miR-497 inhibits the growth and metastasis of SGC-7901 human gastric cancer anoikis resistant cells via blocking Wnt/β-catenin signaling pathway / 细胞与分子免疫学杂志

Objective To investigate the effects of microRNA497 (miR-497) on the metastasis of gastric cancer and its possible molecular mechanism. Methods SGC-7901 gastric cancer parent cells were cultured in an ultra-low adhesion environment, and the anoikis resistance model of SGC-7901 cells was created after re-adhesion. Clone formation assay, flow cytometry, TranswellTM test and scratch healing test were used to detect the differences of biological behavior compared with their parent cells. Fluorescence quantitative PCR was performed to detect the expression of miR-497. Western blot analysis was used to detect the changes of key proteins of Wnt/β-catenin signaling pathway and epithelial mesenchymal transformation (EMT) related proteins such as vimentin and E-cadherin. Parent cells and anoikis resistant SGC-7901 cells were transfected with miR-497 inhibitor or miR-497 mimic, and CCK-8 assay was used to detect the proliferation activity. TranswellTM invasion assay was performed to detect the invasion ability of cells. TranswellTM migration test and scratch healing assay was used to determine the migration ability. Western blot analysis was used to detect the expressions of Wnt1, β-catenin, vimentin and E-cadherin. By transfecting miR-497 mimic into the anoikis resistance SGC-7901 cells and inoculating them subcutaneously in nude mice, the changes in the volume and mass of tumor tissues were measured and recorded. Western blot analysis was used to determine the expressions of Wnt1, β-catenin, vimentin and E-cadherin of tumor tissues. Results Compared with the parent cells, the anoikis resistance SGC-7901 gastric cancer cells had faster proliferation rate, stronger colony formation, lower apoptosis rate, stronger invasion and migration ability. The expression of miR-497 was significantly decreased. After down-regulation of miR-497, the proliferation ability, invasion and migration ability were significantly enhanced. The expressions of Wnt1, β-catenin and vimentin increased significantly, while E-cadherin decreased notably. The results of up-regulation miR-497 were the opposite. The tumor growth rate, tumor volume and mass of miR-497 overexpression group were significantly lower than those of control group. The expressions of Wnt1, β-catenin and vimentin decreased significantly, while the expression of E-cadherin increased significantly. Conclusion The expression of miR-497 is low in the anoikis resistance SGC-7901 cells. miR-497 can inhibit the growth and metastasis of gastric cancer cells by blocking Wnt/β-catenin signaling pathway and EMT.

rBMSCs/ITGA5B1 Promotes Human Vascular Smooth Muscle Cell Differentiation via Enhancing Nitric Oxide Production

BACKGROUND AND OBJECTIVES: Previous studies have shown that integrins alpha5beta1 (ITGA5B1) gene-modified rat bone marrow mesenchymal stem cells (rBMSCs) could prevent cell anoikis and increase the nitric oxide (NO) production. Here we examined the capability of rBMSCs/ITGA5B1 on the phenotype modulation of Human Pulmonary Artery Smooth Muscle Cell (HPASMC) in vitro. METHODS AND RESULTS: The synthetic (dedifferentiated) phenotype of HPASMC was induced by monocrotaline (MCT, 1μM) for 24 h and then co-cultured with rBMSCs/ITGA5B1 in a transwell culture system. The activation of NO/cGMP (nitric oxide/Guanosine-3′, 5′-cyclic monophosphate) signaling was investigated in HPASMC. The changes of pro-inflammatory factors, oxidative stress, vasodilator, vasoconstrictor, contractile and synthetic genes, and the morphological changes of HPASMC were investigated. The results of this study showed that the NO/cGMP signal, endothelial nitric oxide synthase (eNOS) expression, the expression of the vasoprotective genes heme oxygenase-1 (HMOX1) and prostaglandin-endoperoxide synthase 2 (PTGS2) were increased, but the expression of transforming growth factor-β1 (TGF-β1), CCAAT/enhancer-binding proteins delta (Cebpd), Krüppel-like factor 4 (KLF4), and activating transcription factor 4 (ATF4) were reduced in MCT treated HPASMC co-cultured with rBMSCs/ITGA5B1. The synthetic smooth muscle cells (SMCs) phenotype markers thrombospondin-1, epiregulin and the vasoconstrictor endothelin (ET)-1, thromboxane A2 receptor (TbxA2R) were down-regulated, whereas the contractile SMCs phenotype marker transgelin expression was up-regulated by rBMSCs/ITGA5B1. Furthermore, rBMSCs/ITGA5B1 promoted the morphological restoration from synthetic (dedifferentiation) to contractile (differentiation) phenotype in MCT treated HPASMC. CONCLUSIONS: rBMSCs/ITGA5B1 could inhibit inflammation and oxidative stress related genes to promote the HPASMC cell differentiation by activation NO/cGMP signal.

Autophagy and its relationship with tumor proliferation, invasion, and treatment / 华西口腔医学杂志

Autophagy is a highly conservative biological behavior in eukaryotic cells. This dynamic process involves "wrapping" cytoplasmic components and combining with lysosomes in cells for catabolism. The catabolic effect of autophagy can eliminate toxic substances in cells, maintain homeostasis in the intracellular environment, and produce small molecules, such as amino acids, which nourish cells, thereby allowing them to survive. Autophagy can inhibit the occurrence of tumors by maintaining homeostasis in the intracellular environment. However, it can promote the proliferation, invasion, and metastasis of malignant tumor cells. Autophagy can regulate the microenvironment of tumor cells and has an important role in a series of processes, such as anoikis, tumor dormancy, and epithelial-mesenchymal transition.

Role of CD24 in anoikis resistance of ovarian cancer cells / 华中科技大学学报（医学）（英德文版）

This study examined the effect of CD24 on anoikis of ovarian cancer cells. The expression of CD24 was detected by RT-PCR and Western blotting in ovarian cancer cells with high metastatic potential (HO-8910PM cells) and low metastatic potential (A2780 cells). Cell viability and cell proliferation were detected by MTT assay in suspension culture and adhesion culture. Soft agar culture was used to observe the colony formation. Anoikis was flow cytometrically detected. The results showed that the expression levels of CD24 mRNA and protein were significantly higher in HO-8910PM cells than in A2780 cells (P<0.01). In the suspension culture and soft agar culture, the HO-8910PM cells formed larger and more colonies (35.33 ± 5.51 vs. 16.67 ± 4.04; P<0.01), and showed a stronger resistance to anoikis than A2780 cells did (cell apoptosis rate: 5.93% ± 2 .38% vs. 16.32% ± 2.00%; P<0.01). After treated with CD24 monoclonal antibodies, the number of colony formed in HO-8910PM and A2780 cells was significantly decreased (9.33 ± 2.52 and 8.00 ± 2.00, respectively), and the anoikis rate of the two cell lines was also markedly increased (23.11% ± 2.87% and 28.36% ± 2.29%, respectively). Our study suggested that CD24 may play an important role in the development of anoikis resistance and CD24 can be used as a new therapeutic target to induce anoikis and inhibit metastasis in ovarian cancer.

Regulatory effect of MTA1 on the anoikis of human prostate cancer cells / 中华男科学杂志

<p><b>OBJECTIVE</b>To study the effects of MTA1 small interfering RNA (siRNA) on the anchorage-independent growth and anoikis of prostate cancer cell line PC-3.</p><p><b>METHODS</b>After transfection of human prostate cancer PC-3 cells by MTA1 siRNA, we detected the expression of the MTA1 gene by real-time PCR and Western blot, the anchorage-independent growth of the cells by clone formation in soft agar, and their anoikis by DNA fragmentation assay and flow cytometry.</p><p><b>RESULTS</b>Compared with the control group, MTA1 siRNA transfection significantly decreased the mRNA and protein levels of MTA1, inhibited the anchorage-independent growth of the PC-3 cells, and induced their anoikis, all in a dose- and time-dependent manner (r = 0.935, P = 0.001; r = 0.901, P = 0.0005; r = 0.916, P = 0.0003).</p><p><b>CONCLUSION</b>MTA1 siRNA can inhibit the anchorage-independent growth of prostate cancer cells by inducing their anoikis.</p>

miR-124 suppresses multiple steps of breast cancer metastasis by targeting a cohort of pro-metastatic genes in vitro / 癌症

Metastasis is a multistep process involving modification of morphology to suit migration, reduction of tumor cell adhesion to the extracellular matrix, increase of cell mobility, tumor cell resistance to anoikis, and other steps. MicroRNAs are well-suited to regulate tumor metastasis due to their capacity to repress numerous target genes in a coordinated manner, thereby enabling their intervention at multiple steps of the invasion-metastasis cascade. In this study, we identified a microRNA exemplifying these attributes, miR-124, whose expression was reduced in aggressive MDA-MB-231 and SK-3rd breast cancer cells. Down-regulation of miR-124 expression in highly aggressive breast cancer cells contributed in part to DNA hypermethylation around the promoters of the three genes encoding miR-124. Ectopic expression of miR-124 in MDA-MB-231 cells suppressed metastasis-related traits including formation of spindle-like morphology, migratory capacity, adhesion to fibronectin, and anoikis. These findings indicate that miR-124 suppresses multiple steps of metastasis by diverse mechanisms in breast cancer cells and suggest a potential application of miR-124 in breast cancer treatment.

PAUF promotes adhesiveness of pancreatic cancer cells by modulating focal adhesion kinase

Pancreatic cancer is a notorious disease with a poor prognosis and low survival rates, which is due to limited advances in understanding of the molecular mechanism and inadequate development of effective treatment options over the decades. In previous studies, we demonstrated that a novel soluble protein named pancreatic adenocarcinoma up-regulated factor (PAUF) acts on tumor and immune cells and plays an important role in metastasis and progression of pancreatic cancer. Here we show that PAUF promotes adhesiveness of pancreatic cancer cells to various extracellular matrix (ECM). Our results further support a positive correlation of activation and expression of focal adhesion kinase (FAK), a key player in tumor cell metastasis and survival, with PAUF expression. PAUF-mediated adhesiveness was significantly attenuated upon blockade of the FAK pathway. Moreover, PAUF appeared to enhance resistance of pancreatic cancer cells to anoikis via modulation of FAK. Our results suggest that PAUF-mediated FAK activation plays an important role in pancreatic cancer progression.

Screening metastasis-associated genes from anoikis resistant A549 lung cancer cells by human genome array / 中国肺癌杂志

<p><b>BACKGROUND AND OBJECTIVE</b>As a barrier to metastases, cells normally undergo apoptosis after they lose contact with their extra cellular matrix (ECM). This process has been termed "anoikis". Tumour cells that acquire malignant potential have developed mechanisms to resist anoikis and thereby survive after detachment from their primary site while traveling through the lymphatic and circulatory systems. This "anoikis resistance" is considered the first step to tumor metastases. The aim of this study was to screen metastasis-associated genes from anoikis resistant and adherent growth A549 lung cancer cell by Human Genome Array.</p><p><b>METHODS</b>Establish anoikis resistant A549 lung cancer cell lines by using poly-hydroxyethyl methacrylate resin processed petri dishes, which causes cell free from adherent. The different expressed gene between anoikis resistant A549 cell and adherent growth A549 cell was tested using human V2.0 whole-genome oligonucleotide microarray, a product of Capitalbio Corporation, Beijing. Screen metastasis-associated genes.</p><p><b>RESULTS</b>745 different expressed genes were screened, including 63 highly metastasis-associated genes.</p><p><b>CONCLUSION</b>The successfully established anoikis resistant A549 cell lines and screened different expressed genes provide us basis for further research on metastasis of lung cancer.</p>

Relationship between the expression of TrkB and anoikis resistance in nasopharyngeal carcinoma line / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To study the regulation of anoikis by tyrosine kinase receptor B (TrkB) in human nasopharyngeal carcinoma lines. METHODS; Expression levels of TrkB and brain-derived neurotrophic factor (BDNF) were evaluated by RT-PCR and Western blot. Colony formation ability of C666-1 was observed in soft agar. Proliferation rate and apoptosis, that change in cells by treating the TrkB inhibitor K252a and specificity ligand BDNF respectively under suspension culture, were measured by cell counting kit-8 (CCK-8) assay and the flow cytometry assay. The expression change of TrkB, BDNF and phosphorylation of serine threonine kinase (p-Akt) were investigated by Western blot analysis.</p><p><b>RESULTS</b>TrkB and BDNF were identified in C666-1 cells. C666-1 cells could be decreased the proliferation of colony in soft agar by effect of K252a, but BDNF could make the colony prolific. K252a can inhibit the expression of TrkB in C666-1, and prevent p-Akt activation. And exogenous BDNF stimulated up-regulation TrkB and p-Akt, induced anoikis resistance.</p><p><b>CONCLUSION</b>TrkB inhibits anoikis in nasopharyngeal carcinoma cells. Inhibiton of TrkB by K252a can induce anoikis, and may prove particularly effective in treatment of nasopharyngeal carcinoma.</p>

[Effect of hydrostatic pressure on PC12 cell line]

The present study was designed to investigate the effect of hydrostatic pressure on cell viability, apoptosis induction, morphology and cell-substrate interactions of PC 12 cells. PC 12 as a neuronal cell line maintained in RPMI 1640 culture medium supplemented with 10% fetal bovine serum. PC 12 cells were subjected to hydrostatic pressure. Experimental pressure condition was 100mmHg set above atmospheric pressure for 2 h. Controls were treated identically except for the application of pressure. Dye exclusion was used for viability assay, TUNEL staining was used for apoptosis detection. Cell area was assessed as morphometry and then cell adhesion, extension and migration were investigated. Hydrostatic pressure had not changed viability of cells. It induced apoptosis in PC 12 cells. In addition, hydrostatic pressure reduced cell area, adhesion, extension and migration ability of these cells [P<0.05]. Hydrostatic pressure may induce apoptosis in PC 12 cells as a result of inappropriate cell to substrate adhesion. Thus it is suggest that occurring apoptosis in these cells be an anoikis cell death induced by loss of attachment to the substrate

PTEN induces anoikis through its phosphatase activity in hepatocellular carcinoma cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effect and mechanisms of tumor suppressor gene PTEN on the induction of anoikis of hepatocellular carcinoma SMMC-7721 cells.</p><p><b>METHODS</b>SMMC-7721 cells were transfected with GFP plasmids containing wild-type PTEN or phosphatase inactivating mutant PTEN (C124A-PTEN) in vitro; The PTEN expression and the phosphorylation levels of focal adhesion kinase (FAK) and protein kinase B (PKB/Akt) were detected by Western blotting; Flow cytometry assay and laser scanning confocal microscopy were used to analyze apoptosis in adherent and non-adherent cells.</p><p><b>RESULTS</b>Compared with the control, PTEN expression in the cells transfected with wild-type PTEN increased to 248%, while the phosphorylation level of FAK and Akt decreased 65.2% and 89.1%, respectively; and the anoikis percentage increased from 9.5% to 31.3%. In the cells transfected with C124A-PTEN, neither the phosphorylation of FAK and Akt nor the anoikis percentage had obviously changed, although the PTEN expression enhanced dramatically in comparison with the control.</p><p><b>CONCLUSION</b>Through its phosphatase activity, tumor suppressor gene PTEN can suppress the phosphorylation of FAK and Akt, and induce anoikis in hepatocellular carcinoma cells.</p>

Influence of hepatocyte growth factor on biological characteristics of bone marrow-derived mesenchymal stem cells / 中国实验血液学杂志

Hepatocyte growth factor (HGF) is one of major growth factors in the bone marrow microenvironments with which the proliferation, differentiation and migration of bone marrow-derived mesenchymal stem cells were closely contacted. However, its roles in the regulation of proliferation, differentiation and migration of bone marrow-derived mesenchymal stem cells remain unclear. This study was aimed to investigate the effect of HGF on biological characteristics of bone marrow-derived mesenchymal stem cells. Expression of c-Met, the receptor for HGF was detected by immunohistochemistry assay, cell proliferation was determined by MTT, activity of ALP was quantitatively assayed, cell migration and anoikis-induced MSC apoptosis were analyzed. The results showed that HGF not influenced the proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells. Treatment of bone marrow-derived mesenchymal stem cells with recombinant human hepatocyte growth factor resulted in inhibition of anoikis-induced apoptosis. HGF significantly stimulated the migration of bone marrow-derived mesenchymal stem cells. Both PI-3 kinase and MAPK kinase were proved to be involved in HGF-induced migration. It is concluded that HGF/c-Met signal regulates the apoptosis and migration of bone marrow-derived mesenchymal stem cells.

COX-2 inhibits anoikis by activation of the PI-3K/Akt pathway in human bladder cancer cells

Cyclooxygenase-2 (COX-2) has been reported to be associated with tumor development and progression as well as to protect cells from apoptosis induced by various cellular stresses. Through a tetracycline-regulated COX-2 overexpression system, we found that COX-2 inhibits detachment-induced apoptosis (anoikis) in a human bladder cancer cell line, EJ. We also found that the inhibition of anoikis by COX-2 results from activation of the PI-3K/Akt pathway as evidenced by suppression of the COX-2 effect on anoikis by a PI-3K inhibitor, LY294002. Furthermore, COX-2 enhanced Mcl-1 expression in the anoikis process, implying that Mcl-1 also may be involved in mediating the survival function of COX-2. Together, these results suggest that COX-2 inhibits anoikis by activation of the PI-3K/Akt pathway and probably by enhancement of Mcl-1 expression in human bladder cancer cells. This anti- anoikis effect of COX-2 may be a part of mechanisms to promote tumor development and progression.

Modulação da metilação de DNA por óxido nítrico durante a carcinogênese do melanoma murino / Modulation of DNA methylation by nitric oxide during mirine melanoma carcinogenesis

Células em um tecido estabelecem contatos com outras células e com o meio circunjacente. Qualquer abalo nestas interações é percebido e desencadeia uma série de eventos que tendem à homeostase. A perda de contato de uma célula com sua matriz extracelular dispara reações que culminam em morte celular (apoptose), resultando na homeostase do tecido de origem. Este tipo de apoptose por perda de adesão à matriz é chamada de anoikis. Entre os mediadores da morte por anoikis encontram-se os radicais livres, já que a perda do contato célula-matriz leva a um estresse químico. Entretanto, em concentrações ótimas, os radicais livres exercem função de moléculas sinalizadoras intracelulares relevantes em vários processos distintos, como sobrevivência e proliferação celulares. Uma vez que propicia a sobrevivência celular, a resistência ao anoikis é considerada uma aquisição importante para o processo carcinogênico, tanto em suas fases iniciais quanto nas tardias (por exemplo, na metastatização). Radicais livres têm sido amplamente associados à carcinogênese como agentes mutagênicos graças à propriedade de interagir diretamente coma as bases nitrogenadas do DNA. É relativamente menor o número de publicações associando radicais livres com modificações epigenéticas em tumorigênese. Por epigenética entende-se um evento capaz de influenciar a expressão gênica sem alterar a seqüência de nucleotídeos do DNA. Recentemente foi desenvolvido pelo grupo liderado pela Dra. Miriam Jasiulionis (UNIFESP) um modelo de transformação tumoral a partir da resistência ao anoikis. A linhagem de melanócitos melan-a de camundongos C57BL/6 foi usada naquele modelo experimental, que consiste no seguinte: células na concentração de 1 x1 05 por ml foram submetidas ao cultivo em suspensão por 96h. Após este período de tempo, as células remanescentes (0,1 por cento) foram re-aderidas, a população foi expandida e as células foram submetidas a um novo ciclo de cultivo em suspensão por 96h. Após 2 e 4 ciclos de adesão/suspensão, as células viáveis foram expandidas e donadas por diluição limitante, originando linhagens "intermediárias", não tumorigênicas in vivo. Após 5 ciclos e outra clonagem, foram obtidas linhagens designadas de melanoma por serem tumorigênicas in vivo, representados pelos cones Tm1 e Tm5. onsiderando que a falta de contato com a matriz seja um fator de estresse, o presente trabalho propôs investigar o papel do estresse como modulador de eventos epigenéticos que ocorreriam nas fases precoces da transformação de melanócitos murinos. Células da linhagem murina normal de melanócitos melan-a foram submetidas ao cultivo independente de ancoragem por até 24h e foram comparadas com células em condições de adesão quanto a indicadores de estresse, como radicais livres (ânion superóxido, peróxido de hidrogênio, óxido nítrico) e homocisteína, produtos da peroxidação de lipídeos (malondialdeído) e da defesa antioxidante celular (glutationa). Ao longo do tempo em suspensão, as células apresentaram aumento gradual em todos os indicadores de estresse testados, com exceção de homocisteína. Com o intuito de estudar a participação de mecanismos epigenéticos na transformação celular, a metilação global do DNA das células em suspensão foi mensurada por duas metodologias distintas e comparadas com a de células ancoradas e com a das linhagens intermediárias e tumorigênicas geradas após sucessivos ciclos de cultivo em suspensão/adesão. O nível global de metilação do DNA também foi crescente em relação ao tempo em suspensão. Como o uso do inibidor de NO sintase reverteu a hipermetilação decorrente do impedimento de adesão, concluiu-se que óxido nítrico modula a metilação de DNA podendo, assim, participar da tran-sformação tumoral.

The Role of Phosphatase and Tensin Homolog Deleted on Chromosome Ten (PTEN) in Lung Cancer

Lung cancer remains the primary cause of cancer-related death in the world, and the number of cases continues to increase. Like any other human cancer, the development of lung cancer is associated with the activation of oncogenes or inactivation of tumor suppressor genes. Phosphatase and tensin homolog, deleted on chromosome ten (PTEN), is a part of a complex signaling system that affects a variety of important cell functions. PTEN opposes the action of phosphatidylinositol 3-kinase (PI3-kinase) by dephosphorylating the signaling lipid phosphatidylinositol 3, 4, 5-triphosphate (PIP3). In addition, it displays weak tyrosine phosphatase activity, which may down modulate the signaling pathways involving focal adhesion kinase (FAK) or Shc. Functions for PTEN have been identified in the regulation of many normal cell processes, including growth, adhesion, migration, invasion and apoptosis. PTEN appears to play particularly important roles in regulating anoikis (apoptosis of cells after loss of contact with extracellular matrix) and cell migration. Many studies have suggested that the loss of PTEN expression occurs commonly in primary lung cancers and correlates with the histological type. Regulation of PTEN expression may provide a new preventive and therapeutic modality in primary lung cancer. However, there is, in our opinion, a need for further study of this gene