molecular targets of UVB are involved in bypassing the proliferative arrest of transformed melanocytes without alerting a cellular immune response.     

Ajoene inhibits both primary tumor growth and metastasis of B16/BL6 melanoma cells in C57BL/6 mice

Ajoene is an organosulphur compound derived from garlic with important effects on several membrane-associated processes such as platelet aggregation, as well as being cytotoxic for tumor cell lines in vitro. In the present study, we investigated the effect of ajoene on different cell types in vitro, as well as its inhibitory effects on both primary tumors and metastasis in a mouse model. We found ajoene to inhibit tumor cell growth in vitro, but also to inhibit strongly metastasis to lung in the B16/BL6 melanoma tumor model in C57BL/6 mice. As far as we are aware, this is the first report of the anti-metastatic effect of ajoene. Ajoene also inhibited tumor-endothelial cell adhesion, as well as the in vivo TNF-α response to lipopolysaccharide. Possible mechanisms of its antitumoral activity are discussed in the light of these results.     

Genetic control of hepatocarcinogenesis in C57BL/6J and C3H/HeJ inbred mice

Treatment of newborn male C3H/HeJ mice with N, N-dietnyl-nitrosamine(DEN) or N-ethyl-N-nitrosourea (ENU) resulted in the inductionof hepatocellular adenomas and carcinomas with a mean numberof tumors per animal that was 20-to 50-fold higher than thatfor similarly treated C57BL/6J male mice. We used two methodsto study the genetic basis for this difference in susceptibilityto liver tumor induction. Analysis of DEN-induced liver tumormultiplicities as a quantitative genetic trait in segregatingcrosses between C3H/HeJ and C57BL/6J mice indicated that allelicdifferences for at least two loci contributed to the highersensitivity to hepatocarcinogenesis of C3H/HeJ mice relativeto C57BL/6J mice. However, a single locus, which we have denotedHcs (hepato-carcinogen sensitivity), was responsible for 85%of the difference in susceptibility. The C57BL/6J and C3H/HeJalkies at this locus were semi-dominant. This result was confirmedby analysis of hepatocarcinogenesis by ENU in BXH (C57BL/6J? C3H/HeJ) recombinant inbred mice. Four of the nine recombinantinbred strains studied were highly susceptible to the inductionof liver tumors by ENU, three of the strains exhibited the resistantphenotype of the C57BL/6J parent, and two of the strains wereof intermediate sensitivity to hepatocarcinogenesis. Newbornmale C3H/HeJ and C57BL/6J mice did not significantly differin the extent of ethylation of hepatic DNA, or in the relativelevels of N-7-ethylguanine or O⁶-ethylguanine after treatmentwith [1-¹⁴C]DEN.     

Characterization of env gene recombination in x-ray--induced thymomas of C57BL/6 mice

A role for specific recombination events at the env region of endogenous murine leukemia virus (MuLV) sequences in radiation carcinogenesis in C57BL mice has been suggested by a number of studies. We characterized env-related cell surface antigens from a primary, x-ray--induced, and several transplanted C57BL/6 thymomas of viral and radiation etiologies by immunoprecipitation and two-dimensional peptide mapping. DNA from lymphoma cells was also analyzed by Southern blotting for evidence of mink cell focus-forming (MCF) type env recombination events. Although gp70 molecules with novel structural determinants were found on all transplanted lymphomas examined, expression of novel env antigens was variable among these lymphomas, and there was a lack of correlation of characteristic MCF-type env recombination events in endogenous retrovirus DNA sequences with novel env antigens on lymphoma cell surfaces. Neither novel gp70 antigens nor MCF-type env provirus recombinant structures were consistent features of the C57BL/6 thymomas of radiation etiology examined in this study, even though MCF-type env recombination events have been suggested as etiologically significant in MuLV-mediated lymphomagenesis in both RadLV and x-ray--induced tumors in C57BL/6 mice.     

双氢青蒿素对Lewis肺癌小鼠的抑瘤作用及其机制探讨

目的探讨双氢青蒿素对Lewis肺癌小鼠的抑瘤效应及其作用机制。方法C57BL/6J小鼠皮下接种3LL细胞（2&#215;10^6）50只,随机分为5组,分别为生理盐水组、阳性对照顺铂组、双氢青蒿素高、中、低剂量组,检测各组小鼠的体重变化和抑瘤率,应用流式细胞术进行TUNEL分析和凋亡相关基因及其表达改变的检测。结果双氢青蒿素中、高剂量组体重较生理盐水组增加明显,其抑瘤率分别为53.5%及59.2%;流式细胞术TUNEL检测结果显示,其作用细胞凋亡和坏死同时存在。FCM法分析其对肺癌组织凋亡相关基因检测表明中、高剂量组Fas、p53等凋亡基因上调,Bcl-2抑制凋亡基因下调。结论双氢青蒿素对Lewis肺癌小鼠肺癌生长具有一定的抑制作用,能促进肿瘤细胞凋亡,其机制可能与调控细胞凋亡相关基因的表达有关。     

Relationship of spontaneous regional lymph node metastases to dose of local irradiation of primary B16 melanomas

We evaluated the effects of local X-irradiation on microscopic or small macroscopic primary melanomas in the feet of C57BL/6 mice and the subsequent development of spontaneous femoral lymph node (LN) metastases. Doses of 30, 40, 55, 62.5, or 72.5 Gy often cured the foot tumor and metastases to regional femoral lymph nodes were relatively uncommon. Doses of 3.75, 7.5, 10, 15, and 20 Gy were associated with a dose-dependent regrowth delay of the foot tumor treated at microscopic size. Foot melanomas that were not cured spread to regional femoral LNs more frequently (P less than 0.001). The relative risk of developing femoral LN metastasis increased 2.55 times for each 1-mm increase in the anteroposterior diameter of the primary foot tumor in mice with 20 days of primary tumor exposure and increased 4.87 times for each 1-mm increase in mice with 100 days of primary tumor exposure. Although tumors treated with subcurative doses of irradiation had a longer period of time to metastasize to regional LNs for each 1-mm increase in primary tumor size, this variable alone did not account for the increased incidence of metastasis seen with irradiation.     

Reduction of pulmonary metastases of B16 melanoma by human recombinant interleukin 2 and lymphokine-activated killer cells in immunosuppressed C57BL/6 mice receiving anticancer agent

The immunosuppressive effect of a water-soluble nitrosourea derivative, 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), was evaluated in terms of the cytotoxicity of spleen lymphocytes, and the restoring effect of lymphokine-activated killer (LAK) cells and/or human recombinant interleukin-2 (rIL-2) on the cytotoxicities of spleen lymphocytes was examined in ACNU-treated C57BL/6 mice. In addition, we tested whether the administration of LAK cells and/or rIL-2 could reduce the increased numbers of pulmonary metastases in ACNU-treated mice. The maximum effective dose of ACNU suppressed the cytotoxicity of spleen lymphocytes and pretreatment with ACNU enhanced the induction of artificial pulmonary metastases. The administration of LAK cells and/or human rIL-2 restored the cytotoxicity of spleen lymphocytes against YAC-1 and syngeneic B-16 melanoma cells in ACNU-treated mice, and these treatments partially suppressed the increased numbers of artificial pulmonary metastases of B-16 melanoma cells in ACNU-treated mice. These results are extremely important in providing a rationale for the introduction of adoptive immunotherapy using LAK cells and rIL-2 in patients with advanced cancer who are being treated with anticancer agent(s).     

X-linked dominant growth suppression of transplanted tumors in C57BL/6J-scid mice

Tumor susceptibility, angiogenesis, and immune response differ between mouse strains. We, therefore, examined the growth rates of tumor xenografts in three genetically isolated strains of severe combined immunodeficient mice (C.B-17, C57BL/6J, and C3H). Tumors grew at significantly reduced rates in the C57BL/6J-scid strain. Engrafting bone marrow from the C57BL/6J-scid strain onto C.B-17-scid mice did not transfer the slow-growing tumor phenotype to the recipient mice; this counters the supposition that the slow-growing tumor phenotype is caused by a greater immune response to the xenograft in the C57BL/6J-scid strain. To establish the inheritance pattern of the slow-growing tumor phenotype, we reciprocally crossed C.B-17-scid mice and C57BL/6J-scid mice. Tumor growth was suppressed in all of the F1 progeny except the male mice derived from the cross between C.B-17-scid female and C57BL/6J-scid male mice. The F1 male mice that received the X chromosome from the C.B-17 strain displayed a fast-growing tumor phenotype. These results confirm that there are significant strain differences in capacity to support the growth of tumor xenografts. In addition, these results reveal the existence of a dominant allele involved in host suppression of tumor growth on the X chromosome of C57BL/6J mice.     

Increased invasion and spontaneous metastasis of BL6 melanoma with inhibition of the desmoplastic response in C57 BL/6 mice

BL6 melanoma cells injected s.c. in 18-month C57 BL/6 mice elicit a markedly fibrotic response similar in myofibroblast and collagen composition to that characterizing the desmoplastic response of human breast carcinoma. This host response can be quantitated by measuring hydroxyproline (total collagen) and incorporation of i.p.-injected [14C]proline into collagenase-sensitive protein (new collagen synthesis). Inhibition (70%) of the desmoplastic response can be achieved by daily injections of L-3,4-dehydroproline. Inhibiting the response in this manner promotes local invasion of tumor and increases the incidence of spontaneous pulmonary metastasis. 10(5) BL6 melanoma cells produce tumor nodules with a mean diameter of 1.5 +/- 0.5 cm and mean collagen content of 36 +/- 15 mg/g wet tissue at 4 weeks and 10% incidence of pulmonary metastasis at 7 weeks. L-3,4-dehydroproline produces nodules with a mean diameter of 2.3 +/- 0.5 cm and mean collagen content of 12 +/- 2 mg/g with a 40% incidence of metastasis. L-3,4-dehydroproline exerts a selective effect on myofibroblast collagen synthesis in vitro and no effect on [3H]thymidine uptake, doubling time, and viability of BL6 cells and myofibroblasts. Furthermore, this drug exerts no effect on BL6 invasion and metastasis in 6-week C57 BL/6 mice, hosts which exhibit a negligible desmoplastic response.     

Marked granulocytosis in C57BL/6 mice bearing a transplanted BMT-11 fibrosarcoma

When a 3-methylcholanthrene (CAS: 56-49-5)-induced fibrosarcoma, BMT-11, and its eight clones were transplanted sc into syngeneic C57BL/6 mice, leukemoid reaction characterized by a progressive increase in peripheral white blood cells (WBCs) and by splenomegaly was observed as the tumors grew. The WBC count reached about forty-fold of the normal level, and more than 90% of WBCs were found to be polymorphonuclear leukocytes. The increase in WBCs was correlated with tumor size, and its count decreased to normal level within 7 days after surgical excision of subcutaneous tumors. Moreover, a high level of colony-stimulating activity was detected in the supernatant of BMT-11 culture. These results suggest that the colony-stimulating factor produced by BMT-11 cells caused granulocytosis in mice. This is the first report of the marked degree of granulocytosis induced by a transplanted tumor in C57BL/6 strain mice.