Paulus Tsui

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-α) (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-α and EGFR mRNA binding sites, about their respective AUG initiation codons. These bispecifics differ in their 5’ to 3’ tandem orientation (TGF-α/EGFR [MR12] and EGFR/TGF-α [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.

Treatment of prostate and breast tumors employing mono- and bi-specific antisense oligonucleotides targeting apoptosis inhibitory proteins clusterin and bcl-2

Antisense oligonucleotides (oligos) have demonstrated their efficacy in inhibiting the growth of prostate and breast tumor cells. Previous studies employed first generation, phosphorothioated, cDNA oligos synthesized complimentary to mRNA encoding transforming growth factor-alpha (TGF-α), epidermal growth factor receptor (EGFR), the anti-apoptosis protein bcl-2, and the androgen receptor (AR). In an effort to construct oligos with greater than one mRNA binding site, bi-specifics have been developed which target combinations of the above proteins, and these have been shown at least as effective as the mono-specific oligos from which their sequences were derived. While all bi-specifics have inhibitory effects, which can be enhanced by the combined administration of an additional chemotherapeutic agent, those bi-specifics which target bcl-2 and EGFR were reported to be the most effective. The experiments presented here are an effort to evaluate a new group of bi-specifics whose targets include the chaperone protein clusterin, whose expression is up regulated in many tumors and activity is known to inhibit apoptosis. Of particular interest were those bi-specifics constructed to target both clusterin and bcl-2 (also an apoptosis inhibitory protein). Cell lines targeted included both prostate LNCaP and PC-3, as well as the breast derived MCF-7. In order to identify agents which enhance oligo activity, but contribute less toxicity, oligos were tested both alone and in combination with either the immune inhibitor Rapamycin, or the chemotherapeutic (and more toxic) Taxol. Results indicate that bi-specifics targeting clusterin are statistically effective, and are similarly enhanced by Rapamycin, or Taxol. When bi-specifics including clusterin as a target, were tested against LNCaP and MCF-7 cells, the level of activity was intermediate between that of the mono-specific compounds tested separately. In experiments which compared both, bi-specifics which included a target for clusterin had inhibitory activity similar to the previously described bi-specifics directed towards bcl-2 and EGFR.

Correlation Between PSMA and VEGF Expression as Markers for LNCaP Tumor Angiogenesis

Our aim is the identification and correlation of changes in tumor-associated protein expression which results from therapy. LNCaP tumors, excised from nude mice treated either by orchiectomy or with the chemotherapeutic agent paclitaxel, were evaluated for the expression of proteins and receptors associated with growth, differentiation, and angiogenesis using immunohistologic procedures. Compared to untreated control tumors, both treatments reduced the expression of vascular endothelial growth factor (VEGF), prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), androgen receptor (AR), and epidermal growth factor receptor (EGFR). The effect of paclitaxel treatment on AR expression was the most significant (P = .005). Of particular interest was identifying a significant correlation (P < .000801) between PSMA and VEGF expression regardless of treatment modality. These altered expressions suggest that PSMA may also be a marker for angiogenesis and could represent a target for deliverable agents recognizing either prostatic tumors or endothelial development. Cell surface PSMA would then present a unique target for treatment of patients early in their development of prostatic metastases.

Synergistic effects of combination therapy employing antisense oligonucleotides with traditional chemotherapeutics in the PC-3 prostate cancer model.

Combination therapy including antisense oligonucleotides (ODNs) with traditional chemotherapeutic agents offers potential benefits by increasing the effectiveness of the chemotherapeutics, reducing their effective dosage, and simultaneously reducing toxicity. Previously we have reported that antisense ODNs specific for transforming growth factor-α (TGF-α) and its binding site, the epidermal growth factor receptor (EGFR) (MR1 and MR2, respectively), are effective against the PC-3 in vitro and in vivo prostate cancer models. In this series we evaluated these antisense ODNs in various combinations and treatment cycles with paclitaxel (Taxol), cyclophosphamide (Cytoxan), mitoxantrone, carboplatin, cisplatin, and oxaliplatin in order to identify synergistic effects.We found that when either of the ODNs were simultaneously administered with Taxol, no synergistic activity was noted. However, when sequentially administered in a series 1 d apart, a pretreatment with the ODN directed against TGF-α (6.64 µm) followed by Taxol (5 nm) had significantly (p<0.001) greater activity than these agents similarly administered in the reverse order or simultaneously.When Cytoxan was administered in sequence with both ODNs significantly increased growth inhibition was obtained compared to when Cytoxan was administered alone. A 1 d treatment of PC-3 cells with Cytoxan followed the next day with MR1 was significantly more effective (p<0.0001). The reverse order, a pretreatment with MR1 followed by Cytoxan, also resulted in significant additional inhibition (p=0.0004). Similarly sequenced, MR2 followed by Cytoxan, was also significantly more effective (p=0.0014) than Cytoxan treatment alone.For mitoxantrone, which was administered in combination therapy with ODNs: mitoxantrone with MR1 was significantly more inhibitory than the combination of both MR1 and MR2 ODNs (p=0.006) and also mitoxantrone administered alone (p=0.0012). Mitoxantrone administered with MR1 was not significantly different from mitoxantrone given in combination with MR2. Although mitoxantrone and MR2 was statistically (p=00015) more inhibitory than mitoxantrone alone, this combination was barely more effective (p=0.04) than the MR1 ODN administered alone.When the ODN directed against TGF-α (MR1) was added for a single day in combination with carboplatin, cisplatin, and oxaliplatin, the results were significantly more effective than either platinated agent alone (p<0.001,=0.0005,<0.0001 respectively), and more effective than similarly administered MR2 with these agents.We conclude that combination therapy, which includes antisense ODNs directed against TGF-α and EGFR, can significantly increase the effects produced by chemotherapeutics alone in the treatment of prostate cancer.