Radiation Sensitivity Of Cells Research Articles

Inhibition of ubiquitin conjugating enzyme UBE2C reduces proliferation and sensitizes breast cancer cells to radiation, doxorubicin, tamoxifen and letrozole

The objective of this study was to determine radiation, doxorubicin, tamoxifen and letrozole sensitivity of breast cancer cells in response to functional inhibition of the ubiquitin conjugating enzyme UBE2C. Taqman Real time PCR was performed to measure UBE2C levels in breast cancer cell lines and control HBL100 and HEK293T cells. A dominant negative form of UBE2C (DN-UBE2C) was used to functionally inhibit wild type UBE2C. Cell proliferation and anchorage independent growth were measured by colorimetric and soft agar assays, respectively. Radiation, doxorubicin, tamoxifen and letrozole responses of the cell lines were assessed by colorimetric and clonogenic assays. Overexpression of UBE2C was observed in all breast cancer cell lines tested using quantitative real time PCR. UBE2C expression was found to be highest in MDAMB231 and relatively lowest in MCF7 cells, compared to control cells. Both the growth rate and the anchorage independent growth of MCF7 and MDAMB231 cells transfected with DN-UBE2C were significantly reduced compared to cells transfected with vector alone. MCF7 and MDAMB231 cells expressing DN-UBE2C were significantly more sensitive to different doses of radiation and doxorubicin compared to both wild type and vector alone transfected cells. In addition, DN-UBE2C transfected MCF7 cells were more sensitive to inhibition by tamoxifen and letrozole compared to wild type and vector alone transfected cells. Our results show that inhibition of UBE2C sensitizes breast cancer cells to radiation, doxorubicin and hormone blocking agents. UBE2C may, therefore, serve as a potential therapeutic target aimed at inducing radiation and chemo sensitization.

Role of p53, Bax, p21, and DNA-PKcs in radiation sensitivity of HCT-116 cells and xenografts

Molecular factors that dictate tumor response to ionizing radiation in rectal cancer are not well described. We investigated the contribution of p53, p21, Bax, and DNA-PKcs in response to ionizing radiation in an isogeneic colorectal cancer system in vitro and in vivo. HCT-116 DNA-PKcs(-/-) cells and xenografts were radiosensitive compared with wild-type (WT) HCT-116 cells. HCT-116 p53(-/-) cells and tumor xenografts displayed a radioresistant phenotype. Separately, p21 or Bax deficiency was associated with a radiosensitive phenotype in vitro and in vivo. In vivo, Bax deficiency led to increased tumor necrosis and decreased microvessel density. In vitro, HCT-116 Bax(-/-) cells had decreased levels of vascular endothelial growth factor. HCT-116 WT cells had a more radioresistant phenotype after pancaspase inhibition, but pancaspase inhibition did not alter radiosensitivity in HCT-116 Bax(-/-) cells subjected to ionizing radiation. There was no difference in cell growth in HCT-116 WT cells subjected to transient apoptosis-inducing factor (AIF) inhibition; however, HCT-116 Bax(-/-) cells treated with AIF siRNA followed by ionizing radiation had a significant survival advantage compared with control-treated cells, implicating AIF in the radiosensitivity of Bax(-/-) cells. These data might be used along with other markers to predict response to radiation in patients with rectal cancer.

BUB1 and BUBR1 inhibition decreases proliferation and colony formation, and enhances radiation sensitivity in pediatric glioblastoma cells

Glioblastoma (GBM) is a very aggressive and lethal brain tumor with poor prognosis. Despite new treatment strategies, patients' median survival is still lower than 1 year in most cases. The expression of the BUB gene family has demonstrated to be altered in a variety of solid tumors, pointing to a role as putative therapeutic target. The purpose of this study was to determine BUB1, BUB3, and BUBR1 gene expression profiles in glioblastoma and to analyze the effects of BUB1 and BUBR1 inhibition combined or not with Temozolomide and radiation in the pediatric SF188 GBM cell line. For gene expression analysis, 8 cell lines and 18 tumor samples were used. The effect of BUB1 and BUBR1 inhibition was evaluated using siRNA. Apoptosis, cell proliferation, cell cycle kinetics, micronuclei formation, and clonogenic capacity were analyzed after BUB1 and BUBR1 inhibition. Additionally, combinatorial effects of gene inhibition and radiation or Temozolomide (TMZ) treatment were evaluated through proliferation and clonogenic capacity assays. We report the upregulation of BUB1 and BUBR1 expression and the downregulation of BUB3 in GBM samples and cell lines when compared to white matter samples (p < 0.05). Decreased cell proliferation and colony formation after BUB1 and BUBR1 inhibition were observed, along with increased micronuclei formation. Combinations with TMZ also caused cell cycle arrest and increased apoptosis. Moreover, our results demonstrate that BUB1 and BUBR1 inhibition sensitized SF188 cells to γ-irradiation as shown by decreased growth and abrogation of colony formation capacity. BUB1 and BUBR1 inhibition decreases proliferation and shows radiosensitizing effects on pediatric GBM cells, which could improve treatment strategies for this devastating tumor. Collectively, these findings highlight the potentials of BUB1 and BUBR1 as putative therapeutic targets for glioblastoma treatment.

ALK Inhibitor PF02341066 (Crizotinib) Increases Sensitivity to Radiation in Non–Small Cell Lung Cancer Expressing EML4-ALK

Crizotinib (PF02341066) is a tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK) that has been shown to selectively inhibit growth of cancer cells that harbor the EML4-ALK fusion found in a subset of patients with non-small cell lung cancer (NSCLC). While in clinical trials, PF02341066 has shown a significant therapeutic benefit as a single agent; the effectiveness of combining it with other therapeutic modalities including ionizing radiation remains unknown. To further elucidate the role of PF02341066 in tumor inhibition, we examined its effects alone and in combination with radiation on downstream signaling, apoptosis, and radiosensitivity in two NSCLC cell lines in vitro: H3122, which harbors the EML4-ALK fusion, and H460, which does not. We also examined the in vivo effects of PF02341066 in H3122 mouse xenografts. In the H3122 cell line, PF02341066 inhibited phosphorylation of ALK and its downstream effectors: AKT, ERK, and STAT3. H3122 cells treated with a combination of PF02341066 and radiation showed an increase in cellular apoptosis and were sensitized to radiation therapy (dose enhancement ratio, 1.43; P < 0.0001). Moreover, in an H3122 xenograft model, the combined treatment resulted in greater tumor growth inhibition than either treatment alone (P < 0.05). None of these effects was observed in the EML4-ALK-negative H460 cells. Our findings indicate that PF02341066 acts as a radiation sensitizer in cells harboring the EML4-ALK fusion, providing a rationale for a clinical trial combining ALK inhibitor with radiation in the NSCLCs expressing ALK.

Valosin-containing protein regulates the proteasome-mediated degradation of DNA-PKcs in glioma cells

DNA-dependent protein kinase (DNA-PK) has an important role in the repair of DNA damage and regulates the radiation sensitivity of glioblastoma cells. The VCP (valosine-containing protein), a chaperone protein that regulates ubiquitin-dependent protein degradation, is phosphorylated by DNA-PK and recruited to DNA double-strand break sites to regulate DNA damage repair. However, it is not clear whether VCP is involved in DNA-PKcs (DNA-PK catalytic subunit) degradation or whether it regulates the radiosensitivity of glioblastoma. Our data demonstrated that DNA-PKcs was ubiquitinated and bound to VCP. VCP knockdown resulted in the accumulation of the DNA-PKcs protein in glioblastoma cells, and the proteasome inhibitor MG132 synergised this increase. As expected, this increase promoted the efficiency of DNA repair in several glioblastoma cell lines; in turn, this enhanced activity decreased the radiation sensitivity and prolonged the survival fraction of glioblastoma cells in vitro. Moreover, the VCP knockdown in glioblastoma cells reduced the survival time of the xenografted mice with radiation treatment relative to the control xenografted glioblastoma mice. In addition, the VCP protein was also downregulated in ∼25% of GBM tissues from patients (WHO, grade IV astrocytoma), and the VCP protein level was correlated with patient survival (R2=0.5222, P<0.05). These findings demonstrated that VCP regulates DNA-PKcs degradation and increases the sensitivity of GBM cells to radiation.

NVP-BEZ235, a novel dual PI3K/mTOR inhibitor, enhances the radiosensitivity of human glioma stem cells in vitro

NVP-BEZ235 is a novel dual PI3K/mTOR inhibitor and shows dramatic effects on gliomas. The aim of this study was to investigate the effects of NVP-BEZ235 on the radiosensitivity and autophagy of glioma stem cells (GSCs) in vitro. Human GSCs (SU-2) were tested. The cell viability and survival from ionizing radiation (IR) were evaluated using MTT and clonogenic survival assay, respectively. Immunofluorescence assays were used to identify the formation of autophagosomes. The apoptotic cells were quantified with annexin V-FITC/PI staining and flow cytometry, and observed using Hoechst 33258 staining and fluorescence microscope. Western blot analysis was used to analyze the expression levels of proteins. Cell cycle status was determined by measuring DNA content after staining with PI. DNA repair in the cells was assessed using a comet assay. Treatment of SU-2 cells with NVP-BEZ235 (10-320 nmol/L) alone suppressed the cell growth in a concentration-dependent manner. A low concentration of NVP-BEZ235 (10 nmol/L) significantly increased the radiation sensitivity of SU-2 cells, which could be blocked by co-treatment with 3-MA (50 μmol/L). In NVP-BEZ235-treated SU-2 cells, more punctate patterns of microtubule-associated protein LC3 immunoreactivity was observed, and the level of membrane-bound LC3-II was significantly increased. A combination of IR with NVP-BEZ235 significantly increased the apoptosis of SU-2 cells, as shown in the increased levels of BID, Bax, and active caspase-3, and decreased level of Bcl-2. Furthermore, the combination of IR with NVP-BEZ235 led to G1 cell cycle arrest. Moreover, NVP-BEZ235 significantly attenuated the repair of IR-induced DNA damage as reflected by the tail length of the comet. NVP-BEZ235 increases the radiosensitivity of GSCs in vitro by activating autophagy that is associated with synergistic increase of apoptosis and cell-cycle arrest and decrease of DNA repair capacity.

A potential role for resveratrol as a radiation sensitizer for melanoma treatment

BackgroundRadiotherapy (XRT) is used to improve local control of melanoma and for palliation of metastatic disease. Clinical use of XRT for melanoma is often limited by extent of disease and the relative radioresistance of melanoma may limit the effectiveness of XRT. Our group and others have previously shown that resveratrol (RSV) enhances radiation sensitivity in radioresistant prostate cancer cell lines. Material and MethodsIn this study, the effects of XRT in combination with RSV on radioresistant melanoma lines, SK-Mel-5 and HTB-65, were evaluated by assessment of proliferation and apoptosis. Clonogenic assay, comparison of proliferating cell nuclear antigen staining, Quick Cell Proliferation assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining and caspase-3 activity assay were used to assess proliferation and apoptosis, as appropriate. ResultsWe found that the percentage of colonies, proliferating cell nuclear antigen + cells and the optical density value of melanoma cells were decreased after addition of RSV to XRT (XRT/RSV). TUNEL + cells and the relative caspase-3 activity in melanoma cells were increased after addition of RSV to XRT (XRT/RSV). We investigated the possible molecular mechanisms of decreased proliferation and increased apoptosis by using reverse transcriptase-polymerase chain reaction and immunohistochemical staining. The anti-proliferative effect of XRT/RSV correlated with decreased expression of pro-proliferative molecule cyclin B, cyclin D, cdk2 and cdk4. The pro-apoptotic effect of XRT/RSV correlated with decreased expression of the anti-apoptotic molecule FLIP, Bcl-2, and survivin. ConclusionThese data suggest that RSV enhances radiation sensitivity of melanoma cells by inhibiting proliferation and promoting apoptosis. Resveratrol may have a potential role as a radiation sensitizer for melanoma treatment.

Are mouse lens epithelial cells more sensitive to γ-irradiation than lymphocytes?

In this pilot study we compared for the first time the radiation sensitivity of mouse lens epithelial cells (LECs) and mouse lymphocytes. We freshly prepared LECs and lymphocytes and irradiated them with γ-rays (137Cs; doses ranging from 0.25 to 2 Gy). DNA damage and repair were evaluated by alkaline comet assay and γH2AX foci assay. Using the comet assay, we observed a dose-dependent increase in DNA damage in both cell types. The faster formation of single- and double-strand breaks in LECs of C57BL/6 mice at doses below 1 Gy needs to be confirmed in other mouse strains. Immunofluorescence for γH2AX foci showed a higher degree of lesions in LECs from C57BL/6J mice compared to those of JF1 mice and to lymphocytes of both strains. Correspondingly, repair of DNA damage proceeded faster in LECs of C57BL/6J mice compared to LECs of JF1 mice and lymphocytes of both strains. It is obvious that the lymphocytes of both strains repaired DNA lesions more slowly than the corresponding LECs. In conclusion, our results demonstrate that LECs of C57Bl/6 mice show a steeper dose–response than lymphocytes in both types of experiments. It shows that both test systems are able to be used also at doses below 0.25 Gy. The observed difference in DNA repair between the LECs from C57BL/6J mice compared to the LECs from JF1 mice and to the lymphocytes of both strains warrants further experiments to identify the underlying molecular mechanisms.

Role of P53, Bax, P21, and DNA PKcs in Radiation Sensitivity of HCT-116 Cells

Role of P53, Bax, P21, and DNA PKcs in Radiation Sensitivity of HCT-116 Cells

ABT-737 induces Bim expression via JNK signaling pathway and its effect on the radiation sensitivity of HeLa cells.

ABT-737 is a BH3 mimetic small molecule inhibitor that can effectively inhibit the activity of antiapoptotic Bcl-2 family proteins including Bcl2, Bcl-xL and Bcl-w, and further enhances the effect of apoptosis by activating the proapoptotic proteins (t-Bid, Bad, Bim). In this study, we demonstrate that ABT-737 improved the radiation sensitivity of cervical cancer HeLa cells and thereby provoked cell apoptosis. Our results show that ABT-737 inhibited HeLa cell proliferation and activated JNK and its downstream target c-Jun, which caused the up-regulation of Bim expression. Blockade of JNK/c-Jun signaling pathway resulted in significant down-regulation of ABT-737-induced Bim mRNA and protein expression level. Also, ABT-737 could evoke the Bim promoter activity, and enhance the radiation sensitivity of HeLa cells via JNK/c-Jun and Bim signaling pathway. Our data imply that combination of ABT-737 and conventional radiation therapy might represent a highly effective therapeutic approach for future treatment of cervical cancer.

Abstract P5-03-02: Targeting mRNA Translation to Enhance the Radiosensitivity of Inflammatory Breast Cancer Stem Cells

Abstract Purpose/Objective(s): Inflammatory breast cancer (IBC) is a highly aggressive and radiation resistant malignancy with a dismal prognosis despite multimodality therapy, including ionizing radiation. We have previously shown that the unique pathogenic properties of IBC result in part from over-expression of translation initiation factor eIF4G1, which is part of the eIF4F translation initiation complex, along with eIF4E and eIF4A. eIF4F is regulated by mTOR, providing a promising target for anti-cancer therapeutics. We demonstrated that protein synthesis is highly regulated during IR by the DNA-damage response (DDR) pathway through mTOR signaling. Many key proteins required for the DDR pathway are encoded by mRNAs that require high levels of the eIF4F complex and mTOR activity for their efficient translation. We hypothesized that upregulation of eIF4F in IBC plays a crucial role in the radio-resistance of disease. Materials/Methods: Experiments were conducted in IBC SUM149 cells. eIF4G1, eIF4E and eIF4A were silenced through the generation of stable cell lines that express tetracycline-inducible shRNAs. eIF4A was also inhibited using the pharmacologic investigational inhibitor DAMD-PatA. Radiation sensitivity in vitro was determined by cell survival assay. Tumor xenografts were generated by the injection of stable shRNA inducible cell lines into nude mice. IBC SUM149 cancer stem cells (CSC) from both in vitro and in vivo experiments were analyzed by a combination of cell surface marker analysis, mammosphere formation and Aldefluor assays. Results: We show that moderate inhibition by silencing of individual components (or by pharmacologic inhibition of eIF4A) of the eIF4F complex prevents IBC xenograft tumor growth and strongly enhances radiosensitivity. In contrast to results obtained for non-transformed breast epithelial cells, reducing the high levels of eIF4G1 in epithelial IBC cells in 2D cultures provides no enhancement in radiation sensitivity. Rather, SUM149 IBC cells harbor a substantial population of CSCs, which are the cells that are strongly dependent on high levels of eIF4G1, and which are selectively radio-sensitized as a result of eIF4G1-silencing. CSCs also require eIF4E and eIF4A activity in order to survive radiation treatment. We also demonstrate that silencing of eIF4G1 radio-sensitizes the stem cell population within IBC tumor xenografts. Radio-resistance of IBC cells is likely mediated by differential responses to the DDR in the stem-cell populations and by selective mRNA translation of proteins involved in the DDR pathway. Conclusions: Our results demonstrate that regulation of mRNA translation plays an important role in conferring radio-resistance to advanced breast cancers, particularly by allowing the survival of the CSC compartment. While inhibition of eIF4F enhances radiation sensitivity in non-transformed cells, this process is abrogated in IBC due to enrichment of a radiation resistant CSC population, demonstrating translational control of the breast cancer stem cell population, and providing a novel understanding of the role of the regulation of mRNA translation in radiation resistance of breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-02.

Inhibition of EZH2 suppresses self-renewal and induces radiation sensitivity in atypical rhabdoid teratoid tumor cells

Overexpression of the Polycomb repressive complex 2 (PRC2) subunit Enhancer of Zeste 2 (EZH2) occurs in several malignancies, including prostate cancer, breast cancer, medulloblastoma, and glioblastoma multiforme. Recent evidence suggests that EZH2 may also have a role in rhabdoid tumors. Atypical teratoid/rhabdoid tumor (ATRT) is a rare, high-grade embryonal brain tumor that occurs most commonly in young children and carries a very poor prognosis. ATRTs are characterized by absence of the chromatin remodeling protein SMARCB1. Given the role of EZH2 in regulating epigenetic changes, we investigated the role of EZH2 in ATRT. Microarray analysis was used to evaluate expression of EZH2 in ATRT tumor samples. We used shRNA and a chemical inhibitor of EZH2 to examine the impact of EZH2 inhibition on cell growth, proliferation, and tumor cell self-renewal. Here, we show that targeted disruption of EZH2 by RNAi or pharmacologic inhibition strongly impairs ATRT cell growth, suppresses tumor cell self-renewal, induces apoptosis, and potently sensitizes these cells to radiation. Using functional analysis of transcription factor activity, we found the cyclin D1-E2F axis to be repressed after EZH2 depletion in ATRT cells. Our observations provide evidence that EZH2 disruption alters cell cycle progression and may be an important new therapeutic target, particularly in combination with radiation, in ATRT.

Inhibition of cyclin-dependent kinase 6 suppresses cell proliferation and enhances radiation sensitivity in medulloblastoma cells

Medulloblastoma accounts for 20% of all primary pediatric intracranial tumors. Current treatment cures 50-80% of patients but is associated with significant long-term morbidity and thus new therapeutic targets are needed. One such target is cyclin-dependent kinase6 (CDK6), a serine/threonine kinase that plays a vital role in cell cycle progression and differentiation. CDK6 is overexpressed in medulloblastoma patients and is associated with an adverse prognosis. To investigate the role of CDK6 in medulloblastoma, we assayed the effect of CDK6 inhibition on proliferation by depleting expression with RNA interference (RNAi) or by inhibiting kinase function with a small molecule inhibitor, PD0332991. Cell proliferation was assessed by colony focus assay or by the xCELLigence system. We then investigated the impact of CDK6 inhibition on differentiation of murine neural stem cells by immunofluorescence of relevant markers. Finally we evaluated the effects of PD0332991 treatment on medulloblastoma cell cycle and radiosensitivity using colony focus assays. Gene expression analysis revealed that CDK6 mRNA expression is higher than normal cerebellum in fifteen out of sixteen medulloblastoma patient samples. Inhibition of CDK6 by RNAi significantly decreased medulloblastoma cell proliferation and colony forming potential. Interestingly, CDK6 inhibition by RNAi increased differentiation in murine neural stem cells. PD0332991 treatment significantly decreased medulloblastoma cell proliferation and led to a G0/G1 cell cycle arrest. Furthermore, PD0332991 pretreatment sensitized medulloblastoma cells to ionizing radiation. Our findings suggest that targeting CDK6 with small molecule inhibitors may prove beneficial in the treatment of medulloblastoma, especially when combined with radiation.

A Direct Measurement of the Radiation Sensitivity of Normal Mouse Bone Marrow Cells1

A Direct Measurement of the Radiation Sensitivity of Normal Mouse Bone Marrow Cells¹

Monascuspiloin Enhances the Radiation Sensitivity of Human Prostate Cancer Cells by Stimulating Endoplasmic Reticulum Stress and Inducing Autophagy

Prostate cancer is a very common cancer among males. Traditional treatments for prostate cancer have limited efficacy; therefore, new therapeutic strategies and/or new adjuvant drugs must be explored. Red yeast rice (RYR) is a traditional food spice made in Asia by fermenting white rice with Monascus purpureus Went yeast. Accumulating evidence indicates that RYR has antitumor activity. In this study, PC-3 cells (human prostate cancer cells) were used to investigate the anti-cancer effects of ionizing radiation (IR) combined with monascuspiloin (MP, a yellow pigment isolated from Monascus pilosus M93-fermented rice) and to determine the underlying mechanisms of these effects in vitro and in vivo. We found that IR combined with MP showed increased therapeutic efficacy when compared with either treatment alone in PC-3 cells. In addition, the combined treatment enhanced DNA damage and endoplasmic reticulum (ER) stress. The combined treatment induced primarily autophagy in PC-3 cells, and the cell death that was induced by the combined treatment was chiefly the result of inhibition of the Akt/mTOR signaling pathways. In an in vivo study, the combination treatment showed greater anti-tumor growth effects. These novel findings suggest that the combined treatment could be a potential therapeutic strategy for prostate cancer.

Increased aldehyde reductase expression mediates acquired radioresistance of laryngeal cancer cells via modulating p53

The main obstacle to cure tumors by radiotherapy has been ascribed to tumor radioresistance. To determine the mechanisms underlying resistance to irradiation, it is essential to compare proteins differentially expressed from radiotherapy-sensitive and -resistant cancer cells. Aldehyde reductase (AKR1A1) was recently identified as increased in radioresistant laryngeal cancer cells by comparative proteomics approach. Here, we provide the mechanism of AKR1A1-mediated radioresistance via p53 regulation in laryngeal cancer cells. AKR1A1 induction was correlated with the radioresistant phenotype of laryngeal cancer HEp-2 cells. AKR1A1 depletion with siRNA significantly enhanced radiation sensitivity of radioresistant HEp-2 cells by promoting radiation-induced cell death and accelerated radiation-mediated inhibition of cell proliferation, without affecting either the PI3K-Akt or MAPK-ERK pathways. Intriguingly, AKR1A1 depletion induced phosphorylation of p53 at serine 15 and G2/M transition in response to irradiation. We further found that AKR1A1 interacted with p53 and this interaction was dramatically increased in the irradiated radioresistant cells compared with the control cells. AKR1A1 expression also regulated p53 stability in response to irradiation. Furthermore, AKR1A1 depletion only sensitized HCT116 cells expressing p53 to irradiation and not p53-deficient cells. Therefore, our data suggest that radiation-inducible AKR1A1 contributes to acquired radioresistance of laryngeal cancer cells by suppressing p53 activation through inhibitory interaction.

In vivo Importance of Homologous Recombination DNA Repair for Mouse Neural Stem and Progenitor Cells

We characterized the in vivo importance of the homologous recombination factor RAD54 for the developing mouse brain cortex in normal conditions or after ionizing radiation exposure. Contrary to numerous homologous recombination genes, Rad54 disruption did not impact the cortical development without exogenous stress, but it dramatically enhanced the radiation sensitivity of neural stem and progenitor cells. This resulted in the death of all cells irradiated during S or G2, whereas the viability of cells irradiated in G1 or G0 was not affected by Rad54 disruption. Apoptosis occurred after long arrests at intra-S and G2/M checkpoints. This concerned every type of neural stem and progenitor cells, showing that the importance of Rad54 for radiation response was linked to the cell cycle phase at the time of irradiation and not to the differentiation state. In the developing brain, RAD54-dependent homologous recombination appeared absolutely required for the repair of damages induced by ionizing radiation during S and G2 phases, but not for the repair of endogenous damages in normal conditions. Altogether our data support the existence of RAD54-dependent and -independent homologous recombination pathways.

The chemopreventive and clinically used agent curcumin sensitizes HPV- but not HPV+ HNSCC to ionizing radiation, in vitro and in a mouse orthotopic model

Radiation therapy (RT) plays a critical role in the local-regional control of head and neck squamous cell carcinoma (HNSCC). However, the efficacy of RT in treating HNSCC is limited by severe normal tissue toxicity, predominantly mucositis. One pharmacological approach for increasing the clinical response to RT is the use of radiation response modifiers that preferentially sensitize tumor cells. Previously we demonstrated that curcumin, a natural plant polyphenol, increased the radiation sensitivity of HNSCC cells and that the observed sensitization was dependent on curcumin-mediated inhibition of thioredoxin reductase 1 (TxnRd1) a key cytosolic regulator of redox-dependent signaling. Here, we examined curcumin-induced radiation sensitization in HNSCC cell lines with differing HPV status and expressing different levels of TxnRd1, in vitro. The intrinsic radiation resistance of the HPV- cell lines was significantly higher than the HPV+ cell lines used in our study. Notably, all of the HPV- cell lines expressed high levels of TxnRd1 and exhibited higher intrinsic resistance to RT. While curcumin was effective at increasing the radiation response of the resistant HPV- cell lines it had no effect on the HPV+ cells. Based on these findings we employed an orthotopic, HPV- HNSCC tumor model in athymic nude mice to examine the effect of combining curcumin with fractionated RT, in vivo. The combination of curcumin feeding and fractionated RT had a significant effect on tumor doubling time and overall animal survival. We therefore propose that curcumin and RT should be considered as a first line treatment of HPV- HNSCC.

Abstract LB-210: Abrogation of mutant TP53 radiation resistance by lithium induced CTNNB1 (β-catenin) activation in medulloblastoma.

Abstract Introduction: Medulloblastoma (MB) is the most common paediatric malignant brain neoplasm. 30 % of patients will reccur and die of their tumour. TP53 mutations confer radiation resistance; we have recently demonstrated that somatic mutations in TP53 are predictive of poor survival in patients with MB. Patients with both TP53 and WNT activating CTNNB1 (β-catenin) mutations demonstrate improved survival compared to patients with TP53 mutations alone. WNT pathway is abrogated in 10% of MB. Lithium Chloride (LiCl) treatment constitutively activates WNT pathway by inhibition of Gsk3β-mediated degradation of β-catenin. Radiation therapy is key treatment in MB; hence we hypothesized that activation of WNT/β-catenin signalling can rescue radiation resistance of TP53 mutant phenotype, sensitize MB cells to radiation and can be achieved with administration of LiCl. Methods: Human MB, TP53 wild type (wt) and mutant (mut), all CTNNB1 wt cell lines were used to investigate radiation response and effect of LiCl on cell survival by clonogenic assays. Functional roles of TP53 and CTNNB1 mutations in response to LiCl/radiation treatments were assessed by transfection of mutant genes on a wild type background. Double strand (DS) DNA damage-repair was assessed by γH2AX immunofluorescence (IF). Normal neuronal stem cells were used to assess effects of LiCl/radiation on normal brain tissue by clonogenic assay. Results: TP53 mut MB cells were resistant to radiation compared to TP53 wt cells by clonogenic assay (p&amp;lt;0.01), accompanied by more efficient DNA damage response manifested by lower numbers of γH2AX foci. Transfection of R175H dominant-negative TP53 mutation to wt cells resulted in increased clonogenic potential post-radiation (p&amp;lt;0.01). MB cells transfected with CTNNB1 S33Y mutation revealed increased radiosenstivity especially in TP53 mut MB (p&amp;lt;0.01). LiCl treatment resulted in nuclear translocation of β-catenin and signalling activation by luciferase assay, which is consistent with activation of WNT pathway. Treatment with LiCl increased radiation sensitivity of both wt and mut TP53 cells (p&amp;lt;0.01; p&amp;lt;0.05 respectively), accompanied by increased DS DNA damage and delayed repair in both lines by γH2AX IF. LiCl treatment of normal neuronal stem cells did not sensitize them to radiation suggesting that LiCl is a tumour specific treatment. Conclusions: Constitutive activation of β-catenin by LiCl sensitizes TP53 mut MB cells to radiation possibly through activation of β-catenin/TCF mediated transcription and impaired DS DNA repair response. These findings may explain survival advantage in CTNNB1 mut MB patients harbouring TP53 mutation. Knowing safety of LiCl and its neuroprotective capacity in normal neuronal cells, oral administration of LiCl may represent an attractive novel therapy for MB patients. Investigations of the molecular mechanisms governing this phenomenon are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-210. doi:1538-7445.AM2012-LB-210

Polo-like kinase 1 (PLK1) inhibition suppresses cell growth and enhances radiation sensitivity in medulloblastoma cells

BackgroundMedulloblastoma is the most common malignant brain tumor in children and remains a therapeutic challenge due to its significant therapy-related morbidity. Polo-like kinase 1 (PLK1) is highly expressed in many cancers and regulates critical steps in mitotic progression. Recent studies suggest that targeting PLK1 with small molecule inhibitors is a promising approach to tumor therapy.MethodsWe examined the expression of PLK1 mRNA in medulloblastoma tumor samples using microarray analysis. The impact of PLK1 on cell proliferation was evaluated by depleting expression with RNA interference (RNAi) or by inhibiting function with the small molecule inhibitor BI 2536. Colony formation studies were performed to examine the impact of BI 2536 on medulloblastoma cell radiosensitivity. In addition, the impact of depleting PLK1 mRNA on tumor-initiating cells was evaluated using tumor sphere assays.ResultsAnalysis of gene expression in two independent cohorts revealed that PLK1 mRNA is overexpressed in some, but not all, medulloblastoma patient samples when compared to normal cerebellum. Inhibition of PLK1 by RNAi significantly decreased medulloblastoma cell proliferation and clonogenic potential and increased cell apoptosis. Similarly, a low nanomolar concentration of BI 2536, a small molecule inhibitor of PLK1, potently inhibited cell growth, strongly suppressed the colony-forming ability, and increased cellular apoptosis of medulloblastoma cells. Furthermore, BI 2536 pretreatment sensitized medulloblastoma cells to ionizing radiation. Inhibition of PLK1 impaired tumor sphere formation of medulloblastoma cells and decreased the expression of SRY (sex determining region Y)-box 2 (SOX2) mRNA in tumor spheres indicating a possible role in targeting tumor inititiating cells.ConclusionsOur data suggest that targeting PLK1 with small molecule inhibitors, in combination with radiation therapy, is a novel strategy in the treatment of medulloblastoma that warrants further investigation.