Antisense protein kinase A RIalpha acts synergistically with hydroxycamptothecin to inhibit growth and induce apoptosis in human cancer cells: molecular basis for combinatorial therapy.

Abstract

The increased expression of RIalpha, the regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase type I (PKA-I), has been correlated with cancer cell growth. An antisense oligonucleotide targeting the RIalpha subunit of PKA (antisense RIalpha) induces cell growth arrest, apoptosis, and differentiation in a variety of cancer cell lines in vitro and in tumors in vivo. This study investigated the utility of a combinatorial therapy consisting of the RNA-DNA second-generation RIalpha antisense HYB0165 (Gem231) and the cytotoxic drug hydroxycamptothecin (HCPT), which inhibits topoisomerase I. LS-174T colon carcinoma and PC3M androgen-insensitive prostate cancer cells were used as experimental models. The antitumor and apoptotic activities of Gem231 and HCPT, singly and in combination, were measured by cell growth assay, synergism quotient, cell morphology, nuclear morphology, levels of PKA R and C subunits, anti- and proapoptotic proteins, and PKA activity ratio. In a synergistic fashion, Gem231 and HCPT induced growth arrest, apoptosis, and changes in cell morphology; down-regulated RIalpha expression; down-regulated Bcl-2 and promoted its hyperphosphorylation; up-regulated the proapoptotic proteins Bax and Bad; and promoted hypophosphorylation of Bad. Antisense Gem231, but not HCPT, increased the PKA activity ratio, which measures the degree of PKA activation. The results showed that PKA-I activation by Gem231 and topoisomerase I inhibition by HCPT are responsible at the molecular level for the synergistic effects of tumor cell apoptosis and growth inhibition. These results demonstrated the molecular basis for the use of Gem231 and HCPT as combinatorial therapy to treat human cancer.