130-nm albumin-bound paclitaxel enhances tumor radiocurability and therapeutic gain.

PURPOSE: 130-nm albumin-bound paclitaxel (nab-paclitaxel) is a novel solvent-free albumin-bound paclitaxel, designed to avoid solvent-related toxicity. Nab-paclitaxel has been successfully introduced into the clinic but its radiation-enhancing potential has not yet been evaluated. We conducted a preclinical evaluation of the radiation-modulating effects of nab-paclitaxel in tumor and normal tissues. EXPERIMENTAL DESIGN: Mice bearing syngeneic ovarian or mammary carcinomas were treated with nab-paclitaxel, radiation, or combination of both. Nab-paclitaxel was administered at 90 mg/kg, 1.5 times the maximum tolerated dose for solvent-based paclitaxel. End points were antitumor efficacy (growth delay, radiocurability, and cellular effects) and normal tissue toxicity (gut and skin). RESULTS: Nab-paclitaxel showed single-agent antitumor efficacy against both tumor types and acted as a radiosensitizer. Combined with radiation, nab-paclitaxel produced supra-additive effects when given before radiation. Nab-paclitaxel significantly increased radiocurability by reducing the dose yielding 50% tumor cure (TCD(50)) from 54.3 to 35.2 Gy. Tumor histology following nab-paclitaxel treatment was characterized by pronounced necrotic and apoptotic cell death and mitotic arrest. Nab-paclitaxel did not increase normal tissue radioresponse. CONCLUSIONS: Nab-paclitaxel exhibited strong antitumor efficacy against both tumors as a single agent and it improved radiotherapy in a supra-additive manner. These improved effects were achieved without increased normal tissue toxicity to either rapidly or slowly proliferating normal tissues although the drug dose was 1.5 times higher than the maximum tolerated dose of solvent-based paclitaxel. These preclinical findings show that combining nab-paclitaxel with radiotherapy would improve the outcome of taxane-based chemoradiotherapy. This novel taxane is thus a good candidate for testing in clinical chemoradiotherapy trials.

Importance of maintenance therapy in C225-induced enhancement of tumor control by fractionated radiation.

PURPOSE: C225 strongly enhances tumor radioresponse when given concurrently with radiotherapy. We investigated whether additional therapeutic benefit could be achieved by continuing maintenance treatment with C225 after the completion of fractionated radiotherapy. METHODS AND MATERIALS: A431 xenografts were treated with local irradiation or combined with C225 by two different schedules: (1) 6 h before the first dose of irradiation and at 3-day intervals for a total of 3 doses during the 7-day fractionated radiotherapy, or (2) 6 doses of C225 given both during radiotherapy and continuing for 3 additional doses after radiotherapy. Tumor cure was assessed by the radiation dose yielding local tumor control in 50% of animals (TCD50), and time to recurrence was also determined. RESULTS: Both treatment schedules increased radiocurability as evidenced by reductions in TCD50, but the effect was greater when C225 was given both during and after radiotherapy. C225 reduced the TCD50 of 83.1 (73.2-124.8) Gy by radiation only to 46.2 (39.1-57.5) Gy when given during radiotherapy and to 30.8 (22.2-38.0) Gy when given during and after radiotherapy. Dose modification factors were 1.8 when C225 was given during radiotherapy and 2.7 when given both during and after radiotherapy. C225 was also effective in delaying the onset of tumor recurrences, and was more effective when given as both concurrent and maintenance therapy. CONCLUSIONS: Data showed that C225 strongly enhanced the curative effect of fractionated radiation, and its effect was greater if administration was extended beyond the end of radiotherapy. This important finding may influence future designs of clinical trials combining anti-EGFR (anti-epidermal growth factor receptor) agents with radiotherapy.

Endostatin improves radioresponse and blocks tumor revascularization after radiation therapy for A431 xenografts in mice.

PURPOSE: Clinical trials of antiangiogenic agents used alone for advanced malignancy have been disappointing but preclinical studies suggest that the addition of radiation therapy could improve antitumor efficacy. To test the hypothesis that antiangiogenic therapy combined with radiation therapy can overcome the limitations of antiangiogenic monotherapy, we studied the effects of endostatin combined with radiation on the growth and vascularization of A431 human epidermoid carcinomas growing intramuscularly in the legs of mice. METHODS AND MATERIALS: Mice with established A431 human epidermoid leg tumors were treated with radiation, endostatin, both radiation and endostatin, or vehicle control. The experiment was repeated and mice from each group were killed at 2, 7, and 10 days after irradiation so that tumor tissue could be obtained to further analyze the kinetics of the antitumor, antivascular, and antiangiogenic response to therapy. RESULTS: Endostatin enhanced the antitumor effects of radiation, and prolonged disease-free survival was observed in the combined treatment group. Endothelial cell proliferation was increased in tumors after irradiation but was blocked by the concurrent administration of endostatin, and the combination of endostatin with radiation enhanced endothelial cell apoptosis within 48 h after irradiation. Expression of vascular endothelial growth factor, interleukin-8, and matrix metalloproteinase-2 were increased in tumors after irradiation, and this increase was blocked by concurrent administration of endostatin. CONCLUSION: These data indicate that endostatin can block tumor revascularization after radiation therapy and thereby augment radioresponse.

CpG oligodeoxynucleotides are potent enhancers of radio- and chemoresponses of murine tumors.

BACKGROUND AND PURPOSE: Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine-guanine (CpG) motifs bind to Toll-like receptor 9 (TLR9) and stimulate both innate and adaptive immune reactions and possess anti-tumor activity. We recently reported that CpG ODN 1826 strongly enhances radioresponse of both immunogenic [Milas L, Mason K, Ariga H, et al. CpG oligodeoxynucleotide enhances tumor response to radiation. Cancer Res 2004;64:5074-7] and non-immunogenic [Mason KA, Ariga H, Neal R, et al. Targeting toll-like receptor-9 with CpG oligodeoxynucleotides enhances tumor response to fractionated radiotherapy. Clin Cancer Res 2005;11:361-9] murine tumors. Using two immunogenic murine tumors, a fibrosarcoma (FSa) and a mammary carcinoma (MCa-K), the present study explored whether CpG ODN 1826 also improves the response of murine tumors to the chemotherapeutic agent docetaxel (DOC). MATERIALS AND METHODS: CpG ODN 1826 (100 microg) was given sc three times: when leg tumors were 6mm, when they grew to 8mm and again 1 week later. DOC (33 mg/kg iv) and local tumor radiation (10Gy) were given when tumors were 8mm. Effects of the treatments were assayed by tumor growth delay, defined as days for tumors to grow from 8 to 12 mm in diameter. RESULTS: Treatment with CpG ODN 1826 resulted in strongly enhanced response of FSa tumors to radiation and MCa-K tumors to the chemotherapeutic agent DOC. Enhancement of tumor treatment response was demonstrated by a strong prolongation in the primary tumor treatment endpoint, tumor growth delay. Coincidentally, this treatment also resulted in a higher rate of tumor cure than that observed after tumor radiotherapy or chemotherapy alone. When all three agents were combined the effect was comparable to that of the combination of CpG ODN 1826 with radiation in the case of FSa or of the combination of CpG ODN 1826 with DOC in the case of MCa-K. CONCLUSION: Overall results show that CpG ODN 1826 can markedly improve tumor response to radiation and chemotherapy (DOC), suggesting that CpG ODNs have potential to be beneficial when used singly or in combination with other standard treatment modalities such as taxane chemotherapy, radiotherapy or both.

Targeting toll-like receptor 9 with CpG oligodeoxynucleotides enhances tumor response to fractionated radiotherapy.

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs detected by Toll-like receptor 9 of dendritic cells and B cells have potent immunomodulatory effects. CpG oligodeoxynucleotides induce cytokines, activate natural killer cells, and elicit T-cell responses leading to antitumor effects, including improved efficacy of chemotherapeutic agents and, as we reported recently, synergy between CpG oligodeoxynucleotide 1826 and single-dose radiotherapy of an immunogenic mouse fibrosarcoma. The present study extends this finding to the fractionated radiotherapy of the fibrosarcoma tumor and assesses the ability of CpG oligodeoxynucleotide 1826 to increase the radioresponse of a tumor (nonimmunogenic fibrosarcoma). The experiments used a murine immunogenic fibrosarcoma tumor, fibrosarcoma growing in the leg of mice, and response to radiotherapy was assessed by tumor growth delay and tumor cure rate (TCD50, radiation dose yielding 50% tumor cure). Multiple s.c. peritumoral or i.t. administrations of CpG oligodeoxynucleotide 1826 at a dose of 100 microg per mouse were given when established tumors were 6 mm in diameter. Local tumor irradiation was initiated when tumors grew to 8 mm in diameter; radiation was delivered in 1 to 9 Gy fractions given twice daily separated by 6 to 7 hours for 5 consecutive days to achieve a total dose of 10 to 90 Gy. CpG oligodeoxynucleotide 1826, given as a single agent, had only a small antitumor effect, but it dramatically enhanced fibrosarcoma response to radiotherapy. Although 83.1 (79.2-90.0) Gy total dose were needed to achieve tumor cures in 50% of mice treated with radiotherapy alone, only 23.0 (11.5-32.7) Gy total dose were needed in mice treated with both CpG oligodeoxynucleotide 1826 and radiotherapy. The magnitude of potentiation of tumor radioresponse at the TCD50 level was by a factor of 3.61, a much higher value than that (a factor of 1.93) that we reported for single-dose radiotherapy. Mice cured of their tumors by combined CpG oligodeoxynucleotide 1826 plus radiotherapy were highly resistant to s.c. tumor take or development of tumor nodules in the lung from i.v. injected tumor cells when rechallenged with fibrosarcoma cells 100 to 120 days after the treatment, suggesting the development of a memory response. CpG oligodeoxynucleotide 1826 also increased radioresponse of the nonimmunogenic fibrosarcoma tumor by a factor of 1.41 and 1.73 when CpG oligodeoxynucleotide 1826 was given s.c. and i.t., respectively. These findings show that CpG oligodeoxynucleotides are highly potent enhancers of tumor response to both single-dose and fractionated radiation and as such have potential to improve clinical radiotherapy.

CpG oligodeoxynucleotide enhances tumor response to radiation.

CpG oligodeoxynucleotides (ODNs) are synthetic DNA sequences containing unmethylated cytosine-guanine motifs with potent immunomodulatory effects. Via Toll-like receptor 9 agonism of dendritic cells and B cells, CpG ODNs induce cytokines, activate natural killer cells, and elicit vigorous T-cell responses that lead to significant antitumor effects, including improved efficacy of chemotherapeutic agents. On the basis of these properties of CpG ODNs, we tested whether they also could enhance tumor response to radiotherapy. Using an immunogenic mouse tumor, designated FSa, the response to radiotherapy was assayed by tumor growth delay and tumor cure rate (TCD(50), radiation dose yielding 50% tumor cure rate). Treatments were initiated when established tumors were either 6 or 8 mm in diameter. CpG ODN as a single agent given s.c. peritumorally had little effect on tumor growth; however, it dramatically enhanced tumor growth delay in response to single-dose radiation by a factor of 2.58-2.65. CpG ODN also dramatically improved tumor radiocurability, reducing the TCD(50) by a factor of 1.93, from 39.6 (36.1-43.1) Gy to 20.5 (14.3-25.7) Gy. The CpG ODN-induced enhancement of tumor radioresponse was diminished in tumor-bearing mice immunocompromised by sublethal whole-body radiation. Tumors treated with CpG ODN and radiation showed histologic changes characterized by increased necrosis, heavy infiltration by host inflammatory cells (lymphocytes and granulocytes), and reduced tumor cell density. These results show that CpG ODNs are potent enhancers of tumor radioresponse and as such have potential to improve clinical radiotherapy.

The Withers Archive: online availability of H. Rodney Withers' data.

We have collected lab notebooks from Rod Wither's many years of experimentation, from laboratories in Houston and Los Angeles, as well as from several of his collaborators in the USA and overseas. The contents have been digitized, and in this note we explain the mechanism that has been set up to make the 'Withers Archive' available online.

Juvenile Nasopharyngeal Angiofibroma Presenting with Acute Airway Obstruction.

We describe a case of a 24-year-old male presenting urgently with a juvenile nasopharyngeal angiofibroma (JNA) with difficulty breathing, inability to swallow, and respiratory distress following throat swelling. The swelling was reduced with administration of dexamethasone and the JNA was surgically resected within 48 hours. This presentation was atypical given the acuity of presentation and the patient's older age.

Tumor Induction in Mice After Localized Single- or Fractionated-Dose Irradiation: Differences in Tumor Histotype and Genetic Susceptibility Based on Dose Scheduling.

PURPOSE: To investigate differences in tumor histotype, incidence, latency, and strain susceptibility in mice exposed to single-dose or clinically relevant, fractioned-dose γ-ray radiation. METHODS AND MATERIALS: C3Hf/Kam and C57BL/6J mice were locally irradiated to the right hindlimb with either single large doses between 10 and 70 Gy or fractionated doses totaling 40 to 80 Gy delivered at 2-Gy/d fractions, 5 d/wk, for 4 to 8 weeks. The mice were closely evaluated for tumor development in the irradiated field for 800 days after irradiation, and all tumors were characterized histologically. RESULTS: A total of 210 tumors were induced within the radiation field in 788 mice. An overall decrease in tumor incidence was observed after fractionated irradiation (16.4%) in comparison with single-dose irradiation (36.1%). Sarcomas were the predominant postirradiation tumor observed (n=201), with carcinomas occurring less frequently (n=9). The proportion of mice developing tumors increased significantly with total dose for both single-dose and fractionated schedules, and latencies were significantly decreased in mice exposed to larger total doses. C3Hf/Kam mice were more susceptible to tumor induction than C57BL/6J mice after single-dose irradiation; however, significant differences in tumor susceptibilities after fractionated radiation were not observed. For both strains of mice, osteosarcomas and hemangiosarcomas were significantly more common after fractionated irradiation, whereas fibrosarcomas and malignant fibrous histiocytomas were significantly more common after single-dose irradiation. CONCLUSIONS: This study investigated the tumorigenic effect of acute large doses in comparison with fractionated radiation in which both the dose and delivery schedule were similar to those used in clinical radiation therapy. Differences in tumor histotype after single-dose or fractionated radiation exposures provide novel in vivo evidence for differences in tumor susceptibility among stromal cell populations.

Antimetastatic effectiveness of amifostine therapy following surgical removal of Sa-NH tumors in mice.

The effects of dose per fraction on the ability of amifostine exposure to elevate angiostatin levels in the serum of mice and to inhibit spontaneous metastases formation using the well-characterized murine Sa-NH sarcoma were investigated. Amifostine was administered intraperitoneally at doses of 50, 100, or 200 mg/kg every other day for 6 days to C3Hf/Kam mice until tumors reached an average size of 8 mm in diameter. Amifostine was again administered immediately following surgical removal of the tumor-bearing limbs by amputation, and then once more 2 days later. Nontumor-bearing control animals were treated using the same dosing and surgery schedules. The average number of pulmonary metastases per animal was determined for each experimental group. A significant reduction (P <.05) in the average number of pulmonary metastases was observed only in the group of animals exposed to a dose per fraction of 50 mg/kg. A dose of 100 mg/kg was less effective while 200 mg/kg had no effect on metastases formation in this study. The effects of amifostine exposure on serum levels of the angiogenesis inhibitor angiostatin were also determined using Western analysis. Correlating with the antimetastatic effect measured, exposure of animals to 50 mg/kg of amifostine resulted in a four-fold enhanced serum level of angiostatin above control levels. This phenomenon occurred in both tumor-bearing as well as nontumor-bearing animals. In contrast, a dose of 200-mg/kg amifostine administered intraperitoneally under these conditions had no measurable effect on angiostatin serum levels in this animal system. The enhanced ability of relatively low doses of amifostine to inhibit spontaneous metastases formation suggests that effective antimetastatic therapies with amifostine can be designed with minimal toxic side effects. While the dose responses for angiostatin production and metastases inhibition by amifostine are well correlated, the precise mechanism of action underlying these phenomena is unclear but is suggestive of a redox driven process(es).

Poly(L-glutamic acid)-paclitaxel conjugate is a potent enhancer of tumor radiocurability.

PURPOSE: Conjugating drugs with polymeric carriers is one way to improve selective delivery to tumors. Poly (L-glutamic acid)-paclitaxel (PG-TXL) is one such conjugate. Compared with paclitaxel, its uptake, tumor retention, and antitumor efficacy are increased. Initial studies showed that PG-TXL given 24 h before or after radiotherapy enhanced tumor growth delay significantly more than paclitaxel. To determine if PG-TXL-induced enhancement is obtained in a more clinically relevant setting, we investigated PG-TXL effects on tumor cure. METHODS AND MATERIALS: Mice bearing 7-mm-diameter ovarian carcinomas were treated with PG-TXL at an equivalent paclitaxel dose of 80 mg/kg, single dose or 5 daily fractions of radiation or both PG-TXL and radiation. Treatment endpoint was TCD(50) (radiation dose yielding tumor control in 50% of mice). Acute radioresponse of jejunum, skin, and hair was determined for all treatments. RESULTS: PG-TXL dramatically improved tumor radioresponse, reducing TCD(50) of single-dose irradiation from 53.9 (52.2-55.5) Gy to 7.5 (4.5-10.7) Gy, an enhancement factor (EF) of 7.2. The drug improved the efficacy of fractionated irradiation even more, reducing the TCD(50) of 66.6 (62.8-90.4) Gy total fractionated dose to only 7.9 (4.3-11.5) Gy, for an EF of 8.4. PG-TXL did not affect normal tissue radioresponse resulting from either single or fractionated irradiation. CONCLUSION: PG-TXL dramatically potentiated tumor radiocurability after single-dose or fractionated irradiation without affecting acute normal tissue injury. To our knowledge, PG-TXL increased the therapeutic ratio of radiotherapy more than that previously reported for other taxanes, thus, PG-TXL has a high potential to improve clinical radiotherapy.

The epidermal growth factor receptor mediates radioresistance.

PURPOSE: The epidermal growth factor (EGF) receptor is frequently overexpressed in malignant tumors, and its level is correlated with increased cellular resistance to ionizing radiation. However, no precedent studies have investigated whether expression of EGF receptor would by itself confer on cancer cells resistance to radiation. The current study is aimed to address this question. METHODS AND MATERIALS: A full-length human EGF receptor expression vector was transfected into the OCA-I murine ovarian carcinoma cells for stable clones expressing various levels of EGF receptors. Apoptosis and cell clonogenic survival assays were used to evaluate the sensitivity of the resulting cell clones to ionizing radiation. RESULTS: OCA-I cell clones expressing various levels of EGF receptor (OCA-I EGFR) were obtained. These clones showed an EGF receptor level-dependent increase in resistance to ionizing radiation, measured by apoptosis and cell clonogenic survival assays. Compared with the results for parental OCA-I and control vector-transfected OCA-I cells at the 10% cell survival level, the radioresistance was increased by a factor of 1.60 for EGFR-C5 (high level of EGF receptor expression), 1.37 for EGFR-C3 (intermediate level of EGF receptor expression), and 1.28 for EGFR-C1 (low level of EGF receptor expression). Treatment of the OCA-I EGF receptor transfectants with the anti-EGF receptor monoclonal antibody C225 downregulated the levels of EGF receptor, reduced the phosphorylation levels of EGF receptor downstream substrates (such as Akt and MAPK), and reversed the cellular radioresistance. CONCLUSION: Our results demonstrate that overexpression of the EGF receptor conferred cellular resistance to ionizing radiation. The EGF receptor is thus a valid target for potential radiosensitization.

C225 antiepidermal growth factor receptor antibody enhances the efficacy of docetaxel chemoradiotherapy.

PURPOSE: C225 anti-EGFR (epidermal growth factor receptor) antibody has been shown to enhance tumor response to radiation and a number of chemotherapeutic agents. Because of increased use of concurrent chemoradiotherapy in cancer treatment, it is important to determine whether C225 enhances also the antitumor efficacy of radiation when combined with chemotherapy. This study assessed the effect of C225 on tumor response when combined with docetaxel plus single or fractionated radiation. METHODS AND MATERIALS: MDA468 human adenocarcinoma and A431 human epidermoid carcinoma cells growing as xenografts in the right hind leg of nude mice were used. Mice bearing 8-mm tumors were treated with C225 antibody at a dose of 1 mg given i.p. once, twice, or three times 3 days apart, 10 or 30 mg/kg docetaxel given i.v., and/or local tumor irradiation of 8 or 10 Gy single dose or fractionated irradiation consisting of 2 Gy daily for 5 days. When all three agents were combined, C225 was given 6 h before or 18 h after docetaxel, and radiation was given 24 h after docetaxel. The treatment end point was tumor growth delay. RESULTS: C225 enhanced the antitumor efficacy of docetaxel, local tumor irradiation, and docetaxel combined with radiation. The response of both MDA468 and A431 carcinomas was enhanced. The enhancement factors ranged from 1.19 to 8.52, the degree of the enhancement depending on experimental conditions such as administration of multiple vs. single dose C225 or single or fractionated irradiation. C225 given twice or 3 times was more effective than when administered as a single dose. The effect of C225 was more pronounced when combined with single than fractionated irradiation with or without docetaxel. The triple-agent therapy was more effective than a single agent or double combination therapies, expressed by both increased tumor growth delay and the rate of tumor cure. CONCLUSIONS: Our results show that C225 anti-EGFR antibody is a potent enhancer of tumor response to docetaxel or radiation as single agents, and to docetaxel when combined with radiation. Thus, these findings provide strong preclinical evidence in support of combination of anti-EGFR blockade with chemoradiotherapy.

Potentiation of tumor response to radiation or chemoradiation by selective cyclooxygenase-2 enzyme inhibitors.

Cyclooxygenase-2 (COX-2) is an enzyme expressed primarily in pathologic states, such as inflammatory disorders and cancer, where it mediates prostaglandin production. Its overexpression is associated with more aggressive biologic tumor behavior and adverse patient outcome. Increasing evidence shows that agents that selectively inhibit COX-2 enhance tumor response to radiation or chemotherapeutic agents. This article gives an overview of some of this evidence. In addition, we describe new results showing that celecoxib, a selective COX-2 inhibitor, enhanced response of A431 human tumor xenografts in nude mice to radiation by an enhancement factor (EF) of 1.43 and to the chemotherapeutic agent docetaxel by an EF of 2.07. Celecoxib also enhanced tumor response when added to the combined docetaxel plus radiation treatment (EF = 2.13). Further experiments showed that selective COX-2 inhibitors enhanced tumor cell sensitivity to ionizing radiation, involving inhibition of cellular repair from radiation damage and cell cycle redistribution as mechanisms for some cell types. The results show that selective COX-2 inhibitors have the potential to improve tumor radiotherapy or radiochemotherapy, and this therapeutic strategy is currently under clinical testing.

Relationship between cyclin D1 expression and poor radioresponse of murine carcinomas.

PURPOSE: We recently reported that overexpression of epidermal growth factor receptor (EGFR) positively correlated with radioresistance of murine carcinomas. Because cyclin D1 is a downstream sensor of EGFR activation, the present study investigated whether a relationship exists between the extent of cyclin D1 expression and in vivo radiocurability of murine tumors. We further investigated the influence of radiation on cyclin D1 expression and the expression of p27, an inhibitor of the cyclin D1 downstream pathway, as well as the relationship of these molecular determinants to cell proliferation and induced apoptosis in tumors exposed to radiation. METHODS AND MATERIALS: Cyclin D1 expression was assayed in nine carcinomas syngeneic to C3Hf/Kam mice using Western blot analysis. These tumors greatly differed in their radioresponse as assessed by TCD(50). The expression of cyclin D1 and p27 proteins was determined by Western blotting. Cell proliferative activity in tumors was determined by proliferating cell nuclear antigen (PCNA) immunochemistry. The effect of irradiation on the expression of cyclin D1 or p27 proteins and on PCNA positivity was determined in the radiosensitive OCa-I and in the radioresistant SCC-VII tumors. RESULTS: Cyclin D1 expression varied among tumors by 40-fold, and its magnitude positively correlated with poorer tumor radioresponse (higher TCD(50) values). The level of cyclin D1 expression paralleled that of EGFR. A 15-Gy dose reduced constitutive expression of cyclin D1 in the radiosensitive OCa-I tumors, but had no influence on expression of cyclin D1 in the radioresistant SCC-VII tumors. In contrast, 15 Gy increased the expression of p27 in radiosensitive tumors and reduced it in radioresistant tumors. Radiation induced no significant apoptosis or change in the percentage of PCNA-positive (proliferating) cells in SCC-VII tumors with high cyclin D1 levels, but it induced significant apoptosis and a decrease in the percentage of proliferating cells in OCa-I tumors with low cyclin D1 expression. CONCLUSION: Our findings show a positive correlation between cyclin D1 expression and tumor radioresistance. The expression of cyclin D1 and p27 was modified by radiation and was associated with cellular response to radiation, but this depended on the pretreatment level of cyclin D1 expression. These findings may have important clinical implications: The pretreatment assessment of cyclin D1 expression could serve as a useful predictor of radiotherapy outcome and assist in selecting an effective treatment modality.

Flavopiridol increases therapeutic ratio of radiotherapy by preferentially enhancing tumor radioresponse.

PURPOSE: Recently we reported that inhibition of cyclin-dependent kinases (cdks) by flavopiridol enhanced the radiation response of murine ovarian carcinoma cells in culture. The purpose of this investigation was to extend these studies to in vivo tumor models and test whether flavopiridol increases the therapeutic ratio of radiotherapy. METHODS AND MATERIALS: Three transplantable syngeneic mouse tumors were used: mammary carcinoma (MCa-29), ovarian carcinoma (OCa-I), and a lymphoma (Ly-TH). Tumor treatment endpoints included growth delay, cure, and spontaneous lung metastases (OCa-I tumor). The normal tissue endpoint was survival of jejunal crypt cells quantified microscopically. A range of flavopiridol doses from 0.625 to 5.0 mg/kg were given systemically once or twice daily over 5, 10, or 20 days. Combined therapy flavopiridol treatments were initiated either several days before or shortly after the start of single dose or daily fractionated radiotherapy. RESULTS: The major findings of this study are that all three tumors treated with flavopiridol alone responded by tumor growth delay. Two of the tumors (MCa-29 and Ly-TH) responded in a schedule-dependent manner with larger radiation enhancement factors when flavopiridol treatment was started a few hours after irradiation (radioenhancement factors [EF] Ly-TH = 2.04, EF MCa-29 = 1.50 for single dose irradiation). When combined with fractionated irradiation (2.6 Gy daily for 10 or 20 days), flavopiridol enhanced the response of the MCa-29 tumor by a factor of 1.25-1.46. A fractional radiation dose of 6 Gy in combination with flavopiridol produced a 62.5% cure rate compared with 25% tumor cure for radiation alone. A novel finding of this study was the demonstration of antimetastatic activity of flavopiridol in addition to its effect on the local primary tumor. Both the incidence and absolute number of lung metastasis were reduced when flavopiridol followed surgical removal of the large (10 mm) primary leg tumor. The normal jejunum treated with flavopiridol and radiation responded in a schedule independent manner and the degree of radioenhancement (EF, 1.05-1.06) was much less than for any of the tumors studied. CONCLUSIONS: Therapeutic gain was achieved when flavopiridol treatment was initiated either before or after the start of radiotherapy. Flavopiridol shows promising clinical potential administered alone or in combination with other cytotoxic agents, including both chemotherapy and radiotherapy.

Mitigation and treatment of radiation-induced thoracic injury with a cyclooxygenase-2 inhibitor, celecoxib.

PURPOSE: To test whether a cyclooxygenase-2 inhibitor (celecoxib) could reduce mortality resulting from radiation-induced pneumonitis. METHODS AND MATERIALS: Celecoxib was given to mice twice daily for 40 consecutive days starting on the day of local thoracic irradiation (LTI) or 40 or 80 days later. C3Hf/KamLaw mice were observed for morbidity, and time to death was determined. Results were analyzed using the Cox proportional hazards model. RESULTS: Timing of celecoxib relative to LTI determined efficacy. A significant reduction in time to death was achieved only when celecoxib was started 80 days after LTI, corresponding to the time when pneumonitis is expressed. For these mice the reduction in mortality was quantified as a hazard ratio for mortality of treated vs untreated of 0.36 (95% confidence interval [CI] 0.24-0.53), thus significantly less than 1.0. Correspondingly, the median lethal dose for treated mice (12.9 Gy; 95% CI 12.55-13.25 Gy) was significantly (P=.026) higher than for untreated mice (12.4 Gy; 95% CI 12.2-12.65 Gy). CONCLUSIONS: Celecoxib significantly reduced lung toxicity when administered months after LTI when the deleterious effects of radiation were expressed. The schedule-dependent reduction in fatal pneumonitis suggests that celecoxib could be clinically useful by reintroduction of treatment months after completion of radiation therapy. These findings may be important for designing clinical trials using cyclooxygenase-2 inhibitors to treat radiation-induced lung toxicity as a complement to concurrent radiation therapy of lung cancers.

CpG plus radiotherapy: a review of preclinical works leading to clinical trial.

Studies performed three decades ago in our laboratory supported the hypothesis that radiation efficacy may be augmented by bacterial extracts that stimulate non-specific systemic antitumor immune responses. Application to the clinic was halted by unacceptable side effects and toxicities resulting from exposure to whole bacterial pathogens. Later scientific advances demonstrated that DNA isolated from bacteria was immunostimulatory and could be reproduced with synthetic oligodeoxynucleotides (ODNs), thus fueling the transition from bugs to drugs. Unmethylated CpG motifs within bacterial DNA induce activation of Toll-like receptor 9 and subsequently activate antigen-specific cellular immune responses. CpG ODNs have demonstrated favorable toxicity profiles in phase I clinical trials. We showed that this potent immunoadjuvant can be used in combination with radiation therapy to enhance local and systemic responses of several murine tumors. Studies demonstrated that enhanced tumor response is mediated in part by the host immune system. Antitumor efficacy was diminished in immunocompromised mice. Animals cured by combination of radiation and CpG ODN were resistant to subsequent tumor rechallenge. This body of work contributes to our understanding of the dynamic interplay between tumor irradiation and the host immune system and may facilitate translation to clinical trials.

INO-1001, a novel inhibitor of poly(ADP-ribose) polymerase, enhances tumor response to doxorubicin.

Poly(ADP-ribose) synthetase inhibitor, INO-1001, is known to sensitize cells to radiation in vitro by inhibiting the repair of DNA damage. Recent evidence has suggested that PARP inhibition may also be a way of selectively targeting p53 deficient cancer cells. The present study tested INO-1001 for its in vivo effect on the chemoresponse of two p53 deficient tumors, human breast cancer MDA-MB-231 and murine mammary carcinoma MCa-K. Doxorubicin was used as the DNA damaging agent and tumor growth delay assay was used as the endpoint. Results showed that INO-1001 was highly effective in enhancing the anti-tumor effects of Doxorubicin for both MDA-MB-231 (EF=1.88) and MCa-K (EF=1.64). We conclude that PARP inhibitor INO-1001 has high potential for enhancing the anti-tumor effects of chemotherapy agents such as Doxorubicin against p53 deficient breast cancer.