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Murine Intestinal Crypt Survival after Combined Taxol plus Radiation Exposure
GYNECOLOGIC ONCOLOGY ARTICLE NO.
63, 180–183 (1996)
0303
Murine Intestinal Crypt Survival after Combined Taxol plus Radiation Exposure S. L. LOJUN, M.D.,* C. P. SIGDESTAD, PH.D.,† A. M. CONNOR, PH.D.,‡ W. J. SPANOS, JR., M.D.,† AND J. R. BOSSCHER, M.D.§ *Department of Obstetrics, Gynecologic and Reproductive Sciences, Division of Gynecologic Oncology, University of California/Mount Zion Cancer Center, San Francisco, California 94120; and §Division of Gynecological Oncology, †Department of Radiation Oncology, and ‡Department of Diagnostic Radiology, James Graham Brown Cancer Center, School of Medicine, University of Louisville, Louisville, Kentucky 40292 Received January 2, 1996
Taxol is active against a number of cancers, some of which are also radiosensitive. The role of combined Taxol and radiotherapy has not been established. A theoretical benefit exists, however, due to the G2/M phase specificity of Taxol, and radiation-sensitizing effects have been observed in vitro. This study focused on the combined Taxol/radiation effect on murine intestinal epithelium. HA/ICR random-bred male mice were used to examine the temporal effect of injecting Taxol (40 mg/kg) before and after irradiation (12.5 Gy). The microcolony assay was used to determine the effect of single and combined modalities. Control groups consisted of no treatment, drug solvent only (Cremophor/ethanol/saline), radiation only, and Taxol only. A complete radiation dose–response study of treatment with and without Taxol was performed 12 hr after injection. Preirradiation Taxol appears not to enhance intestinal cell killing, and may even offer some protection when injected at 4 hr prior to irradiation. Postirradiation Taxol was found to enhance radiation response most dramatically at times greater than 12 hr. Dose–response data appeared to confirm the increased radiation effect of combined treatment. In conclusion, Taxol administered prior to irradiation results in either no sensitization or possible protection of the normal tissue response. Postirradiation Taxol appears to sensitize intestinal crypts when injected at least 12 hr after gamma irradiation. These data suggest that care should be exercised when using Taxol after irradiation. q 1996 Academic Press, Inc.
INTRODUCTION
Paclitaxel (Taxol) is a naturally occurring chemotherapeutic drug isolated from the Pacific yew tree, Taxus brevifolia. Several clinical trials involving Taxol have demonstrated marked activity against many solid tumors, including, advanced ovarian cancer, metastatic breast cancer, head and neck cancer, esophageal cancer, non-small cell lung cancer Presented at the 27th Annual Meeting of the Society of Gynecologic Oncologists, New Orleans, LA, February 10–14, 1996.
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(NSCLC), and melanoma [1–3]. Taxol has even been active against platinum-resistant ovarian cancer and doxorubicinresistant breast cancer. The mechanism of activity involves the binding of tubulin, thus promoting microtubule formation and inhibiting depolymerization of microtubules [4–6]. This perturbation may result in deregulation of microtubule-related functions such as mitosis, cell proliferation, intracellular transport, maintenance of cell shape, cellular mobility, cellular attachment, and modulation of interactions with cell surface receptors. This may ultimately result in programmed cell death (apoptosis), cytolysis, or arrest of cells in the G2 mitotic phase of the cell cycle. In addition to the intrinsic cytotoxic activity of Taxol, the specificity for the G2/M phase of the cell cycle renders a theoretical propensity for radiosensitizing activity, given the G2/M cell cycle phase to be the most radiosensitive. Therefore, the concomitant use of radiotherapy and Taxol could result in increased cytotoxic effectiveness or decreased radiation dose required for equal effectiveness. This is the basis for ongoing phase I and II trials involving combined Taxol and radiotherapy in primary brain tumors and advanced NSCLC [7, 8]; however, this combination treatment raises the concern for increased toxicity to normal tissues. Ideally, normal tissues would be relatively spared by selective toxicity to tumor cells and/or by decreased required radiation doses. This may not be the case, and normal tissue toxicity may limit this modality. Gastrointestinal toxicity is often the rate-limiting toxic effect of radiation treatment in pelvic malignancies, due to the chronic or late effects of radiation-induced changes. Therefore, to evaluate toxicity in normal gastrointestinal tissue, the in vivo use of concomitant Taxol and radiation was studied in the murine model, with intestinal crypt survival used as the outcome measure. While this technique quantitates the degree of radiation and chemotherapy induced bowel toxicity, it is a measure of acute toxicity; however,
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FIG. 1. Effect of time of Taxol injection on crypt survival with a radiation dose of 12.5 Gy. Taxol was administered six times prior to irradiation and six times following irradiation. In addition, the effects of the Taxol solvent (Cremophor) and Taxol / solvent were determined.
the acute changes in murine crypt survival may help predict patterns of late toxicity. A relatively large dose of Taxol, compared with human clinical use, was used to demonstrate a severe and measurable degree of gastrointestinal toxicity. The relatively high doses of radiation and Taxol were used in an attempt to establish presence of radiosensitization, effect of timing and sequence of treatments, and dose–response relationship. The data may lead to a better understanding of tissue toxic effects as well as tumor cytotoxicity with combined use of radiation and Taxol. MATERIALS AND METHODS
Animals. HA/ICR male mice 6–8 weeks old were used. They were housed in standards cages, held in a laminarairflow climate. All animals were kept on acid water (pH 2.7) to prevent growth of Pseudomonas, and fed Purina mouse chow ad libidum. Conditions of temperature, humidity, and light/dark cycle were standardized to prevent any variation in natural circadian rhythm. Taxol. Taxol was provided by Bristol–Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey. Taxol was dissolved in absolute ethanol at a concentration of 48 mg/ml, then diluted with Cremophor EL to 24 mg/ml for storage. Immediately prior to use, the Taxol was diluted with 0.9% NaCl to 8 mg/ml. Irradiation procedure. Animals were placed in a Plexiglas container and irradiated with a cobalt-60 teletherapy unit at a dose rate of 1.6 Gy/min.
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Time of injection relative to irradiation. Taxol was administered by an single intraperitoneal injection at a dose of 40 mg/kg. Irradiation dose was 12.5 Gy. Treatment times were varied at time points prior to irradiation (46, 24, 8, 4, 2, 1, and 0.5 hr), and time points postirradiation (0.0, 0.5, 3, 6, 12, 28, and 48 hr). Four or five animals per time point were used. Controls consisted of no treatment, solvent only, Taxol plus solvent, and irradiation only. Animals were sacrificed 4 days post-treatment and jejunal tissue samples were obtained for crypt survival assay using Withers’ method [9, 10], which has been described in detail in previous publications [11, 12]. Crypts per circumference were counted by three different investigators in blinded fashion. Crypt survival curves. A radiation dose–response study was performed using Withers’ microcolony assay [9, 10]. Taxol treatment time was set at 12 hr postirradiation, and Taxol dose remained at 40 mg/kg, injected intraperitoneally (IP). Five animals were treated at each of five different radiation dose points, ranging from 9 to 13.2 Gy. The control group was treated with solvent only (IP) 12 hr following irradiation. Five animals were injected with solvent at each of five different radiation dose points, ranging from 11 to 16.1 Gy. Animals were sacrificed 4 days post-treatment, and jejunal tissue samples were taken to determine surviving crypts per circumference by Withers’ method. Crypt survival curves were constructed and standard regression analysis was performed to determine linearity, parallelism, and correlation.
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RESULTS
Overall, the regimen of 12.5 Gy, 40 mg/kg IP Taxol, and Cremophor eluant was fairly well tolerated, with minimal morbidity observed. The timing of Taxol treatment relative to irradiation was found to be especially critical for jejunal crypt survival in this murine model (Fig. 1). Cytotoxicity was reduced, i.e., crypt survival enhanced, when Taxol was administered 8 hr or less prior to irradiation. The maximal number of crypts per circumference was observed at the 2hr preirradiation time point. At this time point, 90 crypts/ circumference were observed, compared with 58 crypts/circumference in the radiation-only control group. Cytotoxicity was enhanced, i.e., crypt survival reduced, when Taxol was administered at the time of or up to 48 hr after irradiation. Reduction in crypt survival was especially marked 12 hr postirradiation, and continued to be observed at time points up to 48 hr. Crypt survival curves were generated with Taxol administered 12 hr postirradiation. (Fig. 2). Crypt survival was shown to vary exponentially as a function of radiation dose. The survival curves were parallel, with a D10 ratio of 1.16. DISCUSSION
FIG. 2. Jejunal crypt survival curves at Day 4 for mice treated with Taxol or radiation only. The curves were significantly parallel, and the SEM for each point was less than 5%.
These results are quite novel in that both cytotoxic enhancement and cytotoxic protection were observed in vivo, dependent on the timing of Taxol treatment with respect to irradiation. Several investigators have examined preirradiation Taxol using in vitro models, with varying results. Radiation-sensitizing effects have been reported for several cell lines, including HL-60 (leukemia) [13]; BG-1, SKOV-3, OVCAR-3 (ovarian cancer) [14, 15]; 618, Kernohan grade III (astrocytoma) [168n]; SK-MEL, HTB-72 (melanoma) [18]; and several others including cervical cancer. Interestingly, all of these studies involved treatment of cell cultures with Taxol prior to irradiation, and these observations are contrary to the cytotoxic protection observed in this study, using preirradiation Taxol in the mouse model. Stromberg et al. [19], however, failed to demonstrate radiosensitizing effects with preirradiation Taxol using cell lines MCF-7 (breast cancer), DUT-145 (prostate cancer), and HT-29 (colon cancer). Liebmann et al. observed similar findings in the case of MCF-7 cells [20]; and Geard and Jones failed to find radiation enhancement using several cervical cancer cell lines (SiHa, HTB-35, HTB-31, C-33A) [18, 21]. Mason et al. investigated murine gastrointestinal toxicity with combined Taxol and radiation, using preirradiation Taxol only [22]. This study supported additive effects only, with no radiosensitization observed. As well, a protective effect was observed when mice were treated 24 hr prior to irradiation. One hypothesis offered by Mason et al. for the observed radioprotection is a regenerative overshoot induced by Taxol, and this may indeed explain or partially explain the
radioprotection observed in this study. The enhanced cytotoxicity observed in the postirradiation treatment groups is intriguing, and there are no reports of Taxol/radiation used in this sequence. The enhancement may simply represent additive effects of Taxol; however, there may be some chemosensitization of cells surviving radiation, perhaps related to injured cells remaining in the G2/M cell cycle phase, where Taxol is thought to exert its effect. In fact, Donaldson et al. showed that Taxol exhibits a threefold increase in cytotoxicity when cells are already in late G2 phase, with apoptotic DNA fragmentation occurring 12–14 hr after exposure to drug [23]. This lends support to the present finding of possible radiation-induced chemosensitization, perhaps as a result of cell cycle redistribution. Other mechanisms may, however, be responsible; e.g., radiation and Taxol signal pathways may interact, or may contribute, to this observation. As well, Cremophor itself in high concentrations is somewhat cytotoxic, and has been shown to induce cell cycle blockade distinct from that produced by Taxol [24]. This study suggests that the safety of combination radiation/Taxol might be limited by gastrointestinal toxicity if radiation dose is not reduced; however, the present study employed a Taxol dose significantly larger than is used clinically establish an observable effect. Therefore, due to the difference between experimental and clinical radiation and drug doses, the conclusions regarding bowel toxicity may be limited. This finding of increased bowel toxicity is similar to in
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vivo investigations using mice treated with other chemotherapeutic agents (methotrexate, cyclophosphamide, doxorubicin, carmustine) in combination with radiation [25]. In fact, a recent phase I trial of Taxol and radiation therapy for advanced NSCLC was limited largely by esophagitis [8]. Other safety concerns exist for combination therapy when considering long-term survivors. Hei et al. [26, 27], as well as Liebmann et al. [28], have shown that Taxol enhances the oncogenic transforming potential of gamma irradiation in vitro; therefore, the development of secondary malignancies may be a realistic concern. It is important to note that the intraperitoneal route of administration, compared with the more common intravenous route, limits comparisons in some cases, due to the pharmacokinetic differences. In addition, this study involved a relatively small number of animals, but SEMs were very small. Further investigation is warranted to evaluate this temporal sequence of radiation/Taxol, the role of cell cycle kinetics, and other mechanisms involved in the resultant cell death. It is hoped that future studies may result in improved treatments for tumors sensitive to both radiation and Taxol. ACKNOWLEDGMENTS The authors thank the University of Louisville Division of Gynecologic Oncology and Department of Radiation Oncology for support of this study.
REFERENCES 1. Rowinsky, E. K., Cazenave, L. A., and Donehower, R. C. Taxol: A novel investigational antineoplastic agent, J. Natl Cancer Inst. 82, 1247–1259 (1990). 2. Rowinsky, I. K., Gilbert, M., and McGuire, W. P. Sequences of Taxol and cisplatin: A phase I and pharmacologic study, J. Clin. Oncol. 9, 1692–1703 (1991). 3. Donehower, R. C., and Rowinsky, E. K. An overview of experience with Taxol (paclitaxel) in the USA, Cancer Treat. Rev. 19, 63–78 (1993). 4. Manfredi, J. J., and Horwitz, S. B. Taxol: An antimitotic agent with a new mechanism of action, Pharmacol. Ther. 25, 83–125 (1984). 5. Kumar, N. Taxol-induced polymerization of purified tubulin: Mechanism of action, J. Biol. Chem. 256, 10435–10441 (1981). 6. Schiff, P. B., Fant, J., and Horwitz, S. B. Promotion of microtubule assembly in vitro by Taxol, Nature 277, 665–667 (1979). 7. Glantz, M., Walhberg, L., Kim, L., Akerley, W., Mills, P., and Choy, H. Phase I study of weekly TAXOL and concurrent cranial radiation in adults with primary brain tumors. Proc. Am. Soc. Clin Oncol. 13, 183 (1994). 8. Hainsworth, J. D., Miller, P., Menchise, A., Raefsky, E., and Greco, F. A. Treatment of locally advanced non-small cell lung cancer (NSCLC) with Taxol (1-hour infusion), cisplatin, etoposide, and radiation therapy (RT): A Phase II trial, Proc. Am. Soc. Clin. Oncol. 13, 1172 (1994). 9. Withers, J. R., and Elkind, M. M. Microcolony survival assay for cells of mouse intestinal mucosa exposed to radiation, Int. J. Radiat. Biol. 17, 261–267 (1970).
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10. Withers, J. R., Mason, K., Reid, B. O., Dubravsky, N., Barkely, H. T., Brown, N. W., and Smathers, J. B. Response of mouse intestine to neutrons and gamma rays in relation to dose fractionation and division cycle, Cancer 34, 39–47 (1974). 11. Sigdestad, C., P., and Scott, R. M. The effect of WR-2721 on intestinal crypt survival: 1.4-MeV X-rays, Radiat. Res. 62, 267–275 (1975). 12. Hagemann, R. F., Sigdestad, C. P., and Lesher, S. Intestinal crypt survival and total and per crypt levels of proliferative cellularity following irradiation: Single X-ray exposure, Radiat. Res. 46, 533–546 (1971). 13. Choy, J., Rodriguez, F. F., Koester, S., Hilsenbeck, S., and Von Hoff, D. D. Investigation of Taxol as a potential radiation sensitizer, Cancer 71, 3774–3778 (1993). 14. Steren, A., Sevin, B. U., Perras, J., Angioli, R., Nguyen, H., Guerra, L., Koechli, O., and Averette, H. E. Taxol sensitizes human ovarian cancer cells to radiation, Gynecol. Oncol. 48, 252–258 (1993). 15. Steren, A., Sevin, B. U., Perras, J., Ramos, R., Angioli, R., Nguyen, H., Koechli, O., and Averette, H. E. Taxol as a radiation sensitizer: A flow cytometric study, Gynecol. Oncol. 50, 89–93 (1993). 16. Tishler, R. B., Geard, C. R., Hall, E. J., and Schiff, P. B. Taxol sensitizes human astrocytoma cells to radiation, Cancer Res. 52, 3495–3497 (1992). 17. Tishler, R. B., Schiff, P. B., Geard, G. R., and Hall, E. J. Taxol: A novel radiation sensitizer, Radiat. Oncol. Biol. Phys. 22, 613–617 (1992). 18. Geard, C. R., Jones, J. M., and Schiff, P. B. Taxol and radiation, Monogr. Natl. Cancer Inst. 15, 89–94 (1993). 19. Stromberg, J. S., Lee, Y. J., Armour, E. P., Martinez, A. A., and Corry, P. M. Lack of radiosensitization after paclitaxel treatment of three human carcinoma cell lines, Cancer 75, 2262–2268 (1995). 20. Liebmann, J., Cook, J. A., Fisher, J., and Teague, D. In vitro studies of Taxol as a radiation sensitizer in human tumor cells, J. Natl. Cancer Inst. 16, 441–446 (1994). 21. Geard, C. R., and Jones, J. M. Radiation and Taxol effects on synchronized human cervical carcinoma cells, Int. J. Radiat. Oncol. Biol. Phys. 29, 565–569 (1994). 22. Mason, K. A., Milas, L., and Peters, L. J. Effect of paclitaxel (Taxol) alone and in combination with radiation on the gastrointestinal mucosa, Int. J. Radiat. Oncol. Biol. Phys. 32, 1381–1389 (1995). 23. Donaldson, K. L., Goolsby, G. L., and Wahl, A. F. Cytotoxicity of the anticancer agents cisplatin and Taxol during cell proliferation and the cell cycle, Int. J. Cancer 57, 847–855 (1994). 24. Liebmann, J., Cook, J. A., Lipschultz, C., Teague, D., Fisher, J., and Mitchell, J. B. The influence of Cremophor EL on the cell cycle effects of paclitaxel (Taxol) in human tumor cell lines, Cancer Chemother. Pharmacol. 33, 331–339 (1994). 25. Schenken, L. L., Burholt, D. R., Hagemann, R. F., and Lesher, S. The modification of gastrointestinal tolerance and responses to abdominal irradiation by chemotherapeutic agents, Radiology 120, 417–420 (1976). 26. Hei, T. K., and Hall, E. J. Taxol, radiation, and oncogenic transformation, Cancer Res. 53, 1368–1372 (1993). 27. Hei, T. K., Piao, C. Q., Geard, C. R., and Hall, E. J. Taxol and ionizing radiation: Interaction and mechanisms, Int. J. Radiat. Oncol. Biol Phys. 29, 267–271 (1994). 28. Liebmann, J., Cook, J. A., Fisher, J., Teague, D., and Mitchell, J. B. Changes in radiation survival curve parameters in human tumor and rodent cells exposed to paclitaxel (Taxol), Int. J. Radiat. Oncol. Biol. Phys. 29, 559–564 (1994).
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0303
Murine Intestinal Crypt Survival after Combined Taxol plus Radiation Exposure S. L. LOJUN, M.D.,* C. P. SIGDESTAD, PH.D.,† A. M. CONNOR, PH.D.,‡ W. J. SPANOS, JR., M.D.,† AND J. R. BOSSCHER, M.D.§ *Department of Obstetrics, Gynecologic and Reproductive Sciences, Division of Gynecologic Oncology, University of California/Mount Zion Cancer Center, San Francisco, California 94120; and §Division of Gynecological Oncology, †Department of Radiation Oncology, and ‡Department of Diagnostic Radiology, James Graham Brown Cancer Center, School of Medicine, University of Louisville, Louisville, Kentucky 40292 Received January 2, 1996
Taxol is active against a number of cancers, some of which are also radiosensitive. The role of combined Taxol and radiotherapy has not been established. A theoretical benefit exists, however, due to the G2/M phase specificity of Taxol, and radiation-sensitizing effects have been observed in vitro. This study focused on the combined Taxol/radiation effect on murine intestinal epithelium. HA/ICR random-bred male mice were used to examine the temporal effect of injecting Taxol (40 mg/kg) before and after irradiation (12.5 Gy). The microcolony assay was used to determine the effect of single and combined modalities. Control groups consisted of no treatment, drug solvent only (Cremophor/ethanol/saline), radiation only, and Taxol only. A complete radiation dose–response study of treatment with and without Taxol was performed 12 hr after injection. Preirradiation Taxol appears not to enhance intestinal cell killing, and may even offer some protection when injected at 4 hr prior to irradiation. Postirradiation Taxol was found to enhance radiation response most dramatically at times greater than 12 hr. Dose–response data appeared to confirm the increased radiation effect of combined treatment. In conclusion, Taxol administered prior to irradiation results in either no sensitization or possible protection of the normal tissue response. Postirradiation Taxol appears to sensitize intestinal crypts when injected at least 12 hr after gamma irradiation. These data suggest that care should be exercised when using Taxol after irradiation. q 1996 Academic Press, Inc.
INTRODUCTION
Paclitaxel (Taxol) is a naturally occurring chemotherapeutic drug isolated from the Pacific yew tree, Taxus brevifolia. Several clinical trials involving Taxol have demonstrated marked activity against many solid tumors, including, advanced ovarian cancer, metastatic breast cancer, head and neck cancer, esophageal cancer, non-small cell lung cancer Presented at the 27th Annual Meeting of the Society of Gynecologic Oncologists, New Orleans, LA, February 10–14, 1996.
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(NSCLC), and melanoma [1–3]. Taxol has even been active against platinum-resistant ovarian cancer and doxorubicinresistant breast cancer. The mechanism of activity involves the binding of tubulin, thus promoting microtubule formation and inhibiting depolymerization of microtubules [4–6]. This perturbation may result in deregulation of microtubule-related functions such as mitosis, cell proliferation, intracellular transport, maintenance of cell shape, cellular mobility, cellular attachment, and modulation of interactions with cell surface receptors. This may ultimately result in programmed cell death (apoptosis), cytolysis, or arrest of cells in the G2 mitotic phase of the cell cycle. In addition to the intrinsic cytotoxic activity of Taxol, the specificity for the G2/M phase of the cell cycle renders a theoretical propensity for radiosensitizing activity, given the G2/M cell cycle phase to be the most radiosensitive. Therefore, the concomitant use of radiotherapy and Taxol could result in increased cytotoxic effectiveness or decreased radiation dose required for equal effectiveness. This is the basis for ongoing phase I and II trials involving combined Taxol and radiotherapy in primary brain tumors and advanced NSCLC [7, 8]; however, this combination treatment raises the concern for increased toxicity to normal tissues. Ideally, normal tissues would be relatively spared by selective toxicity to tumor cells and/or by decreased required radiation doses. This may not be the case, and normal tissue toxicity may limit this modality. Gastrointestinal toxicity is often the rate-limiting toxic effect of radiation treatment in pelvic malignancies, due to the chronic or late effects of radiation-induced changes. Therefore, to evaluate toxicity in normal gastrointestinal tissue, the in vivo use of concomitant Taxol and radiation was studied in the murine model, with intestinal crypt survival used as the outcome measure. While this technique quantitates the degree of radiation and chemotherapy induced bowel toxicity, it is a measure of acute toxicity; however,
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FIG. 1. Effect of time of Taxol injection on crypt survival with a radiation dose of 12.5 Gy. Taxol was administered six times prior to irradiation and six times following irradiation. In addition, the effects of the Taxol solvent (Cremophor) and Taxol / solvent were determined.
the acute changes in murine crypt survival may help predict patterns of late toxicity. A relatively large dose of Taxol, compared with human clinical use, was used to demonstrate a severe and measurable degree of gastrointestinal toxicity. The relatively high doses of radiation and Taxol were used in an attempt to establish presence of radiosensitization, effect of timing and sequence of treatments, and dose–response relationship. The data may lead to a better understanding of tissue toxic effects as well as tumor cytotoxicity with combined use of radiation and Taxol. MATERIALS AND METHODS
Animals. HA/ICR male mice 6–8 weeks old were used. They were housed in standards cages, held in a laminarairflow climate. All animals were kept on acid water (pH 2.7) to prevent growth of Pseudomonas, and fed Purina mouse chow ad libidum. Conditions of temperature, humidity, and light/dark cycle were standardized to prevent any variation in natural circadian rhythm. Taxol. Taxol was provided by Bristol–Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey. Taxol was dissolved in absolute ethanol at a concentration of 48 mg/ml, then diluted with Cremophor EL to 24 mg/ml for storage. Immediately prior to use, the Taxol was diluted with 0.9% NaCl to 8 mg/ml. Irradiation procedure. Animals were placed in a Plexiglas container and irradiated with a cobalt-60 teletherapy unit at a dose rate of 1.6 Gy/min.
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Time of injection relative to irradiation. Taxol was administered by an single intraperitoneal injection at a dose of 40 mg/kg. Irradiation dose was 12.5 Gy. Treatment times were varied at time points prior to irradiation (46, 24, 8, 4, 2, 1, and 0.5 hr), and time points postirradiation (0.0, 0.5, 3, 6, 12, 28, and 48 hr). Four or five animals per time point were used. Controls consisted of no treatment, solvent only, Taxol plus solvent, and irradiation only. Animals were sacrificed 4 days post-treatment and jejunal tissue samples were obtained for crypt survival assay using Withers’ method [9, 10], which has been described in detail in previous publications [11, 12]. Crypts per circumference were counted by three different investigators in blinded fashion. Crypt survival curves. A radiation dose–response study was performed using Withers’ microcolony assay [9, 10]. Taxol treatment time was set at 12 hr postirradiation, and Taxol dose remained at 40 mg/kg, injected intraperitoneally (IP). Five animals were treated at each of five different radiation dose points, ranging from 9 to 13.2 Gy. The control group was treated with solvent only (IP) 12 hr following irradiation. Five animals were injected with solvent at each of five different radiation dose points, ranging from 11 to 16.1 Gy. Animals were sacrificed 4 days post-treatment, and jejunal tissue samples were taken to determine surviving crypts per circumference by Withers’ method. Crypt survival curves were constructed and standard regression analysis was performed to determine linearity, parallelism, and correlation.
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RESULTS
Overall, the regimen of 12.5 Gy, 40 mg/kg IP Taxol, and Cremophor eluant was fairly well tolerated, with minimal morbidity observed. The timing of Taxol treatment relative to irradiation was found to be especially critical for jejunal crypt survival in this murine model (Fig. 1). Cytotoxicity was reduced, i.e., crypt survival enhanced, when Taxol was administered 8 hr or less prior to irradiation. The maximal number of crypts per circumference was observed at the 2hr preirradiation time point. At this time point, 90 crypts/ circumference were observed, compared with 58 crypts/circumference in the radiation-only control group. Cytotoxicity was enhanced, i.e., crypt survival reduced, when Taxol was administered at the time of or up to 48 hr after irradiation. Reduction in crypt survival was especially marked 12 hr postirradiation, and continued to be observed at time points up to 48 hr. Crypt survival curves were generated with Taxol administered 12 hr postirradiation. (Fig. 2). Crypt survival was shown to vary exponentially as a function of radiation dose. The survival curves were parallel, with a D10 ratio of 1.16. DISCUSSION
FIG. 2. Jejunal crypt survival curves at Day 4 for mice treated with Taxol or radiation only. The curves were significantly parallel, and the SEM for each point was less than 5%.
These results are quite novel in that both cytotoxic enhancement and cytotoxic protection were observed in vivo, dependent on the timing of Taxol treatment with respect to irradiation. Several investigators have examined preirradiation Taxol using in vitro models, with varying results. Radiation-sensitizing effects have been reported for several cell lines, including HL-60 (leukemia) [13]; BG-1, SKOV-3, OVCAR-3 (ovarian cancer) [14, 15]; 618, Kernohan grade III (astrocytoma) [168n]; SK-MEL, HTB-72 (melanoma) [18]; and several others including cervical cancer. Interestingly, all of these studies involved treatment of cell cultures with Taxol prior to irradiation, and these observations are contrary to the cytotoxic protection observed in this study, using preirradiation Taxol in the mouse model. Stromberg et al. [19], however, failed to demonstrate radiosensitizing effects with preirradiation Taxol using cell lines MCF-7 (breast cancer), DUT-145 (prostate cancer), and HT-29 (colon cancer). Liebmann et al. observed similar findings in the case of MCF-7 cells [20]; and Geard and Jones failed to find radiation enhancement using several cervical cancer cell lines (SiHa, HTB-35, HTB-31, C-33A) [18, 21]. Mason et al. investigated murine gastrointestinal toxicity with combined Taxol and radiation, using preirradiation Taxol only [22]. This study supported additive effects only, with no radiosensitization observed. As well, a protective effect was observed when mice were treated 24 hr prior to irradiation. One hypothesis offered by Mason et al. for the observed radioprotection is a regenerative overshoot induced by Taxol, and this may indeed explain or partially explain the
radioprotection observed in this study. The enhanced cytotoxicity observed in the postirradiation treatment groups is intriguing, and there are no reports of Taxol/radiation used in this sequence. The enhancement may simply represent additive effects of Taxol; however, there may be some chemosensitization of cells surviving radiation, perhaps related to injured cells remaining in the G2/M cell cycle phase, where Taxol is thought to exert its effect. In fact, Donaldson et al. showed that Taxol exhibits a threefold increase in cytotoxicity when cells are already in late G2 phase, with apoptotic DNA fragmentation occurring 12–14 hr after exposure to drug [23]. This lends support to the present finding of possible radiation-induced chemosensitization, perhaps as a result of cell cycle redistribution. Other mechanisms may, however, be responsible; e.g., radiation and Taxol signal pathways may interact, or may contribute, to this observation. As well, Cremophor itself in high concentrations is somewhat cytotoxic, and has been shown to induce cell cycle blockade distinct from that produced by Taxol [24]. This study suggests that the safety of combination radiation/Taxol might be limited by gastrointestinal toxicity if radiation dose is not reduced; however, the present study employed a Taxol dose significantly larger than is used clinically establish an observable effect. Therefore, due to the difference between experimental and clinical radiation and drug doses, the conclusions regarding bowel toxicity may be limited. This finding of increased bowel toxicity is similar to in
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TAXOL AND RADIATION EFFECT ON MURINE INTESTINE
vivo investigations using mice treated with other chemotherapeutic agents (methotrexate, cyclophosphamide, doxorubicin, carmustine) in combination with radiation [25]. In fact, a recent phase I trial of Taxol and radiation therapy for advanced NSCLC was limited largely by esophagitis [8]. Other safety concerns exist for combination therapy when considering long-term survivors. Hei et al. [26, 27], as well as Liebmann et al. [28], have shown that Taxol enhances the oncogenic transforming potential of gamma irradiation in vitro; therefore, the development of secondary malignancies may be a realistic concern. It is important to note that the intraperitoneal route of administration, compared with the more common intravenous route, limits comparisons in some cases, due to the pharmacokinetic differences. In addition, this study involved a relatively small number of animals, but SEMs were very small. Further investigation is warranted to evaluate this temporal sequence of radiation/Taxol, the role of cell cycle kinetics, and other mechanisms involved in the resultant cell death. It is hoped that future studies may result in improved treatments for tumors sensitive to both radiation and Taxol. ACKNOWLEDGMENTS The authors thank the University of Louisville Division of Gynecologic Oncology and Department of Radiation Oncology for support of this study.
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