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Synergistic effect between doxorubicin and a low dose of all-trans-retinoic acid in MCF-7 breast cancer cell line
Cancer Letters 116 (1997) 103–110
Synergistic effect between doxorubicin and a low dose of all-transretinoic acid in MCF-7 breast cancer cell line Salvatore Toma a , c ,*, Giuseppina Maselli b , c, Giuseppe Dastoli d , e, Elena De Francisci c, Patrizia Raffo a , c a
National Institute for Cancer Research (IST), Department of Medical Oncology, University of Genoa, Genoa, Italy b C.A.R.S.O., Cancer Research Center, University of Bari, Bari, Italy c Pre-clinical Oncology Laboratory, Advanced Biotechnology Center (ABC), Genoa, Italy d ROCHE s.p.a., Milan 20100, Italy e Hoffman-La Roche, Basel, Switzerland Received 12 December 1996; revision received 27 February 1997; accepted 27 February 1997
Abstract The effect of doxorubicin (DOX) used in combination with a low dose (10−7 M) of all-trans-retinoic acid (tRA) was tested on MCF-7 breast carcinoma cell line. Both drugs are able to inhibit cell proliferation in these cells in a dose-dependent way. The combined treatment with DOX and tRA was more effective in inhibiting cell growth than each of the two compounds alone. This was evidenced in the following experimental conditions: pre-treatments with tRA, for 72 h or 18 h, before DOX incubation; post-treatment with tRA for 18 h after DOX incubation. A consistent synergism was reached by 72 h pre-treatment with tRA and also with brief pre- and post-treatments, but only if tRA was also present during DOX incubation (cotreatments). The mechanisms involved in this interaction between chemotherapeutics and differentiating agents are as yet unclear and should be evaluated further. Published by Elsevier Science Ireland Ltd. Keywords: Doxorubicin; All-trans-retinoic acid; Synergistic effect; Breast cancer
1. Introduction Human mammary carcinoma is a heterogeneous entity in terms of sensitivity to cytotoxic agents. The most active single drug [1] in this disease is doxorubicin (DOX), a powerful antitumoral drug, acting
* Corresponding author. Tel.: +39 10 5600670/5737511/5737505; fax: +39 10 5737505.
Published by Elsevier Science Ireland Ltd. PII S0304- 3835 (97 )0 0169- 9
through a series of mechanisms: intercalation in DNA molecule, breaking of DNA strands by interaction with topoisomerase II, free radicals formation [2] and alterations of cell permeability. On the other hand, retinoic acid derivatives, a group of natural and synthetic compounds structurally related to vitamin A, have been demonstrated in various systems as potential agents of chemoprevention and treatment of human cancers [3]. All-trans retinoic acid (tRA), in particular, inhibits the growth of a num-
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ber of cell lines derived from human cancers, including several breast cancer cell lines [4–6]. On these bases, we have investigated the possible increase of the in vitro effects of DOX used in combination with tRA in terms of inhibition of proliferation in a human breast cancer cell line. For this purpose, MCF-7 cells (estrogen receptor positive, ER+) were chosen since it is known that retinoids have profound effects on the growth of this cell line [7–9].
was dissolved in phosphate buffered saline (PBS) solution at a concentration of 1 mg/ml and stored at −20°C; serial dilutions of this drug were performed in culture medium, immediately before each experiment, to obtain the required final concentrations. Retinoid manipulations were carried out in subdued yellow light, whereas all procedures with doxorubicin were performed in a dark room. 2.2. Cell culture
2. Materials and methods 2.1. Chemicals All-trans retinoic acid (Hoffman-La-Roche Ltd., Basel, Switzerland) was dissolved in absolute ethanol (Farmitalia Carlo Erba, Milan, Italy) at a concentration of 10−2 M and then diluted in culture medium to the required final concentration. Retinoid stock solutions were kept at −20°C. Doxorubicin (Farmitalia Carlo Erba, Milan, Italy)
Human mammary carcinoma cell line MCF-7 (ER + with epidermal phenotype) was used in this study. Cells were routinely cultured in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum, 1 mM l-glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin and 1% non-essential amino acids (ICN-Biomedicals). Cells were grown as a monolayer in T75 flasks (Falcon, Becton Dickinson and Co., NJ) in a humid atmosphere with 5% CO2 at 37°C. For proliferation analysis, cells were seeded in
Fig. 1. Effect of DOX on the growth of MCF-7 cell line. Cells were incubated with various concentrations of DOX for 1 h. Cell number was evaluated on the 7th, 10th, 13th and 15th day. Results are expressed as mean of three separate experiments.
S. Toma et al. / Cancer Letters 116 (1997) 103–110
105
six-well tissue culture plates at a concentration of 5 × 103 cells/well with 3 ml of specific medium. Cells were allowed to attach for 24 h and then incubated in medium with the appropriate concentration of drugs.
after and during DOX incubation (Post and Co-treatment). After exposure to drugs, cells were incubated in complete medium until the end of the experiment, renewing it every 2 days.
2.3. Drug treatment
2.4. Proliferation analysis
Cells were incubated with different concentrations of DOX, ranging from 0.06 to 3 mg/ml, for 1 h. Treatment with tRA, at a fixed concentration of 10−7 M, was performed in different ways: (1) for 72 h before DOX incubation (Long Pre-treatment); (2) for 18 h before DOX incubation (Short Pre-treatment); (3) for 18 h after DOX incubation (Post-treatment); (4) for 18 h before and during DOX incubation (Short Pre- and Co-treatment); (5) for 18 h
Cell proliferation was evaluated by growth curves. Cell numbers were determined using a hematocytometer every 2 days after drug treatment. Each experiment was performed in triplicate. Statistical significance was calculated using Student’s t-test. We compared the percentage of surviving cells (in respect to control) obtained with single agent treatment and that achieved with various drug combinations. Synergism was defined as: %AB/
Fig. 2. Growth inhibition of MCF-7 cells treated with tRA 10−7 M for 72 h before DOX incubation at different drug concentrations: (a) 3 mg/ ml; (b) 1 mg/ml; (c) 0.3 mg/ml; (d) 0.06 mg/ml. Histograms show the percentage of survival of DOX-, tRA- and DOX + tRA-treated cells with respect to untreated cells. Results are expressed as mean ± SD of three separate experiments.
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Fig. 3. Growth inhibition of MCF-7 cells treated with tRA 10−7 M for 18 h before DOX incubation at different drug concentrations: (a) 3 mg/ ml; (b) 1 mg/ml; (c) 0.3 mg/ml; (d) 0.06 mg/ml. Histograms show the percentage of survival of DOX-, tRA- and DOX + tRA-treated cells with respect to untreated cells. Results are expressed as mean ± SD of three separate experiments.
%A × %B , 1. Additivity was defined as: %AB/ %A × %B = 1.
3. Results As reported in Fig. 1, DOX was a potent dosedependent antiproliferative agent in MCF-7 cell line (77% proliferation inhibition after 1 h treatment with 3 mg/ml); (DOX ID50 = 1 mg/ml). As we have previously shown [9], tRA is able to inhibit MCF-7 cells proliferation in a dose- and timedependent way. According to this, in the present study either short (18 h) or long (72 h) treatments with 10 − 7 M tRA alone induced a proliferation inhibition, which ranged between 25% and 42%. Moreover, DOX cell growth inhibition resulted regularly dose-dependent, with a major variability of the effect at the maxi-
mum testable dosage. The results are reported in Figs. 2–6. Worthy of note, tRA was able in all treatment types to potentiate the antiproliferative action of DOX. This effect of tRA was mainly marked against low doses of DOX, thus appearing to favor an application in the clinical field. In particular, when cells were incubated with 10 − 7 M tRA for 72 h before DOX administration (Long Pre-treatment), we demonstrated an antiproliferative effect potentiation in respect of the most effective dose of DOX alone (P = 0.021 for DOX 0.3 mg/ml), reaching the level of synergism (see Fig. 2). Synergism was reached also in brief pre- and post-treatment experiments, but only when 10−7 M tRA was also present during DOX exposure (Co-treatments), as shown in Figs. 5 and 6. In contrast, no synergistic effect could be detected
S. Toma et al. / Cancer Letters 116 (1997) 103–110
107
Fig. 4. Growth inhibition of MCF-7 cells treated with tRA 10−7 M for 18 h after DOX incubation at different drug concentrations: (a) 3 mg/ml; (b) 1 mg/ml; (c) 0.3 mg/ml; (d) 0.06 mg/ml. Histograms show the percentage of survival of DOX-, tRA- and DOX + tRA-treated cells with respect to untreated cells. Results are expressed as mean ± SD of three separate experiments.
in experiments where brief pre- and post-treatments with tRA were not superimposed on DOX incubation; although also in this case the effect of DOX was again increased by tRA. In fact, as shown by Figs. 3 and 4, when 10−7 M tRA treatment was performed for a brief period (18 h) either before or after DOX incubation, the action of the antibiotic was significantly increased (with DOX doses of 1 mg/ml, P = 0.014 and P = 0.020 for pre- and post-treatment, respectively).
4. Discussion DOX and tRA, used alone, have been shown to inhibit cell growth of mammary carcinoma cell lines by various authors [10–14]. Regarding tRA, the present observations are in agreement with our previous studies confirming the antiproliferative efficacy of this
drug, and its time- and dose-dependent activity, in MCF-7 cell line [9]. The rationale to associate these agents in order to increase their antiproliferative effect is that tRA treatment in this cellular model can produce growth inhibition without inhibiting DNA synthesis and cell cycle progression [9], thus maintaining cells sensitive to cytotoxic drugs. Confirming this hypothesis, our findings demonstrate that the associated treatment with tRA and DOX is able to potentiate single agent effects in different experimental conditions. In particular, a critical role seems to be played by the duration of tRA exposition, as shown by long pre-treatment experiments, or by simultaneous presence of tRA during DOX incubation, as observed in both short pre- and post-treatments. We remark that there is no preferential sequence of treatment, tRA being effective in poten-
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S. Toma et al. / Cancer Letters 116 (1997) 103–110
Fig. 5. Growth inhibition of MCF-7 cells treated with tRA 10−7 M for 18 h before and during DOX incubation at median drug concentrations: (a) 1 mg/ml, Pre-treatment; (b) 1 mg/ml, Pre/Co-treatment; (c) 0.3 mg/ml, Pre-treatment; (d) 0.3 mg/ml, Pre/Co-treatment. Histograms show the percentage of survival of DOX-, tRA- and DOX + tRA-treated cells with respect to untreated cells. Results are expressed as mean ± SD of three separate experiments.
tiating antiproliferative effects both when used before and after DOX; DOX concentration seems not to be crucial in combined treatment, except for the highest dose used (3 mg/ml). These data seem to indicate that different mechanisms may be involved in this augmented effect, such as modulation by tRA of DOX cellular intake, metabolism, and interaction with DNA and topoisomerase II. A better knowledge of tRA mechanisms of drugs modulation may be achieved by combining tRA with cytotoxic agents displaying a specific mechanism of action, such as actinomicin D, or nitrosoureas, or topoisomerase inhibitors. Of great interest may be also the evaluation of inhibition of AP-1 complex, a possible mechanism of tRA action in this cellular model. Our in vitro results are extended by several inves-
tigators to other cellular models, like squamous carcinoma cells [15], small cell lung cancer line [16] and ovarian teratocarcinoma cells [17] exposed to various cytotoxic agents in combination with tRA, and also in vivo preclinical data offer promising results. Only a few reports on clinical studies have examined the combination of retinoids and chemotherapeutics, showing interesting results only in skin [18] and in head and neck [19,20] cancer. We remark that the low tRA concentration used in our study (10−7 M) corresponds to an in vivo plasmatic concentration of 30 ng/ml, pharmacologically reachable in clinical treatment without excessive sideeffects. Further in vitro and in vivo studies are therefore needed to better investigate the molecular mechanisms involved in the interaction among chemothera-
S. Toma et al. / Cancer Letters 116 (1997) 103–110
109
Fig. 6. Growth inhibition of MCF-7 cells treated with tRA 10 − 7 M for 18 h after and during DOX incubation at median drug concentrations: (a) 1 mg/ml, Post-treatment; (b) 1 mg/ml, Post/Co-treatment; (c) 0.3 mg/ml, Post-treatment; (d) 0.3 mg/ml, Post/Co-treatment. Histograms show the percentage of survival of DOX-, tRA- and DOX + tRA-treated cells with respect to untreated cells. Results are expressed as mean ± SD of three separate experiments.
peutics and differentiating agents and the possible impact of their use as a novel approach in clinical practice.
[3]
[4]
Acknowledgements This work was partially supported by Roche s.p.a., Milano, Italy. We thank Dr. Taningher for his helpful criticism and suggestions.
References [1] Haskell, C.M. (1995). Systemic treatment for metastatic breast cancer. In: Cancer Treatment, pp. 354–368. Editor: C.M. Haskell. Saunders, Philadelphia, PA. [2] Lown, J.W. (1985) Molecular mechanisms of action of anti-
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[6]
[7]
[8]
cancer agents involving free radical intermediates, Free Rad. Biol. Med., 1, 225–264. Sporn, M.B. and Roberts, A.B. (1983) Role of retinoids in differentiation and carcinogenesis, Cancer Res., 43, 3034– 3040. Lotan, R. (1979) Different susceptibilities of human melanoma and breast carcinoma cell lines to retinoic acid-induced growth inhibition, Cancer Res., 39, 1014–1019. Monn, R.C., Mehta, R.G. and Detrisac, C.J. (1992) Retinoids as chemopreventive agents for breast cancer, Cancer Detect. Prev., 16 (1), 73–79. Kurie, J.M., Lippmann, S.M. and Hong, W.K. (1994) Potential of retinoids in cancer prevention, Cancer Treat. Rev., 20, 1–10. Lacroix, A. and Lippman, M.E. (1980) Binding of retinoids to human breast cancer cell lines and their effects on cell growth, J. Clin. Invest., 65, 586–591. Ueda, H., Takenawa, T., Millan, J.C., Gesell, M.S. and Brandes, D. (1980) The effect of retinoids on proliferative capacities and macromolecular synthesis in human breast cancer MCF-7 cells, Cancer (Philadelphia), 46, 2203–2209.
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[9] Toma, S., Isnardi, L., Raffo, P., Dastoli, G., De Francisci, E., Riccardi, L., Palumbo, R., and Bollag, W. (1997) Effects of all-trans-retinoic acid and 13-cis-retinoic acid on breast cancer cell lines: growth inhibition and apoptosis induction. Int. J. Cancer, 70, in press. [10] Sinha, B.K., Katki, A.G., Batist, G., Cowan, K.H. and Myers, C.E. (1987) Adriamycin-stimulated hydroxyl radical formation in human breast tumor cells, Biochem. Pharmacol., 36, 793–796. [11] Doroshow, J. (1986) Prevention of doxorubicin-induced killing of MCF-7 human breast cancer cells by oxygen radical scavengers and iron chelating agents, Biochem. Biophys. Res. Commun., 135, 330–335. [12] Wetherall, N.T. and Taylor, C.M. (1986) The effects of retinoid treatment and antiestrogens on the growth of T47D human breast cancer cells, Eur. J. Cancer Clin. Oncol., 22 (1), 53–59. [13] Marth, C., Bock, G. and Daxenbichler, G. (1985) Effects of 4hydroxyphenyl retinamide and retinoic acid on proliferation and cell cycle of cultured human breast cancer cells, J. Natl. Cancer Inst., 75 (5), 871–875. [14] Toma, S., Monteghirfo, S., Tasso, P., Nicolo`, G., Spadini, N., Palumbo, R. and Molina, F. (1994) Antiproliferative and synergistic effect of interferon alpha-2a, retinoids and their association in established human cancer cell lines, Cancer Lett., 82, 209–216.
[15] Sacks, P.G., Harris, D. and Chou, T.-C. (1995) Modulation of growth and proliferation in squamous cell carcinoma by retinoic acid: a rationale for combination therapy with chemotherapeutic agents, Int. J. Cancer, 61, 409–415. [16] Doyle, L.A., Giangiulo, D., Hussain, A., Park, H.-J., Yen, R.W.C. and Borges, M. (1989) Differentiation of human variant small cell lung cancer cell lines to a classic morphology by retinoic acid, Cancer Res., 49, 6745–6751. [17] Ruppert, K.L., Masters, J.R.W., Kneuchel, R., Seegers, S., Tain-sky, M.A., Hofstaedter, F. and Buettner, R. (1992) The effect of retinoic acid on chemosensitivity of PA-1 human teratocarcinoma cells and its modulation by an activated N-ras oncogene, Int. J. Cancer, 51, 646–651. [18] Prutkin, L. (1992) Antitumor activity of vitamin A acid and fluorouracil used in combination on the skin tumor, keratocarcinoma, Cancer Res., 33, 128–133. [19] Komiyama, S., Hiroto, I., Ryu, S., Nakashima, T., Kuwano, M. and Endo, H. (1978) Synergistic combination therapy of 5-fluorouracil, vitamin A and cobalt-60 radiation upon head and neck tumors, Oncology, 35, 253–257. [20] Thatcher, N., Blackledge, G. and Crowther, D. (1980) Advanced recurrent squamous cell carcinoma of the head and neck. Results of a chemotherapeutic regimen with adriamycin, bleomycin, 5-fluorouracil, methotrexate and vitamin A, Cancer, 46, 1324–1328.
Synergistic effect between doxorubicin and a low dose of all-transretinoic acid in MCF-7 breast cancer cell line Salvatore Toma a , c ,*, Giuseppina Maselli b , c, Giuseppe Dastoli d , e, Elena De Francisci c, Patrizia Raffo a , c a
National Institute for Cancer Research (IST), Department of Medical Oncology, University of Genoa, Genoa, Italy b C.A.R.S.O., Cancer Research Center, University of Bari, Bari, Italy c Pre-clinical Oncology Laboratory, Advanced Biotechnology Center (ABC), Genoa, Italy d ROCHE s.p.a., Milan 20100, Italy e Hoffman-La Roche, Basel, Switzerland Received 12 December 1996; revision received 27 February 1997; accepted 27 February 1997
Abstract The effect of doxorubicin (DOX) used in combination with a low dose (10−7 M) of all-trans-retinoic acid (tRA) was tested on MCF-7 breast carcinoma cell line. Both drugs are able to inhibit cell proliferation in these cells in a dose-dependent way. The combined treatment with DOX and tRA was more effective in inhibiting cell growth than each of the two compounds alone. This was evidenced in the following experimental conditions: pre-treatments with tRA, for 72 h or 18 h, before DOX incubation; post-treatment with tRA for 18 h after DOX incubation. A consistent synergism was reached by 72 h pre-treatment with tRA and also with brief pre- and post-treatments, but only if tRA was also present during DOX incubation (cotreatments). The mechanisms involved in this interaction between chemotherapeutics and differentiating agents are as yet unclear and should be evaluated further. Published by Elsevier Science Ireland Ltd. Keywords: Doxorubicin; All-trans-retinoic acid; Synergistic effect; Breast cancer
1. Introduction Human mammary carcinoma is a heterogeneous entity in terms of sensitivity to cytotoxic agents. The most active single drug [1] in this disease is doxorubicin (DOX), a powerful antitumoral drug, acting
* Corresponding author. Tel.: +39 10 5600670/5737511/5737505; fax: +39 10 5737505.
Published by Elsevier Science Ireland Ltd. PII S0304- 3835 (97 )0 0169- 9
through a series of mechanisms: intercalation in DNA molecule, breaking of DNA strands by interaction with topoisomerase II, free radicals formation [2] and alterations of cell permeability. On the other hand, retinoic acid derivatives, a group of natural and synthetic compounds structurally related to vitamin A, have been demonstrated in various systems as potential agents of chemoprevention and treatment of human cancers [3]. All-trans retinoic acid (tRA), in particular, inhibits the growth of a num-
104
S. Toma et al. / Cancer Letters 116 (1997) 103–110
ber of cell lines derived from human cancers, including several breast cancer cell lines [4–6]. On these bases, we have investigated the possible increase of the in vitro effects of DOX used in combination with tRA in terms of inhibition of proliferation in a human breast cancer cell line. For this purpose, MCF-7 cells (estrogen receptor positive, ER+) were chosen since it is known that retinoids have profound effects on the growth of this cell line [7–9].
was dissolved in phosphate buffered saline (PBS) solution at a concentration of 1 mg/ml and stored at −20°C; serial dilutions of this drug were performed in culture medium, immediately before each experiment, to obtain the required final concentrations. Retinoid manipulations were carried out in subdued yellow light, whereas all procedures with doxorubicin were performed in a dark room. 2.2. Cell culture
2. Materials and methods 2.1. Chemicals All-trans retinoic acid (Hoffman-La-Roche Ltd., Basel, Switzerland) was dissolved in absolute ethanol (Farmitalia Carlo Erba, Milan, Italy) at a concentration of 10−2 M and then diluted in culture medium to the required final concentration. Retinoid stock solutions were kept at −20°C. Doxorubicin (Farmitalia Carlo Erba, Milan, Italy)
Human mammary carcinoma cell line MCF-7 (ER + with epidermal phenotype) was used in this study. Cells were routinely cultured in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum, 1 mM l-glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin and 1% non-essential amino acids (ICN-Biomedicals). Cells were grown as a monolayer in T75 flasks (Falcon, Becton Dickinson and Co., NJ) in a humid atmosphere with 5% CO2 at 37°C. For proliferation analysis, cells were seeded in
Fig. 1. Effect of DOX on the growth of MCF-7 cell line. Cells were incubated with various concentrations of DOX for 1 h. Cell number was evaluated on the 7th, 10th, 13th and 15th day. Results are expressed as mean of three separate experiments.
S. Toma et al. / Cancer Letters 116 (1997) 103–110
105
six-well tissue culture plates at a concentration of 5 × 103 cells/well with 3 ml of specific medium. Cells were allowed to attach for 24 h and then incubated in medium with the appropriate concentration of drugs.
after and during DOX incubation (Post and Co-treatment). After exposure to drugs, cells were incubated in complete medium until the end of the experiment, renewing it every 2 days.
2.3. Drug treatment
2.4. Proliferation analysis
Cells were incubated with different concentrations of DOX, ranging from 0.06 to 3 mg/ml, for 1 h. Treatment with tRA, at a fixed concentration of 10−7 M, was performed in different ways: (1) for 72 h before DOX incubation (Long Pre-treatment); (2) for 18 h before DOX incubation (Short Pre-treatment); (3) for 18 h after DOX incubation (Post-treatment); (4) for 18 h before and during DOX incubation (Short Pre- and Co-treatment); (5) for 18 h
Cell proliferation was evaluated by growth curves. Cell numbers were determined using a hematocytometer every 2 days after drug treatment. Each experiment was performed in triplicate. Statistical significance was calculated using Student’s t-test. We compared the percentage of surviving cells (in respect to control) obtained with single agent treatment and that achieved with various drug combinations. Synergism was defined as: %AB/
Fig. 2. Growth inhibition of MCF-7 cells treated with tRA 10−7 M for 72 h before DOX incubation at different drug concentrations: (a) 3 mg/ ml; (b) 1 mg/ml; (c) 0.3 mg/ml; (d) 0.06 mg/ml. Histograms show the percentage of survival of DOX-, tRA- and DOX + tRA-treated cells with respect to untreated cells. Results are expressed as mean ± SD of three separate experiments.
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Fig. 3. Growth inhibition of MCF-7 cells treated with tRA 10−7 M for 18 h before DOX incubation at different drug concentrations: (a) 3 mg/ ml; (b) 1 mg/ml; (c) 0.3 mg/ml; (d) 0.06 mg/ml. Histograms show the percentage of survival of DOX-, tRA- and DOX + tRA-treated cells with respect to untreated cells. Results are expressed as mean ± SD of three separate experiments.
%A × %B , 1. Additivity was defined as: %AB/ %A × %B = 1.
3. Results As reported in Fig. 1, DOX was a potent dosedependent antiproliferative agent in MCF-7 cell line (77% proliferation inhibition after 1 h treatment with 3 mg/ml); (DOX ID50 = 1 mg/ml). As we have previously shown [9], tRA is able to inhibit MCF-7 cells proliferation in a dose- and timedependent way. According to this, in the present study either short (18 h) or long (72 h) treatments with 10 − 7 M tRA alone induced a proliferation inhibition, which ranged between 25% and 42%. Moreover, DOX cell growth inhibition resulted regularly dose-dependent, with a major variability of the effect at the maxi-
mum testable dosage. The results are reported in Figs. 2–6. Worthy of note, tRA was able in all treatment types to potentiate the antiproliferative action of DOX. This effect of tRA was mainly marked against low doses of DOX, thus appearing to favor an application in the clinical field. In particular, when cells were incubated with 10 − 7 M tRA for 72 h before DOX administration (Long Pre-treatment), we demonstrated an antiproliferative effect potentiation in respect of the most effective dose of DOX alone (P = 0.021 for DOX 0.3 mg/ml), reaching the level of synergism (see Fig. 2). Synergism was reached also in brief pre- and post-treatment experiments, but only when 10−7 M tRA was also present during DOX exposure (Co-treatments), as shown in Figs. 5 and 6. In contrast, no synergistic effect could be detected
S. Toma et al. / Cancer Letters 116 (1997) 103–110
107
Fig. 4. Growth inhibition of MCF-7 cells treated with tRA 10−7 M for 18 h after DOX incubation at different drug concentrations: (a) 3 mg/ml; (b) 1 mg/ml; (c) 0.3 mg/ml; (d) 0.06 mg/ml. Histograms show the percentage of survival of DOX-, tRA- and DOX + tRA-treated cells with respect to untreated cells. Results are expressed as mean ± SD of three separate experiments.
in experiments where brief pre- and post-treatments with tRA were not superimposed on DOX incubation; although also in this case the effect of DOX was again increased by tRA. In fact, as shown by Figs. 3 and 4, when 10−7 M tRA treatment was performed for a brief period (18 h) either before or after DOX incubation, the action of the antibiotic was significantly increased (with DOX doses of 1 mg/ml, P = 0.014 and P = 0.020 for pre- and post-treatment, respectively).
4. Discussion DOX and tRA, used alone, have been shown to inhibit cell growth of mammary carcinoma cell lines by various authors [10–14]. Regarding tRA, the present observations are in agreement with our previous studies confirming the antiproliferative efficacy of this
drug, and its time- and dose-dependent activity, in MCF-7 cell line [9]. The rationale to associate these agents in order to increase their antiproliferative effect is that tRA treatment in this cellular model can produce growth inhibition without inhibiting DNA synthesis and cell cycle progression [9], thus maintaining cells sensitive to cytotoxic drugs. Confirming this hypothesis, our findings demonstrate that the associated treatment with tRA and DOX is able to potentiate single agent effects in different experimental conditions. In particular, a critical role seems to be played by the duration of tRA exposition, as shown by long pre-treatment experiments, or by simultaneous presence of tRA during DOX incubation, as observed in both short pre- and post-treatments. We remark that there is no preferential sequence of treatment, tRA being effective in poten-
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S. Toma et al. / Cancer Letters 116 (1997) 103–110
Fig. 5. Growth inhibition of MCF-7 cells treated with tRA 10−7 M for 18 h before and during DOX incubation at median drug concentrations: (a) 1 mg/ml, Pre-treatment; (b) 1 mg/ml, Pre/Co-treatment; (c) 0.3 mg/ml, Pre-treatment; (d) 0.3 mg/ml, Pre/Co-treatment. Histograms show the percentage of survival of DOX-, tRA- and DOX + tRA-treated cells with respect to untreated cells. Results are expressed as mean ± SD of three separate experiments.
tiating antiproliferative effects both when used before and after DOX; DOX concentration seems not to be crucial in combined treatment, except for the highest dose used (3 mg/ml). These data seem to indicate that different mechanisms may be involved in this augmented effect, such as modulation by tRA of DOX cellular intake, metabolism, and interaction with DNA and topoisomerase II. A better knowledge of tRA mechanisms of drugs modulation may be achieved by combining tRA with cytotoxic agents displaying a specific mechanism of action, such as actinomicin D, or nitrosoureas, or topoisomerase inhibitors. Of great interest may be also the evaluation of inhibition of AP-1 complex, a possible mechanism of tRA action in this cellular model. Our in vitro results are extended by several inves-
tigators to other cellular models, like squamous carcinoma cells [15], small cell lung cancer line [16] and ovarian teratocarcinoma cells [17] exposed to various cytotoxic agents in combination with tRA, and also in vivo preclinical data offer promising results. Only a few reports on clinical studies have examined the combination of retinoids and chemotherapeutics, showing interesting results only in skin [18] and in head and neck [19,20] cancer. We remark that the low tRA concentration used in our study (10−7 M) corresponds to an in vivo plasmatic concentration of 30 ng/ml, pharmacologically reachable in clinical treatment without excessive sideeffects. Further in vitro and in vivo studies are therefore needed to better investigate the molecular mechanisms involved in the interaction among chemothera-
S. Toma et al. / Cancer Letters 116 (1997) 103–110
109
Fig. 6. Growth inhibition of MCF-7 cells treated with tRA 10 − 7 M for 18 h after and during DOX incubation at median drug concentrations: (a) 1 mg/ml, Post-treatment; (b) 1 mg/ml, Post/Co-treatment; (c) 0.3 mg/ml, Post-treatment; (d) 0.3 mg/ml, Post/Co-treatment. Histograms show the percentage of survival of DOX-, tRA- and DOX + tRA-treated cells with respect to untreated cells. Results are expressed as mean ± SD of three separate experiments.
peutics and differentiating agents and the possible impact of their use as a novel approach in clinical practice.
[3]
[4]
Acknowledgements This work was partially supported by Roche s.p.a., Milano, Italy. We thank Dr. Taningher for his helpful criticism and suggestions.
References [1] Haskell, C.M. (1995). Systemic treatment for metastatic breast cancer. In: Cancer Treatment, pp. 354–368. Editor: C.M. Haskell. Saunders, Philadelphia, PA. [2] Lown, J.W. (1985) Molecular mechanisms of action of anti-
[5]
[6]
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