Anchorage-independent growth of breast carcinoma cells is mediated by serum exosomes

Ochieng, Josiah; Pratap, Siddharth; Khatua, Atanu K.; Sakwe, Amos M.

doi:10.1016/j.yexcr.2009.03.010

Cited by 81 publications

(92 citation statements)

References 47 publications

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“…About 10 mg of Pedersen fetuin-A, fractionated fetal bovine serum as described recently (26), or human serum (Sigma) in a total volume of 1.0 ml were layered on the step gradient. This was then centrifuged at 35,000 rpm (Beckman, SW40Ti rotor) for 18 h at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…Cell lysates or various fractions of glycerol gradients were separated in 4 -12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were subsequently probed with the indicated antibodies and revealed by enhanced chemiluminescence as described previously (26).…”

Section: Methodsmentioning

confidence: 99%

“…Uptake of Labeled Purified Fetuin-A by BT-549 Breast Carcinoma Cells-Purified fetuin-A (fraction S1) was labeled with rhodamine isothiocyanate (Sigma) as described previously (26). For real time imaging of rhodamine-labeled purified fetuin, BT-Gal3 cells were transfected with GFP-annexin A2.…”

Section: Methodsmentioning

confidence: 99%

“…We recently showed that fetuin-A is equally distributed between the exosome-free and exosome-enriched fractions of bovine serum (26). We therefore sought to examine whether fetuin-A in the fractionated serum could be separated from its contaminants by floating these fractions on glycerol step gradients.…”

Section: Ultrafiltration and Wheat Germ Agglutinin Purification Ofmentioning

confidence: 99%

“…We therefore sought to examine whether fetuin-A in the fractionated serum could be separated from its contaminants by floating these fractions on glycerol step gradients. To do this, bovine exosome-free serum and exosome-enriched serum were prepared as described recently (26). These were layered on glycerol step gradients, separated by centrifugation, and then fractionated from the top.…”

Section: Ultrafiltration and Wheat Germ Agglutinin Purification Ofmentioning

confidence: 99%

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Fetuin-A (α2HS-Glycoprotein) Is a Major Serum Adhesive Protein That Mediates Growth Signaling in Breast Tumor Cells

Sakwe

Koumangoye

Goodwin

et al. 2010

Journal of Biological Chemistry

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The identity of the cell adhesive factors in fetal bovine serum, commonly used to supplement growth media, remains a mystery due to the plethora of serum proteins. In the present analyses, we showed that fetuin-A, whose function in cellular attachment in tissue culture has been debated for many years, is indeed a major serum cell attachment factor particularly for tumor cells. We are able to report this because of a new purification strategy that has for the first time given us a homogeneous protein band in colloidal Coomassie-stained gels that retains biological activity. The tumor cells adhered to immobilized fetuin-A and not ␣ 2 -macroglobulin, its major contaminant. The interaction of cells with fetuin-A was driven mainly by Ca 2؉ ions, and cells growing in regular medium supplemented with fetal bovine serum were just as sensitive to loss of extracellular Ca 2؉ ions as cells growing in fetuin-A. Fractionation of human serum revealed that cell attachment was confined to the fractions that had fetuin-A. Interestingly, the tumor cells also took up fetuin-A and secreted it back to the medium using an unknown mechanism that can be observed in live cells. The attachment of tumor cells to fetuin-A was accompanied by phosphatidylinositol 3-kinase/Akt activation that was down-regulated in cells that lack annexin-A6, one of the cell surface receptors for fetuin-A. Taken together, our data show the significance of fetuin-A in tumor cell growth mechanisms in vitro and open new research vistas for this protein.Serum, particularly fetal bovine serum, is widely used as a supplement in culture media that is required for growth of most cells in culture (for a review, see Ref. 1). In the absence of serum, most cells fail to adhere properly, spread, and grow on culture dishes. Serum has a plethora of adhesion (2-4) and growth factors (5). The prevailing assumption over the past two decades has been that integrins are the major cellular receptors for adhesion in cell cultures (6). Cellular adhesion to extracellular adhesion molecules such as vitronectin, fibronectin, and laminin using integrins requires the divalent ions Mg 2ϩ /Mn 2ϩ (7). In addition, cell signaling cues for spreading and growth mechanisms in anchorage-dependent cells emanate from the interaction of cells with the adhesion molecules (8). Although it is easy to define such signals in situations where cells are allowed to adhere to known purified extracellular matrix proteins such as fibronectin and collagen (9), adhesion in the presence of fetal bovine serum is complex in that any one of the myriad attachment proteins and or growth factors has the potential to mediate adhesion and growth signals.Ever since it was first purified and described, fetuin-A/ ahsg 2 was suspected of being the principal cell adhesion molecule in serum (1, 10). Fetuin-A isolated and purified from serum by the Pedersen method (hereafter referred to as Pedersen fetuin-A) demonstrated cell adhesive properties in the presence of divalent ions (11). The Pedersen fetuin-A, however, is not cons...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Ultrafiltration and Wheat Germ Agglutinin Purification Ofmentioning

confidence: 99%

Section: Ultrafiltration and Wheat Germ Agglutinin Purification Ofmentioning

confidence: 99%

See 3 more Smart Citations

Fetuin-A (α2HS-Glycoprotein) Is a Major Serum Adhesive Protein That Mediates Growth Signaling in Breast Tumor Cells

Sakwe

Koumangoye

Goodwin

et al. 2010

Journal of Biological Chemistry

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Harnessing the Natural Healing Power of Colostrum: Bovine Milk‐Derived Extracellular Vesicles from Colostrum Facilitating the Transition from Inflammation to Tissue Regeneration for Accelerating Cutaneous Wound Healing

Kim

Han

et al. 2021

Adv Healthcare Materials

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As wound healing is an extremely complicated process, consisting of a cascade of interlocking biological events, successful wound healing requires a multifaceted approach to support appropriate and rapid transitions from the inflammatory to proliferative and remodeling phases. In this regard, here the potential use of bovine milk extracellular vesicles (EVs) to enhance wound healing is investigated. The results show that milk EVs promote fibroblast proliferation, migration, and endothelial tube formation. In particular, milk EVs derived from colostrum (Colos EVs) contain various anti‐inflammatory factors facilitating the transition from inflammation to proliferation phase, as well as factors for tissue remodeling and angiogenesis. In an excisional wound mouse model, Colos EVs promote re‐epithelialization, activate angiogenesis, and enhance extracellular matrix maturation. Interestingly, Colos EVs are further found to be quite resistant to freeze‐drying procedures, maintaining their original characteristics and efficacy for wound repair after lyophilization. These findings on the superior stability and excellent activity of milk Colos EVs indicate that they hold great promise to be developed as anti‐inflammatory therapeutics, especially for the treatment of cutaneous wounds.

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Fetuin‐A (alpha 2HS glycoprotein) modulates growth, motility, invasion, and senescence in high‐grade astrocytomas

et al. 2016

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Glioblastomas (high‐grade astrocytomas) are highly aggressive brain tumors with poor prognosis and limited treatment options. In the present studies, we have defined the role of fetuin‐A, a liver‐derived multifunctional serum protein, in the growth of an established glioblastoma cell line, LN229. We hereby demonstrate that these cells synthesize ectopic fetuin‐A which supports their growth in culture in the absence of serum. We have demonstrated that a panel of tissue microarray (TMA) of glioblastomas also express ectopic fetuin‐A. Knocking down fetuin‐A using shRNA approach in LN229, significantly reduced their in vitro growth as well as growth and invasion in vivo. The fetuin‐A knockdown subclones of LN229 (A and D) also had reduced motility and invasive capacity. Treatment of LN229 cells with asialofetuin (ASF), attenuated their uptake of labeled fetuin‐A, and induced senescence in them. Interestingly, the D subclone that had ~90% reduction in ectopic fetuin‐A, underwent senescence in serum‐free medium which was blunted in the presence of purified fetuin‐A. Uptake of labeled exosomes was attenuated in fetuin‐A knockdown subclones A and D. Taken together, the studies demonstrate the impact of fetuin‐A as significant node of growth, motility, and invasion signaling in glioblastomas that can be targeted for therapy.

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Anchorage-independent growth of breast carcinoma cells is mediated by serum exosomes

Cited by 81 publications

References 47 publications

Fetuin-A (α2HS-Glycoprotein) Is a Major Serum Adhesive Protein That Mediates Growth Signaling in Breast Tumor Cells

Fetuin-A (α2HS-Glycoprotein) Is a Major Serum Adhesive Protein That Mediates Growth Signaling in Breast Tumor Cells

Harnessing the Natural Healing Power of Colostrum: Bovine Milk‐Derived Extracellular Vesicles from Colostrum Facilitating the Transition from Inflammation to Tissue Regeneration for Accelerating Cutaneous Wound Healing

Fetuin‐A (alpha 2HS glycoprotein) modulates growth, motility, invasion, and senescence in high‐grade astrocytomas

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