Radiation-induced Growth Delay Research Articles

Inhibition of the Translation Initiation Factor eIF4A Enhances Tumor Cell Radiosensitivity.

A fundamental component of cellular radioresponse is the translational control of gene expression. Because a critical regulator of translational control is the eukaryotic translation initiation factor 4F (eIF4F) cap binding complex, we investigated whether eIF4A, the RNA helicase component of eIF4F, can serve as a target for radiosensitization. Knockdown of eIF4A using siRNA reduced translational efficiency, as determined from polysome profiles, and enhanced tumor cell radiosensitivity as determined by clonogenic survival. The increased radiosensitivity was accompanied by a delayed dispersion of radiation-induced γH2AX foci, suggestive of an inhibition of DNA double-strand break repair. Studies were then extended to (-)-SDS-1-021, a pharmacologic inhibitor of eIF4A. Treatment of cells with the rocaglate (-)-SDS-1-021 resulted in a decrease in translational efficiency as well as protein synthesis. (-)-SDS-1-021 treatment also enhanced the radiosensitivity of tumor cell lines. This (-)-SDS-1-021-induced radiosensitization was accompanied by a delay in radiation-induced γH2AX foci dispersal, consistent with a causative role for the inhibition of double-strand break repair. In contrast, although (-)-SDS-1-021 inhibited translation and protein synthesis in a normal fibroblast cell line, it had no effect on radiosensitivity of normal cells. Subcutaneous xenografts were then used to evaluate the in vivo response to (-)-SDS-1-021 and radiation. Treatment of mice bearing subcutaneous xenografts with (-)-SDS-1-021 decreased tumor translational efficiency as determined by polysome profiles. Although (-)-SDS-1-021 treatment alone had no effect on tumor growth, it significantly enhanced the radiation-induced growth delay. These results suggest that eIF4A is a tumor-selective target for radiosensitization.

Combination of anthracyclines and anti-CD47 therapy inhibit invasive breast cancer growth while preventing cardiac toxicity by regulation of autophagy.

A perennial challenge in systemic cytotoxic cancer therapy is to eradicate primary tumors and metastatic disease while sparing normal tissue from off-target effects of chemotherapy. Anthracyclines such as doxorubicin are effective chemotherapeutic agents for which dosing is limited by development of cardiotoxicity. Our published evidence shows that targeting CD47 enhances radiation-induced growth delay of tumors while remarkably protecting soft tissues. The protection of cell viability observed with CD47 is mediated autonomously by activation of protective autophagy. However, whether CD47 protects cancer cells from cytotoxic chemotherapy is unknown. We tested the effect of CD47 blockade on cancer cell survival using a 2-dimensional high-throughput cell proliferation assay in 4T1 breast cancer cell lines. To evaluate blockade of CD47 in combination with chemotherapy in vivo, we employed the 4T1 breast cancer model and examined tumor and cardiac tissue viability as well as autophagic flux. Our high-throughput screen revealed that blockade of CD47 does not interfere with the cytotoxic activity of anthracyclines against 4T1 breast cancer cells. Targeting CD47 enhanced the effect of doxorubicin chemotherapy in vivo by reducing tumor growth and metastatic spread by activation of an anti-tumor innate immune response. Moreover, systemic suppression of CD47 protected cardiac tissue viability and function in mice treated with doxorubicin. Our experiments indicate that the protective effects observed with CD47 blockade are mediated through upregulation of autophagic flux. However, the absence of CD47 in did not elicit a protective effect in cancer cells,but it enhanced macrophage-mediated cancer cell cytolysis. Therefore, the differential responses observed with CD47 blockade are due to autonomous activation of protective autophagy in normal tissue and enhancement immune cytotoxicity against cancer cells.

Abstract 3785: Olaparib increases the effectiveness of radiation in hypoxic tumor cells in xenograft models of human non-small-cell lung cancer

Abstract Purpose: Poly (ADP-ribose) polymerase (PARP) detects DNA single strand breaks and facilitates their repair. Recently, PARP inhibitors have been shown to potentiate radiotherapy in mouse models of human non-small-cell lung cancer (NSCLC) by compromising DNA repair and increasing tumor vascular perfusion. This study examined radiosensitization by PARP inhibition in xenograft models of NSCLC with differing levels of hypoxia. Methods: For in vitro studies, human NSCLC cell lines Calu-6 and Calu-3 were treated with olaparib (5 µM) or vehicle prior to ionizing radiation (IR). Cytotoxicity was evaluated by clonogenic survival assays and DNA damage repair was assessed by γH2AX foci immunofluorescence staining. For in vivo efficacy studies, Calu-6 or Calu-3 subcutaneous xenografts (100 mm3) were established in 6-8 week old female BALB/c nude mice. Animals (n=48) were then randomized into four groups (i) control, (ii) olaparib (50 mg/kg, d1-3), (iii) IR (10 Gy, d1) and (iv) combination treatment. Subsequent tumor growth was monitored for up to 42 days. In parallel, for pharmacodynamic studies, tumor bearing animals were sacrificed 24h and 72h after treatment (n=3 for each treatment and time point). Tumor sections were evaluated by immunohistochemistry (IHC). Results: In vitro, both NSCLC cell lines were significantly radiosensitized by olaparib. Sensitization enhancement ratios were 1.9 (Calu-6) and 2.2 (Calu-3). Furthermore, olaparib markedly inhibited DNA damage repair in both Calu-6 and Calu-3 cells 24h post-IR (p<0.01). Growth of Calu-6 and Calu-3 xenografts was reduced by IR alone, but unaffected by olaparib treatment alone. Surprisingly, although olaparib significantly increased radiation-induced growth delay in Calu-6 xenografts (p<0.01), there was no significant effect in Calu-3 xenografts, in contrast to the in vitro data. IHC staining for vascularity (CD31) and hypoxia (CA9) showed that Calu-3 xenografts were well-oxygenated without evidence of necrotic tissue, whereas Calu-6 xenografts had large areas of hypoxia surrounding regions of necrosis. RAD51 expression (a protein involved in homologous recombination, HR) was selectively down-regulated in hypoxic regions of Calu-6 tumors. 24h and 72h after IR, olaparib treated Calu-6 xenografts displayed significantly higher levels of γH2AX (DNA double strand breaks) and cleaved caspase 3 (apoptosis) staining in CA9 positive (hypoxic) cells compared with the hypoxic regions in xenografts treated with IR alone. Conclusion: Our data identify a novel mechanism that contributes to radiosensitization by PARP inhibition in NSCLC. Olaparib selectively increased DNA damage and apoptosis in radioresistant hypoxic tumor cells with decreased HR potential. Our data also suggest that tumor hypoxia may be a potential biomarker of differential benefit from the combination of olaparib with radiotherapy. Citation Format: Yanyan Jiang, Tom Verbiest, Aoife M. Devery, Sivan M. Bokobza, Anika M. Weber, Anderson J. Ryan. Olaparib increases the effectiveness of radiation in hypoxic tumor cells in xenograft models of human non-small-cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3785. doi:10.1158/1538-7445.AM2014-3785

Abstract 2434: Therapeutic targeting of CD47 regulates cell bioenergetics and autophagy to reduce breast tumor growth and protect against anthracycline-mediated cardiac toxicity

Abstract Even though, molecular profiling of breast cancer has allowed for the development of new therapeutic drugs, cytotoxic chemotherapy remains a primary mode of therapy for many breast cancer patients including those diagnosed with triple negative breast cancer. Pre-clinical and clinical studies demonstrate that increased expression of CD47 is associated with poor prognosis in several types of cancer, including breast tumors. Our published evidence shows that blockade of CD47 enhances radiation-induced growth delay of tumors while remarkably protecting soft tissues from death associated to ionizing radiation. We now show that CD47 blockade significantly sensitizes breast tumors to anthracycline chemotherapy while protecting cardiac tissue from the off target effects of this drug. 4T1B breast cancer cells were implanted in the mammary fat-pad of Balb/C mice and treated with saline, doxorubicin, CD47 morpholino (CD47M), or CD47M with doxorubicin. Tumors of mice treated with saline tripled in size. Doxorubicin treatment caused a reduction in tumor volume and weight, but combining CD47 blockade and doxorubicin further reduced tumor volume by over 60%. The reduction of tumor growth was associated with an increased in PINK1 and Parkin gene expression, indicating activation of mitochondrial turnover by mitophagy. Further studies using the Seahorse XF-24 analyzer indicated that blockade of CD47 reduced glycolytic and mitochondrial metabolism measured as the extracellular acidification rate and oxygen consumption rate in 4T1B cells and human MDA-MB-231 cells. The regulation of glycolytic mechanism is associated with a reduction in glucose uptake and reduction in Glut-1 expression in breast tumors. Metastatic spread of tumors to lungs was observed in saline treated animals. Immunohistological analysis indicated that these lesions express CD47. Moreover, we observed positive immunoreactivity to CD47 in clinical samples of metastatic breast carcinoma indicating that CD47 signaling may play an important role in tumor spread and its targeting could lead to reduction of metastatic burden. One of the most common side effects of doxorubicin chemotherapy is cardiac toxicity. Blockade of CD47 in tumor bearing mice protected cardiac tissue indicated by reduction in fibrosis and cell death. This was associated by an increase in autophagy gene expression as demonstrated by the observed increase in ATG5 and ATG7. Therefore blockade CD47 enhances doxorubicin reduction of breast tumor growth in a syngeneic tumor model indicating that CD47 potentiates anthracycline-mediated breast tumor therapy while protecting normal tissue from death associated with cytotoxic therapy. Citation Format: David R. Soto-Pantoja, John M. Sipes, Arunima Ghosh, Maria J. Merino, David D. Roberts. Therapeutic targeting of CD47 regulates cell bioenergetics and autophagy to reduce breast tumor growth and protect against anthracycline-mediated cardiac toxicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2434. doi:10.1158/1538-7445.AM2014-2434

Abstract C219: Novel MET/FGFR/AXL kinase inhibitor S49076 exerts radiosensitizing activity in vitro and in vivo.

Abstract Background: S49076 was developed as an oral ATP-competitive inhibitor of MET receptor and of other kinases involved in tumor development. It is being assessed as single agent in phase I study. We examined its potential as a radiosensitizer in non-MET dependent tumor models. Experimental Procedures: Cell viability and clonogenic survival assays were performed in two non small-cell lung carcinoma (NSCLC) cells (H460 and A549) and in breast cancer cells MDA-MB-231. Then, the drug was tested in combination with ionizing irradiation (IR) in vitro. The combination was assessed in subcutaneous tumor xenografts and in NSCLC orthotopic models of luciferine-expressing H460luc and A549luc tumors. Immunohistochemical correlates were performed and the activity of S49076 was assessed in a lung metastases model of intravenous injections of A549 cells. Results: The drug as single agent inhibited proliferation (IC50 ranging from 0.50 μM to 1.1 μM) and clonogenic survival (IC50 ranging from 0.34 μM to 0.86 μM). Transcriptomic analyses showed that antitumor effect was associated with modulation of molecular pathways involved in radiation response (p53 and ATM). S49076 and IR inhibited synergistically clonogenic survival in H460 cells: sensitivity enhancement ratio at 0.6 μM was 2 for a single radiation dose of 2 Gy. Combination was additive in A549 and MDA-MB-231 cells. S49076 at 50 mg/kg twice daily enhanced radiation-induced growth delay in subcutaneous tumor xenografts, accompanied with an increase in TUNEL staining. Dose enhancement factors were 3.2 and 1.6 in H460 and A549 tumors, respectively. In lung orthotopic models, S49076 generated significant delay in time course of bioluminescent signal and improved median estimated survival, with significant benefit of combination over either IR or S49076 alone. Finally, treatment with S49076 improved significantly median survival in the lung metastases model (14 versus 22 days, p < 0.001). Conclusions: Altogether, these data suggest that S49076 has a potential as radiosensitizer in non MET-dependent cell lines, and that this effect is accompanied with a modulation of apoptosis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C219. Citation Format: Cyrus Chargari, Céline Clémenson, Michele Mondini, Charles Ferte, Anne Jacquet-Bescond, Valérie Cattan, Stéphane Depil, Eric Deutsch. Novel MET/FGFR/AXL kinase inhibitor S49076 exerts radiosensitizing activity in vitro and in vivo. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C219.

Effects of ionizing radiation in combination with Erufosine on T98G glioblastoma xenograft tumours: a study in NMRI nu/nu mice

BackgroundErufosine is a promising anticancer drug that increases the efficacy of radiotherapy in glioblastoma cell lines in vitro. Moreover, treatment of nude mice with repeated intraperitoneal or subcutaneous injections of Erufosine is well tolerated and yields drug concentrations in the brain tissue that are higher than the concentrations required for cytotoxic drug effects on glioblastoma cell lines in vitro.MethodsIn the present study we aimed to evaluate the effects of a combined treatment with radiotherapy and Erufosine on growth and local control of T98G subcutaneous glioblastoma xenograft-tumours in NMRI nu/nu mice.ResultsWe show that repeated intraperitoneal injections of Erufosine resulted in a significant drug accumulation in T98G xenograft tumours on NMRI nu/nu mice. Moreover, short-term treatment with 5 intraperitoneal Erufosine injections caused a transient decrease in the growth of T98G tumours without radiotherapy. Furthermore, an increased radiation-induced growth delay of T98G xenograft tumours was observed when fractionated irradiation was combined with short-term Erufosine-treatment. However, no beneficial drug effects on fractionated radiotherapy in terms of local tumour control were observed.ConclusionsWe conclude that short-term treatment with Erufosine is not sufficient to significantly improve local control in combination with radiotherapy in T98G glioblastoma xenograft tumours. Further studies are needed to evaluate efficacy of extended drug treatment schedules.

Metformin kills and radiosensitizes cancer cells and preferentially kills cancer stem cells

The anti-cancer effects of metformin, the most widely used drug for type 2 diabetes, alone or in combination with ionizing radiation were studied with MCF-7 human breast cancer cells and FSaII mouse fibrosarcoma cells. Clinically achievable concentrations of metformin caused significant clonogenic death in cancer cells. Importantly, metformin was preferentially cytotoxic to cancer stem cells relative to non-cancer stem cells. Metformin increased the radiosensitivity of cancer cells in vitro, and significantly enhanced the radiation-induced growth delay of FSaII tumors (s.c.) in the legs of C3H mice. Both metformin and ionizing radiation activated AMPK leading to inactivation of mTOR and suppression of its downstream effectors such as S6K1 and 4EBP1, a crucial signaling pathway for proliferation and survival of cancer cells, in vitro as well as in the in vivo tumors. Conclusion: Metformin kills and radiosensitizes cancer cells and eradicates radioresistant cancer stem cells by activating AMPK and suppressing mTOR.

Proteomics of the Radioresistant Phenotype in Head-and-Neck Cancer: Gp96 as a Novel Prediction Marker and Sensitizing Target for Radiotherapy

Radiotherapy is an integral part of the treatment modality for head-neck cancer (HNC), but in some cases the disease is radioresistant. We designed this study to identify molecules that may be involved in this resistance. Two radioresistant sublines were established by fractionated irradiation of the HNC cell lines, to determine differentially proteins between parental and radioresistant cells. Proteomic analysis and reverse-transcription polymerase chain reaction were used to identify and confirm the differential proteins. The siRNA knockdown experiments were applied to examine cellular functions of a radioresistant gene, with investigation of the alterations in colonogenic survival, cell cycle status, and reactive oxygen species levels. Xenografted mouse tumors were studied to validate the results. IN all, 64 proteins were identified as being potentially associated with radioresistance, which are involved in several cellular pathways, including regulation of stimulus response, cell apoptosis, and glycolysis. Six genes were confirmed to be differentially expressed in both radioresistant sublines, with Gp96, Grp78, HSP60, Rab40B, and GDF-15 upregulated, and annexin V downregulated. Gp96 was further investigated for its functions in response to radiation. Gp96-siRNA transfectants displayed a radiation-induced growth delay, reduction in colonogenic survival, increased cellular reactive oxygen species levels, and increased proportion of the cells in the G2/M phase. Xenograft mice administered Gp96-siRNA showed significantly enhanced growth suppression in comparison with radiation treatment alone (p = 0.009). We identified 64 proteins and verified 6 genes that are potentially involved in the radioresistant phenotype. We further demonstrated that Gp96 knockdown enhances radiosensitivity both in cells and in vivo, which may lead to a better prognosis of HNC treatment.

Abstract 488: cSrc is a radioresistance molecule in cancer radiotherapy: cSrc-siRNA transfection augments tumor cell radiosensitivity in vitro and chemical inhibition of Src kinases increases tumor response to radiation

Abstract Background: cSrc is often over-expressed in cancer cells including head and neck squamous cell carcinoma (HNSCC) and is implicated in promoting cell survival, proliferation, angiogenesis, cell adhesion and motility. Its role in tumor resistance to cytotoxic agents, such as radiation, is less clear. Present study investigated (1) the relationship between the level of activated cSrc expression and the degree of cell radioresistance and (2) the role of cSrc in the cellular response to radiation, using both in vitro and in vivo models. Materials and methods: Ten HNSCC lines (FaDu, HN-5, UMSCC-1, HN-30, MDA1386, UMSCC-17B, UMSCC-47, Sqccy-1, SN2 and PCI15B) were assessed for their sensitivity to ionizing radiation by clonogenic survival and for expression levels of active Src kinases using phospho-kinase antibody array kit (Proteome ProfilerTM, R&D Systems, Inc., Minneapolis, MN). cSrc expression levels were validated by Western blot. cSrc-siRNA was transfected by electroporation to suppress cSrc expression, after which the cells were assessed for radiosensitivity. We also tested the effect of dasatinib, an inhibitor of Src kinases, on in vitro cell radiosensitivity, and on the growth of tumor xenografts in nude mice. Results: HNSCC lines exhibited a wide range of both radiosensitivity and levels of phosphorylated Src family kinases. There was a highly positive correlation (r=0.883) between SF2 values (cell survival at 2 Gy) and the degree of basal phosphorylated cSrc kinase expression. As the latter increased the ability of cells to survive radiation, increased. Inhibition of cSrc expression by cSrc-siRNA in 2 cell lines tested (FaDu and UMSCC1) resulted in a significant increase in their radiosensitivity, suggesting a causal relationship between cSrc and cell radioresistance. Inhibition of cSrc by dasatinib also resulted in increase of radiosensitivity of 3 cell lines tested (FaDu, HN-5, UMSCC-1). Additionally, dasatinib enhanced in vivo tumor response to radiation as shown by a marked increase in radiation-induced growth delay of FaDu xenografts. Conclusions: Our data showed that (1) a strong correlation exists between the expression level of cSrc and cell radiosensitivity of HNSCCs, (2) inhibition of cSrc expression by siRNA can render tumor cells more radiosensitive, implicating a causal relationship between cSrc and radioresistance, and (3) dasatinib enhanced both the in vitro tumor cell radiosensitivity and in vivo tumor response to radiation. These results suggest that targeting cSrc signaling network may have potential to improve the efficacy of radiotherapy. Supported in part by Bristol-Myers Squibb Company Sponsored Agreement (PI: U. Raju), NIH PO-1 Grant CA-06294 (PI: KK. Ang) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 488.

Synthesis and Evaluation of Nicotinoylamino Acid Compounds as Radiosensitizer

Abstract Nicotinoylamino acid compounds 4, 5, 9a, 9b, 9c, 10a and 10b were synthesized with nicotinoyl chloride or nicotinoyl azide as acetylating agents of amino acid esters or amino acids. The compounds were tested for their radiosensitizing activity in Leukemia cell line(L1210) and compared with nicotinamide; among them, compounds 9a and 9c showed significant radiosensitizing effects, the sensitizer enhancement ratio(SER) was 1.64 and 1.58, respectively, while nicotinamide did not show good radiosensitizing effect under the same conditions. Compound 9c was alone tested for radiosensitization in LA 795 cell-bearing T-739 mice, or hyperthermia and breathing carbogen(5%CO2+95%O2) were together tested for radiosensitization. The results showed that radiation-induced growth delay was enhanced by 9c alone or by the combination of hypertheimia and carbogen. The tumor-bearing mice were irradiated locally by total 10 Gy, and the tumors grew to three times that of the original volume in an average of 5.8 d. The mice were given i.p. compound 9c at 1000 mg/kg 60 min before irradiation and treated at 43 °C for 30 min after irradiation or treated with breathing carbogen for 5 min before radiation or with hyperthmia(43 °C) for 30 min after irradiation; the time required for the tumor to grow to three times the orginal volume was in an average of 12.9 and 13 d, respectively.

Differentially expressed genes in radioresistant nasopharyngeal cancer cells: gp96 and GDF15

Radiotherapy is the major treatment modality for nasopharyngeal cancer (NPC), but in some cases, the disease is radioresistant. We designed this study to identify genes that may be involved in this resistance. We first established two radioresistant subclone cell lines derived from NPC parental cell lines (NPC-076 and NPC-BM1) by treating the cells with four rounds of sublethal ionizing radiation. cDNA microarray analysis was then done, comparing the two resistant cell lines with their corresponding parental cell lines. Seven genes were found to be up-regulated in radioresistant subclones, including gp96 and GDF15, which had shown highest overexpressions. We constructed small interfering RNA plasmids (gp96si and GDF15si) and transfected them into NPC cells to knock down these genes and examine whether this changed their response to radiation. Both gp96si and GDF15si transfectants had radiation-induced growth delay and reduction in colonogenic survival compared with control cells. Knockdown of either gp96 or GDF15 increased the proportion of the cells in G(2)-M phase, the most radiosensitive phase of the cell cycle. We have therefore identified at least two genes, gp96 and GDF15, involved in radioresistance of NPC cell lines and showed that knockdown of the genes enhances radiosensitivity.

Chemosensitization and radiosensitization of human lung and colon cancers by antimitotic agent, ABT-751, in athymic murine xenograft models of subcutaneous tumor growth

ABT-751 is an orally active antimitotic agent that is currently in Phase II clinical trials. This agent binds to the colchicine site on ss-tubulin and inhibits polymerization of microtubules. This disruption of microtubule dynamics leads to a block in the cell cycle at the G2/M phase, and promotes apoptosis. ABT-751, as a single agent, has antitumor activity against a series of xenograft models including non-small cell lung cancer (NSCLC) and colon cancer. The current studies were conducted to determine whether ABT-751 enhances antitumor activity of standard cytotoxic therapies currently in clinical use. Efficacy of ABT-751, in combination with cisplatin, 5-FU, and radiation, was evaluated in the Calu-6 NSCLC, HT-29 colon, and HCT-116 colon carcinoma xenograft models, respectively. Tumor-bearing athymic mice were treated with ABT-751 orally once a day at 75 or 100 mg/kg/day on a 5-days-on, 5-days-off schedule for two cycles. Efficacy of ABT-751 at 100 mg/kg/day was tested in combination with cisplatin at its maximum tolerable dose (MTD) (10 mg/kg/day, i.p. x1) in Calu-6 tumor-bearing athymic mice. The percent treated/control (%T/C) tumor volume ratios on day 38 were 35, 37, and 6, and the percent tumor growth delay (%TGD) values were 71, 65, and 188 for cisplatin, ABT-751 and the combination groups, respectively. HT-29 colon tumors were used to test ABT-751 in combination with an MTD of 5-FU, 30 mg/kg/day, i.p., q.d. x5. The %T/C ratios on day 38 were 22, 28, and 5 and the %TGD values were 75, 75, and 150 for 5-FU, ABT-751, and the combination groups, respectively. Treatment of HCT-116 colon carcinoma tumors with ABT-751, concurrent with the radiation treatment, was able to both enhance radiation-induced tumor regression, and delay the time to recurrence and progression. Growth curves allowed calculation of enhancement of radiation-induced growth delay (defined as the additional time required for a treated tumor to reach four times its original size) of 2, 9, and 12 days, for ABT-751 alone, radiation alone, and the combination, respectively. Collectively, these studies demonstrate that ABT-751 enhanced efficacy of standard cytotoxic therapies in a variety of tumor xenograft models, and that enhancement was at least additive in all systems.

Radiosensitization of tumor cells by modulation of ATM kinase

Purpose: To elucidate the relationship between the radiation-induced activation of ataxia telangiectasia mutated (ATM) kinase, G2 arrest and the caffeine-induced radiosensitization.Method: RKO cells (human colorectal cancer cells) and ATM kinase over-expressing RKO/ATM cells were used. The cellular radiosensitivity was determined with clonogenic survival assay and the cell cycle progression, including G2 arrest, was studied with flow cytometry. The activity of ATM kinase, check point 2 (Chk2) kinase and cycline B1/cell division cycle 2 (Cdc2) kinase was investigated. The radiosensitivity of RKO xenografts grown in nude mice was studied.Results: RKO/ATM cells were radioresistant as compared with RKO cells. There was a greater increase in ATM kinase activity and G2 arrest in RKO/ATM cells than in RKO cells. Caffeine also sensitized both RKO cells and RKO/ATM cells to radiation. The caffeine treatment suppressed the radiation-induced activation of ATM kinase, suppressed the activation of Chk2 kinase and inhibited the accumulation of cells in G2 phase. The activity of cycline B1/Cdc2 kinase increased earlier but decayed rapidly in the presence of caffeine. Caffeine enhanced radiation-induced growth delay of RKO xenografts.Conclusions: Caffeine inhibited the radiation-induced activation of ATM kinase, thereby preventing the accumulation of cells in G2 phase. Consequently, radiosensitivity of cells increased in the presence of caffeine both in vitro and in vivo.

DNA-dependent Protein Kinase Is a Molecular Target for the Development of Non-Cytotoxic Radiation Sensitizing Drugs

DNA-dependent Protein Kinase Is a Molecular Target for the Development of Non-Cytotoxic Radiation Sensitizing Drugs

Enhanced response to radiotherapy in tumours deficient in the function of hypoxia-inducible factor-1

Background and purpose To test the hypothesis that deficiency in expression of the transcription factor, HIF-1, renders tumours more radioresponsive than HIF-1 proficient tumours. Patients and methods Tumours comprising mouse hepatoma cells lacking HIF-1β (and thereby HIF-1 function) were grown in nude mice and radiation-induced growth delay compared with that seen for wild-type tumours and tumours derived from HIF-1β negative cells where HIF-1 function had been restored. Results The xenografts that lack HIF-1 activity take longer to establish their growth and are more radioresponsive than both parental xenografts and those with restored HIF-1 function. Pre-treatment of the HIF-1 deficient xenografts with the hypoxic radiosensitizer misonidazole, had little effect on radioresponse. In contrast this treatment radiosensitized the parental xenografts. In spite of this, no difference in oxygenation status was found between the tumour types as measured by Eppendorf O 2-electrodes and by binding of the hypoxic cell marker NITP. Admixing wild type and HIF-1 deficient cells in the same tumour at ratios of 1 in 10 and 1 in 100 restores the growth of the mixed tumours to that of a 100% HIF-1 proficient cell population. However, when comparing the effects of radiation on the mixed tumours, radioresponsiveness is maintained in those tumours containing the high proportion of HIF-1 deficient cells. Conclusions The differences in radioresponse do not correlate with tumour oxygenation, suggesting that the hypoxic cells within the HIF-1 deficient tumours do not contribute to the outcome of radiotherapy. Thus, hypoxia impacts on tumour radioresponsiveness not simply because of the physio-chemical mechanism of oxygen with radiation-induced radicals causing damage ‘fixation’, but also because hypoxia/HIF-1 promotes expression of genes that allow tumour cells to survive under these adverse conditions. Further, the results from the cell mixing experiments uncouple the growth promoting effects of HIF-1 and the underlying mechanism by which HIF-1 may increase radiation resistance in solid tumours.

Effects of Hypoxic Cell Radiosensitizer Doranidazole (PR-350) on the Radioresponse of Murine and Human Tumor Cells in vitro and in vivo

We have investigated the radiosensitizing effect of doranidazole, a hypoxic cells radiosensitizer, using SCCVII tumor cells of C3H mice and CFPAC-1 and MIA PaCa-2 human pancreatic tumor cells. The radiosensitivity of hypoxic SCCVII cells in vitro increased with 1 mM doranidazole by a factor of 1.34 and 1.68, when determined by clonogenic survival and micronucleus (MN) formation, respectively. The radiation-induced growth delay of SCCVII tumors was significantly enhanced and the TCD(50/120) was reduced by a factor of 1.33 when 200 mg/kg doranidazole was injected, i.v., 20 min prior to tumor irradiation. The in vivo-in vitro excision assay showed that radiosensitivity of SCCVII cells in vivo increased by a factor of 1.47 with 200 mg/kg doranidazole. The radiation-induced growth delay of CFPAC-1 xenografts in nude mice was significantly enhanced and the TCD(50/90) was reduced by a factor of 1.30 by 200 mg/kg doranidazole. On the other hand, 200 mg/kg of doranidazole exerted no influence on the radiation-induced growth delay in MIA PaCa-2 xenografts. The tumor oxygenation status, as determined with an oxygen sensitive needle probe and the immunohistological study using pimonidazole, indicated that MIA PaCa-2 tumors are better oxygenated than CFPAC-1 tumors. The relatively well-oxygenated status in MIA PaCa-2 tumor may account for the lack of radiosensitization by doranidazole. It is concluded that the magnitude of radiosensitization of tumors by doranidazole is dependent on the oxygenation status of the tumors and that doranidazole may be useful in increasing the response of hypoxic human pancreatic tumor to IORT.

Radiosensitization of Murine Ehrlich Ascites Tumor by a Combination of 2-deoxy-D-Glucose and 6-Aminonicotinamide

Enhanced radiosensitizing effects of a combination of 2-deoxy-D-glucose (2-DG), a glycolytic inhibitor and 6-aminonicotinamide (6-AN) an analogue of nicotinamide, which inhibits hexose monophosphate shunt (HMP) have been demonstrated in vitro. The purpose of the present studies is to investigate in vivo effects of this combination in Ehrlich ascites tumor (EAT) bearing mice. EAT tumor was grown in Swiss albino strain A mice. Treatment induced growth delay and tumor free animal survival were evaluated as parameters of radiation response. Focal irradiation of the tumor with a single fraction of 10 Gy induced a moderate delay in tumor growth but did not lead to complete regression of the tumor. Intravenous administration of either 6-AN or 2-DG immediately before irradiation enhanced radiation-induced growth delay with a cure rate of 45%. However, administration of a combination of 2-DG (2 g/kg b.wt.) and 6-AN (2 mg/kg b.wt.) immediately before irradiation led to complete regression of tumor in 80% animals resulting in survival of more than 300 days. A similar response (approximately 80%) was observed when 2-DG dose was reduced to 1 g/kg in combination with 6-AN. It is concluded that 6-AN enhances the radiosensitizing effects of 2-DG and the combination may have potential application in improving radiotherapy of tumors.

Maximizing therapeutic gain with gemcitabine and fractionated radiation

Purpose/Objective: The nucleoside analogue gemcitabine inhibits cellular repair and repopulation, induces apoptosis, causes tumor growth delay, and enhances radiation-induced growth delay. After single doses of drug and radiation, maximum enhancement of tumor response was obtained when gemcitabine preceded radiation by at least 24 h. Conversely, the cellular radioresponse of the normal gastrointestinal epithelium was slightly protected when gemcitabine and radiation were separated by 24 h. This differential response created a time frame within which therapeutic gain could be maximized. In our present investigation, we sought to define the most therapeutically beneficial scheme of gemcitabine administration when combined with fractionated radiotherapy. Methods and Materials: C3Hf/Kam mice were given identical drug and radiation schedules of administration, and both normal tissue (jejunal mucosa) and tumor (Sa-NH) responses were measured. Irradiation was given once per day for 5 days in normal tissue and tumor growth delay studies and twice per day for the tumor cure endpoint. A total dose of 25 mg/kg gemcitabine was given i.p. in 1 of 3 schedules: a single dose of 25 mg/kg 24 h before the start of fractionated irradiation, 12.5 mg/kg 24 h before the first and third radiation doses, or 24 h before each of 5 radiation doses. Groups of mice bearing 7- or 8-mm diameter tumors were treated with gemcitabine alone or in combination with fractionated irradiation under ambient or hypoxic conditions. The survival response of the jejunal mucosa was quantified by the microcolony assay and histologically by quantifying apoptosis, mitosis, S-phase fraction, and crypt cellularity. Results: For tumor growth delay, dose-modifying factors (DMFs) were similar (1.34–1.46) for all 3 schedules of drug administration. In contrast, the response of the jejunum was strongly dependent on the schedule of gemcitabine administration. A single dose of gemcitabine before the start of fractionated radiotherapy resulted in slight radioprotection (DMF 0.96). Two doses and 5 daily doses of gemcitabine enhanced radiation response by factors of 1.09 and 1.23, respectively. Major factors affecting the response of the jejunal mucosa were apoptotic death of S-phase cells exposed to gemcitabine and cell cycle synchrony of surviving cells. Tumor reoxygenation was found to be a major mechanism for tumor radioenhancement, in addition to those reported earlier. Conclusion: All 3 schedules of drug administration produced therapeutic gain; however, when gemcitabine was given more than once in a 5-fraction radiation treatment schedule, normal tissue toxicity increased. The highest therapeutic gain (1.4) was achieved by giving a single dose of gemcitabine (25 mg/kg) 24 h before the start of fractionated radiotherapy.

Radiosensitization of two murine tumours with mild temperature hyperthermia and carbogen breathing

Purpose: To test the individual and combined effects of local mild temperature hyperthermia (MTH) at 41.5 C for 60min and carbogen breathing on tumour radiosensitivity. Materials and methods: The FSall fibrosarcoma of C3H mice and the SCK mammary carcinoma of A/J mice were used. The effect of various treatments on tumour cell survival was determined using the in vivo/in vitro tumour excision assay, and the radiobiological hypoxic fraction was calculated. The tumour radiation sensitivity was tested with the tumour growth delay assay. Results: The radiobiological hypoxic cell fraction (HF) in control FSall and SCK tumours was 0.45 and 0.78, respectively, and these values decreased to 0.12 in FSall tumours and 0.22 in SCK tumours when determined immediately after the tumours were treated with MTH. The HF was 0.32 in FSall tumours and 0.33 in SCK tumours after carbogen breathing was applied. When tumours were treated with MTH and the animals breathed carbogen the HF decreased to 0.03-0.04 in both FSall and SCK tumours. MTH treatment alone had only a small effect on tumour growth, but MTH treatment applied before irradiating the tumours significantly increased the radiation-induced tumour growth delay. Carbogen breathing modestly improved the radiation-induced tumour growth delay while the combination of MTH treatment and carbogen breathing caused the largest increase in radiation-induced tumour growth delay. Conclusions: MTH treatment alone and combined with carbogen breathing substantially increased the tumour radiation response probably through an increase in the tumour oxygenation status.

The Prostaglandin E 1 Analog, Misoprostol, a Normal Tissue Protector, Does Not Protect Four Murine Tumors In Vivo from Radiation Injury

The clinical development of radioprotectors, such as misoprostol, to protect normal tissue during cancer treatment must proceed with the assurance that tumors are not protected similarly or significantly. To provide data on this critical question, radiation-induced growth delay with or without the presence of misoprostol was measured in four murine tumors grown in the flanks of mice: the Lewis lung carcinoma, M-5076 ovarian sarcoma, FSA and NFSA. The effect of misoprostol on the tumor control dose (TCD50) of radiation was measured in FSA-bearing mice with or without prior treatment with the nonsteroidal anti-inflammatory agent, indomethacin. Misoprostol did not influence the in vivo growth of any of the four tumors, nor did it protect any of the tumors from radiation-induced growth delay. Likewise, there was no increase in the radiation TCD50 to treat the FSA in vivo in control or indomethacin-treated tumor-bearing mice. To measure any possible influence of tumor burden on the protective effect of misoprostol on normal tissue in mice, the protective effect of misoprostol on the survival of intestinal clonogenic cells was measured in M-5076-bearing mice and found to be the same as in non-tumor-bearing mice. These data suggest that misoprostol protects normal tissue in mice without protecting at least four experimental murine tumors. The data support the contention that misoprostol can achieve therapeutic gain by protecting normal tissues without protecting tumors.