- Email: [email protected]
P1, in vivo, whereas induction of altered topoisomerase MDR (at-MDR) is not - an in vivo31P MR spectroscopic study of human SCLC tumor xenografts
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114
ESTABLISHMENT
IN VITRO EVALUATION OF ANTIMICROTUBULE AGENTS IN HUMAN SMALL CELL LUNG CANCER
AND CHARACTERIZATION OF SN38 (AN ACTIVE METABOLITE OF CPT-l&RESISTANT HUMAN SMALL CELL LUNG CANCER (SCLC) CELL LINE. T. Shibayama, T. Ohnoshi, H. Ueoka, M. Tabata, M. Chikamori, T .Matsumura, I. Kimura. Department of Medicine, Okayama University Medical School, Okayama, Japan. An SN38resistant subline, SBC-3/SN38, was developed from human SCLC cell line, SBC-3, by continuous exposure to increasing concentrations of SN38 in culture. SBC-3/SN38 was 74.5-fold more resistant to SN38 than SBC-3 in terms of IC50 determined by MIT assay. SBC3/SN38 was highly cross-resistant to CPT-11 and topotecan, partially cross-resistant to adriamycin, etoposide and methotrexate, and not cross-resistant to vinca alkaloids, cisplatin, and 5.FU. Of interest, SBC-3/SN38 showed collateral sensitivity to bleomycin. Topoisomerase I (Top0 I) activity in SBC3/SN38 determined by relaxation of pUC was reduced to a half, and Topo I content by Western blotting was reduced to l/.5 in comparison with those in SBC-3. Topo II activity determined by decatenation of kinetoplast DNA in SBC-3/SN38 was also reduced to a level of 50% comapring with that in SBC-3. IntracelluIar accumulation of SN38 in SBC-3/SN38 was significantly lower than that in SBC-3, however, an overexpression of P-glycoprotein was not detected in SBC-3iSN38. These results indicate that decrease of intracellular accumulation of SN38 and decrease of Topo I activity may play a major role in developing resistance to SN38.
(SCLC) CELL LINES. N. Takigawa, T. Ohnoshi, H. Ueoka, K. Kiura, M. Tabata, T. Shibayama, I. Kimura. Department of Medicine, Okayama University Medical School, Okayama, Japan We examined the cytotoxic activity of antirnicrotnbule agents using SCLC cell lines, SBC-2, -3, -4, -7, which were established in our laboratories. The IC5Os of taxol for these cell lines determined by MIT assay ranged from 1.3nM to 7.5nM. Those of taxotere, rhizosin, vinorelbine and vindesine were 0.74.12nM, 0.41-1.6nM, 2.3.18nM and 0.96-3.0nM, respectively. These IC50 values were significantly lower than those of adriamycin (ADM:19-200nM), etoposide (ETP:28031,00OnM), and cisplatin (CDDP:660-16,OOOnM). Cross-resistance of these agents to ADM, ETP and CDDP was investigated using ADM-resistant SCLC subline;SBC3/ADM, ETP-resistant SCLC subIine;SBC-3/ETP, and CDDP-resistant SCLC subline;SBC-UCDDP. The IC50 ratio of the resistant subline to the parent subline were
determined as follows: SBC-3IADM SBC-YETP SBC-UCDDP 110 1.0 Taxol 1,400 350 220 1.3 Taxotere 0.89 Rhizosin 2.9 1.1 511 94 1.1 Vinorelbine 620 290 0.63 Vindesine These results indicate that the antimicrotubule agents are sensitive for SCLC cell lines and not cross resistant to CDDP. Of interest, rhizoxin showed only partial cross-resistance to ADM, and no cross-resistance to ETP. Rhizosin may have a clinical usefulness relapsing or resistant SCLC.
as a salvage treatment for
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116
lnducaiandP~medidedMullihugresisEance~~ asoci&dtia&cmmeinATP/P,inin,wherashdwimn tapoisanersseMDA(atMDFi)isrKlt-aninti”PMRspecbpscopicstudy dhumanSCLCtunorxenogafls CA. Krfstensen, P.E.G. Kristjansen, P.B. Jensen, M. Sehastad, M. SpangThomsen, 6. Quismfff Dept. of Oncology, Rigshospitafet, Dept. of Pathology, Sundby Hospital, and Tumor Pathology Lab. UPAf B NMR-Center, University of Copenhagen, Denmark. In order to elucidate the relationship between inductionof Pgp-MDR and energy metabolism measured by “P MR spectroscopy we studied the two resistant SCLC lines, NCI-H69/DAU (with PgpMDR expression) and OCNYHAJM (with at-MDR resistance), both established in vitro. and their corresponding wildtypas, NCI-H69 and OC-NYH. The four cell lines were inoculatedand seriallytransptanted as solid tumor lines in nude mice (NMRI nu/nu). The macroscopicalgrowth was characterized using the transformed Gompertz function. Tumor doubling time (TD) (at 396 mm”) of OC-NYH and OC-NYH/VM was 4 days, whereas TD was 2 and 6 days for NCI-H69 and NCIH69/DAU, respectively.The steady state energy metabolismwas anaiysed by weekty non-invasivein viva 3’P-MRspectroscopy of the tumors startingwhen they reached a volume of 269300 mm’. Groups of 7-10 indffMual tumors of each type were examined. A Vivospecspectrometer (Otsuka Electronics,Inc.) wkh a wide-bore 4.7 T magnet (Magnex, Inc.) was used, applying a 12 mm diameter surface coil tuned to the 3’P frequency located over the tumor. The coil size, location, and acquisition parameters optimized tumor signal with minimal contributionfrom the animal except the skin covering the tumor. Steady state ATP/P, ratio was three times higher (pcO.01, Wilcoxon twosample test) in the wildtypeNCI-H69 than in NCI-H69iDAU,whilethere was no differencebetween the energetic status of OC-NYH and OC-NYH/VM tumors. In conclusion, development of Pgpmecliated resistance was aeeociaterl with a decrease in the steady State ATP/P, ratio in the SCLC tumor NCI-H69. Tneae results might reflect differences in the energy demand between the two mechanisms of resistance, Pgp- and at-MDR.
HIGH DOSE ETOPOSIDE (VP-X) COMBINEDWlTR TOPOISOMERASEII ANTAGONISTS. MODELS FOR DIFFRRENTIALPROTECTION OF NORMAL.TISSUES VERSUS TUMOR CELLS. B. Helm, M. Sehested, P.B. Jensen. Dept. of Oncology, Bigshospitalet,Copenhagen 2100 Denmark. The epipcdophyllotoxinesVP-16 and VM-26 are probably the most importantdrugs in the treatmentof small cell lung cancer (SCLC). The drugs are wed in maximally tolerateddoses and the toxicity of the drugs precludes significant dose increments. The cell killingme&attismof VP-16 involves the enzyme topaisomemse Il (top+ II) which is convertedinto a toxic DNA binding protein. This cell hill can be antagonizeaby distinct drug types. We have previously demonstrated that the intercalatingdrug aclantbicia and the cardioprotecting agent ICBP-187 (ICW) antagottizz the cytototicity of VP-16 in vitro. We have studied 2 possible ways of utilizing this antagonismas a means of differentiallyprotectingnormal tissue. 1. Utilizationof the blood brain barrier (BBB). Etoposide transversesthe BBB and ICBP is not expected to, thusthe drug combinationcould enableBhigher dose of etoposide to patientswith brain m&stases. In order to study this possibilitywe examined the in viva protection of ICBP on high dose etopmsidein healthymice. Toxicity in healthymice (female ND) Mean survival after 60 days of observation ICRF(12Smg/hg)+VP-16 100% VP-16 Wmglkg 27% ICRF(lZSmg/kg)+VP-16 5.5% VP-16 140 trig/kg 9% All dmgs were given once i.p. Thus the combinationmakes it possible to increasethe dose of VP-16. 2. Utilizstimtof the extracellularacidity ia solid tumors. Etoposideis an unchargedmolecule unaffected by extracellularpH @HJ. We have screened B numberof weak base intercalatingagents for their effect on VP-16 mediatedcell kill. We found that the intetzalatingdrug chlmuqttineinhibitedetoposide inducedDNAtopa II cleavablecomplex formationand also fully atttagottkd cytotoxicityat pH 7.4. In contrast, no protection is seen at pH 6.8. Being B weak base, chloroquine accumulationdepends on pH as it is tm@ extmcellulmilyat low pH_. Accordingly, the combiiion of the two compmmcbdirects cytotoxicity to area.3with low pI& Conclusion:We present 2 differentpreclinicalmodels for improvingthe selectivityof tumor cell hill by VP-16.
114
ESTABLISHMENT
IN VITRO EVALUATION OF ANTIMICROTUBULE AGENTS IN HUMAN SMALL CELL LUNG CANCER
AND CHARACTERIZATION OF SN38 (AN ACTIVE METABOLITE OF CPT-l&RESISTANT HUMAN SMALL CELL LUNG CANCER (SCLC) CELL LINE. T. Shibayama, T. Ohnoshi, H. Ueoka, M. Tabata, M. Chikamori, T .Matsumura, I. Kimura. Department of Medicine, Okayama University Medical School, Okayama, Japan. An SN38resistant subline, SBC-3/SN38, was developed from human SCLC cell line, SBC-3, by continuous exposure to increasing concentrations of SN38 in culture. SBC-3/SN38 was 74.5-fold more resistant to SN38 than SBC-3 in terms of IC50 determined by MIT assay. SBC3/SN38 was highly cross-resistant to CPT-11 and topotecan, partially cross-resistant to adriamycin, etoposide and methotrexate, and not cross-resistant to vinca alkaloids, cisplatin, and 5.FU. Of interest, SBC-3/SN38 showed collateral sensitivity to bleomycin. Topoisomerase I (Top0 I) activity in SBC3/SN38 determined by relaxation of pUC was reduced to a half, and Topo I content by Western blotting was reduced to l/.5 in comparison with those in SBC-3. Topo II activity determined by decatenation of kinetoplast DNA in SBC-3/SN38 was also reduced to a level of 50% comapring with that in SBC-3. IntracelluIar accumulation of SN38 in SBC-3/SN38 was significantly lower than that in SBC-3, however, an overexpression of P-glycoprotein was not detected in SBC-3iSN38. These results indicate that decrease of intracellular accumulation of SN38 and decrease of Topo I activity may play a major role in developing resistance to SN38.
(SCLC) CELL LINES. N. Takigawa, T. Ohnoshi, H. Ueoka, K. Kiura, M. Tabata, T. Shibayama, I. Kimura. Department of Medicine, Okayama University Medical School, Okayama, Japan We examined the cytotoxic activity of antirnicrotnbule agents using SCLC cell lines, SBC-2, -3, -4, -7, which were established in our laboratories. The IC5Os of taxol for these cell lines determined by MIT assay ranged from 1.3nM to 7.5nM. Those of taxotere, rhizosin, vinorelbine and vindesine were 0.74.12nM, 0.41-1.6nM, 2.3.18nM and 0.96-3.0nM, respectively. These IC50 values were significantly lower than those of adriamycin (ADM:19-200nM), etoposide (ETP:28031,00OnM), and cisplatin (CDDP:660-16,OOOnM). Cross-resistance of these agents to ADM, ETP and CDDP was investigated using ADM-resistant SCLC subline;SBC3/ADM, ETP-resistant SCLC subIine;SBC-3/ETP, and CDDP-resistant SCLC subline;SBC-UCDDP. The IC50 ratio of the resistant subline to the parent subline were
determined as follows: SBC-3IADM SBC-YETP SBC-UCDDP 110 1.0 Taxol 1,400 350 220 1.3 Taxotere 0.89 Rhizosin 2.9 1.1 511 94 1.1 Vinorelbine 620 290 0.63 Vindesine These results indicate that the antimicrotubule agents are sensitive for SCLC cell lines and not cross resistant to CDDP. Of interest, rhizoxin showed only partial cross-resistance to ADM, and no cross-resistance to ETP. Rhizosin may have a clinical usefulness relapsing or resistant SCLC.
as a salvage treatment for
115
116
lnducaiandP~medidedMullihugresisEance~~ asoci&dtia&cmmeinATP/P,inin,wherashdwimn tapoisanersseMDA(atMDFi)isrKlt-aninti”PMRspecbpscopicstudy dhumanSCLCtunorxenogafls CA. Krfstensen, P.E.G. Kristjansen, P.B. Jensen, M. Sehastad, M. SpangThomsen, 6. Quismfff Dept. of Oncology, Rigshospitafet, Dept. of Pathology, Sundby Hospital, and Tumor Pathology Lab. UPAf B NMR-Center, University of Copenhagen, Denmark. In order to elucidate the relationship between inductionof Pgp-MDR and energy metabolism measured by “P MR spectroscopy we studied the two resistant SCLC lines, NCI-H69/DAU (with PgpMDR expression) and OCNYHAJM (with at-MDR resistance), both established in vitro. and their corresponding wildtypas, NCI-H69 and OC-NYH. The four cell lines were inoculatedand seriallytransptanted as solid tumor lines in nude mice (NMRI nu/nu). The macroscopicalgrowth was characterized using the transformed Gompertz function. Tumor doubling time (TD) (at 396 mm”) of OC-NYH and OC-NYH/VM was 4 days, whereas TD was 2 and 6 days for NCI-H69 and NCIH69/DAU, respectively.The steady state energy metabolismwas anaiysed by weekty non-invasivein viva 3’P-MRspectroscopy of the tumors startingwhen they reached a volume of 269300 mm’. Groups of 7-10 indffMual tumors of each type were examined. A Vivospecspectrometer (Otsuka Electronics,Inc.) wkh a wide-bore 4.7 T magnet (Magnex, Inc.) was used, applying a 12 mm diameter surface coil tuned to the 3’P frequency located over the tumor. The coil size, location, and acquisition parameters optimized tumor signal with minimal contributionfrom the animal except the skin covering the tumor. Steady state ATP/P, ratio was three times higher (pcO.01, Wilcoxon twosample test) in the wildtypeNCI-H69 than in NCI-H69iDAU,whilethere was no differencebetween the energetic status of OC-NYH and OC-NYH/VM tumors. In conclusion, development of Pgpmecliated resistance was aeeociaterl with a decrease in the steady State ATP/P, ratio in the SCLC tumor NCI-H69. Tneae results might reflect differences in the energy demand between the two mechanisms of resistance, Pgp- and at-MDR.
HIGH DOSE ETOPOSIDE (VP-X) COMBINEDWlTR TOPOISOMERASEII ANTAGONISTS. MODELS FOR DIFFRRENTIALPROTECTION OF NORMAL.TISSUES VERSUS TUMOR CELLS. B. Helm, M. Sehested, P.B. Jensen. Dept. of Oncology, Bigshospitalet,Copenhagen 2100 Denmark. The epipcdophyllotoxinesVP-16 and VM-26 are probably the most importantdrugs in the treatmentof small cell lung cancer (SCLC). The drugs are wed in maximally tolerateddoses and the toxicity of the drugs precludes significant dose increments. The cell killingme&attismof VP-16 involves the enzyme topaisomemse Il (top+ II) which is convertedinto a toxic DNA binding protein. This cell hill can be antagonizeaby distinct drug types. We have previously demonstrated that the intercalatingdrug aclantbicia and the cardioprotecting agent ICBP-187 (ICW) antagottizz the cytototicity of VP-16 in vitro. We have studied 2 possible ways of utilizing this antagonismas a means of differentiallyprotectingnormal tissue. 1. Utilizationof the blood brain barrier (BBB). Etoposide transversesthe BBB and ICBP is not expected to, thusthe drug combinationcould enableBhigher dose of etoposide to patientswith brain m&stases. In order to study this possibilitywe examined the in viva protection of ICBP on high dose etopmsidein healthymice. Toxicity in healthymice (female ND) Mean survival after 60 days of observation ICRF(12Smg/hg)+VP-16 100% VP-16 Wmglkg 27% ICRF(lZSmg/kg)+VP-16 5.5% VP-16 140 trig/kg 9% All dmgs were given once i.p. Thus the combinationmakes it possible to increasethe dose of VP-16. 2. Utilizstimtof the extracellularacidity ia solid tumors. Etoposideis an unchargedmolecule unaffected by extracellularpH @HJ. We have screened B numberof weak base intercalatingagents for their effect on VP-16 mediatedcell kill. We found that the intetzalatingdrug chlmuqttineinhibitedetoposide inducedDNAtopa II cleavablecomplex formationand also fully atttagottkd cytotoxicityat pH 7.4. In contrast, no protection is seen at pH 6.8. Being B weak base, chloroquine accumulationdepends on pH as it is tm@ extmcellulmilyat low pH_. Accordingly, the combiiion of the two compmmcbdirects cytotoxicity to area.3with low pI& Conclusion:We present 2 differentpreclinicalmodels for improvingthe selectivityof tumor cell hill by VP-16.