Urokinase induces receptor mediated brain tumor cell migration and invasion

The plasminogen activation (PA) system plays an important role in tumor invasion by initiating pericellular proteolysis of the extracellular matrix (ECM) and inducing cell migration. Malignant brain tumors overexpress PA members and characteristically invade by migrating on ECM-producing white matter tracts and blood vessel walls. To determine whether urokinase-type plasminogen activator (uPA) and its receptor (uPAR) directly modulate the migration of brain tumor cells, we examined six human brain tumor cell lines, 2 astrocytomas (SW1088, SW1783), 2 medullobastomas (Daoy, D341Med), and 2 glioblastomas (U87MG, U118MG), for their surface uPAR expression, endogenous PA activity, and functional proteolytic activity by an ECM-degradation assay. Migration on Transwell membranes and invasion of Matrigel was then tested by pre-incubating the cells with increasing concentrations of either uPA, the proteolytically inactive amino-terminal fragment (ATF) of uPA, or the uPAR cleaving enzyme, phosphatidylinositol-specific phospholipase C (PI-PLC).All of the cell lines, except D341Med, express surface uPAR protein and uPA activity. High levels of uPAR and uPA activity correlated with cellular degradation of ECM, cell migration, and Matrigel invasion. Cell migration and invasion were enhanced by uPA or ATF in a dose dependent manner, while PI-PLC treatment abolished the uPA effect and inhibited migration and invasion. We conclude that ligation of uPAR by uPA directly induces brain tumor cell migration, independent of uPA-mediated proteolysis; and in concert with ECM degradation, markedly enhances invasion. Conversely, removing membrane bound uPAR from the surface of the cells studied inhibited their ability to migrate and invade even in the presence of proteolytically active uPA.     

Antibody and lectin target podoplanin to inhibit oral squamous carcinoma cell migration and viability by distinct mechanisms

Podoplanin (PDPN) is a unique transmembrane receptor that promotes tumor cell motility. Indeed, PDPN may serve as a chemotherapeutic target for primary and metastatic cancer cells, particularly oral squamous cell carcinoma (OSCC) cells that cause most oral cancers. Here, we studied how a monoclonal antibody (NZ-1) and lectin (MASL) that target PDPN affect human OSCC cell motility and viability. Both reagents inhibited the migration of PDPN expressing OSCC cells at nanomolar concentrations before inhibiting cell viability at micromolar concentrations. In addition, both reagents induced mitochondrial membrane permeability transition to kill OSCC cells that express PDPN by caspase independent nonapoptotic necrosis. Furthermore, MASL displayed a surprisingly robust ability to target PDPN on OSCC cells within minutes of exposure, and significantly inhibited human OSCC dissemination in zebrafish embryos. Moreover, we report that human OSCC cells formed tumors that expressed PDPN in mice, and induced PDPN expression in infiltrating host murine cancer associated fibroblasts. Taken together, these data suggest that antibodies and lectins may be utilized to combat OSCC and other cancers that express PDPN.     

Inhibition of erythropoietin signalling destroys xenografts of ovarian and uterine cancers in nude mice

We have recently shown that malignant tumours from the ovary and uterus expressed erythropoietin (Epo) and its receptor (EpoR), and that deprivation of Epo signal in tumour blocks induced death of malignant cells and capillary endothelial cells in vitro (Yasuda et al, submitted). These in vitro results prompted us to examine the effect of Epo-signal withdrawal on tumours in vivo. RT-PCR analysis demonstrated the expression of mRNAs for Epo and EpoR in the transplants of uterine and ovarian tumours in nude mice. Then we injected locally anti-Epo antibody or soluble form of EpoR into the transplants. At 12 h, 1, 7 or 14 days after the injection, all transplants were resected and examined macro- and microscopically. Tumour size was reduced in Epo signal-deprived transplants. Immunohistochemical examinations revealed destruction of Epo-responding malignant and capillary endothelial cells through apoptotic death. The degree of tumour regression correlated well with the dose and frequency of the injections. Control xenografts with saline injection or needle insertion showed well-developed tumour masses. This Epo response pathway will have profound implications for our understanding of the development and progression of malignant tumours and for the use of Epo-signal deprivation as an effective therapy.     

siRNA沉默雄激素受体对人结肠癌HCT-8细胞增殖、迁移及侵袭的影响

目的:利用小干扰RNA(siRNA)沉默人结肠癌HCT-8细胞中雄激素受体(androgen receptor,AR)的表达,观察AR敲除对癌细胞增殖、迁移和侵袭能力的影响,初步探讨AR在结肠癌中的功能.方法:利用qRT-PCR和Western blot检测HCT-8细胞及正常人肠上皮细胞中AR的表达;siRNA转染HCT-8细胞沉默AR表达,利用qRT-PCR和Western blot检测转染效率;流式细胞术检测沉默AR后对HCT-8细胞周期的影响;CCK-8法检测沉默AR后对HCT-8细胞增殖的影响;Transwell实验检测沉默AR后对HCT-8细胞迁移及侵袭的影响.结果:AR在人结肠癌HCT-8细胞中高表达;siRNA能有效沉默HCT-8细胞中AR的mRNA及蛋白的表达.与对照组相比,沉默AR表达能够明显抑制HCT-8细胞的增殖、迁移和侵袭能力.结论:AR在人结肠癌HCT-8中高表达并能够促进癌细胞的增殖、迁移和侵袭能力.     

慢病毒介导的αB-晶状体蛋白基因沉默对子宫内膜癌ISK细胞增殖和迁移能力的影响

目的:研究αB-晶状体蛋白(CRYAB)基因在子宫内膜癌增殖和迁移中发挥的作用.方法:利用慢病毒介导的RNA干扰技术,沉默子宫内膜癌ISK细胞系CRYAB基因.通过CCK-8、Transwell和细胞划痕实验分别检测CRYAB基因沉默后细胞增殖和迁移能力的变化.结果:Real-time PCR和Western blot结果均表明成功构建稳定转染的细胞株.CCK-8实验结果显示CRYAB病毒转染组与对照组相比,细胞的增殖能力显著降低(P<0.05);Transwell迁移实验和细胞划痕实验结果显示,病毒转染组的细胞迁移率明显低于对照组(P<0.01).结论:慢病毒干扰载体能有效抑制CRYAB基因在子宫内膜癌细胞系ISK中的表达,进而抑制细胞增殖和迁移.证实CRYAB在子宫内膜癌侵袭和转移中发挥重要作用.     

Lack of functional erythropoietin receptors of cancer cell lines

Erythropoietin (Epo) therapy reduces red cell transfusion requirements and improves the quality of life of anemic cancer patients receiving chemotherapy. However, there is concern that Epo may promote tumor growth. We investigated by real-time RT-PCR, immunofluorescence microscopy, Western blotting and cell growth analysis whether human cancer cell lines (SH-SY5Y, MCF7, HepG2, U2-OS, HeLa, HEK293T, RCC4, HCT116, 7860wt and SW480) possess functional Epo receptors (EpoR). We detected EpoR mRNA in all cell lines. Neither hypoxia nor Epo treatment altered the level of EpoR mRNA expression. Four commonly used commercial antibodies proved to be unsuitable for immunoblot procedures because they cross-reacted with several proteins unrelated with EpoR. Depending on the antibody used, EpoR was localized to the plasma membrane, the cytoplasm or the nucleus. Experiments with small interfering RNA showed that EpoR protein was not expressed by the tumor cells except by UT7/Epo leukemia cells, which served as an EpoR positive control line, and by cells transfected with the human EpoR gene. Apart from UT7/Epo, none of the tumor cell lines responded to Epo treatment with phosphorylation of signaling molecules or with cell proliferation.     

Inhibition of triple-negative and Herceptin-resistant breast cancer cell proliferation and migration by Annexin A2 antibodies

Background:

Annexin A2 (AnxA2), a calcium-dependent phospholipid binding protein, is abundantly present at the surface of triple-negative and Herceptin-resistant breast cancer cells. Interactions between cell-surface AnxA2 and tyrosine kinase receptors have an important role in the tumour microenvironment and act together to enhance tumour growth. The mechanism supporting this role is still unknown.

Methods:

The membrane function of AnxA2 was blocked by incubating cells with anti-AnxA2 antibodies. Western blotting, immunoprecipitation, immunofluorescence, 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), flow cytometry, Clonogenic, and wound-healing assays were performed in this study.

Results:

We demonstrate that AnxA2 interacts with epidermal growth factor receptor (EGFR) at the cell surface and has an important role in cancer cell proliferation and migration by modulating EGFR functions. Blocking AnxA2 function at the cell surface by anti-AnxA2 antibody suppressed the EGF-induced EGFR tyrosine phosphorylation and internalisation by blocking its homodimerisation. Furthermore, addition of AnxA2 antibody significantly inhibited the EGFR-dependent PI3K-AKT and Raf-MEK-ERK downstream pathways under both EGF-induced and basal growth conditions, resulting in lower cell proliferation and migration.

Conclusions:

These findings suggest that cell-surface AnxA2 has an important regulatory role in EGFR-mediated oncogenic processes by keeping EGFR signalling events in an activated state. Therefore, AnxA2 could potentially be used as a therapeutic target in triple-negative and Herceptin-resistant breast cancers.     

Restoration of plakoglobin expression in bladder carcinoma cell lines suppresses cell migration and tumorigenic potential

The reduction or loss of plakoglobin expression in late-stage bladder cancer has been correlated with poor survival where upregulation of this catenin member by histone deacetylase inhibitors has been shown to accompany tumour suppression in an in vivo model. In this study, we directly addressed the question of the role of plakoglobin in bladder tumorigenesis following restoration, or knockdown of expression in bladder carcinoma cell lines. Restoration of plakoglobin expression resulted in a reduction in migration and suppression of tumorigenic potential in vivo. Immunocytochemistry revealed cytoplasmic and membranous localisation of plakoglobin in transfectants with < 1% of cells displaying detectable nuclear localisation of plakoglobin. siRNA knockdown experiments targeting plakoglobin, revealed enhanced migration in all cell lines in the presence and absence of E-cadherin expression. In bladder cell lines expressing low levels of plakoglobin and desmoglein-2, elevated levels of desmoglein-2 were detected following restoration of plakoglobin expression in transfected cell lines. Analysis of wnt signalling revealed no activation event associated with plakoglobin expression in the bladder model. These results show that plakoglobin acts as a tumour suppressor gene in bladder carcinoma cells and the silencing of plakoglobin gene expression in late-stage bladder cancer is a primary event in tumour progression.     

肿瘤细胞迁移的动力

肿瘤细胞迁移是肿瘤转移的关键步骤。细胞挤出、趋化性、趋物性、趋触性和渗流提供了肿瘤细胞迁移的动力支持,通过变形虫样运动和追逐的迁移方式,肿瘤细胞完成近乎直线的迁移过程。研究肿瘤细胞迁移的动力可以提供阻断转移的新策略     

CXCR4/CXCL12 expression and signalling in kidney cancer

CXCL12 (SDF-1), a CXC-chemokine, and its specific receptor, CXCR4, have recently been shown to be involved in tumourgenesis, proliferation and angiogenesis. Therefore, we analysed CXCL12alpha/CXCR4 expression and function in four human kidney cancer cell lines (A-498, CAKI-1, CAKI-2, HA-7), 10 freshly harvested human tumour samples and corresponding normal kidney tissue. While none of the analysed tumour cell lines expressed CXCL12alpha, A-498 cells were found to express CXCR4. More importantly, real-time RT-PCR analysis of 10 tumour samples and respective adjacent normal kidney tissue disclosed a distinct and divergent downregulation of CXCL12alpha and upregulation of CXCR4 in primary tumour tissue. To prove that the CXCR4 protein is functionally active, rhCXCL12alpha was investigated for its ability to induce changes of intracellular calcium levels in A-498 cells. Moreover, we used cDNA expression arrays to evaluate the biological influence of CXCL12alpha. Comparing gene expression profiles in rhCXCL12alpha stimulated vs unstimulated A-498 kidney cancer cells revealed specific regulation of 31 out of 1176 genes tested on a selected human cancer array, with a prominent stimulation of genes involved in cell-cycle regulation and apoptosis. The genetic changes reported here should provide new insights into the developmental paths leading to tumour progression and may also aid the design of new approaches to therapeutic intervention.     

Inhibitors of BCR signalling interrupt the survival signal mediated by the micro‐environment in mantle cell lymphoma

Several studies provide evidences for mantle cell lymphoma (MCL) cell survival relying on B‐cell receptor (BCR)‐mediated signalling pathways, whereas the nature of this activation is unknown. Significant progress in MCL treatment is achieved through therapies targeting BCR‐associated kinases, i.e., Ibrutinib and Fostamatinib, inhibitors of BTK and SYK, respectively. Our study addresses survival signals emanating from the BCR or the tumour environment and how inhibiting BCR signalling effectors might impact these survival signals. We found that BTK was constitutively activated and that SYK phosphorylation was highly increased and sustained upon BCR activation of primary MCL cells. Moreover, MCL cells from leukaemic patients secreted high amount of IL‐1β, IL‐6, IL‐8 and CCL5. Activation of the BCR induced (i) cell survival, (ii) STAT3 activation and (iii) increased autocrine secretion of IL‐1β, IL‐6, IL‐8, CCL5, IL‐10, TNFα and VEGF. Specific inhibition of BTK by Ibrutinib or SYK by Fostamatinib (R406) reversed these protective effects and decreased both basal and BCR‐induced autocrine cytokine secretions associated with STAT3 phosphorylation. Interestingly, targeting BTK and SYK prevented and inhibited BCR‐induced MCL cell adhesion to human bone marrow stromal cells (HMSCs) in short‐ and long‐term co‐culture. We demonstrated that BCR‐induced survival relies on autocrine secretion of IL‐1β, TNFα and CCL5 that might facilitate adhesion of MCL cells to HMSC. Treatment with Ibrutinib or Fostamatinib blocked the chemotactic signal thus increasing apoptosis.     

钠尿肽受体A在食管鳞状细胞癌中的表达及对食管癌细胞迁移和侵袭能力的影响

目的 探讨钠尿肽受体A(NPRA)在食管鳞状细胞癌中的表达及其对食管癌细胞迁移和侵袭的影响.方法 采用免疫组织化学染色法检测119例食管鳞状细胞癌组织和91例癌旁组织中NPRA的表达情况,并分析食管鳞状细胞癌组织中NPRA表达情况与患者临床特征及预后的关系.采用蛋白质印迹法(Westernblot)检测食管癌细胞株Ec...     

Piperlongumine promotes autophagy via inhibition of Akt/mTOR signalling and mediates cancer cell death

Background:

The Akt/mammalian target of rapamycin (mTOR) signalling pathway serves as a critical regulator of cellular growth, proliferation and survival. Akt aberrant activation has been implicated in carcinogenesis and anticancer therapy resistance. Piperlongumine (PL), a natural alkaloid present in the fruit of the Long pepper, is known to exhibit notable anticancer effects. Here we investigate the impact of PL on Akt/mTOR signalling.

Methods:

We examined Akt/mTOR signalling in cancer cells of various origins including prostate, kidney and breast after PL treatment. Furthermore, cell viability after concomitant treatment with PL and the autophagy inhibitor, Chloroquine (CQ) was assessed. We then examined the efficacy of in vivo combination treatment using a mouse xenograft tumour model.

Results:

We demonstrate for the first time that PL effectively inhibits phosphorylation of Akt target proteins in all tested cells. Furthermore, the downregulation of Akt downstream signalling resulted in decrease of mTORC1 activity and autophagy stimulation. Using the autophagy inhibitor, CQ, the level of PL-induced cellular death was significantly increased. Moreover, concomitant treatment with PL and CQ demonstrated notable antitumour effect in a xenograft mouse model.

Conclusions:

Our data provide novel therapeutic opportunities to mediate cancer cellular death using PL. As such, PL may afford a novel paradigm for both prevention and treatment of malignancy.     

TRPV3 inhibits colorectal cancer cell proliferation and migration by regulating the MAPK signaling pathway

BackgroundThe aim of this study was to investigate the inhibiting effect of transient receptor potential vanilloid 3 (TRPV3) on the proliferation and migration of colorectal cancer (CRC) cells and to explore the underlying mechanism.MethodsA microarray dataset from the publicly available Gene Expression Omnibus (GEO) database was used to investigate the prognostic value of TRPV3 in CRC. In addition, 100 CRC tissue samples were collected at our center to further validate its prognostic value at the protein level. Cell proliferation ability was detected by Cell Counting Kit-8 (CCK-8) assay, and cell migration ability was detected by transwell assay. Gene set variation analysis (GSVA) was performed to identify the potential pathways regulated by TRPV3.ResultsBased on the largest microarray dataset ({"type":"entrez-geo","attrs":{"text":"GSE39582","term_id":"39582"}}GSE39582), low expression of TRPV3 was found to be significantly associated with poor prognosis in CRC patients, and this result was successfully validated at our cancer center. Functional experiments showed that knockdown of TRPV3 enhanced cell proliferation and migration, while enforced TRPV3 expression exhibited the opposite effect. GSEA based on public microarray data revealed that the mitogen-activated protein kinase (MAPK) signaling pathway was notably activated in patients with low expression of TRPV3. Further experiments in vivo confirmed that TRPV3 silencing promoted cell proliferation and migration by activating the MAPK signaling pathway.ConclusionsLow expression of TRPV3, which stimulates cell proliferation and migration by provoking the MAPK signaling pathway, indicated poor prognosis in CRC patients.