Bispecific antisense oligonucleotides having binding sites directed against an autocrine regulated growth pathway and bcl-2 for the treatment of prostate tumors.
Antisense oligonucleotides (oligos) against transforming growth factor-alpha (TGF-alpha) (MR1) and its binding site, the epidermal growth factor receptor (EGFR) (MR2), are efficacious against PC-3 and LNCaP prostate tumors. To enhance activity and aid in simultaneous delivery, "bispecific" 39-mer oligos were constructed containing portions of both MR1 and MR2 sequences. The first pair contained truncated sequences recognizing TGF-alpha and EGFR mRNA binding sites, about their respective AUG initiation codons. These bispecifics differ in their 5' to 3' tandem orientation (TGF-alpha/EGFR [MR12] and EGFR/TGF-alpha [MR21] sequences). A second pair was constructed having complementary sequences for EGFR and bcl-2 (EGFR/bcl-2 [MR24] and bcl-2/EGFR [MR42]). All bispecifics were tested in vitro against PC-3 and LNCaP prostate tumor cells, and compared to mono-specific oligos from which they were derived. The purpose of this study was: (1) to evaluate bispecific antitumor activity; (2) to identify dominant sequences; (3) to identify effects of binding site orientation; and (4) to determine whether bispecifics are more effective when targeting one versus different growth-dependent pathways. Comparisons were made between oligos tested against either PC-3 or LNCaP cells incubated for 2 d with the agents followed by 2 d in their absence. The first PC-3 cell experiment demonstrated that bispecific MR12 and MR21 oligos are at least as effective as their mono-specific counterparts and that the MR21 bispecific orientation is more effective than the MR1 mono-specific by 64% (p = 0.014). It also suggested that the sequence directed against EGFR contributed most to bispecific activity, particularly in the MR21 orientation. In a second PC-3 study a second bispecific pair of 37-mer oligos was constructed containing bases complementary to mRNA encoding EGFR and the apoptosis-associated protein bcl-2 (MR4). MR24 was constructed with the EGFR complementary site at the 5' end (EGFR/bcl-2), and MR42, containing the opposite orientation (bcl-2/EGFR). Each contained the dominant EGFR activity identified previously. MR1, MR2, MR4, MR12, MR21, MR24, and MR42 (1X and 2X in concentration) were cultured with cells and compared to controls. Each oligo significantly inhibited growth of PC-3 cells. MR42 was most effective and significantly better than MR1 (p = 0.0128), MR2 (p = 0.021), MR4 (p = 0.0002), and MR12 (p = 0.0032). 2X MR24 and 2X MR42 were better than their 1X concentration counterparts, but the differences were not significant. In a similar experiment MR1, MR2, MR4, MR12, MR21, MR24, and MR42 were cultured with LNCaP cells and compared to lipofectin-containing controls. Each oligo significantly inhibited the growth of LNCaP cells. Again, MR42 was most effective and significantly better than MR2 (p = 0.021) and MR4 (p = 0.038). MR24 was significantly better than MR2 (p = 0.048). Bispecific oligos are a significant advance in antisense technology and could play a role in treating prostate cancer, particularly if combined with traditional chemotherapeutics.
Rubenstein
Marvin
M
Division of Cellular Biology, Hektoen Institute of Medicine, 2100 W. Harrison Street, Chicago, IL, 60612, USA. DrMarv@Prodigy.net
Tsui
Paulus
P
Guinan
Patrick
P
D000970:Antineoplastic Agents / Q000737:chemistry / Q000627:therapeutic use*; D019898:Autocrine Communication / Q000187:drug effects*; D001483:Base Sequence; D001665:Binding Sites; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000627:therapeutic use*; D011471:Prostatic Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016211:Transforming Growth Factor alpha / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D016376:Oligonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D016211:Transforming Growth Factor alpha; D066246:ErbB Receptors