Combining an FPTase inhibitor with cisplatin facilitates induction of apoptosis in human A549 lung cancer cells.

Despite great efforts to develop efficacious curative treatments, the prognosis for lung cancer patients is poor. In the present study we compared the effects of cisplatin (CP), a strong DNA damaging compound, with those of roscovitine (ROSC), a selective inhibitor of cyclin-dependent kinases (CDKs), on wt p53-positive human A549 lung adenocarcinoma cells harboring a mutated K-RAS gene. Asynchronously growing A549 cells were relatively resistant to CP treatment for 24 h, but after exposure to CP at sufficiently high doses (> or = 20 microM) an accumulation of S-arrested cells was observed. However, after post-incubation of CP-treated cells in a drug-free medium for a further 48 h the number of living cells was markedly reduced. Combining CP with L-744,832, a small molecule FPTase inhibitor (FTI), slightly enhanced its anti-proliferative effect. Interestingly, FTI sensitized A549 cells to CP-induced apoptosis. ROSC inhibited A549 cells at the G/M transition, resulting in a marked decrease in the number of viable cells within 24 h, and prolonged treatment with ROSC for 48 h reduced the frequency of living cells by inducing apoptosis. The effects of ROSC (unlike those of CP) were more strongly enhanced by inhibition of the Ras protein processing pathway. Our preliminary results indicate that functional p53 contributes to the outcome of the therapy in human A549 cells by certain anti-cancer drugs.

Reconstitution of human MCF-7 breast cancer cells with caspase-3 does not sensitize them to action of CDK inhibitors.

Human MCF-7 breast cancer cells are resistant to pro-apoptotic stimuli due to caspase-3 inactivation. On the other hand, they should be sensitive to agents like selective pharmacological inhibitors of cyclin-dependent kinases (CDKs) that (re)activate p53 tumor suppressor protein because they harbor intact p53 pathways. In this study we examined whether reconstitution of caspase-3 in MCF-7 cells sensitizes them to inhibitors of CDKs, by analyzing the effects of roscovitine (ROSC) and olomoucine (OLO), two closely related selective pharmacological CDK inhibitors, on both mother MCF-7 cells and a secondary mutant line, MCF-7.3.28 that stably expresses human caspase-3. The results show that ROSC is, as expected, much more potent than OLO. Surprisingly; however, ROSC and OLO reduced proliferation of parental MCF-7 cells more strongly than caspase-3-proficient counterparts. Both inhibitors arrest human breast cancer cells at the G(2)-phase of the cell cycle. Analysis of cell-cycle regulators by immunoblotting revealed that ROSC strongly induces p53 protein activity by inducing its phosphorylation at Ser46 in the MCF-7 cells lacking caspase-3, but not in caspase-3-proficient cells. Furthermore, reconstitution of caspase-3 in MCF-7 cells neither elevates the mitochondrial apoptosis rate nor significantly increases caspases activity upon ROSC treatment. However, the stabilization of p53 in response to DNA damaging agents is the same in both caspase negative and positive MCF-7 cells. Cytotoxic agents induce caspase-3-dependent apoptosis in caspase-3-proficient cells. These results indicate that reconstitution of MCF-7 cancer cells with caspase-3 sensitize them to the action of DNA damaging agents but not to ATP-like pharmacological inhibitors of CDKs.