Synergistic inhibition of human cancer cell growth by cytotoxic drugs and mixed backbone antisense oligonucleotide targeting protein kinase A

Abstract

Protein kinase A type I plays a key role in neoplastic transformation, conveying mitogenic signals of different growth factors and oncogenes. Inhibition of protein kinase A type I by antisense oligonucleotides targeting its RIalpha regulatory subunit results in cancer cell growth inhibition in vitro and in vivo. A novel mixed backbone oligonucleotide HYB 190 and its mismatched control HYB 239 were tested on soft agar growth of several human cancer cell types. HYB 190 demonstrated a dose-dependent inhibition of colony formation in all cell lines whereas the HYB 239 at the same doses caused a modest or no growth inhibition. A noninhibitory dose of each mixed backbone oligonucleotide was used in OVCAR-3 ovarian and GEO colon cancer cells to study whether any cooperative effect may occur between the antisense and a series of cytotoxic drugs acting by different mechanisms. Treatment with HYB 190 resulted in an additive growth inhibitory effect with several cytotoxic drugs when measured by soft agar colony formation. A synergistic growth inhibition, which correlated with increased apoptosis, was observed when HYB 190 was added to cancer cells treated with taxanes, platinum-based compounds, and topoisomerase II selective drugs. This synergistic effect was also observed in breast cancer cells and was obtained with other related drugs such as docetaxel and carboplatin. Combination of HYB 190 and paclitaxel resulted in an accumulation of cells in late S-G2 phases of cell cycle and marked induction of apoptosis. A cooperative effect of HYB 190 and paclitaxel was also obtained in vivo in nude mice bearing human GEO colon cancer xenografts. These results are the first report of a cooperative growth inhibitory effect obtained in a variety of human cancer cell lines by antisense mixed backbone oligonucleotide targeting protein kinase A type I-mediated mitogenic signals and specific cytotoxic drugs.

Figure 1

Dose-dependent effect of the RIα antisense MBO HYB 190 and its control sequence HYB 239 in different cancer cell lines. (A) HYB 190. (B) HYB 239. Data represent means and standard errors of three different experiments with each performed in triplicate.

Figure 2

Effect of different cytotoxic drugs and antisense RIα MBO on the growth of OVCAR-3 cancer cells. (A) HYB 190 (0.1 μM) in combination with paclitaxel or cisplatin. (B) HYB 239 (1 μM) in combination with paclitaxel or cisplatin. (C) HYB 190 (0.1 μM) in combination with doxorubicin or etoposide. (D) HYB 239 (1 μM) in combination with doxorubicin or etoposide. The drugs were used at the following doses: a–c, 1, 2.5, and 5 nM paclitaxel; d–f, 5, 10, and 50 ng/ml cisplatin; a–c, 0.01, 0.05, and 0.1 μg/ml doxorubicin; d–f, 0.05, 0.1, and 1 μg/ml etoposide. Data are expressed as percentage growth inhibition in reference to the growth of untreated control cells. The open portion of the bars represents the percentage growth inhibition values for HYB 190 (A and C) or HYB 239 (B and D). The striped or squared portion of the bars represents the percentage growth inhibition values for the cytotoxic drugs as indicated in the respective legends. The height of the bars on the left represents the sum of the individual agents’ effects and the expected percentage growth inhibition if drugs are additive when used in combination. The total height of the solid bar indicates the actual observed growth inhibition when drugs were used in combination. Therefore, the differences between the heights of the paired bars reflect the magnitude of synergism of growth inhibition. The data represent means and standard errors of triplicate determination of at least two experiments.

Figure 3

Flow-cytometric analysis of the effect of 1 nM paclitaxel and/or 0.1 μM HYB 190 on the induction of apoptosis in OVCAR-3 cells. Apoptotic cells are present in the area indicated by a bar on the left side of each histogram. The numbers in each panel represent the percent of apoptotic cells calculated by flowcytometric analysis (25). Data represents one of three different experiments showing similar results.

Figure 4

Effect of the treatment with paclitaxel and/or HYB 190 on the growth of GEO human colon cancer xenografts in nude mice. The administration of each single agent alone or in sequential schedule is described in Materials and Methods. Paclitaxel at 400 μg/dose and HYB 190 at 200 μg/dose.