Histone deacetylase inhibitors induce G2-checkpoint arrest and apoptosis in cisplatinum-resistant ovarian cancer cells associated with overexpression of the Bcl-2-related protein Bad

Trichostatin A produces predominantly G, cell-cycle blockade and differentiation of the cisplatinum-sensitive A2780 ovarian cancer cell line. Given the propensity of ovarian tumors to become resistant to cisplatinum, often leading to cross-resistance to other agents, we have extended these observations by examining how the emergence of resistant phenotypes in A2780 cells affects the actions of histone deacetylase (HDAC) inhibitors. Trichostatin A exposure (100 ng/mL, 24 hours) induced ultrastructural differentiation of the intrinsically cisplatinum-resistant A2780-9M subline, with the reappearance of intercellular junctions and lumina containing primitive microvilli. Similar trichostatin A exposure in the acquired resistance A2780CP cells produced minimal differentiation consisting of occasional weak intercellular junctions. Independent of the differences in trichostatin A-induced differentiation, in both resistant sublines trichostatin A produced a similar reduction in cell viability, by > 90%, within 5 days of treatment. Diminished viability in both A2780-9M and CP cells was associated with the absence of cell cycle arrest in G(1), resulting in predominant G(2)-checkpoint arrest accompanied by a 10- to 20-fold increase in Annexin V binding and the reemergence of apoptosis. Similar cell cycle arrests and apoptosis were also observed using other HDAC inhibitors and in other resistant ovarian cancer: cell lines (OVCAR-3 and SK-OV-3). Trichostatin A-induced apoptosis in resistant cells is. in sharp contrast to its effects on the parental cisplatinum-sensitive A2780 and normal MRC-5 fibroblast cell lines (predominant cycle arrest in G(1) with no detectable apoptosis). Western immunoblot analysis indicated trichostatin A triggers apoptosis in resistant ovarian cancer cells. via p53-independent activation of the intrinsic mitochondrial pathway, commensurate with induction of the Bcl-2-related protein Bad. These results suggest cisplatinum resistance alters the effects of HDAC inhibition through a shift in cell cycle arrest from the G(1) to the G(2) checkpoint and reactivation of the intrinsic mitochondrial apoptotic cascade.