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.     

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.     

C57BL/6小鼠B16黑色素瘤细胞脾转移机制

 目的　研究C57BL／6小鼠B16黑色素瘤细胞脾转移与血行转移的相关性。方法　细胞悬液接种法制备C57BL／6小鼠B16黑色素瘤细胞的荷瘤模型，HE染色法观察各组织器官中转移瘤细胞的有无及其生长状态，伊文思蓝尾静脉注射法观察蓝染部位与肿瘤转移阳性部位间的相关性。结果　接种成瘤率100 ％。12只小鼠中有9只出现脾转移，均同时伴有不同程度的局部淋巴结转移， 其中3只尚伴有肺转移。脾转移较肺转移出现早、概率高。大体观脾转移阳性部位位于脾背侧段约全长1／4区域。镜下观脾内转移瘤细胞呈散在分布，生长受抑并向成熟分化。伊文思蓝蓝染部位位于脾背侧段约全长1／4区域，与肿瘤转移阳性部位一致。结论　C57BL／6小鼠的B16黑色素瘤细胞脾转移先于其他脏器转移，其转移途径为血行转移。     

Generation of phenotypic diversity in the B16 mouse melanoma relative to spontaneous metastasis

Serial s.c. transplantation of the B16 melanoma in syngeneic mice for nearly 30 monthly generations effected gradual changes in the incidence of spontaneous pulmonary metastasis. Cell lines derived from s.c. tumors and from secondary tumor growths in the lungs were comparably variable in metastatic predilection and also differed in ability to produce tumor colonies in the lungs following i.v. injection of cultured cells. Some lines metastasized to the lungs from s.c. tumors and also colonized the lungs; others were metastatic but noncolonizing, colonizing and nonmetastatic, or nonmetastatic and noncolonizing ("null"). Metastatic and colonizing activities of all cell lines except the most potent colonizers were unstable in culture and during s.c. growth. Over 150 clones and subclones were obtained from B16 melanoma cell lines, and four distinct categories were defined on the basis of dissemination-related phenotypic characteristics: slow-growing null (Ns); rapid-growing null; metastatic; and colonizing. No single cell belonged to more than one category at the same time, but interconversions occurred rapidly and consistently during growth in vitro and in vivo. Progenitor Ns cells generated metastatic cells in culture and in tumors and became rapid-growing null cells and colonizers solely within tumors. Metastatic activity was transient, with cells reverting back to an Ns phenotype in culture and s.c. or converting to rapid-growing null cells and colonizers in vivo. Only potent colonizers were stable, an apparent end result of phenotypic diversification, but formation or proliferation of these cells within tumors was somehow regulated. Comparable heterogeneity was generated within lung metastases, except that reversion of metastatic cells to Ns cells and regeneration of metastatic activity were not demonstrated; the result was a progressive loss of metastatic cells within developing metastases.     

Rubratoxin A specifically and potently inhibits protein phosphatase 2A and suppresses cancer metastasis

(Cancer Sci 2010; 101: 743–750) Although cytostatin analog protein phosphatase 2A (PP2A)‐specific inhibitors are promising candidates of a new type of anticancer drug, their development has been hindered because of their liability. To find new classes of PP2A‐specific inhibitors, we conducted a screening with microbial metabolites and found that rubratoxin A, a classical mycotoxin, is a highly specific and potent inhibitor of the enzyme. While rubratoxin A inhibits PP2A at Ki = 28.7 nm , it hardly inhibited any other phosphatases examined. Rubratoxin B, a close analog, also specifically but weakly inhibits PP2A at Ki = 3.1 μm . The inhibition of intracellular PP2A in cultured cells is obviously observed with 20 μm rubratoxin A treatment for 3 h, inducing the overphosphorylation in PP2A substrate proteins. Although rubratoxins and cytostatin differ in the apparent structures, these compounds share similarities in the structures in detail and PP2A‐binding manners. Rubratoxin A showed higher suppression of tumor metastasis and reduction of the primary tumor volume than cytostatin in mouse experiments. As a successor of cytostatin analogs, rubratoxin A should be a good compound leading to the development of antitumor drugs targeting PP2A.     

Site-induced differences in spontaneous metastasis of MO4 mouse fibrosarcoma cells

MO4 mouse fibrosarcoma cells metastasized to the lungs and to the regional lymph nodes from a subcutaneous tumour in the pinna when they were derived from a tumour in the tail or from lung metastases. In contrast, no metastases were found when MO4 cells were derived from a tumour in the pinna or from the parent cell line. Cells from tumours in the pinna produced metastatic tumours when they were implanted in the tail but not in the pinna. The type of implant-spheroidal cell aggregate, freshly cut tumour fragment or precultured tumour fragment- had no influence on the metastatic behaviour of the resulting tumour. These observations confirm the concept that local factors at the growth site of a tumour influence the metastatic behaviour of that tumour.     

Effect of glucose stress conditions in BL6T murine melanoma cells

Elevated expression of stress proteins can be a characteristic of human cancer and may be involved in the development of resistance to some types of chemotherapeutic agent. In this paper, the effect of physiological stress conditions, such as glucose deprivation, was investigated in overexpressing nPKCdelta murine melanoma BL6 (BL6T) cells. Glucose stress conditions decreased the proliferative capacity, increasing the percentage of BL6T cells in the G0/G1 phase of the cell cycle. Furthermore, under such conditions, nPKCdelta, whose subcellular localization is cell cycle dependent, showed a cytoplasmic and perinuclear localization by immunohistochemistry, this being typical for cells in G0/G1 phase. Moreover, these cells expressed GRP-78, a known stress protein. On the other hand, glucose depletion enhanced intracellular melanin as well as tyrosinase activity and expression. In summary, these data demonstrate that stress conditions can modify the biological characteristics of BL6T cells, and therefore can select a quiescent cellular population.     

Statins improve survival by inhibiting spontaneous metastasis and tumor growth in a mouse melanoma model

Metastatic melanoma is a life-threatening disease for which no effective treatment is currently available. In melanoma cells, Rho overexpression promotes invasion and metastasis. However, the effect of statins on spontaneous metastasis and tumor growth remains unclear. In the present study, we investigated the mechanism of statin-mediated tumor growth and metastasis inhibition in an in vivo model. We found that statins significantly inhibited spontaneous metastasis and tumor growth. Statins inhibited the mRNA expression and enzymatic activities of matrix metalloproteinases (MMPs) in vivo and also suppressed the mRNA and protein expression of very late antigens (VLAs). Moreover, statins inhibited the prenylation of Rho as well as the phosphorylation of LIM kinase, serum response factor (SRF), and c-Fos downstream of the Rho signaling pathway. In addition, statins enhanced p53, p21, and p27 expression and reduced phosphorylation of cyclin-dependent kinase and expression of cyclin D1 and E2. These results indicate that statins suppress Rho signaling pathways, thereby inhibiting tumor metastasis and growth. Furthermore, statins markedly improved the survival rate in a metastasis model, suggesting that statins have potential clinical applications for the treatment of metastatic cancers.     

A spontaneous murine melanoma lung metastasis comprised of host x tumor hybrids

Cells from a lung metastasis, arising from Cloudman S91 melanoma cells implanted s.c. in the tail of a BALB/c nu/nu mouse, were comprised chiefly of host x tumor hybrids. These lung metastasis cells showed: (a) 30-40% increased DNA content; (b) resistance to 10(-4) M hypoxanthine, 4 x 10(-7) M aminopterin, and 1.6 x 10(-5) M thymidine (HAT) + G418; and (c) the presence in genomic DNA of genes for both wt and albino tyrosinase, reflecting the DBA/2J (Cloudman S91) and BALB/c mouse genotypes, respectively. Individual clones of lung metastasis cells expressed enhanced pigmentation, motility, and responsiveness to MSH/IBMX, a behavior similar to that recently reported for artificially generated melanoma x macrophage fusion hybrids. These similarities suggested that the host fusion partner generating the lung metastasis hybrids might have been a macrophage, although formal proof for this was not possible. The results provide the first direct evidence that host x tumor hybridization could serve as an initiating mechanism for melanoma metastasis.     

Effect of melanoma stem cells on melanoma metastasis

Cancer stem cells (CSCs) are involved in the metastatic process, the resistance of many types of cancer to therapeutic treatments and consequently the onset of recurrences. The CSC concept therefore significantly extends our understanding of melanoma biology. More recently, melanoma stem cells (MSCs) have been described in melanoma as expressing specific biomarkers. These primitive melanoma cells are not only capable of self-renewal and differentiation plasticity, but may also confer virulence via immune evasion and multidrug resistance, and potentially, via vasculogenic mimicry and transition to migratory and metastasizing derivatives. This review will present the specific biomarkers of MSCs, including CD133, ATP binding cassette subfamily B member 5, CD271, CD20 and aldehyde dehydrogenase, which can regulate the transduction of tumor-related signals. These signal molecules can reversely act on tumor cells and regulate tumor angiogenesis, leading to the occurrence of melanoma metastasis. Targeting these specific biomarkers could inhibit the progression of melanoma and may help the development of novel therapeutic strategies for melanoma.     

The effect of poly (aspartic acid-co-lactic acid) nanospheres on the lung metastasis of B16BL6 melanoma cells by intravenous administration

Poly (aspartic acid-co-lactic acid) (PAL) has been investigated as a new biodegradable material for Drug Delivery Systems (DDS). Similar to the poly (lactic acid-co-glycolic acid) (PLGA) nanospheres, the PAL nanospheres can control-release encapsulated drugs by hydrolysis and adhere to the mucous membranes to improve the drug absorption. In this study, the vitamin encapsulated PAL nanospheres were applied on mice to examine their effect on tumor metastasis and safety as an injectable DDS material for anti-cancer and other drugs. In the experiment, 6 C57BL/6 mice per group were intravenously administered with B16BL6 melanoma cells (1 x 10(5) per mouse) and non-encapsulated PAL nanospheres or pro-vitamin encapsulated nanospheres respectively, while the control group was administered with B16BL6 cells alone. Two weeks later, the lungs of the mice were excised and metastatic foci on the lung surface were counted. The melanoma cell metastasis to lungs was prevented by intravenous co-injection of B16BL6 melanoma cells with 1.7 microg of pro-vitamin E encapsulated PAL nanospheres. Its metastatic foci count (mean +/- SD) was 127+/-80, which was better than the control (246+/-95, p<0.02). Also, applying the pro-vitamin C and pro-vitamin A encapsulated PAL nanospheres as well as the non-encapsulated PAL nanospheres slightly decreased the number of metastasis colonies in the lungs as compared to that of the control. These results suggested that PAL nanospheres did not promote the lung metastasis of B16BL6 melanoma cells. Thus, the PAL nanospheres are safe material for injection applications.     

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.     

Inhibitory effect of tumor necrosis serum on the metastasis of B-16 mouse melanoma cells

The inhibitory effect of tumor necrosis serum (TNS) on the artificial metastasis of B-16 melanoma cells and the spontaneous metastasis of Lewis lung carcinoma cells was investigated. The results obtained were as follows: 1) When tumor necrosis serum (TNS) was administered either 20 min or 2 days after injection of B-16 melanoma cells, a very strong inhibitory effect (99%) relative to the control was noted. 2) TNS showed a 60% inhibitory effect on spontaneous metastasis, and the weight of the original tumor regressed by 60%. 3) No histological changes in normal tissues were observed microscopically following TNS injection. The above results confirm that TNS is extremely effective in preventing metastasis.     

组胺对C57BL/6小鼠B16黑色素瘤转移行为的影响

背景与目的:肿瘤生物治疗中微血管壁通透性是大分子抗体偶联药物进入瘤体的主要屏障.组胺可通过增加微血管壁通透性和组织液的生成促进抗体偶联物在肿瘤局部的富集.本研究观察组胺所致微血管壁通透性的增加和组织液的生成增多是否会增加肿瘤血行及/或淋巴的转移.方法:采用瘤细胞悬液接种法将小鼠黑色素瘤细胞B16注入小鼠左上肢腋部皮下,建立C57BL/6小鼠荷瘤模型.实验组接种第6天起,背部皮下注射300 mg/kg组胺,隔日一次,共5次,对照组注射等体积生理盐水.组织化学方法检测肿瘤细胞在各脏器的转移情况.分别用Student t-test和四格表资料确切概率法分析组胺对肿瘤细胞增殖和转移的作用.结果:接种成瘤率100%.300 mg/kg组胺隔日一次皮下注射能明显抑制肿瘤的生长,实验组与对照组瘤重分别为(5.26 1.55)g和(6.96±1.31)g,二者间差异有统计学意义(P＜0.01).实验组与对照组淋巴结转移率分别为33.3%和75.0%,血行转移率分别为25.0%和75.0%,差异均有统计学意义(P＜0.05).结论:组胺能够抑制C57BL/6小鼠B16黑色素瘤的血行和淋巴转移,该抑制作用部分与其抑制瘤细胞的增殖有关.     

Overexpression of novel protein kinase C delta in BL6 murine melanoma cells inhibits the proliferative capacity in vitro but enhances the metastatic potential in vivo

In this study we analysed the effect of overexpressing novel protein kinase C delta isoform (n-PKC delta) on melanin synthesis and metastatic potential in the highly metastatic BL6 murine melanoma cells. The proliferative capacity in vitro and into matrigel in vivo were also examined. Although murine melanocytes express the n-PKC delta isoform, BL6 cells do not express this isoform at levels detectable by Western blot analysis. In untransfected and transfected cells we also studied the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), a modulator of specific isoforms of PKC, and of bryostatin 1, a potent immunomodulator and antineoplastic drug and a partial agonist of PKC. Our results demonstrate a pivotal role for this isoform in melanin synthesis and the close relationship between n-PKC delta expression and its association with the particulate fraction, melanogenesis and metastatic potential. In fact, heterogeneous BL6 cells overexpressing n-PKC delta and all the clones isolated showed increased intracellular melanin and metastatic capacity. TPA and bryostatin 1 decreased n-PKC delta expression, the intracellular melanin level and metastatic capacity in both cell lines. Therefore both treatments were able to abolish the effects of overexpressing n-PKC delta.     

Acridine orange inhibits pulmonary metastasis of mouse osteosarcoma

Although the survival of patients with osteosarcoma has improved following development of chemotherapy and surgery, the presence of pulmonary metastases indicate a poor prognosis. We developed photodynamic and radiodynamic therapies with acridine orange (AO-PDT and AO-RDT) for minimally invasive surgery to treat musculoskeletal sarcomas and reported a good clinical outcome of local control and limb function. We investigated the effect of AO-PDT using flash-wave light (FWL) on pulmonary metastasis of mouse osteosarcoma. In in vitro and in vivo studies, AO alone and AO-PDT significantly inhibited cell invasion and the growth of pulmonary metastases from primary mouse osteosarcoma. AO may have a specific metastasis-inhibitory effect, different from the effect of AO-PDT. The fluorovisualization effect on pulmonary metastases following intravenous AO administration showed that pulmonary metastases localized on the lung surface were recognized as brilliant green lesions. In conclusion, AO-PDT using FWL inhibited cell invasion and pulmonary metastases in mouse osteosarcoma; therefore, this treatment modality might be applicable for treating pulmonary metastasis from malignant musculoskeletal tumors in humans.     

Enhanced spontaneous metastasis of mouse carcinoma cells transfected with an activated c-Ha-ras-1 gene

To investigate whether the presence of an activated ras oncogene influences the ability of tumour cells to metastasize, the c-Ha-ras-1 oncogene cloned from EJ/T24 cells was introduced into MT1 Cl.5/7 mouse mammary carcinoma cells. Since the MT1 Cl.5/7 cells are already tumorigenic but have a low metastatic capacity, this experimental design allows a distinction to be made between the effects of the ras gene on metastasis and tumorigenicity. MT1 Cl.5/7 containing the EJ c-Ha-ras-1 metastasized more readily and to more tissue sites than control cells (2.8 sites/mouse vs 0.9 sites/mouse). The metastases expressed the EJ c-Ha-ras-1 p21 ras proteins; however, one metastasis was discovered that had lost the expression of the c-Ha-ras-1 gene. When these cells were re-tested for metastasis, the rate of metastasis was indistinguishable from that of controls. This observation, coupled with a demonstration that lung colonization potential following intravenous inoculation is unaffected by the presence of the activated ras gene, argues that the effect of mutant ras genes is exerted on the ability of cells to escape from the primary tumour, rather than on a survival in the circulatory systems and ability to seed a second site.