Combination studies of gemcitabine with UCN-01 or staurosporine

1990

Gemcitabine (GEM) is an excellent compound to be combined with other agents. The action of GEM may be affected by protein kinase C (PKC) inhibitors, such as staurosporine (STS) and 7-hydroxystaurosporine (UCN-01), which may modulate deoxycytidine kinase (dCK) essential for activation of GEM, or modulate the cell cycle and apoptosis by inhibition of cyclin dependent kinase 2 (cdk2) activity. We evaluated the combination of GEM with STS and UCN-01 focusing on growth inhibition, apoptosis induction, cell cycle and the role of the p53 protein. Lovo colon cancer cell line variants with wild type (Lovo B2), non-functional (Lovo Li) and mutant (Lovo 175x2) p53 were used for this purpose. Combinations of GEM with UCN-01 or STS were most effective when GEM was added prior to STS or UCN-01. For 1.0 μM GEM, a gradual time dependent increase of cells in S phase was observed in all Lovo variants. Exposure to 0.05 μM STS initially induced accumulation of cells in the G₂/M phase (after 24 hr), while 48 hr exposure showed accumulation in the S phase. An arrest in G₀/G₁ was observed after 24 hr exposure to 0.5 μM UCN-01, but after 48 hr cells accumulated in the S phase, associated with an induction of free E2F1 expression in Lovo B2, but not in Lovo 175x2. Simultaneous exposure to GEM with UCN-01 or STS showed an average cell cycle distribution of that of each drug alone. In sequential drug exposure, the cell cycle distribution was determined by the first drug. Induction of apoptosis by UCN-01 was 2-fold higher in Lovo 175x2 compared to other Lovo variants. Additive effects in induction of apoptosis were observed at the simultaneous addition of GEM and UCN-01. GEM prior to UCN-01 caused a 2-fold higher induction of apoptosis than the sum of each drug alone in Lovo 175x2 cells, in contrast to the other lines. In Lovo 175x2, no change in p53 was observed while in Lovo B2 single drug exposure to GEM upregulated p53 about 4-fold at 48 hr, but after exposure to STS or UCN-01 a slight decrease was found. In simultaneous drug exposure, drugs contributed equally to p53 expression leading to about 2-fold upregulation. In sequential drug exposure, the first drug given dominated in the p53 induction, leading to no change in p53 expression when UCN-01 or STS were added first, although a high extent of apoptosis was found in this sequence. Since also in the Lovo 175x2 cells a similar and higher extent of apoptosis was found than in the wt p53, induction of apoptosis follows a p53 independent pathway. In conclusion, although UCN-01 interferes with cdk2 and PKC, the action of both UCN-01 and STS in combination with GEM seems to be related to cell cycle effects by preventing cells to progress in cell cycle after GEM exposure, leading to more cell death.