Curcumin inhibits cell motility and alters microfilament organization and function in prostate cancer cells

Curcumin is a dietary phytochemical associated with anti-tumorigenic effects, but the mechanisms by which it inhibits cancer cell growth and metastasis are not completely understood. For example, little information is available regarding the effects of curcumin on cytoskeletal organization and function. In this study, time-lapse video and immunofluorescence labeling methods were used to demonstrate that curcumin significantly alters microfilament organization and cell motility in PC-3 and LNCaP human prostate cancer cells in vitro. Curcumin rapidly arrests cell movements and subsequently alters cell shape in the highly motile PC-3 cell line, but has a less noticeable effect on the relatively immobile LNCaP cell line. Stress fibers are augmented, and the overall quantity of f-actin appears to increase in both types of cells following curcumin treatment. Cytochalasin B (CB) disrupts microfilament organization in both cell lines, and causes vigorous membrane blebbing in PC-3 cells, but not LNCaP cells. Pre-treatment of cells with curcumin suppresses changes in microfilament organization caused by CB, and blocks PC-3 membrane blebbing. At least some of the effects of curcumin appear to be mediated by protein kinase C (PKC), as treatment with the PKC inhibitor bisindolylmaleimide inhibits the ability of curcumin to block CB-induced membrane blebbing. These findings demonstrate that curcumin exerts significant effects on the actin cytoskeleton in prostate cancer cells, including altering microfilament organization and function. This is a novel observation that may represent an important mechanism by which curcumin functions as a chemopreventative agent, and as an inhibitor of angiogenesis and metastasis.     

Altered cellular morphology and microfilament array in ataxia-telangiectasia fibroblasts

Cells derived from individuals with the ataxia-telangiectasia syndrome demonstrate a number of unusual properties. They are highly sensitive to the lethal effects of ionizing radiation and also fail to demonstrate the normal inhibition of DNA synthesis associated with this type of DNA-damaging agent. Additionally, a number of ataxia-telangiectasia lymphoblastoid lines have been shown to have an unusual regulation of the cellular actin levels. However, the primary lesion causing ataxia-telangiectasia is unknown. In this paper we report an altered cellular morphology in three ataxia-telangiectasia fibroblast lines, but not in a number of control fibroblast lines. Investigation of the cytoskeleton using antibodies against certain cytoskeletal proteins revealed a difference in the microfilament pattern from ataxia-telangiectasia fibroblasts compared to controls. Ataxia fibroblasts showed a microfilament stress fiber pattern that appeared to have a more well defined and abundant array of stress fibers than control fibroblasts. In contrast, no differences were observed in the microtubule array, nor in the vinculin patterns between any of the cell lines. In addition to the differences in the microfilament patterns, ataxia-telangiectasia fibroblasts differed in their ability to recover from microfilament disruption by dimethyl sulfoxide. Control fibroblasts returned to a normal cellular state in a shorter time compared to ataxia fibroblasts, as judged by indirect immunofluorescence using antiactin. These results provide further evidence for a cytoskeletal anomaly in ataxia-telangiectasia.     

Differential effects of cellular fibronectin and plasma fibronectin on ovarian cancer cell adhesion, migration, and invasion

Summary The main form of fibronectin (FN) encountered by tumor cells in vivo is cellular FN (cFN), which differs structurally and functionally from the commonly used plasma FN (pFN). We compared the effects of cFN and pFN on the ovarian carcinoma lines OVCAR-3 and SKOV-3 and on cultures of normal ovarian surface epithelium, which is the precursor of the epithelial ovarian carcinomas. Ovarian surface epithelial cells and SKOV-3 cells attached and spread faster on cFN than on pFN. On cFN, SKOV-3 migration was enhanced compared with pFN or plastic. In a matrigel transfilter assay, cFN strongly inhibited SKOV-3 invasion, whereas pFN did not. In contrast to SKOV-3, OVCAR-3 cells adhered faster on FN than on plastic but did not discriminate between cFN and pFN, and they did not migrate or invade matrigel either with or without FN. In both carcinoma lines, proliferation was unaffected by either FN. The results show profound differences in the responses to cFN and pFN by two invasive ovarian carcinoma lines. Because cFN is the main type that cancer cells encounter in vivo, extrapolations from culture data to in vivo events should preferably be based on studies using this form of FN.     

A simple cell motility assay demonstrates differential motility of human periodontal ligament fibroblasts, gingival fibroblasts, and pre-osteoblasts

During periodontal regeneration, multiple cell types can invade the wound site, thereby leading to repair. Cell motility requires interactions mediated by integrin receptors for the extracellular matrix (ECM), which might be useful in guiding specific cell populations into the periodontal defect. Our data demonstrate that fibroblasts exhibit differential motility when grown on ECM proteins. Specifically, gingival fibroblasts are twice as motile as periodontal ligament fibroblasts, whereas osteoblasts are essentially non-motile. Collagens promote the greatest motility of gingival fibroblasts in the following order: collagen III>collagen V>collagen I. Differences in motility do not correlate with cell proliferation or integrin expression. Osteoblasts display greater attachment to collagens than does either fibroblast population, but lower motility. Gingival fibroblast motility on collagen I is generally mediated by α2 integrins, whereas motility on collagen III involves α1 integrins. Other integrins (α10 or α11) may also contribute to gingival fibroblast motility. Thus, ECM proteins do indeed differentially promote the cell motility of periodontal cells. Because of their greater motility, gingival fibroblasts have more of a potential to invade periodontal wound sites and to contribute to regeneration. This finding may explain the formation of disorganized connective tissue masses rather than the occurrence of the true regeneration of the periodontium. This research was supported by the Louisiana Board of Regents through the Millennium Trust Health Excellence Fund, HEF-(2000-05)-04.     

Cyclic amp and cell morphology in cultured fibroblasts. Effects on cell shape, microfilament and microtubule distribution, and orientation to substratum

The change in shape of 3T3 and L929 cells due to Bt2cAMP treatment is accompanied by altered intracellular distribution of microfilaments and microtubules. Bt2cAMP added to cells in low density culture causes (a) microfilaments to accumulate in bundles near the plasma membrane, mainly at the cell periphery, and (b) microtubules to accumulate beneath these microfilament bundles. In narrow cell processes that form characteristically in Bt2cAMP-treated L cells, microtubules accumulate in parallel arrays near the center of these processes. A new simple method for evaluating the relative distance of the cell from its underlying substratum is desribed. In normal medium, 3T3 cells attach to their substratum near the nucleus and at the tips of cell processes, bridging irregularities in the plastic surface. With Bt2cAMP treatment, attachment occurs at the cell edge and at many isolated points under the cytoplasm, and the cells conform more closely to irregularities of the underlying substratum. A model of the mechanism by which cAMP modulates cell shape is presented.     

Interferon effects on the growth and division of human fibroblasts.

The overall rate of proliferation of human fibroblasts in culture is reduced at interferon concentrations greater than 40 international reference units (U)/ml. Inhibition is near maximal at 640 U/ml, at which concentration the doubling time between 24 and 72 h after beginning of treatment is increased 2–3 times over the control value. Inhibition of cell proliferation was not readily reversible upon removal of interferon and refeeding of cultures. Study of the mitotic behavior of individual cells showed that the first intermitotic interval after beginning of treatment with interferon (640 U/ml) was prolonged in about two-thirds of the cells. In this fraction, many cells failed to divide again after the second post-treatment mitosis, while others exhibited a progressively increasing intermitotic interval with subsequent divisions. One-third of the interferon-treated fibroblasts initially divided at a rate similar to the rate of proliferation of control cells, but subsequently these cells also slowed down and finally stopped dividing. After treatment at 640 U/ml for 3 days, the rates of DNA, RNA, and protein synthesis were depressed to 86, 75, and 64% of control values, respectively. However, the interferon-treated fibroblasts had grown larger than control cells as indicated by the following parameters: cell attachment area, 165%; volume, 131%; DNA content, 130% and protein content, 150%. Thus, interferon does not prevent cell growth, but interferes with cell division.     

Cytochalasin B-induced alterations of insulin binding and microfilament organization in cultured fibroblasts

The effect of cytochalasin B (CB) on insulin binding has been investigated in confluent cultures of chick embryo fibroblasts. Time- and dose-dependent increases in binding of [¹²⁵I]insulin was observed after incubation of fibroblasts with CB. At 10 μg/ml, CB caused a 2-fold increase in binding, due to an increase in the number of binding sites from 9.3 × 10³ to 2.0 × 10⁴ per cell. Removal of CB from the growth medium was accompanied by a decrease in [¹²⁵I]insulin binding to control values in 24 h. Increase in the binding of insulin in CB-treated CEF was also accompanied by enhancement of insulin to stimulation of [³H]thymidine incorporation into acid-insoluble material. CB treatment also caused disorganization and disappearance of microfilament bundles and changes in cell shape from flat, with a few blebs and folds on the cell surface, to round with numerous blebs and folds. The data from this study suggest that changes in the number of surface insulin-binding sites may be related to the state of organization of cytoskeletal structures in chick embryo fibroblasts.     

Opposite effects of overexpressed myosin Va or heavy meromyosin Va on vesicle distribution, cytoskeleton organization, and cell motility in nonmuscle cells

Myosin Va, an actin-based motor protein that transports intracellular cargos, can bundle actin in vitro. Whether myosin Va regulates cellular actin dynamics or cell migration remains unclear. To address this, we compared Chinese Hamster Ovary (CHO) cells that stably express GFP fused to either full length mouse myosin Va (GFP-M5) or heavy meromyosin Va (GFP-M5Delta). GFP-M5 and GFP-M5Delta co-immunoprecipitate with CHO myosin Va and serve as overexpression of wild-type and dominant negative mutants of myosin Va. Compared to non-expressing control cells, GFP-M5-overexpressing cells have peripheral endocytic vesicles, spread slowly after plating, as well as produce robust interior actin stress fibers, myosin II bundles, and focal adhesions. However, these cells display normal cell migration and lamellipodial dynamics. In contrast, GFP-M5Delta-expressing cells have perinuclear endocytic vesicles, produce thin interior actin and myosin bundles and contain no interior focal adhesions. In addition, these cells spread rapidly, migrate slowly and display reduced lamellipodial dynamics. Similarly, neurite outgrowth is compromised in neurons cultured from transgenic Drosophila that express M5Delta-dsRed and in neurons cultured from Drosophila that produce a tailless version of endogenous myosin V. Together, these data suggest that myosin Va overexpression induces actin bundles in vivo whereas the tailless version fails to bundle actin and disrupts cell motility.     

Fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells in vitro

Human umbilical vein endothelial cells (ECs) have been shown to attach to a substratum of fibrinogen (fg). Later, ECs undergo spreading, organization of thick microfilament bundles of the stress fiber type, and formation of focal contacts (adhesion plaques) that correspond to accumulation of vinculin at the cytoplasmic aspect of the ventral membrane. The rate of attachment to fg and the type of spreading is virtually identical to that obtained on substrata coated with fibronectin (FN). Antibodies to fg, but not to FN, prevent EC adhesion to fg; conversely, antibodies to FN, but not to fg, prevent adhesion of ECs to a FN-coated substratum. The removal of residual FN contamination from fg preparations by means of DEAE-cellulose chromatography does not result in any difference in EC adhesion on fg. Moreover, pretreatment of cells with inhibitors of synthesis and release of proteins does not impair their adhesion capacity on an fg-coated substratum. In contrast, human arterial smooth muscle cells do not adhere and spread on fg substrata but do so on FN. The synthetic peptides (Gly-Arg-Gly-Asp[GRGD] and Gly-Arg-Gly-Asp-Ser-Pro[GRGDSP]) containing the tripeptide Arg-Gly-Asp (RGD), originally found to be responsible for the cell binding activity of FN, have been found to inhibit EC spreading and the redistribution of their cytoskeleton, including the formation of stress fibers and the localization of vinculin either on fg or on FN. Conversely, the synthetic peptide Arg-Gly-Gly (RGG) was completely uneffective in inhibiting the adhesion and the sequence of events leading to spreading and cytoskeletal organization. These results indicate that ECs, but not smooth muscle cells, specifically adhere and spread on an fg substratum and this occurs by recognition mechanisms similar to those reported for FN.     

Binding of plasma fibronectin to cell layers of human skin fibroblasts

Human plasma fibronectin bound to confluent cell layers of cultured human-skin fibroblasts in two distinct pools. Initial binding of fibronectin occurred in a deoxycholate-soluble pool (Pool I). Binding in Pool I was reversible and reached a steady state after 3 h. After longer periods of incubation, fibronectin became bound in a deoxycholate-insoluble pool (Pool II). Binding in Pool II was irreversible and proceeded at a linear rate for 30 h. After 30 h of incubation, a significant proportion of fibronectin bound in Pool II was present as disulfide-bonded multimers. HT1080 cells, a human sarcoma cell line, did not bind fibronectin in either pool. Also, isolated cell matrices prepared by deoxycholate extraction did not bind fibronection. Binding of fibronectin in Pool I of normal fibroblasts occurred via specific, saturable receptors. There were 128,000 binding sites per cell, and KDiss was 3.6 X 10(-8) M. Fluorescence microscopic localization of fibronectin bound in Pool I and Pool II was performed using fluorescein-conjugated fibronectin. Fluorescent staining in Pool I was present in a punctate pattern and in short, fine fibrils. Pool II fluorescence was exclusively in coarse, dense fibrils. These data indicate that plasma fibronectin may become incorporated into the tissue extracellular matrix via specific cell-surface receptors.     

Interferon enhances fibronectin expression in various cell types.

Fibronectin (FN), a normal plasma and extracellular matrix glycoprotein, plays a significant role in various phases of wound healing. At wound site FN is synthesized locally by various cell types involved in the healing process (viz. epithelial, endothelial, fibroblast and macrophage cells) or deposited from the plasma. The present study was undertaken to investigate the in vitro effect of IFN on FN synthesis as well as release in the culture medium by various cell types. Indirect immunofluorescence and immunoelectron microscopy studies, using specific antibodies, revealed that IFN treatment resulted in significantly more staining for FN as compared to untreated control cells. Metabolic labeling with 35S-methionine, immunoprecipitation and SDS-page studies showed an increase in FN synthesis and release by IFN treated cells. In addition, to determine whether this increased synthesis was reflected at mRNA levels, poly (A)+ RNA was isolated from human lung epithelial cells (A549) and probed with FN specific cDNA. We found that IFN treatment increased the level of FN mRNA.     

Immunocytochemical localization of procollagen and fibronectin in human fibroblasts: effects of the monovalent ionophore, monensin

The monovalent ionophore monensin inhibits the secretion of both procollagen and fibronectin from human fibroblasts in culture. The distribution of these proteins in control and inhibited (5 x 10(-7) M monensin) cells has been studied by immunofluorescence microscopy. In control cells, both antigens are present throughout the cytoplasm and in specific deposits in a region adjacent to the nucleus, which we identify as a Golgi zone by electron microscopy. Treatment of cells with monensin causes intracellular accumulation of procollagen and fibronectin, initially in the juxta-nuclear region and also subsequently in peripheral regions. Electron microscope studies reveal that in such cells the juxta-nuclear Golgi zone becomes filled with a new population of smooth-membraned vacuoles and that normal Golgi complexes are not found. Immunocytochemically detected procollagen and fibronectin are localized in the region of these vacuoles, whereas more peripheral deposits correspond to the dilated cisternae of rough endoplasmic reticulum, which are also caused by monensin. Procollagen and fibronectin are often codistributed in these peripheral deposits. Accumulation of exportable proteins in Golgi-related vacuoles is consistent with previous analyses of the monensin effect. The subsequent development of dilated rough endoplasmic reticulum also containing accumulated proteins may indicate that there is an additional blockade at the exit from the endoplasmic reticulum, or that the synthesized proteins exceed the capacity of the Golgi compartment and that their accumulation extends into the endoplasmic reticulum.     

Functional changes in cellular fibronectin from late passage fibroblasts in vitro

Analysis of fibronectin synthesized by human fibroblasts, at different times during serial subcultivation, reveals functional differences. Fibronectin isolated from late passage cells is defective in promoting cell adhesion, cell spreading, and the formation of focal contacts. These changes are not the result of an inability of late passage cells to interact with fibronectin, since late passage cells become adhesive and form focal contacts in the presence of fibronectin isolated from early passage cells. Therefore, we conclude that late passage cellular fibronectin derived from late passage cells cannot support the cell substrate interactions.     

Effects of bunaftine on morphology,microfilament integrity,and mitotic activity in cultured human fibroblasts and HeLa cells

Summary Human fibroblasts and HeLa cells were treated with bunaftine (N-butyl-N-/2-(diethylamino)ethyl/-1-naphthalenecarboxamide) in vitro. At concentrations of 0.5–2.0 mM, the drug caused contraction and rounding of the cells with loss of microvilli-like processes. Aggregates of dense, partly granular, partly fibrillar material formed in the cytoplasm and the rough endoplasmic reticulum became vesiculated. Immunofiuorescence microscopy with DNase I and anti-DNase I demonstrated that bundles of actin filaments were disrupted, forming rings, coils, and granules. Filaments stained with antibodies to vimentin (fibroblasts) and prekeratin (HeLa cells) showed less characteristic rearrangements, probably related to the rounding up of the cells. 0.4 mM bunaftine increased and 0.8–1.0 mM markedly decreased the percentage of mitotic cells, without accumulation of cells in any particular stage of mitosis. The drug may arrest the cell cycle at some point before mitosis; it may have a critical concentration above which the arrest becomes permanent.These results suggest that bunaftine interferes with the integrity of microfilament bundles in a different manner from that of cytochalasins. It does not cause any depletion of cellular ATP, indicating that its effect is not a result of inhibition of cell metabolism. It is proposed that bunaftine may be used as a complement to cytochalasins in studies of the microfilament system of the cell. The possible binding of bunaftine to actin or myosin and further details of its mechanism of action remain to be elucidated.     

Effects of microfilament disrupters on microfilament distribution and morphology in maize root cells

Maize root tip cells were examined for the distribution of actin microfilaments in various cell types and to determine the effects of microfilament disrupters. Fluorescence microscopy on fixed, stabilized, squashed cells using the F-actin specific probe, rhodamine-labelled phalloidin, allowed for a three-dimensional visualization of actin microfilaments. Microfilaments were observed as long, meandering structures in root cap cells and meristematic cells, while those in immature vascular parenchyma were abundant in the thin band of cytoplasm and were long and less curved. By modifying standard electron microscopic fixation procedures, microfilaments in plant cells could be easily detected in all cell types. Treatment with cytochalasin B, cytochalasin D and lead acetate, compounds that interfere with microfilament related processes, re-organized the microfilaments into abnormal crossed and highly condensed masses. All the treatments affected not only the microfilaments but also the accumulation of secretory vesicles. The vivid demonstration of the effects of all of these microfilament disrupters on the number and size of Golgi vesicles indicates that these vesicles may depend on microfilaments for intracellular movement.     

Effects of microfilament disrupters on microfilament distribution and morphology in maize root cells

Summary Maize root tip cells were examined for the distribution of actin microfilaments in various cell types and to determine the effects of microfilament disrupters. Fluorescence microscopy on fixed, stabilized, squashed cells using the F-actin specific probe, rhodamine-labelled phalloidin, allowed for a three-dimensional visualization of actin microfilaments. Microfilaments were observed as long, meandering structures in root cap cells and meristematic cells, while those in immature vascular parenchyma were abundant in the thin band of cytoplasm and were long and less curved. By modifying standard electron microscopic fixation procedures, microfilaments in plant cells could be easily detected in all cell types. Treatment with cytochalasin B, cytochalasin D and lead acetate, compounds that interfere with microfilament related processes, re-organized the microfilaments into abnormal crossed and highly condensed masses. All the treatments affected not only the microfilaments but also the accumulation of secretory vesicles. The vivid demonstration of the effects of all of these microfilament disrupters on the number and size of Golgi vesicles indicates that these vesicles may depend on microfilaments for intracellular movement.     

The distribution of fibronectin in attachment sites of chick fibroblasts

Primary chick heart fibroblasts were cultured on glass coverslips and examined by reflection contrast microscopy and then by indirect immunofluorescence microscopy using affinity purified antifibronectin antibody. Fibronectin was found to be primarily localized in areas of ‘close’ contact and in intensely staining fibrous webs that were usually external to the cytoplasmic matrix. Focal contacts, in which there is intimate contact between the cell and the substratum by localized attachment, failed to react with the anti-fibronectin antibody despite a variety of extraction procedures designed to increase the accessibility of antibody to this type of attachment site. It is concluded that in chick fibroblasts, fibronectin participates in cell attachment at areas of close contact and fibrillar attachment sites, but is often excluded from the attachment pads of focal contacts.     

Generation of cortactin floxed mice and cellular analysis of motility in fibroblasts

Cortactin is an F‐actin binding protein that has been suggested to play key roles in various cellular functions. Here, we generated mice carrying floxed alleles of the cortactin (Cttn) gene (Cttn^flox/flox mice). Expression of Cre recombinase in mouse embryonic fibroblasts (MEFs) isolated from Cttn^flox/flox embryos depleted cortactin within days, without disturbing F‐actin distribution and localization of multiple actin‐binding proteins. Cre‐mediated deletion of Cttn also did not affect cell migration. To obtain mice with a Cttn null allele, we next crossed Cttn^flox/flox mice with transgenic mice that express Cre recombinase ubiquitously. Western blot and immunocytochemical analysis confirmed complete elimination of cortactin expression in MEFs carrying homozygously Cttn null alleles. However, we found no marked alteration of F‐actin organization and cell migration in Cttn null‐MEFs. Thus, our results indicate that depletion of cortactin in MEFs does not profoundly influence actin‐dependent cell motility. genesis 47:638–646, 2009. © 2009 Wiley‐Liss, Inc.     

Quantitative model of cell cycle arrest and cellular senescence in primary human fibroblasts

Primary human fibroblasts in tissue culture undergo a limited number of cell divisions before entering a non-replicative "senescent" state. At early population doublings (PD), fibroblasts are proliferation-competent displaying exponential growth. During further cell passaging, an increasing number of cells become cell cycle arrested and finally senescent. This transition from proliferating to senescent cells is driven by a number of endogenous and exogenous stress factors. Here, we have developed a new quantitative model for the stepwise transition from proliferating human fibroblasts (P) via reversibly cell cycle arrested (C) to irreversibly arrested senescent cells (S). In this model, the transition from P to C and to S is driven by a stress function γ and a cellular stress response function F which describes the time-delayed cellular response to experimentally induced irradiation stress. The application of this model based on senescence marker quantification at the single-cell level allowed to discriminate between the cellular states P, C, and S and delivers the transition rates between the P, C and S states for different human fibroblast cell types. Model-derived quantification unexpectedly revealed significant differences in the stress response of different fibroblast cell lines. Evaluating marker specificity, we found that SA-β-Gal is a good quantitative marker for cellular senescence in WI-38 and BJ cells, however much less so in MRC-5 cells. Furthermore we found that WI-38 cells are more sensitive to stress than BJ and MRC-5 cells. Thus, the explicit separation of stress induction from the cellular stress response, and the differentiation between three cellular states P, C and S allows for the first time to quantitatively assess the response of primary human fibroblasts towards endogenous and exogenous stress during cellular ageing.     

Interferon induces tryptophanyl-tRNA synthetase expression in human fibroblasts.

A cDNA clone complementary to an interferon (IFN)-induced mRNA was isolated and used to characterize the regulation of expression of its RNA by the IFNs and to identify the protein its RNA encodes. This cDNA hybridizes to IFN-induced 3.1- and 2.3-kilobase mRNAs that are synthesized in response to both IFN-alpha and IFN-gamma. IFN-gamma induces the sustained accumulation of these mRNAs while IFN-alpha induces their transient accumulation. Cycloheximide (50 micrograms/ml) failed to inhibit the induction of these mRNAs by either IFN-alpha or IFN-gamma, suggesting that their induction does not require de novo protein synthesis. DNA sequence analysis of this cDNA reveals that it encodes a protein of Mr 53,168 that has sequence homology with and the biological activity of a tryptophanyl-tRNA synthetase, an enzymatic activity that has been demonstrated to play a role in and be modulated by the growth of cells. Elevated levels of this enzyme may be involved in the cell growth inhibitory activity of the IFNs.