A novel role of ribonuclease inhibitor in regulation of epithelial-to-mesenchymal transition and ILK signaling pathway in bladder cancer cells

Human ribonuclease inhibitor (RI) is a cytoplasmic acidic protein possibly involved in biological functions other than the inhibition of RNase A and angiogenin activities. We have previously shown that RI can inhibit growth and metastasis in some cancer cells. Epithelial-mesenchymal transition (EMT) is regarded as the beginning of invasion and metastasis and has been implicated in the metastasis of bladder cancer. We therefore postulate that RI regulates EMT of bladder cancer cells. We find that the over-expression of RI induces the up-regulation of E-cadherin, accompanied with the decreased expression of proteins associated with EMT, such as N-cadherin, Snail, Slug, vimentin and Twist and of matrix metalloprotein-2 (MMP-2), MMP-9 and Cyclin-D1, both in vitro and in vivo. The up-regulation of RI inhibits cell proliferation, migration and invasion, alters cell morphology and adhesion and leads to the rearrangement of the cytoskeleton in vitro. We also demonstrate that the up-regulation of RI can decrease the expression of integrin-linked kinase (ILK), a central component of signaling cascades controlling an array of biological processes. The over-expression of RI reduces the phosphorylation of the ILK downstream signaling targets p-Akt and p-GSK3β in T24 cells. We further find that bladder cancer with a high-metastasis capability shows higher vimentin, Snail, Slug and Twist and lower E-cadherin and RI expression in human clinical specimens. Finally, we provide evidence that the up-regulation of RI inhibits tumorigenesis and metastasis of bladder cancer in vivo. Thus, RI might play a novel role in the development of bladder cancer through regulating EMT and the ILK signaling pathway.     

Downregulation of integrin-linked kinase inhibits epithelial-to-mesenchymal transition and metastasis in bladder cancer cells

Integrin-linked kinase (ILK) is a multifunctional serine/threonine kinase in cytoplasm. Recent studies showed that cancer patients with increased ILK expression had low survival, poor prognosis and increased metastasis. Although the causes of ILK overexpression remain to be fully elucidated, accumulating evidence suggests that its oncogenic capacity derives from its regulation of several downstream targets that provide cells with signals that promote proliferation, survival and migration. However, the mechanisms underlying tumor metastasis by ILK is still not fully understood. Epithelial–mesenchymal transition (EMT) is a critical event of cancer cells that triggers invasion and metastasis. We recently reported that knockdown of ILK inhibited the growth and induced apoptosis in human bladder cancer cells. Therefore, we postulate that ILK might involve in EMT. Here we further investigate the function of ILK with RNA interference in bladder cancer cells. Knockdown of ILK impeded an EMT with low Vimentin, Snail, Slug and Twist as well as high E-cadherin expression in vivo and vitro. In addition, we found that knockdown of ILK inhibited cell proliferation, migration and invasion as well as changed cell morphology, adhesion and rearranged cytoskeleton in vitro. We also demonstrated that ILK siRNA inhibited phosphorylation of downstream signaling targets Akt and GSK3β, increased expression of nm23-H1, as well as reduced expression of MMP-2 and MMP-9 in vivo and vitro. Furthermore, downregulation of ILK could increase expression of Ribonuclease inhibitor (RI), an important acidic cytoplasmic protein with many functions. Finally, the effects of ILK siRNA on bladder cancer cell phenotype and invasiveness translate into suppression for tumorigenesis and metastasis in vivo. Taken together, our findings highlight that ILK signaling pathway plays a novel role in the development of bladder cancer through regulating EMT. ILK could be a promising diagnostic marker and therapeutic target for bladder cancer.     

转染人核糖核酸酶抑制因子基因对B16黑色瘤细胞及肿瘤转移的影响(英)

人核糖核酸酶抑制因子(human ribonuclease inhibitor, RI)是一种细胞质中分子质量为50 ku的酸性糖蛋白.RI能抑制核糖核酸酶A（RNase A）的活性, RNase A与血管生成因子（angiogenin,Ang）的氨基酸有着高度保守的同源序列.Ang是RNase A超家族的一员,RI通过与RNase A和Ang的紧密结合而抑制其活性.血管生成及新血管的形成, 是肿瘤发生和转移的必要条件.所以抗血管生成将是一种很有希望的对抑制肿瘤生长和转移的有效方法.实验显示RI能有效地抑制肿瘤诱导血管的生成.RI由含有许多亮氨酸重复序列的多肽组成.含有这样重复序列的100多种蛋白质显示了广泛的功能,包括细胞周期调节,DNA修复,对细胞外基质相互作用以及抑制酶活性等.RI被认为是胚胎发育,创伤愈合及肿瘤发生中新血管形成的一种调节因子.RI定位于染色体的11p15.5,与ras基因邻近,在肿瘤病人中经常存在染色体11p15.5部位的变异和异常.RI可能与细胞的生长和分化有关, 因此,RI 可能还具有尚未知的生物学作用.为了进一步了解RI的潜在功能以及探讨RI与肿瘤浸润、转移的关系, 将人的核糖核酸酶抑制因子基因的cDNA通过逆转录包装细胞PA317,并转染到B16小鼠黑色瘤细胞中, 用转染空载体和未转染的B16细胞作为对照.通过PCR, RT-PCR, 蛋白质免疫印迹, 免疫荧光分析鉴定,获得稳定表达人核糖核酸酶抑制因子的细胞株.结果显示, 转染的RI基因在体外能显著地抑制细胞增殖和细胞迁移,增加了细胞的粘附以及改善细胞的恶性形态,B16,B16 pLNCX,B16 pLNCX-RI 3种细胞的倍增时间分别为(24.98±0.16) h, (25.62±0.28) h, (32.64±1.11) h.与对照组相比,转RI的细胞粘附率增加17.8%和19.5%而迁移降低了61.4%和60%.转RI的细胞比对照组细胞较平展,核仁和分裂相较少,胞质嗜碱性减弱,提示细胞增殖活性降低和恶性表型的改善. 将3种B16细胞静脉注射到C57BL/6小鼠中, 结果表明, 转染RI基因的实验组显著地抑制了肿瘤的转移, 与两个对照组相比,荷瘤小鼠有更长的存活时间, 少得多的转移节结, 更低的肿瘤血管密度和肺重量.结果显示,RI的表达可能与黑色瘤的转移有关, 提示RI能显著地抑制肿瘤的转移,可能由于其与抑制血管作用,增加细胞粘附,降低细胞迁移及增殖有关.     

过表达核糖核酸酶抑制因子通过调节ILK/PI3K/AKT信号途径抑制小鼠黑色素瘤B16-F10细胞体内外生长

核糖核酸酶抑制因子（ribonuclease inhibitor,RI）是胞浆内的一种酸性蛋白质.已有研究证明,RI与核糖核酸酶A（RNaseA）和血管生成素（angiogenin,ANG）结合可抑制其活性.本室前期实验证实,RI可有效抑制某些肿瘤的生长和转移. 然而,RI抑制肿瘤的分子机制尚不清楚. 本研究探讨RI对小鼠黑色素瘤B16-F10细胞生长和凋亡的影响及其机制. MTT法结合流式细胞术分析结果证明,RI基因稳定转染导致B16+F10黑色素瘤细胞S期阻滞,抑制B16-F10黑色素瘤细胞增殖. Annexin V/PI结合流式细胞术结果显示,RI过表达引起细胞凋亡.与此相一致,蛋白质印迹分析显示,过表达RI引起抗凋亡分子Bcl-2表达下调,而Bax上调,同时伴有Pro-casepase 3激活. C57BL/ 6小鼠移植成瘤实验显示,与对照相比,转染RI的B16-F10细胞形成的肿瘤重量显著减少,同时伴有肿瘤组织微血管密度降低.提示RI过表达能抑制微血管生成. 此外,体内外组织/细胞免疫化学和蛋白质印迹结果揭示,过表达RI可显著抑制整合素连接激酶(integrin-linked kinase,ILK)下游靶分子Akt和GSK-3β的磷酸化,并降低β-联蛋白的表达.研究结果证明,过表达RI可通过抑制ILK/ PI3K/AKT信号通路,促进细胞凋亡,引起S期阻滞,并抑制血管生成,从而显著抑制小鼠黑色素瘤B16-F10细胞在体内、外的生长.上述结果提示,RI可能是治疗黑色素瘤的有效分子靶点.     

Inhibition of melanoma growth and metastasis by combination with (-)-epigallocatechin-3-gallate and dacarbazine in mice.

(-)-Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, was shown to have cancer chemopreventive activity. In this study, we examined the antimetastatic effects of EGCG or the combination of EGCG and dacarbazine on B16-F3m melanoma cells in vitro and in vivo. First, the antimetastatic potentials of five green tea catechins were examined by soft agar colony formation assay, and the results show that EGCG was more effective than the other catechins in inhibiting soft agar colony formation. Second, EGCG dose-dependently inhibited B16-F3m cell migration and invasion by in vitro Transwell assay. Third, EGCG significantly inhibited the spread of B16-F3m cells on fibronectin, laminin, collagen, and Matrigel in a dose-dependent manner. In addition, EGCG significantly inhibited the tyrosine phosphorylation of focal adhesion kinase (FAK) and the activity of matrix metalloproteinase-9 (MMP-9). In animal experiments, EGCG alone reduced lung metastases in mice bearing B16-F3m melanomas. However, a combination of EGCG and dacarbazine was more effective than EGCG alone in reducing the number of pulmonary metastases and primary tumor growths, and increased the survival rate of melanoma-bearing mice. These results demonstrate that combination treatment with EGCG and dacarbazine strongly inhibits melanoma growth and metastasis, and the action mechanisms of EGCG are associated with the inhibition of cell spreading, cell-extracellular matrix and cell-cell interactions, MMP-9 and FAK activities.     

Potential ability of hot water adzuki (Vigna angularis) extracts to inhibit the adhesion, invasion, and metastasis of murine B16 melanoma cells

The 40% ethanol eluent of the fraction of hot-water extract from adzuki beans (EtEx.40) adsorbed onto DIAION HP-20 resin has many biological activities, for example, antioxidant, antitumorigenesis, and intestinal alpha-glucosidase suppressing activities. This study examined the inhibitory effect of EtEx.40 on experimental lung metastasis and the invasion of B16-BL6 melanoma cells. EtEx.40 was found significantly to reduce the number of tumor colonies. It also inhibited the adhesion and migration of B16-BL6 melanoma cells into extracellular matrix components and their invasion into reconstituted basement membrane (matrigel) without affecting cell proliferation in vitro. These in vivo data suggest that EtEx.40 possesses a strong antimetastatic ability, which might be a lead compound in functional food development.     

miR-146b Reverses epithelial-mesenchymal transition via targeting PTP1B in cisplatin-resistance human lung adenocarcinoma cells

Epithelial-mesenchymal transformation (EMT) is associated with drug resistance in human lung adenocarcinoma cells, but its specific mechanism has not been clarified. In this study, we investigated the effect of miRNA-146b on EMT in cisplatin (DDP) resistant human lung adenocarcinoma cells and the corresponding mechanism. Cisplatin resistant (CR) human lung adenocarcinoma cells (A549/DDP and H1299/DDP) were established, and the EMT characteristics and invasion and metastasis ability of CR cells were determined by tumor cell-related biological behavior experiments. The role of miR-146b in EMT of CR cells was determined by in vitro functional test. The targeted binding of miR-146b to protein tyrosine phosphatase 1B (PTP1B) was verified by biological information and double luciferin gene reporting experiments. The effect of miR-146b on tumor growth and EMT phenotype in vivo was investigated by establishing the xenotransplantation mouse model. Compared with the control group, H1299/DDP and A549/DDP cells showed the enhanced EMT phenotypes, invasion and migration ability. Besides, miR-146b was lowly expressed in H1299/DDP and A549/DDP cells. More importantly, overexpressed miR-146b could specifically bind to PTP1B, thus inhibiting the EMT process and ultimately reducing CR in H1299/DDP and A549/DDP cells. Finally, overexpressed miR-146b observably inhibited tumor growth in xenograft model mice and inhibited the EMT phenotype of A549/DDP cells in vivo by regulating the expressions of EMT-related proteins. Overexpressed miR-146b could reverse the EMT phenotype of CR lung adenocarcinoma cells by targeting PTP1B, providing new therapeutic directions for CR of lung adenocarcinoma cells.     

Thujone inhibits lung metastasis induced by B16F-10 melanoma cells in C57BL/6 mice

The antimetastatic potential of thujone, a naturally occurring monoterpene, was evaluated. Metastasis was induced in C57BL/6 mice by injecting highly metastatic B16F-10 melanoma cells through the lateral tail vein. Administration of thujone (1 mg·(kg body weight)(-1)), prophylactically and simultaneously with tumor induction, inhibited tumor nodule formation in the lungs by 59.45% and 57.54%, respectively, with an increase in the survival rate (33.67% and 32.16%) of the metastatic tumor bearing animals. These results correlated with biochemical parameters such as lung collagen hydroxyproline, hexosamine and uronic acid contents, serum sialic acid and γ-glutamyl transpeptidase levels, and histopathological analysis. Treatment with thujone downregulated the production of proinflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and granulocyte-monocyte colony-stimulating factor. Thujone administration downregulated the expression of matrix metalloproteinase (MMP)-2, MMP-9, extracellular signal-regulated kinase (ERK)-1, ERK-2, and vascular endothelial growth factor (VEGF) and also upregulated the expression of nm-23, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 in the lung tissue of metastasis-induced animals. Treatment with thujone inhibited the activity of MMP-2 and MMP-9 in gelatin zymographic analysis. Thujone treatment significantly inhibited the invasion of B16F-10 melanoma cells across the collagen matrix in a Boyden chamber. Thujone also inhibited the adhesion of tumor cells to collagen-coated microtire plate wells and the migration of B16F-10 melanoma cells across a polycarbonate filter in vitro. These results indicate that Thujone can inhibit the lung metastasis of B16F-10 cells through inhibition of tumor cell proliferation, adhesion, and invasion, as well as by regulating expression of MMPs, VEGF, ERK-1, ERK-2, TIMPs, nm23, and levels of proinflammatory cytokines and IL-2 in metastatic animals.     

Antitumor effects of human ribonuclease inhibitor gene transfected on B16 melanoma cells

Human ribonuclease inhibitor (RI) is a cytoplasmic acidic protein. The experiment demonstrated that it might effectively inhibit tumor-induced angiogenesis and inhibit tumor growth. Ribonuclease inhibitor is constructed almost entirely of leucine-rich repeats, which might be involved in unknown biological effects besides inhibiting RNase A and angiogenin activities. The exact molecular mechanism of antitumor on ribonuclease inhibitor remains unclear so far. In order to further understand the function of ribonuclease inhibitor and investigate the relationship with tumor growth, our study established a transfection of human ribonuclease inhibitor cDNA into the murine B16 cells by the retroviral packaging cell line PA317. The cell line transfected with a stably high expression of ribonuclease inhibitor was identified. We found that the transfected ribonuclease inhibitor could obviously inhibit cell proliferation, regulate cell cycle and induce cell apoptosis in vitro. Mice that were injected with the B16 cells transfected RI cDNA showed a significant inhibition of the tumor growth with lighter tumor weight, lower density of microvessels, longer latent periods, and survival time than those in the other two control groups. In conclusion, the results reveal the novel mechanism that antitumor effect of ribonuclease inhibitor is also associated with inducing apoptosis, regulating cell cycle and inhibiting proliferation besides antiangiogenesis. These results suggest that ribonuclease inhibitor might be a candidate of tumor suppressor gene in some tissues. RI could become a target gene for gene therapy. Our study may be of biological and clinical importance.     

Knockdown of ribonuclease inhibitor expression with siRNA in non-invasive bladder cancer cell line BIU-87 promotes growth and metastasis potentials

Human ribonuclease inhibitor (RI) is a cytoplasmic acidic protein. RI is constructed almost entirely of leucine-rich repeats, which might be involved in some unknown biological functions like other structurally similar proteins besides inhibiting RNase A and angiogenin activities. Our previous experiments demonstrated that up-regulating RI might effectively inhibit some tumor growth and metastasis. However, the down-regulating RI influence on the tumor does not have any report until now, the mechanisms underlying antitumor of RI have not been fully understood. In this study, the efficient RNA interferences of RI were constructed using a plasmid vector and identified with RT-PCR, Western blot and Immunocytochemistry, then were transfected into non-invasive bladder cancer BIU-87 cells. We demonstrated that knockdown RI expression in BIU-87 cells could obviously change the cell morphology, rearrange the microfilaments and extend the lamellipodia, as well as enhance proliferation, increase migration, invasion and matrix metalloprotease level, and also reduce adhesion in vitro. BALB/C nude mice that were injected with the BIU-87 cells transfected RI siRNA showed a significant facilitation of the tumor with heavier tumor weight, higher density of microvessels, lower nm23-H1 and E-Cadherin expressions than those in the control group. Taken together, these experiments suggest that knockdown of RI could promote growth and metastasis potentials of BIU-87 cells. Our present findings reveal the novel mechanism that anti-tumor effect of RI is also involved in suppressing growth and metastasis, besides antiangiogenesis. The results show that RI may be a therapeutic target protein for bladder cancer and may be of biological importance.     

Marine algal fucoxanthin inhibits the metastatic potential of cancer cells

Metastasis is major cause of malignant cancer-associated mortality. Fucoxanthin has effect on various pharmacological activities including anti-cancer activity. However, the inhibitory effect of fucoxanthin on cancer metastasis remains unclear. Here, we show that fucoxanthin isolated from brown alga Saccharina japonica has anti-metastatic activity. To check anti-metastatic properties of fucoxanthin, in vitro models including assays for invasion, migration, actin fiber organization and cancer cell–endothelial cell interaction were used. Fucoxanthin inhibited the expression and secretion of MMP-9 which plays a critical role in tumor invasion and migration, and also suppressed invasion of highly metastatic B16-F10 melanoma cells as evidenced by transwell invasion assay. In addition, fucoxanthin diminished the expressions of the cell surface glycoprotein CD44 and CXC chemokine receptor-4 (CXCR4) which play roles in migration, invasion and cancer–endothelial cell adhesion. Fucoxanthin markedly suppressed cell migration in wound healing assay and inhibited actin fiber formation. The adhesion of B16-F10 melanoma cells to the endothelial cells was significantly inhibited by fucoxanthin. Moreover, in experimental lung metastasis in vivo assay, fucoxanthin resulted in significant reduction of tumor nodules. Taken together, we demonstrate, for the first time, that fucoxanthin suppresses metastasis of highly metastatic B16-F10 melanoma cells in vitro and in vivo.     

Dimethylfumarate inhibits tumor cell invasion and metastasis by suppressing the expression and activities of matrix metalloproteinases in melanoma cells

NF-κB acts as a signal transducer during tumor progression, cell invasion, and metastasis. Dimethylfumarate (DMF) is reported to inhibit tumor necrosis factor-α-induced nuclear entry of NF-κB/p65. However, only a few reports suggest that DMF inhibits tumor metastasis; also the molecular mechanisms underlying the inhibition of metastasis are poorly understood. We investigated the inhibition of tumor invasion and metastasis by DMF in a melanoma cell line, B16BL6. DMF inhibited B16BL6 cell invasion and metastasis by suppressing the expression and activities of MMPs. DMF also inhibited the nuclear entry of NF-κB/p65, thus inhibiting B16BL6 cell invasion and metastasis. These results suggest that DMF is potentially useful as an anti-metastatic agent for the treatment of malignant melanoma.     

Inhibition by tyroserleutide (YSL) on the invasion and adhesion of the mouse melanoma cell

Tyroserleutide (YSL) is an active, low-molecular-weight polypeptide, comprised of three amino acids, that has shown antitumor effects on human hepatocarcinoma BEL-7402 in vitro and in vivo. In this study, we evaluated the inhibition of YSL on invasion and adhesion of the mouse B16-F10 melanoma cell line by injecting B16-F10 cells into the tail veins of C57BL/6 mice to establish an experimental lung metastasis model. YSL inhibited B16-F10 cell metastasis to lung, reducing the number and area of metastasis lesions. When we treated B16-F10 cells with YSL (0.01, 0.1, 1, 10, or 100 microg/mL) in vitro, we found that YSL inhibited the proliferation of B16-F10 cells with a 28.11% rate of inhibition. YSL significantly decreased the adhesiveness of B16-F10 cells to Matrigel with a 29.15% inhibition rate; YSL also significantly inhibited the invasion of B16-F10 cells, producing an inhibition of 35.31%. By analyses with Western blot and real-time RT-PCR, we found that YSL markedly inhibited the expression of ICAM-1 in B16-F10 cells. These data suggest that YSL inhibits the growth, invasion, and adhesion of B16-F10 cells.     

Silencing of Diphthamide Synthesis 3 (Dph3) Reduces Metastasis of Murine Melanoma

Melanoma is the most dangerous skin cancer due to its highly metastatic potential and resistance to chemotherapy. Currently, there is no effective treatment for melanoma once it is progressed to metastatic stage. Therefore, further study to elucidate the molecular mechanism underlying the metastasis of melanoma cells is urgently required for the improvement of melanoma treatment. In the present study, we found that diphthamide synthesis 3 (Dph3) is involved in the metastasis of B16F10 murine melanoma cells by insertional mutagenesis. We demonstrated that Dph3 disruption impairs the migration of B16F10 murine melanoma cells. The requirement of Dph3 in the migration of melanoma cells was further confirmed by gene silencing with siRNA in vitro. In corresponding to this result, overexpression of Dph3 significantly promoted the migratory ability of B16F10 and B16F0 melanoma cells. Moreover, down regulation of Dph3 expression in B16F10 melanoma cells strikingly inhibits their cellular invasion and metastasis in vivo. Finally, we found that Dph3 promotes melanoma migration and invasion through the AKT signaling pathway. To conclude, our findings suggest a novel mechanism underlying the metastasis of melanoma cells which might serve as a new intervention target for the treatment of melanoma.     

Novel TGF-β1 inhibitor antagonizes TGF-β1-induced epithelial-mesenchymal transition in human A549 lung cancer cells

Transforming growth factor β1 (TGF-β1), a multifunctional cytokine, is known to promote tumor invasion and metastasis and induce epithelial-mesenchymal transition (EMT) in various cancer cells. Inhibition of TGF-β1 signaling is a new strategy for cancer therapy. Most cancer cells display altered or nonfunctional TGF-β1 signaling; hence, TGF-β1 inhibitors exert limited effects on these cells. Recent studies have suggested that developing a TGF-β1 inhibitor from natural compounds is a key step to create novel therapeutic agents. This study aimed to develop a new anti-TGF-β1 therapy for cancer. We found an improved analog of chalcones, compound 67, and investigated its effects in vitro. We demonstrated the inhibitory role of compound 67 through migration and invasion assays on TGF-β1-induced EMT of human A549 lung cancer cells. Compound 67 inhibited TGF-β1-induced smad2 phosphorylation, suppressed TGF-β1-induced EMT markers, matrix metalloproteinase-2 (MMP-2) and MMP-9, and inhibited migration and invasion of A549 cells. The study results showed that compound 67 is useful to prevent tumor growth and metastasis.     

MiR-144 Inhibits Uveal Melanoma Cell Proliferation and Invasion by Regulating c-Met Expression

MicroRNAs (miRNAs) are a group endogenous small non-coding RNAs that inhibit protein translation through binding to specific target mRNAs. Recent studies have demonstrated that miRNAs are implicated in the development of cancer. However, the role of miR-144 in uveal melanoma metastasis remains largely unknown. MiR-144 was downregulated in both uveal melanoma cells and tissues. Transfection of miR-144 mimic into uveal melanoma cells led to a decrease in cell growth and invasion. After identification of two putative miR-144 binding sites within the 3'' UTR of the human c-Met mRNA, miR-144 was proved to inhibit the luciferase activity inMUM-2B cells with a luciferase reporter construct containing the binding sites. In addition, the expression of c-Met protein was inhibited by miR-144. Furthermore, c-Met-mediated cell proliferation and invasion were inhibited by restoration of miR-144 in uveal melanoma cells. In conclusion, miR-144 acts as a tumor suppressor in uveal melanoma, through inhibiting cell proliferation and migration. miR-144 might serve as a potential therapeutic target in uveal melanoma patients.     

Antimetastatic activity of Sulforaphane

The effect of Sulforaphane on the inhibition of lung metastasis induced by B16F-10 melanoma cells was studied in C57BL/6 mice by three different modalities of administration-simultaneous, prophylactic and after tumour developed. Of this simultaneous mode of Sulforaphane administration was found to be most effective. There was 95.5% inhibition of lung tumour nodule formation and 94.06% increase in the life span of metastatic tumour bearing animals. Highly elevated levels of lung hydroxyproline, lung uronic acid, lung hexosamine, serum sialic acid and serum gamma-glutamyl transpeptidase (GGT) in the metastatic control animals was found to be significantly lowered in the Sulforaphane treated animals. Histopathological analysis of lung tissues also correlated with these results. In the in vitro system Sulforaphane showed a significant inhibition in the invasion of B16F-10 melanoma cells across the collagen matrix. (3)H-thymidine proliferation assay showed that Sulforaphane could inhibit the proliferation of B16F-10 melanoma cells in vitro. Gelatin zymographic analysis showed that Sulforaphane could inhibit the activation of matrix metalloproteinases. These findings suggest that Sulforaphane reduced the invasion of B16F-10 melanoma cells by the inhibition of activation of matrix metalloproteinases, thereby inhibiting lung metastasis.     

Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways

In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities of matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKCα and PKCδ phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.     

Down-regulation of Gli-1 inhibits hepatocellular carcinoma cell migration and invasion

Glioma-associated oncogene homolog-1 (Gli-1) is considered a marker of Hedgehog pathway activation and is associated with the progression of several cancers. We have previously reported that Gli-1 was correlated with invasion and metastasis in hepatocellular carcinoma (HCC). However, the exact roles and mechanisms of Gli-1 in HCC invasion are unclear. In this study, we found that small interfering RNA mediated down-regulation of Gli-1 expression significantly suppressed adhesion, motility, migration, and invasion of both SMMC-7721 and SK-Hep1 cells. Furthermore, down-regulation of Gli-1 significantly reduced expressions and activities of both matrix metalloproteinase (MMP)-2 and MMP-9. In addition, we found that down-regulation of Gli-1 resulted in up-regulation of E-cadherin and concomitant down-regulation of Snail and Vimentin, consistent with inhibition of epithelial-mesenchymal transition (EMT). Taken together, our results suggest that down-regulation of Gli-1 suppresses HCC cell migration and invasion likely through inhibiting expressions and activations of MMP-2, 9 and blocking EMT.