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Experimental Therapeutics, Preclinical Pharmacology |
Cattedra di Oncologia Medica, Dipartimento di Endocrinologia e Oncologia Molecolare e Clinica, Università Federico II, 80131 Napoli, Italy [G. T., R. C., G. P., S. P., A. R. B., F. C.]; Hybridon Inc., Cambridge, Massachusetts 02139 [S. A.]; and University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030 [J. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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regulatory subunit results in cancer cell growth inhibition in vitro and in vivo. We have recently shown that a mixed backbone oligonucleotide targeting RI can cooperatively inhibit human cancer cell growth when combined with selected cytotoxic drugs. In the present study, we have used HYB 165, a novel DNA/RNA hybrid mixed backbone oligonucleotide that exhibits improved pharmacokinetic and bioavailability properties in vivo and is presently undergoing Phase I trials. We have shown that HYB 165 exhibits a dose-dependent inhibitory effect on ZR-75-1 cells and a cooperative activity with docetaxel, a cytotoxic drug active in breast cancer. The antiproliferative activity is accompanied by increased apoptosis, as compared with each single agent. On the basis of our previous demonstration of a structural and functional relation between PKAI and epidermal growth factor receptor, we have performed a double blockade of these pathways using HYB 165 in combination with monoclonal antibody (MAb) C225, an anti-epidermal growth factor receptor chimeric MAb. The two compounds determined a cooperative growth inhibitory effect on ZR-75-1 cells and increased apoptosis. To study whether different biological agents and cytotoxic drugs can interact together, low doses of HYB 165, MAb C225, and docetaxel were combined causing an even greater cooperative effect toward growth inhibition. Finally, we have demonstrated that each single agent is able to induce bcl-2 phosphorylation and that the three agents, used in combination at suboptimal doses, determine a greater degree of bcl-2 phosphorylation and cause apoptosis of the majority of ZR-75-1 cells. These findings provide the basis for a novel strategy of treatment of breast cancer patients because both HYB 165 and MAb C225 are presently under clinical evaluation. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and EGF), and is overexpressed in the majority of human cancers, correlating with worse clinicopathological features and prognosis in breast and ovarian cancer patients (1, 2, 3, 4, 5, 6, 7)
. Conversely, PKAII is preferentially expressed in normal tissues and seems to be involved in cell growth arrest and differentiation (1, 2, 3, 4, 5, 6, 7, 8)
. Down-regulation of PKAI by different unmodified or PS-antisense oligodeoxynucleotides targeting its RI subunit causes cell growth arrest and differentiation in a wide variety of cancer cell lines (9, 10, 11)
and inhibition of growth of human colon cancer xenografts in nude mice (12)
. It has been demonstrated that PS- oligos, although they exhibit sequence-specific antitumor activity in vitro and in vivo, may have also toxic side effects in animal models and humans (13)
. To overcome these limits, MBOs have been synthesized recently. This novel class of modified oligos has shown a significant reduction of side effects in vivo compared with PS- oligos (13
, 14)
. We have recently shown that an antisense RI MBO containing methylphosphonate linkages exerts a synergistic inhibitory effect when added to paclitaxel, cisplatin, doxorubicin, or etoposide on the growth of several human cancer cell lines (15)
.
EGF-like growth factors such as TGF and amphiregulin bind to the extracellular domain of the EGFR, activating its intracellular tyrosine kinase domain, and control human breast cancer cell growth through autocrine and paracrine mechanisms (16
, 17)
. Enhanced expression of TGF and/or EGFR has been detected in human breast carcinomas and is generally associated with poor prognosis in breast cancer patients (16
, 18)
. In the past years, a large number of studies has disclosed a functional relationship between the EGFR-mediated signaling and PKA (19)
. Recently, we have demonstrated that RI has a structural interaction with the ligand-activated EGFR through the Grb2 protein, allowing a cross-talk between PKAI and EGFR (5
, 19)
. On this basis, we have proposed and shown that a double blockade of EGFR and PKAI, caused by the chimeric MAb C225 (20)
and a selective PKAI inhibitor, results in a cooperative growth inhibitory effect in different cancer cell types, providing the rationale for a novel therapeutic strategy (19
, 21
, 22)
.
Recent studies have demonstrated that the intracellular signaling cascade plays a role in the induction of apoptosis. On this regard, it has been demonstrated that phosphorylation of bcl-2 and induction of apoptosis caused by the microtubule-interacting drugs paclitaxel and vincristine involves mitogenic signaling proteins such as raf-1 (23) . Most recently, it has been demonstrated that the bcl-2 phosphorylation induced by paclitaxel and vincristine is mediated by PKA (24) .
In the present study, we have used for the first time HYB 165, a novel hybrid antisense RI MBO containing 2'-O-methyl-ribonucleotides, which exhibits improved pharmacokinetic properties and better bioavailability in vivo and is now entering clinical evaluation in Phase I trials (25)
. The aim of our study was to evaluate whether this MBO exerts any cooperative effect with docetaxel, a very effective drug against breast cancer (26)
, and/or MAb C225, on the growth of ZR-75-1 human breast cancer cells, which have an activated TGF/EGFR autocrine pathway (16)
.
We have demonstrated that the novel MBO HYB 165 inhibits the growth of ZR-75-1 at submicromolar concentrations, exhibits a synergistic growth inhibitory activity with docetaxel or MAb C225 and, remarkably, that an even higher degree of cooperativity can be obtained when the three agents are used together. Moreover, we have demonstrated that HYB 165, as well as docetaxel and MAb C225, induces bcl-2 phosphorylation and that the cooperative antiproliferative effect of the three agents together, used at suboptimal doses, results in increased expression of phosphorylated bcl-2 and apoptosis. Our studies provide a rationale for the translation of this combination strategy in a clinical setting.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MBOs.
The oligonucleotides targeted against the NH2-terminal codons 8–13 of the RI regulatory subunit of PKA (12)
used in the study are HYB 165, GCGUGCCTCCTCACUGGC and HYB 508, GCAUGCTTCCACACAGGC. The different oligos contain PS- internucleotide linkages (identified by roman type for the nucleosides flanking each position) and 2'-O-Methylribonucleosides modifications (identified by italics type). HYB 508 is a control oligonucleotide for HYB 165, containing four mismatched nucleosides, as underlined. The oligonucleotides have been synthesized by the protocol described earlier (14)
. The identity and purity of the oligonucleotides was confirmed by 31P NMR, capillary gel electrophoresis, hybridization melting temperature, and A269:mass ratio (14)
.
Cell Growth Experiments.
ZR-75-1 cells were maintained in DMEM (Flow Laboratories, Irvine, Scotland) and supplemented with 10% heat-inactivated fetal bovine serum, 20 mM HEPES (pH 7.4), 5 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (Flow Laboratories). Cells were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37°C. For cell growth experiments in soft agar, 104 cells/well were seeded in 24-multiwell cluster dishes, as previously described, (4)
and treated with different concentrations of docetaxel (day 0). The antisense RI MBOs were added after 12 h (day 1) and on day 3. For the experiments with the MBOs in combination with the MAb C225, cells were treated with various concentrations of MAb C225 and/or the antisense oligos on day 1 and day 3. Twelve days after the last treatment, cells were stained with nitroblue tetrazolium (Sigma), and colonies >0.05 mm were counted (11)
.
Western Blot Analysis.
Total cell lysates (50 µg) were fractionated through 7.5% or 12% SDS-polyacrylamide gels, transferred to nitrocellulose filters, incubated with specific MAbs, followed by horseradish peroxidase antiserum (Bio-Rad Laboratories, Milano, Italy). Immunoreactive proteins were visualized by enhanced chemiluminescence (Amersham International, England), as described previously (11)
. Antihuman RI mouse MAb was from Transduction Laboratories (Lexington, KY). Antihuman bcl-2 mouse MAb was from Santa Cruz Biotechnology (Santa Cruz, CA).
Flow-Cytometric Analysis of Cell Cycle and Apoptosis.
Cells seeded in monolayer in 6-well dish clusters were treated with docetaxel (day 0). The MBO and/or the MAb C225 were added after 24 h (day 1), and the treatment was repeated on day 3. After 4 days, cells were harvested, fixed in 70% ethanol, washed in PBS, and mixed with RNase (Sigma Chemicals, Milan, Italy) and a propidium iodide solution (Sigma). DNA content for cell cycle analysis and apoptotic cell death was analyzed after the method previously reported (27)
, using a FACScan flow-cytometer (Becton Dickinson, Mountain View, CA) coupled with a Hewlett Packard computer. Cell cycle data analysis was performed by the CELL-FIT program (Becton Dickinson) and apoptotic cell death was analyzed using the FL2H signal in logarithmic scale by the LYSYS software (Becton Dickinson).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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regulatory subunit of PKAI (12)
. When ZR-75-1 human breast cancer cells growing in soft agar were treated with the 2'-O-methyl-RNA-modified MBO HYB 165, a dose-dependent inhibition of colony formation was observed, whereas HYB 508, a control mismatched oligo, caused a little or no growth inhibition at the same doses tested (Fig. 1A)
.
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protein expression, whereas HYB 508 did not affect RI levels (Fig. 1B)
.
We have previously shown that a different MBO antisense RI exerts a synergistic effect with a different class of cytotoxic drugs, including taxanes. Docetaxel is one of the most active cytotoxic drugs in the treatment of breast cancer patients. Docetaxel has a dose-dependent inhibitory effect on soft agar growth of ZR-75-1 cells at doses ranging between 0.01 and 0.3 nM(Fig. 2A)
. We have selected two suboptimal doses of HYB 165, 0.1 µM and 0.5 µM, which cause about 5% and 10–15% cell growth inhibition, respectively, to study whether any cooperative effect could be obtained with different doses of docetaxel. We have found such cooperative effect at all doses tested, obtaining a stronger synergistic effect with lower doses of docetaxel (Fig. 2A)
. In fact, 0.1 µM HYB 165, combined with 0.01 nM docetaxel, which alone causes about 15% growth inhibition, determined over 40% cell growth inhibition. This cooperative effect corresponds to a synergism quotient of 
2.0, defined as the net growth inhibitory effect of a drug combination divided by the sum of the net individual drug effects (28)
. The same dose of docetaxel combined with 0.5 µM HYB 165, which could determine about 30% growth inhibition if an additive effect is expected, caused about 70% inhibition of colony formation. Conversely, no more than an additive effect was observed when ZR-75-1 cells were treated with docetaxel in combination with all doses of the control mismatched MBO, HYB 508 (Fig. 2A)
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12%, at the dose of 0.25 µg/ml, and 80%, at the dose of 2.5 µg/ml (Fig. 2, B and C)
. When we used HYB 165 at the suboptimal doses of 0.1 µM or 0.5 µM, we observed a supra-additive growth inhibitory effect with all doses of MAb C225. A particularly relevant cooperativity was obtained when the lowest dose of MAb C225, 0.25 µM, was combined to either 0.1 µM HYB 165 or 0.5 µM HYB 165. In contrast, no cooperative inhibitory effect was obtained when MAb C225 was used in combination with the control MBO HYB 508 (Fig. 2B)
.
When we treated ZR-75-1 cells with MAb C225 in combination with different doses of docetaxel, ranging between 0.01 and 0.3 nM, we observed a cooperative inhibitory effect with 0.25 µg/ml MAb C225, whereas a mostly additive effect was achieved when MAb C225 was used at the higher dose (Fig. 2C)
.
Because these data suggest that each single agent can cooperatively inhibit cell growth when used in combination, we then studied whether the three different classes of compounds, anti-RI MBO, MAb C225, and docetaxel, used together could still retain a cooperative growth inhibitory effect. We found that combination of the three agents at the lowest doses caused an even greater synergistic effect than that observed combining two agents. In fact, although each single agent used alone determined an average 10% inhibition of colony formation, the combination of the three altogether caused over 90% inhibition of soft agar growth of ZR-75-1 cells, achieving a synergism quotient of about 3.0 (Fig. 3)
.
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. When either compound was used in combination with any other, an additive effect could be obtained. Remarkably, an additive effect was still obtained when the three compounds were used altogether, causing almost 80% of apoptotic cell death. Moreover, surviving cells appeared prevalently accumulated in G2-M (Fig. 4)
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. Therefore, these results correlate with the cooperative effect observed on the apoptosis.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit have shown promising results in inhibiting human cancer cell growth in vitro and in vivo (9, 10, 11, 12
, 15)
. In the present study, we have demonstrated that HYB 165, a novel DNA/RNA hybrid MBO with improved pharmacokinetic and bioavailability properties in vivo, exhibits a dose-dependent inhibitory effect on soft agar growth of ZR-75-1 breast cancer cells.
We have also shown that HYB 165, but not its control oligo HYB 508, has a synergisitic inhibitory effect on ZR-75-1 colony formation when used in combination with docetaxel, a cytotoxic drug very active in breast cancer patients (26) . The antiproliferative activity is accompanied by an additive apoptotic effect, as compared with each single agent alone, and by cell accumulation in G2-M phase, suggesting a prevalence of docetaxel-induced effect on cell cycle regulation.
A relevant role in tumor pathogenesis and progression is played by the EGFR (16, 17, 18)
, therefore, several tools have been devised to selectively inhibit the EGFR-mediated signaling pathway (29)
. It has been demonstrated that the chimeric anti-EGFR MAb C225 is able to inhibit the growth of several cancer cell types and to cooperate with cytotoxic drugs in vitro and in vivo (30
, 31)
. Moreover, the growth inhibitory effect is associated to G0–G1 arrest and, in some cases, to induction of apoptosis (32)
. We have shown here that MAb C225 is able to induce bcl-2 phosphorylation and apoptosis in ZR-75-1 cells. On the basis of our previous demonstration of the interactions between the EGFR and PKAI (19)
, we have used HYB 165 in combination with the anti-EGFR antibody MAb C225, demonstrating a marked synergistic antiproliferative effect on ZR-75-1 soft agar growth. When used in combination with HYB 165, a cooperative apoptotic effect and cell accumulation in G0–G1 phases were observed. These data further support the hypothesis and recent demonstration that the double blockade of PKAI and EGFR tyrosine kinase pathways may represent an important therapeutic approach (19
, 21)
. We have recently shown that the same MAb C225 exerts a synergisitic inhibition of human renal cancer cells growth in vitro and in vivo, when combined with another MBO antisense, RI (22)
. Therefore, such cooperative effect is a more general phenomenon not restricted to the breast cancer cells.
Finally, we have demonstrated that a greater degree of cooperativity can be obtained combining all three agents together, achieving an almost complete suppression of breast cancer cell growth with low doses of each single agent. This effect is associated with an increase of the phosphorylated bcl-2 protein and induction of apoptotic cell death in 80% of ZR-75-1 cells.
A recent study has demonstrated for the first time that PKA is directly implicated in the mechanism of apoptosis and in bcl-2 phosphorylation and inactivation by microtubule interacting agents (24) . We have hypothesized that the selective inhibition of PKAI, by interfering with the transduction of mitogenic signaling, may induce phosphorylation of bcl-2 and subsequent apoptosis. We have shown here that HYB 165, as well as the taxane docetaxel, increases the expression of phosphorylated bcl-2 and induces apoptosis. Moreover, it causes cell accumulation in G0–G1, supporting our previous demonstration of a role for PKAI in the G1>S transition (2 , 19) .
This is the first study of a relevant cooperative antitumor effect obtained by combining a specific cytotoxic drug, an anti-EGFR antibody, and a second generation oligonucleotide targeting PKAI. Moreover, we have shown that the growth inhibitory effect is associated to increase of bcl-2 phosphorylation and apoptosis.
It has been proposed that a new approach to increase antitumor activity of cytotoxic drugs is their combination with biological agents, rather than enhancing their doses and their toxicity (33) . On this regard, several studies have recently demonstrated a cooperative effect of cytotoxic drugs with antisense oligonucleotides, inhibiting the expression and function of mitogenic proteins (15 , 34, 35, 36) . Our results demonstrate that this concept can be further improved by combining cytotoxics and biological agents that selectively target signal transduction pathways and that interfere with different, but related, targets. In this respect, because HYB 165 has recently entered clinical evaluation and MAb C225 is in Phase II trials, their combination with docetaxel may represent an opportunity to translate these findings into a novel strategy for the treatment of breast cancer patients.
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FOOTNOTES |
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1 Supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (AIRC) and from the Consiglio Nazionale delle Ricerche (CNR) Target Project on Biotechnologies. 
2 To whom requests for reprints should be addressed, Cattedra di Oncologia Medica, Dipartimento di Endocrinologia e Oncologia Molecolare e Clinica, Università di Napoli Federico II, Via Pansini, 5, 80131 Napoli, Italy. Phone: 39-081-7462061; Fax: 39-081-7462066.
3 The abbreviations used are: PKA, protein kinase A; TGF, transforming growth factor ; EGF, epidermal growth factor; EGFR, EGF receptor; PS-, phosphorothioate; MBO, mixed-backbone oligonucleotide; MAb, monoclonal antibody.
Received 8/ 5/98; revised 11/ 8/98; accepted 11/25/98.
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REFERENCES |
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subunit of protein kinase A controls serum dependency and entry into cell cycle of human mammary epithelial cells. Oncogene, 9: 3233-3240, 1994.[Medline]
production in Ki-ras-transformed rat fibroblasts. FEBS Lett., 242: 363-367, 1989.[Medline]
transformation of mammary epithelial cells by restoration of the normal mRNA patterns for cAMP-dependent protein kinase regulatory subunit isoforms which show disruption upon transformation. J. Biol. Chem., 265: 1016-1020, 1990. subunit of protein kinase A (PKA) binds to Grb2 and allows PKA interaction with the activated EGF-receptor. Oncogene, 14: 923-928, 1997.[Medline]
subunit of cyclic AMP-dependent protein kinase induces growth inhibition in human cancer cells. Cancer Res., 53: 868-872, 1993. subunit of the cAMP-dependent protein kinase induces growth inhibition of human mammary epithelial cells transformed by c-Ha-ras and c-erbB-2 protooncogenes. Int. J. Cancer, 53: 438-443, 1993.[Medline]
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, HYB 165; 
, HYB 165 + Docetaxel; 
, Docetaxel; 
, HYB 508; 
, HYB 508 + Docetaxel. B, HYB 165 (0.1 µM; a–d) and 0.5 µM HYB 165 (e and f) or 0.5 µM HYB 508 (i–l) in combination with MAb C225 at doses of 0.25 µg/ml (a, e, and i), 0.5 µg/ml (b, f, and j), 1 µg/ml (c, g, and k), and 2.5 µg/ml (d, h, and l). 
, and 
