Upon oxidative stress, the antiapoptotic Hsp60/procaspase-3 complex persists in mucoepidermoid carcinoma cells.
Hsp60, a mitochondrial chaperonin highly conserved during evolution, has been found elevated in the cytosol of cancer cells, both in vivo and in vitro, but its role in determining apoptosis during oxidative stress (OS) has not yet been fully elucidated. The aim of the present work was to study the effects of OS on Hsp60 levels and its interactions with procaspase- 3 (p-C3) and p53 in tumor cells. NCI-H292 (mucoepidermoid carcinoma) cells were exposed to various concentrations of hydrogen peroxide (H2O2) for 24 hours. Cell viability was determined by Trypan blue and MTT assays. DNA damage was assessed by the Comet assay, and apoptosis was measured by the AnnexinV cytofluorimetric test. Exposure to increasing concentrations of H2O2 resulted in a reduction of cell viability, DNA damage, and early apoptotic phenomena. Hsp60, p-C3, p53, and p21 were assessed by Western blotting and immunocytochemistry before and after OS. Hsp60 and p-C3 were present before and after OS induction. Immunoprecipitation experiments showed an Hsp60/p-C3 complex before OS that persisted after it, while an Hsp60/p53 complex was not detected in either condition. The presence of wild type (wt) p53 was confirmed by RT-PCR, and p21 detection suggested p53 activation after OS. We postulate that, although OS may induce early apoptosis in NCI-H292 cells, Hsp60 exerts an anti-apoptotic effect in these cells and, by extension, it may do so in other cancer cells.
Campanella
C
C
Human Anatomy Section, Department of Experimental Medicine, University of Palermo, via del Vespro 129, 90127 Palermo, Italy.
Bucchieri
F
F
Ardizzone
N M
NM
Marino Gammazza
A
A
Montalbano
A
A
Ribbene
A
A
Di Felice
V
V
Bellafiore
M
M
David
S
S
Rappa
F
F
Marasà
M
M
Peri
G
G
Farina
F
F
Czarnecka
A M
AM
Conway de Macario
E
E
Macario
A J L
AJ
Zummo
G
G
Cappello
F
F
D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D018277:Carcinoma, Mucoepidermoid / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D053148:Caspase 3 / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D018834:Chaperonin 60 / Q000378:metabolism*; D020552:Comet Assay; D004247:DNA / Q000187:drug effects; D004249:DNA Damage; D005562:Formazans / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007150:Immunohistochemistry; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D018384:Oxidative Stress; D013778:Tetrazolium Salts / Q000378:metabolism; D014343:Trypan Blue / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics
D018834:Chaperonin 60; D005562:Formazans; D013778:Tetrazolium Salts; D016159:Tumor Suppressor Protein p53; C079782:MTT formazan; D004247:DNA; D006861:Hydrogen Peroxide; D053148:Caspase 3; D014343:Trypan Blue