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Increased Lipiodol uptake in hepatocellular carcinoma possibly due to increased membrane fluidity by dexamethasone and tamoxifen
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Nuclear Medicine and Biology 37 (2010) 777 – 784 www.elsevier.com/locate/nucmedbio
Increased Lipiodol uptake in hepatocellular carcinoma possibly due to increased membrane fluidity by dexamethasone and tamoxifen Stéphanie Beckera,b,c,d,⁎, Valérie Ardissona,b,c , Nicolas Lepareura,b,c , Odile Sergentc,e , Sahar Bayatf , Nicolas Noiretc,g , François Gaboriaub , Bruno Clémentb , Evelyne Boucherb,h , Jean-Luc Raoulb,c,h , Etienne Garina,b,c a
Department of Nuclear Medicine, Centre E. Marquis, F-35042 Rennes, France b INSERM U 991, Rennes, F-35033 France c European University of Brittany, F-35000 Rennes, France d Department of Nuclear Medicine, Centre H. Becquerel, F-76038 Rouen, France e UPRES EA SeRAIC, IFR 140, University of Rennes 1, F-35043 Rennes, France f INSERM U936 Department of Biostatistics, CHRU Pontchaillou, F-35033 Rennes, France g Ecole Nationale Supérieure de Chimie de Rennes, UMR CNRS 6226, F-35708 Rennes, France h Department of Medical Oncology, Centre E. Marquis, F-35042 Rennes, France Received 22 January 2010; received in revised form 16 March 2010; accepted 31 March 2010
Abstract Introduction: Lipiodol is used as a vector for chemoembolization or internal radiotherapy in unresectable hepatocellular carcinomas (HCCs). The aim of this study is to improve the tumoral uptake of Lipiodol by modulating membrane fluidizing agents to optimize the effectiveness of Lipiodol vectorized therapy. Methods: The effect of dexamethasone and tamoxifen on membrane fluidity was studied in vitro by electron paramagnetic resonance applied to rat hepatocarcinoma cell line N1S1. The tumoral uptake of Lipiodol was studied in vivo on rats with HCC, which had been previously treated by dexamethasone and/or tamoxifen, after intra-arterial administration of 99mTc-SSS-Lipiodol. Results: The two molecules studied here exhibit a fluidizing effect in vitro which appears dependent on time and dose, with a maximum fluidity obtained after 1 hr at concentrations of 20 μM for dexamethasone and 200 nM for tamoxifen. In vivo, while the use of dexamethasone or tamoxifen alone tends to lead to increased tumoral uptake of Lipiodol, this effect does not reach levels of significance. On the other hand, there is a significant increase in the tumoral uptake of 99mTc-SSS-Lipiodol in rats pretreated by both dexamethasone and tamoxifen, with a tumoral uptake (expressed in % of injected activity per g of tumor) of 13.57±3.65% after treatment, as against 9.45±4.44% without treatment (Pb.05). Conclusions: Dexamethasone and tamoxifen fluidify the N1S1 cells membrane, leading to an increase in the tumoral uptake of Lipiodol. These drugs could be combined with chemo-Lipiodol-embolization or radiolabeled Lipiodol, with a view to improving the effectiveness of HCCs therapy. © 2010 Elsevier Inc. All rights reserved. Keywords: Hepatocarcinoma; Fluidity; Lipiodol; Dexamethasone; Tamoxifen
1. Introduction
⁎ Corresponding author. Department of Nuclear Medicine, Centre Henri Becquerel, 76000 Rouen, France. Tel.: +33 2 32 08 22 56; fax: +33 2 32 08 25 50. E-mail address: [email protected] (S. Becker). 0969-8051/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nucmedbio.2010.03.013
Hepatocellular carcinoma (HCC) is the fifth most common cancer by order of frequency and represents the third most important cause of death by cancer worldwide. Its incidence has been rising over the last ten years [1,2]. The prognosis is poor and a curative treatment is only possible in
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less than 20 to 30% of the cases. A loco-regional palliative treatment, as chemoembolization, is recommended for patients who are not candidates for a curative treatment, provided they are unaffected by extra-hepatic progression and show a preserved hepatic function [3]. Treatment by 131I-Lipiodol is an alternative to chemoembolization, since this radiopharmaceutical shows a similar effectiveness and a better tolerance [4,5]. Researches are in progress on the development of rhenium-188-radiolabeled Lipiodol [6,7], which is less constraining to handle than iodine-131, and this method could represent a promising alternative to the use of 131 I-Lipiodol. Although radioembolization can also be carried out by means of yttrium-90labeled microspheres [8], this technique is more complex as well as expensive. For more advanced forms of HCC, the use of sorafenib leads to a significant increase in the total survival (10.7 months under sorafenib versus 7.9 months without treatment) [9], but with a prognosis that remains very poor. Future researches will focus on the use of a combination of a loco-regional treatment (chemoembolization or radioembolization) with an antiangiogenic treatment such as sorafenib. Optimizing the tumor targeting of Lipiodol is also a promising approach to improve the therapeutic effectiveness of intra-arterial vectorized treatments using Lipiodol. Since some studies have shown that fluidizing agents produce an increased penetration of drugs into cancerous cells and liposomes [10,11], we propose that changing the membrane fluidity should make possible to increase the cellular permeability with respect to Lipiodol. The aim of this study was to investigate the impact of membrane fluidity on the tumor uptake of Lipiodol. We chose to test the effect of dexamethasone (fluidizing) [12–16] and tamoxifen (rigidifying at low doses and fluidizing at high doses) [17,18], because of their good tolerance and their frequent use in oncology.
2.2. Culture treatments Cell densities were adjusted to 1×106/ml. Dexamethasone (D4902, Sigma-Aldrich, Saint Quentin Fallavier, France) and tamoxifen (T9262, Sigma-Aldrich, Saint Quentin Fallavier, France) were dissolved in ethanol and added to the incubation medium at a final ethanol concentration not exceeding 0.2%. Cell suspensions were prepared in 20 ml volumes with various concentrations of dexamethasone (10–20 μM) or tamoxifen (5–200 nM), and then incubated at 37°C in 5% CO2 in air for various time intervals (0.5–24 h), or with 20 μM dexamethasone and 200 nM tamoxifen for 1 h. Cell suspensions containing 0.2% ethanol and untreated culture were used as controls. After incubation, the cells were washed twice in DMEM and once in phosphate-buffered saline. 2.3. Measurement of membrane fluidity The fluidity of N1S1 cell membranes was determined by a spin-labeling method using EPR, as described previously in primary rat hepatocytes [20]. At the end of each incubation period, the lipid bilayer of N1S1 membranes was spin labeled by incubation, for 15 min at 37°C, of N1S1 with 42 μg/ml 12-DSA (Sigma-Aldrich, France), a fatty acid exhibiting a stable nitroxide radical ring at the C12-position. Cells were then washed three times with phosphate-buffered saline to remove the free spin label. The resulting pellet was then transferred to a disposable glass capillary. The EPR spectra of labeled samples were acquired at ambient temperature on a Brucker 106 EPR spectrometer operating at 3495 G centre field, 20 mW microwave power, 9.82 GHz microwave frequency, 1.771G modulation amplitude and 100 kHz modulation frequency. The fluidity of the labeled membrane is quantified by calculating the order parameter S according to equations described previously [21]. An increase in the value of S is interpreted as a decrease in membrane fluidity, whereas a decrease of this parameter reflects an increase in membrane fluidity.
2. Materials and methods 2.1. Cell culture The N1S1 cell line from Novikoff hepatocarcinoma in rat (purchased from American Type Culture Collection, Rockville, MD, USA, in 2008) was used for electron paramagnetic resonance (EPR) analysis and tumoral induction. It was established initially from a hepatocarcinoma induced in a male Sprague–Dawley rat, by ingestion of 4-dimethylaminoazobenzene [19]. Cells were seeded at densities of 1×106 cells in 75-cm2 Falcon flasks and cultured in a medium composed of 75% Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% horse serum and 5% foetal calf serum (Cambrex Biosciences, Verviers, France). After 3 days, these cultures allowed us to obtain 25–30×106 cells per flask. Routinely, more than 90% of isolated cells are found to exclude trypan blue. The cells were kept at 37°C in an atmosphere of 5% CO2 and 95% air.
2.4. Experimental animals The experiments were performed on 38 female Sprague Dawley rats weighing 180–200 g (Depré, Saint Doulchard, France) in compliance with the French regulations in force (law 0189.4 of January 24, 1990). The animals were kept in cages with corn-cob bedding, using 12-h light/12-h dark cycles and were given free access to rat food (Alphadry SDS-Dietex, France) and tap water. Tumoral inoculation and intra-arterial injection of 99mTcSSS-Lipiodol — prepared according to a previously described technique [22] — were carried out as described previously [23]. 2.4.1. Rat treatments Ten days after tumoral inoculation, the dexamethasone group (n=11) was treated by subcutaneous injection of dexamethasone (100 μg/day per 100 g body weight in
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0.25 ml physiological salt solution) over a period of 4 days. The last injection was carried out 1 hour before injection of 99m Tc-SSS-Lipiodol. In the tamoxifen group (n=9), the rats were tube-fed with a dose of 10 mg/kg tamoxifen dissolved by sonication in 0.25 ml of physiological salt solution and Tween 80 (9/1, v/v). Two tube-feedings were carried out at an interval of 24 h, the day before and 2 h before the injection of 99m Tc-SSS-Lipiodol, to obtain a plasma concentration of tamoxifen of about 60 ng/ml (approximately 200 nM) [24]. Lastly, a third group of rats (n=9) was treated by an association of dexamethasone and tamoxifen according to same method as described above. These three groups were compared with a series of untreated control rats (n=9). 2.4.2. Biodistribution studies The rats were euthanized (anesthesia by xylazine and ketamine, then intracardiac injection with 0.1 ml KCl) 24 h after injection of 2.6 MBq of 99mTc-SSS-Lipiodol. Tumor, liver and lungs were then removed, their total activity being measured on a well counter (Auto gamma Cobra II, Packard)
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and expressed as a percentage of the injected activity per gram of tissue (% IA/g). 2.5. Statistical analysis Statistical analyses were performed with SPSS 13 (SPSS, Chicago, IL, USA). Data are expressed as means±S.D. Multiple comparisons among the groups are performed using analysis of variance followed by the parametric test t or the nonparametric Wilcoxon or Mann–Whitney test, depending on the results of the normality test. Differences are considered significant at Pb.05.
3. Results 3.1. Effect of dexamethasone or tamoxifen or both on membrane fluidity 3.1.1. As a function of concentration No significant difference can be detected between the membrane fluidity of control cells and cells incubated in the presence of 0.2% ethanol.
Fig. 1. Effect of dexamethasone or tamoxifen concentration on membrane fluidity. Results expressed as order parameter S, with values corresponding to the means and standard deviations of three independent experiments. Δ is the percentage variation of the S factor compared to the untreated cells. (A) Changes in membrane fluidity of rat hepatocarcinoma cells following treatment by 10, 15 and 20 μM dexamethasone for 1 h. (B) Changes in membrane fluidity of rat hepatocarcinoma cells following treatment by 5, 50 and 200 nM tamoxifen for 1 h. Treated cells versus control cells: *Pb.05; **Pb.01.
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The effects of the concentration of drugs on the membrane fluidity of N1S1 cells treated by 10, 15 and 20 μM dexamethasone or 5, 50 and 200 nM of tamoxifen for 1 h, are expressed in terms of order parameter S (means and standard deviations) and are quantified as a percentage variation of the order parameter S compared to the controls (Fig. 1). There is a significant increase in the membrane fluidity of the hepatocytes of treated rats compared to the controls at all concentrations. This effect appears at concentrations of 10 μM dexamethasone (+3.84%, Pb.01) and 5 nM tamoxifen (+3.65%, Pb.05). The fluidizing effect of the two drugs appears concentration dependent. Indeed, higher concentrations lead to an increase in fluidity, with an increased percentage variation of +8.65% for 20 μM of dexamethasone (Pb.01) and from +8.16% for 200 nM of tamoxifen (Pb.01). In the same way, there is a significant difference between the fluidities obtained for dexamethasone at concentrations of 10–20 μM (Pb.05) and for tamoxifen at concentrations of 5 and 200 nM (Pb.05).
3.1.2. As a function of time The effects of kinetics on the membrane fluidity of cells N1S1 are presented for exposures of 20 μM dexamethasone at 0.5, 1, 2 and 24 h or 200 nM tamoxifen at 1 h and 24 h (Fig. 2). These concentrations were selected since they allowed us to obtain an increase in optimal fluidity at 1 h. The two drugs exhibit a fluidizing effect that appears to be time dependent. In rat hepatocytes treated with dexamethasone we note an early significant increase in fluidity compared to the controls, the effect beginning to appear after 30 min incubation (+4.67%, Pb.01). Incubations of 1 h, 2 h and 24 h lead to a significant increase in fluidity compared to the controls: +8.65% at 1 h (Pb.01), +7.76% at 2 h (Pb.01) and +6.92% at 24 h (Pb.01). There is also a significant difference between measured fluidities at 1 h and 2 h compared to values measured at 30 min (Pb.05). On the other hand, there is no significant difference between fluidities measured at 1, 2 and 24 h, nor any difference between measurements at 30 min and 24 h.
Fig. 2. Effect of dexamethasone or tamoxifen on membrane fluidity as a function of time. Results expressed as order parameter S, with values corresponding to the means and standard deviations of three independent experiments. Δ is the percentage variation of the S factor compared to the untreated cells. (A) Variation of membrane fluidity of rat hepatocarcinoma cells following treatment by 20 μM dexamethasone for 30 min, 1 h, 2 h and 24 h. (B) Variation of membrane fluidity of rat hepatocarcinoma cells following treatment by 200 nM tamoxifen for 1 and 24 h. Treated cells versus control cells: *Pb.05; **Pb.01.
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Fig. 3. Membrane fluidity of rat hepatocarcinoma cells following treatment by 20 μM dexamethasone and 200 nM tamoxifen for 1 hour. Results expressed as order parameter S, with values corresponding to the means and standard deviations of three independent experiments. Δ is the percentage variation of the S factor compared to the untreated cells. Treated cells versus control cells: **Pb.01.
In rat hepatocytes treated with tamoxifen a fluidizing effect persists at 24 h compared to the controls (Pb.05), but it is significantly weaker than that observed at 1 h (Pb.01). Finally, we observe an optimal fluidizing effect at 1 h of incubation for a concentration of 20 μM dexamethasone and 200 nM tamoxifen. 3.2. Combined effect of dexamethasone and tamoxifen on membrane fluidity To look for a possible synergistic effect of these two drugs on membrane fluidity, we incubated N1S1 cells in the presence of 20 μM dexamethasone and 200 nM tamoxifen for 1 h (Fig. 3). The results show a significant reduction in the S factor compared to the controls: −6.85% (Pb.01). On the other
hand, we do not observe any significant difference in the percentage variation of the S factor after treatment by both molecules compared to the result observed after treatment by dexamethasone alone or tamoxifen alone. 3.3. Effect of prior treatment by dexamethasone or tamoxifen or both on biodistribution of 99mTc-SSS-Lipiodol The biodistribution of 99mTc-SSS-Lipiodol is expressed as percentage activity injected per gram of tissue (% AI/g). The results are acquired 24 h after injection of the 99mTcSSS-Lipiodol into the tumor, nontumoral liver and lungs. After treatment by dexamethasone, the mean radioactivity present in the tumor is higher than in controls (13.78±5.65% AI/g as against 9.45±4.44%AI/g), but this difference is nonsignificant (P=.078) (Fig. 4).
Fig. 4. Biodistribution of 99mTc-SSS-Lipiodol at 24 hours, in the liver, tumor and lungs, in groups of rats pre-treated by dexamethasone (n=11), tamoxifen (n=9) or dexamethasone and tamoxifen (n=9) compared with a control group (n=9). The results are expressed as a percentage of activity injected per gram of tissue (means and standard deviations). *Pb.05; **Pb.01.
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In addition, the dexamethasone group shows a pulmonary uptake significantly higher than that observed in the reference group (5.71% AI/g against 2.56% AI/g) (Pb.05). After treatment by tamoxifen, there is a tendency to increased tumor uptake of 99mTc-SSS-Lipiodol compared to the controls (15.59±9.25% AI/g against 9.45±4.44% AI/g), but this difference is not statistically significant (P=.1) (Fig. 4). There is no significant difference in the biodistribution of the tracer in the lungs. After combination of both molecules (dexamethasone and tamoxifen), there is a significant increase in the tumor uptake of 99mTc-SSS-Lipiodol in the treated group compared to the controls (13.57±3.65% AI/g as against 9.45±4.44% AI/g, respectively, Pb.05) (Fig. 4). We also find a more intense pulmonary uptake in the treated group compared to the controls (5.64±2.27% AI/g, as against 2.56±2.02% AI/g, Pb.01).
4. Discussion Lipiodol is an oil made up of polyunsaturated esters enriched in iodine. After its administration by hepatic intraarterial injection, this oil is distributed mainly within the tumor, liver and lungs, taking advantage of the hypervasculature of the tumor at the expense of the hepatic artery, and receiving few or no portal blood. While the exact mechanism of uptake of the Lipiodol at the level of the hepatocarcinoma is not fully elucidated, several authors have described an uptake onto the membrane of cancerous cells [25,26], as well as intracellular penetration [25–28]. Few data have been published on the possible optimization of tumor targeting of Lipiodol. One of the investigated approaches makes use of the eventual effect on the embolization mechanism, by varying the viscosity of the Lipiodol. In this way, De Baere et al. [29] showed that the preparation of some types of emulsion (i.e., water-in-oil with large droplets) significantly increases the degree of tumor uptake. However, while this approach is already routinely used in chemoembolization, it is not possible with radiolabeled Lipiodol for reasons of radiation protection (the emulsions are unstable and must be prepared at the patient's bedside). The second explored approach involves the use of vasoconstrictor agents. Studies report an increase in the tumor uptake of microspheres labeled with yttrium-90 after administration of angiotensin II, with an increase in the tumor-to-non tumoral liver ratio from 1.5 to 3.3 [30,31]. This approach is little used in practice because of the increased risk of hypertension. No studies have yet been carried out to investigate the influence of membrane fluidity of tumoral cells on the intracellular uptake of Lipiodol. HCC is one of the best characterized solid tumors as regards membrane composition and fluidity. Compared to normal hepatocytes, hepatocarcinoma cells (either with fast
or slow division) have lower membrane fluidity because of an increase in the phospholipid/cholesterol level [32,33]. In this study, we used a rat hepatocarcinoma model to evaluate the effect of dexamethasone and tamoxifen on the penetration of 99mTc-SSS-Lipiodol. According to our in vitro study, both molecules show a fluidizing effect that appears to be dependent on dose and time, with a maximum fluidity observed at 1 h for concentrations of 20 μM dexamethasone and 200 nM tamoxifen. In the case of dexamethasone, these results are in agreement with the literature. Indeed, the majority of studies report an increase in the fluidity of plasma and intracellular membranes in various cell lines [12–16]. While the mechanism of action of dexamethasone on membrane fluidity remains unclear, several authors agree that there is a modification of the lipid composition of the membranes following treatment by dexamethasone, thus leading to an increase in fluidity. Indeed, they describe an enhanced degree of unsaturation of the fatty acids, with, in particular, an increase in the level of arachidonic and linoleic acids [14–16]. On the other hand, these authors failed to note any variation of the phospholipid/cholesterol level on the studied membranes. Another hypothesis is that lipophilic glucocorticoids are intercalated in the double-layered lipid molecules and can thus disturb membrane fluidity. In our model, this direct membrane mechanism probably occurred for the very short incubation times, while inhibiting effect of phospholipase A2 by glucocorticoids could contribute to the increase in fluidity for longer incubation times. Indeed, phospholipase A2 activity is responsible for the elevation of arachidonic acid concentration in membranes. An increase in such a polyunsaturated fatty acid can fluidify plasma membrane. We also show there is a fluidizing effect of tamoxifen on the N1S1 cells, which is time and dose dependent, with a fluidity maximum observed at 1 hr a concentration of 200 nM. In the literature, the data are contradictory concerning the effects of tamoxifen on membrane fluidity. Several studies report a stabilizing effect on the membrane, attributed to a reduction in the mobility of the lipids by incorporation of tamoxifen into the hydrophobic part of the membrane [34,35]. While most of these studies are related to artificial membranes (dimyristoylphosphatidylcholine, dipalmitoylphospatidylcholine or distearoylphosphatidylcholine), some are also focused on cells of neoplasic mammalian lines. On the contrary, some authors report an increase in membrane fluidity after exposure to tamoxifen [36,37]. Several hypothesis regarding these contradictory results have been advanced. Most authors agree on the importance of the membrane nature used in the different studies. For instance, Engelke et al. [37] show, for the same tamoxifen concentration, a rigidifying effect on artificial membranes and a fluidizing effect on retinal epithelial membrane, with a significant decrease in cholesterol content. They explain these results by a cellular compensation mechanism induced by the direct stabilizing effect of
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tamoxifen on membrane lipids, while in artificial liposomes being without synthetic enzymes, the observed rigidifying effect could be attributed to this direct physical action of tamoxifen on membranes. The effect of these fluidizing agents on the tumoral penetration of 99m Tc-SSS-Lipiodol was tested in vivo on a rat hepatocarcinoma model. The mean values of tumor uptake after treatment by dexamethasone or tamoxifen appear higher than observed in controls, but the difference is not statistically significant. This can probably be explained by a large dispersion of the values which reflects the individual variability of tumor uptake. This phenomenon is well known in clinical practice, and is related, at least partly, to tumoral vascularization. In addition, there may be a considerable inter-individual variance of the action of these of two drugs. On the other hand, there is a significant increase in the tumor uptake of 99mTc-SSS-Lipiodol (+44%) in rats treated with combined dexamethasone+tamoxifen compared to the controls (Pb.05). This suggests a synergistic action of dexamethasone and tamoxifen in vivo which would lead to an increase in the tumoral incorporation of 99mTc-SSSLipiodol in the rat hepatocarcinomas. The differences between in vitro and in vivo results could be explained by the model used. Indeed, the cell is a simple model which is free from any staggering effect, such as vasculature. Yet, dexamethasone has a well known role on vascular tonus and permeability. It potentiates noradrenaline and angiotensine II peripheral action [38]. Angiotensine II is a powerful vasoconstrictor agent, acting mainly on normal vessels and not on tumoral vessels [39]. This, as a consequence, increases tumoral blood flow [30,31]. In our study, the increase in tumor uptake observed in vivo after treatment with dexamethasone and tamoxifen could be in part explained by the vascular effect of dexamethasone on healthy hepatic vessels, giving rise to an increase in tumoral blood flow. These results could have some important implications in therapy for increasing the intratumoral penetration of anticancer drugs or radioactive isotopes. Such an approach would make it possible either to increase or prolong the retention of chemotherapy in the case of chemoembolization with Lipiodol. Alternatively, it could greatly increase the tumoral dosimetry using radiolabeled Lipiodol. This last point is particularly important, since the concept of threshold dose required to obtain a tumoral response has been described in the case of 131I-Lipiodol [40]. However, we also need to take into account the possible capacity of dexamethasone and tamoxifen to increase the permeability and secondary accumulation of Lipiodol in healthy tissues, in particular the lungs. Indeed, we report a significant increase in pulmonary uptake in rats treated with dexamethasone or dexamethasone+tamoxifen compared to the controls. This result can be related to an increase in the membrane fluidity of pulmonary cells due to the action of dexamethasone, such as described in previous studies [41].
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5. Conclusion In conclusion, results obtained by electron paramagnetic resonance show that dexamethasone and tamoxifen have a fluidizing effect in vitro on rat hepatocarcinoma cells. This effect appears time and dose dependent, with a maximum fluidity obtained at 1 hr for concentrations of 20 μM dexamethasone and 200 nM tamoxifen. In vivo, there is a significant increase (+44%) in the tumor uptake of 99mTcSSS-Lipiodol in rats treated by combined dexamethasone +tamoxifen compared to the controls (Pb.05). The increase in membrane fluidity, which involves a prior treatment by easily used drugs, could thus have interesting implications for optimizing the therapeutic effect of radiolabeled Lipiodol and chemoembolization, either used alone or jointly with antiangiogenic drugs. References [1] Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics 2002. CA Cancer J Clin 2005;55:74–108. [2] Bosch FX, Ribes J, Díaz M, Cléries R. Primary liver cancer: worldwide incidence and trends. Gastroenterology 2004;127:S5–16. [3] Bruix J, Sherman M, Practice Guidelines Committee, American Association for the Study of Liver Diseases. Management of hepatocellular carcinoma. Hepatology 2005;42:1208–36. [4] Raoul JL, Guyader D, Bretagne JF, Heautot JF, Duvauferrier R, Bourguet P, et al. Prospective randomized trial of chemoembolisation versus intra-arterial injection of 131I-Lipiodol iodized oil in the treatment of hepatocellular carcinoma. Hepatology 1997;26: 1156–61. [5] Boucher E, Garin E, Guylligomarc'h A, Olivié D, Boudjema K, Raoul JL. Intra-arterial injection of iodine-131-labeled Lipiodol for treatment of hepatocellular carcinoma. Radiother Oncol 2007;82:76–82. [6] Bernal P, Raoul JL, Stare J, Sereegotov E, Sundram FX, Kumar A, et al. International Atomic Energy Agency-sponsored multination study of intra-arterial rhenium-188-labeled Lipiodol in the treatment of inoperable hepatocellular carcinoma: results with special emphasis on prognostic value of dosimetric study. Semin Nucl Med 2008;38: S40–5. [7] Garin E, Noiret N, Malbert CH, Lepareur N, Roucoux A, CauletMaugendre S, et al. Development and biodistribution of 188Re-SSS Lipiodol following injection into hepatic artery of healthy pig. Eur J Nucl Med Mol Imaging 2004;31:542–6. [8] Salem R, Lewandowski RJ, Atassi B, Gordon SC, Gates VL, Barakat O, et al. Treatment of unresectable hepatocellular carcinoma with use of 90Y microspheres (TheraSphere): safety, tumor response, and survival. J Vasc Interv Radiol 2005;16:1627–39. [9] Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008;359:378–90. [10] Preetha A, Huilgol N, Banerjee R. Effect of fluidizing agents on paclitaxel penetration in cervical cancerous monolayer membranes. J Membrane Biol 2007;219:83–91. [11] Auner BG, O'Neill MAA, Valenta C, Hadgraft J. Interaction of phloretin and 6-ketocholestanol with DPPC-liposomes as phospholipid model membranes. Int J Pharm 2005;294:149–55. [12] Johnston D, Melnykovych G. Effects of dexamethasone on the fluorescence polarisation of diphenylhexatriène in HeLa cells. Biochim Biophys Acta 1980;596:320–4. [13] Boullier JA, Melnykovych G, Barias BG. A photobleaching recovery study of glucocorticoid effects on lateral mobilities of a lipid analog in S3G HeLa cell membranes. Biochim Biophys Acta 1982;692:278–86.
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[28] Iwai K, Maeda H, Konno T. Use of oily contrast medium for selective drug targeting to tumor: enhanced therapeutic effect and X-ray image. Cancer Res 1984;44:2115–21. [29] De Baere T, Zhang X, Aubert B, Harry G, Lagrange C, Ropers J, et al. Quantification of tumor uptake of iodized oils and emulsions of iodized oils: experimental study. Radiology 1996;201:731–5. [30] Burton MA, Gray BN, Coletti A. Effect of angiotensin II on blood flow in the transplanted sheep squamous cell carcinoma. Eur J Cancer Clin Oncol 1988;24:1373–6. [31] Sasaki Y, Imaoka S, Hasegawa Y, Nakano S, Ishikawa O, Ohigashi H, et al. Changes in distribution of hepatic blood flow induced by intraarterial infusion of angiotensin II in human hepatic cancer. Cancer 1985;55:311–6. [32] Schuller A, Solis-Herruzo JA, Moscat J, Fernandez-Checa JC, Municio AM. The fluidity of liver plasma membranes from patients with different types of liver injury. Hepatology 1986;6:714–7. [33] Mahler SM, Wilce PA, Shanley BC. Studies on regenerating liver and hepatoma plasma membranes II Membrane fluidity and enzyme activity. Int J Biochem 1988;20:613–9. [34] Clarke R, Van den Berg H, Murphy R. Reduction of the membrane fluidity of human breast cancer cells by tamoxifen and 17β-estradiol. J Natl Cancer Inst 1990;82:1702–5. [35] Engelke M, Bojarski P, Bloss R, Diehl H. Tamoxifen perturbs lipid bilayer order and permeability: comparison of DSC, fluorescence anisotropy, Laurdan generalized polarization and carboxyfluorescein leakage studies. Biophys Chem 2001;90:157–73. [36] Dicko A, Morissette M, Ben Ameur SB, Pézolet M, Di Paolo T. Effect of estradiol and tamoxifen on brain membranes: Investigation by infrared and fluorescence spectroscopy. Brain Res Bull 1999;49: 401–5. [37] Engelke M, Tykhonova S, Zorn-Kruppa M, Diehl H. Tamoxifen induces changes in the lipid composition of the retinal pigment epithelium cell line D407. Pharmacol Toxicol 2002;91:13–21. [38] Grünfeld JP, Eloy L. Glucocorticoids modulate vascular reactivity in the rat. Hypertension 1987;10:608–18. [39] Burton MA, Gray BN, Self GW, Heggie JC, Towensend PS. Manipulation of experimental rat and rabbit liver tumor blood flow with angiotensin II. Cancer Res 1985;45:5390–3. [40] Becker S, Laffont S, Vitry F, Rolland Y, Lecloirec J, Boucher E, et al. Dosimetric evaluation and therapeutic response to internal radiation therapy of hepatocarcinomas using iodine-131-labelled Lipiodol. Nucl Med Commun 2008;29:815–25. [41] Rooney SA, Gobran L, Gross I, Wai-lee TS, Nardone LL, Motoyama EK. Studies on pulmonary surfactant. Effects of cortisol administration to fetal rabbits on lung phospholipid content, composition and biosynthesis. Biochim Biophys Acta 1976;450:121–30.
Nuclear Medicine and Biology 37 (2010) 777 – 784 www.elsevier.com/locate/nucmedbio
Increased Lipiodol uptake in hepatocellular carcinoma possibly due to increased membrane fluidity by dexamethasone and tamoxifen Stéphanie Beckera,b,c,d,⁎, Valérie Ardissona,b,c , Nicolas Lepareura,b,c , Odile Sergentc,e , Sahar Bayatf , Nicolas Noiretc,g , François Gaboriaub , Bruno Clémentb , Evelyne Boucherb,h , Jean-Luc Raoulb,c,h , Etienne Garina,b,c a
Department of Nuclear Medicine, Centre E. Marquis, F-35042 Rennes, France b INSERM U 991, Rennes, F-35033 France c European University of Brittany, F-35000 Rennes, France d Department of Nuclear Medicine, Centre H. Becquerel, F-76038 Rouen, France e UPRES EA SeRAIC, IFR 140, University of Rennes 1, F-35043 Rennes, France f INSERM U936 Department of Biostatistics, CHRU Pontchaillou, F-35033 Rennes, France g Ecole Nationale Supérieure de Chimie de Rennes, UMR CNRS 6226, F-35708 Rennes, France h Department of Medical Oncology, Centre E. Marquis, F-35042 Rennes, France Received 22 January 2010; received in revised form 16 March 2010; accepted 31 March 2010
Abstract Introduction: Lipiodol is used as a vector for chemoembolization or internal radiotherapy in unresectable hepatocellular carcinomas (HCCs). The aim of this study is to improve the tumoral uptake of Lipiodol by modulating membrane fluidizing agents to optimize the effectiveness of Lipiodol vectorized therapy. Methods: The effect of dexamethasone and tamoxifen on membrane fluidity was studied in vitro by electron paramagnetic resonance applied to rat hepatocarcinoma cell line N1S1. The tumoral uptake of Lipiodol was studied in vivo on rats with HCC, which had been previously treated by dexamethasone and/or tamoxifen, after intra-arterial administration of 99mTc-SSS-Lipiodol. Results: The two molecules studied here exhibit a fluidizing effect in vitro which appears dependent on time and dose, with a maximum fluidity obtained after 1 hr at concentrations of 20 μM for dexamethasone and 200 nM for tamoxifen. In vivo, while the use of dexamethasone or tamoxifen alone tends to lead to increased tumoral uptake of Lipiodol, this effect does not reach levels of significance. On the other hand, there is a significant increase in the tumoral uptake of 99mTc-SSS-Lipiodol in rats pretreated by both dexamethasone and tamoxifen, with a tumoral uptake (expressed in % of injected activity per g of tumor) of 13.57±3.65% after treatment, as against 9.45±4.44% without treatment (Pb.05). Conclusions: Dexamethasone and tamoxifen fluidify the N1S1 cells membrane, leading to an increase in the tumoral uptake of Lipiodol. These drugs could be combined with chemo-Lipiodol-embolization or radiolabeled Lipiodol, with a view to improving the effectiveness of HCCs therapy. © 2010 Elsevier Inc. All rights reserved. Keywords: Hepatocarcinoma; Fluidity; Lipiodol; Dexamethasone; Tamoxifen
1. Introduction
⁎ Corresponding author. Department of Nuclear Medicine, Centre Henri Becquerel, 76000 Rouen, France. Tel.: +33 2 32 08 22 56; fax: +33 2 32 08 25 50. E-mail address: [email protected] (S. Becker). 0969-8051/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nucmedbio.2010.03.013
Hepatocellular carcinoma (HCC) is the fifth most common cancer by order of frequency and represents the third most important cause of death by cancer worldwide. Its incidence has been rising over the last ten years [1,2]. The prognosis is poor and a curative treatment is only possible in
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less than 20 to 30% of the cases. A loco-regional palliative treatment, as chemoembolization, is recommended for patients who are not candidates for a curative treatment, provided they are unaffected by extra-hepatic progression and show a preserved hepatic function [3]. Treatment by 131I-Lipiodol is an alternative to chemoembolization, since this radiopharmaceutical shows a similar effectiveness and a better tolerance [4,5]. Researches are in progress on the development of rhenium-188-radiolabeled Lipiodol [6,7], which is less constraining to handle than iodine-131, and this method could represent a promising alternative to the use of 131 I-Lipiodol. Although radioembolization can also be carried out by means of yttrium-90labeled microspheres [8], this technique is more complex as well as expensive. For more advanced forms of HCC, the use of sorafenib leads to a significant increase in the total survival (10.7 months under sorafenib versus 7.9 months without treatment) [9], but with a prognosis that remains very poor. Future researches will focus on the use of a combination of a loco-regional treatment (chemoembolization or radioembolization) with an antiangiogenic treatment such as sorafenib. Optimizing the tumor targeting of Lipiodol is also a promising approach to improve the therapeutic effectiveness of intra-arterial vectorized treatments using Lipiodol. Since some studies have shown that fluidizing agents produce an increased penetration of drugs into cancerous cells and liposomes [10,11], we propose that changing the membrane fluidity should make possible to increase the cellular permeability with respect to Lipiodol. The aim of this study was to investigate the impact of membrane fluidity on the tumor uptake of Lipiodol. We chose to test the effect of dexamethasone (fluidizing) [12–16] and tamoxifen (rigidifying at low doses and fluidizing at high doses) [17,18], because of their good tolerance and their frequent use in oncology.
2.2. Culture treatments Cell densities were adjusted to 1×106/ml. Dexamethasone (D4902, Sigma-Aldrich, Saint Quentin Fallavier, France) and tamoxifen (T9262, Sigma-Aldrich, Saint Quentin Fallavier, France) were dissolved in ethanol and added to the incubation medium at a final ethanol concentration not exceeding 0.2%. Cell suspensions were prepared in 20 ml volumes with various concentrations of dexamethasone (10–20 μM) or tamoxifen (5–200 nM), and then incubated at 37°C in 5% CO2 in air for various time intervals (0.5–24 h), or with 20 μM dexamethasone and 200 nM tamoxifen for 1 h. Cell suspensions containing 0.2% ethanol and untreated culture were used as controls. After incubation, the cells were washed twice in DMEM and once in phosphate-buffered saline. 2.3. Measurement of membrane fluidity The fluidity of N1S1 cell membranes was determined by a spin-labeling method using EPR, as described previously in primary rat hepatocytes [20]. At the end of each incubation period, the lipid bilayer of N1S1 membranes was spin labeled by incubation, for 15 min at 37°C, of N1S1 with 42 μg/ml 12-DSA (Sigma-Aldrich, France), a fatty acid exhibiting a stable nitroxide radical ring at the C12-position. Cells were then washed three times with phosphate-buffered saline to remove the free spin label. The resulting pellet was then transferred to a disposable glass capillary. The EPR spectra of labeled samples were acquired at ambient temperature on a Brucker 106 EPR spectrometer operating at 3495 G centre field, 20 mW microwave power, 9.82 GHz microwave frequency, 1.771G modulation amplitude and 100 kHz modulation frequency. The fluidity of the labeled membrane is quantified by calculating the order parameter S according to equations described previously [21]. An increase in the value of S is interpreted as a decrease in membrane fluidity, whereas a decrease of this parameter reflects an increase in membrane fluidity.
2. Materials and methods 2.1. Cell culture The N1S1 cell line from Novikoff hepatocarcinoma in rat (purchased from American Type Culture Collection, Rockville, MD, USA, in 2008) was used for electron paramagnetic resonance (EPR) analysis and tumoral induction. It was established initially from a hepatocarcinoma induced in a male Sprague–Dawley rat, by ingestion of 4-dimethylaminoazobenzene [19]. Cells were seeded at densities of 1×106 cells in 75-cm2 Falcon flasks and cultured in a medium composed of 75% Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% horse serum and 5% foetal calf serum (Cambrex Biosciences, Verviers, France). After 3 days, these cultures allowed us to obtain 25–30×106 cells per flask. Routinely, more than 90% of isolated cells are found to exclude trypan blue. The cells were kept at 37°C in an atmosphere of 5% CO2 and 95% air.
2.4. Experimental animals The experiments were performed on 38 female Sprague Dawley rats weighing 180–200 g (Depré, Saint Doulchard, France) in compliance with the French regulations in force (law 0189.4 of January 24, 1990). The animals were kept in cages with corn-cob bedding, using 12-h light/12-h dark cycles and were given free access to rat food (Alphadry SDS-Dietex, France) and tap water. Tumoral inoculation and intra-arterial injection of 99mTcSSS-Lipiodol — prepared according to a previously described technique [22] — were carried out as described previously [23]. 2.4.1. Rat treatments Ten days after tumoral inoculation, the dexamethasone group (n=11) was treated by subcutaneous injection of dexamethasone (100 μg/day per 100 g body weight in
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0.25 ml physiological salt solution) over a period of 4 days. The last injection was carried out 1 hour before injection of 99m Tc-SSS-Lipiodol. In the tamoxifen group (n=9), the rats were tube-fed with a dose of 10 mg/kg tamoxifen dissolved by sonication in 0.25 ml of physiological salt solution and Tween 80 (9/1, v/v). Two tube-feedings were carried out at an interval of 24 h, the day before and 2 h before the injection of 99m Tc-SSS-Lipiodol, to obtain a plasma concentration of tamoxifen of about 60 ng/ml (approximately 200 nM) [24]. Lastly, a third group of rats (n=9) was treated by an association of dexamethasone and tamoxifen according to same method as described above. These three groups were compared with a series of untreated control rats (n=9). 2.4.2. Biodistribution studies The rats were euthanized (anesthesia by xylazine and ketamine, then intracardiac injection with 0.1 ml KCl) 24 h after injection of 2.6 MBq of 99mTc-SSS-Lipiodol. Tumor, liver and lungs were then removed, their total activity being measured on a well counter (Auto gamma Cobra II, Packard)
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and expressed as a percentage of the injected activity per gram of tissue (% IA/g). 2.5. Statistical analysis Statistical analyses were performed with SPSS 13 (SPSS, Chicago, IL, USA). Data are expressed as means±S.D. Multiple comparisons among the groups are performed using analysis of variance followed by the parametric test t or the nonparametric Wilcoxon or Mann–Whitney test, depending on the results of the normality test. Differences are considered significant at Pb.05.
3. Results 3.1. Effect of dexamethasone or tamoxifen or both on membrane fluidity 3.1.1. As a function of concentration No significant difference can be detected between the membrane fluidity of control cells and cells incubated in the presence of 0.2% ethanol.
Fig. 1. Effect of dexamethasone or tamoxifen concentration on membrane fluidity. Results expressed as order parameter S, with values corresponding to the means and standard deviations of three independent experiments. Δ is the percentage variation of the S factor compared to the untreated cells. (A) Changes in membrane fluidity of rat hepatocarcinoma cells following treatment by 10, 15 and 20 μM dexamethasone for 1 h. (B) Changes in membrane fluidity of rat hepatocarcinoma cells following treatment by 5, 50 and 200 nM tamoxifen for 1 h. Treated cells versus control cells: *Pb.05; **Pb.01.
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The effects of the concentration of drugs on the membrane fluidity of N1S1 cells treated by 10, 15 and 20 μM dexamethasone or 5, 50 and 200 nM of tamoxifen for 1 h, are expressed in terms of order parameter S (means and standard deviations) and are quantified as a percentage variation of the order parameter S compared to the controls (Fig. 1). There is a significant increase in the membrane fluidity of the hepatocytes of treated rats compared to the controls at all concentrations. This effect appears at concentrations of 10 μM dexamethasone (+3.84%, Pb.01) and 5 nM tamoxifen (+3.65%, Pb.05). The fluidizing effect of the two drugs appears concentration dependent. Indeed, higher concentrations lead to an increase in fluidity, with an increased percentage variation of +8.65% for 20 μM of dexamethasone (Pb.01) and from +8.16% for 200 nM of tamoxifen (Pb.01). In the same way, there is a significant difference between the fluidities obtained for dexamethasone at concentrations of 10–20 μM (Pb.05) and for tamoxifen at concentrations of 5 and 200 nM (Pb.05).
3.1.2. As a function of time The effects of kinetics on the membrane fluidity of cells N1S1 are presented for exposures of 20 μM dexamethasone at 0.5, 1, 2 and 24 h or 200 nM tamoxifen at 1 h and 24 h (Fig. 2). These concentrations were selected since they allowed us to obtain an increase in optimal fluidity at 1 h. The two drugs exhibit a fluidizing effect that appears to be time dependent. In rat hepatocytes treated with dexamethasone we note an early significant increase in fluidity compared to the controls, the effect beginning to appear after 30 min incubation (+4.67%, Pb.01). Incubations of 1 h, 2 h and 24 h lead to a significant increase in fluidity compared to the controls: +8.65% at 1 h (Pb.01), +7.76% at 2 h (Pb.01) and +6.92% at 24 h (Pb.01). There is also a significant difference between measured fluidities at 1 h and 2 h compared to values measured at 30 min (Pb.05). On the other hand, there is no significant difference between fluidities measured at 1, 2 and 24 h, nor any difference between measurements at 30 min and 24 h.
Fig. 2. Effect of dexamethasone or tamoxifen on membrane fluidity as a function of time. Results expressed as order parameter S, with values corresponding to the means and standard deviations of three independent experiments. Δ is the percentage variation of the S factor compared to the untreated cells. (A) Variation of membrane fluidity of rat hepatocarcinoma cells following treatment by 20 μM dexamethasone for 30 min, 1 h, 2 h and 24 h. (B) Variation of membrane fluidity of rat hepatocarcinoma cells following treatment by 200 nM tamoxifen for 1 and 24 h. Treated cells versus control cells: *Pb.05; **Pb.01.
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Fig. 3. Membrane fluidity of rat hepatocarcinoma cells following treatment by 20 μM dexamethasone and 200 nM tamoxifen for 1 hour. Results expressed as order parameter S, with values corresponding to the means and standard deviations of three independent experiments. Δ is the percentage variation of the S factor compared to the untreated cells. Treated cells versus control cells: **Pb.01.
In rat hepatocytes treated with tamoxifen a fluidizing effect persists at 24 h compared to the controls (Pb.05), but it is significantly weaker than that observed at 1 h (Pb.01). Finally, we observe an optimal fluidizing effect at 1 h of incubation for a concentration of 20 μM dexamethasone and 200 nM tamoxifen. 3.2. Combined effect of dexamethasone and tamoxifen on membrane fluidity To look for a possible synergistic effect of these two drugs on membrane fluidity, we incubated N1S1 cells in the presence of 20 μM dexamethasone and 200 nM tamoxifen for 1 h (Fig. 3). The results show a significant reduction in the S factor compared to the controls: −6.85% (Pb.01). On the other
hand, we do not observe any significant difference in the percentage variation of the S factor after treatment by both molecules compared to the result observed after treatment by dexamethasone alone or tamoxifen alone. 3.3. Effect of prior treatment by dexamethasone or tamoxifen or both on biodistribution of 99mTc-SSS-Lipiodol The biodistribution of 99mTc-SSS-Lipiodol is expressed as percentage activity injected per gram of tissue (% AI/g). The results are acquired 24 h after injection of the 99mTcSSS-Lipiodol into the tumor, nontumoral liver and lungs. After treatment by dexamethasone, the mean radioactivity present in the tumor is higher than in controls (13.78±5.65% AI/g as against 9.45±4.44%AI/g), but this difference is nonsignificant (P=.078) (Fig. 4).
Fig. 4. Biodistribution of 99mTc-SSS-Lipiodol at 24 hours, in the liver, tumor and lungs, in groups of rats pre-treated by dexamethasone (n=11), tamoxifen (n=9) or dexamethasone and tamoxifen (n=9) compared with a control group (n=9). The results are expressed as a percentage of activity injected per gram of tissue (means and standard deviations). *Pb.05; **Pb.01.
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In addition, the dexamethasone group shows a pulmonary uptake significantly higher than that observed in the reference group (5.71% AI/g against 2.56% AI/g) (Pb.05). After treatment by tamoxifen, there is a tendency to increased tumor uptake of 99mTc-SSS-Lipiodol compared to the controls (15.59±9.25% AI/g against 9.45±4.44% AI/g), but this difference is not statistically significant (P=.1) (Fig. 4). There is no significant difference in the biodistribution of the tracer in the lungs. After combination of both molecules (dexamethasone and tamoxifen), there is a significant increase in the tumor uptake of 99mTc-SSS-Lipiodol in the treated group compared to the controls (13.57±3.65% AI/g as against 9.45±4.44% AI/g, respectively, Pb.05) (Fig. 4). We also find a more intense pulmonary uptake in the treated group compared to the controls (5.64±2.27% AI/g, as against 2.56±2.02% AI/g, Pb.01).
4. Discussion Lipiodol is an oil made up of polyunsaturated esters enriched in iodine. After its administration by hepatic intraarterial injection, this oil is distributed mainly within the tumor, liver and lungs, taking advantage of the hypervasculature of the tumor at the expense of the hepatic artery, and receiving few or no portal blood. While the exact mechanism of uptake of the Lipiodol at the level of the hepatocarcinoma is not fully elucidated, several authors have described an uptake onto the membrane of cancerous cells [25,26], as well as intracellular penetration [25–28]. Few data have been published on the possible optimization of tumor targeting of Lipiodol. One of the investigated approaches makes use of the eventual effect on the embolization mechanism, by varying the viscosity of the Lipiodol. In this way, De Baere et al. [29] showed that the preparation of some types of emulsion (i.e., water-in-oil with large droplets) significantly increases the degree of tumor uptake. However, while this approach is already routinely used in chemoembolization, it is not possible with radiolabeled Lipiodol for reasons of radiation protection (the emulsions are unstable and must be prepared at the patient's bedside). The second explored approach involves the use of vasoconstrictor agents. Studies report an increase in the tumor uptake of microspheres labeled with yttrium-90 after administration of angiotensin II, with an increase in the tumor-to-non tumoral liver ratio from 1.5 to 3.3 [30,31]. This approach is little used in practice because of the increased risk of hypertension. No studies have yet been carried out to investigate the influence of membrane fluidity of tumoral cells on the intracellular uptake of Lipiodol. HCC is one of the best characterized solid tumors as regards membrane composition and fluidity. Compared to normal hepatocytes, hepatocarcinoma cells (either with fast
or slow division) have lower membrane fluidity because of an increase in the phospholipid/cholesterol level [32,33]. In this study, we used a rat hepatocarcinoma model to evaluate the effect of dexamethasone and tamoxifen on the penetration of 99mTc-SSS-Lipiodol. According to our in vitro study, both molecules show a fluidizing effect that appears to be dependent on dose and time, with a maximum fluidity observed at 1 h for concentrations of 20 μM dexamethasone and 200 nM tamoxifen. In the case of dexamethasone, these results are in agreement with the literature. Indeed, the majority of studies report an increase in the fluidity of plasma and intracellular membranes in various cell lines [12–16]. While the mechanism of action of dexamethasone on membrane fluidity remains unclear, several authors agree that there is a modification of the lipid composition of the membranes following treatment by dexamethasone, thus leading to an increase in fluidity. Indeed, they describe an enhanced degree of unsaturation of the fatty acids, with, in particular, an increase in the level of arachidonic and linoleic acids [14–16]. On the other hand, these authors failed to note any variation of the phospholipid/cholesterol level on the studied membranes. Another hypothesis is that lipophilic glucocorticoids are intercalated in the double-layered lipid molecules and can thus disturb membrane fluidity. In our model, this direct membrane mechanism probably occurred for the very short incubation times, while inhibiting effect of phospholipase A2 by glucocorticoids could contribute to the increase in fluidity for longer incubation times. Indeed, phospholipase A2 activity is responsible for the elevation of arachidonic acid concentration in membranes. An increase in such a polyunsaturated fatty acid can fluidify plasma membrane. We also show there is a fluidizing effect of tamoxifen on the N1S1 cells, which is time and dose dependent, with a fluidity maximum observed at 1 hr a concentration of 200 nM. In the literature, the data are contradictory concerning the effects of tamoxifen on membrane fluidity. Several studies report a stabilizing effect on the membrane, attributed to a reduction in the mobility of the lipids by incorporation of tamoxifen into the hydrophobic part of the membrane [34,35]. While most of these studies are related to artificial membranes (dimyristoylphosphatidylcholine, dipalmitoylphospatidylcholine or distearoylphosphatidylcholine), some are also focused on cells of neoplasic mammalian lines. On the contrary, some authors report an increase in membrane fluidity after exposure to tamoxifen [36,37]. Several hypothesis regarding these contradictory results have been advanced. Most authors agree on the importance of the membrane nature used in the different studies. For instance, Engelke et al. [37] show, for the same tamoxifen concentration, a rigidifying effect on artificial membranes and a fluidizing effect on retinal epithelial membrane, with a significant decrease in cholesterol content. They explain these results by a cellular compensation mechanism induced by the direct stabilizing effect of
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tamoxifen on membrane lipids, while in artificial liposomes being without synthetic enzymes, the observed rigidifying effect could be attributed to this direct physical action of tamoxifen on membranes. The effect of these fluidizing agents on the tumoral penetration of 99m Tc-SSS-Lipiodol was tested in vivo on a rat hepatocarcinoma model. The mean values of tumor uptake after treatment by dexamethasone or tamoxifen appear higher than observed in controls, but the difference is not statistically significant. This can probably be explained by a large dispersion of the values which reflects the individual variability of tumor uptake. This phenomenon is well known in clinical practice, and is related, at least partly, to tumoral vascularization. In addition, there may be a considerable inter-individual variance of the action of these of two drugs. On the other hand, there is a significant increase in the tumor uptake of 99mTc-SSS-Lipiodol (+44%) in rats treated with combined dexamethasone+tamoxifen compared to the controls (Pb.05). This suggests a synergistic action of dexamethasone and tamoxifen in vivo which would lead to an increase in the tumoral incorporation of 99mTc-SSSLipiodol in the rat hepatocarcinomas. The differences between in vitro and in vivo results could be explained by the model used. Indeed, the cell is a simple model which is free from any staggering effect, such as vasculature. Yet, dexamethasone has a well known role on vascular tonus and permeability. It potentiates noradrenaline and angiotensine II peripheral action [38]. Angiotensine II is a powerful vasoconstrictor agent, acting mainly on normal vessels and not on tumoral vessels [39]. This, as a consequence, increases tumoral blood flow [30,31]. In our study, the increase in tumor uptake observed in vivo after treatment with dexamethasone and tamoxifen could be in part explained by the vascular effect of dexamethasone on healthy hepatic vessels, giving rise to an increase in tumoral blood flow. These results could have some important implications in therapy for increasing the intratumoral penetration of anticancer drugs or radioactive isotopes. Such an approach would make it possible either to increase or prolong the retention of chemotherapy in the case of chemoembolization with Lipiodol. Alternatively, it could greatly increase the tumoral dosimetry using radiolabeled Lipiodol. This last point is particularly important, since the concept of threshold dose required to obtain a tumoral response has been described in the case of 131I-Lipiodol [40]. However, we also need to take into account the possible capacity of dexamethasone and tamoxifen to increase the permeability and secondary accumulation of Lipiodol in healthy tissues, in particular the lungs. Indeed, we report a significant increase in pulmonary uptake in rats treated with dexamethasone or dexamethasone+tamoxifen compared to the controls. This result can be related to an increase in the membrane fluidity of pulmonary cells due to the action of dexamethasone, such as described in previous studies [41].
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5. Conclusion In conclusion, results obtained by electron paramagnetic resonance show that dexamethasone and tamoxifen have a fluidizing effect in vitro on rat hepatocarcinoma cells. This effect appears time and dose dependent, with a maximum fluidity obtained at 1 hr for concentrations of 20 μM dexamethasone and 200 nM tamoxifen. In vivo, there is a significant increase (+44%) in the tumor uptake of 99mTcSSS-Lipiodol in rats treated by combined dexamethasone +tamoxifen compared to the controls (Pb.05). The increase in membrane fluidity, which involves a prior treatment by easily used drugs, could thus have interesting implications for optimizing the therapeutic effect of radiolabeled Lipiodol and chemoembolization, either used alone or jointly with antiangiogenic drugs. References [1] Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics 2002. CA Cancer J Clin 2005;55:74–108. [2] Bosch FX, Ribes J, Díaz M, Cléries R. Primary liver cancer: worldwide incidence and trends. Gastroenterology 2004;127:S5–16. [3] Bruix J, Sherman M, Practice Guidelines Committee, American Association for the Study of Liver Diseases. Management of hepatocellular carcinoma. Hepatology 2005;42:1208–36. [4] Raoul JL, Guyader D, Bretagne JF, Heautot JF, Duvauferrier R, Bourguet P, et al. Prospective randomized trial of chemoembolisation versus intra-arterial injection of 131I-Lipiodol iodized oil in the treatment of hepatocellular carcinoma. Hepatology 1997;26: 1156–61. [5] Boucher E, Garin E, Guylligomarc'h A, Olivié D, Boudjema K, Raoul JL. Intra-arterial injection of iodine-131-labeled Lipiodol for treatment of hepatocellular carcinoma. Radiother Oncol 2007;82:76–82. [6] Bernal P, Raoul JL, Stare J, Sereegotov E, Sundram FX, Kumar A, et al. International Atomic Energy Agency-sponsored multination study of intra-arterial rhenium-188-labeled Lipiodol in the treatment of inoperable hepatocellular carcinoma: results with special emphasis on prognostic value of dosimetric study. Semin Nucl Med 2008;38: S40–5. [7] Garin E, Noiret N, Malbert CH, Lepareur N, Roucoux A, CauletMaugendre S, et al. Development and biodistribution of 188Re-SSS Lipiodol following injection into hepatic artery of healthy pig. Eur J Nucl Med Mol Imaging 2004;31:542–6. [8] Salem R, Lewandowski RJ, Atassi B, Gordon SC, Gates VL, Barakat O, et al. Treatment of unresectable hepatocellular carcinoma with use of 90Y microspheres (TheraSphere): safety, tumor response, and survival. J Vasc Interv Radiol 2005;16:1627–39. [9] Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008;359:378–90. [10] Preetha A, Huilgol N, Banerjee R. Effect of fluidizing agents on paclitaxel penetration in cervical cancerous monolayer membranes. J Membrane Biol 2007;219:83–91. [11] Auner BG, O'Neill MAA, Valenta C, Hadgraft J. Interaction of phloretin and 6-ketocholestanol with DPPC-liposomes as phospholipid model membranes. Int J Pharm 2005;294:149–55. [12] Johnston D, Melnykovych G. Effects of dexamethasone on the fluorescence polarisation of diphenylhexatriène in HeLa cells. Biochim Biophys Acta 1980;596:320–4. [13] Boullier JA, Melnykovych G, Barias BG. A photobleaching recovery study of glucocorticoid effects on lateral mobilities of a lipid analog in S3G HeLa cell membranes. Biochim Biophys Acta 1982;692:278–86.
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