Overexpression of NaV 1.6 channels is associated with the invasion capacity of human cervical cancer

Functional activity of voltage-gated sodium channels (VGSC) has been associated to the invasion and metastasis behaviors of prostate, breast and some other types of cancer. We previously reported the functional expression of VGSC in primary cultures and biopsies derived from cervical cancer (CaC). Here, we investigate the relative expression levels of VGSC subunits and its possible role in CaC. Quantitative real-time PCR revealed that mRNA levels of Na(V) 1.6 α-subunit in CaC samples were ～40-fold higher than in noncancerous cervical (NCC) biopsies. A Na(V) 1.7 α-subunit variant also showed increased mRNA levels in CaC (～20-fold). All four Na(V) β subunits were also detected in CaC samples, being Na(V) β1 the most abundant. Proteins of Na(V) 1.6 and Na(V) 1.7 α-subunits were immunolocalized in both NCC and CaC biopsies and in CaC primary cultures as well; however, although in NCC sections proteins were mainly relegated to the plasma membrane, in CaC biopsies and primary cultures the respective signal was stronger and widely distributed in both cytoplasm and plasma membrane. Functional activity of Na(V) 1.6 channels in the plasma membrane of CaC cells was confirmed by whole-cell patch-clamp experiments using Cn2, a Na(V) 1.6-specific toxin, which blocked ～30% of the total sodium current. Blocking of sodium channels VGSC with tetrodotoxin and Cn2 did not affect proliferation neither migration, but reduced by ～20% the invasiveness of CaC primary culture cells in vitro assays. We conclude that Na(V) 1.6 is upregulated in CaC and could serve as a novel molecular marker for the metastatic behavior of this carcinoma.     

Epidermal growth factor potentiates <Emphasis Type="Italic">in vitro</Emphasis> metastatic behaviour of human prostate cancer PC-3M cells: involvement of voltage-gated sodium channel

Background  

Although a high level of functional voltage-gated sodium channel (VGSC) expression has been found in strongly metastatic human and rat prostate cancer (PCa) cells, the mechanism(s) responsible for the upregulation is unknown. The concentration of epidermal growth factor (EGF), a modulator of ion channels, in the body is highest in prostatic fluid. Thus, EGF could be involved in the VGSC upregulation in PCa. The effects of EGF on VGSC expression in the highly metastatic human PCa PC-3M cell line, which was shown previously to express both functional VGSCs and EGF receptors, were investigated. A quantitative approach, from gene level to cell behaviour, was used. mRNA levels were determined by real-time PCR. Protein expression was studied by Western blots and immunocytochemistry and digital image analysis. Functional assays involved measurements of transverse migration, endocytic membrane activity and Matrigel invasion.     

Therapeutic approaches targeting prostate cancer progression using novel voltage-gated ion channel blockers

The early detection and treatment of prostate cancer have increased survival and improved clinical outcomes. The nature of the disease and pathologic understaging result in a high proportion of patients developing locally recurrent disease or distant metastases. The development of prostate cancer the time from tumor initiation and progression to invasive carcinoma often begins in men in the fourth or fifth decades of life and extends across decades. This prolonged window highlights the tremendous clinical impact that early intervention with therapeutic agents that selectively target the invasive and metastatic potential of the prostate cancer cell could have on patient survival and quality of life. Our research is currently focused on the development and testing of novel voltage-gated ion channel blockers. The expression of voltage-gated sodium channels (VGSCs) was recently associated with the metastatic behavior of prostate cancer cells. In these studies, VGSC blockers altered prostate cancer cell morphology and arrested prostate cancer cell migration. Clinically, one of the most widely used sodium channel blockers is phenytoin. We have used rational drug design based on the phenytoin binding site in a VGSC to make novel sodium channel blockers with enhanced activity and minimal acute toxicity. Our initial studies in vitro demonstrate enhanced binding of the compounds to the sodium channel and increased inhibition of prostate cancer cell growth in culture and in soft agarose compared with phenytoin. These derivatives are currently being tested for their antitumor activity in human prostate cancer xenografts.     

电压门控钠离子通道表达对宫颈癌细胞增殖侵袭转移作用的研究

  目的  探讨电压门控钠离子通道(voltage-gated sodium channels, VGSCs)在宫颈癌细胞中的表达、分布及其对该细胞增殖、侵袭能力的影响。  方法  用Western blot了解VGSCs在不同宫颈癌细胞株中表达水平, 采用免疫荧光检测VGSCs蛋白在细胞中的定位。分别用MTT法和Matrigel法检测VGSCs对宫颈癌细胞的增生和侵袭作用。以半定量RT-PCR和RNAi方法确认宫颈癌细胞中VGSC表达亚型及其功能。  结果  在检测的4种宫颈癌细胞株中, ME180细胞表达较高水平VGSCs蛋白, 其存在于细胞膜和细胞质中。VGSCs抑制剂-河豚毒素(TTX)对ME180细胞增生无影响(P > 0.05), 但呈浓度依赖性抑制细胞的Matrigel侵袭(P < 0.05)。在ME180细胞中检测到Nav 1.2、Nav 1.6和Nav 1.7三种VGSCs亚型, 其中Nav 1.6 mRNA占总mRNA的78%, RNAi抑制Nav 1.6mRNA表达后可降低细胞侵袭达52%(P < 0.05)。  结论  VCSCs在宫颈癌ME180细胞株高表达, Nav 1.6为其主要表达亚型, 增加了体外癌细胞的侵袭转移。      

Interaction between human cancer cells and cultured murine endothelial cells, and its relationship with metastatic potential

The hematogenous metastasis of cancer consists of a multistep process. It is surmised that a number of interactions between cancer and endothelial cells occur, with cell adhesion molecules playing certain roles in this process. The authors conducted an investigation on the interaction between human cancer cells and cultured murine endothelial cells (F-2 cells) in vitro, and on its relationship with the metastatic activity of cancer cells in vivo. A correlation was found between the degree of expression of carbohydrate antigens on the cell surface and adhesion of cancer cells to F-2 cells. Five of 13 examined cell lines showed liver metastasis after inoculation to the spleen of nude mice. These cell lines showed not only a strong binding activity to F-2 cells but implantation in F-2 cells in vitro was also observed. These findings suggest that adhesion to, and implantation in endothelial cells are necessary for the induction of distant metastasis. Treatment with antibodies against carbohydrate antigens inhibited the formation of liver metastasis in nude mice. It is possible that strategies to interfere with the function of cell adhesion molecules may be formulated to result in the decreased distant metastasis of cancer.     

Voltage-gated sodium ion channels in prostate cancer: expression and activity

BACKGROUND: High levels of voltage-gated sodium channels (VGSCs) have been previously associated with the invasiveness of rat and human prostate cancer (Pca) cell lines. MATERIALS AND METHODS: 4 normal prostate and 80 clinical Pca specimens on a tissue microarray slide were examined by immunohistochemistry using anti-sodium channel (III-IV linker region) antibody. The effects of a VGSC-opener and VGSC-blockers on the in vitro proliferation of 4 human Pca cell lines was examined. RESULTS: Fifty-five% (44 out of 80) Pca specimens showed higher VGSC levels compared to normal, with 14 (of 44) showing increased focal staining. VGSC-opener veratrine (1-50 microg/mL), increased growth of PC3, DU145, LNCaP and MDA-PCA-2B Pca cells. VGSC-blockers, flunarizine (IC50=2 microg/mL) and riluzole (IC50=10-30 microg/mL) caused dose-dependent growth-inhibition of all four cell lines. Western analysis of cell extracts showed VGSC-immunoreactivity in the 4 Pca cell lines. CONCLUSION: Our results indicate increased expression of VGSCs in Pca and VGSC involvement in Pca growth.     

TRPM7 Is Required for Breast Tumor Cell Metastasis

TRPM7 encodes a Ca(2+)-permeable nonselective cation channel with kinase activity. TRPM7 has been implicated in control of cell adhesion and migration, but whether TRPM7 activity contributes to cancer progression has not been established. Here we report that high levels of TRPM7 expression independently predict poor outcome in breast cancer patients and that it is functionally required for metastasis formation in a mouse xenograft model of human breast cancer. Mechanistic investigation revealed that TRPM7 regulated myosin II-based cellular tension, thereby modifying focal adhesion number, cell-cell adhesion and polarized cell movement. Our findings therefore suggest that TRPM7 is part of a mechanosensory complex adopted by cancer cells to drive metastasis formation. Cancer Res; 72(16); 4250-61. ?2012 AACR.     

Activation of nuclear factor-kappa B by linear ubiquitin chain assembly complex contributes to lung metastasis of osteosarcoma cells

NF-κB is involved in the metastasis of malignant cells. We have shown that NF-κB activation is involved in the pulmonary metastasis of LM8 cells, a highly metastatic subclone of Dunn murine osteosarcoma cells. Recently, it was determined that a newly identified type of polyubiquitin chain, a linear polyubiquitin chain, which is specifically generated by the linear ubiquitin chain assembly complex (LUBAC), plays a critical role in NF-κB activation. Here, we have evaluated the roles of LUBAC-mediated NF-κB activation in the development of lung metastasis of osteosarcoma cells. All three components of LUBAC (HOIL-1L, HOIP and SHARPIN) were highly expressed in LM8 cells compared to Dunn cells. Attenuation of LUBAC expression by stable knockdown of HOIL-1L in LM8 cells significantly suppressed NF-κB activity, invasiveness in?vitro and lung metastasis. Induction of intracellular adhesion molecule-1 (ICAM-1) expression by LUBAC is involved in cell retention in the lungs after an intravenous inoculation of tumor cells. Moreover, we found that knockdown of LUBAC decreased not only the number but also the size of the metastatic nodules of LM8 cells in the lungs. These results indicate that LUBAC-mediated NF-κB activation plays crucial roles in several steps involved in metastasis, including extravasation and growth of osteosarcoma cells in the lung, and that suppression of LUBAC-mediated linear polyubiquitination activity may be a new approach to treat this life-threatening disease of young adolescents.     

Combined treatment with verapamil,a calcium channel blocker,and B428, a synthetic uPA inhibitor,impairs the metastatic ability of a murine mammary carcinoma

Urokinase plasminogen activator (uPA) and metalloproteinases (MMP) play key roles in invasion and metastasis, degrading extracellular matrix compounds and modulating tumor cell motility. Their regulation is an attractive therapeutic target for controlling tumor metastasis. Previously we have demonstrated that urokinase overexpression in murine mammary tumor cells is regulated by a Ca2+-dependent pathway and that blockage of Ca2+ channels by verapamil partially inhibited their invasive and metastatic ability. Moreover, the catalytic inhibition of uPA by a synthetic uPA inhibitor B428 reduced local tumor invasiveness but not tumor cell dissemination. We evaluated the effect of a combined treatment with verapamil and B428 on the murine mammary carcinoma F3II behavior in vivo and in vitro. In vivo administration of the combined treatment was not associated to an overt toxicity. Only the daily combined treatment, beginning after tumor take, reduced the incidence and the number of spontaneous lung metastasis, while no differences were found in the subcutaneous growth of the primary tumor. Interestingly, a remarkable reduction in plasma MMP-9 activity was found associated to metastasis impairment. In addition, the number of experimental lung metastases was also significantly diminished, with respect to the control group, only when both compounds were co-administered daily, beginning three days after i.v. tumor cell injection. In vitro, both compounds, either separately or combined, could inhibit secreted uPA activity. F3II cell migration was significantly inhibited by incubation with 50 microM verapamil, 15 microM B428 or the co-treatment with 7.5 microM B428 + 25 microM verapamil. The cell spread was also significantly reduced when F3II cells were exposed to the compounds, with an additive effect when B428 + verapamil combination was used. The combination of two compounds acting through different molecular targets may be useful to improve the control of metastatic dissemination.     

Establishment of two new scirrhous gastric cancer cell lines: analysis of factors associated with disseminated metastasis.

Determination of the differences between cell lines which are derived from a primary tumour and a disseminated metastatic lesion from the same patient may aid in elucidating the factors associated with disseminated metastases. We report on the establishment and characterisation of two new scirrhous gastric cancer cell lines, designated OCUM-2M and OCUM-2D, derived from a 49-year-old female. OCUM-2M was derived from a primary gastric tumour, and OCUM-2D was derived from a sample of disseminated metastasis. The two cell lines were derived from the same patient. We investigated biological differences between the two cell lines to study mechanisms involved in disseminated metastasis. The growth activity of OCUM-2D cells as determined by doubling time and tumorigenicity was greater than that of OCUM-2M cells. The level of epidermal growth factor receptor (EGFR) expression in OCUM-2D cells was about twice that of OCUM-2M cells and the growth of OCUM-2D cells was stimulated more by epidermal growth factor (EGF) than that of OCUM-2M cells. The invasive activity of OCUM-2D cells was higher than that of OCUM-2M cells and was increased after addition of transforming growth factor-beta 1 (TGF-beta 1). An increase in the number of attached and spreading cells was found following the addition of 10 ng ml-1 TGF-beta 1. These findings suggest that high growth and invasive activity may play an important role in disseminated metastasis and that EGF and TGF-beta 1, which affect the growth and invasive activity of OCUM-2D cells, might be factors associated with metastasis in scirrhous gastric carcinoma. The two cell lines OCUM-2M and OCUM-2D should be beneficial for analysing mechanisms of tumour progression.     

Tumor invasion induced by oxidative stress is dependent on membrane ADAM 9 protein and its secreted form

Oxidative stress plays a role in the regulation of cancer cell metastasis which involves cell invasion and adhesion that could be supported by ADAM proteins through the activities of their metalloprotease and disintegrin domains. We hypothesized that oxidative stress could act through the induction of ADAM9 protein in some cancer cells. Indeed, Western blot analysis for ADAM9 performed on A549 cells exposed to H(2) O(2) reveals a dose-dependent induction of two proteins (80 and 68 kDa) correlated with a sharp increase of the ADAM protease activity measured in supernatant while the activity measured on the cell layer was slightly affected. The 80kDa protein corresponds to the mature form of ADAM9. Immunoprecipitation analysis performed on concentrated supernatants revealed that the 68 kDa protein is a secreted form of ADAM9. When exposed to H(2) O(2) , A549 cells cocultured with confluent endothelial vascular cells resulted in a 5.5 fold (p < 0.001) increase in the number of adherent cells. Similarly, matrigel assay revealed a 3.25 fold (p < 0.01) increase in the number of invasive cells. The suppression of ADAM9 expression by specific small interfering RNA reduced oxidative stress-induced invasiveness and adhesiveness. These functions could be mediated by an interaction between ADAM9 and β1 integrin because each of them were inhibited when the experiment is performed in presence of mAbs targeting ADAM9 ectodomain or β1-integrin. These results emphasize the importance of oxidative stress in the regulation of cancer cell metastasis and suggest that ADAM9 and its secreted isoform can be important determinants in the ability of cancer cells to disseminate.     

Cyclin-dependent kinase 5 activity controls cell motility and metastatic potential of prostate cancer cells

We show here that cyclin-dependent kinase 5 (CDK5), a known regulator of migration in neuronal development, plays an important role in prostate cancer motility and metastasis. P35, an activator of CDK5 that is indicative of its activity, is expressed in a panel of human and rat prostate cancer cell lines, and is also expressed in 87.5% of the human metastatic prostate cancers we examined. Blocking of CDK5 activity with a dominant-negative CDK5 construct, small interfering RNA, or roscovitine resulted in changes in the microtubule cytoskeleton, loss of cellular polarity, and loss of motility. Expression of a dominant-negative CDK5 in the highly metastatic Dunning AT6.3 prostate cancer cell line also greatly impaired invasive capacity. CDK5 activity was important for spontaneous metastasis in vivo; xenografts of AT6.3 cells expressing dominant-negative CDK5 had less than one-fourth the number of lung metastases exhibited by AT6.3 cells expressing the empty vector. These results show that CDK5 activity controls cell motility and metastatic potential in prostate cancer.     

Natural killer cell tumoricidal activity and deterioration of lung tumor metastasis in silicotic mice and stressed silicotic mice

Aim of this study is to explore the role of splenic NK cells in the lung metastasis in the silicotic mice. The number of metastatic foci increased 1.5-fold and 1.8-fold in the silicotic and stressed silicotic mice compared with the control mice. Treatment with an immunomodulator reduced the rate of tumor metastasis in silicotic mice with or without stresses, while their NK cell activity was normalized. Decrease of NK cell activity on the day of tumor inoculation but not on the post-inoculation days seems to be a major factor for predicting the extent of tumor metastasis in the silicotic and stressed silicotic mice.     

Inhibition of experimental and spontaneous pulmonary metastasis of murine RCT (+) sarcoma by beta-cyclodextrin-benzaldehyde

The effect of beta-cyclodextrin-benzaldehyde (CDBA) on pulmonary metastasis in C3H/He mice was examined. In experimental metastasis that was induced by iv injection of 1 X 10(6) RCT (+) cells, the highest inhibition was observed in the mice that were treated daily with CDBA (5 mg/day) for 1 week before tumor cell inoculation and further treated for 3 weeks after inoculation, when compared with those in other experimental groups that were given only pretreatment or posttreatment. The inhibitory effect was dose-dependent. In spontaneous metastasis that was induced by sc injection of 3 X 10(6) RCT (+) cells, the inhibition of metastasis was also observed in the mice treated with CDBA (5 mg/day) in the same manner as described above. However, the development of the primary tumor was not inhibited. CDBA-treated tumor-bearing mice showed almost as much NK activity as normal mice. Furthermore, although injection of 5-fluorouracil suppressed this activity to about 50% of that in normal mice, the combined treatment with CDBA could maintain the NK cell activity at the normal level. The results suggested that the inhibition of pulmonary metastasis might be induced by a combined effect of CDBA; that is, the direct inhibition of tumors and the maintenance of NK cell activity.     

肿瘤患者外周血NK细胞活性与sIL-2R水平的研究

目的探讨肿瘤患者外周血NK细胞活性与血清sIL-2R的临床意义。方法应用LDH释放法及双抗体夹心酶联免疫吸附试验(ELISA)对40例正常人、114例恶性肿瘤患者进行了外周血NK细胞活性和血清sIL-2R水平检测。结果与正常对照组相比,肿瘤患者外周血NK细胞活性显著降低(P<0.01),血清sIL-2R水平明显增高(P<0.01);上述改变在伴转移患者中更明显,与不伴转移者相比,差异显著(P<0.001)。肿瘤患者外周血NK细胞活性与血清sIL-2R水平呈负相关(r=-0.473,P<0.001)。治疗有效的转移癌患者NK活性明显增强(P<0.05),sIL-2R水平显著降低(P<0.01);无效者变化不明显(P>0.05)。结论血清中异常增高的sIL-2R可能是导致肿瘤患者NK细胞活性低下的原因之一。NK细胞活性和sIL-2R可作为研究肿瘤发生、发展及转移的重要免疫学指标,监测肿瘤患者外周血中两者的变化有助于评估疗效和预后。     

循环肿瘤细胞簇在肿瘤转移中的研究进展

肿瘤转移是肿瘤相关死亡的主要原因,大量研究显示循环肿瘤细胞（circulating tumor cells,CTCs)在肿瘤转移中起着重要作用。越来越多的研究表明循环肿瘤细胞簇（circulating tumor cells clusters,CTC clusters）与单循环肿瘤细胞相比转移能力更强。分离检测技术的不断进步为深入研究循环肿瘤细胞簇提供了便利。本文就循环肿瘤细胞簇在肿瘤转移中的研究进展作一综述。     

Efficacy of oncolytic reovirus against human gastric cancer with peritoneal metastasis in experimental animal model

The prognosis of gastric cancer patients with peritoneal dissemination is extremely poor, and the development of an effective treatment is necessary. The aim of this study was to investigate the efficacy of oncolytic reovirus against peritoneal metastasis in human gastric cancer using an experimental animal model. Four human gastric cancer cell lines, including MKN45p, NUGC4, MKN7 and KatoIII, a normal NIH3T3 cell line as a control, and reovirus serotype 3, were used in this study. We evaluated the cytopathic effect of reovirus and the Ras activity in each gastric cancer cell line in vitro. To evaluate oncolytic efficacy in vivo, reovirus (1x10(8) PFU) was administered into the peritoneal cavity of nude mice on days 7, 8 and 9 after inoculation with MKN45p cells. Mean volume of ascites and the total number and weight of the peritoneal tumors were measured after sacrifice. After reovirus infection, cytopathic effect was observed in all four gastric cancer cell lines, but not in the control cells. Ras activation assay showed that Ras activity in all four gastric cancer cell lines increased to a higher level than that in the control cells. In the animal model experiments, mean volume of ascites and the total number and weight of the peritoneal tumors in the reovirus treatment group were significantly lower than those in the control group. In conclusions, intraperitoneal administration of reovirus could be useful as a new modality against peritoneal metastasis in gastric cancer.