Departments of Neurosurgery (WZ, HY, NS, SN) and Biochemistry (YN), Gifu University School of Medicine, Gifu City, Japan Neurosurgery 31; 725-730, 1992 ABSTRACT: THE ANTIESTROGEN DRUG tamoxifen, which is used extensively in the treatment of breast cancer, has also been reported to inhibit the proliferation of some estrogen receptor-negative cell lines, including malignant glioma in vitro. To explore the possible role of tamoxifen in the treatment of malignant glioma, we have investigated its effects on cell growth and radiosensitivity in C6 glioma cells using a colony-forming assay and a tetrazoliumformazan growth rate assay. Pretreatment of C6 cells with tamoxifen resulted in dose-dependent inhibition of cell growth and enhancement of the antitumor effects of ACNU and irradiation. The radiosensitivity of the treated cells was enhanced by the administration of 5 µmol/L of tamoxifen either before and during irradiation or continuously before, during, and after irradiation [37% survival dose (Do) = 2.68 ± 0.19 and 2.64 ± 0.04 Gy, respectively, P < 0.01)], as compared with controls (Do = 3.79 ± 0.25 Gy). In addition, protein kinase C activity was also inhibited by tamoxifen at the concentration in which the radiosensitivity was augmented in C6 cells. Taken together, our results demonstrate a synergistic effect of tamoxifen with radiation on intracellular damage in C6 glioma cells, which may in part be due to the inhibition of protein kinase C, suggesting that tamoxifen serves as a useful agent in combination therapy of glioma. KEY WORDS: Glioma cells; Protein kinase C; Radiosensitization; Tamoxifen Despite treatment with conventional surgical, radiotherapeutic, and chemotherapeutic modalities, patients with malignant gliomas (glioblastoma multiform and anaplastic astrocytoma) have a poor prognosis (10,11). One reason is the resistance of gliomas to irradiation or chemotherapeutic agents. An important goal in the management of malignant glioma is to increase the cellular sensitivity to anticancer drugs and irradiation. Tamoxifen, (Z)-2[p-(1,2-diphenyl-1butenyl)phenoxy]-N,N-dimethylethylamine citrate (1:1), a synthetic, nonsteroidal antiestrogen, has been used widely in the treatment of postmenopausal women with estrogen receptor (ER)-positive breast cancer (3,16). It has been generally acknowledged that
MATERIALS AND METHODS Cell culture C6 cells (JCRB, Tokyo, Japan), were cultured in Dulbecco's modified Eagle's minimum essential medium (DMEM) supplemented with 10% fetal calf serum (FCS) at 37°C in a humidified atmosphere of air and 10% CO2. For experiments, cells were seeded in culture dishes measuring 60 mm in diameter (Corning, Corning, NY) at a density of 3 × 105 cells/dish for irradiation; in dishes measuring 100 mm in diameter for PKC activity assay (2 × 106 cells/dish); or in 96-well culture plates (Falcon, Oxnard, CA) for microculture tetrazolium (MTT) assay (103 cells/well). Tamoxifen exposure and irradiation In the experiments, tamoxifen was added to the the cell culture either 1 hour before and during irradiation, or continuously 1 hour before, and during and after irradiation. The C6 cell culture was grown exponentially in monolayers and irradiated with 7 MeV (Microtron MM22, Scanitronix AB, Husbyborg, Uppsala, Sweden) at a dose rate of 5 Gy/min. Singledose exposures ranged from 0 to 10 Gy. After irradiation, the cells were washed twice with Ca2+and Mg2+-free phosphate-buffered saline, trypsinized, counted, and placed in a culture dish measuring 60 mm in diameter for clonogenic assay or in a 96-well microtiter plate for MTT assay. Microculture tetrazolium assay The effect of tamoxifen on cell proliferation was
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AUTHOR(S): Zhang, Wei, M.D.; Yamada, Hiromu, M.D.; Sakai, Noboru, M.D.; Niikawa, Shuji, M.D.; Nozawa, Yoshinori, M.D., Ph.D.
the effect of this agent and its metabolites on such tumors is largely mediated by competitive inhibition of estrogen binding to its receptors. However, several lines of evidence suggest that tamoxifen has a variety of other antiproliferative effects (4,14). This drug also inhibits the growth of some ERnegative cell lines, including U138 and two other malignant glioma lines in vitro (4,14,17), suggesting that tamoxifen can inhibit cell proliferation by pathways independent of the estrogen receptor. In one report, in fact, 13% of patients with ER-negative tumors responded to tamoxifen (9). It has recently been reported that tamoxifen inhibits protein kinase C (PKC) by interfering with the activity of the catalytic subunit of the enzyme (12,14,15). Tamoxifen can also penetrate the blood-brain barrier (20), which suggests a possible role for it in the treatment of malignant glioma (17). On the other hand, because the radiosensitivity of cells is likely related to the growth stages and gene expression within cells (19), and because tamoxifen induces several biological changes to affect cell growth, we hypothesize that tamoxifen influences cellular sensitivity to radiation. Therefore, to explore the possibility that tamoxifen is a potent drug in the combination therapy of glioma, we performed experiments to investigate its effects on the response to radiation of C6 glioma cells grown as an exponential monolayer culture. We also investi-gated the effect of tamoxifen on PKC activity and on the cytotoxicity of ACNU in this cell line.
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Neurosurgery 1992-98 October 1992, Volume 31, Number 4 725 Enhancement of Radiosensitivity by Tamoxifen in C6 Glioma Cells Experimental Study
The 37% survival dose (Do) was calculated from the slope of the exponential cell-killing line. Statistical differences between control and test values were analyzed by Student's t test, and all P values are twosided.
Clonogenic assay Clonogenic assays were performed by seeding 200 to 500 cells per 60-mm culture dish and incubating them in 10% FCS-DMEM at 37°C (10% CO2/90% air). After 12 days, the cells were fixed with 10% formalin and counterstained with 0.5% crystal violet. Only colonies containing 50 or more cells were scored. Plating efficiency was determined for unirradiated controls treated in the same way and maintained in the same conditions, and the surviving fraction was then calculated.
RESULTS Antiproliferative and cytotoxic effects of tamoxifen After the cell culture had been exposed for 4 days to tamoxifen with or without ACNU (30 µg/ml) or had been irradiated (8 Gy), the number of viable cells was determined using MTT. As shown in Figure 1, treatment of C6 cells with increasing concentrations of tamoxifen caused a dose-dependent inhibition of growth, with complete inhibition occurring at a concentration greater than 12.5 µmol/L. Moreover, when the cells were incubated with tamoxifen and ACNU for 4 days, increased cytotoxicity was observed. In the presence of ACNU alone, the proliferation of C6 cells was 44.8% that of the control culture; when tamoxifen was also present in the culture, the cell growth was 40.6% or 15.9% that of the control (6.25 or 12.5 µmol/L of tamoxifen, respectively). Tamoxifen also showed a synergistic effect on cell impairment with irradiation in C6 cells: When 8 Gy only was applied, cell growth was 65.1% that of the control cultures; when 6.25 µmol/L of tamoxifen alone was applied, cell growth was 61.9% that of the controls; when 6.25 µmol/L of tamoxifen was administered in conjunction with 8 Gy irradiation, cell growth decreased to 51.7% that of the control cultures (P < 0.01 and P < 0.05, respectively).
Data analysis The experiments were performed in triplicate or duplicate and repeated at least twice. Radiation survival curves were plotted as logarithms of the surviving fraction against radiation dose, and analyzed using the single-hit multitarget model (8).
Effect of tamoxifen on cellular sensitization to radiation Figure 2 shows the enhanced effect of tamoxifen on the radiosensitivity of exponentially growing C6 cells. The dose-response curve for irradiation indicates the existence of a shoulder that implies a quasithreshold dose (Dq) at all doses, and exponential cell-killing at higher doses than Do, the 37% survival dose. Based on dose-response curves corresponding to the multitarget single-hit model, C6 cells treated with 5 µmol/L of tamoxifen either before and during irradiation, or continuously before, during, and after irradiation showed higher radiosensitivity (2.68 ± 0.19 Gy and 2.64 ± 0.04 Gy at Do values,
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Protein kinase C isolation and assay C6 cells treated with or without tamoxifen for 1 hour were washed twice with cold phospate-buffered saline and scraped in phosphate-buffered saline. The homogenate was prepared by sonication of the cell pellets in "Buffer A," an extraction buffer containing 20 mmol/L of Tri-HCl (pH 7.5), 5 mmol/L of ethylenediamine-tetraacetic acid, 10 mmol/L of ethyleneglycol-tetraacetic acid, 10 mmol/L of benzamidine, 0.5 mmol/L of phenylmethylsulfonyl fluoride, and 2 µg/ml of leupeptin, at 4°C. After being centrifuged at 100,000 g for 1 hour, the cell pellet was resuspended in Buffer A, and the membrane-bound PKC was extracted from the pellet by stirring it for 30 minutes in 0.5% nonionic detergent (Nonidet P-40, Sigma, St. Louis, MO) and centrifuging it at 100,000 g for another hour. The cytosolic and membrane fractions were then subjected to DE-52 cellulose chromatography, and the enzyme activity was determined by measuring the incorporation of 32P from [γ-32P]adenosine triphosphate into synthetic peptide for 15 minutes at 25°C using the PKC assay system.
Chemicals Tamoxifen, (Z)-2-[p-(1,2-diphenyl)phenoxy]-N,Ndimethylethylamine citrate (1:1), and MTT, 3-(4,5dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide, were purchased from the Sigma Chemical Co. (St. Louis, MO). ACNU, 1-(4-amino-2-methyl-5primidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride was purchased from the Sankyo Pharmaceutical Co. (Tokyo, Japan). The protein kinase C assay system was obtained from Amersham International plc. (Amersham, England). Dulbecco's modified Eagle's minimum essential medium and fetal calf serum were obtained from GIBCO (Grand Island, NY). All other chemicals used were of the highest grade commercially available.
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evaluated based on the growth rate assay in which the cell number was quantified by tetrazolium dye reduction, as described by Alley et al. (2). In brief, cells were seeded into 96-well microtiter plates at a density of 1000 cells/well in 100-µl volumes of DMEM supplemented with 10% FCS and incubated at 37°C. After 24 hours of incubation, 100 µl/well of DMEM, either with or without test agents, was dispensed within appropriate wells. For determination of the effect of irradiation, the cells were seeded in 200 µl of medium (5% FCS-DMEM) with or without tamoxifen. MTT was prepared at 1 mg/ml in DMEM and on Day 6, 50 µl was added to the microculture wells. After 4 hours of incubation at 37°C, all the media were removed from each well, and 150 µl of 100% dimethylsulfoxide was added to solubilize the MTT-formazan product. After thorough mixing, the absorbance of each well was measured using a microculture plate reader at 570 nm (Dynatech MR5000, Dynatech AG ZUG, Denkendorf, Germany).
DISCUSSION The results presented in this study demonstrate that tamoxifen enhances radiosensitivity in C6 cells, raising the possibility that this drug could be used in combination therapy of malignant glioma. Patients with brain tumor often undergo radiotherapy and chemotherapy after surgery. As the radiosensitivity of tumor cells can be affected significantly by the position of the cell cycle phase and by the cells' proliferative state, further understanding of the effects of irradiation and combined drugs on glioma cells will be necessary. The nonsteroidal antiestrogen tamoxifen has long been used in the treatment of human ER-positive breast carcinoma, and the importance of the interaction between tamoxifen and the estrogen receptor as a component of the mechanism of inhibition of tamoxifen-mediated growth is well documented (5,18). However, this agent also inhibits the growth of some ER-negative cell lines (4,14), including glioma cells (17), suggesting that not all of its biological effects are mediated by the estrogen receptor. However, the precise mechanism is not fully understood. It has been reported that whereas concentrations of tamoxifen in the nanomolar range are sufficient to inhibit the binding of estrogen to its receptor at the physiological level, inhibition of the growth of human mammary cell lines, such as MCF-7 and ZR-75-1, requires micromolar concentrations of the drug (5,18). Indeed, tamoxifen-treated breast cancer patients have micromolar levels of tamoxifen in their plasma (1,6,7), and an average of 25 ng of tamoxifen per milligram of protein in their tumor tissue (7). The data presented here also show an antiproliferative and cytotoxic effect in tamoxifentreated C6 cells in a dose-dependent manner at micromolar concentrations (Fig. 1). After pretreatment of C6 cells with tamoxifen, ACNUinduced cytotoxicity was markedly enhanced. Furthermore, when C6 cells were treated with
Received, November 19, 1991. Accepted, April 3, 1992. Reprint requests: Wei Zhang, M.D., Department of Neurosurgery, Gifu University School of Medicine, 40 Tsukasamachi, Gifu City, 500, Japan. REFERENCES: (1-20) 1. 2.
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Adam HK, Douglas EJ, Kemp JV: The metabolism of tamoxifen in humans. Biochem Pharmacol 28:145-147, 1979. Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwinski MJ, Fine DL, Abbott BJ, Mayo JG, Shoemaker RH, Boyd MR: Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res 48:589-601, 1988. Bianco AR, De Placido S, Gallo C, Pagliarulo C, Marinelli A, Petrella G, D'Istria M, Delrio G: Adjuvant therapy with tamoxifen in operable breast cancer: 10 year results of the Naples (GUN) study. Lancet 2:1095-1099,
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Effects of tamoxifen on protein kinase C activity To evaluate the possible role of PKC in the tamoxifen-induced radiosensitization in glioma cells, the effect of tamoxifen on PKC activity was investigated at concentrations that enhanced radiosensitivity. As shown in Figure 4, pretreatment with 5 µmol/L of tamoxifen for 1 hour caused a substantial decrease in PKC activity to 65% of the control value (P < 0.01), with an increased sensitivity to radiation (surviving fraction, 32.28 ± 3.04 for irradiation with 8 Gy alone and 20.89 ± 1.07 for tamoxifen in conjunction with 8 Gy irradiation, P < 0.02), whereas 100 nmol/L of tamoxifen had no significant effects on radiosensitization and inhibited PKC activity only slightly.
tamoxifen either before and during, or continuously before, during, and after irradiation, a dose-dependent shift was observed in the survival curve for irradiation (Fig. 2), suggesting that tamoxifen at clinically relevant concentrations is also a useful radiosensitizer in the treatment of brain tumor. The cellular events that increase the radiosensitivity of tamoxifen-treated C6 cells are probably multifold. Recently, it has become increasingly apparent that one of the antitumor mechanisms of tamoxifen is via inhibition of PKC (14,15,17) , a key enzyme in cellular regulation, including proliferation, differentiation, and gene expression (13) . Tamoxifen inhibits PKC by interacting with its regulatory domain, but not with the active site of the enzyme (14). To gain more insight into the mechanisms by which tamoxifen enhances radiosensitivity in glioma cells, we examined whether tamoxifen inhibits PKC activity at the same concentration needed to enhance radiosensitivity. As shown in Figure 4, 5 µmol/L of tamoxifen caused a significant inhibition of PKC in C6 cells, while 100 nmol/L of the drug only induced a slight inhibition in PKC activity, which correlates with its relative potency in radiosensitization in these cells: 5 µmol/L but not 100 nmol/L of tamoxifen remarkably enhanced the cellular response to irradiation. In addition, we recently also obtained the interesting result that inhibitors of PKC, such as staurosporine, could enhance the radiosensitivity of C6 cells (unpublished data). It may thus be concluded that the effects of tamoxifen on C6 cell proliferation and radiosensitization may depend in part on inhibition of PKC. In conclusion, the results obtained in this study, together with the fact that tamoxifen can penetrate the blood-brain barrier, suggest that tamoxifen may play a certain useful role in the treatment of malignant glioma, although the precise molecular mechanism remains to be explored.
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respectively) compared with the radiation killing curve in the controls (Do, 3.79 ± 0.25 Gy) (P < 0.01). However, 5 µmol/L of tamoxifen alone had no significant effect on the cell growth (Fig. 3). Furthermore, as shown in Figures 1 and 3, the enhanced radiosensitivity of C6 cells was also dependent on the dose of tamoxifen.
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COMMENTS Zhang and colleagues present data obtained in vitro demonstrating decreased survival of C6 cells that have been exposed to tamoxifen and irradiation in combination as compared with either tamoxifen or irradiation alone. Suggestion of a synergistic effect is seen most clearly at the lowest doses of tamoxifen (5 µmol/L), at which the drug alone had no significant effect on survival in the hands of these investigators. The ability to enhance the efficacy of radiation, the most effective mode of therapy for malignant primary brain tumors, would be a significant advance. The authors are correct in dwelling on the point that we do not understand how tamoxifen alters radiosensitivity. The analysis of protein kinase C, an enzyme known to have second messenger activity in multiple cellular metabolic pathways, is a reasonable and currently popular approach to understanding the mechanism of this compound. The effects reported here are largely additive; the combined treatment is not much better than either alone, and therefore, the treatment is not classically synergistic. In this sense, these data are disappointing, as these circumstances are ideal, in that the experiments were carried out in vitro. Treatment with tamoxifen during irradiation is appealing, because the compound is relatively familiar and its side effects well known. This does not justify proceeding without caution. If this is a reasonable intervention, a similar effect should be observed under proper circumstances in a simple in vivo implanted glioma model. Provided the authors can provide such appropriate follow-up, a clinical trial would be an appropriate next step. A more important implication of this work is that the modulation of protein kinase C by tamoxifen and other mechanisms in vitro may potentially allow us to understand better the effect of irradiation on this enzyme and in general. Such an insight could then allow a more rationally based combination of irradiation with an agent whose assets exceed familiarity and easy availability. Jeffrey Olson Atlanta, Georgia
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Lien EA, Solheim E, Ueland PM: Distribution of tamoxifen and its metabolites in rat and human tissues during steady-state treatment. Cancer Res 51:4837-4844, 1991. Pollack IF, Randall MS, Kristofik MP, Kelly RH, Selker RG, Vertosick PT: Effect of
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Frank T. Vertosick Pittsburgh, Pennsylvania
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Zhang et al. have studied the effect of tamoxifen alone and in combination with either a chemotherapeutic agent (ACNU) or radiation upon the C6 rat glioma line. Tamoxifen was found to have a direct cytotoxic effect, as measured by a tetrazolium viability assay. Moreover, tamoxifen was a mild radiosensitizer when used in micromolar concentrations. The authors provide some evidence, although not conclusive, that these effects are mediated by inhibition of protein kinase C. Based on their observations, the authors suggest that tamoxifen therapy may be clinically effective in treating human gliomas. The authors contribute to a rapidly developing area of neuro-oncology; however, their work must be interpreted cautiously. Drawing any broad conclusion from the behavior of a single immortalized cell line of nonhuman origin is impossible. Moreover, the concentrations of tamoxifen used are fairly high. Tamoxifen levels in brain and brain tumors during oral therapy are on the order of 1 to 2 µmol/L (1), while Zhang et al. use concentrations of up to 50 µmol/L in their study. The authors report radiosensitization resulting from administration of 5 µmol/L of tamoxifen, well above what can typically be achieved in patients. By contrast, our initial study showed inhibition of tritiated thymidine uptake (but not cytotoxicity) in human gliomas exposed to nanomolar concentrations of tamoxifen (2). Very high concentrations can lead to cell detachment and nonspecific cytotoxicity. Recent work in our laboratory suggests that the major metabolite of tamoxifen, Ndesmethyltamoxifen (DMT), is significantly more effective against human glioma lines in vitro than is tamoxifen. DMT is a PKC inhibitor tenfold more potent than tamoxifen and reaches brain, tumor, and serum levels that are twice as high. DMT is cytotoxic for glioma lines at nanomolar concentrations (data submitted for publication). Thus, in vitro studies using tamoxifen alone may not reflect the clinical effect of the drug's metabolites. I agree that trials of orally administered tamoxifen in glioma patients are warranted. We have seen some benefit in patients with very advanced disease (3), but have only recently started using it in patients at earlier stages of disease. Tamoxifen is well tolerated, even in very ill patients. The work of Zhang et al. suggests that starting the drug at diagnosis and continuing it through radiotherapy (and indefinitely thereafter if some response is seen) may be the optimal treatment strategy.
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Figure 1. Dose-dependence of the antiproliferative and cytotoxic effects of tamoxifen in C6 cells. Following a 4-day incubation with tamoxifen alone (•, 3.125-50 µmol/L), or with tamoxifen plus ACNU (♦, 30 µg/ml), or tamoxifen plus a single dose of irradiation ([solid up-triangle], 8 Gy), the number of viable cells was determined using MTT, as described in Materials and Methods. The data shown (mean ± standard error, n = 4) are typical of three independent experiments that yielded similar results. The differences between the tamoxifen-treated cells and the cells exposed to tamoxifen and 8 Gy of radiation or 30 µg/ml of ACNU were not statistically significant at the tamoxifen doses greater than 12.5 µmol/L, but were so at 6.25 µmol/L (P < 0.05).
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Figure 2. Radiation dose-response curves of C6 cells: •, control cells; [solid up-triangle], cells pretreated with 5 µmol/L of tamoxifen for 1 hour before and during irradiation; [hollow up-triangle], cells treated continuously with 5 µmol/L of tamoxifen, i.e., 1 hour before and during irradiation as well as during the clonogenic assay period. The results are means of three independent assays, each performed in duplicate or triplicate.
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Figure 3. Dose-dependence of tamoxifen in radiosensitization of C6 cells. •, control cells treated with tamoxifen alone; [solid up-triangle], cells treated with tamoxifen for 1 hour before and during irradiation at 8 Gy; [hollow up-triangle], cells treated continuously with tamoxifen 1 hour before and during and after irradiation at 8 Gy. The data shown are the means of two separate assays, each performed in triplicate.
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Figure 4. Effects of tamoxifen on radiosensitization and protein kinase C activity in C6 cells: [plus in box], the surviving fraction for cells pretreated with tamoxifen 1 hour before and during irradiation at 8 Gy; [shaded box], percentage of PKC activity for cells treated with tamoxifen for 1 hour and then assayed as described in the Materials and Methods. The results represent the mean ± the standard error from two independent experiments, each performed in duplicate. (* and **, significantly different from controls not treated with tamoxifen; * P < 0.02; ** P < 0.01).
MATERIALS AND METHODS Cell culture C6 cells (JCRB, Tokyo, Japan), were cultured in Dulbecco's modified Eagle's minimum essential medium (DMEM) supplemented with 10% fetal calf serum (FCS) at 37°C in a humidified atmosphere of air and 10% CO2. For experiments, cells were seeded in culture dishes measuring 60 mm in diameter (Corning, Corning, NY) at a density of 3 × 105 cells/dish for irradiation; in dishes measuring 100 mm in diameter for PKC activity assay (2 × 106 cells/dish); or in 96-well culture plates (Falcon, Oxnard, CA) for microculture tetrazolium (MTT) assay (103 cells/well). Tamoxifen exposure and irradiation In the experiments, tamoxifen was added to the the cell culture either 1 hour before and during irradiation, or continuously 1 hour before, and during and after irradiation. The C6 cell culture was grown exponentially in monolayers and irradiated with 7 MeV (Microtron MM22, Scanitronix AB, Husbyborg, Uppsala, Sweden) at a dose rate of 5 Gy/min. Singledose exposures ranged from 0 to 10 Gy. After irradiation, the cells were washed twice with Ca2+and Mg2+-free phosphate-buffered saline, trypsinized, counted, and placed in a culture dish measuring 60 mm in diameter for clonogenic assay or in a 96-well microtiter plate for MTT assay. Microculture tetrazolium assay The effect of tamoxifen on cell proliferation was
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AUTHOR(S): Zhang, Wei, M.D.; Yamada, Hiromu, M.D.; Sakai, Noboru, M.D.; Niikawa, Shuji, M.D.; Nozawa, Yoshinori, M.D., Ph.D.
the effect of this agent and its metabolites on such tumors is largely mediated by competitive inhibition of estrogen binding to its receptors. However, several lines of evidence suggest that tamoxifen has a variety of other antiproliferative effects (4,14). This drug also inhibits the growth of some ERnegative cell lines, including U138 and two other malignant glioma lines in vitro (4,14,17), suggesting that tamoxifen can inhibit cell proliferation by pathways independent of the estrogen receptor. In one report, in fact, 13% of patients with ER-negative tumors responded to tamoxifen (9). It has recently been reported that tamoxifen inhibits protein kinase C (PKC) by interfering with the activity of the catalytic subunit of the enzyme (12,14,15). Tamoxifen can also penetrate the blood-brain barrier (20), which suggests a possible role for it in the treatment of malignant glioma (17). On the other hand, because the radiosensitivity of cells is likely related to the growth stages and gene expression within cells (19), and because tamoxifen induces several biological changes to affect cell growth, we hypothesize that tamoxifen influences cellular sensitivity to radiation. Therefore, to explore the possibility that tamoxifen is a potent drug in the combination therapy of glioma, we performed experiments to investigate its effects on the response to radiation of C6 glioma cells grown as an exponential monolayer culture. We also investi-gated the effect of tamoxifen on PKC activity and on the cytotoxicity of ACNU in this cell line.
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Neurosurgery 1992-98 October 1992, Volume 31, Number 4 725 Enhancement of Radiosensitivity by Tamoxifen in C6 Glioma Cells Experimental Study
The 37% survival dose (Do) was calculated from the slope of the exponential cell-killing line. Statistical differences between control and test values were analyzed by Student's t test, and all P values are twosided.
Clonogenic assay Clonogenic assays were performed by seeding 200 to 500 cells per 60-mm culture dish and incubating them in 10% FCS-DMEM at 37°C (10% CO2/90% air). After 12 days, the cells were fixed with 10% formalin and counterstained with 0.5% crystal violet. Only colonies containing 50 or more cells were scored. Plating efficiency was determined for unirradiated controls treated in the same way and maintained in the same conditions, and the surviving fraction was then calculated.
RESULTS Antiproliferative and cytotoxic effects of tamoxifen After the cell culture had been exposed for 4 days to tamoxifen with or without ACNU (30 µg/ml) or had been irradiated (8 Gy), the number of viable cells was determined using MTT. As shown in Figure 1, treatment of C6 cells with increasing concentrations of tamoxifen caused a dose-dependent inhibition of growth, with complete inhibition occurring at a concentration greater than 12.5 µmol/L. Moreover, when the cells were incubated with tamoxifen and ACNU for 4 days, increased cytotoxicity was observed. In the presence of ACNU alone, the proliferation of C6 cells was 44.8% that of the control culture; when tamoxifen was also present in the culture, the cell growth was 40.6% or 15.9% that of the control (6.25 or 12.5 µmol/L of tamoxifen, respectively). Tamoxifen also showed a synergistic effect on cell impairment with irradiation in C6 cells: When 8 Gy only was applied, cell growth was 65.1% that of the control cultures; when 6.25 µmol/L of tamoxifen alone was applied, cell growth was 61.9% that of the controls; when 6.25 µmol/L of tamoxifen was administered in conjunction with 8 Gy irradiation, cell growth decreased to 51.7% that of the control cultures (P < 0.01 and P < 0.05, respectively).
Data analysis The experiments were performed in triplicate or duplicate and repeated at least twice. Radiation survival curves were plotted as logarithms of the surviving fraction against radiation dose, and analyzed using the single-hit multitarget model (8).
Effect of tamoxifen on cellular sensitization to radiation Figure 2 shows the enhanced effect of tamoxifen on the radiosensitivity of exponentially growing C6 cells. The dose-response curve for irradiation indicates the existence of a shoulder that implies a quasithreshold dose (Dq) at all doses, and exponential cell-killing at higher doses than Do, the 37% survival dose. Based on dose-response curves corresponding to the multitarget single-hit model, C6 cells treated with 5 µmol/L of tamoxifen either before and during irradiation, or continuously before, during, and after irradiation showed higher radiosensitivity (2.68 ± 0.19 Gy and 2.64 ± 0.04 Gy at Do values,
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Protein kinase C isolation and assay C6 cells treated with or without tamoxifen for 1 hour were washed twice with cold phospate-buffered saline and scraped in phosphate-buffered saline. The homogenate was prepared by sonication of the cell pellets in "Buffer A," an extraction buffer containing 20 mmol/L of Tri-HCl (pH 7.5), 5 mmol/L of ethylenediamine-tetraacetic acid, 10 mmol/L of ethyleneglycol-tetraacetic acid, 10 mmol/L of benzamidine, 0.5 mmol/L of phenylmethylsulfonyl fluoride, and 2 µg/ml of leupeptin, at 4°C. After being centrifuged at 100,000 g for 1 hour, the cell pellet was resuspended in Buffer A, and the membrane-bound PKC was extracted from the pellet by stirring it for 30 minutes in 0.5% nonionic detergent (Nonidet P-40, Sigma, St. Louis, MO) and centrifuging it at 100,000 g for another hour. The cytosolic and membrane fractions were then subjected to DE-52 cellulose chromatography, and the enzyme activity was determined by measuring the incorporation of 32P from [γ-32P]adenosine triphosphate into synthetic peptide for 15 minutes at 25°C using the PKC assay system.
Chemicals Tamoxifen, (Z)-2-[p-(1,2-diphenyl)phenoxy]-N,Ndimethylethylamine citrate (1:1), and MTT, 3-(4,5dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide, were purchased from the Sigma Chemical Co. (St. Louis, MO). ACNU, 1-(4-amino-2-methyl-5primidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride was purchased from the Sankyo Pharmaceutical Co. (Tokyo, Japan). The protein kinase C assay system was obtained from Amersham International plc. (Amersham, England). Dulbecco's modified Eagle's minimum essential medium and fetal calf serum were obtained from GIBCO (Grand Island, NY). All other chemicals used were of the highest grade commercially available.
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evaluated based on the growth rate assay in which the cell number was quantified by tetrazolium dye reduction, as described by Alley et al. (2). In brief, cells were seeded into 96-well microtiter plates at a density of 1000 cells/well in 100-µl volumes of DMEM supplemented with 10% FCS and incubated at 37°C. After 24 hours of incubation, 100 µl/well of DMEM, either with or without test agents, was dispensed within appropriate wells. For determination of the effect of irradiation, the cells were seeded in 200 µl of medium (5% FCS-DMEM) with or without tamoxifen. MTT was prepared at 1 mg/ml in DMEM and on Day 6, 50 µl was added to the microculture wells. After 4 hours of incubation at 37°C, all the media were removed from each well, and 150 µl of 100% dimethylsulfoxide was added to solubilize the MTT-formazan product. After thorough mixing, the absorbance of each well was measured using a microculture plate reader at 570 nm (Dynatech MR5000, Dynatech AG ZUG, Denkendorf, Germany).
DISCUSSION The results presented in this study demonstrate that tamoxifen enhances radiosensitivity in C6 cells, raising the possibility that this drug could be used in combination therapy of malignant glioma. Patients with brain tumor often undergo radiotherapy and chemotherapy after surgery. As the radiosensitivity of tumor cells can be affected significantly by the position of the cell cycle phase and by the cells' proliferative state, further understanding of the effects of irradiation and combined drugs on glioma cells will be necessary. The nonsteroidal antiestrogen tamoxifen has long been used in the treatment of human ER-positive breast carcinoma, and the importance of the interaction between tamoxifen and the estrogen receptor as a component of the mechanism of inhibition of tamoxifen-mediated growth is well documented (5,18). However, this agent also inhibits the growth of some ER-negative cell lines (4,14), including glioma cells (17), suggesting that not all of its biological effects are mediated by the estrogen receptor. However, the precise mechanism is not fully understood. It has been reported that whereas concentrations of tamoxifen in the nanomolar range are sufficient to inhibit the binding of estrogen to its receptor at the physiological level, inhibition of the growth of human mammary cell lines, such as MCF-7 and ZR-75-1, requires micromolar concentrations of the drug (5,18). Indeed, tamoxifen-treated breast cancer patients have micromolar levels of tamoxifen in their plasma (1,6,7), and an average of 25 ng of tamoxifen per milligram of protein in their tumor tissue (7). The data presented here also show an antiproliferative and cytotoxic effect in tamoxifentreated C6 cells in a dose-dependent manner at micromolar concentrations (Fig. 1). After pretreatment of C6 cells with tamoxifen, ACNUinduced cytotoxicity was markedly enhanced. Furthermore, when C6 cells were treated with
Received, November 19, 1991. Accepted, April 3, 1992. Reprint requests: Wei Zhang, M.D., Department of Neurosurgery, Gifu University School of Medicine, 40 Tsukasamachi, Gifu City, 500, Japan. REFERENCES: (1-20) 1. 2.
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Effects of tamoxifen on protein kinase C activity To evaluate the possible role of PKC in the tamoxifen-induced radiosensitization in glioma cells, the effect of tamoxifen on PKC activity was investigated at concentrations that enhanced radiosensitivity. As shown in Figure 4, pretreatment with 5 µmol/L of tamoxifen for 1 hour caused a substantial decrease in PKC activity to 65% of the control value (P < 0.01), with an increased sensitivity to radiation (surviving fraction, 32.28 ± 3.04 for irradiation with 8 Gy alone and 20.89 ± 1.07 for tamoxifen in conjunction with 8 Gy irradiation, P < 0.02), whereas 100 nmol/L of tamoxifen had no significant effects on radiosensitization and inhibited PKC activity only slightly.
tamoxifen either before and during, or continuously before, during, and after irradiation, a dose-dependent shift was observed in the survival curve for irradiation (Fig. 2), suggesting that tamoxifen at clinically relevant concentrations is also a useful radiosensitizer in the treatment of brain tumor. The cellular events that increase the radiosensitivity of tamoxifen-treated C6 cells are probably multifold. Recently, it has become increasingly apparent that one of the antitumor mechanisms of tamoxifen is via inhibition of PKC (14,15,17) , a key enzyme in cellular regulation, including proliferation, differentiation, and gene expression (13) . Tamoxifen inhibits PKC by interacting with its regulatory domain, but not with the active site of the enzyme (14). To gain more insight into the mechanisms by which tamoxifen enhances radiosensitivity in glioma cells, we examined whether tamoxifen inhibits PKC activity at the same concentration needed to enhance radiosensitivity. As shown in Figure 4, 5 µmol/L of tamoxifen caused a significant inhibition of PKC in C6 cells, while 100 nmol/L of the drug only induced a slight inhibition in PKC activity, which correlates with its relative potency in radiosensitization in these cells: 5 µmol/L but not 100 nmol/L of tamoxifen remarkably enhanced the cellular response to irradiation. In addition, we recently also obtained the interesting result that inhibitors of PKC, such as staurosporine, could enhance the radiosensitivity of C6 cells (unpublished data). It may thus be concluded that the effects of tamoxifen on C6 cell proliferation and radiosensitization may depend in part on inhibition of PKC. In conclusion, the results obtained in this study, together with the fact that tamoxifen can penetrate the blood-brain barrier, suggest that tamoxifen may play a certain useful role in the treatment of malignant glioma, although the precise molecular mechanism remains to be explored.
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respectively) compared with the radiation killing curve in the controls (Do, 3.79 ± 0.25 Gy) (P < 0.01). However, 5 µmol/L of tamoxifen alone had no significant effect on the cell growth (Fig. 3). Furthermore, as shown in Figures 1 and 3, the enhanced radiosensitivity of C6 cells was also dependent on the dose of tamoxifen.
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COMMENTS Zhang and colleagues present data obtained in vitro demonstrating decreased survival of C6 cells that have been exposed to tamoxifen and irradiation in combination as compared with either tamoxifen or irradiation alone. Suggestion of a synergistic effect is seen most clearly at the lowest doses of tamoxifen (5 µmol/L), at which the drug alone had no significant effect on survival in the hands of these investigators. The ability to enhance the efficacy of radiation, the most effective mode of therapy for malignant primary brain tumors, would be a significant advance. The authors are correct in dwelling on the point that we do not understand how tamoxifen alters radiosensitivity. The analysis of protein kinase C, an enzyme known to have second messenger activity in multiple cellular metabolic pathways, is a reasonable and currently popular approach to understanding the mechanism of this compound. The effects reported here are largely additive; the combined treatment is not much better than either alone, and therefore, the treatment is not classically synergistic. In this sense, these data are disappointing, as these circumstances are ideal, in that the experiments were carried out in vitro. Treatment with tamoxifen during irradiation is appealing, because the compound is relatively familiar and its side effects well known. This does not justify proceeding without caution. If this is a reasonable intervention, a similar effect should be observed under proper circumstances in a simple in vivo implanted glioma model. Provided the authors can provide such appropriate follow-up, a clinical trial would be an appropriate next step. A more important implication of this work is that the modulation of protein kinase C by tamoxifen and other mechanisms in vitro may potentially allow us to understand better the effect of irradiation on this enzyme and in general. Such an insight could then allow a more rationally based combination of irradiation with an agent whose assets exceed familiarity and easy availability. Jeffrey Olson Atlanta, Georgia
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Frank T. Vertosick Pittsburgh, Pennsylvania
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Zhang et al. have studied the effect of tamoxifen alone and in combination with either a chemotherapeutic agent (ACNU) or radiation upon the C6 rat glioma line. Tamoxifen was found to have a direct cytotoxic effect, as measured by a tetrazolium viability assay. Moreover, tamoxifen was a mild radiosensitizer when used in micromolar concentrations. The authors provide some evidence, although not conclusive, that these effects are mediated by inhibition of protein kinase C. Based on their observations, the authors suggest that tamoxifen therapy may be clinically effective in treating human gliomas. The authors contribute to a rapidly developing area of neuro-oncology; however, their work must be interpreted cautiously. Drawing any broad conclusion from the behavior of a single immortalized cell line of nonhuman origin is impossible. Moreover, the concentrations of tamoxifen used are fairly high. Tamoxifen levels in brain and brain tumors during oral therapy are on the order of 1 to 2 µmol/L (1), while Zhang et al. use concentrations of up to 50 µmol/L in their study. The authors report radiosensitization resulting from administration of 5 µmol/L of tamoxifen, well above what can typically be achieved in patients. By contrast, our initial study showed inhibition of tritiated thymidine uptake (but not cytotoxicity) in human gliomas exposed to nanomolar concentrations of tamoxifen (2). Very high concentrations can lead to cell detachment and nonspecific cytotoxicity. Recent work in our laboratory suggests that the major metabolite of tamoxifen, Ndesmethyltamoxifen (DMT), is significantly more effective against human glioma lines in vitro than is tamoxifen. DMT is a PKC inhibitor tenfold more potent than tamoxifen and reaches brain, tumor, and serum levels that are twice as high. DMT is cytotoxic for glioma lines at nanomolar concentrations (data submitted for publication). Thus, in vitro studies using tamoxifen alone may not reflect the clinical effect of the drug's metabolites. I agree that trials of orally administered tamoxifen in glioma patients are warranted. We have seen some benefit in patients with very advanced disease (3), but have only recently started using it in patients at earlier stages of disease. Tamoxifen is well tolerated, even in very ill patients. The work of Zhang et al. suggests that starting the drug at diagnosis and continuing it through radiotherapy (and indefinitely thereafter if some response is seen) may be the optimal treatment strategy.
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Figure 1. Dose-dependence of the antiproliferative and cytotoxic effects of tamoxifen in C6 cells. Following a 4-day incubation with tamoxifen alone (•, 3.125-50 µmol/L), or with tamoxifen plus ACNU (♦, 30 µg/ml), or tamoxifen plus a single dose of irradiation ([solid up-triangle], 8 Gy), the number of viable cells was determined using MTT, as described in Materials and Methods. The data shown (mean ± standard error, n = 4) are typical of three independent experiments that yielded similar results. The differences between the tamoxifen-treated cells and the cells exposed to tamoxifen and 8 Gy of radiation or 30 µg/ml of ACNU were not statistically significant at the tamoxifen doses greater than 12.5 µmol/L, but were so at 6.25 µmol/L (P < 0.05).
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Figure 2. Radiation dose-response curves of C6 cells: •, control cells; [solid up-triangle], cells pretreated with 5 µmol/L of tamoxifen for 1 hour before and during irradiation; [hollow up-triangle], cells treated continuously with 5 µmol/L of tamoxifen, i.e., 1 hour before and during irradiation as well as during the clonogenic assay period. The results are means of three independent assays, each performed in duplicate or triplicate.
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Figure 3. Dose-dependence of tamoxifen in radiosensitization of C6 cells. •, control cells treated with tamoxifen alone; [solid up-triangle], cells treated with tamoxifen for 1 hour before and during irradiation at 8 Gy; [hollow up-triangle], cells treated continuously with tamoxifen 1 hour before and during and after irradiation at 8 Gy. The data shown are the means of two separate assays, each performed in triplicate.
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Figure 4. Effects of tamoxifen on radiosensitization and protein kinase C activity in C6 cells: [plus in box], the surviving fraction for cells pretreated with tamoxifen 1 hour before and during irradiation at 8 Gy; [shaded box], percentage of PKC activity for cells treated with tamoxifen for 1 hour and then assayed as described in the Materials and Methods. The results represent the mean ± the standard error from two independent experiments, each performed in duplicate. (* and **, significantly different from controls not treated with tamoxifen; * P < 0.02; ** P < 0.01).
