1. Combined treatment of Graffi cancer cells with the new anti-neoplastic agent erufosine and doxorubicin. In vivo study.
 Veselina Uzunova1, Ani Georgieva2, Sonia Apostolova1, Iva Ugrinova3, Reneta Toshkova2, Martin R. Berger4, Rumiana Tzoneva1
1Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Sofia, Bulgaria; 2Institute of experimental morphology, pathology and anthropology with museum, Bulgarian Academy of Sciences, Sofia, Bulgaria; 3 Institute of Molecular Biology „Roumen Tsanev“, Bulgarian Academy of Sciences, Sofia, Bulgaria; 4 German cancer Research center, Heidelberg, Germany
Background: Doxorubicin is a widely used anticancer drug in many different cancers because of its pleiotropic effects although exhibiting serious side effects. In order to minimize the side effects of doxorubicin is used erufosine which is derived from a new class of anti-cancer agents termed alkylphosphocholines, which show high antineoplastic activity in cancer cells but low side effects in normal tissues.
Experimental groups
Group no.
Treatment group
Dosage (mg/100g) 1
Untreated control 2
Erufosinea
1.5 3
Erufosineb 4
Erufosinea +Doxorubicinea 5
Erufosineb +Doxorubicineb 6
Doxorubicinea
Results and discussion: In the present work we tried to minimize the side effects of doxorubicin by combining low to moderate doses of doxorubicin (below IC50) with erufosine.
In vitro experiments revealed that EPC3 in concentration lower and equal to IC50 has an additive effect on cytotoxicity of Dox. That additive effect increased with the time.
The antitumor effect of EPC and Dox was assessed in Graffi tumor bearing hamsters. The mean volume of tumors in hamsters treated with combinations of EPC3 and Dox is significantly lower compared to untreated control (100 % inhibition of tumor growth on 8th and 15th day). Graphs are shifted to the right from that of the controls. The best effect of decreasing of tumor volume is noted in tumor-bearing hamsters which were treated with a combination of the two drugs before on day 0. The tendency of decreasing the tumor volume in this group was kept to the end of the treatment Tumor volume in the city . 3 is the smallest of all stages of the study (up to 30 day ). This reduction in tumor volume was related to the prolongation of the mean survival time (MST) (Fig. 7). Since the MST for control animals is 24.8 ± 3.5 days, for hamsters-bearing tumors treated with combination of EPC3 and DOX from day 0 or day 11 (tumor volume 1 cm2) the significant increase of MST is ±4.7 days and 38 ± 4.6 days, respectively. The lethality tumor-bearing hamsters treated with anticancer agents was found to decrease for all groups between 25th and 40th day of treatment comparing to the control (Fig.6). The most significant decrease in lethality was observed in hamsters treated with EPC3 and DOX combination( Figure 6 ). As can be seen from the graph the lowest mortality was found for animals treated with both agents from day 0.
The obtained results showed a significant increase in the cytotoxic effect of the combination of EPC3 and doxorubicin administered at IC50 dose for in vitro experiment and on lower dose than IC50 for in vivo therapeutic experiments in comparison to using anti-tumor agents alone.
Conclusion. The combined application of the conventional chemotherapeutic drug doxorubicin with the new anti-cancer agent erufosine may have therapeutic potential for the treatment of patients with chemo-resistant metastatic breast cancer with minimal side effects.
Acknowledgements: The work is supported by grants DFNI/BO2/5 and DNTS/Germany/01/4
References:
[1] Toshkova R., N. Manolova, E. Gardeva, M. Ignatova, L. Yossifova, I. Rashkov, M. Alexandrov. International Journal of Pharmaceutics, 2010, 400 (1-2):
[2] Chung SD, Chen KH, Tu CC, Chen Y. , Urology. 2009 Jan;73(1):212-3.
Table 1
Fig. 1 Experimental design of in vivo experiment
Materials and methods:
Antineoplastic agents: Erufosine (EPC3) was kindly provided by Prof. H. Eibl (MPI for Biophysical Chemistry, Gottingen, Germany. Doxorubicin hydrochloride (Dox) was from Sigma.
Cells: Primary Graffi tumor cells were isolated from Graffi myeloid tumor induced in Golden syrian hamsters as described elsewhere [1]. The cell culture was incubated in RPMI 1640 culture medium at 37oC and 5% CO2.
Erufosine and Doxorubicin treatment in vitro
Graffi tumor cells (1x104 cells/mL) were seeded onto glass coverslips (d = 12 mm) pleased in a sterile 24-well tissue culture plates and incubated for 24 h to obtain good cell adhesion. After the incubation the cells were treated with different concentrations of erufosine (IC50, ½ IC50 and ¼ IC50) and doxorubicin (IC50) alone or in a combination and were additionally incubated for 24 h or 48 h. Untreated tumor cells were used as a control.
MTT assay was used to determine the in vitro cytotoxicity of erufosine and doxorubicin in the applied concentrations.
Experimental animals - Golden Syrian hamsters, 2–4 months old, weighing approximately 100 g were purchased from a breeding base Oncology Center, Sofia. The animals were kept under standard conditions in individual plastic cages with free access to food and water. All studies were performed in accordance with the Guide for Care and Use of Laboratory Animals.
Tumor model -A Graffi myeloid tumor was primary created by the Graffi-virus in new-born hamsters and maintained monthly in vivo by subcutaneous transplantation of viable tumor cells (2×106/mL PBS) in the interscapular area of hamsters, for keeping the tumor’s survival [1]. The tumor is 100% cancerous, and the animals die usually up to the 30th day after transplantation. Spontaneous regression in this experimental tumor model was not observed. For the present experiment 2x104 viable Graffi tumor cells per animal were transplanted subcutaneously (s.c.) on hamsters.
Therapy and dosages: The experimental design is summarized in Table 1 and Fig.1 Erufosine and Doxorubicin was administrated s.c. twice a week for 4 weeks. The single dosage for Erufosine correspond to 30µM/kg and for Doxorubicin to 0.1µM/kg.
Evaluation and statistics: Tumor growth was monitored by recording tumor volume per animal. The total tumor volume was measured using a caliper and was calculated by the formula: V = A·B2/2, where A—major axis; B—minor axis [2]. All data for in vitro and in vivo experiments are given as the mean ± standard deviation. Significance testing was performed using one-way analysis of variance followed by Bonferroni’s post hoc test. Values of *p<0.05, **p<0.01 and ***p<0.001 were considered significant.
Fig.2 Cytotoxic effect of EPC3 and Dox on Graffi myeloid tumor cells after 24 hours of exposure.
Fig.3 Cytotoxic effect of EPC3 and Dox on Graffi myeloid tumor cells after 84 hours of exposure.
Fig.5 Tumor volume in GTBH after treatment with EPC3 and DOX
Fig.6 Lethality (%) of GTBH, treated with EPC3 + Dox
Fig.4 Mean survival time of Graffi tumor bearing hamsters (GTBH), treated with EPC3 and Doxorubicin