Radiation-induced Cell Cycle Arrest Research Articles

Genome-wide analyses of lung cancer after single high-dose radiation at five time points (2, 6, 12, 24, and 48h).

Background: An increasing number of clinicians are experimenting with high-dose radiation. This study focuses on the genomic effects of high-dose single-shot radiotherapy and aims to provide a dynamic map for non-small cell lung cancer (NSCLC). Methods: We used whole-transcriptome sequencing to understand the evolution at molecular levels in A549 and H1299 exposed to 10 Gy X-rays at different times (2, 6, 12, 24, and 48h) in comparison with the no radiation group. Ingenuity pathway analysis, ceRNA analysis, enrichment analysis, and cell cycle experiments are performed for molecular analyses and function analyses. Results: Whole-transcriptome sequencing of NSCLC showed a significant dynamic change after radiotherapy within 48h. MiR-219-1-3p and miR-221-3p, miR-503-5p, hsa-miR-455-5p, hsa-miR-29-3p, and hsa-miR-339-5p were in the core of the ceRNA related to time change. GO and KEGG analyses of the top 30 mRNA included DNA repair, autophagy, apoptosis, and ferroptosis pathways. Regulation of the cell cycle-related transcription factor E2F1 might have a key role in the early stage of radiotherapy (2.6h) and in the later stage of autophagy (24 and 48h). Functions involving different genes/proteins over multiple periods implied a dose of 10Gy was related to the kidney and liver pathway. Radiation-induced cell cycle arrest at the G2/M phase was evident at 24h. We also observed the increased expression of CCNB1 at 24h in PCR and WB experiments. Conclusion: Our transcriptomic and experimental analyses showed a dynamic change after radiation therapy in 48h and highlighted the key molecules and pathways in NSCLC after high-dose single-shot radiotherapy.

EXTH-90. HDAC INHIBITOR, CAY10603, IS A POTENT RADIOSENSITIZER IN MENINGIOMAS

Abstract Developing and evaluating potential radiosensitisers to enhance therapeutic efficacy are urgently needed due to the lack of efficient chemotherapy for meningioma. Histone deacetylase (HDACs) expression is generally increased in many cancer types and regulate the expression of numerous proteins involved in tumorigenesis. Targeting HDAC using HDAC inhibitor (HDACi) represent promising radiosensitisers that affect various biological processes, such as cell survival, apoptosis, and DNA repair. Recently, one of the HDAC inhibitor, AR42 monotherapy trials for patients with neurofibromatosis type2 started Nov 2021. However, we were not able to detect the actual HDAC downregulation after applying AR42 (0.5-2 μM) in immortalised meningioma. Hence, we investigated whether pretreatment with the hydroxamate-based HDAC6 inhibitor, Cay10603, impacts HDAC expression, radiation-induced DNA double-strand break (DSB) induction, cell survival, and cell cycle arrest in both immortalised and human meningioma cell lines. Cay10603 at nanomolar level (100 nM) was treated prior to high energy x-ray irradiation (2 Gy) by a medical linear accelerator (LINAC). We found that surviving rate was synergistically decreased after combination treatment of Cay10603 and radiation. Combination therapy induced DNA damage with activation of histone gH2AX and increased G2/M arrest compared to drug or radiation alone. To focus on the mechanisms of action of HDAC6 inhibition followed by radiation, we further investigated nuclear localisation of beta-catenin levels. The results showed the beta-catenin in the nucleus was suppressed after combination therapy. Our findings demonstrate a potential therapeutic strategy of Cay10603 to improve the radiosensitization for meningiomas.

The Molecular Effects of a PARP-1/-2 Inhibitor on Proton and Photon Radiotherapy in Head and Neck Cancer Cells

<h3>Purpose/Objective(s)</h3> The responses of human head and neck cancer (HNSCC) cells to photon (XRT) and proton (PRT) radiotherapy can be enhanced by a poly-(ADP-ribose) polymerase (PARP)-1 and PARP-2 inhibitor (PARPi) with undefined underlying mechanisms. We hypothesize that elucidating these mechanisms will facilitate a personalized selection of PARPi in combination with XRT or PRT. Herein, the effects of PARPi on XRT and PRT induced cell death, cell cycle distribution, and related protein expression in HNSCC cells were defined. <h3>Materials/Methods</h3> Human papillomavirus (HPV)-negative HN5, SqCC/Y1, and HPV-positive UPCI-SCC-154, UM-SCC-47 cell lines were tested. Cells were exposed to a 4 Gy PRT or XRT with or without PARPi (1 µM, 1 hour [h] before radiation). Cell cycle distribution, cell necrosis and apoptosis (staining with Annexin-VFITC) were determined using terminal deoxy-nucleotidyltransferase dUTP nickend labeling assay and were analyzed by flow cytometry. Cell mitotic catastrophe (co-staining for cytoplasm with γ- tubulin for nucleus with Dapi), and senescence (assessed using SA-β-gal) were examined. Protein expressions were determined by western blot or by flow cytometry. <h3>Results</h3> PARPi significantly increased XRT and PRT induced cellular senescence at 6 days in the two HPV+ cell lines, with higher percentages of senescence in PRT than XRT cells (<i>P</i> all < 0.05); in the two HPV- cell lines, the PARPi significantly increased the ratio of XRT-induced senescence (<i>P</i> all < 0.01). PARPi caused significantly more cells to undergo mitotic catastrophe after PRT (in 3 cell lines) or XRT (in the two HPV- cell lines), with higher rates of mitotic catastrophe induced by PARPi in PRT vs. XRT group (<i>P</i> all < 0.05). The PARPi led to a trend (non-significant) of increased cell necrosis at 48 h after PRT (in 3 cell lines) or XRT (in 2 cell lines). No effect of PARPi on XRT or PRT induced apoptosis was noted. At 24 h, PARPi significantly prolonged XRT (in two HPV- cell lines) and PRT (in HN5 cells) induced cell cycle arrest at G2/M phase; trends of this prolongation were noted after PRT in SqCC/Y1 and UPCI-SCC-154 cells, and after XRT in UPCI-SCC-154 cells. PARPi caused trends of increasing ratios of specific protein (53BP1 and RAD51 [DNA repair related]; Ki-67 [cell cycle related]) abundant cells at 24 h after PRT (in two HPV- cell lines); and increased cleaved caspase-3 expression after XRT or PRT in four cell lines tested. <h3>Conclusion</h3> In conclusion, PARPi induced higher levels of mitotic catastrophe in combination with PRT versus XRT, promoted XRT and PRT induced senescence, while it led to a trend of increased cell necrosis after PRT and XRT. These upregulating effects of PARPi were cell line dependent. Cell line based delayed DNA damage repair and increase in radiation-induced cell cycle arrest at G2/M phase may have served as parts of the underlying mechanisms. Further studies on personalized therapy of XRT or PRT in combination with PARPi to improve the efficacy of radiotherapy are warranted.

B-Myb participated in ionizing radiation-induced apoptosis and cell cycle arrest in human glioma cells

As a common treatment of human glioma, ionizing radiation (IR) was reported to result in cell cycle arrest. However, the mechanisms underlying IR-induced abnormal cell cycle remain largely unclear. Here we found that IR caused an elevated expression of B-Myb and cell cycle-related proteins, as well as G2/M phase arrest in U251 cells instead of U87 cells. However, the knockdown of B-Myb by small interfering RNAs ameliorated the increasing of cell cycle-related proteins and G2/M phase arrest induced by IR. Further analysis demonstrated that decreased-B-Myb enhanced the sensitivity of U251 cells to IR. Moreover, the establishment of H1299 cell line proved that B-Myb expression was associated with the status of p53. Immunoprecipitation (IP) and chromatin immunoprecipitation (CHIP) assay results indicated that mutant p53 and SP1 regulated the expression of B-Myb via different mechanisms. This study not only elucidated the role of B-Myb in IR-induced cell cycle alternation, but also provided insight into mechanism of B-Myb expression.

Dual Inhibition of PARP and the Intra-S/G2 Cell Cycle Checkpoints Results in Highly Effective Radiosensitization of HPV-Positive HNSCC Cells.

In head and neck squamous cell carcinoma (HNSCC), tumors positive for human papillomavirus (HPV) represent a distinct biological entity with favorable prognosis. An enhanced radiation sensitivity of these tumors is evident in the clinic and on the cellular level when comparing HPV-positive and HPV-negative HNSCC cell lines. We could show that the underlying mechanism is a defect in DNA double-strand break repair associated with a profound and sustained G2 arrest. This defect can be exploited by molecular targeting approaches additionally compromising the DNA damage response to further enhance their radiation sensitivity, which may offer new opportunities in the setting of future de-intensified regimes. Against this background, we tested combined targeting of PARP and the DNA damage-induced intra-S/G2 cell cycle checkpoints to achieve effective radiosensitization. Enhancing CDK1/2 activity through the Wee1 inhibitor adavosertib or a combination of Wee1 and Chk1 inhibition resulted in an abrogation of the radiation-induced G2 cell cycle arrest and induction of replication stress as assessed by γH2AX and chromatin-bound RPA levels in S phase cells. Addition of the PARP inhibitor olaparib had little influence on these endpoints, irrespective of checkpoint inhibition. Combined PARP/Wee1 targeting did not result in an enhancement in the absolute number of residual, radiation induced 53BP1 foci as markers of DNA double-strand breaks but it induced a shift in foci numbers from S/G2 to G1 phase cells. Most importantly, while sole checkpoint or PARP inhibition induced moderate radiosensitization, their combination was clearly more effective, while exerting little effect in p53/G1 arrest proficient normal human fibroblasts, thus indicating tumor specificity. We conclude that the combined inhibition of PARP and the intra-S/G2 checkpoint is a highly effective approach for the radiosensitization of HPV-positive HNSCC cells and may represent a viable alternative for the current standard of concomitant cisplatin-based chemotherapy. In vivo studies to further evaluate the translational potential are highly warranted.

RETRACTED ARTICLE: LncRNA SNHG12 regulates the radiosensitivity of cervical cancer through the miR-148a/CDK1 pathway

BackgroundRadiation resistance is a major obstacle to the prognosis of cervical cancer (CC) patients. Many studies have confirmed that long non-coding RNAs (lncRNAs) are involved in the regulation of radiosensitivity of cancers. However, whether small nucleolar RNA host gene 12 (SNHG12) regulates the radiosensitivity of CC remains unknown.MethodsQuantitative real-time polymerase chain reaction was used to measure the expression levels of SNHG12 and microRNA-148a (miR-148a). The radiosensitivity of cells was evaluated by clonogenic assay. Flow cytometry and caspase-3 activity assay were performed to assess the apoptosis ability and cell cycle distribution of cells. Besides, dual-luciferase reporter and RNA immunoprecipitation assay were used to verify the interaction between miR-148a and SNHG12 or cyclin-dependent kinase 1 (CDK1). Also, the protein levels of CDK1, CCND1 and γ-H2AX were detected by western blot analysis. Furthermore, in vivo experiments were conducted to verify the effect of SNHG12 on CC tumor growth. Ki-67 and TUNEL staining were employed to evaluate the proliferation and apoptosis rates in vivo. The hematoxylin and eosin (HE) staining were employed to evaluate the tumor cell morphology.ResultsSNHG12 was upregulated in CC tissues and cells, and its knockdown improved the radiosensitivity by promoting the radiation-induced apoptosis and cell cycle arrest of CC cells. Also, miR-148a could be sponged by SNHG12 and could target CDK1. MiR-148a inhibitor or CDK1 overexpression could invert the promotion effect of silenced-SNHG12 on CC radiosensitivity. Meanwhile, SNHG12 interference reduced the tumor growth of CC, increased miR-148a expression, and inhibited CDK1 level in vivo.ConclusionLncRNA SNHG12 promoted CDK1 expression to regulate the sensitivity of CC cells to radiation through sponging miR-148a, indicating that SNHG12 could be used as a potential biomarker to treat the radiotherapy resistance of CC patients.

Abstract A52: Deciphering MET-dependent modulation of global cellular responses to DNA damage by quantitative phosphoproteomics reveals Ser1016 as a novel MET phosphosite that regulates cellular response to irradiation

Abstract There is emerging evidence that DNA damage response (DDR) intertwines with signaling pathways downstream of oncogenic receptor tyrosine kinases (RTKs). Two complementary phosphoproteomic approaches revealed global networks and identified and validated core phosphosites that participate in a crosstalk between the MET RTK and the DDR, including a newly discovered phosphosite on MET itself, Ser1016. DNA-PK phosphorylates Ser1016, and this phosphorylation is increased following irradiation. Cell lines as well as animal models expressing Ser1016Ala phosphodeficient receptor exhibit greater sensitivity to irradiation. MET Ser1016Ala mutants fail to undergo proper radiation-induced cell cycle arrest by bypassing the G2 checkpoint with a premature entry into mitosis and abnormal mitotic spindles, leading to a lower proliferation rate of Ser1016-deficient cells. Regulation of MET activity through Ser1016 phosphorylation by a DDR core kinase represents a critical event involved in DNA repair signaling through a growth factor receptor that frequently functions as a tumor driver. Citation Format: Jonas P. Koch, Ariel Bensimon, Selina M. Roth, Matúš Medo, Paola Francica, Eleonora Orlando, Rahel Riedo, Jacopo Gavini, Aurélie Quintin, Astrid A. Glück, Deborah M. Stroka, Andree Blaukat, Daniel M. Aebersold, Yitzhak Zimmer, Ruedi Aebersold, Michaela Medová. Deciphering MET-dependent modulation of global cellular responses to DNA damage by quantitative phosphoproteomics reveals Ser1016 as a novel MET phosphosite that regulates cellular response to irradiation [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A52.

X irradiation induced colonic mucosal injury and the detection of apoptosis through PARP-1/p53 regulatory pathway

This study aimed to explore whether PARP-1 regulatory pathway mediated X irradiation induced cell cycle arrest and apoptosis or not. In this regard, colonic mucosal injury caused by whole-body X-irradiation induced apoptosis through PARP-1, caspase 3 and p53 regulatory pathway were evaluated in experimental rat models. Eighteen Wistar albino rats were divided into three groups. Two radiation groups received 8.3 Gy dose of whole-body X-irradiation as a single dose and the control group received physiological saline intraperitoneally. Radiation groups were sacrificed after 6 h and 4 days of irradiation. PARP-1 and caspase 3 expression in the nuclei of colonic crypt cells significantly increased 6 h after irradiation, and declined 4 days after irradiation. In conflict with other studies that reported p53 as not being expressed widely in colonic mucosa, in our study the expressions of p53 were elevated both in the cytoplasm and in the nucleus of the crypt cells, especially 6 h after irradiation. In the radiation groups, colonic mucosal injury score was significantly elevated compared with that of the control group. Our data demonstrated that PARP-1, caspase-3 and p53 expression increased in colonic mucosa 6 h after irradiation.

Time- and Dose-Dependent Effects of Ionizing Irradiation on the Membrane Expression of Hsp70 on Glioma Cells.

The major stress-inducible protein Hsp70 (HSPA1A) is overexpressed in the cytosol of many highly aggressive tumor cells including glioblastoma multiforme and presented on their plasma membrane. Depending on its intracellular or membrane localization, Hsp70 either promotes tumor growth or serves as a target for natural killer (NK) cells. The kinetics of the membrane Hsp70 (mHsp70) density on human glioma cells (U87) was studied after different irradiation doses to define the optimal therapeutic window for Hsp70-targeting NK cells. To maintain the cells in the exponential growth phase during a cultivation period of 7 days, different initial cell counts were seeded. Although cytosolic Hsp70 levels remained unchanged on days 4 and 7 after a sublethal irradiation with 2, 4 and 6 Gy, a dose of 2 Gy resulted in an upregulated mHsp70 density in U87 cells which peaked on day 4 and started to decline on day 7. Higher radiation doses (4 Gy, 6 Gy) resulted in an earlier and more rapid onset of the mHsp70 expression on days 2 and 1, respectively, followed by a decline on day 5. Membrane Hsp70 levels were higher on cells in G2/M than in G1; however, an irradiation-induced cell cycle arrest on days 4 and 7 was not associated with an increase in the mHsp70 density. Extracellular Hsp70 concentrations in the supernatant of irradiated cells were significantly higher than sham (0 Gy) irradiated cells on days 4 and 7, but not on day 1. Functionally, elevated mHsp70 densities were associated with a significantly better lysis by Hsp70-targeting NK cells. In summary, the kinetics of changes in the mHsp70 density upon irradiation on tumor cells is time- and dose-dependent.

Involvement of TGF-β and ROS in G1 Cell Cycle Arrest Induced by Titanium Dioxide Nanoparticles Under UVA Irradiation in a 3D Spheroid Model.

BackgroundAs one of the most widely produced engineered nanomaterials, titanium dioxide nanoparticles (nano-TiO2) are used in biomedicine and healthcare products, and as implant scaffolds; therefore, the toxic mechanism of nano-TiO2 has been extensively investigated with a view to guiding application. Three-dimensional (3D) spheroid models can simplify the complex physiological environment and mimic the in vivo architecture of tissues, which is optimal for the assessment of nano-TiO2 toxicity under ultraviolet A (UVA) irradiation.Methods and ResultsIn the present study, the toxicity of nano-TiO2 under UVA irradiation was investigated in 3D H22 spheroids cultured in fibrin gels. A significant reduction of approximately 25% in spheroid diameter was observed following treatment with 100 μg/mL nano-TiO2 under UVA irradiation after seven days of culture. Nano-TiO2 under UVA irradiation triggered the initiation of the TGF-β/Smad signaling pathway, increasing the expression levels of TGF-β1, Smad3, Cdkn1a, and Cdkn2b at both the mRNA and protein level, which resulted in cell cycle arrest in the G1 phase. In addition, nano-TiO2 under UVA irradiation also triggered the production of reactive oxygen species (ROS), which were shown to be involved in cell cycle regulation and the induction of TGF-β1 expression.ConclusionNano-TiO2 under UVA irradiation induced cell cycle arrest in the G1 phase and the formation of smaller spheroids, which were associated with TGF-β/Smad signaling pathway activation and ROS generation. These results reveal the toxic mechanism of nano-TiO2 under UVA irradiation, providing the possibility for 3D spheroid models to be used in nanotoxicology studies.

Single high dose irradiation induces cell cycle arrest and apoptosis in human hepatocellular carcinoma cells through the Ras/Raf/MEK/ERK pathways

Purpose: Stereotactic body radiation therapy (SBRT) is emerging as a new noninvasive treatment in patients with primary liver carcinoma or liver-confined metastatic cancer. However, the radiobiological targets remain a subject of debate. Here, we investigated the potential biological effects of the radiation on the human hepatocellular carcinoma HepG2 cells.Materials and methods: Firstly, HepG2 cells were divided into three groups: control group, 3.5 Gy*8f group (L group), and 15 Gy*1f group (H group). After treatment, cell proliferation was examined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and plate colony formation assays. Cell cycle and apoptosis were assessed using propidium iodide and Hoechst 33258 staining, respectively. Furthermore, the mechanisms underlying irradiation-induced cell cycle arrest and cell apoptosis were investigated by Western blot assay.Results: Irradiation could effectively inhibit the proliferation and colony formation of HepG2 cells, and the single high dose irradiation showed stronger inhibitory effects. Irradiation-induced cell cycle arrest at G2/M phase in HepG2 cell, during which the expression levels of cyclin B1, CDK1, and p-CDK1 proteins were downregulated, whereas expression of p21 was upregulated in the irradiated HepG2 cells. After irradiation, typical morphological changes of apoptosis in HepG2 cells were observed; the number of cell apoptosis and the expression of apoptosis associated proteins were significantly increased in HepG2 cells by high dose irradiation compared with low dose irradiation. Additionally, compared with low dose irradiation, high dose irradiation significantly downregulated the phosphorylated proteins in the Ras/Raf/MEK/ERK signaling pathway.Conclusions: Our results suggest that irradiation applied in SBRT, particularly single high dose irradiation, mediates its anti-tumor effects by inducing cell cycle arrest and apoptosis via modulation of the Ras/Raf/MEK/ERK signaling pathway.

Abstract 2917: Inhibition of Annexin A6 sensitizes triple negative breast cancer cells to radiation

Abstract Introduction: Despite vast evidence that tumor genetics play a dominant role in determining radiosensitivity and ultimate benefit from radiation therapy (RT), current RT delivery algorithms have not successfully incorporated tumor biology. Instead, clinical decisions regarding the use of RT are still based on features of tumor aggressiveness such as stage, grade, degree of metastasis and type of surgery. We provide evidence showing that most aggressive basal-like TNBC cells characteristically expresses low levels of the Ca2+-dependent membrane-binding tumor suppressor Annexin A6 (AnxA6) as compared to mesenchymal-like TNBC cell lines. We have also shown that depletion of AnxA6 in TNBC cells induced severe morphological changes from the slow growing mesenchymal-like to rapidly growing basal-like phenotypes and that this was not only associated with enhanced anchorage independent cell proliferation, but significantly inhibited cell adhesion, motility and invasiveness. Given that rapidly proliferating cells are more radiosensitive, we hypothesized that the expression status of AnxA6 in triple negative breast cancer cells underlies their response to RT. Materials and Methods: For clonogenic assay, cells were grown to 70-80% confluency then were allowed to settle and attach for 8 hours before being exposed to irradiation at various doses. Dishes were then incubated at 37oC in 5% CO2 in a humidified incubator to form colonies for 14 days. Colonies (50 cells or more) were stained with crystal violet and enumerated under a Fisher Stereomaster II 10X stereoscopic microscope. Radiation induced cell cycle arrest was assessed by flow cytometry. Results: Our studies show that depletion of AnxA6, significantly increased the sensitivity of triple negative breast cancer cells via up-regulation of cell cycle pathways as demonstrated by lack of G2/M cell cycle arrest in cells with low level of AnxA6. Additionally, we demonstrate that knockdown of AnxA6 enhanced radiation induced DNA damage. Conclusions: Our findings demonstrate that reduced expression of AnxA6 enhances radiation sensitivity of triple negative breast cancer cells. Note: This abstract was not presented at the meeting. Citation Format: Gladys N. Nangami, Amos Sakwe, Josiah Ochieng. Inhibition of Annexin A6 sensitizes triple negative breast cancer cells to radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2917.

Delayed Captopril Administration Mitigates Hematopoietic Injury in a Murine Model of Total Body Irradiation

The increasing potential for accidental radiation exposure from either nuclear accidents or terrorist activities has escalated the need for radiation countermeasure development. We previously showed that a 30-day course of high-dose captopril, an ACE inhibitor, initiated 1–4 h after total body irradiation (TBI), improved Hematopoietic Acute Radiation Syndrome (H-ARS) and increased survival in mice. However, because of the time likely required for the deployment of a stockpiled radiation countermeasure to a radiation mass casualty site, there is a need for therapies that can be administered 24–48 hours after initial exposure. Using C57BL/6 mice exposed to an LD50-80/30 of 60Co TBI (7.75–7.9 Gy, 0.615 Gy/min), we show that low-dose captopril administration, initiated as late as 48 h post-TBI and continued for 14 days, significantly enhanced overall survival similarly to high-dose, rapid administration. Captopril treatment did not affect radiation-induced cell cycle arrest genes or the immediate loss of hematopoietic precursors. Reduced mortality was associated with the recovery of bone marrow cellularity and mature blood cell recovery at 21–30 days post-irradiation. Captopril reduced radiation-induced cytokines EPO, G-CSF, and SAA in the plasma. Finally, delayed captopril administration mitigated brain micro-hemorrhage at 21 days post-irradiation. These data indicate that low dose captopril administered as late as 48 h post-TBI for only two weeks improves survival that is associated with hematopoietic recovery and reduced inflammatory response. These data suggest that captopril may be an ideal countermeasure to mitigate H-ARS following accidental radiation exposure.

Porous platinum nanoparticles as a high-Z and oxygen generating nanozyme for enhanced radiotherapy in vivo

Radiotherapy (RT), along with surgery and chemotherapy, is a major modality of cancer therapy. Nevertheless, insufficient deposition of radiation energy in tumors and hypoxia-associated radioresistance remain the greatest challenges in RT. Here, we propose porous platinum nanoparticles as a new nanomedicine platform for solving these two problems at the same time using a single agent. Because of the combined advantages of a high-Z element and oxygen generation capability, porous platinum nanoparticles can significantly increase radiation-induced DNA damage, ROS stress, and cell cycle arrest by effectively depositing X-ray radiation energy within the cancer cells. Further, porous platinum nanoparticles increase tumor oxygenation by converting endogenic H2O2 to O2, thus greatly enhancing RT with no apparent in vivo toxicity to animals. This study presents a new nanomedicine strategy based on the use of porous high-Z metal nanoparticles with oxygen generation function for the synergistic enhancement of RT in cancer treatment.

Cerium Oxide Nanoparticles Re-establish Cell Integrity Checkpoints and Apoptosis Competence in Irradiated HaCat Cells via Novel Redox-Independent Activity.

Cerium oxide nanoparticles (CNPs) are potent radical scavengers protecting cells from oxidative insults, including ionizing radiation. Here we show that CNPs prevent X-ray-induced oxidative imbalance reducing DNA breaks on HaCat keratinocytes, nearly abating mutagenesis. At the same time, and in spite of the reduced damage, CNPs strengthen radiation-induced cell cycle arrest and apoptosis outcome, dropping colony formation; notably, CNPs do not possess any intrinsic toxicity toward non-irradiated HaCat, indicating that they act on damaged cells. Thus CNPs, while exerting their antioxidant action, also reinforce the stringency of damage-induced cell integrity checkpoints, promoting elimination of the “tolerant” cells, being in fact radio-sensitizers. These two contrasting pathways are mediated by different activities of CNPs: indeed Sm-doped CNPs, which lack the Ce3+/Ce4+ redox switch and the correlated antioxidant action, fail to decrease radiation-induced superoxide formation, as expected, but surprisingly maintain the radio-sensitizing ability and the dramatic decrease of mutagenesis. The latter is thus attributable to elimination of damaged cells rather than decreased oxidative damage. This highlights a novel redox-independent activity of CNPs, allowing selectively eliminating heavily damaged cells through non-toxic mechanisms, rather reactivating endogenous anticancer pathways in transformed cells.

Overexpression of the Drosophila ATR homologous checkpoint kinase Mei-41 induces a G2/M checkpoint in Drosophila imaginal tissue

BackgroundDNA damage generally results in the activation of ATM/ATR kinases and the downstream checkpoint kinases Chk1/Chk2. In Drosophila melanogaster, the ATR homologue meiotic 41 (mei-41) is pivotal to DNA damage repair and cell cycle checkpoint signalling. Although various mei-41 mutant alleles have been analyzed in the past, no gain-of-function allele is yet available. To fill this gap, we have generated transgenic flies allowing temporal and tissue-specific induction of mei-41.ResultsOverexpression of mei-41 in wing and eye anlagen affects proliferation and a G2/M checkpoint even in the absence of genomic stress. Similar consequences were observed following the overexpression of the downstream kinase Grapes (Grp) but not of Loki (Lok), encoding the respective Drosophila Chk1 and Chk2 homologues, in agreement with their previously reported activities. Moreover, we show that irradiation induced cell cycle arrest was prolonged in the presence of ectopic mei-41 expression. Similar to irradiation stress, mei-41 triggered the occurrence of a slower migrating form of Grp, implying specific phosphorylation of Grp in response to either signal. Using a p53R-GFP biosensor, we further show that overexpression of mei-41 was sufficient to elicit a robust p53 activation in vivo.ConclusionWe conclude that overexpression of the Drosophila ATR homologue mei-41 elicits an effectual DNA damage response irrespective of irradiation.

Abstract 841: Protection of normal tissues by DIM (3,3'-Diindolylmethane) during radiation therapy

Abstract 96 Normal 0 false false false EN-US ZH-CN X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-fareast-language:EN-US;} Despite advances in precision targeting of tumors by radiation therapy, local recurrence is still a serious problem due to the inability to sometimes deliver a sufficiently high dose of radiation to achieve tumor control without unacceptable late normal tissue and organ toxicity. DIM (3,3'-diindolylmethane), a commercially available dietary supplement, has long been studied as a cancer prevention and anti-tumor agent. We are investigating DIM's differential effects after radiation in tumor and normal tissues using a mouse model approach. We monitored tumor growth after localized radiation in a tumor engraftment model of MDA-MB-231 human breast carcinoma cells using immunocompromised NSG mice. The fractionated regimen consists of a total dose of 24 Gy in four fractions (6 Gy per fraction given every other day) with administration of DIM or vehicle at one hour prior to each fraction. DIM showed no significant effect on the growth of irradiated tumors, i.e. DIM did not affect the treatment efficacy of radiation on tumors. On the other hand, we are investigating the molecular mechanism of DIM's radio-protective effects in normal tissues. Our transcriptomic data indicate that DIM treatment increases expression of some stress-responsive genes without causing DNA damage. Results to date indicate that DIM might delay radiation-induced cell cycle arrest and apoptosis. The ultimate goal is to develop DIM as a clinical radioprotector in order to improve the therapeutic index by allowing higher doses of radiation to improve local tumor control by reducing late dose-limiting normal tissue toxicity by radiation.&amp;lt;!–EndFragment–&amp;gt; Citation Format: Renxiang Chen, Lijun Li, Loretta Y. Lin, Albert J. Fornace, Heng-Hong Li. Protection of normal tissues by DIM (3,3'-Diindolylmethane) during radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 841.

Ionizing Radiation Induces Morphological Changes and Immunological Modulation of Jurkat Cells.

Impairment or stimulation of the immune system by ionizing radiation (IR) impacts on immune surveillance of tumor cells and non-malignant cells and can either foster therapy response or side effects/toxicities of radiation therapy. For a better understanding of the mechanisms by which IR modulates T-cell activation and alters functional properties of these immune cells, we exposed human immortalized Jurkat cells and peripheral blood lymphocytes (PBL) to X-ray doses between 0.1 and 5 Gy. This resulted in cellular responses, which are typically observed also in naïve T-lymphocytes in response of T-cell receptor immune stimulation or mitogens. These responses include oscillations of cytosolic Ca2+, an upregulation of CD25 surface expression, interleukin-2 and interferon-γ synthesis, elevated expression of Ca2+ sensitive K+ channels and an increase in cell diameter. The latter was sensitive to inhibition by the immunosuppressant cyclosporine A, Ca2+ buffer BAPTA-AM, and the CDK1-inhibitor RO3306, indicating the involvement of Ca2+-dependent immune activation and radiation-induced cell cycle arrest. Furthermore, on a functional level, Jurkat and PBL cell adhesion to endothelial cells was increased upon radiation exposure and was highly dependent on an upregulation of integrin beta-1 expression and clustering. In conclusion, we here report that IR impacts on immune activation and functional properties of T-lymphocytes that may have implications in both toxic effects and treatment response to combined radiation and immune therapy in cancer patients.

Radiosensitization of Prostate Cancer Cells by 2-Deoxyglucose

Prostate cancer is the most common malignancy of men. Treatment options include radiotherapy with or without hormonal manipulation and radical prostatectomy. However, there is no effective treatment for disseminated disease. A hallmark of malignancy is abnormal metabolism which also confers survival advantages and contributes to resistance to therapy. In response to exposure to ionizing radiation, metabolic pathways are activated which can protect the cell from irreversible injury. Tumor cell glycolytic activity is elevated and correlates with aggressiveness and radio resistance, indicating that targeting glucose metabolism may sensitize cancer cells to radiation. We have demonstrated that the clonogenic kill of PC3 cells induced by exposure to x-rays was enhanced by the glycolytic inhibitor 2-deoxyglucose (2DG). In contrast, treatment with 2DG failed to inhibit growth of multicellular spheroids derived from LNCaP cells. However, 2DG treatment, in the absence of irradiation, induced similar toxicity to PC3 and LNCaP cells cultured as monolayers. Radiation-induced cell cycle arrest was prevented by the simultaneous administration of 2DG in both cell lines, indicating a possible mechanism underlying sensitization. Therefore, we hypothesise that observed differences in cellular response to incubation with 2DG in the presence or absence of ionizing radiation resulted from variation in metabolic processes between tumor cell types. We conclude that inhibition of glucose metabolism by 2DG is an effective method for sensitizing prostate cancer cells to experimental radiotherapy and that this may occur by preventing DNA repair during radiation-induced cell cycle arrest.

Ascorbyl stearate and ionizing radiation potentiate apoptosis through intracellular thiols and oxidative stress in murine T lymphoma cells

Ascorbyl stearate (Asc-s) is a derivative of ascorbic acid with better anti-tumour efficacy compared to its parent compound ascorbic acid. In this study, we have examined radio-sensitizing effect of Asc-s in murine T cell lymphoma (EL4) cells at 4 Gy. Asc-s and radiation treatment reduced cell proliferation, induced apoptosis in a dose dependent manner by arresting the cells at S/G2-M phase of cell cycle. It also decreased the frequency of cancer stem cells per se, with significantly higher decrease in combination with radiation treatment./Further, Asc-s and radiation treatment increased the level of reactive oxygen species (ROS), drop in mitochondrial membrane potential (MMP) and increased caspase-3 activity resulting in apoptosis of EL4 cells. Further it also significantly decreased GSH/GSSG ratio due to binding of Asc-s with thiols. The increase in oxidative stress induced by Asc-s and radiation treatment was abrogated by thiol antioxidants in EL4 cells. Interestingly, this redox modulation triggered significant increase in protein glutathionylation in a time dependent manner. Asc-s treatment resulted in glutathionylation of IKK, p50-NF-kB and mutated p53, thereby inhibiting cancer progression during oxidative stress. Asc-s quenches GSH ensuing Asc-s + GSH adduct thereby further modulating GSH/GSSG ratio as evident from HPLC and docking studies. The anti-tumour effect of Asc-s along with radiation was studied by injecting EL4 cells in synegenicC57/BL6 male mice. Intraperitoneal injection of Asc-s followed by radiation exposure at 4 Gy to the tumour bearing mice resulted in radio-sensitization which is evident from significant regression of tumour as evident from tumour burden index. The survival study supports the data that Asc-s pre-treatment enhances radio-sensitization in murine lymphoma. Our data, suggest that Asc-s and ionizing radiation induced cell cycle arrest and apoptosis by perturbing redox balance through irreversible complexes of thiols with Asc-s, disturbed mitochondrial membrane permeability and activation of caspase-3 in EL4 cells.