Clinical Cancer Research The Future of Cancer Research: Science and Patient Impact
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cho, Y. S.
Right arrow Articles by Cho-Chung, Y. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cho, Y. S.
Right arrow Articles by Cho-Chung, Y. S.
Clinical Cancer Research Vol. 9, 1171-1178, March 2003
© 2003 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Antisense Protein Kinase A RI{alpha} Acts Synergistically with Hydroxycamptothecin to Inhibit Growth and Induce Apoptosis in Human Cancer Cells

Molecular Basis for Combinatorial Therapy

Yee Sook Cho and Yoon S. Cho-Chung1

Cellular Biochemistry Section, Basic Research Laboratory, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892-1750


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: The increased expression of RI{alpha}, the regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase type I (PKA-I), has been correlated with cancer cell growth. An antisense oligonucleotide targeting the RI{alpha} subunit of PKA (antisense RI{alpha}) induces cell growth arrest, apoptosis, and differentiation in a variety of cancer cell lines in vitro and in tumors in vivo. This study investigated the utility of a combinatorial therapy consisting of the RNA-DNA second-generation RI{alpha} antisense HYB0165 (Gem231) and the cytotoxic drug hydroxycamptothecin (HCPT), which inhibits topoisomerase I.

Experimental Design: LS-174T colon carcinoma and PC3M androgen-insensitive prostate cancer cells were used as experimental models. The antitumor and apoptotic activities of Gem231 and HCPT, singly and in combination, were measured by cell growth assay, synergism quotient, cell morphology, nuclear morphology, levels of PKA R and C subunits, anti- and proapoptotic proteins, and PKA activity ratio.

Results: In a synergistic fashion, Gem231 and HCPT induced growth arrest, apoptosis, and changes in cell morphology; down-regulated RI{alpha} expression; down-regulated Bcl-2 and promoted its hyperphosphorylation; up-regulated the proapoptotic proteins Bax and Bad; and promoted hypophosphorylation of Bad. Antisense Gem231, but not HCPT, increased the PKA activity ratio, which measures the degree of PKA activation.

Conclusion: The results showed that PKA-I activation by Gem231 and topoisomerase I inhibition by HCPT are responsible at the molecular level for the synergistic effects of tumor cell apoptosis and growth inhibition. These results demonstrated the molecular basis for the use of Gem231 and HCPT as combinatorial therapy to treat human cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
cAMP2 -dependent PKA is the primary mediator of cAMP action in mammalian cells (1) . Researchers interested in the regulation of cell growth by cAMP have focused on the two isoforms of PKA, type I (PKA-I) and type II (PKA-II), which differ in their regulatory subunits, RI and RII, respectively (2) . Recent experimental evidence has shown that the balanced, intracellular expression between the two isoforms may play a critical role in the control of cell growth and differentiation, pointing to distinct functions for PKA-I and PKA-II. PKA-I is only transiently overexpressed in normal cells in response to the physiological stimulation of cell proliferation (3 , 4) . However, this isoform is constitutively overexpressed in cancer cells and is associated with a poor prognosis in human cancers of different cell types (5, 6, 7, 8, 9) . Conversely, PKA-II is preferentially expressed in normal, differentiated tissues (9, 10, 11) .

Antisense ODNs targeted against RI{alpha}, which differ in sequence or chemical modification, promote a sequence- and target-specific reduction in RI{alpha} mRNA and protein levels and inhibit cancer cell growth in a wide variety of different cell types both in vitro and in vivo (10, 11, 12, 13, 14, 15) . Growth inhibition is accompanied by changes in cell morphology and apoptosis.

In a recent study using cDNA microarrays, we have shown that in a sequence-specific manner, antisense RI{alpha} induces molecular signatures of cell differentiation (16) . These signatures underlie the reverse transformation observed in tumor cells treated with RI{alpha} antisense, leading to a new, reverted phenotype in which the tumor no longer grows. Unlike conventional chemotherapy regimens, which depend on the maximum tolerated dose of a given drug to achieve optimal tumor-cell kill, treatment regimens involving antisense ODNs may rely more on an optimal biological dose. The ultimate goal for therapeutic ODNs is their use as biological gene modulators for a long period of time with minimal-to-no toxicity. In that case, antisense ODNs would resemble cytostatic rather than cytotoxic drugs. As such, these ODNs can induce tumor cells to differentiate or revert, eventually leading to apoptosis, and can reduce or eliminate the chances of relapse in cancer patients after initial treatment. Thus, drugs such as RI{alpha} antisense can be used in combination with conventional cytotoxic drugs at their nontoxic, minimum doses.

The development of adjuvant chemotherapy has improved response rates for advanced or recurrent malignant diseases, but the overall impact of this therapy on survival has been minimal. Moreover, chemotherapy is complicated by toxicity and by the development of drug resistance. The development of new chemotherapeutic agents and new combination regimens is thus highly desirable. In recent years, antisense ODNs in combination with a variety of cytotoxic drugs (17, 18, 19, 20) have been studied in vitro and in vivo, and several antisense ODNs are now being tested as monotherapies and in combination with several cytotoxic drugs in human clinical trials (21, 22, 23) .

CPT analogues are a family of cytotoxic cancer therapeutic agents that inhibit topoisomerase I (24 , 25) . CPT is an S-phase-specific cytotoxic agent that induces cellular responses such as G2 arrest and transient inhibition of cell division attributable to the formation of reversible topoisomerase I-drug-DNA complexes (26, 27, 28, 29) . HCPT is more active and less toxic than other CPT analogues (30, 31, 32, 33, 34, 35) . Both in vitro and in vivo models have shown that CPT and its natural 10-hydroxy analogue, HCPT, have significant antitumor activity against human breast cancer (30) . The therapeutic effects of oral administration of HCPT and CPT have also been demonstrated in severe combined immunodeficient mice bearing human breast cancer xenografts (30) .

In the present study, we aimed to identify conditions that yield highly favorable interactions between the RNA-DNA second-generation antisense RI{alpha} Gem231 and the topoisomerase I inhibitor, HCPT. We chose HCPT for the present study as a model of topoisomerase-I inhibitor because it is readily accessible, and it is not a prodrug like CPT-11 (irinotecan), which requires in vivo activation to function. The results from this study will provide a basis for later in vivo analyses, which will be more predictive of clinical responses to this combination. Gem231 has been undergoing Phase-I and Phase-II clinical trials (21, 22, 23 , 36) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
Campothecin was obtained from Sigma Chemical Co. (St. Louis, MO) and dissolved in DMSO. Cell culture medium, fetal bovine serum, MEM nonessential amino acids, antibiotic-antimycotic, and phosphocellulose disk were purchased from Life Technologies, Inc. (Rockville, MD). cAMP, kemptide, and protein kinase inhibitor were purchased from Sigma (St. Louis, MO). [{gamma}-32P]ATP was purchased from ICN (Costa Mesa, CA). Antibodies against C{alpha}, RI{alpha}, RIIß, and Bad were purchased from Transduction Laboratories (Lexington, KY). Bcl-2, Bak, Bax, and phospho-Bad antibodies were obtained from Upstate Biotechnology (Lake Placid, NY). Anti-actin antibody was obtained from Oncogene Research Products (Cambridge, MA).

Second Generation Oligonucleotides.
The oligonucleotides used in the present study are RNA-DNA, mixed backbone ODNs. Gem231, 5-[GCGU]GCCTCCTCAC[UGGC]-3', is targeted against codons 8–13 of the RI{alpha} regulatory subunit of PKA (12) and contains four 2'-O-methyl-oligoribonucleosides (RNA) at both the 5'- and 3'-ends (bracketed). Control oligonucleotides are HYB0295, 5'[GCAU]GCTTCCACAC[AGGC]-3', a 4-base mismatched (underlined) form of Gem231, and HYB0674, 5'[NNNN]NNNNNNN[NNNN]-3', a scrambled sequence oligonucleotide, containing segments of 2'-O-methyl-RNA (bracketed) and N = A, T, C, or G. All of the internucleotide linkages are phosphorothioate. These oligonucleotides were kindly provided by Dr. Sudhir Agrawal (Hybridon, Inc.).

Cell Culture.
PC3M cells were grown in RMPI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 0.1 mM MEM nonessential amino acids (pH 7.4), and antibiotic-antimycotic. LS174T cells were grown in MEM (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal bovine serum, 0.1 nM MEM nonessential amino acids (pH 7.4, 2 mM glutamine, and antibiotic-antimycotic. The cells were maintained under a humidified atmosphere of 95% air-5% CO2 at 37°C.

Cell Growth Assay.
Cells were plated at 2 x 105 cells in 60-mm culture dishes. After 12 h of seeding, cells were washed with fresh medium, and Gem231 antisense RI{alpha} was added to culture medium, with or without HCPT, at different concentrations. To increase the delivery of oligonucleotides into the cells, transfection reagents N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl ammonium methyl sulfate (DOTAP) was used according to the manufacturer’s instructions (Roche Diagnostics Corporation). Mismatched and scrambled-sequence ODNs were used as control ODNs. Cell growth was assessed by total cell counts. Cells were harvested at the indicated times and counted with a ZI Coulter counter (Coulter Company, Miami, FL). The results were expressed as the mean cell number per dish, ± SE.

Western Blot Analysis.
Cells were seeded at a density of 106 cells per 100 mm plate, and Gem231 (100 nM) and/or HCPT (10 nM) was added. Cells were washed twice with ice-cold PBS, then scraped into Buffer 10 [20 mM Tris/HCl, 10 mM NaCl, 5 nM MgCl2, 1% NP40, 0.5% sodium deoxycholate, 100 µM pepstain, 100 µM antipain, 10 µM chymostain, 10 µg/ml leupeptin, 0.5 mM PMSF, 5 µg/ml trypsin inhibitor, and 1 mM benzamidine (pH 7.5)]. Cell extracts were subjected to Western analysis using antibodies against Bcl-2, Bak, Bax, or phospho-Bad, and the R and C subunits of PKA.

Morphological Determination of Apoptotic Nuclei.
Cells were grown on glass coverslips and treated as described above. To examine whole cell morphology, cells were washed with PBS, fixed with 70% methanol for 5 min, and stained with Giemsa (Bio-Rad) for 30 min. Coverslips were then rinsed with PBS and mounted with 80% glycerol in PBS. Cells were photographed using a Zeiss Axiovert 25 CFL inverted microscope. Morphological changes characteristic of apoptosis were observed by staining cell nuclei with Hoechst 33258 (Sigma). Coverslips were gently rinsed with PBS, fixed with 3.7% formaldehyde for 10 min, and stained with 1 µM Hoechst 33258 in PBS for 15 min. Coverslips were rinsed with PBS, and mounted with SlowFade antifade mounting medium (Molecular Probes, Eugene, OR). The slides were observed under a Zeiss Axiovert 25 CFL inverted microscope. Cells were determined to be apoptotic if they showed condensed chromatin or apparently fragmented nuclei. Apoptosis was quantified as the percentage of apoptotic nuclei per 103 nuclei.

PKA Assay.
Cells were seeded at a density of 106 cells per 100-mm plate and treated with Gem231 (100 nM) with or without HCPT (10 nM) for 2 days. Cells were washed twice with ice-cold PBS, scraped into Buffer 10, and lysed on ice. The PKA activity was measured by the method described previously (37) . Cell extracts (10 µg of protein) were added to 50 µl of the reaction mixture [50 mM Tris-HCI (pH 7.5), 20 µM kemptide (a Ser peptide: Leu-Arg-Arg-Ala-Ser-Leu-Gly; Life Technologies, Inc.), 1.2 µM [{gamma}-32P]ATP (100–200 cpm/pmol), 10 mM MgCl2, 1 mM DTT] with or without 5 µM cAMP, and with or without 20 µM protein kinase inhibitor, and incubated for 5 min at 30°C. Forty µl of the reaction mixture were spotted onto phosphocellulose disks and washed three times in 1.5% phosphoric acid. Filters were air-dried, then cells were counted in a liquid scintillation counter (Beckman, Columbia, MD). One unit of PKA activity was defined as the amount of enzyme that transferred 1 pmol of 32P from [{gamma}-32P]ATP to recovered protein in 5 min at 30°C in the standard assay system.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Growth Inhibition by Antisense RI{alpha} and HCTP.
To determine whether LS-174T colon carcinoma and PC3M androgen-insensitive prostate cancer cells responded to antisense RI{alpha} (Gem231) and topoisomerase I-inhibitor HCPT used in combination, we first generated dose-response curves for each agent. HCPT inhibited cell growth in a dose-dependent manner for both cell lines (Figs. 1ACitation and 2ACitation ). Gem231 inhibited growth for both LS-174T (Fig. 1B)Citation and PC3M (Fig. 2B)Citation cells with IC50 of 200 nM and time-dependent growth inhibition exhibiting 40–50% growth inhibition by 48-h exposure to 200 nM Gem231, as shown previously (13 , 14) .



View larger version (42K):
[in this window]
[in a new window]
  		Fig. 1. Synergistic effects of Gem231 [antisense (AS) ODN) and HCPT on growth inhibition in LS-174T cells. A, cells were treated with 100 nM AS ODN or control ODN (HYB0295) in the presence of various concentrations of HCPT for 2 days. B, cells were treated with 10 nM HCPT in the presence of various concentrations of AS ODN for 2 days. The data are expressed as percentage growth inhibition in comparison with untreated cells. Data (mean ± SE) represent one of three separate experiments that gave similar results. Insets, the synergism quotient at each concentration of drug combination. The synergism quotient was defined as the net effect of a drug combination on growth inhibition divided by the sum of the net individual drug effect. {square}, AS ODN; , HCPT; , AS ODN + HCPT; , control ODN: , control ODN + HCPT. Inset: •, AS ODN + HCPT; {circ}, control ODN + HCPT. Control ODNs, HYB0295 and HYB0674, gave essentially the same results.

 


View larger version (38K):
[in this window]
[in a new window]
  		Fig. 2. Synergistic effects of Gem231 [antisense (AS) ODN] and HCPT on growth inhibition in PC3M cells. A, cells were treated with 100 nM AS ODN or control ODN (HYB0295) in the presence of various concentrations of HCPT for 2 days. B, cells were treated with 10 nM HCPT in the presence of various concentrations of AS ODN for 2 days. The data are expressed as percentage growth inhibition in comparison with untreated cells. Data (mean ± SE) represent one of three separate experiments that gave similar results. The insets show the synergism quotient (see Fig. 1Citation symbols for columns and inset). Control ODNs, HYB0295 and HYB0674, gave essentially the same results.

 
We next examined the interactive effects of Gem231 and HCPT on growth inhibition at the 48-hour exposure time point. Low doses of Gem231 acted synergistically with a given low dose of HCPT and vice versa in both cell lines (Figs. 1Citation and 2Citation ). For example, antisense Gem231 singly, at a dose of 100 nM, exhibited 12–16% growth inhibition in both cell lines, whereas control ODN exerted less than 3% growth inhibition, and HCPT at 10 nM exerted 16–18% growth inhibition in these cells. Strikingly, when these two compounds were combined, growth inhibition reached 60–70%.

This combined effect was observed only when antisense Gem231, but not control ODNs HYB0295 or HY0674, was added with HCPT (Figs. 1Citation and 2Citation ). We examined the synergism quotients of the interactive effects of Gem231 and HCPT by keeping the concentration of one drug constant and varying the concentration of the other drug and vice versa (Figs. 1Citation and 2Citation , inset). It was observed that antisense Gem231 and HCPT at low doses in combination brought about a marked synergism in growth inhibition (Figs. 1Citation and 2Citation ). The synergism of these two compounds in antitumor activity was indifferent, however, when they were added in a sequential manner under the short-term culture (48-h) conditions (data not shown).

The combination of 100 nM Gem231 and 10 nM HCPT yielded the largest synergistic effect (synergism quotient 2.3–2.5; Figs. 1Citation and 2Citation , inset). These doses of 100 nM Gem231 and 10 nM HCPT are at much lower (3–5-fold) concentrations than their maximal plasma levels achievable in animal models in vivo (15 , 30) , indicating potential clinical relevance. We, therefore, used these doses in the following experiments.

Effects on Cell Morphology and Apoptosis.
The morphology of PC3M cells treated with Gem231 (100 nM) and HCPT (10 nM) was examined by Giemsa staining (Fig. 3A)Citation and by nuclear staining with Hoechst 33258 (Fig. 3B)Citation . Although treatment with either Gem231 or HCPT alone produced little or no effect on cell morphology at these low doses, the combined agents resulted in cells exhibiting a flattened shape and an increased cytoplasm:nucleus ratio. In addition, Gem231 and HCPT together induced changes indicative of apoptosis, such as chromatin condensation, nuclear fragmentation (Fig. 3B)Citation , and increased apoptotic nuclei count (Fig. 3, C and D)Citation . Thus, at doses that are ineffective individually, these agents together induced changes in cell morphology and apoptosis.



View larger version (46K):
[in this window]
[in a new window]
  		Fig. 3. Synergistic effects of Gem231 [antisense (AS) ODN] and HCPT on cell morphology and apoptosis. A, cell morphology; B, apoptotic nuclei; C, apoptotic nuclei count in PC3M cells: {circ}, control ODN; {square}, AS ODN; {triangleup}, HCPT; {blacksquare}, AS ODN + HCPT; {blacktriangleup}, control ODN + HCPT); D, apoptotic nuclei count in LS-174-T cells. Symbols are the same as those in C. Untreated control cells (saline), cells treated with AS ODN (100 nM), or control ODN (HYB0295), and HCPT (10 nM) for 2 days were examined for cell morphology and apoptosis (see "Materials and Methods"). Whole cell and nuclear morphology (x180). Data represent one of three separate experiments that gave similar results. Cells treated with mismatched control ODN (HYB0295) exhibited the same morphology and nuclear appearance as those of saline-treated control cells.

 
Effects on the Down-Regulation of RI{alpha} and the Up-Regulation of RIIß.
We next determined the effects of antisense Gem231 and HCPT on the levels of the R subunits and C subunit of PKA in LS-174T and PC3M cell extracts. Western analyses revealed that the combined treatment of Gem231 (100 nM) and HCPT (10 nM) resulted in a marked down-regulation of RI{alpha}, as well as in the up-regulation of RIIß, in both LS-174T and PC3M cells (Fig. 4)Citation . Individually, Gem231 and HCPT had little or no effect on RI{alpha} and RIIß protein levels at these low doses (Fig. 4)Citation . The combined effects were sequence specific; the four-base mismatched (HYB0295) or scrambled (HYB0674) sequence control ODNs had no effect on the levels of RI{alpha} and RIIß, either singly or in combination with HCPT (data not shown). The synergistic effect was also target specific; Gem231 and HCPT did not alter the RII{alpha} and C{alpha} subunit levels, either singly or in combination (Fig. 4)Citation .



View larger version (62K):
[in this window]
[in a new window]
  		Fig. 4. Effects of antisense (AS) ODN and/or HCPT on the R and C subunits of PKA in LS-174T or PC3M cells. Cells were plated at 106 cells per 100 mm plate and treated with AS ODN (100 nM) and/or HCPT (10 nM) for 2 days. Samples of total cellular protein (20 µg) were subjected to Western analysis using antibodies against C{alpha}, RI{alpha}, RII{alpha}, or RIIß. Lane 1, untreated (saline) control cells; Lane 2, AS ODN-treated cells; Lane 3, HCPT-treated cells; Lane 4, AS ODN-plus-HCPT-treated cells.

 
Effect on PKA Activity Ratio.
We have previously shown that the antisense-induced down-regulation of RI{alpha} brings about an increase in the PKA activity ratio, which measures how much PKA is in the active form (free versus total; Ref. 14 ). We, therefore, determined the ratio in cells treated with antisense Gem231 (100 nM) and HCPT (10 nM), each singly and combined. As shown in Table 1Citation , antisense RI{alpha} Gem231 treatment substantially increased the PKA activity ratio in a time-dependent manner in LS-174T cells, as was previously shown in PC3M cells (14) . This increase in PKA activity ratio paralleled the increase in free C activity (catalytic subunit of PKA; Table 1Citation ). The mismatched- or scrambled-sequence control ODN had no effect on the PKA activity ratio (data not shown). In contrast to antisense RI{alpha} Gem231, HCPT had no substantial effect on the PKA activity ratio (Table 2)Citation . The small increase in the activity ratio reflected the decrease of total PKA activity, but free C activity remained unaltered during HCPT treatment (Table 2)Citation . The addition of HCPT to Gem231 did not further affect the activity ratio beyond that elicited by Gem231 alone (Table 3)Citation . As expected, there was no synergistic effect on PKA activity ratio in the combined treatment of Gem231 and HCPT (Table 3)Citation .


View this table:
[in this window]
[in a new window]
  		Table 1 PKA activity ratio in LS-174T cells treated with Gem231 RI{alpha} antisense ODN

To examine PKA activity, the cells were seeded at a density of 106 cells per 100-mm plate and treated with antisense ODN (100 nM). At the indicated times, cells were harvested, and the PKA assay was performed (see "Materials and Methods"). Zero time indicates untreated control cells. The mismatched control ODN had no effect on either free or total activity throughout the experimental time period. Data represent one of three independent experiments that gave similar results.

 

View this table:
[in this window]
[in a new window]
  		Table 2 PKA activity ratio in HCPT-treated LS-174T cells

To examine PKA activity, cells were seeded at a density of 106 cells per 100-mm plate and treated with 10 nM HCPT. At the indicated times, cells were harvested, and subjected to the PKA assay (see "Materials and Methods").

 

View this table:
[in this window]
[in a new window]
  		Table 3 PKA activity ratio in LS-174T cells treated with Gem231 RI{alpha} antisense ODN (AS ODN) and/or HCPT

To examine PKA activity, the cells were seeded at a density of 106 cells per 100-mm plate and treated with AS ODN (100 nM) and/or HCPT (10 nM) for 48 h. Cells were harvested and subjected to the PKA assay (see "Materials and Methods").

 
Effect on the Induction of Proapoptotic Proteins Bax and Bad and Down-Regulation of Antiapoptotic Bcl-2 Protein.
Some proteins within the Bcl-2 family, including Bcl-2 and Bcl-XL, inhibit apoptosis, whereas others such as Bax, Bak, and Bad promote apoptosis. It has been shown that microtubule-damaging drugs induce Bcl-2 hyperphosphorylation and apoptosis in cancer cells, and that such hyperphosphorylation is mediated by activated PKA (38) .

We examined the interactive effect of Gem231 (100 nM) and HCPT (10 nM) on the expression of Bcl-2 family proteins. In both cell lines, antisense Gem231 and HCPT each induced expression of the Bax and Bad. In combination, these two agents induced expression of these proteins at an even higher level (Fig. 5)Citation . Conversely, Bcl-2 was markedly down-regulated by Gem231 and HCPT in combination (Fig. 5)Citation , more so than by either agent alone. Gem231 and HCPT also exerted a synergistic effect on Bcl-2 hyperphosphorylation in PC3M cells. However, Bcl-2 hyperphosphorylation was not detected in LS-174T cells (Fig. 5)Citation . Hypophosphorylation of Bad was also enhanced after treatment with the Gem231/HCPT combination (Fig. 5)Citation . Such hypophosphorylation of Bad will promote apoptosis, because the phosphorylated form of Bad is antiapoptotic (39) . In comparison, the mismatched control ODN could not induce these effects. These data confirm the above findings that the combination of Gem231 and HCPT exerts synergistic effects on the induction of apoptosis in both LS-174T colon cancer and PC3M prostate cancer cells.



View larger version (75K):
[in this window]
[in a new window]
  		Fig. 5. Effects of Gem231 [antisense (AS)ODN) and/or HCPT on apoptotic protein expression in LS-174T and PC3M cells. Cells were plated at 106 cells per 100 mm plate and treated with AS ODN (100 nM) and/or HCPT (10 nM) for 2 days. Samples of total cellular protein (40 µg) were subjected to Western analysis using anti-Bcl-2, Bad, and Bax monoclonal antibodies. Lane 1, untreated (saline) control cells; Lane 2, AS ODN-treated cells; Lane 3, HCPT-treated cells; Lane 4, AS ODN-plus-HCPT-treated cells.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have demonstrated that the apoptosis and cell morphology of LS-174T colon carcinoma and PC3M androgen-insensitive prostate cancer cells can be regulated by the combination of Gem231, an RNA-DNA second-generation antisense targeting the RI{alpha} regulatory subunit of PKA, and HCPT, a topoisomerase I inhibitor. These effects were concentration dependent and synergistically activated by the two agents. The growth inhibition induced by HPB0165 and HCPT correlated with down-regulation of the RI{alpha} protein, PKA activation, induction of phenotypic change, apoptosis, and the modulation of Bcl-2 family protein expression. This is the first report showing that a combination of antisense RI{alpha} with a topoisomerase-I-inhibiting cytotoxic drug suppresses growth by inducing apoptosis. The most important result of this investigation is the observation that HCPT enhances the growth-inhibitory effects of antisense RI{alpha} Gem231 in cancer cells in a dose-dependent manner. Comparison of growth inhibition induced individually by HCPT or Gem231 with a combination of these two agents showed a substantial enhancement of growth inhibition at clinically relevant concentrations. On the basis of our study, it is clear that these drugs act in a supra-additive or synergistic fashion and may be valuable for combinatorial therapy.

Many of the effects of antisense RI{alpha} result from the modulation of gene expression. In a recent study using cDNA microarrays, we showed that in a sequence-specific manner, antisense RI{alpha} exerts multisite genomic modulation (16) . Antisense-directed depletion of the RI{alpha} subunit of PKA thus modulates the signal transduction signatures of multiple pathways beyond the cAMP pathway. This is not surprising, because the intracellular signaling pathways are interrelated and interdependent, and cross-talk occurs even among pathways that are in opposition, such as the negative versus the positive regulation pathway. Antisense, therefore, blocks RI{alpha} expression and ultimately remodels the total intracellular trafficking network, which results in the reversion of the tumor phenotype to a normal-like phenotype in which cells stop growing (16) . cAMP response element (CRE)-directed transcription (40) , which is triggered by the activation of PKA via antisense-depletion of RI{alpha} (13 , 14 , 41) , may play a role in this process. Indeed, the PKA activity ratio was specifically increased by RI{alpha} antisense Gem231, but not by HCPT. This indicates that when Gem231 and HCPT treatments are combined, Gem231 contributes to the enhancement of growth inhibition and apoptosis through the cAMP signaling cascade.

The role of protein kinases has been studied in cell cycle regulation in a variety of cell types. In normal fibroblasts, the selective inhibition of various protein kinases, including PKA, produces growth arrest at different checkpoints along the G1 phase of the cell cycle (42) . In LS-174T colon carcinoma cells (43) and 60 human promyelocytic leukemia cells (44) , RI{alpha} antisense brought about the accumulation of cells in the S phase of the cell cycle, indicating inappropriate entry of the cells into S phase. The expression of cyclin E peaked throughout the S and G2-M phases of the cell cycle in antisense-treated HL-60 cells (44) . The level of RI{alpha} increases sharply in the fractions of cells enriched in the G1 to early S phase and in late S phase to early G2-M phase in centrifugally elutriated HL-60 cells (44) . Antisense-induced suppression of RI{alpha} may thus deregulate the cell cycle at two critical points: one at the entry into S phase, and the other at the G2-M phase (3 , 43 , 44) . This deregulation may have caused the accumulation of cells in S phase observed after antisense RI{alpha} treatment (43 , 44) . On the other hand, HCPT, which is an S-phase-specific cytotoxic agent, induces G2 arrest (26, 27, 28, 29) . Thus, we postulate that Gem231 and HCPT in combination would greatly enhance the cytotoxic effect (G2 arrest) of HCPT because of the increased recruitment of cells into S phase (S-phase arrest) by Gem231. In this study, however, the effect of sequential administration of Gem231 and HCPT was not found in the short-term (48-h) combination culture assays. It is likely that the sequential effect can be better observed in long-term growth such as soft agar growth or in in vivo animal model systems, if indeed Gem321 and HCPT cooperate in their mechanism of action of antitumor activity at the level of cell cycle control.

In the present study, we have shown that, at more clinically relevant doses, the combined effects of antisense RI{alpha} (Gem231) and the topoisomerase-I inhibitor HCPT cause a decrease in the level of RI{alpha} protein and growth inhibition in cancer cells with minimal or no side effects or toxicity. Because RI{alpha} expression is enhanced in human cancer cell lines and primary tumors (5, 6, 7, 8, 9) , it is a target for cancer diagnosis and therapy. Our findings demonstrate that antisense Gem231 and HCPT synergistically induce apoptosis as is evident from nuclear morphology, the up-regulation of pro-apoptotic proteins Bax and Bad, and the down-regulation of antiapoptotic proteins Bcl-2 and pBad. Thus, combined treatment with Gem231 and HCPT turns on signals for the blockade of cancer cell survival, suggesting its therapeutic potential against human cancer.


   ACKNOWLEDGMENTS
 
We thank Cheryl Pellerin of Palladian Partners, Inc., who provided editorial support under contract number NO2-BC-76212/C2700212 with the National Cancer Institute.


   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 To whom requests for reprints should be addressed, at National Cancer Institute, Building 10, Room 5B05, 9000 Rockville Pike, Bethesda, MD 20892-1750. Phone: (301) 496-4020; Fax: (301) 496-2443; E-mail: chochung{at}helix.nih.gov Back

2 The abbreviations used are: cAMP, cyclic AMP; PKA, protein kinase A; ODN, oligodeoxynucleotide; CPT, camptothecin; HCPT, hydroxycamptothecin. Back

Received 7/ 1/02; revised 10/25/02; accepted 11/18/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Krebs E. G., Beavo J. A. Phosphorylation-dephosphorylation of enzymes. Rev. Biochem., 48: 923-939, 1979.[CrossRef][Medline]
  2. Beebe S. J., Corbin J. D. Cyclic nucleotide-dependent protein kinases Beebe S. J. Corbin J. D. eds. . The Enzymes: Control by Phosphorylation, Vol. 17 part A: 43-111, Academic Press New York 1986.
  3. Tortora G., Pepe S., Bianco C., Baldassarre G., Budillon A., Clair T., Cho-Chung Y. S., Bianco A. R., Ciardiello F. The RI{alpha} subunit of protein kinase A controls serum dependency and entry into cell cycle of human mammary epithelial cells. Oncogene, 9: 3233-3240, 1994.[Medline]
  4. Tortora G., Pepe S., Cirafici A. M., Ciardiello F., Porcellini A., Clair T., Colletta G., Cho-Chung Y. S., Bianco A. R. Thyroid-stimulating hormone-regulated growth and cell cycle distribution of thyroid cells involve type I isozyme of cyclic AMP-dependent protein kinase. Cell Growth Differ., 4: 359-365, 1993.[Abstract]
  5. Miller W. R., Hulme M. J., Cho-Chung Y. S., Elton R. A. Types of cyclic AMP binding proteins in human breast cancers. Eur. J. Cancer, 29A: 989-991, 1993.[CrossRef]
  6. Miller W. R., Watson D. M. A., Jack W., Chetty U., Elton R. A. Tumor cyclic AMP binding proteins: An independent prognostic factor for disease recurrence and survival in breast cancer. Breast Cancer Res Treat., 26: 89-94, 1993.[CrossRef][Medline]
  7. Simpson B. J. B., Ramage A. D., Hulme M. J., Burns D. J., Katsaros D., Langdon S. P., Miller W. R. Cyclic adenosine 3', 5'-monophosphate-binding proteins in human ovarian cancer: correlations with clinicopathological features. Clin. Cancer Res., 2: 201-206, 1996.[Abstract/Free Full Text]
  8. McDaid H. M., Cairns M. T., Atkinson R. I., McAleer S., Harkin D. P., Gilmore P., Johnston P. G. Increased expression of the RI{alpha} subunit of the cAMP-dependent protein kinase A is associated with advanced stage of ovarian cancer. Br. J. Cancer, 79: 933-939, 1999.[CrossRef][Medline]
  9. Cho-Chung Y. S., Pepe S., Clair T., Budillon A., Nesterova M. cAMP-dependent protein kinase: role in normal and malignant growth. Crit. Rev. Oncol. Hematol., 21: 33-61, 1995.[Medline]
  10. Cho-Chung Y. S., Nesterova M., Pepe S., Lee G. R., Noguchi K., Srivastava R. K., Srivastava A. R., Alper O., Park Y. G., Lee Y. N. Antisense DNA-targeting protein kinase A-RI{alpha} subunit: a novel approach to cancer treatment. Front Biosci., 4: D898-907, 1999.[Medline]
  11. Tortora G., Ciardiello F. Targeting of epidermal growth factor receptor and protein kinase A: molecular basis and therapeutic applications. Ann. Oncol., 11: 777-783, 2000.[Free Full Text]
  12. Nesterova M., Cho-Chung Y. S. A single-injection protein kinase A-directed antisense treatment to inhibit humor growth. Nat. Med., 1: 528-633, 1995.[CrossRef][Medline]
  13. Nesterova M., Cho-Chung Y. S. Oligonucleotide sequence-specific inhibition of gene expression, tumor growth inhibition, and modulation of cAMP signaling by an RNA-DNA hybrid antisense targeted to protein kinase A RI{alpha} subunit. Antisense Nucleic Acid Drug Dev., 10: 423-433, 2000.[Medline]
  14. Cho Y. S., Kim M.-K., Tan L., Srivastava R., Agrawal S., Cho-Chung Y. S. Protein kinase A RI{alpha} antisense inhibition of PC3M prostate cancer growth: Bcl-2 hyperphosphorylation, Bax up-regulation, and Bad-hypophosphorylaton. Clin. Cancer Res., 8: 607-614, 2002.[Abstract/Free Full Text]
  15. Wang H., Cai Q., Zeng X., Yu D., Agrawal S., Zhang M. Q. Antitumor activity and pharmacokinetics of a mixed-backbone antisense oligonucleotide targeted to the RI{alpha} subunit of protein kinase A after oral administration. Proc. Natl. Acad. Sci. USA, 96: 13989-13994, 1999.[Abstract/Free Full Text]
  16. Cho Y. S., Kim M.-K., Cheadle C., Neary C., Becker K. G., Cho-Chung Y. S. Antisense DNAs as multisite genomic modulators identified by DNA microarray. Proc. Natl. Acad. Sci. USA, 98: 9819-9823, 2001.[Abstract/Free Full Text]
  17. Zangemeister-Wittke U., Schenker T., Luedke G. H., Stahel R. A. Synergistic cytotoxicity of bcl-2 antisense oligodeoxynucleotides and etoposide, doxorubicin and cisplatin on small-cell lung cancer cell lines. Br. J. Cancer., 78: 1035-1042, 1998.[Medline]
  18. Wang H., Zeng X., Oliver P., Le L. P., Chen J., Chen L., Zhou W., Agrawal S., Zhang R. MDM2 oncogene as a target for cancer therapy: an antisense approach. Int. J. Oncol., 15: 653-660, 1999.[Medline]
  19. Tortora G., Caputo R., Damiano V., Bioance R., Peppe S., Bianco A. R., Jiang Z., Agrawal S., Ciardiello F. Synergistic inhibition of human cancer cell growth by cytotoxic drugs and mixed backbone antisense oligonucleotides targeting protein kinase A. Proc. Natl. Acad. Sci. USA, 94: 12586-12591, 1997.[Abstract/Free Full Text]
  20. Tortora G., Caputo R., Pomatico G., Pepe S., Bianco A. R., Agrawal S., Mendelsohn J., Ciardiello F. Cooperative inhibitory effect of novel mixed backbone oligonucleotide targeting protein kinase A in combination with docetaxel and anti-epidermal growth factor-receptor antibody on human breast cancer cell growth. Clin. Cancer Res., 5: 875-881, 1999.[Abstract/Free Full Text]
  21. Agrawal S., Kandimalla E. R. Antisense therapeutics: is it as simple as complementary base recognition?. Mol. Med. Today, 6: 72-81, 2000.[CrossRef][Medline]
  22. Uhlmann, E. Oligonucleotide technologies: synthesis, production, regulations and applications. 29–30th Nov 2000. Hamburg, Germany. Expert Opin. Biol. Ther., 1: 319–328, 2001.
  23. Cho-Chung Y. S. Antisense and therapeutic oligonucleotides: toward a gene-targeting cancer clinic. Expert Opin. Ther. Patents, 10: 1711-1724, 2000.[CrossRef]
  24. Wall M. E., Wani M. C., Cook C. E., Palmer K. H., McPhail A. T., Sim G. A. Plant antitumor agents: I. The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata. J. Am. Chem. Soc., 88: 3888-3890, 1966.[CrossRef]
  25. Wall M. E., Wani M. C. Camptothecin and analogs: from discovery to clinic Potmesol M. Pinedo H. eds. . Camptothecins: New Anticancer Agents, 21-41, CRC Press Boca Raton, FL 1995.
  26. Tsao Y. P., D’Arpa P., Liu L. F. The involvement of active DNA synthesis in camptothecin-induced G2 arrest: altered regulation of p34cdc2/cyclin B. Cancer Res., 52: 1823-1829, 1992.[Abstract/Free Full Text]
  27. Tsao Y. P., Russo A., Nyamuswa G., Silber R., Liu L. F. Interaction between replication forks and topoisomerase I-DNA cleavable complexes: studies in a cell-free SV40 DNA replication system. Cancer Res., 53: 5908-5914, 1993.[Abstract/Free Full Text]
  28. Ryan A. J., Squires S., Strutt H. L., Evans A., Johnson R. T. Different fates of camptothecin-induced replication fork-associated double-strand DNA breaks in mammalian cells. Carcinogenesis (Lond.), 15: 823-828, 1994.[Abstract/Free Full Text]
  29. Binaschi M., Zunino F., Capranico G. Mechanism of action of DNA topoisomerase inhibitors. Stem Cells, 13: 369-379, 1995.[Abstract]
  30. Zhang R., Cai Q., Lindsey J. R., Li Y., Chambless B., Naguib F. N. M. Antitumor activity and pharmacokinetics following oral administration of natural product DNA topoisomerase inhibitors 10-hydroxycampthothecin and camptothecin in SCID mice bearing human breast cancer xenografts. Int. J. Oncol., 10: 1147-1156, 1997.
  31. Potmesil M., Glovanella B. C. Preclinical development of 20(S)-camptothecin, 9-aminocamptothecin, and other analogues Potmesol M. Pinedo H. eds. . Camptothecins: New Anticancer Agents, 41-57, CRC Press Boca Raton, FL 1995.
  32. Cai Q., Lindsey J. R., Zhang R. Regression of human colon cancer xenografts in SCID mice following oral administration of water-insoluble camptothecins. Int. J. Cancer, 10: 953-960, 1997.
  33. Wani M. C., Wall M. E. Plant antitumor agents II. The structure of two new alkaloids from Camptotheca acuminata. J. Org. Chem., 51: 1364-1367, 1969.[CrossRef]
  34. Ling Y. H., Perez-Soler R., Tseng M. T. Effect of DNA topoisomerase I inhibitor, 10-hydroxycamptothecin, on the structure and function of nuclei and nuclear matrix in bladder carcinoma MBT-2 cells. Anticancer Res., 13: 1613-1618, 1993.[Medline]
  35. Ling Y. H., Xu B. Inhibition of phosphorylation of histone H1 and H3 induced by 10- hydroxycamptothecin. DNA topoisomerase I inhibitor, in murine ascites hepatoma cells. Acta Pharmacol. Sin., 14: 546-550, 1993.
  36. Chen H. X., Marshall J. L., Ness E., Martin R. R., Dvorchik B., Rizvi N., Marquis I., McKinlay M., Dahut W., Hawkins M. J. A safety and pharmacokinetic study of a mixed-backbone oligonucleotide (Gem231) targeting the type I protein kinase A by 2-hour infusions in patients with refractory solid tumors. Clin. Cancer Res., 6: 1259-1266, 2000.[Abstract/Free Full Text]
  37. Cho Y. S., Park Y. G., Lee Y. N., Kim M. K., Bates S., Tan L., Cho-Chung Y. S. Extracellular protein kinase A as a cancer biomarker: its expression by tumor cells and reversal by a myristate-lacking C{alpha} and RIIß subunit overexpression. Proc. Natl. Acad. Sci. USA, 97: 835-840, 2000.[Abstract/Free Full Text]
  38. Srivastava R. K., Srivastava A. R., Korsmeyer S. J., Nesterova M., Cho-Chung Y. S., Longo D. L. Involvement of microtubules in the regulation of Bcl2 phosphorylation and apoptosis through cyclic AMP-dependent protein kinase. Mol. Cell. Biol., 18: 3509-3517, 1998.[Abstract/Free Full Text]
  39. Harada H., Becknell B., Wilm M., M., M., Huang L. J., Taylor S. S., Scott J. D., Korsmeyer S. J. Phosphorylation and inactivation of BAD by mitochondria-anchored protein kinase A. Mol. Cell, 3: 413-422, 1999.[CrossRef][Medline]
  40. Roesler W. J., Vandenbark G. R., Hanson R. W. Cyclic AMP and the induction of eukaryotic gene transcription. J. Biol. Chem., 263: 9063-9066, 1988.[Free Full Text]
  41. Neary C. L., Cho-Chung Y. S. Nuclear translocation of the catalytic subunit of protein kinase A induced by an antisense oligonucleotide directed against the RI{alpha} regulatory subunit. Oncogene, 20: 8019-8024, 2001.[CrossRef][Medline]
  42. Gadbois D. M., Crissman H. A., Tobey R. A., Bradbury E. M. Multiple kinase arrest points in the G1 phase of nontransformed mammalian cells are absent in transformed cells. Proc. Natl. Acad. Sci. USA, 89: 8626-8630, 1992.[Abstract/Free Full Text]
  43. Nesterova M., Noguchi K., Park Y. G., Lee Y. N., Cho-Chung Y. S. Compensatory stabilization of RIIß protein, cell cycle deregulation, and growth arrest in colon and prostate carcinoma cells by antisense-directed down-regulation of protein kinase A RI{alpha} protein. Clin. Cancer Res., 6: 3434-3441, 2000.[Abstract/Free Full Text]
  44. Cho-Chung Y. S., Nesterova M., Kondrashin A., Noguchi K., Srivastava R. K., Pepe S. Antisense-protein kinase A: a single-gene-based therapeutic approach. Antisense and Nucleic Acid Drug Dev., 7: 217 1997.[Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
H. Yang, C. J. Lee, L. Zhang, M. D. Sans, and D. M. Simeone
Regulation of transforming growth factor {beta}-induced responses by protein kinase A in pancreatic acinar cells
Am J Physiol Gastrointest Liver Physiol, July 1, 2008; 295(1): G170 - G178.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cho, Y. S.
Right arrow Articles by Cho-Chung, Y. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cho, Y. S.
Right arrow Articles by Cho-Chung, Y. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online