CPA7, a Novel Stat3 Inhibitor Radiosensitizes Prostate Cancer Cells

Proceedings of the 47th Annual ASTRO Meeting

survival in Ugi-expressing U251 and HT29 cells as compared with empty vector controls. During drug treatment, the former cells incorporate and retain dUMP in genomic DNA. These findings support the suggestion that stable incorporation of deoxyuridine monophosphate into mammalian DNA is at least as toxic to the cells as is ineffective U-BER. In marked contrast to these results, we show that Ugi expression reduces the ability of FUdR to potentiate the lethality of ionizing radiation exposures in the engineered U251 and HT29 cells. We propose a model in which repair of radiation-induced DNA DSB by the HR pathway is aborted by U-BER action on uracil deoxynucleotides incorporated during the strand elongation step. Inhibiting U-BER, we suggest, allows for the completion of DSB repair by HR through synthesis of stable, uracil DNA-containing repair “patches”. In support of this view, we show that FUdR does not sensitize radiation lethality to the usual degree in cells engineered to be deficient in HR--an example of “pharmacological-genetic epistasis”. Conclusions: Design or discovery of small molecule inhibitors of U-BER is a rational approach to improve the anti-tumor activity of FUdR and related agents. The different molecular basis for cell killing when uracil deoxynucleotides are stably incorporated into genomic DNA, as opposed to when U-BER is proficient, may lead to a novel spectrum of clinical activity, and co-administration of a U-BER inhibitor may restore sensitivity to killing for certain tumors that have developed resistance to FUdR exposure alone. We also suggest that HR is a rational target for the development of novel radiosensitizing agents for clinical use.

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CPA7, a Novel Stat3 Inhibitor Radiosensitizes Prostate Cancer Cells

M. Sekharam,1 J.F. Torres-Roca,1 J. Turkson,1 H. Kay,3 S. Zhang,1 R. Jove,1 D.P. Calvin2 Interdisciplinary Oncology, H Lee Moffitt Cancer Center, Tampa, FL, 2Radiation Oncology Program, Lakeland Regional Cancer Center, Lakeland, FL, 3College of Public Health, University of South Florida, Tampa, FL

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Purpose/Objective: In previous studies we described CPA7, a platinum-containing compound, as a novel inhibitor of Stat3. CPA7 inhibited cell growth and induced apoptosis exclusively in malignant cells that expressed activated Stat3. Since CPA7 is a platinum derivative (platinum IV complex), we hypothesized that it could represent a novel radiosensitizer. Materials/Methods: DU145 and LNCaP prostate cancer cells were used in this study. DU145 cells express constitutive activation of Stat3, while LnCap cell do not. In radiosensitization experiments, cells were treated with several concentrations of CPA7 (1–5 micromolar), 4 hours prior to the delivery of ionizing radiation (IR). Cytotoxicity was measured with four different approaches: clonogenic assay, Trypan Blue exclusion, MTT and TUNEL. Stat3 nuclear binding activity was determined using electrophoretic mobility shift assay (EMSA). Survivin, Bcl-XL and Mcl-1 levels were determined by western immunoblot assay. Results: CPA7 induced a dose-dependent cytotoxic effect in DU145 but not LNCaP prostate cancer cells. We performed radiosensitizing experiments in DU145 cells. Cells treated with CPA7 (2 micromolar) were radiosensitized when compared to untreated cells (SF2 CPA7 ⫹ IR vs. IR alone, 0.52 vs. 0.74). CPA7 treatment increased the baseline apoptotic rate to 6.6% of cells at 24 hours, compared to 2% of cells that were untreated or treated with IR alone. However when radiation and CPA7 were combined, 15.7% of cells were found apoptotic at 24 hours, indicative of a synergistic effect. Similar cell viability at 48 hours was determined in cells treated with CPA 7 (85%) or IR alone (91%) when compared to untreated controls (90%). In contrast combined treatment with CPA 7 and IR resulted in 63% viability at 48 hours, also consistent with a synergistic effect. Since Stat3 has been shown to directly bind and regulate the survivin promoter, we sought to determine whether survivin levels were being altered in our experiments. Indeed, CPA7 induced down-regulation of survivin, 24 hours after treatment. Interestingly, survivin expression levels returned to baseline at 48 hours. Furthermore, we determined whether other downstream effectors in the Stat3 pathway were affected by CPA7. Bcl-XL and Mcl-1, two anti-apoptotic proteins were both downregulated in CPA7 treated cells after 48 hours of incubation. Conclusions: CPA7, a platinum IV complex compound, induced cytotoxicity and radiosensitization of DU145 but not of LNCaP cells. A synergistic effect between CPA7 and ionizing radiation was demonstrated by SF2, apoptosis and cellular viability determination. CPA7 induced downregulation of survivin, Bcl-XL and Mcl-1, which are all downstream effectors of the Stat3 pathway. These data, taken together suggest that CPA 7 may be inducing its radiosensitizing effect by inhibiting the Stat3 signaling pathway.

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Assessment of Early Therapeutic Effects on VX2 Rabbit Carcinoma Using FDG-microPET

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K. Ishii, M. Hosono,1 Y. Wada,2 S. Kondo,1 Y. Takada,1 T. Tada,1 T. Okamura,1 M. Maeda,3 Y. Watanabe,2 Y. Inoue1 Radiology, Osaka City University Graduate School of Medicine, Osaka, Japan, 2Physiology, Osaka City University Graduate School of Medicine, Osaka, Japan, 3Anatomy, Osaka City University Graduate School of Medicine, Osaka, Japan

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Purpose/Objective: The aim of this study was to determine the potential role of FDG-PET for evaluating the prediction for primary effects of radiotherapy and/or hyperthermia on tumor bearing rabbits using high resolution FDG-microPET. Materials/Methods: Twenty-eight VX2 xenografts in the thigh of rabbits were divided into five groups; radiotherapy at a single doses of 10, 20 and 30 Gy, hyperthermia (43 degrees C, 1 hour) and combination of radiotherapy and hyperthermia (10 Gy ⫹ 43 degrees C, 1 hour). Primary effect was estimated by comparing the initial tumor volume with that at 7 days after treatment. PET images were obtained for 120 min after the FDG injection using microPET P4 system at pretreatment, 24 hours and 7 days after treatment. For the quantitative evaluation of FDG-PET, tumor/muscle (T/M) ratios, retention index [RI⫽(T/M ratio at 120 min ⫺ T/M ratio at 60 min ) / T/M ratio at 60 min]] and time activity curve (TAC) were acquired. Results: T/M ratio of the various treatments changed dose-dependently compared to untreated tumor. In order to clarify the relationship between early treatment effect and FDG uptake, the xenografts were divided into responder group (PR⫹NC group, n⫽14) and non-responder group (PD group, n⫽14). NC was considered to be responder, because tumor growth of untreated tumor was extremely rapid. In responder group, T/M ratio was 5.0⫾1.3, and 6.8⫾1.9 at 24 hours and 7 days after treatment, respectively. In non-responder group, T/M ratio was 8.0⫾3.0, and 8.8⫾2.5 at each time point. T/M ratio in responder group

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