- Email: [email protected]
Effects of 8-chloro-cyclic adenosine monophosphate on the growth and sensitivity to doxorubicin of multidrug-resistant tumour cell lines
Pharmacological Research, Vol. 30, No. 1.1994
81
EFFECTS OF S-CHLORO-CYCLIC ADENOSINE MONOPHOSPHATE ON THE GROWTH AND SENSITIVITY TO DOXORUBICIN OF MULTIDRUG-RESISTANT TUMOUR CELL LINES N. BORSELLINO,
M. CRESCIMANNO, and N. D’ALESSANDRO
V. LEONARD1
Institute of Pharmacology, Palermo University Medical School, Policlinico P. Giaccone, 90127 Palermo, Italy. Received
in final
form 20 April 1994
SUMMARY We examined the in vitro effects of %chloro-adenosine 3’:5’-monophosphate (8Cl-CAMP), a reportedly stable, potent and site-selective analogue of CAMP, on the proliferation and sensitivity to doxorubicin (DXR) of two mouse cell lines, the B16 melanoma and Friend leukaemia, both as wild-type (B16, FLC) and DXRresistant (B16/DXR, FLC/DXR) variants. The latter strains had characteristics of ‘typical’ multidrug resistance (MDR), including the over-expression of Pglycoprotein. Encouragingly, 8-Cl-CAMP affected almost equally the growth of the chemosensitive and chemoresistant variants of both cell lines. Its activity proved to be much more elevated on cells cultivated with fresh rather than heatinactivated calf serum. In fact, the I& values for B16 and B16/DXR were about 4.7 PM in fresh serum and 215 ,u~ in heat-inactivated serum; the I& values for FLC and FLC/DXR were about 12 PM in fresh serum and 70~~ in heatinactivated serum. Furthermore, experiments with B16 showed that cotreatments with isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, or adenosine deaminase (ADA) greatly reduce the activity of 8-Cl-CAMP bringing it to comparable levels in fresh and heat-inactivated serum. These results indicate that the antiproliferative effects of 8-Cl-CAMP may be due principally to metabolites formed by the enzymic activities of the serum, most probably including 8-chloroadenosine (8-Cl-adenosine), as suggested by other authors. Moreover, the dose-response curves and the I& values of the latter compound for the various cell lines were compatible with those observed for 8-Cl-CAMP in fresh serum. Finally, there was no evidence that 8-Cl-CAMP, either in the presence of fresh or heat-inactivated serum, or 8-Cl-adenosine may increase the sensitivity to DXR of the MDR variants of B16 melanoma and Friend leukaemia. KEY WORDS: %chloro-adenosine multidrug resistance.
3’5’ monophosphate,
%chloro-adenosine,
doxorubicin,
0 1994The Italian PharmacologicalSociety
Pharmacological Research, Vol. 30, No. I,1994
82
INTRODUCTION
The frequent occurrence of drug resistance represents one of the major obstacles to more effective cancer treatment; the phenomenon is often of multiple type (Multidrug Resistance, MDR) and.associated with various mechanisms, especially the over-expression at the tumour cell level of a multidrug efflux pump known as P-glycoprotein [for review, see, for example refs 1, 21. Thus, at present, considerable research effort is being made to develop therapeutic modalities which are capable of overcoming this problem, either by producing antiproliferative effects in drug-resistant populations or by conferring or restoring drug-sensitivity. With view to this, great interest has been directed towards the cyclic adenosine 3’:5’-monophosphate (CAMP) system [3]. In fact, the cyclic nucleotide is known to control cell growth and differentiation via its interaction with different types of protein kinases [for review, see 41. On the other hand, it seems that these enzymes are also involved in the modulation of MDR; in particular, type I CAMP-dependent protein kinase might promote the expression of P-glycoprotein [5,6], perhaps at transcriptional level [6]. Among the CAMP analogues, one in particular which is of potential interest in clinical antitumour therapy is 8-chloro-CAMP (8-Cl-CAMP), which seems to act through a preferential binding to the regulatory subunit RI1 of type II CAMP-dependent protein kinase [7,8]; however, other authors [9, 10, 1 l] have argued that the antiproliferative activity of 8-Cl-CAMP might be due rather to its metabolites, principally 8-chloro-adenosine (8-Cl-adenosine), whose mechanisms might be different from the interaction with the CAMP-dependent kinases. Finally, it has recently been reported that 8-Cl-CAMP is able to exert inhibitory effects on the growth of cells with MDR and, at subcytotoxic concentrations, to increase their sensitivity to the antiproliferative effects of agents frequently affected by this process, including doxorubicin (DXR) [6, 121. Taking these premises into account, we examined the effects of 8-Cl-CAMP, and also of 8-Cl-adenosine, on the growth and sensitivity to DXR of two mouse cultured tumour cell lines, the solid B16 melanoma and the liquid Friend erythroleukemia, both as wild-type and selected for resistance to DXR variants. The chemoresistant strains had characteristics of ‘typical’ MDR [1, 21, including cross-resistance to vincristine (VCR) and over-expression of P-glycoprotein [ 131. The experiments with 8-Cl-CAMP were carried out on cells grown on foetal calf serum either heat-inactivated or not; this was done to ascertain whether the preservation of the serum enzymic activities could influence the effects of the compound. In some experiments on B 16 melanoma the antiproliferative activity of 8-Cl-AMPc was studied also in the presence of isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, or adenosine deaminase (ADA) and this with the aim of making a better assessment of the responsibility of 8-Cl-adenosine as a growthinhibitory metabolite of the compound. MATERIALS
AND METHODS
Drugs
8-chloro-cyclic adenosine monophosphate (NSC 614491) was kindly supplied by Dr Nancita A. Lomax (National Cancer Institute, Bethesda, USA). 8-chloro-
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Pharmacological Research, Vol. 30, No. 1, 1994
adenosine was supplied by Biolog Life Science Institute, Bremen, Germany, and doxorubicin hydrochloride by Farmitalia Carlo Erba, Milan, Italy. Isobutylmethylxanthine (IBMX) and calf intestinal mucosa adenosine deaminase (ADA) were from Sigma Chemical Co., St. Louis, MO, USA. Cell lines and cytotoxicity assays
Mouse B16 melanoma cells as parental (B16) or DXR-resistant (B16/DXR) variants and mouse Friend erythroleukaemia cells as parental (FLC) and DXRresistant (FLC/DXR) variants were obtained as previously described [ 131. The cell lines were grown in RPM1 1640 (Gibco, Grand Island, NY, USA) containing 10% foetal calf serum (Gibco), whether heat-inactivated (by heating at 56°C for 30 min) or not, and 1% penicillin and streptomycin; there was a humidified atmosphere of 5% CO2 in air at 37°C. In the case of B16 and B16/DXR 1 mM sodium pyruvate was also added to the culture medium and, in order to maintain the level of resistance, B16/DXR cells were grown in the presence of DXR 15 ng ml-‘. B16 and B16/DXR were subcultured twice a week, FLC and FLC/DXR three times a week. B 16 and B 16/DXR were seeded at 1x10’ ml-’ and FLC and FLC/DXR at 5x lo4 ml-’ in 24-well culture plates (NUNC, Roskilde, Denmark) and in the presence of various concentrations of the drugs: 72 or, in some cases, 144 h after the beginning of the experiment the viable cells were counted through the microscope with Trypan blue exclusion. In the case of B16 and B16/DXR, the cells were harvested by trypsin-EDTA because they had grown as monolayer cultures.
RESULTS The characteristics of the cells used here have already been described [13] and some of them are summarized in Table 1. When compared with their parental counterparts, B16/DXR and FLC/DXR proved to be resistant both to DXR and VCR. The MDR phenotype of these cell lines also included the over-expression of P-glycoprotein, detected by immunocytochemistry or immunoblotting [ 131.
Table I Some characteristics of B16 melanoma and Friend erythroleukaemia DXR-sensitive or -resistant variants IC50DXR (ng ml I*)
) KS0 VCR (ng ml _I*
(RI)
(RI)
Doubling (b)
B16 B I6fDXR
5.5 65.0 (11.8)
24.0 170.0 (7.1)
13.6 11.1
FLC FLC/DXR
5.3 460.2 (86.8)
12.0 155.0 (12.9)
13.3 13.5
*Mean of triplicate experiments RI: index of resistance.
with
SDS
10%.
time*
as
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60
1
1
1
5
1
10
/
I
50 100
I
I
250 500 1000
80 60 40 20 I
0
I
,I
1
10
I
1
50
100
I
500
8-Cl-CAMP (pm) Fig. 1. Effects of 8-Cl-CAMP on the growth of B16, B 16/DXR, FLC and FLC/DXR, either in the presence of heat-inactivated (A and C) or fresh serum (B and D). (A) and (B): 0, B16; ?? , Bl6-DXR. (C) and (D): 0, FLC; W, FLC-DXR. Results are expressed as a percentage of the control cell number and are the mean of at least three independent experiments; SD was regularly less than 10%. For further details see Materials and Methods and Results.
The effects of ~-U-CAMP on the growth of the various cell lines are shown in Fig. 1. B 16 and B 16/DXR were seeded at 50 000 per ml and FLC and FLC/DXR at 100 000 per ml in the presence of a wide range of concentrations of ~-U-CAMP; fresh solutions of the drug were added after 48 h. Control cells received only standard medium. The cells were counted 72 h after the beginning of the experiment. At that time, the numberfsD (per millilitre) of the control cells was
Pharmacological Research, Vol. 30, No. I,1994
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I
I
1
1
5
10
I
/
I
I
50
100
250
500
B-Cl-CAMP (flrn)
Fig. 2. Effects of 8Cl-CAMPfadenosine deaminase (ADA) on B16 cells. The cells were cultivated with heat-inactivated serum and seeded in presence of 8-Cl-CAMP&ADA in a concentration of 3 U/ml; 48 h later the medium was removed and new medium, 8-Cl-CAMP and ADA were added. The cells were counted after 72 or 144 h from the beginning of the experiment; the number (per ml) of the control cells was 910 416243 898 at 72 h and 971 083f 89 340 at 144 h. Results are expressed as a percentage of the control cell number and are the mean of three independent experiments with SD regularly less than 10%. 0, 72 h 8-Cl-CAMP; 0,72 h 8-Cl-cAMP+ADA; W, 144 h 8-Cl-CAMP; 0, 144 h 8-Cl-cAMP+ADA. ADA alone did not affect the growth of the cells (not shown). For further details see Materials and Methods and Results.
853 125f52 570 for B16 grown in heat-inactivated serum and 881 2.50+44 083 for B16 grown in fresh serum; it was 1 181 250+79 733 for B16/DXR grown in heatinactivated serum and 1 15 1 041+59 484 for B 16/DXR grown in fresh serum; it was 2 7 13 28 1f 15 1 69 1 for FLC grown in heat-inactivated serum and 2 168 625f 181 242 for FLC grown in fresh serum; it was 2 604 687f164 994 for FLC/DXR grown in heat-inactivated serum and 1 881 250+94 923 for FLC/DXR grown in fresh serum. The plots of Fig. 1 (in which the results are expressed as a percentage of the control cell numbers) indicate that when the cells were grown in the presence of heat-inactivated serum, 8-Cl-CAMP was active at rather elevated concentrations; the 50% inhibitory concentration (ICJ was about 215 PM in B16 and B16/DXR (Fig. 1A) and about 70~~ in FLC and FLC/DXR (Fig. 1C). However, the I& values turned out to be considerably lower when the cells were grown in fresh serum and were about 4.7 ,LLMfor B 16 and B 16/DXR (Fig. 1B) and about 12 PM for FLC and FLC/DXR (Fig. 1D). In some experiments on B 16 cells (not shown) the activity of 8-Cl-CAMP was also studied in the presence of IBMX 0.25 mM; this phosphodiesterase inhibitor, which alone affected the growth of the cells only marginally, greatly antagonized the antiproliferative effects of the CAMP analogue raising its ICSO to a comparable value (about 700 PM) both in fresh and heat-inactivated serum. The activity of 8-Cl-CAMP on B16 was also virtually abolished following co-exposure to ADA in a concentration of 3 U ml-’ (Fig. 2). Furthermore, we studied the antiproliferative effects of g-Cladenosine on the various cell lines grown in heat-inactivated serum; the dose-
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100 l,?YTYT?L (D)
80 60 40 20 0
0.1
0.5 1
3
10
30
100 300 1000
DXR (ng ml-‘)
Fig. 3. Effects of DXRf8-Cl-CAMP on the growth of B16, B16/DXR (A and B), FLC and FLC/DXR (C and D) cells. (A) and (B): 0, B16:DXR; 0, B16:DXR+&CI-CAMP; W, B16DXR: DXR; 0, Bl6-DXR: DXR+8-Cl-CAMP. (C) and (D): 0, FLC: DXR; 0, FLC:DXR+8Cl-CAMP; W, FLC-DXR: DXR; 0, FLC-DXR: DXR+8-Cl-CAMP. The experiments were carried out in heat-inactivated (A and C) or fresh serum (B and D). ~-U-CAMP was 50,~~ in (A), 1 PM in (B), 15 ,UM in (C) and 2 PM in (D). Results are expressed as a percentage of the control cell number and are the mean of at least three independent experiments with SD regularly less than 10%. Combined 8-Cl-cAMP/DXR data are normalized to account for the inhibitory effects of 8-Cl-CAMP alone (like those noticed in Fig. 1). For further details see Materials and Methods and Results.
pharmacological Research, Vol. 30, No. 1,1994
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response curves of this compound (not shown), of which fresh solutions were added after 0 and 48 h as in the case of 8-Cl-CAMP, were similar in shape to those observed with 8-Cl-CAMP in fresh serum. The IGo of 8-Cl-adenosine proved to be about 2.1 PM for B 16 and B 16/DXR, 0.6 PM for FLC and FLC/DXR. Experiments were also carried out to ascertain if cotreatment with slightly cytotoxic concentrations of 8-Cl-CAMP could influence the sensitivity to DXR of our cell lines (either grown in fresh or in heat-inactivated serum). In the experiments of Fig. 3, the cells were seeded in the presence of 8-Cl-CAMP and fresh 8-Cl-CAMP was added after 48 h; various concentrations of DXR were added 24 h after the beginning of the experiment. The cells were counted 72 h after the beginning of the experiment. The numbers of the control cells were very similar to those already reported for the experiments of Fig. 1 and they are not quoted for the sake of brevity. 8-Cl-CAMP was 50 PM for B16 and B16/DXR grown in heat-inactivated serum (Fig. 3A) and 1 PM for B16 and BlB/DXR grown in fresh serum (Fig. 3B). It was 15 ,u~ for FLC and FLC/DXR grown in heatinactivated serum (Fig. 3C) and 2 PM when the same cells were grown in fresh serum (Fig. 3D). In presenting the combination data of Fig. 3, the slight cytotoxic effects of 8-Cl-CAMP alone (as in the experiments of Fig. 1) were normalized by making these equal to 100%. From the plots of Fig. 3 it can be seen that neither with fresh nor with inactivated serum did 8-Cl-CAMP show synergism with DXR in the drug-resistant tumours; only additive (Fig. 3A, B and C) or slight antagonistic (Fig. 3D) antiproliferative effects were seen. For the sensitive cell lines, slight synergistic effects were encountered in the case of B 16 cells grown in fresh serum (Fig. 3B) and FLC grown in heat-inactivated serum (Fig. 3C); additive effects were noticed in the other two cases (Fig. 3A and D). Finally, we studied the effects of slightly cytotoxic concentrations of 8-Cladenosine on the sensitivity to DXR of the cell lines (data not shown). These experiments were carried out with heat-inactivated serum. In no case was a synergistic antiproliferative effect of the combination of 8-Cl-adenosine with DXR noticed.
DISCUSSION In the present work we started from the reported premise that 8-Cl-CAMP, a stable, potent and site-selective analogue of CAMP [7], may exert potentially useful antiproliferative effects on drug-sensitive and drug-resistant tumour cell populations and, administered at subcytotoxic concentrations, may favourably modify the sensitivity to DXR of MDR tumours [6, 121. We explored these possible activities of the compound in two mouse tumour cell lines, the solid B16 melanoma and the liquid Friend erythroleukaemia, both as DXR-sensitive and DXR-resistant MDR variants. Encouragingly, 8-Cl-CAMP affected the growth of the chemosensitive and chemoresistant variants of both cell lines almost equally. However, the I& values observed in the experiments on cells cultivated with heat-inactivated serum appeared rather high (about 2 15 PM for B 16 and B 16/DXR and about 70 PM for FLC and FLC/DXR), also when compared with the data reported for other tumour
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cell lines where the activity of 8U-CAMP was in the l-25 PM range [7]. It must be stressed that we used a wide range of ~-U-CAMP doses, including low [l-lo ,UM] ones, which, according to Cho-Chung [8], should undergo only minimal degradation, if any (see below). In addition, the cells were exposed to the compound for 72 h. We know that in this period, after a phase of adaptation during which they halve their initial inoculum, untreated B16 and B16-DXR cells produce about four new cell generations; in the same period, FLC and FLC-DXR cells produce about 5-6 new cell generations. Thus, the time of exposure of our cell lines to 8-Cl-CAMP was even longer than that (2-3 generation times longer) reported by others [7, 81 as necessary for the antiproliferative activity of the compound to be apparent. On the other hand, the I& values were considerably reduced (about 4.7 ,UM for B 16 and B 16/DXR and about 12 ,LLM for FLC and FLUDXR) in the presence of fresh serum. Looking for an explanation for these findings, we can cite several papers in which the possible mechanisms of the inhibitory effect of 8-Cl-CAMP on tumour cell growth have been examined. It has been proposed that the compound may act by itself and induce differentiation through a selective interaction with the regulatory subunit RIIP of type II CAMP-dependent kinase, which would translocate to the nucleus where it restores a normal gene transcription pattern [7, 81. Alternatively, 8-Cl-CAMP may form growth-inhibitory metabolites, among which 8-Cl-adenosine is the most important [9, 10, 11, 141; it has also been reported that, in conditions similar to those we adopted, Gibco foetal calf serum, through phosphodiesterase and 5’-nucleotidase activities, hydrolizes about 72% of 8-Cl-CAMP 100,~~ with the production of 8-Cl-adenosine as the principal metabolite; the hydrolysis is only 11% when the same serum is heat-inactivated [9]. Thus, it seems likely that in our cell lines 8-Cl-CAMP acted mainly through its transformation, probably into 8-Cl-adenosine, and this was supported by the virtual abolition of the activity of the compound, both in fresh and heat-inactivated serum, when B16 melanoma cells were coexposed to IBMX, a phosphodiesterase inhibitor, or, more specifically, to ADA, which should convert 8-Cl-adenosine to 8-chloro-inosine; according to Van Lookeren Campagne [9], the latter compound is devoid of a growth-inhibitory activity. The I& values of 8-Cl-adenosine for the cell lines were lower than those of 8-Cl-CAMP in fresh serum, but this result may be compatible with an incomplete transformation of the latter compound in that medium. The other major aspect, i.e. the possibility that subcytotoxic concentrations of 8-Cl-CAMP might favourably modulate the sensitivity of DXR of MDR turnours, was not seen in our experiments either with fresh or heat-inactivated serum. The combination of S-Cl-adenosine with DXR did not even produce synergistic antiproliferative effects. With regard to these results, in other papers [6, 121 g-ClCAMP appeared to be capable of sensitizing to DXR MDR cells whether or not they over-express P-glycoprotein. A rationale has been put forward according to which the RIa regulatory subunit of CAMP-dependent protein kinase is in excess in the MDR cells [6, 12 and see also 51; accordingly, 8-Cl-CAMP would downregulate the RIa subunit and control the processes of MDR, including the Pglycoprotein over-expression, at transcriptional level [6, 121. We are currently not aware whether these critical modifications of the RI subunit levels are present in
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our cell lines; indeed, their growth was affected by 8-Cl-adenosine and, although the mechanisms of the cytotoxic activity of this compound are still not completely known, it is interesting that in a study on normal and neoplastic mouse lung cells it selectively down-regulated the RI and Ca subunits of type I CAMP-dependent protein kinase [14]. On the other hand, it is also possible that our cells have some other factor which would account for their unresponsiveness to 8-Cl-CAMP per se, such as, for example, lack of or alterations to the RI1 isoforms of CAMP-dependent protein kinase subunits, which have frequently been observed in human and mouse tumour cells [14-161. In any case, we conclude that the supposed modulatory activity of 8-Cl-CAMP as a sensitizer to DXR in MDR cell lines cannot be generalized to the relevant models of typical MDR used herein.
ACKNOWLEDGEMENTS This work was partially
supported
by AIRC.
REFERENCES 1. Morrow CS, Cowan KM. Mechanisms and clinical significance of multidrug resistance. Oncology 1988; 2: 55-67. 2. Tsuruo T. Mechanisms of multidrug resistance and implications for therapy. Jup J Cancer Res (Gann) 1988; 79: 285-96. 3. D’Amico C, Crescimanno M, Armata MG, Leonardi V, Palazzoadriano M, D’Alessandro N. Beta-adrenergic influences on doxorubicin-sensitive or -resistant P388 leukemia cells. Anticancer 4. Cho-Chung
5.
6.
7.
8. 9.
10. 11.
12.
Res 1992;
12: 2253-6.
YS. Role of cyclic AMP receptor proteins in growth, differentiation and suppression of malignancy: new approaches to therapy. Canctir Res 1990; 50: 7093-100. Abraham I, Chin KW, Gottesman MM, Majo JK, Sampson KE. Transfection of a mutant subunit gene of CAMP-dependent protein kinase causes increased drug sensitivity and decreased expression of P-glycoprotein. .&p Cell Res 1990; 189: 133-41. Yokozaki H, Budillon A, Clair T, Kelley K, Cowan KH, Rohlff C, Glazer RI, Cho-Chung YS. 8-chloroadenosine 3’-5’-monophosphate as a novel modulator of multidrug resistance. Inr J 0~01 1993; 3: 423-20. Cho-Chung YS. Site-selective 8-chloro-cyclic adenosine 3’, 5’-monophosphate as a biologic modulator of cancer: restoration of normal control mechanisms. J Nat1 Cancer Inst 1989; 81: 982-7. Cho-Chung YS. Letter to the editor. Cancer Res 1991; 51: 6206-7. Van Lookeren Campagne MM, Villalba Diaz F, Jastorff B, Kessin RH. 8chloroadenosine 3’, 5’ monophosphate inhibits the growth of Chinese hamster ovary and Molt-4 cells through its adenosine metabolite. Cancer Res 1991; 51: 1600-5. Kessin RH, Villalba Diaz F, Van Lookeren Campagne MM, Jastorff B. Reply. Cancer Res 1991; 51: 6207-8. Langeveld CH, Jongenelen CAM, Heimans JJ, Stoof JC. Growth inhibition of human glioma cells induced by 8-chloroadenosine, an active metabolite of 8-chloro cyclic adenosine 3’:5’ monophosphate. Cancer Res 1992; 52: 3994-9. Rohlff C, Safa B, Rahman A, Cho-Chung YS, Klecker RW, Glazer RI. Reversal of resistance to adriamycin by 8-chloro-cyclic AMP in adriamycin-resistant HL-60 leukemia cells is associated with reduction of type I cyclic AMP-dependent protein kinase and cyclic AMP response element-binding protein DNA-binding activities. Mel Pharmacol
1993; 43: 372-9.
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N, Crescimanno M, Flandina N, Leonardi V, Rausa L, D’Alessandro N. Antiproliferative and chemomodulatory effects of interferon-gamma on doxorubicinsensitive and -resistant cell lines. Ann-Cancer Drugs 1993; 4: 265-72. 14. Lange-Carter CA, Vuillequez JJ, Malkinson AM. 8chloroadenosine mediates 8-chlorocyclic AMP-induced down-regulation of cyclic AMP-dependent protein kinase in normal and neoplastic mouse lung epithelial cells by a cyclic AMP-independent mechanism. Cancer Res 1993; 53: 593-400. 15. Weber W, Schwoch G, Wielckens K, Gartermann A, Hilz M. CAMP receptor proteins and protein kinases in human lymphocytes: fundamental alterations in chronic lymphocytic leukemia cells. Eur .I Biochem 1981; 120: 585-92. 16. Tortora G, Cho-Chung YS. Type II regulatory subunit of protein kinase restores CAMPdependent transcription in a CAMP-unresponsive cell line. J Biol Chem 1990; 265: 18067-70.
81
EFFECTS OF S-CHLORO-CYCLIC ADENOSINE MONOPHOSPHATE ON THE GROWTH AND SENSITIVITY TO DOXORUBICIN OF MULTIDRUG-RESISTANT TUMOUR CELL LINES N. BORSELLINO,
M. CRESCIMANNO, and N. D’ALESSANDRO
V. LEONARD1
Institute of Pharmacology, Palermo University Medical School, Policlinico P. Giaccone, 90127 Palermo, Italy. Received
in final
form 20 April 1994
SUMMARY We examined the in vitro effects of %chloro-adenosine 3’:5’-monophosphate (8Cl-CAMP), a reportedly stable, potent and site-selective analogue of CAMP, on the proliferation and sensitivity to doxorubicin (DXR) of two mouse cell lines, the B16 melanoma and Friend leukaemia, both as wild-type (B16, FLC) and DXRresistant (B16/DXR, FLC/DXR) variants. The latter strains had characteristics of ‘typical’ multidrug resistance (MDR), including the over-expression of Pglycoprotein. Encouragingly, 8-Cl-CAMP affected almost equally the growth of the chemosensitive and chemoresistant variants of both cell lines. Its activity proved to be much more elevated on cells cultivated with fresh rather than heatinactivated calf serum. In fact, the I& values for B16 and B16/DXR were about 4.7 PM in fresh serum and 215 ,u~ in heat-inactivated serum; the I& values for FLC and FLC/DXR were about 12 PM in fresh serum and 70~~ in heatinactivated serum. Furthermore, experiments with B16 showed that cotreatments with isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, or adenosine deaminase (ADA) greatly reduce the activity of 8-Cl-CAMP bringing it to comparable levels in fresh and heat-inactivated serum. These results indicate that the antiproliferative effects of 8-Cl-CAMP may be due principally to metabolites formed by the enzymic activities of the serum, most probably including 8-chloroadenosine (8-Cl-adenosine), as suggested by other authors. Moreover, the dose-response curves and the I& values of the latter compound for the various cell lines were compatible with those observed for 8-Cl-CAMP in fresh serum. Finally, there was no evidence that 8-Cl-CAMP, either in the presence of fresh or heat-inactivated serum, or 8-Cl-adenosine may increase the sensitivity to DXR of the MDR variants of B16 melanoma and Friend leukaemia. KEY WORDS: %chloro-adenosine multidrug resistance.
3’5’ monophosphate,
%chloro-adenosine,
doxorubicin,
0 1994The Italian PharmacologicalSociety
Pharmacological Research, Vol. 30, No. I,1994
82
INTRODUCTION
The frequent occurrence of drug resistance represents one of the major obstacles to more effective cancer treatment; the phenomenon is often of multiple type (Multidrug Resistance, MDR) and.associated with various mechanisms, especially the over-expression at the tumour cell level of a multidrug efflux pump known as P-glycoprotein [for review, see, for example refs 1, 21. Thus, at present, considerable research effort is being made to develop therapeutic modalities which are capable of overcoming this problem, either by producing antiproliferative effects in drug-resistant populations or by conferring or restoring drug-sensitivity. With view to this, great interest has been directed towards the cyclic adenosine 3’:5’-monophosphate (CAMP) system [3]. In fact, the cyclic nucleotide is known to control cell growth and differentiation via its interaction with different types of protein kinases [for review, see 41. On the other hand, it seems that these enzymes are also involved in the modulation of MDR; in particular, type I CAMP-dependent protein kinase might promote the expression of P-glycoprotein [5,6], perhaps at transcriptional level [6]. Among the CAMP analogues, one in particular which is of potential interest in clinical antitumour therapy is 8-chloro-CAMP (8-Cl-CAMP), which seems to act through a preferential binding to the regulatory subunit RI1 of type II CAMP-dependent protein kinase [7,8]; however, other authors [9, 10, 1 l] have argued that the antiproliferative activity of 8-Cl-CAMP might be due rather to its metabolites, principally 8-chloro-adenosine (8-Cl-adenosine), whose mechanisms might be different from the interaction with the CAMP-dependent kinases. Finally, it has recently been reported that 8-Cl-CAMP is able to exert inhibitory effects on the growth of cells with MDR and, at subcytotoxic concentrations, to increase their sensitivity to the antiproliferative effects of agents frequently affected by this process, including doxorubicin (DXR) [6, 121. Taking these premises into account, we examined the effects of 8-Cl-CAMP, and also of 8-Cl-adenosine, on the growth and sensitivity to DXR of two mouse cultured tumour cell lines, the solid B16 melanoma and the liquid Friend erythroleukemia, both as wild-type and selected for resistance to DXR variants. The chemoresistant strains had characteristics of ‘typical’ MDR [1, 21, including cross-resistance to vincristine (VCR) and over-expression of P-glycoprotein [ 131. The experiments with 8-Cl-CAMP were carried out on cells grown on foetal calf serum either heat-inactivated or not; this was done to ascertain whether the preservation of the serum enzymic activities could influence the effects of the compound. In some experiments on B 16 melanoma the antiproliferative activity of 8-Cl-AMPc was studied also in the presence of isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, or adenosine deaminase (ADA) and this with the aim of making a better assessment of the responsibility of 8-Cl-adenosine as a growthinhibitory metabolite of the compound. MATERIALS
AND METHODS
Drugs
8-chloro-cyclic adenosine monophosphate (NSC 614491) was kindly supplied by Dr Nancita A. Lomax (National Cancer Institute, Bethesda, USA). 8-chloro-
83
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adenosine was supplied by Biolog Life Science Institute, Bremen, Germany, and doxorubicin hydrochloride by Farmitalia Carlo Erba, Milan, Italy. Isobutylmethylxanthine (IBMX) and calf intestinal mucosa adenosine deaminase (ADA) were from Sigma Chemical Co., St. Louis, MO, USA. Cell lines and cytotoxicity assays
Mouse B16 melanoma cells as parental (B16) or DXR-resistant (B16/DXR) variants and mouse Friend erythroleukaemia cells as parental (FLC) and DXRresistant (FLC/DXR) variants were obtained as previously described [ 131. The cell lines were grown in RPM1 1640 (Gibco, Grand Island, NY, USA) containing 10% foetal calf serum (Gibco), whether heat-inactivated (by heating at 56°C for 30 min) or not, and 1% penicillin and streptomycin; there was a humidified atmosphere of 5% CO2 in air at 37°C. In the case of B16 and B16/DXR 1 mM sodium pyruvate was also added to the culture medium and, in order to maintain the level of resistance, B16/DXR cells were grown in the presence of DXR 15 ng ml-‘. B16 and B16/DXR were subcultured twice a week, FLC and FLC/DXR three times a week. B 16 and B 16/DXR were seeded at 1x10’ ml-’ and FLC and FLC/DXR at 5x lo4 ml-’ in 24-well culture plates (NUNC, Roskilde, Denmark) and in the presence of various concentrations of the drugs: 72 or, in some cases, 144 h after the beginning of the experiment the viable cells were counted through the microscope with Trypan blue exclusion. In the case of B16 and B16/DXR, the cells were harvested by trypsin-EDTA because they had grown as monolayer cultures.
RESULTS The characteristics of the cells used here have already been described [13] and some of them are summarized in Table 1. When compared with their parental counterparts, B16/DXR and FLC/DXR proved to be resistant both to DXR and VCR. The MDR phenotype of these cell lines also included the over-expression of P-glycoprotein, detected by immunocytochemistry or immunoblotting [ 131.
Table I Some characteristics of B16 melanoma and Friend erythroleukaemia DXR-sensitive or -resistant variants IC50DXR (ng ml I*)
) KS0 VCR (ng ml _I*
(RI)
(RI)
Doubling (b)
B16 B I6fDXR
5.5 65.0 (11.8)
24.0 170.0 (7.1)
13.6 11.1
FLC FLC/DXR
5.3 460.2 (86.8)
12.0 155.0 (12.9)
13.3 13.5
*Mean of triplicate experiments RI: index of resistance.
with
SDS
10%.
time*
as
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60
1
1
1
5
1
10
/
I
50 100
I
I
250 500 1000
80 60 40 20 I
0
I
,I
1
10
I
1
50
100
I
500
8-Cl-CAMP (pm) Fig. 1. Effects of 8-Cl-CAMP on the growth of B16, B 16/DXR, FLC and FLC/DXR, either in the presence of heat-inactivated (A and C) or fresh serum (B and D). (A) and (B): 0, B16; ?? , Bl6-DXR. (C) and (D): 0, FLC; W, FLC-DXR. Results are expressed as a percentage of the control cell number and are the mean of at least three independent experiments; SD was regularly less than 10%. For further details see Materials and Methods and Results.
The effects of ~-U-CAMP on the growth of the various cell lines are shown in Fig. 1. B 16 and B 16/DXR were seeded at 50 000 per ml and FLC and FLC/DXR at 100 000 per ml in the presence of a wide range of concentrations of ~-U-CAMP; fresh solutions of the drug were added after 48 h. Control cells received only standard medium. The cells were counted 72 h after the beginning of the experiment. At that time, the numberfsD (per millilitre) of the control cells was
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I
I
1
1
5
10
I
/
I
I
50
100
250
500
B-Cl-CAMP (flrn)
Fig. 2. Effects of 8Cl-CAMPfadenosine deaminase (ADA) on B16 cells. The cells were cultivated with heat-inactivated serum and seeded in presence of 8-Cl-CAMP&ADA in a concentration of 3 U/ml; 48 h later the medium was removed and new medium, 8-Cl-CAMP and ADA were added. The cells were counted after 72 or 144 h from the beginning of the experiment; the number (per ml) of the control cells was 910 416243 898 at 72 h and 971 083f 89 340 at 144 h. Results are expressed as a percentage of the control cell number and are the mean of three independent experiments with SD regularly less than 10%. 0, 72 h 8-Cl-CAMP; 0,72 h 8-Cl-cAMP+ADA; W, 144 h 8-Cl-CAMP; 0, 144 h 8-Cl-cAMP+ADA. ADA alone did not affect the growth of the cells (not shown). For further details see Materials and Methods and Results.
853 125f52 570 for B16 grown in heat-inactivated serum and 881 2.50+44 083 for B16 grown in fresh serum; it was 1 181 250+79 733 for B16/DXR grown in heatinactivated serum and 1 15 1 041+59 484 for B 16/DXR grown in fresh serum; it was 2 7 13 28 1f 15 1 69 1 for FLC grown in heat-inactivated serum and 2 168 625f 181 242 for FLC grown in fresh serum; it was 2 604 687f164 994 for FLC/DXR grown in heat-inactivated serum and 1 881 250+94 923 for FLC/DXR grown in fresh serum. The plots of Fig. 1 (in which the results are expressed as a percentage of the control cell numbers) indicate that when the cells were grown in the presence of heat-inactivated serum, 8-Cl-CAMP was active at rather elevated concentrations; the 50% inhibitory concentration (ICJ was about 215 PM in B16 and B16/DXR (Fig. 1A) and about 70~~ in FLC and FLC/DXR (Fig. 1C). However, the I& values turned out to be considerably lower when the cells were grown in fresh serum and were about 4.7 ,LLMfor B 16 and B 16/DXR (Fig. 1B) and about 12 PM for FLC and FLC/DXR (Fig. 1D). In some experiments on B 16 cells (not shown) the activity of 8-Cl-CAMP was also studied in the presence of IBMX 0.25 mM; this phosphodiesterase inhibitor, which alone affected the growth of the cells only marginally, greatly antagonized the antiproliferative effects of the CAMP analogue raising its ICSO to a comparable value (about 700 PM) both in fresh and heat-inactivated serum. The activity of 8-Cl-CAMP on B16 was also virtually abolished following co-exposure to ADA in a concentration of 3 U ml-’ (Fig. 2). Furthermore, we studied the antiproliferative effects of g-Cladenosine on the various cell lines grown in heat-inactivated serum; the dose-
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100 l,?YTYT?L (D)
80 60 40 20 0
0.1
0.5 1
3
10
30
100 300 1000
DXR (ng ml-‘)
Fig. 3. Effects of DXRf8-Cl-CAMP on the growth of B16, B16/DXR (A and B), FLC and FLC/DXR (C and D) cells. (A) and (B): 0, B16:DXR; 0, B16:DXR+&CI-CAMP; W, B16DXR: DXR; 0, Bl6-DXR: DXR+8-Cl-CAMP. (C) and (D): 0, FLC: DXR; 0, FLC:DXR+8Cl-CAMP; W, FLC-DXR: DXR; 0, FLC-DXR: DXR+8-Cl-CAMP. The experiments were carried out in heat-inactivated (A and C) or fresh serum (B and D). ~-U-CAMP was 50,~~ in (A), 1 PM in (B), 15 ,UM in (C) and 2 PM in (D). Results are expressed as a percentage of the control cell number and are the mean of at least three independent experiments with SD regularly less than 10%. Combined 8-Cl-cAMP/DXR data are normalized to account for the inhibitory effects of 8-Cl-CAMP alone (like those noticed in Fig. 1). For further details see Materials and Methods and Results.
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response curves of this compound (not shown), of which fresh solutions were added after 0 and 48 h as in the case of 8-Cl-CAMP, were similar in shape to those observed with 8-Cl-CAMP in fresh serum. The IGo of 8-Cl-adenosine proved to be about 2.1 PM for B 16 and B 16/DXR, 0.6 PM for FLC and FLC/DXR. Experiments were also carried out to ascertain if cotreatment with slightly cytotoxic concentrations of 8-Cl-CAMP could influence the sensitivity to DXR of our cell lines (either grown in fresh or in heat-inactivated serum). In the experiments of Fig. 3, the cells were seeded in the presence of 8-Cl-CAMP and fresh 8-Cl-CAMP was added after 48 h; various concentrations of DXR were added 24 h after the beginning of the experiment. The cells were counted 72 h after the beginning of the experiment. The numbers of the control cells were very similar to those already reported for the experiments of Fig. 1 and they are not quoted for the sake of brevity. 8-Cl-CAMP was 50 PM for B16 and B16/DXR grown in heat-inactivated serum (Fig. 3A) and 1 PM for B16 and BlB/DXR grown in fresh serum (Fig. 3B). It was 15 ,u~ for FLC and FLC/DXR grown in heatinactivated serum (Fig. 3C) and 2 PM when the same cells were grown in fresh serum (Fig. 3D). In presenting the combination data of Fig. 3, the slight cytotoxic effects of 8-Cl-CAMP alone (as in the experiments of Fig. 1) were normalized by making these equal to 100%. From the plots of Fig. 3 it can be seen that neither with fresh nor with inactivated serum did 8-Cl-CAMP show synergism with DXR in the drug-resistant tumours; only additive (Fig. 3A, B and C) or slight antagonistic (Fig. 3D) antiproliferative effects were seen. For the sensitive cell lines, slight synergistic effects were encountered in the case of B 16 cells grown in fresh serum (Fig. 3B) and FLC grown in heat-inactivated serum (Fig. 3C); additive effects were noticed in the other two cases (Fig. 3A and D). Finally, we studied the effects of slightly cytotoxic concentrations of 8-Cladenosine on the sensitivity to DXR of the cell lines (data not shown). These experiments were carried out with heat-inactivated serum. In no case was a synergistic antiproliferative effect of the combination of 8-Cl-adenosine with DXR noticed.
DISCUSSION In the present work we started from the reported premise that 8-Cl-CAMP, a stable, potent and site-selective analogue of CAMP [7], may exert potentially useful antiproliferative effects on drug-sensitive and drug-resistant tumour cell populations and, administered at subcytotoxic concentrations, may favourably modify the sensitivity to DXR of MDR tumours [6, 121. We explored these possible activities of the compound in two mouse tumour cell lines, the solid B16 melanoma and the liquid Friend erythroleukaemia, both as DXR-sensitive and DXR-resistant MDR variants. Encouragingly, 8-Cl-CAMP affected the growth of the chemosensitive and chemoresistant variants of both cell lines almost equally. However, the I& values observed in the experiments on cells cultivated with heat-inactivated serum appeared rather high (about 2 15 PM for B 16 and B 16/DXR and about 70 PM for FLC and FLC/DXR), also when compared with the data reported for other tumour
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cell lines where the activity of 8U-CAMP was in the l-25 PM range [7]. It must be stressed that we used a wide range of ~-U-CAMP doses, including low [l-lo ,UM] ones, which, according to Cho-Chung [8], should undergo only minimal degradation, if any (see below). In addition, the cells were exposed to the compound for 72 h. We know that in this period, after a phase of adaptation during which they halve their initial inoculum, untreated B16 and B16-DXR cells produce about four new cell generations; in the same period, FLC and FLC-DXR cells produce about 5-6 new cell generations. Thus, the time of exposure of our cell lines to 8-Cl-CAMP was even longer than that (2-3 generation times longer) reported by others [7, 81 as necessary for the antiproliferative activity of the compound to be apparent. On the other hand, the I& values were considerably reduced (about 4.7 ,UM for B 16 and B 16/DXR and about 12 ,LLM for FLC and FLUDXR) in the presence of fresh serum. Looking for an explanation for these findings, we can cite several papers in which the possible mechanisms of the inhibitory effect of 8-Cl-CAMP on tumour cell growth have been examined. It has been proposed that the compound may act by itself and induce differentiation through a selective interaction with the regulatory subunit RIIP of type II CAMP-dependent kinase, which would translocate to the nucleus where it restores a normal gene transcription pattern [7, 81. Alternatively, 8-Cl-CAMP may form growth-inhibitory metabolites, among which 8-Cl-adenosine is the most important [9, 10, 11, 141; it has also been reported that, in conditions similar to those we adopted, Gibco foetal calf serum, through phosphodiesterase and 5’-nucleotidase activities, hydrolizes about 72% of 8-Cl-CAMP 100,~~ with the production of 8-Cl-adenosine as the principal metabolite; the hydrolysis is only 11% when the same serum is heat-inactivated [9]. Thus, it seems likely that in our cell lines 8-Cl-CAMP acted mainly through its transformation, probably into 8-Cl-adenosine, and this was supported by the virtual abolition of the activity of the compound, both in fresh and heat-inactivated serum, when B16 melanoma cells were coexposed to IBMX, a phosphodiesterase inhibitor, or, more specifically, to ADA, which should convert 8-Cl-adenosine to 8-chloro-inosine; according to Van Lookeren Campagne [9], the latter compound is devoid of a growth-inhibitory activity. The I& values of 8-Cl-adenosine for the cell lines were lower than those of 8-Cl-CAMP in fresh serum, but this result may be compatible with an incomplete transformation of the latter compound in that medium. The other major aspect, i.e. the possibility that subcytotoxic concentrations of 8-Cl-CAMP might favourably modulate the sensitivity of DXR of MDR turnours, was not seen in our experiments either with fresh or heat-inactivated serum. The combination of S-Cl-adenosine with DXR did not even produce synergistic antiproliferative effects. With regard to these results, in other papers [6, 121 g-ClCAMP appeared to be capable of sensitizing to DXR MDR cells whether or not they over-express P-glycoprotein. A rationale has been put forward according to which the RIa regulatory subunit of CAMP-dependent protein kinase is in excess in the MDR cells [6, 12 and see also 51; accordingly, 8-Cl-CAMP would downregulate the RIa subunit and control the processes of MDR, including the Pglycoprotein over-expression, at transcriptional level [6, 121. We are currently not aware whether these critical modifications of the RI subunit levels are present in
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our cell lines; indeed, their growth was affected by 8-Cl-adenosine and, although the mechanisms of the cytotoxic activity of this compound are still not completely known, it is interesting that in a study on normal and neoplastic mouse lung cells it selectively down-regulated the RI and Ca subunits of type I CAMP-dependent protein kinase [14]. On the other hand, it is also possible that our cells have some other factor which would account for their unresponsiveness to 8-Cl-CAMP per se, such as, for example, lack of or alterations to the RI1 isoforms of CAMP-dependent protein kinase subunits, which have frequently been observed in human and mouse tumour cells [14-161. In any case, we conclude that the supposed modulatory activity of 8-Cl-CAMP as a sensitizer to DXR in MDR cell lines cannot be generalized to the relevant models of typical MDR used herein.
ACKNOWLEDGEMENTS This work was partially
supported
by AIRC.
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