苦参碱衍生物M19对于肿瘤细胞转移的抑制作用

目的:探讨苦参碱衍生物M19对于肝细胞癌和黑色素瘤细胞转移的抑制作用。方法:肝癌细胞Hep3B、MHCC-LM3和黑色素瘤细胞B16F10经M19处理后,检测肿瘤细胞体外迁移能力。建立C57BL/6小鼠黑色素瘤B16F10和裸鼠肝细胞癌MHCC-LM3转移模型,经M19体内给药后,观察肿瘤细胞肝、肺转移情况。结果:M19能浓度依赖性地抑制肝癌细胞和黑色素瘤细胞迁移。M19体内给药使B16F10细胞在小鼠肺的转移减少,且还能抑制MHCC-LM3细胞在裸鼠肺和肝的转移。结论:M19能在体内外抑制肝细胞癌和黑色素瘤细胞的转移。     

Effect of Wf-536, a novel ROCK inhibitor,against metastasis of B16 melanoma

PURPOSE: Rho-associated coiled-coil-forming protein kinase (ROCK) is pivotally involved in invasion by tumor cells and their evolution to metastasis. We have developed a novel inhibitor of ROCK, Wf-536 [(+)-(R)-4-(1-aminoethyl)-N-(4-pyridyl) benzamide monohydrochloride]. In the present study, we investigated its effect on in vitro invasion and in vivo pulmonary metastasis of B16 melanoma. METHODS: The following were evaluated: the anti-invasive effect of Wf-536 against the motility of mouse B16BL6 melanoma cells through a culture insert layered with reconstituted basement membrane (Matrigel); the cytotoxic effect of Wf-536 in the same cell line; the antimetastatic effect of Wf-536, administered by osmotic pump, on spontaneous pulmonary metastasis following subcutaneous injection of B16BL6 melanoma in mice; and the inhibitory effect of orally administered Wf-536, alone or in combination with the antineoplastic drug paclitaxel, on pulmonary metastasis of intravenously injected B16F10 melanoma in mice. RESULTS: Wf-536 inhibited in vitro invasion by B16BL6 cells significantly and in a concentration-dependent manner and displayed an anti-invasive effect under conditions of both chemotaxis and chemokinesis. No cytotoxic effect was observed at any of the concentrations used. In vivo, Wf-536 administration suppressed tumor colony formation on the lung surface in a dose-dependent manner (0.3-3 mg/kg per day), with a metastasis inhibition rate of 95% at 3 mg/kg per day. In experimental metastasis of B16F10 melanoma, oral administration of Wf-536 significantly decreased tumor colony formation in the lung, with an inhibition rate of 41% at 3 mg/kg per day. The inhibition rate of paclitaxel (5 mg/kg per day) was 27%. The combination of Wf-536 and paclitaxel produced a synergistic effect on B16F10 metastasis and a 68% inhibition rate. Wf-536 administration at the doses used did not alter body weight, blood pressure or the health of treated animals as compared to vehicle-treated controls. CONCLUSION: The results suggest that Wf-536 is a potentially valuable drug for preventing tumor metastasis both in monotherapy and in combination with an antineoplastic drug.     

Suramin augments the antitumor and antimetastatic activity of pentoxifylline in B16F10 melanoma

Rapid tumor growth and metastasis are 2 major problems associated with treatment of malignant melanoma. Therefore, drugs that can intervene these processes are of clinical importance. Pentoxifylline (PTX), a methyl xanthine derivative, has been shown to inhibit B16F10 melanoma tumor growth and metastasis. We hypothesized that suramin when combined with PTX enhances its antineoplastic effects, which we have examined using the B16F10 mouse melanoma model. Suramin in simultaneous or sequential combination potentiated the cytotoxic effects of PTX on B16F10 cells. PTX arrested cells in the G0-G1 phase and suramin augmented the effects. Both the drugs inhibited F10 adhesion to laminin, matrigel and collagen type IV and showed enhanced inhibition in combination The combination also demonstrated significantly higher inhibition in cell motility (p = 0.002) and invasion through matrigel (p = 0.005) as compared to the single agents. Suramin synergized with PTX in its effects on secretion of MMP-9 gelatinase. DBA2/J mice implanted with intradermal B16F10 tumor were used as a model to study tumor growth. Animals were intratumorally treated with 50 mg/kg of PTX, 10 mg/kg of suramin and their combinations. Simultaneous administration of the drugs inhibited tumor growth by 5- to 6-folds. Tumor growth was completely blocked in sequential regimen with regression in some cases. The number and size of metastatic nodules on lung was also reduced significantly by the combination treatment. In conclusion, the novel combination of PTX and suramin has synergistic antitumor and antimetastatic activity in B16F10 melanoma and may be a promising approach in treatment of patients suffering from malignant melanoma.     

Long exposure of non-cytotoxic concentrations of methylselenol suppresses the invasive potential of B16F10 melanoma

To assess the inhibitory effects of methylselenol on the invasion of murine B16F10 melanoma cells, we carried out in vivo and in vitro experiments using Se-methylselenocysteine (Se-MSC) and selenomethionine (SeMet), respectively. In an animal experiment, the supplementation of drinking water with Se-MSC (4 ppm Se) led to a significant increase in Se levels in the lung, liver and serum in mice. Mice given a mash diet or water supplemented with Se-MSC (2, 4 and 6 ppm Se in the mash diet, and 2 and 4 ppm Se in the drinking water) displayed an almost completely diminished pulmonary metastasis of B16F10 melanoma cells and an enhanced survival, compared to the control mice which were given a basal diet. Treatment with non-cytotoxic concentrations of SeMet (2.5, 5 and 10 microM plus 0.02 U/ml METase, methioninase) induced a substantial decrease in the expression of integrin alphavbeta3, the FN receptor and adhesion ability to vitronectin (VN) and fibronectin (FN) in B16F10 melanoma cells. Moreover, these compounds suppressed gelatinase activity, invasive ability and wound migration in the culture system. SeMet-METase prevented the conversion of pro-MMP-9 to its active form and decreased pro-MMP-2 activities in a zymogram. The pre-treatment of B16F10 melanoma cells with SeMet-METase led to a decrease in pulmonary metastasis and extended survival in mice injected with tumor cells. Collectively, our results indicate that integrin expression is crucial in promoting the metastatic phenotype in murine B16F10 melanoma cells by supporting specific adhesive and invasive properties, suggesting that Se-MSC effectively reduces the metastasis of B16F10 melanoma cells as a nutritional adjuvant. Methylselenol may also contribute to the suppression of integrin expression.     

基因枪介导的neu基因DNA疫苗抗肿瘤作用的实验研究

目的 探讨DNA疫苗pWRG-neu的皮内免疫,对高表达neu基因的小鼠移植瘤生长和转移的抑制作用。方法 向小鼠黑色素瘤B16F10细胞系转染pcDNA-neu,用有限稀释法筛选一株高表达neu基因的细胞株B16F10-neu。在基因枪介导下,向C57BL／6小鼠导入DNA疫苗pWRG-neu,通过观察免疫动物的生存期,评价DNA疫苗的抗肿瘤作用。分离免疫动物脾细胞,经自体淋巴细胞混合培养实验,分析DNA疫苗体内免疫后机体的CTL应答。结果 筛选到一株高表达neu基因的B16F10-neu细胞株,转基因过程和外源基因的表达没有改变细胞系的增殖特性。用基因枪轰击,进行DNA疫苗pWRG-neu皮内免疫,对小鼠黑色素瘤B16F10-neu进行预防、治疗和抗转移的实验研究,结果表明,DNA疫苗的免疫能够明显推迟移植瘤的生长,延长小鼠生存期,获得明显的抗肿瘤效果。DNA疫苗免疫后可诱导小鼠脾淋巴细胞CTL活性。结论 基因枪介导的DNA疫苗pWRG-neu经皮内免疫,能够有效的诱导机体的细胞免疫应答,预防和治疗小鼠移植瘤的发生,并有一定的预防肿瘤肺转移的作用。     

Effect of Caffeine, a xanthine derivative, in the inhibition of experimental lung metastasis induced by B16F10 melanoma cells

Caffeine, a methyl xanthine derivative, was studied to assess the effect on B16F10 melanoma induced experimental metastasis. Caffeine was administered at a dose of 100 and 50 mg/kg body weight by both routes, to tumour bearing animals. Solid tumour reduction studies with Caffeine showed a significant reduction in tumour volume for 100 mg/kg dose by both oral and i.p. routes. The Caffeine treated metastatic tumour bearing animals significantly (p<0.001) inhibited lung tumour nodules. Serum sialic acid levels and lung hydroxyproline contents in the treated groups were significantly (p<0.001) low, when compared with the untreated control animals. In the present study, our results suggest that Caffeine inhibits solid tumour development and pulmonary experimental metastasis induced by B16F10 melanoma cells, in murine model.     

Antigenic differences among B16 melanoma variants selected for their differing abilities to metastasize: a possible mechanism for effective adjuvant immunotherapy

Most C57BL/6J mice will develop pulmonary metastases four to six weeks after excision of B16 melanoma isografts and begin to die within 40 days. When applied in an adjuvant setting after isograft excision, specific B16 immune RNA (I-RNA) therapy prevents pulmonary metastases and prolongs survival in 50% of treated animals. Since increased in vitro cell-mediated cytotoxicity (CMC) against B16 targets has been demonstrated in animals whose survival is improved by B16 I-RNA therapy, we have proposed that the treatment functioned, at least in part, by specifically altering host CMC in vivo. In this study, C57BL/6J splenocytes were specifically sensitized in vitro by B16 I-RNA exposure and examined for cytotoxic effect on variants of B16 melanoma selected for their differing metastatic potential in vivo. F10 tumor targets (explanted from a B16 melanoma vairant producing frequent pulmonary metastases in vivo) were consistently more sensitive to specific in vitro cytotoxic effect than F1 target cells (from a B16 melanoma variant with low metastatic potential in vivo). Lung metastases (F1 mets) were explanted after intravenous (IV) injection of F1 and used as target cells in the in vitro CMC assays. F1 mets demonstrated greater cytotoxic effect than F1 targets after exposure to B16 I-RNA-treated syngeneic splenocytes. C57BL/6J mice were sacrificed at weekly intervals following adjuvant in vivo B16 I-RNA therapy (after B16 isograft excision), and their splenocytes were shown to be consistently more cytotoxic in vitro to B16 and F10 than to F1 target cells. Splenocytes harvested from mice treated with I-RNA specific to antigenically distinct Lewis lung carcinoma (3LL) after 3LL isograft excision had no cytotoxic effect on any of the B16 variants. When nonsensitized C57BL/6J splenocytes were examined for cytotoxic effect in natural killer (NK) assays or in NK inhibition assays, differences in cytotoxic sensitivity of B16, F10, F1, and F1 mets could not be demonstrated. We conclude, therefore, that specifically sensitized effector cells could distinguish antigenic differences among B16 variants selected for differing in vivo metastatic potiential. These differences were tumor specific and could be demonstrated by cytotoxic splenocytes after either in vivo B16 I-RNA treatment or in vitro B16 I-RNA exposure. The relationship of these antigenic differences to in vivo metastatic potential and to the effectiveness of adjuvant B16 I-RNA therapy is discussed.     

The reversible P2Y12 inhibitor ticagrelor inhibits metastasis and improves survival in mouse models of cancer

Tumor cells use activated platelets to promote their proliferation and metastatic potential. Because platelet activation is largely mediated through ADP engagement of purinergic P2Y12 receptors on platelets, we investigated the potential of the reversible P2Y12 inhibitor ticagrelor, a clinical agent used in the prevention of cardiovascular and cerebrovascular events, to inhibit tumor adhesion and metastasis. In B16‐F10 melanoma intravenous and intrasplenic metastasis models, mice treated with a clinical dose of ticagrelor (10 mg/kg) exhibited marked reductions in lung (84%) and liver (86%) metastases. Furthermore, ticagrelor treatment improved survival compared to saline‐treated animals. A similar effect was observed in a 4T1 breast cancer model, with reductions in lung (55%) and bone marrow (87%) metastases following ticagrelor treatment. In vitro, B16‐F10 cells exhibited decreased interaction with platelets from ticagrelor‐treated mice compared to saline‐treated mice, an effect similar to that observed with blockade of glycoprotein IIbIIIa. Similarly, B16‐F10 cells co‐incubated with platelets from ticagrelor‐treated mice exhibited reduced adhesion to endothelial monolayers compared to those co‐incubated with platelets from saline‐treated animals, an effect also observed in vivo. Interestingly, pretreatment of endothelial monolayers with ticagrelor did not result in reduced tumor cell adhesion. These findings support a role for P2Y12‐mediated platelet activation in promoting metastases, and provide proof‐of‐concept for the clinical use of ticagrelor in the prevention of tumor metastasis.     

B16 melanoma cell arrest in the mouse liver induces nitric oxide release and sinusoidal cytotoxicity: a natural hepatic defense against metastasis

The formation of liver metastases involves interactions between intravascular cancer cells and the hepatic microvasculature. Here we provide evidence that the arrest of intravascular B16F1 melanoma cells in the liver induces a rapid local release of nitric oxide (NO) that causes apoptosis of the melanoma cells and inhibits their subsequent development into hepatic metastases. B16F1 melanoma cells (5 x 10(5)) labeled with fluorescent microspheres were injected into the portal circulation of C57BL/6 mice. The production of NO in vivo was detected by electron paramagnetic resonance spectroscopy ex vivo using an exogenous NO-trapping agent. A burst of NO was observed in liver samples examined immediately after tumor cell injection. The relative electron paramagnetic resonance signal intensity was 667 +/- 143 units in mice injected with tumor cells versus 28 +/- 5 units after saline injection (P < 0.001). Two-thirds of cells arrested in the sinusoids compared with the terminal portal venules (TPVs). By double labeling of B16F1 cells with fluorescent microspheres and a TdT-mediated UTP end labeling assay, we determined that the melanoma cells underwent apoptosis from 4-24 h after arrest. The mean rate of apoptosis was 2-fold greater in the sinusoids than in the TPVs at 4, 8, and 24 h after injection (P < 0.05-0.01). Apoptotic cells accounted for 15.9 +/- 0.8% of tumor cells located in the sinusoids and 7.1 +/- 0.9% of tumor cells in the TPVs. The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester completely blocked the NO burst (P < 0.001) and inhibited the apoptosis of B16F1 cells in the sinusoids by 77%. However, the rate of tumor cell apoptosis in the TPVs was not changed. There were 5-fold more metastatic nodules in the livers of N(G)-nitro-L-arginine methyl ester-treated mice (P < 0.05). The inactive enantiomer N(G)-nitro-D-arginine methyl ester had no effect on the initial NO burst or on apoptosis of tumor cells in vivo. Both annexin V phosphatidylserine plasma membrane labeling and DNA end labeling of apoptotic cells were demonstrated after a 5-min exposure (a time equivalent to the initial transient NO induction in vivo) of B16F1 cells to a NO donor in vitro. These results identify the existence of a natural defense mechanism against cancer metastasis whereby the arrest of tumor cells in the liver induces endogenous NO release, leading to sinusoidal tumor cell killing and reduced hepatic metastasis formation.     

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.     

Beta-elemene inhibits melanoma growth and metastasis via suppressing vascular endothelial growth factor-mediated angiogenesis

Purpose

It was to assess antiangiogenic effect of ??-elemene in?vitro and in?vivo, and it was involved in inhibiting melanoma growth and metastasis, as well as to elucidate its intrinsic mechanism.

Methods

Inhibitive effect of ??-elemene on B16F10 cells was performed by cell proliferation assay. Angiogenesis assays in vitro including rat aortic ring and chick embryo chorioallantoic membrane were used, as well as melanoma growth and metastasis assay in C57BL/6 mice. Vascular endothelial growth factor (VEGF) expression in?vitro and in?vivo was measured respectively by western blot analysis and enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry analysis of CD34 and VEGF expression in primary melanoma was also presented.

Results

??-Elemene inhibited B16BF10 cell proliferation starting from 200???M, but VEGF from 20???M. Both 20 and 50???M ??-elemene in?vitro inhibited VEGF-induced sprouting vessel of rat aortic ring and microvessel formation of chick embryo chorioallantoic membrane. In?vivo, tumor size of primary melanoma in mice intraperitoneally treated with ??-elemene was significantly smaller than that of the control; CD34 expression of primary melanoma was also suppressed; and the metastatic melanoma colonies and content of melanin in lung were detected obviously decreased in mice of ??-elemene-treated groups. Furthermore, results of VEGF expressing in primary melanoma, serum and lung of mice also disclosed that VEGF was inhibited in?vivo.

Conclusions

??-Elemene inhibited melanoma growth and metastasis through suppressing VEGF-mediated angiogenesis. It is a natural potential antiangiogenic agent.     

Granulocytes mediates the Fas-L-associated apoptosis during lung metastasis of melanoma that determines the metastatic behaviour

The survival of tumour cells in a new tissue environment is crucial for tumour metastasis. Factors contributing to the death of tumour cells during metastasis are not completely understood. In murine melanoma model, activation of Fas (CD95, APO-1) signal in tumour cells reduces their lung metastasis potential, which may be associated with an induction of apoptosis in tumours. To elucidate the cellular mechanism, we used a Fas-ligand (Fas-L) specific ribozyme (Fas-L(ribozyme)) to suppress the expression of Fas-L but not Fas or TNF-alpha in B16F10 melanoma cells. The Fas-L(ribozyme)-carrying cells grew slightly faster in vitro with better viability than controls. Suppression of Fas-L in B16F10 melanoma cells by Fas-L(ribozyme) enhanced lung metastasis of the cells in C57BL/6 mice, and that was correlated with reductions in both apoptotic tumour cells and granulocytic infiltration. Mice depleted of granulocytes, but not CD4+ and CD8+ cells, showed a greatly elevated susceptibility to lung metastasis. Moreover, apoptosis in tumour cells was significantly reduced in granulocyte-depleted mice during the course of tumour formation. Taken together, our findings indicate that Fas-L-associated apoptosis in tumour cells determines the metastasis behaviour of melanoma in the lung and this apoptosis is primarily mediated by the cytotoxicity of recruited granulocytes.     

Effect of thrombin treatment of tumor cells on adhesion of tumor cells to platelets in vitro and tumor metastasis in vivo.

Seven different tumor cell lines (human melanoma SK MEL 28; hamster melanoma HM29; murine melanomas B16F10 and amelanotic melanoma B16a; human colon carcinoma HCT8; murine colon carcinoma CT26; and murine Lewis lung carcinoma) were treated with thrombin at 0.5-1 unit/ml and examined for their ability to bind to adherent platelets; HM29 was studied for its ability to bind to fibronectin and von Willebrand factor; CT26, B16F1, B16F10, and B16a were studied for their ability to form pulmonary metastasis after i.v. injection of thrombin-treated tumor cells; CT26 was studied for its ability to grow s.c. Five of 7 thrombin-treated tumor cell lines increased their adhesion to adherent platelets 2-to 3-fold. HM29 increased its adherence to fibronectin and von Willebrand factor 2- to 3-fold. CT26, B16F1, B16F10, and B16a increased experimental pulmonary metastasis 10- to 156-fold. Thrombin-treated CT26 cells demonstrated 2-fold greater growth in vivo after s.c. injection. The mechanism of enhanced adhesion of thrombin-treated tumor cells to platelets required the platelet integrin GPIIb-GPIIIa since it could be inhibited by agents known to block adhesion of ligands to GPIIb-GPIIIa (monoclonal antibody 10E5, tetrapeptide RGDS, disintegrin Albolabrin); as well as a "GPIIb-GPIIIa-like" structure on tumor cells since it could be inhibited by treatment of thrombin-treated tumor cells with 10E5 and RGDS. The thrombin effect on tumor cells was optimum at 1 h of incubation with thrombin, did not require active thrombin on the tumor cell surface, and did not require protein synthesis (not inhibited by cycloheximide). Thus, thrombin-treated tumor cells markedly enhance pulmonary metastasis. It is suggested that this may be secondary to thrombin-induced enhanced adhesion as well as growth of tumor cells.     

Effect of naturally occurring monoterpenes carvone, limonene and perillic acid in the inhibition of experimental lung metastasis induced by B16F-10 melanoma cells

The effects of naturally occurring monoterpenes on lung metastasis induced by B16F-10 melanoma cells were studied in C57BL/6 mice. Administration of monoterpenes such as limonene (100 micromoles/kg body wt. 10 doses i.p.) and perillic acid (50 micromoles/kg body wt 10 doses i.p.) remarkably reduced the metastatic tumour nodule formation by 65% and 67%, respectively. These results correlated with the biochemical parameters such as serum sialic acid, lung collagen hydroxyproline and uronic acid contents. Serum sialic acid level in control group was 126.8 microg/ml serum which was significantly lowered in limonene (49.3 microg/ml serum) and perillic acid treated animals (53.6 microg/ml serum). Uronic acid level was also inhibited to 56% and 39.7% in limonene and perillic acid treated animals, respectively. Histopathological studies also correlated with these above results. Administration of Carvone even at 100 micromoles/kg body wt. did not have any significant effect on the metastatic tumour growth. These results indicate that limonene and perillic acid could inhibit the metastatic progression of B16F-10 melanoma cells in mice.     

Overexpression of Merlin in B16F10 mouse melanoma cells reduces their metastatic activity: role of the cell surface heparan sulfate glycosaminoglycans

Merlin, the protein product of the neurofibromatosis type 2 gene (NF2) acts as a tumor suppressor in mice and humans. In this study, melanoma B16F10 cells were engineered to overexpress the NF2 gene by establishing stable transductants. A cell line overexpressing Merlin (B16F10-M) was generated. When compared to the parental cells, the B16F10-M cells demonstrated differences in their cell surface organization. The overexpressing strain changed its ability to grow in soft agar as well as its cell motility properties. B16F10-M cells were then examined in the in vivo mouse melanoma tumor growth and tumor metastasis models. While tumor growth was marginally affected, the presence of increased Merlin severely reduced the metastastatic ability of the cells. When isolated using specific enzymes with distinct substrate specificity, the cell surface heparan sulfate glycosaminoglycans (HSGAGs) from the overexpressing B16F10-M cells, inhibited the metastatic properties of the parental B16F10 cells. The results obtained provide a causal link between the reorganization/changes to the cell surface HSGAGs by the overexpression of Merlin and the inhibition of the metastatic activity of the mouse melanoma B16F10 cells in vivo.     

Enhancement role of host 12/15-lipoxygenase in melanoma progression

12/15-Lipoxygenase (12/15-LOX) is a non-haeme iron-containing dioxygenase that forms 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) or 15(S)-HETE. Several biological mediators including cytokines, growth factors and lipid metabolites released during tumour cell-endothelial cell adhesion are associated with malignant tumour progression. Here we found that HETEs released from the host organ played a critical role in tumour metastasis. Intravenous injection of B16F10 melanoma cells caused lung nodule formation, which was markedly attenuated in 12/15-LOX null mice. Co-injection of melanoma cells with 12(S)-HETE increased the lung homing activity of B16F10 melanoma cells. In vitro studies showed that 12(S)-HETE and 15(S)-HETE treatment resulted in a concentration-dependent increase of adhesion of B16F10 cells on collagen or fibronectin. The melanoma cell adhesion was then evaluated in pulmonary primary cell culture isolated from wild-type (WT) and 12/15-LOX knockout (KO) mice. It was found that the adhesion of melanoma cells on the epithelial cells isolated from 12/15-LOX null mice was reduced in comparison with those isolated from WT mice. Treatment of 12(S)-HETE increased the pFAK in melanoma cells adhering on collagen-coated slide. The enhancement of adherence elicited by 12(S)-HETE in B16F10 cells could be antagonised by focal adhesion kinase (FAK) inhibitor 14 (FAK inhibitor) or PD98059 (extracellular signal-regulated kinase (ERK) inhibitor). 12(S)-HETE increased the phosphorylation of FAK and ERK in adhering melanoma cells. The FAK phosphorylation induced by 12(S)-HETE was further inhibited by PD98059, indicating that FAK is the downstream target of ERK. The adhesion and lung metastasis of human melanoma cells of C32 in NOD/SCID mice were also potentiated by co-treatment with 12(S)-HETE. These results demonstrate that 12(S)-HETE/15(S)-HETE activates ERK and FAK signalling pathways, thereby upregulates the adhesion and metastatic potential of melanoma cells. The endogenous release of 12(S)-HETE/15(S)-HETE in the host organ may affect the metastatic potential of melanoma.     

Barbigerone Inhibits Tumor Angiogenesis,Growth and Metastasis in Melanoma

Tumor angiogenesis, growth and metastasis are three closely related processes. We therefore investigated theeffects of barbigerone on all three in the B16F10 tumor model established in both zebrafish and mouse models,and explored underlying molecular mechanisms. In vitro, barbigerone inhibited B16F10 cell proliferation,survival, migration and invasion and suppressed human umbilical vascular endothelial cell migration, invasionand tube formation in concentration-dependent manners. In the transgenic zebrafish model, treatment with10μM barbigerone remarkably inhibited angiogenesis and tumor-associated angiogenesis by reducing blood vesseldevelopment more than 90%. In vivo, barbigerone significantly suppressed angiogenesis as measured by H andE staining of matrigel plugs and CD31 staining of B16F10 melanoma tumors in C57BL/6 mice. Furthermore,it exhibited highly potent activity at inhibiting tumor growth and metastasis to the lung of B16F10 melanomacells injected into C57BL/6 mice. Western blotting revealed that barbigerone inhibited phosphorylation of AKT,FAK and MAPK family members, including ERK, JNK, and p38 MAPKs, in B16F10 cells mainly through theMEK3/6/p38 MAPK signaling pathway. These findings suggested for the first time that barbigerone could inhibittumor-angiogenesis, tumor growth and lung metastasis via downregulation of the MEK3/6/p38 MAPK signalingpathway. The findings support further investigation of barbigerone as a potential anti-cancer drug.     

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.     

腺病毒介导的大肠杆菌CD自杀基因体内外对肿瘤杀伤作用的研究

编委会 Editorial Board名誉主编 Honorary Editor-in-chief吴孟超 Wu Meng chao(Second Military Medical University,Shanghai 200433学术顾问 Academic Advisers巴德年 Ba Denian(Chinese Academy of Medical Sciences,Beijing 100730)刘新垣 Liu Xinyuan(Shanghai Institute of Biochemistry,Chinese Academy of Sciences,Shanghai 200031)吴旻 WuMin(Cancer Institute,Chinese Academy of Medical Sciences,Beijing 100021)汤钊猷 Tang Zhaoyou(Shanghai Medical University,Shanghai 200032)主编 Editor-in-chief张友会 Zhang Youhui(Cancer Institute,Chinese Academy of Medical Sciences,Beijing,100021)副主编 Associate Editor-in-chief崔正言 Cul Zhenyan(Department of Immunology,Shandong Academy of Medical Sciences,Jinan 250001)钱振超 Qian Zhenchao(Department of Patho-physiology,Dalian Medical University,Dalian 116027)何球藻 He Qiuzao(Department of Immunology,Shanghai Medical University,Shanghai 200032)董志伟 Dong Zhiwei(Cancer Institute,Chinese Academy of Medical Sciences,Beijing 100021)常务副主编 Managing Editor-in-chief     

p16基因重组质粒的构建及其对人肺癌细胞的抑制作用

表达大肠杆菌胞嘧啶脱胺酶(CD)基因的重组腺病毒AdCD体外转染小鼠黑色素瘤细胞B16F10,结果显示转染了CD基因的B16F10细胞对5-氟胞嘧啶(5FC)的敏感性显著提高.将经AdCD/5FC系统处理的B16F10细胞上清倍比稀释后.加至野生型B16F10细胞中,发现当上清仅占6.25%时即可对野生型B16F10细胞发挥明显的杀伤作用,提示AdCD/5FC介导的旁观者效应可能是通过5FC经CD酶代谢产生的毒性产物扩散而实现的.本实验还观察了CD基因体内转染后的杀伤效果,荷瘤小鼠经注射AdCD并连续10天给予5FC治疗后,与PBS、对照病毒AdLacZ/5FC治疗小鼠比较,小鼠肿瘤生长明显受到抑制,小鼠存活期明显延长.