ABCA1 transporter promotes the motility of human melanoma cells by modulating their plasma membrane organization

BACKGROUND: Melanoma is one of the most aggressive and deadliest skin tumor. Cholesterol content in melanoma cells is elevated, and a portion of it accumulates into lipid rafts. Therefore, the plasma membrane cholesterol and its lateral organization might be directly linked with tumor development. ATP Binding Cassette A1 (ABCA1) transporter modulates physico-chemical properties of the plasma membrane by modifying cholesterol distribution. Several studies linked the activity of the transporter with a different outcome of tumor progression depending on which type. However, no direct link between human melanoma progression and ABCA1 activity has been reported yet. METHODS: An immunohistochemical study on the ABCA1 level in 110 patients-derived melanoma tumors was performed to investigate the potential association of the transporter with melanoma stage of progression and prognosis. Furthermore, proliferation, migration and invasion assays, extracellular-matrix degradation assay, immunochemistry on proteins involved in migration processes and a combination of biophysical microscopy analysis of the plasma membrane organization of Hs294T human melanoma wild type, control (scrambled), ABCA1 Knockout ( ABCA1 KO) and ABCA1 chemically inactivated cells were used to study the impact of ABCA1 activity on human melanoma metastasis processes. RESULTS: The immunohistochemical analysis of clinical samples showed that high level of ABCA1 transporter in human melanoma is associated with a poor prognosis. Depletion or inhibition ofABCA1 impacts invasion capacities of aggressive melanoma cells. Loss of ABCA1 activity partially prevented cellular motility by affecting active focal adhesions formation via blocking clustering of phosphorylated focal adhesion kinases and active integrin ß3. Moreover, ABCA1 activity regulated the lateral organization of the plasma membrane in melanoma cells. Disrupting this organization, by increasing the content of cholesterol, also blocked active focal adhesion formation. CONCLUSION: Human melanoma cells reorganize their plasma membrane cholesterol content and organization via ABCA1 activity to promote motility processes and aggressiveness potential. Therefore, ABCA1 may contribute to tumor progression and poor prognosis, suggesting ABCA1 to be a potential metastatic marker in melanoma.

Advances in biological functions of Ezrin and its effects on tumor metastasis / 中国药理学通报

As a member of the ERM (Ezrin/Radixin/Moesin) protein family, Ezrin is widely distributed in the body. Ezrin acts as a "scaffold" participating in anchorage and interacting between plasma membrane and cytoskeleton. Its special subcel-lular localization is critical for many complex cell processes. Increasing evidence suggests that the abnormal expression, phosphorylation and localization of Ezrin would affect tumor progression. The influence of Ezrin on the morphology of tumor cells during metastasis has gradually attracted the attention of researchers. Further investigations that focus on the mechanism of Ezrin' s influence on different stages of tumor metastasis will be gradually elucidated. In this article, we review the biological functions of Ezrin and its research progress in tumor metastasis, and explain the mechanism of Ezrin-mediated tumor metastasis. It is proposed that strategies targeting Ezrin for tumor metastasis treatment are a promising way to achieve great success in clinic.

Overexpression of CD44 Standard Isoform Upregulates HIF-1α Signaling in Hypoxic Breast Cancer Cells

Cluster of differentiation 44 (CD44), a cell surface receptor for hyaluronic acid (HA), is involved in aggressive cancer phenotypes. Herein, we investigated the role of the CD44 standard isoform (CD44s) in hypoxia-inducible factor-1α (HIF-1α) regulation using MCF7 overexpressing CD44s (pCD44s-MCF7). When pCD44s-MCF7 was incubated under hypoxia, levels of HIF-1α, vascular endothelial growth factor, and the HIF-1α response element-derived luciferase activity were significantly increased compared to those in the control MCF7. Incubation of pCD44s-MCF7 cells with HA further increased HIF-1α accumulation, and the silencing of CD44s attenuated HIF-1α elevation, which verifies the role of CD44s in HIF-1α regulation. In addition, the levels of phosphorylated extracellular signal-regulated kinase (ERK) was higher in hypoxic pCD44s-MCF7 cells, and HIF-1α accumulation was diminished by the pharmacological inhibitors of ERK. CD44s-mediated HIF-1α augmentation resulted in two functional outcomes. First, pCD44s-MCF7 cells showed facilitated cell motility under hypoxia via the upregulation of proteins associated with epithelial-mesenchymal transition, such as SNAIL1 and ZEB1. Second, pCD44s-MCF7 cells exhibited higher levels of glycolytic proteins, such as glucose transporter-1, and produced higher levels of lactate under hypoxa. As a consequence of the enhanced glycolytic adaptation to hypoxia, pCD44s-MCF7 cells exhibited a higher rate of cell survival under hypoxia than that of the control MCF7, and glucose deprivation abolished these differential responses of the two cell lines. Taken together, these results suggest that CD44s activates hypoxia-inducible HIF-1α signaling via ERK pathway, and the CD44s-ERK-HIF-1α pathway is involved in facilitated cancer cell viability and motility under hypoxic conditions.

Role of the CHD7 chromatin remodeler protein in glioblastoma multiformePalavras-chave em inglês Cell motility / Papel do remodelador de cromatina CHD7 em glioblastoma multiforme

Chromatin remodeler proteins exert an important function in promoting dynamic modifications in the chromatin architecture, rendering the transcriptional machinery available to the condensed genomic DNA. Due to this central role in regulating gene transcription, deregulation of these molecular machines may lead to severe perturbations in the normal cell functions. Loss-of-function mutations in the CHD7 gene, a member of the chromodomain helicase DNA-binding (CHD) family, are the major cause of the CHARGE syndrome in humans. The disease is characterized by a variety of congenital anomalies, including malformations of the craniofacial structures, peripheral nervous system, ears, eyes and heart. In this context, several studies have already shown the importance of CHD7 for proper function of the neural stem cells (NSCs). Interestingly, we found that CHD7 mRNA levels are upregulated in gliomas, when compared to normal brain tissue, therefore, we hypothesized that CHD7 might have a role in the pathogenesis of these tumors. To investigate the possible oncogenic role of CHD7 in glioblastoma (GBM), we adopted gain- and loss-of-function approaches in adherent GBM cell lines. Using CRISPR_Cas9 genome editing, we found that CHD7 deletion suppresses anchorage-independent growth and reduces spheroid invasion in human LN-229 cells. Moreover, deletion of CHD7 delayed tumor growth and improved overall survival in an orthotopic xenograft glioma mouse model. Conversely, ectopic overexpression of CHD7 in LN-428 and A172 cells was found to increase cell motility and invasiveness in vitro and LN-428 tumor growth in vivo. RNAseq analysis showed that alterations of CHD7 expression levels promote changes in several molecular pathways and modulate critical genes associated with cell adhesion and locomotion. However, the mechanisms underlying the effects of CHD7 overexpression in glioma tissue are still not understood. Here, we also generated recombinant plasmid with functional CHD7 promoter activity reported by luciferase assay. This powerful tool should enable future studies to determine the direct targeting relationship between different signal transduction pathways and CHD7 geneexpression. In summary, our findings indicate that GBM cells expressing a high level of CHD7 may exist and contribute to tumor infiltration and recurrence. Further studies should warrant important clinical-translational implications of our findings for GBM treatment

As proteínas remodeladoras de cromatina exercem importante papel, promovendo modificações dinâmicas na arquitetura da cromatina e dando acesso à maquinaria transcricional ao DNA genômico condensado. Devido à esta função central na regulação da transcrição gênica, a desregulação dessas máquinas moleculares pode levar a perturbações graves na função normal das células. Assim, por exemplo, mutações do tipo perda de função no gene CHD7, um membro da família "chromodomain helicase DNA-binding" (CHD), são a principal causa da síndrome de CHARGE em humanos. A doença é caracterizada por uma variedade de anomalias congênitas, incluindo malformações das estruturas craniofaciais, sistema nervoso periférico, orelhas, olhos e coração. Neste contexto, vários estudos já mostraram a importância da proteína CHD7 para o funcionamento normal de células-tronco neurais (NSCs). Curiosamente, descobrimos que os níveis de mRNA de CHD7 estão mais fortemente expressos em gliomas, quando comparados ao tecido cerebral normal, portanto, nós hipotetizamos que CHD7 poderia ter um papel na patogênese desses tumores. Para investigar o possível papel oncogênico de CHD7 em glioblastoma (GBM), utilizamos enfoques de ganho e perda de função em linhagens celulares aderentes de GBM. Utilizando a técnica de CRISPR_Cas9 para edição do genoma, demonstramos que a deleção do gene CHD7 suprime o crescimento independente de ancoragem e reduz a invasão de esferóides em células LN-229 humanas de GBM. Além disso, a deleção de CHD7 reduziu o crescimento do tumor e melhorou a sobrevida em modelo de injeção ortotópica xenográfica em camundongo. Por outro lado, verificou-se que a super-expressão ectópica de CHD7 nas células LN-428 e A172 aumenta não só a motilidade celular e a capacidade de invasão in vitro, mas, também, o crescimento do tumor de LN-428 in vivo. A análise de RNA-seq mostrou que o nocauteamento da sequência codificadora de CHD7 e sua super-expressão promovem alterações em diversas vias moleculares, modulando genes críticosassociados à adesão e locomoção celular. No entanto, os mecanismos subjacentes aos efeitos da super-expressão de CHD7 em tecidos de glioma ainda não são compreendidos. Neste trabalho, geramos um plasmídeo recombinante contendo um fragmento da região promotora de CHD7, o qual se mostrou funcional em ensaios de luciferase. Esta ferramenta permitirá que estudos futuros possam identificar a relação direta entre as diferentes vias de transdução de sinal e a expressão do gene CHD7. Em resumo, nossos achados indicam que células de GBM expressando um alto nível de CHD7 podem existir e contribuir para a infiltração e recorrência do tumor. Estudos posteriores deverão avaliar as possíveis implicações dos resultados apresentados neste trabalho para a translação clínica no tratamento de pacientes com GBM

Early bacterial biofilm colonizers in the coastal waters of Mauritius

Background: The past years have witnessed a growing number of researches in biofilm forming communities due to their environmental and maritime industrial implications. To gain a better understanding of the early bacterial biofilm community, microfiber nets were used as artificial substrates and incubated for a period of 24 h in Mauritian coastal waters. Next-generation sequencing technologies were employed as a tool for identification of early bacterial communities. Different genes associated with quorum sensing and cell motility were further investigated. Results: Proteobacteria were identified as the predominant bacterial microorganisms in the biofilm within the 24 h incubation, of which members affiliated to Gammaproteobacteria, Alphaproteobacteria and Betaproteobacteria were among the most abundant classes. The biofilm community patterns were also driven by phyla such as Firmicutes, Bacteroidetes, Chloroflexi, Actinobacteria and Verrucomicrobia. The functional analysis based on KEGG classification indicated high activities in carbohydrate, lipid and amino acids metabolism. Different genes encoding for luxI, lasI, agrC, flhA, cheA and cheB showed the involvement of microbial members in quorum sensing and cell motility. Conclusion: This study provides both an insight on the early bacterial biofilm forming community and the genes involved in quorum sensing and bacterial cell motility.

Effect of PTP4A3 gene on tumor growth and metastasis / 中国病理生理杂志

PTP4A3 is an oncogene,which encodes phosphatase of regenerating liver(PRL)-3 protein that is a metastasis-associated phosphorylase. At present,a number of studies have found that it promotes cell proliferation, cell motility,cell invasion,tumor metastasis and epithelial-mesenchymal transition. Therefore,it is inseparable with the occur-rence and development of cancer. Here,we summarize the relationship between PTP4A3 gene and the occurrence and de-velopment of cancer,the alteration of PTP4A3 gene expression and the functional role of PTP4A3 gene in a variety of canc-ers. This review will help us to understand the correlation between PTP4A3 gene and cancer as well as the mechanism of signaling pathway,providing new insights of PTP4A3 gene targeting strategy for treating cancer.

Important role of ELMO1 in invasion and migration of breast cancer cells induced by IL-8 / 中国药理学通报

Aim To explore the relationship between IL-8 and ELMO1 in breast carcinoma and the mecha-nisms of IL-8 induced invasion and metastasis. Meth-ods Under the IL-8 stimulation, chemotaxis assay was examined to detect the chemotaxis ability of breast cancer cell line MDA-MB-231 and MCF-7 . ELMO1 protein levels in breast cancer cell lines were detected using Western blot. MDA-MB-231 cells were transfect-ed with small RNA interference plasmids in order to downregulate ELMO1 expression, and overexpression plasmids were used to upregulate the expression of EL-MO1 in MCF-7 cells. Matrigel invasion assay and chemotaxis assay were used to detect the in vitro inva-sion and chemotaxis ability of breast cancer cells with IL-8 stimulation. Results IL-8 induced chemotaxis of the different breast cancer cell lines in a dose-depend-ent manner. After transient transfection, Western blot results showed that ELMO 1 protein levels observably decreased in SiELMO1/MDA-MB-231 cells compared with Scr/MDA-MB-231 cells, while the expression of ELMO1 protein levels significantly increased in MCF-7/ELMO1 cells compared with the MCF-7/Con cells;with IL-8 stimulation, SiELMO1/MDA-MB-231 cells showed significantly decreased chemotaxis ability com-pared with Scr/MDA-MB-231 cells. MCF-7/ELMO1 cells showed significantly increased chemotaxis ability compared with MCF-7/Con cells; the invasion assay showed under the stimulation of IL-8 , and the invasion ability was significantly reduced in SiELMO1/MDA-MB-231 cells compared with Scr/MDA-MB-231 cells ( P<0. 05 ) . The invasion ability was significantly en-hanced in MCF-7/ELMO1 cells compared with MCF-7 cells( P<0. 05 ) . Conclusion IL-8 promotes the in-vasion and migration of breast cancer cells MDA-MB-231 and MCF-7 , and ELMO1 plays an important role in IL-8 induced chemotaxis and invasion.

Effect of Carboplatin and Methotrexate on Lipid Levels in the Plasma Membrane of MCF-7 Cells and their Association with Cell Motility.

Aim: We examine in this study the effect of two antitumor drugs metothrexate (MTX) and carboplatin (CPT) on the phospholipid content of plasma membrane of MCF-7 breast cancer cells. MTX is a folate analog that inhibit dihydrofolate reductase. CPT is a platinum-containing antineoplastic drug that inter-chelates DNA and inhibits replication. Methods: MCF-7 breast cancer cells were treated with different concentrations of CPT or MTX. Plasma membranes were purified and their protein contents were measured with and without drug treatment. We extracted the lipids from plasma membranes of drug-treated and control MCF-7 cells, quantitated, and separated by HPLC using a C18 reverse-phase column. The ability of both drugs to affect cell movement was studied using a motility assay. Results: MTX induced 50% cell death at concentrations ranging from 50 to 100 mM. No further decrease in viability was seen above this concentration even at 1 mM. CPT induced 50% cell death at 5 mM. It also showed a range concentration that gives a 50% cell death after 24 hrs of incubation. Protein levels in plasma membranes of treated cells doubled compared to control. Lipid levels in plasma membranes decrease insignificantly after drug treatment. No changes in separation pattern of lipid extracted from membranes were seen after CPT treatment compared to control; however, MTX treatment showed to a single change in elution pattern in peak at 4.36 min. Both drugs had little effect on cell motility causing a decrease of 13.3% and 17.4% with CPT and MTX respectively compared to control. Conclusions: Our results show that both drugs did not affect the amount of lipids but lead to doubling in the protein concentration in the plasma membranes of MCF-7 breast cancer cells. These drugs did not lead to a significant decrease in cell motility compared to control.

Cell Motility Is Decreased in Macrophages Activated by Cancer Cell-Conditioned Medium

Macrophages play a role in innate immune responses to various foreign antigens. Many products from primary tumors influence the activation and transmigration of macrophages. Here, we investigated a migration of macrophages stimulated with cancer cell culture-conditioned medium (CM). Macrophage activation by treatment with CM of B16F10 cells were judged by the increase in protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2). The location where macrophages were at 4 h-incubation with control medium or CM was different from where they were at 5 h-incubation in culture dish. Percentage of superimposed macrophages at every 1 h interval was gradually increased by CM treatment as compared to control. Total coverage of migrated track expressed in coordinates was smaller and total distance of migration was shorter in CM-treated macrophages than that in control. Rac1 activity in CM-treated macrophages was also decreased as compared to that in control. When macrophages were treated with CM in the presence of dexamethasone (Dex), an increase in COX2 protein levels, and a decrease in Rac1 activity and total coverage of migration were reversed. In the meanwhile, biphasic changes were detected by Dex treatment in section distance of migration at each time interval, which was more decreased at early time and then increased at later time. Taken together, data demonstrate that macrophage motility could be reduced in accordance with activation in response to cancer cell products. It suggests that macrophage motility could be a novel marker to monitor cancer-associated inflammatory diseases and the efficacy of anti-inflammatory agents.

The role of Rho GTPases in the regulation of the rearrangement of actin cytoskeleton and cell movement

The rearrangement of the actin cytoskeleton has been shown to play a critical role in the development of transformation and malignant phenotype of cancer cells. Rho family GTPases regulate the arrangement of the actin cytoskeleton. By wound-healing assay, we have found that NIH 3T3 fibroblast cells move towards the wound- gaps by extending filopodial and lamellipdial structures at the leading edge of the moving cells. We have inactivated the function of Rho GTPases of v-Ras transformed NIH 3T3 cells by overexpressing Rho GTPase-activating (RhoGAP) domain of RhoGAP of p190. We have observed that inactivation of Rho, Rac and Cdc42 GTPases by overexpressing RHG causes inhibition of: (i) polymerization of actin to form filaments, (ii) formation of lamellipodia, filopodia and stress fibres, (iii) cell motility, (iv) cell spreading and (v) cell-to-cell adhesions. These results further strengthen the current knowledge on the role of Rho, Rac and Cdc42 GTPases in the regulation of the rearrangement of actin cytoskeleton. Our results, for the first time, demonstrate that RhoGAP domain of RhoGAP could be used to study the molecular mechanism of Ras-mediated signalling in growth, differentiation and carcinogenesis.

The Relationship between Subcellular Localization of Actinin-4 and Cell Motility in Oral Squamous Cell Carcinoma / Oral Science International

To elucidate the mechanism of invasion of oral squamous cell carcinoma, we newly established two different cell lines with a high-motility phenotype (designated HI type) and low-motility phenotype (LI type) from CA-9-22, a human oral squamous cell carcinoma cell line, through cell invasion assay (Boyden chamber assay). When we examined the subcellular localization and protein expression of actinin-4 using these cell lines, although the growth curves were not significantly different between the HI type and LI type, more invasion was seen in the HI-type than in the LI-type on Boyden chamber assay (p<0.0001). Morphologically, a larger number of sharply extended cell processes and spindle formation were observed in the HI-type than in the LI-type, and actinin-4 was mainly distributed in these processes. Western analysis showed that the expression level of actinin-4 was almost equivalent between the HI and LI types. These findings suggest that subcellular localization of actinin-4 might be involved in cell motility and cancer invasion by regulating the actin cytoskeleton at the cell processes in oral squamous cell carcinoma.

A decrease in the expression of CD63 tetraspanin protein elevates invasive potential of human melanoma cells

CD63, which belongs to the tetraspanin membrane proteins, has been proposed to play an important role in inhibiting melanoma metastasis. To determine whether reduction of CD63 expression, which frequently occurs in the malignant progression of human melanoma, is responsible for metastasis promotion, we transfected the antisense CD63 cDNA into MelJuso melanoma cells having endogenous CD63 expression. The antisense CD63 transfectant clones showing decreased CD63 expression displayed increased cell motility, matrix-degrading activity, and invasiveness in vitro when compared with the control transfectant cells. The antisense CD63 cDNA-transfected cells also exhibited altered adhesiveness to extracellular matrix. The results suggest that reduced CD63 expression contributes to the invasive and metastatic ability of human melanoma cells.

Effects of Conjugated Linoleic Acid (CLA) on Matrix Metalloproteinase (MMP) Activity and Cell Motility in Human Colon Cancer Cell Lines

Conjugated linoleic acid (CLA) consists of several geometric isomers of linoleic acid. CLA is found in foods derived from ruminants and exhibits strong anticarcinogenic effects in a variety of animal models. Matrix metalloproteinases (MMPs) play a key role in cancer progression. Specifically, MMP-2 and -9, which hydrolyze the basal membrane type IV collagen, are involved in the initial breakdown of collagen and basement membrane components during tumor growth and invasion. However, the effects of CLA on cancer cell motility and MMP expression and activity are not currently well known. Therefore, the present study examined whether CLA reduces the activity of MMP and cell motility in SW480 and SW620 cells, the human colon cancer cell lines. Gelatin zymography and Western blot analysis revealed that phorbol 12-myristate 13-acetate (PMA) induced the activity and protein expression of Mr 92,000 MMP-9 in both cell lines. To examine whether CLA inhibits the MMP activity, cells were incubated with 100 ngfmL PMA in the presence of various concentrations of CLA. PMA-induced MMP-9 activity was decreased by 20 micrometer CLA in SW480 cells, and by 10 micrometer and 20 micrometer CLA in SW620 cells. Results from the Hoyden chamber assay showed that cell motility was increased by PMA and that PMA-induced cell motility was significantly decreased by 20 micrometer CLA in SW480 cells. These results indicate that CLA may reduce the motility and MMP activity in human colon cancer cells.

Influence of Sodium Nitroprusside on Cell Motility of Outer Hair Cells of Guinea Pigs / 听力学及言语疾病杂志

Objective To study the changes of cell motility of outer hair cells in the cochlear of guinea pigs under treatment of sodium nitroprusside(SNP). Methods By using the whole cell patch clamp recording technique in normal external and internal cell solution, the cell motility of outer hair cells under stimulation of voltage was observed in different concentration of SNP. Results Sodium nitroprusside apparently inhibited outer hair cells' motility when SNP concentration was higher than 100 ?Mol/L ( P