MDA-7 regulates cell growth and radiosensitivity in vitro of primary (non-established) human glioma cells.
We examined the impact of purified bacterially synthesized GST-MDA-7 (IL-24) and ionizing radiation on the proliferation and survival of nonestablished human glioblastoma multiforme (GBM) cells. Glioma cell types expressing mutated PTEN and p53 molecules, activated ERBB1VIII, overexpressing wild type ERBB1 or without receptor overexpression were selected. In MTT assays, GST-MDA-7 caused a dose-dependent reduction in the proliferation of nonestablished glioma cells; however only at higher concentrations did GST-MDA-7 reduce cell viability. The anti-proliferative and cytotoxic effects of GST-MDA-7 were enhanced by radiation in a greater than additive fashion that correlated with JNK1/2/3 activation. The reduction in cell growth and enhancement in cell killing by the combination of GST-MDA-7 and radiation were blocked by an ROS scavenger, N-acetyl cysteine (NAC), a JNK1/2/3 inhibitor SP600125, a pan-caspase inhibitor (zVAD) and by an inhibitor of caspase 9 (LEHD), but not by an inhibitor of caspase 8 (IETD). Low concentrations of either GST-MDA-7 or radiation reduced clonogenic survival, however colony formation ability was significantly further decreased when the two treatments were combined, which was also blocked by inhibition of caspase 9 function. In general agreement with activation of the intrinsic caspase pathway, cell death correlated with reduced BCL-XL expression and with increased levels of the pro-apoptotic proteins BAD and BAX. Inhibition of caspase 9 after combination treatment blunted neither JNK1/2/3 activation nor the enhanced expression of BAD and BAX, but did block caspase 3 cleavage, reduced expression of BCL-XL and inhibition of ERK1/2 activity. In contrast, incubation with NAC blocked JNK1/2/3 activation and cell killing, but not the increases in BAD and BAX expression. These findings argue that after combination treatment JNK1/2/3 activation is a primary pro-apoptotic event and loss of BCL-XL expression and ERK1/2 activity are secondary caspase-dependent processes. This data also argues that GST- MDA-7 induces two parallel pro-apoptotic pathways via ROS-dependent and -independent mechanisms. Infection of primary human astrocytes with a recombinant adenovirus to express MDA-7, Ad.mda-7, but not infection with either Ad.cmv or Ad.mda-7SP- lacking MDA-7 secretion, resulted in the suppression of GBM cell colony formation in soft agar overlay assays, an effect that was enhanced in a greater than additive fashion by radiation. Collectively, our findings demonstrate that MDA-7 reduces proliferation and enhances the radiosensitivity of nonestablished human GBM cells in vitro, and when grown in 3 dimensions, and that sensitization occurs independently of basal EGFR/ERK1/2/AKT activity or the functions of PTEN and p53.
Yacoub
Adly
A
Department of Radiation Oncology, Virginia Commonwealth University, Richmond, Virginia 23298-0058, USA.
Mitchell
Clint
C
Hong
Young
Y
Gopalkrishnan
Rahul V
RV
Su
Zhao-Zhong
ZZ
Gupta
Pankaj
P
Sauane
Moria
M
Lebedeva
Irina V
IV
Curiel
David T
DT
Mahasreshti
Parameshwar J
PJ
Rosenfeld
Myrna R
MR
Broaddus
William C
WC
James
C David
CD
Grant
Steven
S
Fisher
Paul B
PB
Dent
Paul
P
D000111:Acetylcysteine / Q000494:pharmacology; D000256:Adenoviridae / Q000235:genetics; D000276:Adjuvants, Immunologic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects*; D001253:Astrocytes / Q000187:drug effects / Q000528:radiation effects; D001932:Brain Neoplasms / Q000378:metabolism / Q000628:therapy*; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects / Q000528:radiation effects*; D003114:Colony-Forming Units Assay; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D016147:Genes, Tumor Suppressor; D018773:Genes, erbB-1 / Q000502:physiology; D005909:Glioblastoma / Q000378:metabolism / Q000628:therapy*; D006801:Humans; D007378:Interleukins / Q000378:metabolism / Q000494:pharmacology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051059:PTEN Phosphohydrolase; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011836:Radiation Tolerance; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D000099180:Interleukin-24
D000111:Acetylcysteine; D000276:Adjuvants, Immunologic; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases; D004791:Enzyme Inhibitors; D066246:ErbB Receptors; D007378:Interleukins; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; D051059:PTEN Phosphohydrolase; D010744:Phosphoric Monoester Hydrolases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2; D011838:Radiation-Sensitizing Agents; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D000099180:Interleukin-24; C494826:BAX protein, human; C494811:BCL2L1 protein, human; C494918:AKT1 protein, human; C494929:PTEN protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human