l-744832 and Adenocarcinoma

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.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Alkyl and Aryl Transferases; Antineoplastic Agents; Apoptosis; Caspases; Cell Cycle; Cell Proliferation; Cisplatin; Drug Synergism; Drug Therapy, Combination; Flow Cytometry; Humans; Immunoblotting; Lung Neoplasms; Methionine; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Activated ras is known to dysregulate TGF-beta signaling by altering the expression of TGF-beta type II receptor (RII). It is well documented that tumor cells harboring mutant ras are more resistant to radiation than cells with wild-type ras. In this study, we hypothesized that the use of farnesyltransferase inhibitor (FTI, L-744,832) may directly restore TGF-beta signaling through RII expression via ras dependent or independent pathway leading to induction of radiation sensitivity. Two pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2 were used in this study. FTI inhibited farnesylation of Ras protein more significantly in MIA PaCa-2 than BxPC-3 cells. In contrast, MIA PaCa-2 cells were resistant to radiation when compared to BxPC-3 cells. BxPC-3 cells were more resistant to FTI than MIA PaCa-2 cells. In combination treatment, no significant radiosensitizing effect of FTI was observed in BxPC-3 cells at 5 or 10 microM. However, in MIA PaCa-2 cells, a significant radiosensitizing effect was observed at both 5 and 10 microM concentrations (P>0.004). The TGF-beta effector gene p21(waf1/cip1) was elevated in combination treatment in MIA PaCa-2 but not in BxPC-3 cells. In MIA PaCa-2 cells, FTI induced TGF-beta responsive promoter activity as assessed by 3TP-luciferase activity. A further induction of luciferase activity was observed in MIA PaCa-2 cells treated with radiation and FTI. Induction of TGF-beta signaling by FTI was mediated through restoration of the RII expression, as demonstrated by RT-PCR analysis. In addition, re-expression of RII by FTI was associated with a decrease in DNA methyltransferase 1 (DNMT1) levels. Thus, these findings suggest that the L-744,832 treatment restores the RII expression through inhibition of DNMT1 levels causing induction of TGF-beta signaling by radiation and this forms a novel molecular mechanism of radiosensitization by FTI.

Topics: Adenocarcinoma; Alkyl and Aryl Transferases; Apoptosis; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Enzyme Induction; Enzyme Inhibitors; Farnesyltranstransferase; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Methionine; Neoplasm Proteins; Pancreatic Neoplasms; Protein Prenylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins p21(ras); Radiation Tolerance; Radiation-Sensitizing Agents; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Signal Transduction; Transforming Growth Factor beta