H Acker — Page 8 | PubMed Health

PubMedDate not supplied

Effects of epidermal growth factor receptor blocking in cultured glioma spheroids.

A concentration as high as 1 microgram/ml of non-radioactive epidermal growth factor, EGF/was necessary to inhibit effectively the binding of 125I-EGF in glioma U-343MGaC12:6 cells. This concentration blocked the available EGF receptors within 30 minutes in monolayers, while 24 hour treatments were required in spheroids. The effects on growth, incorporation of radioactive thymidine, cell density and on extracellular pH were analysed in spheroids after exposure to 1 microgram/ml EGF. The high EGF concentration did not significantly modify the growth curves for monolayers and small spheroids but increased the volume growth of large spheroids. The increase was partly due to lower cell density and partly to increased proliferation. The EGF treatment gave an increased incorporation of thymidine in spheroids, for at least up to 5 days after the administration, while no effect was seen in monolayers. The cell density decreased after the EGF treatment as seen from morphometric analysis in histological sections and by counting the number of cells per volume unit after trypsinization. The capacity to take up radiolabelled dextran increased, probably due to the decreased cell density. Other EGF-induced changes were also recognized, such as a reduction in extracellular pH by 0.1 units in the central regions of spheroids and an increase in intracellular pH by 0.47 units in analysed monolayer cells. The results showed that it is not possible to block the EGF-receptors without imposing changes in growth and metabolism.

Cell Count↗

PubMedDate not supplied

Induction of cell death by Doxorubicin in multicellular spheroids as studied by confocal laser scanning microscopy.

In the present study the effects of the anticancer drug Doxorubicin (Dox) on necrosis development and cell lethality of multicellular DU-145 spheroids (MCS) were examined. Multicellular spheroids consist of a peripheral rim of proliferating cells, a inner shell of nonproliferating, quiescent cells and a central core of dead cells. After the application of Dox for different time periods dead cell areas and single dead cells in MCS of different size classes were identified using a set of lethal fluorescence dyes, and a confocal laser scanning microscope (CLSM). The distribution of Dox within MCS was examined by determining Dox fluorescence in single cells and cell areas. Outgrowth experiments were performed to show the effects of Dox on cancer cell migration and cell proliferation. The application of low (400 nM) concentrations of Dox over a time period of 2hours resulted in distinct Dox fluorescence staining of the most peripheral cell layers of the MCS. After long term incubation (48hours) cell lethality was most prominent in large spheroids (diameter between 350 and 800 micron) which possess a dead cell core and single dead cells at the periphery. These MCS showed an approximately 120 microm +/- 30 microm increased dead cell core as compared to control MCS. The cytotoxic effect of Dox was lower in MCS of a diameter between 150-350 microm and nearly no cytotoxic effects were found in spheroids smaller than 150 microm in diameter. Dox fluorescence persisted in dead cells for at least three days. During this time the cytotoxic agent leaked slowly from dead cells and penetrated into the layers of quiescent cells and proliferating cells mediating a prolonged cytotoxicity. In conclusion, the most efficient cytotoxic effect on MCS larger than 150 microm in diameter, can be achieved using a Dox concentration of 400 nM, and applying the drug for long incubation periods to allow its accumulation and storage in the dead cell core and in the single dead cells within vital cell layers. Dox is gradually delivered from these storage sites and kills proliferating and quiescent cells when no Dox is present in the external medium.

Antibiotics, Antineoplastic↗