Mechanisms of small globular protein-induced plasma membrane permeability and cytotoxicity in U87-MG human malignant glioblastoma cells.
BACKGROUND
Small globular protein (SGP) is an artificially designed protein that forms small pores on lipid bilayer membranes. The anti-tumor effect of SGP has been reported in several papers. However, the exact mechanism underlying SGP-induced cell death remains to be determined.


METHODS
The mechanisms of SGP-mediated cytotoxicity were examined using cultured malignant glioblastoma cells (U87-MG). Morphological changes were observed under a light microscope. Ultrastructural changes were examined by electron microscopy. Changes in membrane permeability were evaluated by measuring the extracellular release of lactate dehydrogenase (LDH) and intracellular calcium concentration ([Ca2+]i). A new cell viability assay was also conducted using DAPI/calcein fluorescent-double staining methods.


RESULTS
SGP cytotoxic effects were strongly influenced by fetal bovine serum (FBS) concentration in culture media. After 30 min of incubation, cytotoxic effects were noted at SGP concentrations greater than 1.5 microM. [Ca2+]i increased immediately after SGP addition. Extracellular efflux of LDH increased linearly with SGP doses (r(2) =0.936), and occurred even at concentrations less than 1.5 microM. Non-viable and viable cells were simultaneously detected by fluorescent staining with DAPI and calcein-AM, respectively. After 30 min of incubation with SGP, DAPI-positive nuclei were evident at concentrations higher than 1.5 microM. Cultures were then maintained in 10% FBS-containing media for 24 h before addition of calcein-AM. Calcein-positive cells were found at SGP concentrations lower than 1.5 5M. Cells positive for both DAPI and calcein appeared at a concentration of 1.5 microM.


CONCLUSIONS
Our results indicate that SGP induces rapid but transient cytotoxicity, which may be beneficial in local therapy of malignant brain tumors.
Onishi
Hirokazu
H
Department of Neurosurgery, School of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. hirokazu-onishi1031@dream.ocn.ne.jp
Jimi
Shiro
S
Tsugu
Hitoshi
H
Lee
Sannam
S
Fukushima
Takeo
T
D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D001430:Bacteriocins / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000187:drug effects; D005909:Glioblastoma / Q000188:drug therapy* / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D007211:Indoles / Q000493:pharmacokinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry / Q000494:pharmacology*
D000970:Antineoplastic Agents; D001430:Bacteriocins; D007211:Indoles; D011506:Proteins; C007293:DAPI; D007770:L-Lactate Dehydrogenase; D002118:Calcium