The anticancer properties of iron core-gold shell nanoparticles in colorectal cancer cells.

Previously, iron core-gold shell nanoparticles (Fe@Au) have been shown to possess cancer-preferential cytotoxicity in oral and colorectal cancer (CRC) cells. However, CRC cell lines are less sensitive to Fe@Au treatment when compared with oral cancer cell lines. In this research, Fe@Au are found to decrease the cell viability of CRC cell lines, including Caco-2, HT-29, and SW480, through growth inhibition rather than the induction of cell death. The cytotoxicity induced by Fe@Au in CRC cells uses different subcellular pathways to the mitochondria-mediated autophagy found in Fe@Au-treated oral cancer cells, OECM1. Interestingly, the Caco-2 cell line shows a similar response to OECM1 cells and is thus more sensitive to Fe@Au treatment than the other CRC cell lines studied. We have investigated the underlying cell resistance mechanisms of Fe@Au-treated CRC cells. The resistance of CRC cells to Fe@Au does not result from the total amount of Fe@Au internalized. Instead, the different amounts of Fe and Au internalized appear to determine the different response to treatment with Fe-only nanoparticles in Fe@Au-resistant CRC cells compared with the Fe@Au-sensitive OECM1 cells. The only moderately cytotoxic effect of Fe@Au nanoparticles on CRC cells, when compared to the highly sensitive OECM1 cells, appears to arise from the CRC cells' relative insensitivity to Fe, as is demonstrated by our Fe-only treatments. This is a surprising outcome, given that Fe has thus far been considered to be the "active" component of Fe@Au nanoparticles. Instead, we have found that the Au coatings, previously considered only as a passivating coating to protect the Fe cores from oxidation, significantly enhance the cytotoxicity of Fe@Au in certain CRC cells. Therefore, we conclude that both the Fe and Au in these core-shell nanoparticles are essential for the anticancer properties observed in CRC cells.

GM1 expression in caco-2 cells: characterisation of a fundamental passage-dependent transformation of a cell line.

Caco-2 cells, which are known to spontaneously differentiate in cell culture, adopt typical epithelial characteristics and are widely used as a model to study cellular uptake, transport and metabolism processes. However, groups of flat and undifferentiated cells have been observed amid differentiating Caco-2 cell monolayers. In this study, we isolated and characterised these morphologically distinct, flat and island-forming Caco-2 cells. We visualised the undifferentiated cell islands with the aid of optical and electron microscopy and identified mono-sialo-ganglioside one (GM1) as their unique marker. Furthermore, two distinct subpopulations of morphology and GM1 expression were dilution cloned (Caco-2(GM1-) and Caco-2(GM1+) ), leading to the first documented Caco-2 clone that does not show differentiation characteristics. Caco-2(GM1+) cells were flat, non-polarising with extremely low transepithelial electrical resistance (TEER), whereas Caco-2(GM1-) cells showed typical epithelial features and high TEER. Importantly, the proportion of Caco-2(GM1+) cells in a culture increased with passage number and eventually dominated the cell culture. The novel GM1 passage-dependent cell transformation described here shows that careful monitoring is required when performing in vitro cell studies. Therefore, to guarantee consistent and valid experimental data, GM1 expression and the loss of differentiation characteristics should be carefully monitored and the use of fresh cultures should be standard practice.

Caveolae and caveolin-1 in reptilian liver.

Caveolae are plasma-membrane invaginations that, by interacting with membrane-associated molecules such as endothelial nitric oxide synthase and tyrosine kinases, precisely regulate cell-signalling pathways responsible for cell structure and cell function. Indeed, there is widespread evidence that caveolae associate, structurally and functionally, with proteins, lipids and solutes to facilitate transcellular transport of these macromolecules. Caveolin-1, one of the family of membrane proteins that form caveolae, is most prominently expressed in endothelial cells of the vascular bed. Therefore, we have applied advanced electron microscopy as well as molecular biology techniques to study the presence of caveolae and caveolin-1 in the liver sinusoidal endothelium of reptiles. Reptiles are known to store excess lipid in the liver as an energy source for hibernation, and so offer a useful animal model in which to assess the structural and functional implications these subcellular compartments might have on liver sinusoidal endothelial transport. This study demonstrates that caveolae are indeed conserved across vertebrate species, whether mammalian or reptilian. It also presents as first novel data on the presence of caveolin-1-associated, tubular structures located within the cytoplasm of the lizard liver sinusoidal endothelium.

Multifaceted nature of membrane microdomains in colorectal cancer.

Membrane microdomains or lipid rafts are known to be highly dynamic and to act as selective signal transduction mediators that facilitate interactions between the cell's external and internal environments. Lipid rafts play an important mediating role in the biology of cancer: they have been found in almost all existing experimental cancer models, including colorectal cancer (CRC), and play key regulatory roles in cell migration, metastasis, cell survival and tumor progression. This paper explores the current state of knowledge in this field by highlighting some of the pioneering and recent lipid raft studies performed on different CRC cell lines and human tissue samples. From this literature review, it becomes clear that membrane microdomains appear to be implicated in all key intracellular signaling pathways for lipid metabolism, drug resistance, cell adhesion, cell death, cell proliferation and many other processes in CRC. All signal transduction pathways seem to originate directly from those peculiar lipid islands, thereby orchestrating the colon cancer cells' state and fate. As confirmed by recent animal and preclinical studies in different CRC models, continuing to unravel the structure and function of lipid rafts - including their associated complex signaling pathways - will likely bring us one step closer to better monitoring and treating of colon cancer patients.

Unlocking the ultrastructure of colorectal cancer cells in vitro using selective staining.

AIM: To characterise differences between three widely used colorectal cancer cell lines using ultrastructural selective staining for glycogen to determine variation in metastatic properties. METHODS: Transmission electron microscopy was used in this investigation to help identify intracellular structures and morphological features which are precursors of tumor invasion. In addition to morphological markers, we used selective staining of glycogen as a marker for neoplastic cellular proliferation and determined whether levels of glycogen change between the three different cell lines. RESULTS: Ultrastructural analysis revealed morphological differences between the cell lines, as well as differentiation into two sub-populations within each cell line. Caco-2 cells contained large glycogen deposits as well as showing the most obvious morphological changes between the two sub-populations. SW480 cells also contained large glycogen stores as well as deep cellular protrusions when grown on porous filter membranes. HT-29 cells had trace amounts of glycogen stores with few cellular projections into the filter pores and no tight junction formation. CONCLUSION: Morphology indicative of metastatic properties coincided with larger glycogen deposits, providing strong evidence for the use of selective staining to determine the neoplastic properties of cells.

Multi-dimensional correlative imaging of subcellular events: combining the strengths of light and electron microscopy.

To genuinely understand how complex biological structures function, we must integrate knowledge of their dynamic behavior and of their molecular machinery. The combined use of light or laser microscopy and electron microscopy has become increasingly important to our understanding of the structure and function of cells and tissues at the molecular level. Such a combination of two or more different microscopy techniques, preferably with different spatial- and temporal-resolution limits, is often referred to as 'correlative microscopy'. Correlative imaging allows researchers to gain additional novel structure-function information, and such information provides a greater degree of confidence about the structures of interest because observations from one method can be compared to those from the other method(s). This is the strength of correlative (or 'combined') microscopy, especially when it is combined with combinatorial or non-combinatorial labeling approaches. In this topical review, we provide a brief historical perspective of correlative microscopy and an in-depth overview of correlative sample-preparation and imaging methods presently available, including future perspectives on the trend towards integrative microscopy and microanalysis.

Three-dimensional organization of fenestrae labyrinths in liver sinusoidal endothelial cells.

BACKGROUND/AIMS: Liver sinusoidal endothelial cell (LSEC) fenestrae are membrane-bound pores that are grouped in sieve plates and act as a bidirectional guardian in regulating transendothelial liver transport. The high permeability of the endothelial lining is explained by the presence of fenestrae and by various membrane-bound transport vesicles. The question as to whether fenestrae relate to other transport compartments remains unclear and has been debated since their discovery almost 40 years ago. METHODS: In this study, novel insights concerning the three-dimensional (3D) organization of the fenestrated cytoplasm were built on transmission electron tomographical observations on isolated and cultured whole-mount LSECs. Classical transmission electron microscopy and atomic force microscopy imaging was performed to accumulate cross-correlative structural evidence. RESULTS AND CONCLUSIONS: The data presented here indicate that different arrangements of fenestrae have to be considered: i.e. open fenestrae that lack any structural obstruction mainly located in the thin peripheral cytoplasm and complexes of multifolded fenestrae organized as labyrinth-like structures that are found in the proximity of the perinuclear area. Fenestrae in labyrinths constitute about one-third of the total LSEC porosity. The 3D reconstructions also revealed that coated pits and small membrane-bound vesicles are exclusively interspersed in the non-fenestrated cytoplasmic arms.

Monitoring membrane rafts in colorectal cancer cells by means of correlative fluorescence electron microscopy (CFEM).

Detergent-resistant membrane (DRM) rafts have been shown to play a pivotal role in regulating key cell biological processes, such as signal transduction, cellular transport and cell survival. The fine structure of membrane rafts are studied using various different imaging approaches and the outcomes are largely dependent on the detection methodology applied. All these microscopy techniques which employ light-, laser- and photon-optics, electrons as well as atomic force probing are characterized on their turn by their strengths and limitations for membrane raft identification. This explains in part the diversity of definitions available to describe these peculiar membrane structures. We present herewith an alternative and uncomplicated microscopy tool to study fluorescently labelled DRMs with information at the transmission electron microscopical level of the same cell, enabling us to obtain a snapshot of the morpho-functional relationships between the cell's interior and DRMs. The proposed approach of correlative fluorescence electron microscopy (CFEM) can therefore be considered as an additional alternative imaging approach to unravel DRM structure-function relationships from micro- to nanometre length scales, from the cell to the molecule.