Overexpression of lumican affects the migration of human colon cancer cells through up-regulation of gelsolin and filamentous actin reorganization

Cell migration is a multistep process initiated by extracellular matrix components that leads to cytoskeletal changes and formation of different protrusive structures at the cell periphery. Lumican, a small extracellular matrix leucine-rich proteoglycan, has been shown to inhibit human melanoma cell migration by binding to α2β1 integrin and affecting actin cytoskeleton organization. The aim of this study was to determine the effect of lumican overexpression on the migration ability of human colon adenocarcinoma LS180 cells. The cells stably transfected with plasmid containing lumican cDNA were characterized by the increased chemotactic migration measured on Transwell filters. Lumican-overexpressing cells presented the elevated filamentous to monomeric actin ratio and gelsolin up-regulation. This was accompanied by a distinct cytoskeletal actin rearrangement and gelsolin subcellular relocation, as observed under laser scaning confocal microscope. Moreover, LS180 cells overexpressing lumican tend to form podosome-like structures as indicated by vinculin redistribution and its colocalization with gelsolin and actin at the submembrane region of the cells. In conclusion, the elevated level of lumican secretion to extracellular space leads to actin cytoskeletal remodeling followed by an increase in migration capacity of human colon LS180 cells. These data suggest that lumican expression and its presence in ECM has an impact on colon cancer cells motility and may modulate invasiveness of colon cancer.     

Subcellular distribution and expression of cofilin and ezrin in human colon adenocarcinoma cell lines with different metastatic potential

The dynamic reorganization of the actin cytoskeleton is regulated by a number of actin binding proteins (ABPs). Four human colon adenocarcinoma cell lines – parental and three selected sublines, which differ in motility and metastatic potential, were used to investigate the expression level and subcellular localization of selected ABPs. Our interest was focused on cofilin and ezrin. These proteins are essential for cell migration and adhesion. The data received for the three more motile adenocarcinoma sublines (EB3, 3LNLN, 5W) were compared with those obtained for the parental LS180 adenocarcinoma cells and fibroblastic NRK cells. Quantitative densitometric analysis and confocal fluorescence microscopy were used to examine the expression levels and subcellular distribution of the selected ABPs. Our data show distinct increase in the level of cofilin in adenocarcinoma cells accompanied by the reduction of inactive phosphorylated form of cofilin. In more motile cells, cofilin was accumulated at cellular periphery in co-localization with actin filaments. Furthemore, we indicated translocation of ezrin towards the cell periphery within more motile cells in comparison with NRK and parental adenocarcinoma cells.In summary, our data indicate the correlation between migration ability of selected human colon adenocarcinoma sublines and subcellular distribution as well as the level of cofilin and ezrin. Therefore these proteins might be essential for the higher migratory activity of invasive tumor cells.Key words: actin, cofilin, ezrin, colon adenocarcinoma.     

Beta-actin in human colon adenocarcinoma cell lines with different metastatic potential

Human colon adenocarcinoma LS180 parental cell line and selected variants, characterized by different metastatic capacity were used to examine, whether a correlation exists between beta-actin expression, its subcellular distribution and metastatic potential of these cells. Cytosolic fraction (supernatant 105000 x g), isolated from the tumor cells was used as a source for actin quantification. The higher level of beta-actin was observed in the cytosol of three selected sublines to compare with LS180 parental line. Statistically significant increase of beta-actin level in highly motile EB3 cells variant should be underlined to compare with the other sublines. Distinct differences in the phenotype of adenocarcinoma cell variants were found, such as the changes in cells shape, cells spreading and ability to attach to the surface of culture dish. Actin cytoskeleton was visualized with fluorescence microscopy application and microfilaments rhodamine-conjugated phalloidin staining. beta-actin subcellular localization was done by immunofluorescence staining with monoclonal anti-beta actin antibodies. In the elongated cells (LS180, 3LNLN), this isoactin is dispersed in the whole cell body and concentrates in pseudopods and at the leading edges, when in the rounded variant (EB3) beta-actin dominates mainly in cortical ring under cellular membrane and it is also seen in the subtle protrusions. Summary of our former (Nowak et al., 2002, Acta Biochim. Polon., 49: 823) and current data lead to the conclusion that there is a distinct correlation between metastatic capacity of examined human colon adenocarcinoma cells, the state of actin polymerization, actin cytoskeleton organization and beta-actin expression.     

Actin in human colon adenocarcinoma cells with different metastatic potential

Four human colon adenocarcinoma cell line variants with different metastatic potential were used to examine whether a correlation exists between actin level, state of actin polymerization and invasiveness of tumour cells. Monomeric (G), total (T) and filamentous (F) actin were determined in the cytosolic fraction of these cells. A statistically significant decrease in G actin level and increase in the state of actin polymerization (measured by F:G actin ratio) were found in the cytosol of three cell variants with higher metastatic potential and invasiveness (EB3, 3LNLN, 5W) compared with the parental cell line (LS180). Our experimental data lead to the conclusion that there is a correlation between the metastatic capacity of human colon adenocarcinoma cells and the state of actin polymerization.     

Cofilin overexpression affects actin cytoskeleton organization and migration of human colon adenocarcinoma cells

The dynamic reorganization of actin cytoskeleton is regulated by a large number of actin-binding proteins. Among them, the interaction of ADF/cofilin with monomeric and filamentous actin is very important, since it severs actin filaments. It also positively influences actin treadmilling. The activity of ADF/cofilin is reversibly regulated by phosphorylation and dephosphorylation at Ser-3, with the phosphorylated form (P-cofilin) being inactive. Here, we studied the effects of overexpression of cofilin and two cofilin variants in the human colon adenocarcinoma LS180 cell line. We have generated the LS180 cells expressing three different cofilin variants: WT (wild type), Ser 3 Ala (S3A) (constitutively active) or Ser 3 Asp (S3D) (constitutively inactive cofilin). The cells expressing WT cofilin were characterized by abundant cell spreading and colocalization of cofilin with the submembranous F-actin. Similar effects were observed in cells expressing S3A cofilin. In contrast, LS180 cells expressing S3D cofilin remained longitudinal in morphology and cofilin was equally distributed within the cell body. Furthermore, the migration ability of LS180 cells expressing different cofilin mutants was analyzed. In comparison to control cells, we have noticed a significant, approximately fourfold increase in the migration factor value of cells overexpressing WT type cofilin. The overexpression of S3D cofilin resulted in an almost complete inhibition of cell motility. The estimation of actin pool in the cytosol of LS180 cells expressing S3A cofilin has shown a significantly lower level of total actin in reference to control cells. The opposite effect was observed in LS180 cells overexpressing S3D cofilin. In summary, the results of our experiments indicate that phosphorylation “status” of cofilin is a factor affecting the actin cytoskeleton organization and migration abilities of colon adenocarcinoma LS180 cells.     

环状RNA Circ_0001073在恶性黑素瘤中的表达及其对A375细胞恶性表型的影响

目的:通过检测环状RNA Circ_0001073在恶性黑素瘤细胞与正常表皮黑素细胞中的表达差异,及其对恶性黑素瘤A375细胞恶性表型的影响,阐明Circ_001073在恶性黑素瘤细胞中的表达及功能。方法:采用聚合酶链式反应(Real-time quantitative polymerase chain reaction, RT-QPCR)方法检测正常表皮黑素细胞和恶性黑素瘤细胞中Circ_0001073的表达水平;应用小干扰RNAs沉默恶性黑素瘤A375细胞中Circ_0001073表达,5-乙炔基-2'-脱氧尿苷(5-Ethynyl-2'-deoxyuridine,EdU)实验检测沉默Circ_0001073对A375细胞增殖的影响,平板克隆实验检测沉默Circ_0001073对A375细胞克隆形成的影响,细胞划痕实验评估沉默Circ_0001073对A375细胞迁移能力的影响,Transwell实验评估沉默Circ_0001073对A375细胞侵袭能力的影响。结果:qRT-PCR结果显示Circ_0001073在恶性黑素瘤细胞中的表达低于正常表皮黑素HEMa-LP细胞(P0.05)。应用Circ_0001073si RNA转染A375细胞后,Circ_0001073的表达水平较转染Circ_0001073 NC的细胞显著降低(P0.05)。EdU实验结果显示:沉默A375细胞中Circ_0001073的表达后,A375细胞增殖能力显著增强(P0.05);平板克隆实验结果显示:沉默A375细胞中Circ_0001073的表达后,A375细胞克隆形成能力显著增强(P0.05);细胞划痕实验结果显示:沉默A375细胞中Circ_0001073的表达后,细胞迁移能力显著增强(P0.05);Transwell实验结果显示:沉默A375细胞中Circ_0001073的表达后,细胞侵袭能力显著增强(P0.05)。结论:Circ_0001073在恶性黑素瘤细胞中低表达,并可抑制A375细胞的增殖、克隆形成、迁移及侵袭。     

Vasodilator-stimulated Phosphoprotein (VASP) Regulates Actin Polymerization and Contraction in Airway Smooth Muscle by a Vinculin-dependent Mechanism

Vasodilator-stimulated phosphoprotein (VASP) can catalyze actin polymerization by elongating actin filaments. The elongation mechanism involves VASP oligomerization and its binding to profilin, a G-actin chaperone. Actin polymerization is required for tension generation during the contraction of airway smooth muscle (ASM); however, the role of VASP in regulating actin dynamics in ASM is not known. We stimulated ASM cells and tissues with the contractile agonist acetylcholine (ACh) or the adenylyl cyclase activator, forskolin (FSK), a dilatory agent. ACh and FSK stimulated VASP Ser¹⁵⁷ phosphorylation by different kinases. Inhibition of VASP Ser¹⁵⁷ phosphorylation by expression of the mutant VASP S157A in ASM tissues suppressed VASP phosphorylation and membrane localization in response to ACh, and also inhibited contraction and actin polymerization. ACh but not FSK triggered the formation of VASP-VASP complexes as well as VASP-vinculin and VASP-profilin complexes at membrane sites. VASP-VASP complex formation and the interaction of VASP with vinculin and profilin were inhibited by expression of the inactive vinculin mutant, vinculin Y1065F, but VASP phosphorylation and membrane localization were unaffected. We conclude that VASP phosphorylation at Ser¹⁵⁷ mediates its localization at the membrane, but that VASP Ser¹⁵⁷ phosphorylation and membrane localization are not sufficient to activate its actin catalytic activity. The interaction of VASP with activated vinculin at membrane adhesion sites is a necessary prerequisite for VASP-mediated molecular processes necessary for actin polymerization. Our results show that VASP is a critical regulator of actin dynamics and tension generation during the contractile activation of ASM.     

MicroRNA-330 inhibits growth and migration of melanoma A375 cells: In vitro study

Melanoma skin cancer is one of the main causes of male cancer-related deaths worldwide. It has been suggested that miR-330-5p can act as a tumor suppressor in various types of cancers. So, in this study, we replaced miR-330 in melanoma cancer cells by vector-based miR-330 to evaluate the effects of this microRNA on the growth and migration inhibition of melanoma cancer cells, and to determine the molecular mechanisms underlying its action. By using the MTT assay, the IC₅₀ of Geneticin antibiotic was obtained as 460 µg/mL. The results of the qRT-PCR showed the increased expression level of miR-330 and decreased expression levels of MMP-9, CXCR4, Vimentin, melanoma cell adhesion molecule, AKT1, and E2F1 messenger RNA in A375 transfected cells. The cytotoxicity assay results demonstrated the inhibition of cancer cells proliferation. Furthermore, the wound healing test results showed a migration reduction of transfected cells with miR-330 compared with nontransfected ones. In addition, 4′,6-diamidino-2-phenylindoleLB: Luria-Bertani (DAPI) staining revealed the significant nucleus fragmentation in miR-330 replaced cells, which correspond to apoptosis induction in replaced cells. The results showed that increase in miR-330 expression level could significantly inhibit the tumor cell growth and the migration of melanoma cancer cells.     

Lumican affects actin cytoskeletal organization in human melanoma A375 cells

AIMS: Lumican, a small leucine-rich proteoglycan (SLRP), has attracted attention as a molecule of the extracellular matrix possibly involved in signalling pathways affecting cancer cell behaviour. The remodelling of the actin cytoskeleton, induced in response to external stimuli, is crucial for cell motility and intracellular signal transduction. The main goal of this study was to examine the effects of recombinant lumican on actin organization, the state of actin polymerization, actin isoform expression, and their sub-cellular distribution in the A375 human melanoma cell line. MAIN METHODS: Fluorescence and confocal microscopy were used to observe actin cytoskeletal organization and the sub-cellular distribution of cytoplasmic beta- and gamma-actins. The ability of actin to inhibit DNaseI activity was used to quantify actin. Western blotting and real-time PCR were used to determine the expression levels of the actin isoforms. KEY FINDINGS: A375 cells grown on lumican coatings changed in morphology and presented rearranged actin filament organization: from filaments evenly spread throughout the whole cell body to their condensed sub-membrane localization. In the presence of lumican, both actin isoforms were concentrated under the cellular membrane. A statistically significant increase in the total, filamentous, and monomeric actin pools was observed in A375 cells grown on lumican. SIGNIFICANCE: Novel biological effects of lumican, an extracellular matrix SLRP, on the actin pool and organization are identified, which may extend our understanding of the mechanism underlying the inhibitory effect of lumican on the migration of melanoma cells.     

NCAM regulates the proliferation,apoptosis, autophagy,EMT, and migration of human melanoma cells via the Src/Akt/mTOR/cofilin signaling pathway

The neural cell adhesion molecule (NCAM) plays critical roles in multiple cellular processes in neural cells, mesenchymal stem cells, and various cancer cells. However, the effect and mechanism of NCAM in human melanoma cells are still unclear. In this study, we found that NCAM regulated the proliferation, apoptosis, autophagy, migration, and epithelial-to-mesenchymal transition of human melanoma cells by determining the biological behavior of NCAM knockdown A375 and M102 human melanoma cells. Further studies revealed that NCAM knockdown impaired the organization of actin cytoskeleton and reduced the phosphorylation of cofilin, an actin-cleaving protein. When cells were transfected with cofilin S3A (dephosphorylated cofilin), biological behavior similar to that of NCAM knockdown cells was observed. Research on the underlying molecular mechanism showed that NCAM knockdown suppressed activation of the Src/Akt/mTOR pathway. Specific inhibitors of Src and PI3K/Akt were employed to further verify the relationship between Src/Akt/mTOR signaling and cofilin, and the results showed that the phosphorylation level of cofilin decreased following inhibition of the Src/Akt/mTOR pathway. These results indicated that NCAM may regulate the proliferation, apoptosis, autophagy, migration, and epithelial-to-mesenchymal transition of human melanoma cells via the Src/Akt/mTOR/cofilin pathway-mediated dynamics of actin cytoskeleton.     

早孕因子单克隆抗体对黑色素瘤细胞A-375增殖、凋亡的影响

目的:研究早孕因子单克隆抗体(EPF-McAb)对黑色素瘤细胞A-375增殖、凋亡的影响。方法:体外培养黑色素瘤细胞A-375,通过CCK-8实验检测EPF-McAb对A-375细胞增殖的影响;通过流式细胞术检测EPF-McAb对A-375细胞凋亡的影响;Western Blot检测EPF-McAb对A-375细胞EPF蛋白表达的影响。结果:CCK-8结果显示EPF-McAb可以抑制黑色素瘤细胞A-375的增殖,且随着作用时间延长和药物浓度的增加,对A-375细胞增殖的抑制作用也增强;流式细胞术实验结果显示EPF-McAb可以促进黑色素瘤细胞A-375的凋亡,且调亡率随着药物浓度的增加而升高,0.2、0.4、0.8 mg/m L的EPF单抗作用于A-375细胞24 h后,细胞调亡率分别为:14.68%(P0.01)、19.81%(P0.01)、23.97%(P0.01);Western Blot实验结果显示EPF-McAb可以降低黑色素瘤细胞A-375 EPF蛋白的表达。结论:早孕因子单克隆抗体可以抑制黑色素瘤细胞A-375的增殖,并促进其凋亡。     

Regulation of sperm motility by PIP2(4,5) and actin polymerization

Actin polymerization and development of hyperactivated (HA) motility are two processes that take place during sperm capacitation. In previous studies, we demonstrated that the increase in F-actin during capacitation depends upon inactivation of the actin severing protein, gelsolin, by its binding to phosphatydilinositol-4, 5-bisphosphate (PIP₂). Here, we showed for the first time the involvement of PIP₂/gelsolin in human sperm motility before and during capacitation. Activation of gelsolin by causing its release from PIP₂ inhibited sperm motility, which could be restored by adding PIP₂ to the cells. Reduction of PIP₂ synthesis inhibited actin polymerization and motility, and increasing PIP₂ synthesis enhanced these activities. Furthermore, sperm demonstrating low motility contained low levels of PIP₂ and F-actin. During capacitation there was an increase in PIP₂ and F-actin levels in the sperm head and a decrease in the tail. In sperm with high motility, gelsolin was mainly localized to the sperm head before capacitation, whereas in low motility sperm, most of the gelsolin was localized to the tail before capacitation and translocated to the head during capacitation. We also showed that phosphorylation of gelsolin on tyrosine-438 depends on its binding to PIP₂. Activation of phospholipase C by Ca²⁺-ionophore or by activating the epidermal-growth-factor-receptor inhibits tyrosine phosphorylation of gelsolin. In conclusion, the data indicate that the increase of PIP₂ and/or F-actin in the head during capacitation enhances gelsolin translocation to the head. As a result the decrease of gelsolin in the tail allows keeping high level of F-actin in the tail, which is essential for the development of HA motility.     

新霉胺通过抑制血管生成素活性抑制人黑色素瘤细胞增殖、迁移和侵润

新霉胺（neamine）是一种无毒的新霉素（neomycin）降解产物;已有研究证明,其可抑制血管生成素（angiogenin,ANG）诱导的内皮细胞血管生成作用,阻滞异种移植的人结肠腺癌在裸鼠的生长.本研究证明,新霉胺对人黑色瘤细胞株A375的细胞增殖、迁移和侵润作用.MTT法及软琼脂培养显示,新霉胺可明显抑制A375细胞的增殖和集落形成能力. Transwell试验证明,新霉胺可阻滞A375细胞,乃至血管生成素诱导的A375细胞的迁移和侵润能力.此外,免疫荧光揭示新霉胺可阻断血管生成素的核转位,从而抑制血管生成素诱导的A375细胞增殖.上述结果提示,新霉胺可通过抑制血管生成素的核转位,从而抑制肿瘤细胞增殖、迁移和侵润.鉴于与新霉素比较,新霉胺毒性小,因此新霉胺可望作为黑色素瘤治疗的先导药物,颇具开发前景和潜力.     

Antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells

Reactive oxygen species (ROS) generation is linked to dynamic actin cytoskeleton reorganization, which is involved in tumor cell motility and metastasis. Thus, inhibition of ROS generation and actin polymerization in tumor cells may represent an effective anticancer strategy. However, the molecular basis of this signaling pathway is currently unknown. Here, we show that the Ecklonia cava-derived antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Steady-state intracellular ROS levels were higher in malignant B16F10 cells than in parental, nonmetastatic B16F0 cells. Elevation of ROS by H₂O₂ treatment increased migration and invasion ability of B16F0 cells to level similar to that of B16F10 cells, suggesting that intracellular ROS signaling mediates the prometastatic properties of B16 mouse melanoma cells. ROS levels and the cell migration and invasion ability of B16 melanoma cells correlated with Rac1 activation and WAVE2 expression. Overexpression of dominant negative Rac1 and depletion of WAVE2 by siRNA suppressed H₂O₂-induced cell invasion of B16F0 and B16F10 cells. Similarly, dieckol attenuates the ROS-mediated Rac1 activation and WAVE2 expression, resulting in decreased migration and invasion of B16 melanoma cells. In addition, we found that dieckol decreases association between WAVE2 and NADPH oxidase subunit p47^phox. Therefore, this finding suggests that WAVE2 acts to couple intracellular Rac1/ROS signaling to the invasive migration of B16 melanoma cells, which is inhibited by dieckol.     

Interaction of extracellular S100A4 with RAGE prompts prometastatic activation of A375 melanoma cells

S100A4, a member of the S100 protein family of EF‐hand calcium‐binding proteins, is overexpressed in various tumour entities, including melanoma, and plays an important role in tumour progression. Several studies in epithelial and mesenchymal tumours revealed a correlation between extracellular S100A4 and metastasis. However, exact mechanisms how S100A4 stimulates metastasis in melanoma are still unknown. From a pilot experiment on baseline synthesis and secretion of S100A4 in human melanoma cell lines, which are in broad laboratory use, A375 wild‐type cells and, additionally, newly generated A375 cell lines stably transfected with human S100A4 (A375‐hS100A4) or human receptor for advanced glycation endproducts (A375‐hRAGE), were selected to investigate the influence of extracellular S100A4 on cell motility, adhesion, migration and invasion in more detail. We demonstrated that A375 cells actively secrete S100A4 in the extracellular space via an endoplasmic reticulum‐Golgi‐dependent pathway. S100A4 overexpression and secretion resulted in prometastatic activation of A375 cells. Moreover, we determined the influence of S100A4‐RAGE interaction and its blockade on A375, A375‐hS100A4, A375‐hRAGE cells, and showed that interaction of RAGE with extracellular S100A4 contributes to the observed activation of A375 cells. This investigation reveals additional molecular targets for therapeutic approaches aiming at blockade of ligand binding to RAGE or RAGE signalling to inhibit melanoma metastasis.     

Actin ADP‐ribosylation at Threonine148 by Photorhabdus luminescens toxin TccC3 induces aggregation of intracellular F‐actin

Intoxication of eukaryotic cells by Photorhabdus luminescens toxin TccC3 induces cell rounding and detachment from the substratum within a few hours and compromises a number of cell functions like phagocytosis. Here, we used morphological and biochemical procedures to analyse the mechanism of TccC3 intoxication. Life imaging of TccC3‐intoxicated HeLa cells transfected with AcGFP‐actin shows condensation of F‐actin into large aggregates. Life cell total internal reflection fluorescence (TIRF) microscopy of identically treated HeLa cells confirmed the formation of actin aggregates but also disassembly of F‐actin stress fibres. Recombinant TccC3 toxin ADP‐ribosylates purified skeletal and non‐muscle actin at threonine148 leading to a strong propensity to polymerize and F‐actin bundle formation as shown by TIRF and electron microscopy. Native gel electrophoresis shows strongly reduced binding of Thr148‐ADP‐ribosylated actin to the severing proteins gelsolin and its fragments G1 and G1–3, and to ADF/cofilin. Complexation of actin with these proteins inhibits its ADP‐ribosylation. TIRF microscopy demonstrates rapid polymerization of Thr148‐ADP‐ribosylated actin to curled F‐actin bundles even in the presence of thymosin β4, gelsolin or G1–3. Thr148‐ADP‐ribosylated F‐actin cannot be depolymerized by gelsolin or G1–3 as verified by TIRF, co‐sedimentation and electron microscopy and shows reduced treadmilling as indicated by a lack of stimulation of its ATPase activity after addition of cofilin‐1.     

Apoptosis induced by interferon-alpha and antagonized by EGF is regulated by caspase-3-mediated cleavage of gelsolin in human epidermoid cancer cells

We have previously reported that interferon-alpha (IFNalpha) induces apoptosis and EGF can antagonize this effect in human epidermoid cancer KB cells. Since apoptosis occurs together with cytoskeleton reorganization we have evaluated if IFNalpha and EGF could modulate cell remodeling in our experimental conditions. We have found that 48 h 1,000 IU/ml IFNalpha induced structural reorganization of stress fibers and membrane delocalization and partial capping of the actin severing protein gelsolin. The transfection of KB cells with both a wild type (WT) or a C-terminal truncated form of gelsolin caused overexpression of the protein and an increase of both the spontaneous and IFNalpha-induced apoptosis and cell cytoskeletal modifications. In fact, after 48 h of treatment IFNalpha induced 45% of apoptotic cell death in parental cells while an approximately 80% of cell population was apoptotic in transfected cells. These effects occurred together with an increase of the expression and consequent degradation of gelsolin. Again the addition of EGF to IFNalpha-treated transfected cells caused a recovery of the apoptosis. Notably, IFNalpha and EGF did not modify the expression of other molecules associated to cytoskeleton such as focal adhesion kinase and vinculin. In the same experimental conditions IFNalpha induced also gelsolin cleavage that occurred together with caspase-3 activation and release of cytochrome c. All these effects were antagonized by the exposure of IFNalpha-treated KB to 10 nM EGF for the last 12 h. Moreover, the specific inhibition of caspase-3 with 20 microM DEVD completely abrogated apoptosis and gelsolin cleavage induced by IFNalpha. In conclusion, our data are the first demonstration that IFNalpha can induce morphological cell changes that are peculiar of apoptosis onset through the caspase-3-mediated cleavage of gelsolin. Furthermore, we have demonstrated that EGF is able to antagonize these effects through the inhibition of caspase-3 activation.     

The Molecular Chaperone CCT Sequesters Gelsolin and Protects it from Cleavage by Caspase-3

The actin filament severing and capping protein gelsolin plays an important role in modulation of actin filament dynamics by influencing the number of actin filament ends. During apoptosis, gelsolin becomes constitutively active due to cleavage by caspase-3. In non-apoptotic cells gelsolin is activated by the binding of Ca²⁺. This activated form of gelsolin binds to, but is not a folding substrate of the molecular chaperone CCT/TRiC. Here we demonstrate that in vitro, gelsolin is protected from cleavage by caspase-3 in the presence of CCT. Cryoelectron microscopy and single particle 3D reconstruction of the CCT:gelsolin complex reveals that gelsolin is located in the interior of the chaperonin cavity, with a placement distinct from that of the obligate CCT folding substrates actin and tubulin. In cultured mouse melanoma B16F1 cells, gelsolin co-localises with CCT upon stimulation of actin dynamics at peripheral regions during lamellipodia formation. These data indicate that localised sequestration of gelsolin by CCT may provide spatial control of actin filament dynamics.     

miR-223在结肠癌组织中的表达及促进结肠癌细胞迁移侵袭的机制研究

目的:探讨微小RNA-223 (mi R-223)在结肠癌组织中的表达及对结肠癌HT-29细胞侵袭、迁移能力的影响及机制。方法:检测mi R-223在结肠癌组织与癌旁组织中的表达。通过脂质体转染法将mi R-223模拟物(mi R-223 mimics,mi R-223 mimics组)及microRNA无关序列(mi R-223 NC,NC组)转染入结肠癌HT-29细胞。采用Real-time PCR检测转染后细胞中mi R-223和TWIST的表达,Western blot检测TWIST的蛋白表达,Tranwell检测细胞的迁移与侵袭能力。双荧光素酶报告基因检测mi R-223对TWIST基因启动子活性的影响。采用Transwell迁移与侵袭实验检测mi R-223 mimic及Twist si RNA共转染后人结肠癌细胞系HT-29迁移与侵袭能力的变化。结果:与癌旁结肠组织比较,mi R-223在结肠癌组织中呈现明显高表达(P0.05);与空白对照组和mi R-223 NC组比较,转染mi R-223 mimics后的HT-29细胞中的mi R-223表达显著增加(P0.05)。与阴性对照组和空载转染组相比较,mi R-223 mimics转染组穿透的细胞数目明显增加(P0.05),且mi R-223 mimics转染组的细胞侵袭能力显著增强(P0.05)。与mi R-223 NC组和空白对照组比较,转染mi R-223 mimics的HT-29细胞的TWIST基因m RNA和蛋白表达均显著增加(P0.05)。双荧光素酶检验结果显示TWIST为mi R-223的下游靶基因。共转染TWIST si RNA和mi R-223 mimics的结肠癌HT-29细胞的迁移与侵袭能力较单独转染mi R-223 mimics的HT-29细胞显著减弱(P0.05)。结论:mi R-223可能通过上调下游靶基因TWIST水平促进结肠癌HT-29细胞的迁移与侵袭。