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Clinical Cancer Research Vol. 5, 215-220, January 1999
© 1999 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Synergistic Interaction between Paclitaxel and 8-Chloro-adenosine 3',5'-Monophosphate in Human Ovarian Carcinoma Cell Lines1

Hayley M. McDaid and Patrick G. Johnston2

Department of Oncology, Queens University of Belfast, Belfast City Hospital, Belfast, Northern Ireland, United Kingdom BT9 7AB


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Taxol is a unique anticancer agent that is used in treatment of advanced ovarian cancer. Taxol exposure results in the polymerization and stabilization of the microtubule skeleton of eukaryotic cells, hence blocking replication and intracellular motility. 8-Chloro-adenosine 3',5'-monophosphate (8-Cl-cAMP) is a cAMP analogue, currently in Phase II clinical trials, that displays growth inhibition at micromolar concentrations. The aim of this study was to assess the nature of the interaction between 8-Cl-cAMP and paclitaxel using the combination index (CI) method of Chou and Talalay, which uses the median-effect analysis. Two ovarian cancer cell lines, A2780 and OAW42, which differ in sensitivity to both drugs, were tested using the fixed-ratio design using various scheduling regimens. Concurrent exposure of both drugs resulted in highly synergistic interactions in both cell lines. CIs (mean ± SE) with this schedule were 0.182 ± 0.016, 0.315 ± 0.32, and 0.618 ± 0.637 at 20, 50, and 80% cell kill, respectively, in A2780 cells and 0.001 ± 0.0009, 0.016 ± 0.0075, and 0.184 ± 0.168 at 20, 50, and 80% cell kill, respectively, in OAW42 cells. In both cell lines, synergy was effective over a 4-fold log range of concentration for either drug. Sequencing with paclitaxel for 24 h prior to 8-Cl-cAMP was the most effective regimen; it resulted in consistently low CIs of up to the 90% cell kill level for both cell lines. Exposure to 8-Cl-cAMP prior to paclitaxel was the least effective regimen. In conclusion, the combination of paclitaxel and 8-Cl-cAMP is highly synergistic in ovarian carcinoma cell lines, suggesting that 8-Cl-cAMP may stimulate the antitumor effect of the taxanes.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Paclitaxel (Taxol), isolated from the bark of the Pacific yew tree Taxus brevifolia (1) , is a novel anticancer agent that has impressive antitumor activity against a range of human tumors (2) and, in particular, breast (3) and ovarian carcinomas (4) . It was approved for Phase I clinical trial in 1983 despite difficulties regarding its solubility and supply. Currently, it is the most frequently used chemotherapeutic drug in the treatment of recurrent ovarian carcinoma (5) , following its approval by the Food and Drug Administration in 1992.

Unlike other microtubule-binding drugs, Taxol has a unique mechanism of action. It binds specifically to the ß-subunit of tubulin at two sites: amino acid residues 1–31 (6) and 217–231 (7) . This results in the disruption of the microtubule cytoskeleton via the stabilization of tubulin polymers and displaces the normal cycle of tubulin polymerization and depolymerization. Cells exposed to Taxol become growth arrested at the G2-M phase of the cell cycle (8) , or they proceed through replication at greatly reduced rates. It has also been recently demonstrated that, apart from the microtubule system, Taxol also modulates the activities of key proteins that play fundamental roles in cellular signaling, such as Raf-1 (9) , Bcl-2 (10) , mitogen-activated protein kinase (11) , and tumor necrosis factor-{alpha} (12) .

8-Cl-cAMP3 is a cAMP analogue that was approved for Phase I clinical trial by the National Cancer Institute in 1988, representing the first cAMP analogue to enter clinical trials in over 30 years of research in this field. 8-Cl-cAMP has undergone two Phase I trials using continuous low-dose infusion (13 , 14) . The potency of 8-Cl-cAMP has been attributed to its ability to discriminate between the two isoforms of PKA, type I PKA, composed of RI subunits, and type II PKA, which are composed of RII subunits (15 , 16) . Overexpression of type I PKA have been observed in a spectrum of human cancer cell lines and tumors (17) , and for this reason, they are associated with cellular proliferation and transformation. 8-Cl-cAMP is unable to activate type II PKA, resulting in an overall lowering of the type I/type II intracellular ratio, and the restoration of "normal" PKA protein levels in tumor cells (16) .

The acquisition of drug resistance is a common occurrence in ovarian epithelial carcinoma, often leading to chemotherapeutic failure. The term MDR is used to describe the development of cross-resistance to structurally and functionally unrelated drugs following exposure of cells to a single hydrophobic anticancer agent. MDR is characterized by the overexpression of P-glycoprotein, which functions as an energy-dependent efflux pump (18) . Several studies have demonstrated that 8-Cl-cAMP modulates the activity of MDR 1 (19 , 20) , and sequence analysis of the gene has identified two activator protein-2 elements in exon 1a, which mediate transcriptional activation of MDR 1 following PKA activation (21) . These observations provide a rationale for the evaluation of 8-Cl-cAMP in combination with Taxol.

The aims of this study were to investigate whether 8-Cl-cAMP modulates the cytotoxicity of paclitaxel and to investigate whether the combination demonstrated schedule dependency. We assessed this combination in two human ovarian carcinoma cell lines that differ in (a) endogenous type I PKA expression levels and (b) cytotoxicity to both 8-Cl-cAMP and paclitaxel.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
Experiments were conducted using the human ovarian carcinoma cell lines OAW42 (22) and A2780, which were supplied by Dr. R. I. Freshney (Glasgow University, Glasgow, Scotland). All cell lines were maintained in RPMI 1640 (Life Technologies Inc.) supplemented with 10% heat-inactivated FCS (Imperial), penicillin-streptomycin, sodium pyruvate (both 100 mg/ml), and insulin (10 mg/ml) in a humidified 5% CO2 atmosphere.

Drugs.
Paclitaxel (Taxol) was supplied by Bristol Myers Squibb United Kingdom, at a concentration of 6 mg/ml dissolved in dehydrated alcohol and polyethoxylated caster oil, which was stored at room temperature. 8-Cl-cAMP (sodium salt) was obtained from ICN (Irvine, CA). 8-Cl-cAMP was dissolved in sterile PBS to make a stock solution of 1 x 10-3M, which was filter sterilized through a single-use, nonpyrogenic, 0.22-µm filter (Sartorius AG, Goettingen, Germany). The stock solution was stored at -70°C. Drugs were diluted from stock solutions into medium to reconstitute the desired concentrations.

Determination of IC50s for 8-Cl-cAMP and Paclitaxel.
Cells growing in log phase were harvested by trypsinization, washed, and seeded at 2 x 103 cells per well into 96-well plates (Life Technologies, Inc.), and allowed an overnight period for attachment. Each experiment was allocated 10 wells containing drug-free medium for the control. The IC50 doses for 8-Cl-cAMP and paclitaxel were determined for each cell line by exposure to various concentrations of either drug (10-4–10-8M) for 72 h. The medium-containing drug was decanted, and cell numbers were determined using either the Coulter Counter (Coulter Electronics Ltd., Luton, United Kingdom) or the MTT reduction assay described below. Cell numbers (or A) were plotted against the log of the drug concentration, and the IC50 derived as that concentration required for 50% reduction in cell growth. Each experiment was performed on at least three separate occasions.

Median Effect Analysis.
The nature of the interaction observed between 8-Cl-cAMP and paclitaxel was analyzed using the software Calcusyn, which uses the CI method of Chou and Talalay (23) , based on the multiple drug effect equation. This analysis requires (a) that each drug alone has a dose-effect relationship and (b) that at least three or more data points for each single drug are available in each experiment. The constant ratio combination design was chosen to assess the effect of both drugs in combination, in which dose-response curves were determined with both drugs in combination, at a fixed ratio equivalent to the ratio of their IC50s. The advantage to this method is the automatic construction of a fraction affected-CI table, graph, and classic isobologram by the software. CIs of <1 indicate greater than additive effects (synergism; the smaller the value, the greater the degree of synergy), CIs equal to 1 indicate additivity, and CIs >1 indicate antagonism. Each CI ratio represented here is the mean value derived from at least three independent experiments.

Concurrent Exposure to 8-Cl-cAMP and Paclitaxel.
Cells were seeded into 96-well plates, as described previously. For each experiment, 10 wells were used to assess the drug-free control, and 5 wells allocated for each drug treatment. Cells were treated with six different concentrations of the single drugs 8-Cl-AMP and paclitaxel and again with six different concentrations of both drugs, at their equipotent ratio. Cells were exposed for 72 h, after which growth inhibition was measured using the MTT assay.

Sequential Exposure to 8-Cl-cAMP and Paclitaxel.
Using the same experimental setup described above, we treated cells with six different concentrations of the single drugs 8-Cl-cAMP and paclitaxel for 24 h. This period was used for pretreatment to assess the effects of drug sequencing on growth inhibition. This medium was decanted after 24 h, and fresh medium containing either 8-Cl-cAMP or paclitaxel at different concentrations were added to the relative wells for 48 h. Growth inhibition was determined using the MTT assay.

MTT Thiazolyl Blue Test.
Viable cell growth was determined by the MTT reduction assay (24) . MTT (Sigma Chemical Co., St. Louis, MO) was dissolved in sterile distilled water to a stock concentration of 5 mg/ml and stored in aliquots at -20°C. Following treatment, the medium was replaced with drug-free medium, and MTT was added at a 1/10 volume. After incubation for 2–3 h at 37°C, the supernatants were carefully aspirated, and 1/4 volume of DMSO (Sigma) was added to each well, and the plates agitated on a shaker (Rotatest R100, Luckham Ltd., Burgesshill, Suffolk, United Kingdom) for 10 min to dissolve the crystal product. Absorbances were measured at 570 nm using an SLT spectra multiscan (Alpha Analytical). For each experimental set, the mean absorbances ± SE were determined, and the blank readings (wells containing DMSO only) were subtracted to give final values. Growth inhibition was expressed as a percentage of the untreated control, and all values were converted to ratios of 1.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Determination of IC50 Doses with 8-Cl-cAMP and Paclitaxel.
Table 1Citation summarizes the IC50 doses for both A2780 and OAW42 cell lines following treatment with 8-Cl-cAMP and paclitaxel. This allowed a constant ratio combination design to be used for assessment of the drug combinations. A2780 cells were treated at 8-Cl-cAMP concentrations of 20–0.001 µM and paclitaxel concentrations of 3.2–0.00016 µM. OAW42 cells were treated with concentrations of 8-Cl-cAMP ranging from 200 to 0.01 µM and concentrations of paclitaxel ranging from 58 to 0.0029 nM. Because each cell line displayed different sensitivities to both drugs, the fixed molar ratio for both cell lines differed.


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  		Table 1 IC50 doses of 8-Cl-cAMP and paclitaxel

 
Interaction between 8-Cl-cAMP and Paclitaxel.
The dose-response curves for A2780 cells and OAW42 cells exposed to 8-Cl-cAMP, paclitaxel, and the simultaneous combination of both at equipotent ratios for 72 h are shown in Fig. 1, A and BCitation , respectively. For both cell lines, the drug combinations gave a more negative slope than either 8-Cl-cAMP or paclitaxel alone, which suggested an interaction between the two drugs. Because both drugs were cytotoxic as single agents, the precise nature of this interaction was investigated using the median effect analysis of Chou and Talalay (23) .



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  		Fig. 1. Percentage survival as a function of drug concentration for A2780 cells (A) and OAW42 cells (B) exposed to 8-Cl-cAMP ({blacksquare}) for 72 h, paclitaxel (x) for 72 h, and the combination of both drugs at their equipotent ratios ({circ}) for 72 h. Data points, means of at least three independent experiments; bars, SE.

 
Simultaneous (72 h) Exposure to 8-Cl-cAMP and Paclitaxel.
Fig. 2, A and BCitation , shows the CI plots for A2780 cells and OAW42 cells, respectively, which were simultaneously exposed to 8-Cl-cAMP and paclitaxel at equipotent molar ratios for 72 h. The curves demonstrate synergy between both drugs in both cell lines. The CI values are summarized in Table 2Citation , all of which were below 1, indicating greater than additive effects or synergism. At all three levels of cell kill (20, 50, and 80%), the CI ratios were significantly less than 1 (P < 0.05) for both cell lines, although the CI values obtained using higher doses of drugs resulted in higher CI indices. Moreover, the CI values obtained for OAW42 cells indicated a highly synergistic effect for this drug combination. The synergy observed with concurrent 8-Cl-cAMP and paclitaxel exposure for 72 h, for both cell lines, was apparent over a range of 1 log of cell kill and a 4-fold log concentration of both drugs.



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  		Fig. 2. CI as a function of cell kill in A2780 cells (A) and OAW42 cells (B) exposed simultaneously to 8-Cl-cAMP and paclitaxel at their equipotent ratios for 72 h. Data points, means of at least three independent experiments; bars, SE.

 

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  		Table 2 Summary of CI values at 20, 50, and 80% fraction affected

 
Sequential Exposure to 8-Cl-cAMP (24 h) Followed by Paclitaxel (48 h).
Fig. 3, A and BCitation , illustrates the CI plots obtained for A2780 and OAW42 cells, respectively, when cells were initially exposed to 8-Cl-cAMP, followed by paclitaxel for 48 h using the equipotent ratios described earlier. Both cell lines displayed similar effects, wherein CI values were lowest at the 0.02% cell kill level and increased to CI ratios of >1 at high cell kill levels. Synergy was evident at cell kill values of 0.02–50% for A2780 cells and 0.02–80% in OAW42 cells. This regimen became antagonistic at cell kill values exceeding these limits, and the effect was more pronounced in A2780 cells.



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  		Fig. 3. CI as a function of cell kill in A2780 cells (A) and OAW42 cells (B) exposed first to 8-Cl-cAMP for 24 h, followed by paclitaxel for 48 h, at their equipotent ratios.

 
Sequential Exposure to Paclitaxel (24 h) Followed by 8-Cl-cAMP (48 h).
Fig. 4, A and BCitation , illustrates the CI plots obtained for A2780 and OAW42 cells, respectively, when cells were treated with paclitaxel for 24 h followed by 8-Cl-cAMP for 48 h. The effects were similar for both cell lines, and CI values remained consistent at 0–80% cell kill levels. The consistent synergy effect was more apparent in A2780 cells in which CI indices of 0.39–0.62 were obtained from the 20–95% cell kill level. Beyond the 95% cell kill level, the effects were additive. In OAW42 cells, CI values ranged from 0.346 to 0.471 for 20–80% cell kill levels. This regimen became antagonistic at levels exceeding the 95% cell kill boundary, although overall, the median CI ratio obtained was lower for OAW42 cells than it was for A2780 cells.



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  		Fig. 4. CI as a function of cell kill in A2780 cells (A) and OAW42 cells (B) exposed first to paclitaxel for 24 h, followed by 8-Cl-cAMP for 48 h, at their equipotent ratios.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Paclitaxel is one of the most promising chemotherapeutic drugs to enter the clinical arena; however, the optimal dose and schedule for the drug remain to be established and are the subject of ongoing clinical trials (25) . The combination of paclitaxel with platinum compounds is the optimal first-line chemotherapy following primary debulking surgery for ovarian cancer (26) , and in vitro studies have also demonstrated synergism between the two drugs (27) . There is a need to develop innovative approaches for the treatment of advanced primary and recurrent ovarian cancer by developing and identifying clinically active agents that overcome resistance and inhibit oncogenic signal transduction pathways. The purpose of this study was to assess the cytotoxic interaction between 8-Cl-cAMP and paclitaxel in two human ovarian cancer cell lines with a view to defining the potential role, if any, of this combination in future clinical trials.

The data presented here show a highly synergistic interaction between 8-Cl-cAMP and paclitaxel for both cell lines, which displayed significant schedule dependency biased toward preexposure to paclitaxel. Simultaneous and sequential administration of both drugs had synergistic effects in both cell lines that were evident over a 4-fold log concentration of either drug.

Overall, OAW42 cells were more sensitive to the 8-Cl-cAMP/paclitaxel combination, regardless of schedule, and the lowest CI ratios were obtained for the 72 h concurrent exposure. A2780 cells also demonstrated synergy with this drug combination; however, mean CIs were higher than those obtained for OAW42, and the synergistic effect was less potent. Also notable were the consistent CI ratios obtained when cells were preexposed to paclitaxel prior to 8-Cl-cAMP, which remained uniform up to the 90% cell kill level; therefore, we propose that this would be the most acceptable sequence for evaluation in clinical trials.

Plasma concentrations of 8-Cl-cAMP following continuous i.v. infusion in Phase I trial yielded concentrations of 2–5 µM(14) , whereas Taxol yielded plasma concentrations of 0.95–8 µM (275 mg/m2 constant infusion over 24 h–275 mg/m2 over 6 h; Ref. 28 ). Hence, the attainable plasma concentrations for both drugs are well within the range that we have used here to demonstrate synergism. The clinical evaluation of paclitaxel (Taxol) has been impeded by unexpected hypersensitivity reactions observed in some patients. This disadvantage makes a strong case for the introduction of low dose or short infusion times for paclitaxel in combination with 8-Cl-cAMP in clinical trial without compromising the efficacy of the treatment. Furthermore, this regimen would reduce the potentially toxic effects of this synergistic schedule on normal tissue. This study has only addressed the combination of both drugs at their equipotent ratios, although in both cell lines, the dose of paclitaxel evaluated was considerably less than the 8-Cl-cAMP component. The optimal dose schedule for this drug combination remains to be determined in future studies.

While this work was being completed, a similar study was reported in which 8-Cl-cAMP was found to synergize with paclitaxel and cisplatin in a series of human cancer cell lines (29) . This study addressed only the combination of paclitaxel prior to 8-Cl-cAMP in vitro and related the degree of synergism in terms of a quotient defined as the ratio of growth inhibitory effects of the drug combination: the sum of the individual drug effects. Moreover, the concentrations of 8-Cl-cAMP used here to demonstrate synergy were 10–100 times less than those concentrations evaluated in the above study. Nevertheless, both studies confirm the highly synergistic effect of this drug combination and highlight its potential in future clinical trials.

We have not proposed a mechanism for the observed synergy between 8-Cl-cAMP and paclitaxel. Neither cell line expressed P-glycoprotein (data not shown), excluding 8-Cl-cAMP-induced modulation of drug accumulation as a mechanism for the interaction. It has been shown that 8-Cl-cAMP exposure, like Taxol, results in the accumulation of cells at G2-M (30) . It is possible that sequential exposure of Taxol prior to 8-Cl-cAMP simply exposes a greater proportion of cells to the cytotoxic action of 8-Cl-cAMP, resulting in enhanced cell kill effects. However, this does not explain the high degree of synergy observed at low concentrations of both drugs when a G2-M block is not apparent. It has been demonstrated that cytotoxic drugs that disrupt the microtubule skeleton induce Raf-1 activation and Bcl-2 hyperphosphorylation (31) . The level of paclitaxel-induced apoptosis appears to be dependent on Raf-1 kinase activity, and in tumorigenic epithelial cells (which account for >90% of all human malignancies), evidence suggests that high Raf-1 kinase activity protects against paclitaxel-induced cytotoxicity (32 , 33) .

Recent evidence suggests that the paclitaxel-induced hyperphosphorylation and inactivation of Bcl-2 leading to apoptosis is mediated through the cAMP-dependent protein kinase (34) . 8-Cl-cAMP and forskolin, like paclitaxel and vincristine, induced Bcl-2 hyperphosphorylation and the addition of the PKA inhibitor, adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer-cAMP inhibited this effect and prevented the induction of apoptosis. We did not address whether the PKA-dependent hyperphosphorylation of Bcl-2 in these cell lines involved Raf-1, because it has been shown previously that Raf-1 kinase activation is necessary for Bcl-2 inactivation (31) . Cooperativity exists between the PKA and mitogen-activated protein kinase signaling pathways, specifically via Raf-1, the result of which appears to be the triggering of cells into apoptosis. An increase in the proportion of hypodiploid cells following exposure to paclitaxel and 8-Cl-cAMP has been demonstrated, compared to either drug alone (29) .

It has also been shown that the activated catalytic subunit of PKA phosphorylates and partially degrades stathmin (Op18 or p19), resulting in an increase in the cellular content of microtubule polymers (35) . Op18 is a phosphorylation-responsive regulator of microtubule dynamics, which opposes the microtubule-stabilizing activity of microtubule-associated proteins (36) . 8-Cl-cAMP preferentially dissociates type I PKA, leading to an increase in activated catalytic subunits. From these observations, we suggest a model in which 8-Cl-cAMP exposure inactivates Op18, resulting in increased tubulin polymerization. This potential alteration in microtubule dynamics would result in an increased proportion of stabilized tubulin polymers following exposure to Taxol because Taxol only binds to polymers of tubulin. This model provides an interesting alternative mechanism for the synergy we have observed with 8-Cl-cAMP and paclitaxel and also highlights the functional diversity of PKA. This model will form the basis for future mechanistic studies.

In conclusion, we have demonstrated a highly synergistic interaction between 8-Cl-cAMP and paclitaxel in two human ovarian carcinoma cell that is evident over a 4-fold log of either drug concentration. Synergy was evident for all drug combinations evaluated; however, we propose that sequencing with paclitaxel prior to 8-Cl-cAMP exposure would be the most favorable regimen for investigation in future clinical trials due to the consistently high degree of synergy obtained for a range of doses evaluated. These data highlight the potential of antitumor treatments that target the cAMP-dependent protein kinase and suggest that the dual targeting of cooperative signaling systems by different cytotoxic drugs is a powerful technique for improving chemotherapeutic potency.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by grants from the Ulster Cancer Foundation and the Department of Education for Northern Ireland. Back

2 To whom requests for reprints should be addressed, at Department of Oncology, U-Floor, Belfast City Hospital, The Queens University of Belfast, Northern Ireland, United Kingdom BT9 7AB. Phone: 44-1232-263911; Fax: 44-1232-263744; E-mail: oncology{at}qub.ac.uk Back

3 The abbreviations used are: 8-Cl-cAMP, 8-chloro-adenosine 3',5'-monophosphate; PKA, protein kinase A; MDR, multidrug resistance; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CI, combination index. Back

Received 9/ 2/98; accepted 10/23/98.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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