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1010 poster Increased radiosensitizing effect of bleomycin or cisplatin on LPB sarcoma tumor cells...
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Posters $425 Country, Surgery and Radiology and Physical Medicine, Leioa, Vizcaya, Spain 3School of Medicine and Dentistry. University of the Basque Country, Cell Biology, Leioa, Vizcaya, Spain The mucositis is one of the most important toxic effects of radiotherapy on head and neck carcinomas. We have previously demonstrated that L-oxothiazolidine-carboxylate (OTZ, a pro-drug of cysteine) enhances cell proliferation of normal keratinocytes of oral mucosa and protects them against the toxic effects of irradiation. However, when using a radiomodifier of normal cells, is also very important to achieve, at the same time, the maximum effect on tumor cells. Because of this, the purpose of the present study, was to evaluate the effect of OTZ on the radioresponse of the squamous cell carcinoma-25 (SCC-25), a cell line derived from human epithelium of the tongue. Exponentially tumor cells cultured in 25 cm 2 flasks were irradiated (125 - 1000 cGy) with or without pre-treatment with OTZ (0,5 mM, 1 h before X rays). Immediately after irradiation, 5 x 102 - 1 [4 103 cells were seeded in 30-mm plastic dishes and incubated for 14 days. Colonies were counted and surviving fraction was calculated by the ratio of plating efficiency of treated cells to that non-treated cells. Treatment with OTZ alone significantly reduced (1.2-fold) cell proliferation, but not differences were observed in platting efficiency with respect to untreated cells. The radiosensitivity of SCC-25 cell line measured as the survival fraction at 2 Gy (SF2) showed a value of 0.45 _+ 0.03. In comparison to cells irradiated without radiomodifier, pre-treatment with OTZ produced 1.4- fold increase of the radiosensitivity of this tumor cell line (SF2 value of 0.32 +_0.03). The dose modifying factor (DMF) measured for the survival level of 37% showed a value of 1.3. In conclusion, OTZ works as a selective radiomodifier, sensitizing the tumor cell line SCC-25, while at the same time minimizing the effect on normal keratinocytes. Therefore, these studies suggest that OTZ could be a very important treatment to prevent the mucositis induced by irradiation (this work was supported by the University of the Basque Country, project no. 075.327-EB071/98 and JesQs Gangoiti Barrera Foundation). 1010 poster Increased radiosensitizing effect of bleomycin or cisplatin on LPB sarcoma tumor cells and tumors with localy drug delivery method, electroporation St Kran/c, IV/. Cemazar, A. Grosel, S. Gregor Institute of Oncology, Department of Tumor Biology, Ljubljana, Slovenia Electroporation provides a way to increase the intracellular concentration of impermeable molecules through the electropores in the cell membrane. Bleomycin and cisplatin are poorly permeant but potent cytotoxic and radiosensitizing drugs. Bleomycin causes single- and double-strand breaks in DNA by production of a free radical complex utilizing oxygen and ferrous ion. Cisplatin exert its cytotoxic effect by forming intra- or inter-strand crosslinks in DNA and also by crosslinking DNA with chromosomal proteins. Our previous study showed transient reduction of tumor blood flow and therefore lower oxygen level in tumors after the application of electric pulses. Our study was undertaken to determine whether electroporation of mouse LPB sarcoma tumor cells in vitro and tumors in vivo increases radiosensitizing effect of bleomycin or cisplatin. In addition, because it is known that radiation response of tumors is approximately 3-times higher in the presence of oxygen, the effect of electroporation on radioresponse of tumors was also evaluated. LPB sarcoma cells and tumors were treated either with bleomycin, Cisplatin, electroporation or ionizing radiation, and combination of these treatments. In vitro, radioresponse was determined by colony forming assay, while in vivo, treatment effectiven~ess was determined by local tumor control (TCDs0). Time dependence of partial oxygen pressure in LPB tumors after application of electric pulses was measured by electron paramagnetic oxymetry. Electroporation increased radiosenzitizing effect of bleomycin and cisplatin on LPB tumor cells. This combined treatment was more effective when using bleomycin, compared to cisplatin. Electrochemotherapy with bleomycin potentiated cytotoxicity of irradiation by 1.53-fold and by 1.35-fold in the case of electrochemotherapy with cisplatin. Treatment of LPB tumors either with the electric pulses or with bleomycin prior to irradiation had no effect on local tumor control compared to irradiated tumours only. However, the combination of application electric pulses to the tumors and intravenous injection of bleomycin (electrochemotherapy) prior to irradiation resulted in significant enchancement of radioresponse of tumors (EF=I.9). The TCDs0 value was reduced from 23.1 Gy in mice that were irradiated only to 12.4 Gy in mice that were treated with the combination electrochemotherapy and irradiation. Application of electric pulses to the tumors induced profound but transient reduction of tumor oxygenation. Although tumor oxygenation partially restored at the time of irradiation, partial oxygen pressure especially in the tumor center was still low enough to affect radioresponse of tumors alone or in combination with electrochemotherapy. This study demonstrated that electroporation of LPB tumor cells and tumors, as a drug delivery method, increased radiosensitizing effect of bleomycin and cisplatin. 1011 poster Apoptotic-resistance of the human osteosarcoma cell line HS-Os-1 to irradiation is converted to apoptotic- susceptibility by exogenous hydrogen peroxide ~ 1 , T. Kobayashi2, T. Takahashl ~, S. Kariya 1, A. 2 1 4 ~5 Nishioka , H. Seguchi , S. Yoshida , H. Sonobe , T. Meriki IKochi University, Medical School Radiology, Nankoku-shL Japan 2Kochi University, Cell Biology, Nankoku-shL Japan 3Ehime University, Orthopaedic Surgery, Shigenobu-cho, Japan 4National Fukuyama Hospital, Laboratory Medicine, Fukuyama, Japan 5Kochi Univeristy, Laboratory Medicine, Nankoku-shi, Japan We examined the immunocytochemical characteristics of the apoptotic resistance of the HS-Os-1 cells against irradiation in order to clarify its possible implications regarding radiosensitivity. The results showed that these cells lack P53 and Bax protein expression, and strong peroxidase activity was confirmed in the nuclei of the cells. Moreover, SODII (manganese superoxide dismutase) protein expression was gradually increased in spite of irradiation of up to 30 Gy. Therefore, it was concluded that HS-Os-1 cells are originally apoptotic-resistant and that the cells possess a strong ability to scavenge for free radicals. To convert these cells to a state of apoptotic-susceptibility, a powerful oxidant such as hydrogen peroxide was speculated to exert such an effect in terms of the production of hydroxyl radicals in lysosomes in the cells. The origin of the radioresistance of the human osteosarcoma cell line HS-Os-1 was considered to involve a low degree of ROS formation following irradiation, reflecting the strong scavenging ability of these cells for free radicals including hydroxyl radicals. Therefore, we examined the
Transcript of 1010 poster Increased radiosensitizing effect of bleomycin or cisplatin on LPB sarcoma tumor cells...
Posters $425
Country, Surgery and Radiology and Physical Medicine, Leioa, Vizcaya, Spain 3School of Medicine and Dentistry. University of the Basque Country, Cell Biology, Leioa, Vizcaya, Spain The mucositis is one of the most important toxic effects of radiotherapy on head and neck carcinomas. We have previously demonstrated that L-oxothiazolidine-carboxylate (OTZ, a pro-drug of cysteine) enhances cell proliferation of normal keratinocytes of oral mucosa and protects them against the toxic effects of irradiation. However, when using a radiomodifier of normal cells, is also very important to achieve, at the same time, the maximum effect on tumor cells. Because of this, the purpose of the present study, was to evaluate the effect of OTZ on the radioresponse of the squamous cell carcinoma-25 (SCC-25), a cell line derived from human epithelium of the tongue. Exponentially tumor cells cultured in 25 cm 2 flasks were irradiated (125 - 1000 cGy) with or without pre-treatment with OTZ (0,5 mM, 1 h before X rays). Immediately after irradiation, 5 x 102 - 1 [4 103 cells were seeded in 30-mm plastic dishes and incubated for 14 days. Colonies were counted and surviving fraction was calculated by the ratio of plating efficiency of treated cells to that non-treated cells. Treatment with OTZ alone significantly reduced (1.2-fold) cell proliferation, but not differences were observed in platting efficiency with respect to untreated cells. The radiosensitivity of SCC-25 cell line measured as the survival fraction at 2 Gy (SF2) showed a value of 0.45 _+ 0.03. In comparison to cells irradiated without radiomodifier, pre-treatment with OTZ produced 1.4- fold increase of the radiosensitivity of this tumor cell line (SF2 value of 0.32 +_0.03). The dose modifying factor (DMF) measured for the survival level of 37% showed a value of 1.3. In conclusion, OTZ works as a selective radiomodifier, sensitizing the tumor cell line SCC-25, while at the same time minimizing the effect on normal keratinocytes. Therefore, these studies suggest that OTZ could be a very important treatment to prevent the mucositis induced by irradiation (this work was supported by the University of the Basque Country, project no. 075.327-EB071/98 and JesQs Gangoiti Barrera Foundation).
1010 poster
Increased radiosensitizing effect of bleomycin or cisplatin on LPB sarcoma tumor cells and tumors with localy drug delivery method, electroporation
St Kran/c, IV/. Cemazar, A. Grosel, S. Gregor Institute of Oncology, Department of Tumor Biology, Ljubljana, Slovenia Electroporation provides a way to increase the intracellular concentration of impermeable molecules through the electropores in the cell membrane. Bleomycin and cisplatin are poorly permeant but potent cytotoxic and radiosensitizing drugs. Bleomycin causes single- and double-strand breaks in DNA by production of a free radical complex utilizing oxygen and ferrous ion. Cisplatin exert its cytotoxic effect by forming intra- or inter-strand crosslinks in DNA and also by crosslinking DNA with chromosomal proteins. Our previous study showed transient reduction of tumor blood flow and therefore lower oxygen level in tumors after the application of electric pulses.
Our study was undertaken to determine whether electroporation of mouse LPB sarcoma tumor cells in vitro and tumors in vivo increases radiosensitizing effect of bleomycin or cisplatin. In addition, because it is known that radiation response of tumors is approximately 3-times higher in the presence of oxygen, the effect of electroporation on radioresponse of tumors was also evaluated.
LPB sarcoma cells and tumors were treated either with bleomycin, Cisplatin, electroporation or ionizing radiation, and combination of these treatments. In vitro, radioresponse was determined by colony forming assay, while in vivo, treatment effectiven~ess was determined by local tumor control (TCDs0). Time dependence of partial oxygen pressure in LPB tumors after application of electric pulses was measured by electron paramagnetic oxymetry.
Electroporation increased radiosenzitizing effect of bleomycin and cisplatin on LPB tumor cells. This combined treatment was more effective when using bleomycin, compared to cisplatin. Electrochemotherapy with bleomycin potentiated cytotoxicity of irradiation by 1.53-fold and by 1.35-fold in the case of electrochemotherapy with cisplatin. Treatment of LPB tumors either with the electric pulses or with bleomycin prior to irradiation had no effect on local tumor control compared to irradiated tumours only. However, the combination of application electric pulses to the tumors and intravenous injection of bleomycin (electrochemotherapy) prior to irradiation resulted in significant enchancement of radioresponse of tumors (EF=I.9). The TCDs0 value was reduced from 23.1 Gy in mice that were irradiated only to 12.4 Gy in mice that were treated with the combination electrochemotherapy and irradiation. Application of electric pulses to the tumors induced profound but transient reduction of tumor oxygenation. Although tumor oxygenation partially restored at the time of irradiation, partial oxygen pressure especially in the tumor center was still low enough to affect radioresponse of tumors alone or in combination with electrochemotherapy.
This study demonstrated that electroporation of LPB tumor cells and tumors, as a drug delivery method, increased radiosensitizing effect of bleomycin and cisplatin.
1011 poster
Apoptotic-resistance of the human osteosarcoma cell line HS-Os-1 to irradiation is converted to apoptotic- susceptibility by exogenous hydrogen peroxide ~ 1 , T. Kobayashi 2, T. Takahashl ~, S. Kariya 1, A.
2 1 4 ~5 Nishioka , H. Seguchi , S. Yoshida , H. Sonobe , T. Meriki I Kochi University, Medical School Radiology, Nankoku-shL Japan 2Kochi University, Cell Biology, Nankoku-shL Japan 3Ehime University, Orthopaedic Surgery, Shigenobu-cho, Japan 4National Fukuyama Hospital, Laboratory Medicine, Fukuyama, Japan 5Kochi Univeristy, Laboratory Medicine, Nankoku-shi, Japan We examined the immunocytochemical characteristics of the apoptotic resistance of the HS-Os-1 cells against irradiation in order to clarify its possible implications regarding radiosensitivity. The results showed that these cells lack P53 and Bax protein expression, and strong peroxidase activity was confirmed in the nuclei of the cells. Moreover, SODII (manganese superoxide dismutase) protein expression was gradually increased in spite of irradiation of up to 30 Gy. Therefore, it was concluded that HS-Os-1 cells are originally apoptotic-resistant and that the cells possess a strong ability to scavenge for free radicals. To convert these cells to a state of apoptotic-susceptibility, a powerful oxidant such as hydrogen peroxide was speculated to exert such an effect in terms of the production of hydroxyl radicals in lysosomes in the cells. The origin of the radioresistance of the human osteosarcoma cell line HS-Os-1 was considered to involve a low degree of ROS formation following irradiation, reflecting the strong scavenging ability of these cells for free radicals including hydroxyl radicals. Therefore, we examined the
