We have recently shown that
thymoquinone (TQ) is an
antineoplastic drug that induces p53-dependent apoptosis in human
colon cancer cells. This study evaluated the antiproliferative and pro-apoptotic effects of TQ in two human
osteosarcoma cell lines with different p53 mutation status. TQ decreased cell survival dose-dependently and, more significantly, in p53-null MG63 cells (IC(50) = 17 muM) than in p53-mutant
MNNG/HOS cells (IC(50) = 38 muM). Cell viability was reduced more selectively in MG63
tumor cells than in normal human osteoblasts. Flow cytometric analysis showed that TQ induced a much greater increase in the PreG(1) (apoptotic) cell population, but no cell cycle arrest in MG63. G(2)/M arrest in
MNNG/HOS cells was associated with p21(WAF1) upregulation. Using three DNA damage assays, TQ was confirmed to result in a significantly greater extent of apoptosis in p53 null MG63 cells. Although the Bax/Bcl-2 ratios were not differentially modulated in both cell lines, the mitochondrial pathway appeared to be involved in TQ-induced apoptosis in MG63 by showing the cleavage of caspases-9 and -3. Oxidative stress and mitochondrial O(2)(*-) generation in isolated rat mitochondria were enhanced by TQ as measured by the dose-dependent reduction in
aconitase enzyme activity and
Amplex Red oxidation respectively. TQ-induced oxidative damage, reflected by an increase in gamma-H2AX foci and increased
protein expression levels of gamma-H2AX and the
DNA repair enzyme, NBS1, was more pronounced in
MNNG/HOS than in MG63. We suggest that the resistance of
MNNG/HOS cells to drug-induced apoptosis is caused by the up-regulation of p21(WAF1) by the mutant p53 (transcriptional activity was shown by p53
siRNA treatment) which induces cell cycle arrest and allows to repair DNA damage. Collectively, these findings show that TQ induces p53-independent apoptosis in human
osteosarcoma cells. As the loss of p53 function is frequently observed in
osteosarcoma patients, our data suggest the potential clinical usefulness of TQ for the treatment of these
malignancies.