Pharmacological Reports Copyright © 2013 2013, 65, 445452 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences
In vivo effect of oracin on doxorubicin reduction, biodistribution and efficacy in Ehrlich tumor bearing mice
Veronika Hanušová1, Pavel Tomšík2, Lenka Kriesfalusyová3, Alena Pakostová1, Iva Boušová1, Lenka Skálová1
1 Department of Biochemical Sciences, Charles University in Prague, Faculty of Pharmacy, Heyrovského 1203, Hradec Králové, CZ-500 05, Czech Republic
2 Department of Medical Biochemistry, Charles University in Prague, Faculty of Medicine, Šimkova 570, Hradec Králové, CZ-500 38, Czech Republic
3 Radio-Isotope Laboratory, Charles University in Prague, Faculty of Medicine, Šimkova 570, Hradec Králové, CZ-500 38, Czech Republic
Correspondence: Lenka Skálová, e-mail: [email protected]
Abstract: Background: The limitation of carbonyl reduction represents one possible way to increase the effectiveness of anthracycline doxo- rubicin (DOX) in cancer cells and decrease its toxicity in normal cells. In vitro, isoquinoline derivative oracin (ORC) inhibited DOX reduction and increased the antiproliferative effect of DOX in MCF-7 breast cancer cells. Moreover, ORC significantly decreases DOX toxicity in non-cancerous MCF-10A breast cells and in hepatocytes. The present study was designed to test in mice the in vivo effect of ORC on plasma and tissue concentrations of DOX and its main metabolite DOXOL. The effect of ORC on DOX efficacy in mice bearing solid Ehrlich tumors (EST) was also studied. Methods: DOX and DOX + ORC combinations were iv administered to healthy mice. Blood samples, livers and hearts were col- lected during the following 48 h. DOX and DOXOL concentrations were assayed using HPLC. The mice with inoculated EST cells were treated repeatedly iv with DOX and DOX + ORC combinations, and the growth of tumors was monitored. Results: ORC in combination with DOX significantly decreased DOXOL plasma concentrations during four hours after administra- tion, but this significantly affected neither DOX plasma concentrations nor DOX or DOXOL concentrations in the liver and heart at any of intervals tested. In EST bearing mice, ORC did not significantly affect DOX efficacy on tumor growth. However, EST was shown to be an improper model for the testing of ORC efficacy in vivo, as ORC did not inhibit DOXOL formation in EST. Conclusions: In vivo, ORC was able to retard DOXOL formation but was not able to improve DOX efficacy in EST-bearing mice.
Key words: doxorubicin pharmacokinetics, doxorubicinol, CBR1, carbonyl reduction
Pharmacological Reports, 2013, 65, 445452 445 Abbreviations: CBR1 – carbonyl reductase 1, DOX – doxoru- is rapidly absorbed from the gastrointestinal tract, and bicin, DOXOL – doxorubicinol, EST – Ehrlich solid tumor, it possesses good bioavailability. Its acute, subchronic ORC – oracin and chronic toxicity is low, and no cardiotoxicity has been observed in experimental animals [1, 10, 21]. As with DOX, carbonyl reduction is the main metabolic pathway of ORC [25]. In MCF-7 breast cancer cells, Introduction ORC is also metabolized by CBR1, and it is much better substrate for CBR1 than DOX [9]. In our previous in vitro studies, we have demon- Anthracycline antibiotic doxorubicin (DOX) is one of strated that ORC acts as an effective competitive in- the most useful anticancer agents and it still repre- hibitor of DOX reduction in MCF-7 cells, and in this sents the key component in the therapy of many carci- way ORC significantly increased DOX efficacy in the noma types. Unfortunately, DOX is not sufficiently killing of cancer cells [14]. On the other hand, the in- effective in many cases, but increasing the dosage of hibitory effect of ORC on DOX reduction in non- DOX is limited due to its systemic toxicity, particu- cancerous MCF-10A breast cells was almost none in larly cardiotoxicity [4, 15]. It is widely accepted that comparison to cancer cells MCF-7, therefore, ORC DOX cardiotoxicity is caused mainly by DOX- cannot be used to decrease the deactivation of DOX in mediated oxidative stress [24]. In addition, doxoru- normal breast tissue. In viability tests, ORC surpris- bicinol (DOXOL), the main metabolite of DOX, is ingly protected non-tumorous MCF-10A cells and rat thought to contribute to DOX toxicity [19, 23]. hepatocytes against DOX-mediated toxicity, probably DOXOL, which is less cytostatically active but more due to the inhibition of enzymes catalyzing DOX re- cardiotoxic than its parent drug, accumulates in the dox cycling [12]. The obtained data suggest that on heart and participates significantly in the development one hand, ORC could act as an inhibitor of DOX de- of the chronic cumulative cardiotoxicity resulting activation in tumor cells and thus potentiate DOX ef- from DOX therapy. Therefore, the search for possi- ficacy, and on the other hand, ORC could partly pro- bilities of how to increase DOX efficacy in cancer tect non-tumor tissues against DOX toxicity. cells and also minimize the associated toxicities to The present study was designed to test the in vivo effect non-cancerous tissues has become quite an important of ORC on DOX and DOXOL concentrations in plasma, research goal [6]. Many strategies are based on al- as well as in the liver and heart after iv administration to tered DOX metabolism. The enhancing of DOX up- mice of a single dose of DOX alone or ORC + DOX com- take by cancer cells and the metabolic activation of binations. The efficacy of DOX, ORC, and DOX + ORC DOX prodrug within cancer cells have been the main combinations on the growth of solid Ehrlich tumors (EST) approaches [13]. The inhibition of DOX-metabolizing in tumor-bearing mice was tested and compared. enzymes represents another possible strategy [14]. Several carbonyl reducing enzymes participate in DOXOL formation: in the liver primarily carbonyl re- ductase 1 (CBR1), but also partly the aldehyde reduc- Materials and Methods tase AKR1A1 and aldo/keto reductase AKR1C; in the heart mainly AKR1A1 [2, 16, 18]. CBR1 has been identified as the main enzyme catalyzing DOX reduc- Chemicals and reagents tion in MCF-7 breast cancer cells [9]. Moreover, the elevated activity of CBR1 in cancerous tissues as op- DOX was provided by Pharmacia (Upjohn, Italy). posed to normal ones has been reported [20]. Thus the ORC and dihydrooracin were obtained from the inhibitors of CBR1 have been intensively tested, as Research Institute for Pharmacy and Biochemistry their concomitant use with DOX seems to be a possi- (Prague, Czech Republic). ZnSO4 was purchased from ble strategy to increase the cytostatic effect of DOX in Merck Co. (Darmstadt, Germany). Coenzyme NADPH cancer cells [11]. The isoquinoline derivative oracin was supplied by Sigma-Aldrich (Prague, Czech Re- (ORC, 6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo- public). All other chemicals used were of HPLC or 5,6-dihydro-11H-indeno[1,2-c]isoquinoline), which had analytical grade. The stock solutions were prepared in been originally prepared as a potential anticancer double-distilled deionized water or DMSO and stored agent [8], is another promising CBR1 inhibitor. ORC in the dark at 4°C.
446 Pharmacological Reports, 2013, 65, 445452 Doxorubicin and oracin in mice in vivo Veronika Hanuová et al.
Animals try, VUFB, Prague, Czech Republic, and then main- tained in adult female NMRI mice through serial sub- Female NMRI mice weighing 30–33 g were purchased cutaneous transplantation. For the tumor passage, from the Faculty of Medicine in Brno, Czech Repub- 1 volume of tumor homogenate of EST was diluted lic. They were given a standard diet of Altromin 1320 with 2 volumes of physiological saline. On day 0, the (manufactured by Velaz, a.s., Czech Republic) and mice were inoculated subcutaneously with 0.2 ml of water ad libitum. The experiments were designed and the final tumor cell suspension containing 1 × 106 conducted in accordance with European Union rec- cells. The mice bearing the tumor inoculums were ommendations on the handling of experimental ani- then divided into 5 groups: one control group (un- mals and were approved by the Ethics Committee of treated) and four treated groups of 7 animals each. Charles University in the Czech Republic. The second group was treated with DOX alone (one dose 2 mg/kg); the third group was treated with ORC DOX and DOXOL concentrations in blood alone (one dose 20 mg/kg); the fourth group was plasma and tissues after DOX and DOX + ORC treated by a 1 : 2 combination of DOX + ORC (one treatment dose of DOX 2 mg/kg and ORC 4 mg/kg); and the fifth group was treated by a 1 : 10 combination of The mice were divided into four groups (12 animals DOX + ORC (one dose of DOX 2 mg/kg and ORC in each): the first group (control) underwent no treat- 20 mg/kg). All drugs were administered iv in three ment; the second group was treated with DOX alone single doses (the volume of one dose was 0.25 ml per (single iv dose 10 mg/kg); the third group was treated 30 g of body weight) on days 1, 4, and 7 after the with a 1 : 2 combination of DOX + ORC (single iv inoculation of the tumors. dose of DOX 10 mg/kg and ORC 20 mg/kg); and the On the eighth day, all the animals were anesthe- fourth group was treated with a 1 : 5 combination of tized with ether and sacrificed by cervical dislocation. DOX + ORC (single iv dose of DOX 10 mg/kg and The tumors were then excised and weighed. ORC 50 mg/kg). The drugs were applied iv through the tail vein in volumes of 0.25 ml per 30 g of body weight at time 0. Blood samples (45 µl from each Preparation of samples prior to HPLC analysis mouse) were collected by retro-orbital plexus punc- ture or posterior vena cava puncture using heparinized The plasma samples and tissue homogenates were in- blood collection tubes (Drummond Scientific Com- cubated for 15 min at 37°C to allow equilibrium in pany, USA) at 1, 2, 4, 8, 24, and 48 h after treatment. protein binding. The samples were then vortex-mixed During the blood sampling, animals were under ether with the same volume of ZnSO4 solutions (saturated) anesthesia. The blood samples collected were centri- and with a 2.5 times higher volume of acetone for fuged for 5 min at 300 × g for plasma separation. The 15 min at 37°C. After centrifugation, the supernatants plasma samples were then stored at –20°C until analy- obtained were evaporated under nitrogen at room sis. At 8, 24, and 48 h, the mice were sacrificed by temperature. The dried residue was completely dis- cervical dislocation. For each time interval, four mice solved in 100 µl of the mobile phase and injected into from each group were used (n = 4). The liver and the HPLC system for analysis. heart were immediately removed from each and put into a freezer (–80°C). Before analysis, the organs HPLC analysis were thawed and homogenized (150 mg liver/1 ml and 125 mg heart/1 ml) in 0.1 M sodium phosphate The quantification of DOX and DOXOL was per- buffer (pH 7.4) using a Potter-Elvehjem homogenizer formed as described by Václavíková et al. [27] with and sonication with Sonopuls (Bandelin, Germany). the HP 1100 series LC system (Agilent Technologies, Waldbronn, Germany) using a fluorescence detector l l Effect of DOX, ORC and DOX + ORC ( EX = 480 nm, EM = 560 nm). The chromatographic combinations on tumor growth in Ehrlich separation was carried out with a 125 mm × 4 mm Nu- tumor bearing mice cleosil 100-5 C18 column (Macherey-Nagel, Düren, Germany). The mobile phase consisted of a 75 : 25 The solid Ehrlich tumor (EST) was purchased from (v/v) 0.05 M ammonium formate buffer/acetonitrile the Research Institute for Pharmacy and Biochemis- mixture adjusted to pH 4.0. The flow rate was
Pharmacological Reports, 2013, 65, 445452 447 1.2 ml/min. Ten µl of each sample was injected onto Statistical analysis the HPLC column. The compounds were identified with the retention times of reference standards (To- Data are presented as the mean ± SD. Statistical ronto Research Chemicals Inc., North York, Ontario, analysis was carried out using one way analysis of Canada). All experiments were carried out at 25°C in variance (ANOVA) followed by Bonferroni’s test for the dark to avoid the photo-degradation of DOX. multiple comparisons. Statistical significance was acceptable at a level of p < 0.05. Data analysis was performed using GraphPad Prism 5.0. Preparation of subcellular fractions from EST and protein content determination
Tumors for the subcellular fraction preparation were isolated from the untreated EST-bearing mice and were Results stored at –80°C. Before the preparation of the subcellu- lar fractions, the tumors were thawed and quickly ho- mogenized in 0.1 M sodium phosphate buffer (pH 7.4) Plasma concentration of DOX and DOXOL after iv administration of DOX and DOX + ORC using a Potter-Elvehjem homogenizer and subjected to combinations sonication with Sonopuls (Bandelin, Germany). Differ- ential centrifugation of the homogenates was used in DOX alone and DOX + ORC combinations (1 : 2 and the preparation of the subcellular fractions. The cyto- 1 : 5) were administered iv in one single dose to non- solic fractions were obtained as supernatants after the tumor healthy mice, and blood samples were col- last ultracentrifugation at 105,000 × g for 60 min. The lected at 1, 2, 4, 8, 24, and 48 h after administration. subcellular fractions were preserved in aliquots at The concentrations of DOX and DOXOL in the –80°C until further analyses. Protein concentration was plasma after the administration of DOX and DOX determined by a bicinchoninic acid (BCA) assay using + ORC combinations (1 : 2 and 1 : 5) are shown in bovine serum albumin as a standard. Figure 1. No significant differences in plasma concen- trations of the parent drug DOX were found among In vitro assays of inhibitory effect of ORC on the experimental groups at any of time intervals DOX reduction in EST cytosol tested. On the other hand, in first four hours after ad- ministration, plasma concentrations of DOXOL were DOX reduction and its inhibition by ORC were as- significantly lower in the mice treated with DOX sayed in mouse tumor (protein 9.5 mg) cytosolic frac- + ORC combinations than in the mice treated with tions using the following reaction mixture (total vol- DOX alone. A higher ratio of ORC in the DOX ume of 150 µl): 75 µl of cytosolic suspension, 50 µM + ORC combination led to more pronounced inhibi- DOX as a substrate, 1 mM coenzyme NADPH, and tion of DOXOL formation. From 8 to 24 h after ad- 0–1000 µM ORC as an inhibitor in a 0.1 M sodium ministration, DOXOL concentrations were similar in phosphate buffer (pH 7.4). The samples without cyto- all groups. After 48 h, higher concentrations of sol were also tested in order to determine a possible DOXOL were found in the mice treated with DOX non-enzymatic DOX reduction. + ORC combinations than in those treated with DOX Pre-incubation of all reaction mixtures (without cy- alone. tosol) was performed for 5 min at 37°C. Incubation was then started with the addition of cytosol. The Accumulation of DOX and DOXOL in the liver samples were incubated for 60 min at 37°C and all and heart after iv administration of DOX and reactions were stopped by the addition of 75 µl ice- DOX + ORC combinations cooled 96% ethanol and cooling to 0°C. After 5 min of intensive shaking, the samples were centrifuged at DOX alone and DOX + ORC combinations (1 : 2 or 2,300 × g for 15 min and 150 µl of supernatant was 1 : 5) were administered iv in one single dose to transferred to a glass flask and immediately subjected healthy mice without tumors, and their livers and to HPLC analysis. Specific activities were expressed hearts were collected at 8, 24, and 48 h after admini- as pmol of DOXOL formed within min per mg of pro- stration. Amounts of DOX and DOXOL in tissue ho- tein. mogenates were measured, and the data were ex-
448 Pharmacological Reports, 2013, 65, 445452 Doxorubicin and oracin in mice in vivo Veronika Hanuová et al.
Fig. 1. Plasma concentrations of DOX or DOXOL after iv administration of DOX alone or DOX + ORC combina- tions (1:2 or 1:5). Data represent the mean ± SD (n = 4). * Significant differ- ence from treatment with DOX alone. See the Materials and Methods section for further details
Fig. 2. The amount of DOX or DOXOL in the liver after iv administration of DOX alone or DOX + ORC combina- tions (1:2 or 1:5). Data represent the mean ± SD (n = 4). See the Materials and Methods section for further details
Fig. 3. The amount of DOX or DOXOL in the heart after iv administration of DOX alone or DOX + ORC combina- tions (1:2 or 1:5). Data represent the mean ± SD (n = 4). See the Materials and Methods section for further details
pressed as ng/g tissue (see Figs. 2 and 3). When the Effect of DOX, ORC and DOX + ORC amounts of DOX and DOXOL in the liver were com- combinations on tumor growth in pared among experimental groups, no significant dif- EST-bearing mice ferences were observed. A minimal increase in DOX and DOXOL amounts was found in the hearts of mice The anti-tumor activities of DOX, ORC, and combi- treated with DOX + ORC combinations 24 h after ad- nation therapies thereof were evaluated in mice with ministration as compared to DOX-treated mice. Nev- EST. The mice were treated with three doses of DOX, ertheless, no differences were observed at other time ORC and DOX + ORC combinations (1 : 2 and 1 : 10) intervals. at days 1, 4, and 7 after tumor inoculation. The mice
Pharmacological Reports, 2013, 65, 445452 449 Tab. 1. Effect of DOX, ORC and combination DOX with ORC (1 : 2, 1 : 10) to mice inoculated with an Ehrlich solid tumor. NMRI female using HPLC. The effect of ORC on DOX reduction in mice were given 0.2 ml tumor homogenate sc on day 0. Therapy was EST is shown in Figure 4. The inhibitory effect of applied on days 1st, 4th and 7th following tumor inoculation. DOX was administered iv at dose 2 mg/kg and ORC iv at doses 4 or 20 mg/kg. ORC (up to 1,000 µM) was very weak; a pronounced The table presents arithmetic means of tumor weight, and their 95% effect was observed only at high concentrations of confidence interval values are shown as mean ± SD this compound (1,250 and 1,500 µM).
Group Dose n Mean tumor 95%CI (mg/kg) weight (g) Control 0 7 2.49 ± 0.80 1.29–3.77 DOX 2 7 1.45 ± 0.35 0.78–1.89 Discussion ORC 20 7 2.57 ± 0.63 1.58–3.69 DOX + ORC (1 : 2) 2 + 4 7 1.98 ± 0.55 1.24–2.30 Cancer cell mortality has been correlated with both DOX + ORC (1 : 10) 2 + 20 7 1.89 ± 0.52 0.91–2.68 dosage and exposure time, but DOX systemic toxic- ity, especially cardiotoxicity, strongly limits the maxi- mal therapeutic dose of DOX [6]. To overcome this problem, several strategies to increase DOX efficacy were sacrificed on the eighth day, and the weights of in cancer cells as well as decrease its toxicity in nor- the tumors were evaluated after necropsy. Results are mal cells have been intensively studied (reviewed in shown in Table 1. ORC alone had no effect on tumor [13]). The inhibition of DOX-metabolizing enzymes growth, while DOX alone significantly decreased the within cancer cells represents one possible approach. growth of tumors. No statistically significant effect of In MCF-7 breast cancer cells, the isoquinoline de- ORC in combination with DOX on DOX efficacy in rivative ORC acted as a competitive inhibitor of DOX the EST-bearing mice was observed. reduction and thus ORC significantly increased DOX efficacy in the killing of MCF-7 cells [14]. However, Effect of ORC on DOXOL formation in the EST the inhibitory effect of ORC on DOX reduction in cytosolic fractions non-tumor MCF-10A cells and isolated hepatocytes was almost none. Moreover, ORC protected non- The inhibitory effect of ORC on DOX reduction was tumor MCF-10A cells and rat hepatocytes against assayed in cytosolic fractions from EST. A fixed con- DOX-mediated toxicity, probably due to the inhibi- centration of DOX (100 µM) and variable concentra- tion of enzymes catalyzing DOX redox cycling [12]. tions of ORC (0–1,500 µM) were used. After incuba- Based on these promising results, an in vivo study on tion, the amount of DOXOL formed was analyzed the administration of DOX + ORC combinations to mice was designed. Firstly, we wanted to determine if ORC, concomi- tantly administered with DOX, affected DOX and DOXOL plasma concentrations and their accumula- tion in the liver and heart. Single iv doses of DOX alone and DOX + ORC combinations (1 : 2 and 1 : 5) were administered to female NMRI mice. A relatively high dose of DOX was used to allow the quantifica- tion of DOX and its metabolite DOXOL in a small amount of plasma. The DOX and ORC ratios were chosen according to the results obtained in vitro [14]. Blood samples as well as the livers and hearts were collected, and the amounts of DOX and DOXOL were measured. A slight increase in DOX plasma concentration ob- Fig. 4. The inhibitory effect of ORC on DOXOL formation in cytosol served in the first two hours after DOX administration extracted from Ehrlich solid tumor homogenate. The concentration of DOX in the reaction mixture was 100 µM. See the Materials and was followed by a rapid decrease in the next six hours Methods section for further details and a slow decline until the end of the experiment at
450 Pharmacological Reports, 2013, 65, 445452 Doxorubicin and oracin in mice in vivo Veronika Hanuová et al.
48 h. These results are in good agreement with previ- The mice bearing EST were treated with DOX ous studies of DOX pharmacokinetics in a number of alone, ORC alone and DOX + ORC combinations (1:2 species, e.g., a rapid fall of DOX blood levels after and 1:10). The DOX dose was chosen as a suboptimal administration and subsequent slow elimination due dose (our unpublished data; [3]) representing approxi- to DOX distribution into various tissues has been re- mately 40% of the LD10 [17]. In the combinations, ported [5]. Comparing results among experimental a higher ratio of ORC than in the first study (pharma- groups, plasma concentrations of DOX during the cokinetics) was used to achieve a longer period of the first four hours were higher after the administration of diminishing of DOX deactivation. The drugs were ad- DOX + ORC combinations than after the administra- ministered in 3 doses (on days 1, 4, and 7 after tumori- tion of DOX alone, but these results were not statisti- zation). The most pronounced effect was observed in cally significant due to high inter-individual differ- the group treated with DOX alone, while no effect was ences. After the eighth hour, DOX plasma concentra- observed in mice treated with ORC alone. ORC did not tions were similar in all the experimental groups. Also significantly affect DOX efficacy on tumor growth in in the liver and heart, no marked differences in DOX the EST-bearing mice. Therefore, this in vivo study did accumulation among experimental groups were not verify our hypothesis that ORC could increase found. Based on these results, ORC (in combination DOX efficacy in tumor treatment. with DOX) should not significantly influence DOX With the aim of determining the reason for this fail- pharmacokinetics and the tissue distribution of DOX ure, cytosolic fraction from EST homogenate was pre- in humans. pared and the inhibitory effect of ORC on DOX deac- As the main metabolite of DOX, DOXOL is consid- tivation in this cytosol was assayed. Surprisingly, al- ered as less cytostatically active but more cardiotoxic most no inhibition of DOXOL formation by ORC was than the parent drug [23]. Plasma concentrations of observed in EST, while in the previous study ORC DOXOL and its accumulation in the liver and heart were significantly inhibited DOXOL formation in cytosol assayed. In the first four hours after administration, from MCF-7 cells [14]. Although both cells have their plasma concentrations of DOXOL were significantly origins in breast cancer, they differ in species and lower in mice treated with DOX + ORC combinations probably also in expression and in the properties of than in mice treated with DOX alone. The combination their biotransformation enzymes. The lack of inhibi- of DOX + ORC with a higher ratio of ORC caused a tory effect of ORC on DOXOL formation in EST greater decrease of DOXOL concentration in the seems to be one possible explanation for the lack of plasma. These results verify that ORC is able to inhibit effect of ORC on DOX efficacy in EST-bearing mice. DOXOL formation in vivo. Unfortunately, this inhibi- For further in vivo studies of ORC + DOX combina- tion was time-limited: in the eighth hour after admini- tions, nude mice bearing MCF-7 cells should be used. stration, ORC did not decrease DOXOL concentrations Taken together, ORC in combination with DOX in plasma nor in the liver or heart. The reason may lie in administered to mice significantly influenced neither the very rapid decrease of ORC plasma levels and low the plasma concentration of DOX nor its accumula- ORC tissue accumulation; this was observed after a sin- tion in the liver and heart. ORC is able to inhibit gle iv administration in rabbits [10]. The repeated ad- DOXOL formation in vivo, as ORC in combination ministration of ORC may solve this problem, but further with DOX significantly decreased DOXOL plasma testing would be necessary. concentrations during four hours following admini- In the second study, the influence of ORC on DOX stration. This inhibition is time-limited, and thus ORC efficacy in tumor-bearing mice was tested. The solid did not significantly affect DOXOL accumulation in Ehrlich tumor is an easily transplantable, poorly dif- the liver and heart in the eighth hour after administra- ferentiated malignant tumor derived by Paul Ehrlich tion. In EST-bearing mice, ORC did not significantly from a spontaneous murine breast adenocarcinoma. affect DOX efficacy on tumor growth. A consequent The use of such a tumor model in immunocompetent in vitro assay revealed that ORC did not inhibit mice has the advantage of involving the immune sys- DOXOL formation in EST and this fact may be the tem in the inhibition of tumor progression [7, 22]. In reason for the failure of ORC in EST-bearing mice. order to avoid an excessive curability due to the hosts’ For further in vivo studies of ORC + DOX combina- immune response [26] the therapy was started on day tions, MCF7 xenografts as well as repeated ORC ad- 1 after the implantation. ministration should be employed.
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