Combination chemotherapy employing bispecific antisense oligonucleotides having binding sites directed against an autocrine regulated growth pathway and bcl-2 for the treatment of prostate tumors

Abstract

In previous studies we demonstrated that antisense oligonucleotides (oligos) against transforming growth factor-alpha (TGF-α [MR₁]), its binding site the epidermal growth factor receptor (EGFR [MR₂]), and the anti-apoptosis protein bcl-2 (MR₄) are efficacious against prostate tumors. In recent reports we also describe how two of these mRNA directed binding sites can be synthesized sequentially within a single linear complementary strand and administered either in the presence or absence of additional therapeutic agents.

In these continuing experiments “bispecific” oligo pairs were further evaluated in the presence or absence of Cytoxan, Taxol, or DES. One oligo pair recognized the binding sites for TGF-α and EGFR mRNA (TGF-α/EGFR [MR₁₂] and EGFR/TGF-α [MR₂₁]); another pair recognized binding sites for EGFR and bcl-2 (EGFR/bcl-2 [MR₂₄] and bcl-2/EGFR [MR₄₂]). Oligo pairs differ in their linear 5′ to 3′ binding site orientations, and were tested in vitro against PC-3 and LNCaP prostate tumor cell lines. Following cell attachment, incubations were for 2 days with the agents followed by 2 days in their absence.

When tested against PC-3 cells and combined with LD₅₀ Cytoxan, MR₂, MR₄, MR₂₄, MR₄₂ significantly inhibited 47.3, 45.7, 68.3, and 64.9%; with LD₅₀ Taxol MR₂, MR₄, MR₂₄, MR₄₂ significantly inhibited 49.8, 45.8, 64.1, and 59.2%; and with LD₅₀ DES MR₂, MR₄, MR₂₄, MR₄₂ significantly inhibited 66.6, 67.6, 64.3, and 67.2% respectively. Each agent significantly increased the inhibition produced by either oligo alone.

LNCaP cells were also incubated with mono- and bispecific oligos in either the presence or absence of chemotherapeutics. MR₂, MR₄, MR₂₄, MR₄₂ produced significant inhibitions of 57.4, 58.4, 69.4, and 68.6% with LD₅₀ Cytoxan; 70.4, 70.1, 73.6, and 74.0% with LD₅₀ Taxol; and 49.8, 50.1, 59.6, and 53.9%, respectively with LD₅₀ DES.

A complete PC-3 experiment compared MR₁, MR₂, MR₄, MR₁₂, MR₂₁, MR₂₄ and MR₄₂, in the presence of LD₅₀ Cytoxan. Each oligo combined with Cytoxan significantly inhibited: MR₁ by 51.0, MR₂ by 55.0, MR₄ by 58.0; MR₁₂ by 56.0; MR₂₁ by 61.1, MR₂₄ by 65.5 and MR₄₂ by 66.0%. Bispecifics directed against two different pathways, MR_24, and MR₄₂ were the most effective.

A complete LNCaP experiment compared the same series of oligos also in the presence of LD₅₀ Cytoxan. Each oligo combined with Cytoxan significantly inhibited: MR₁ by 49.0, MR₂ by 50.0, MR₄ by 53.0; MR₁₂ by 52.0; MR₂₁ by 58.6, MR₂₄ by 53.9 and MR₄₂ by 58.0%.