Radiation Resistance Of Tumor Cells Research Articles

Activation of NLRP3 inflammasome in lung epithelial cells triggers radiation-induced lung injury

BackgroundRadiation-induced lung injury (RILI) is the most common and serious complication of chest radiotherapy. However, reported radioprotective agents usually lead to radiation resistance in tumor cells. The key to solving this problem is to distinguish between the response of tumor cells and normal lung epithelial cells to radiation damage.MethodsRNA-Seq was used to recognize potential target of alleviating the progression of RILI as well as inhibiting tumor growth. The activation of NLRP3 inflammasome in lung epithelial cells was screened by qRT-PCR, western blotting, immunofluorescence, and ELISA. An in vivo model of RILI and in vitro conditioned culture model were constructed to evaluate the effect of NLRP3/interleukin-1β on fibroblasts activation. ROS, ATP, and (NADP)+/NADP(H) level in lung epithelial cells was detected to explore the mechanism of NLRP3 inflammasome activation. The lung macrophages of the mice were deleted to evaluate the role of lung epithelial cells in RILI. Moreover, primary cells were extracted to validate the results obtained from cell lines.ResultsNLRP3 activation in epithelial cells after radiation depends on glycolysis-related reactive oxygen species accumulation. DPYSL4 is activated and acts as a negative regulator of this process. The NLRP3 inflammasome triggers interleukin-1β secretion, which directly affects fibroblast activation, proliferation, and migration, eventually leading to lung fibrosis.ConclusionsOur study suggests that NLRP3 inflammasome activation in lung epithelial cells is essential for radiation-induced lung injury. These data strongly indicate that targeting NLRP3 may be effective in reducing radiation-induced lung injury in clinical settings.

PVP-Modified Multifunctional Bi2WO6 Nanosheets for Enhanced CT Imaging and Cancer Radiotherapy.

Malignant tumors are one of the main causes of human death. The clinical treatment of malignant tumors is usually surgery, chemotherapy, radiotherapy, and so forth. Radiotherapy, as a traditional and effective treatment method for cancer, is widely used in clinical practice, but the radiation resistance of tumor cells and the toxic side effects to normal cells are still the Achilles heel of radiotherapy. Multifunctional inorganic high-atom nanomaterials are expected to enhance the effect of tumor radiotherapy. Tungsten and bismuth, which contain elements with high atomic coefficients, have strong X-ray energy attenuation capability. We synthesized Bi2WO6 nanosheets (NSs) using a hydrothermal synthesis method and modified polyvinylpyrrolidone (PVP) on their surface to make them more stable. PVP–Bi2WO6 NSs have a variety of effects after absorbing X-rays (such as the photoelectric effect and Compton effect) and release a variety of particles such as photoelectrons, Compton electrons, auger electrons, and so forth, which can react with organic molecules or water in cells, generate a large number of free radicals, and promote cell apoptosis, thereby improving the effect of radiotherapy. We show through γ-H2AX and DCFH-DA probe analysis experiments that PVP–Bi2WO6 NSs can effectively increase cell DNA damage and reactive oxygen species formation under X-ray irradiation. Clone formation analysis showed that PVP–Bi2WO6 NSs can effectively suppress cell colony formation under X-ray irradiation. These versatile functions endow PVP–Bi2WO6 NSs with enhanced radiotherapy efficacy in animal models. In addition, PVP–Bi2WO6 NSs can also be used as contrast agents for X-ray computed tomography (CT) imaging with obvious effects. Therefore, PVP–Bi2WO6 NSs can be used as CT imaging contrast agents and tumor radiotherapy sensitizers and have potential medical applications.

Size-changeable nanoprobes for the combined radiotherapy and photodynamic therapy of tumor

Radiation therapy (RT) and photodynamic therapy (PDT) are promising while challenging in treating tumors. The potential radiation resistance of tumor cells and side effects to healthy tissues restrict their clinical treatment efficacy. Effective delivery of therapeutic agents to the deep tumor tissues would be available for tumor-accurate therapy and promising for the tumor therapy. Thus, developing nanoprobes with effectively delivering radiotherapy sensitizers and photosensitizers to the interior of tumors is needed for the accurate combined RT and PDT of tumor. The size-changeable nanoprobes of Gd2O3@BSA-BSA-Ce6 (BGBC) were synthesized with a crosslinking method. Magnetic resonance imaging (MRI) and in vivo near-infrared (NIR) imaging were measured to evaluate the nanoprobes' tumor accumulation and intratumor penetration effect. The tumor suppression effect of combined RT and PDT with these nanoprobes was also studied for the 4T1 bearing Balb/c mice. The nanoprobes BGBC showed high tumor accumulation and disintegrated into small particles responding to the photo-irradiation-produced reactive oxygen species (ROS), allowing for tumor penetration. Abundant radiotherapy sensitizers and photosensitizers were delivered to the deep tumor tissues, which is available for the accurate therapy of tumor. In addition, the BGBC displayed outstanding MRI and fluorescence imaging effects for evaluating the biodistribution and tumor suppression effect of nanoprobes. Consequently, significant tumor suppression effect was obtained based on the accurate tumor treatment with the combined RT and PDT. The designed size-changeable nanoprobes BGBC showed excellent tumor accumulation and deep tumor penetration, resulting in a significant tumor suppression effect based on the combined RT and PDT. This study provides a novel strategy for dual delivery of radiotherapy sensitizers and photosensitizers into the deep tumor tissues and is promising for the accurate theranostics of tumor.

Role of non-coding RNAs in response of breast cancer to radiation therapy.

Breast cancer ranks as the first common cancer with a high incidence rate and mortality among women. Radiation therapy is the main therapeutic method for breast cancer patients. However, radiation resistance of tumor cells can reduce the efficacy of treatment and lead to recurrence and mortality in patients. Non-coding RNA (ncRNAs) refers to a group of small RNA molecules that are not translated into protein, while they have the ability to modulate the translation of target mRNA. Several studies have reported the altered expression of ncRNAs in response to radiation in breast cancer. NcRNAs have been found to influence on radiation response of breast cancer by regulating various mechanisms, including DNA damage response, cell cycle regulation, cell death, inflammatory response, cancer stem cell and EGFR related pathways. This paper aimed to provide a summary of current findings on ncRNAs dysregulation after irradiation. We also present the function and mechanism of ncRNAs in modulating radiosensitivity or radioresistance of breast cancer cells.

Research Progress in CircRNA and Radiotherapy Resistance of Non-small Cell Lung Cancer

非小细胞肺癌（non-small cell lung cancer, NSCLC）作为临床上较为常见的癌症，主要特点是患者5年生存率低、预后偏差。目前有部分研究认为生存率低、预后偏差是由于环状RNA（circluar RNA, circRNA）引起了放疗抵抗。因此，找出circRNA与NSCLC放疗抵抗之间的联系，可为改善临床治疗提供理论基础。通过查阅相关文献，找出circRNA与NSCLC放疗抵抗的关系。CircRNA在NSCLC细胞的侵袭、转移、增殖和治疗抵抗中起着重要的作用，其中它所诱导的肿瘤细胞放疗抵抗已经成为放射治疗上的一个重点关注问题。CircRNA在NSCLC的放疗抵抗中起着重要作用。

Potential Biomarkers in Diagnosis, Prognosis and Prediction of Treatment Response in Malignant Glioma

Gliomas are the most common type of primary central nervous system malignancies with poor prognosis in adults. There are several challenges in developing a treatment protocol for this malignancy including presence of blood-brain barrier that inhibit drug delivery to brain tissue, drug and radiation resistance of tumor cells, and inter and intra-tumor heterogeneity of glioma. In addition, early treatment assessment is difficult for glioma patients because of phenomenon of pseudo-progression. Due to the challenges involved in treatment and monitoring of treatment response for glioma, it is very helpful to identify specific and non-invasive molecular and imaging markers in order to provide useful prognostic information. The aim of this article is to summarize several potential biological and imaging markers regarding malignant glioma. A brief description of the proteins involved in the glioma signaling pathways is provided in order to introduce potential biological markers. Furthermore, the role of imaging techniques in treatment management is discussed. Finally, correlation between tumor characteristics and values of angiogenesis and physiological factors measured in perfusion magnetic resonance imaging techniques as well as metabolites in MRS, and PET tracer’s uptake is investigated.

MicroRNA-29b regulates the radiosensitivity of esophageal squamous cell carcinoma by regulating the BTG2-mediated cell cycle.

Many patients with esophageal squamous cell carcinoma (ESCC) are inoperable due to old age or advanced stage; thus, radio- and chemotherapy are considered the standard treatments for these patients. However, due to the radiation resistance of tumor cells that may arise during radiotherapy, results are still not satisfactory. The authors' previous studies found that microRNA can affect radiosensitivity, and further microRNA research was conducted to improve the radiosensitivity of ESCC. Cells were treated with silent miR-29b (si-miR-29b). Thereafter,proliferation, colony formation, cell cycle, and apoptosis were determined. The luciferase reporting assay was used to confirm the direct interaction between miR-29b and BTG2. Serum samples and clinical follow-up data of 75elderly or advanced ESCC patients who could not tolerate surgery were collected. The expression level of miR-29 in ESCC serum was closely correlated to radiosensitivity (χ2 =8.36, p < 0.05) and correlated with overall survival (OS; hazard ratio [HR] 0.47, 95% confidence interval [CI] 0.24-0.90). Function assays demonstrated that the number of cell clones increased after radiometry radiation, and the cell cycle was blocked in the G0/G1 phase (from 37.2 to 56.9%) in the si-miR-29b transfection group. Expression of BTG2 was upregulated and expression of cyclin D1 was downregulated (p < 0.05). Transfection of si-BTG2 can reverse this result and restore the expression level of cyclin D1 (p < 0.05). The target gene BTG2 of miR-29b was predicted using abioinformatics tool and confirmed by dual-luciferase reporter assay. Silencing of miR-29b in ESCC cells can increase expression of BTG2 and decrease the level of intracellular cyclin D1, resulting in cell cycle arrest and accumulation in the G0/G1 phase. Because G0/G1-phase cells are insensitive to radiotherapy, the sensitivity of radiotherapy is reduced.

Health Informatics of Composite Nanoparticles as an Effective Contrast Agent in Magnetic Resonance Imaging and for Glioblastoma Radiosensitization

Background: Radiation therapy is a method using radiation to treat tumors, and has become commonly used and most effective treatments for malignant tumors. However, radiotherapy still has the shortcomings of high radiation dose, especially radiation resistance of tumor cells. Multifunctional nanoradiosensitizers can enhance the radiosensitivity of tumor cells and improving the effect of radiotherapy. Hyaluronic acid-functionalized ironbismuth composite particles are an effective contrast agent for magnetic resonance imaging (MRI), and can be used as radiosensitizers. Method: Hyaluronic acid-functionalized iron-bismuth composite nanoparticles HABilOPs can be prepared by a hydrothermal polyol method. In the cytotoxicity experiment, we took human glioblastoma cell line U87MG in logarithmic growth phase and rat vascular smooth muscle cells. We set different mass concentrations of 0, 15, 30, 45, 60, 100, and 300 mg/LHA-BMOPs was cultured for 48 h to calculate the cell proliferation rate. In the histocompatibility experiment, we injected HA-BilOPs solution into the tail vein of ICR mice to observe the pathological changes of mouse organs. In the radiosensitization experiment, we took U87MG cells in logarithmic growth phase for group culture. X-ray irradiation of 0, 3, 6, 9 Gy was given, and the HA-BilOPs group was added with culture medium of different mass concentration of LHA-BilOPs, and the combined group was added to the culture medium of different mass concentration of HA-BilOPs first, and then given X-ray irradiation of 0, 3, 6, 9 Gy. To analyse the features of glioblastoma, the conventional MRI manifestations of glioblastoma were examined. Results: We found that different concentrations of HA-BilOPs had no significant effect on the proliferation of vascular smooth muscle cells and U87MG cells. It was found that no pathological changes occurred in mouse organs after tail vein injection of HA-BilOPs solution. Nanoparticles can be phagocytosed by U87MG cells and it was found that U87MG cell proliferation rate had a significant linear negative correlation with HA-BilOPs mass concentration (0–200 mg/L) and radiation dose (0–9 Gy). At 6 Gy X Under irradiation, 200 mg/L HA-BilOPs reduced the cell proliferation rate to (41± 7)%. Using 100 mg/L HA-BilOPs and 6 Gy X-ray irradiation for colony formation experiments, the proliferation rate of U87MG cells in the combined group was statistically lower than that of the blank control group and radiotherapy group (P &lt; 0.05). Conclusion: Hyaluronic acid-functionalized iron-bismuth composite nanoparticles have a significant radiosensitizing effect on glioblastoma and proved to be a good contrast agent used in MRI.

Semiconductor heterojunction-based radiocatalytic platforms for tumors treatment by enhancing radiation response and reducing radioresistance

Radiotherapy is usually accompanied by side effects to healthy tissue and radiation resistance of tumor cells in clinical application. Therefore, how to enhance radiation effect and reduce radiation resistance is the key problem to enhance radiotherapy efficacy. In recent years, the development of nanomaterials with radiation catalysis function has provided us with opportunities. Here, we report a radiocatalytic platform using inorganic semiconductor nanoheterojunctions WO3@Ag2WO4@chitosan (WO3@Ag2WO4@CS) as radiation sensitizers to improve the radiotherapeutic effect of tumors by enhancing radiosensitization and reducing radioresistance. On one hand, WO3@Ag2WO4@CS with a higher atomic number has better X-ray absorptivity and can deposit a large amount of radiation dose upon X-ray irradiation. In addition, due to the heterojunction structure, a large number of electron-hole pairs can be generated and separated under X-ray trigger. The WO3@Ag2WO4@CS nanoheterojunctions thus can effectively decompose the surrounding hydrogen peroxide/water into free radicals to kill cancer cells by electron-hole pairs. On the other hand, WO3@Ag2WO4@CS nanoheterojunctions also have the ability to consume intracellular GSH through redox reaction, leading to increased ROS level and reduced radioresistance. Our study provides a novel nanoradiosensitizer that can improve the radiotherapeutic efficacy by enhancing the radiation response and reducing radioresistance of tumors.

Depletion of Akt1 and Akt2 Impairs the Repair of Radiation-Induced DNA Double Strand Breaks via Homologous Recombination.

Homologous recombination repair (HRR), non-homologous end-joining (NHEJ) and alternative NHEJ are major pathways that are utilized by cells for processing DNA double strand breaks (DNA-DSBs); their function plays an important role in the radiation resistance of tumor cells. Conflicting data exist regarding the role of Akt in homologous recombination (HR), i.e., the regulation of Rad51 as a major protein of this pathway. This study was designed to investigate the specific involvement of Akt isoforms in HRR. HCT116 colon cancer cells with stable AKT-knock-out and siRNA-mediated AKT-knockdown phenotypes were used to investigate the role of Akt1 and Akt2 isoforms in HR. The results clearly demonstrated that HCT116 AKT1-KO and AKT2-KO cells have a significantly reduced Rad51 foci formation 6 h post irradiation versus parental cells. Depletion of Akt1 and Akt2 protein levels as well as inhibition of Akt kinase activity resulted in an increased number of residual-γH2AX in CENP-F positive cells mainly representing the S and G2 phase cells. Furthermore, inhibition of NHEJ and HR using DNA-PK and Rad51 antagonists resulted in stronger radiosensitivity of AKT1 and AKT2 knockout cells versus wild type cells. These data collectively show that both Akt1 and Akt2 are involved in DSBs repair through HRR.

Replication Stress Drives Constitutive Activation of the DNA Damage Response and Radioresistance in Glioblastoma Stem-like Cells.

Glioblastoma (GBM) is a lethal primary brain tumor characterized by treatment resistance and inevitable tumor recurrence, both of which are driven by a subpopulation of GBM cancer stem-like cells (GSC) with tumorigenic and self-renewal properties. Despite having broad implications for understanding GSC phenotype, the determinants of upregulated DNA-damage response (DDR) and subsequent radiation resistance in GSC are unknown and represent a significant barrier to developing effective GBM treatments. In this study, we show that constitutive DDR activation and radiation resistance are driven by high levels of DNA replication stress (RS). CD133+ GSC exhibited reduced DNA replication velocity and a higher frequency of stalled replication forks than CD133- non-GSC in vitro; immunofluorescence studies confirmed these observations in a panel of orthotopic xenografts and human GBM specimens. Exposure of non-GSC to low-level exogenous RS generated radiation resistance in vitro, confirming RS as a novel determinant of radiation resistance in tumor cells. GSC exhibited DNA double-strand breaks, which colocalized with "replication factories" and RNA: DNA hybrids. GSC also demonstrated increased expression of long neural genes (>1 Mbp) containing common fragile sites, supporting the hypothesis that replication/transcription collisions are the likely cause of RS in GSC. Targeting RS by combined inhibition of ATR and PARP (CAiPi) provided GSC-specific cytotoxicity and complete abrogation of GSC radiation resistance in vitro These data identify RS as a cancer stem cell-specific target with significant clinical potential.Significance: These findings shed new light on cancer stem cell biology and reveal novel therapeutics with the potential to improve clinical outcomes by overcoming inherent radioresistance in GBM. Cancer Res; 78(17); 5060-71. ©2018 AACR.

Abstract 1326: Nonprotective autophagy fails to confer resistance to radiation

Abstract Radiation remains a predominant treatment option for advanced, inoperable lung cancer, however, resistance represents a major barrier against effective therapy. The exact mechanisms through which cancer cells exhibit resistance to radiotherapy still remains unresolved. Traditionally, radiation-induced autophagy is considered as a mechanism of resistance which attenuates the antitumor effects of radiotherapy i.e., autophagy plays a cytoprotective role on cell survival. To examine this concept further, we employed H460 non-small cell lung cancer (NSCLC) cells that were either p53 wild-type (p53 +/+) or p53 null (p53 -/-) to study the contribution of autophagy to radiation sensitivity. While autophagy was markedly higher in p53 +/+ cells (determined by p62 degradation, LC3B conversion, and acidic vesicle formation) the p53 +/+ cells were actually more radiosensitive than their counterpart H460 p53 -/- cells, which showed lower levels of radiation-induced autophagy. Despite modest radiosensitization by chloroquine, alternative pharmacological (3-methly adenine) and genetic (ATG5 silencing) inhibition of autophagy failed to radiosensitize either p53 +/+ or p53 -/- cells, indicating that radiation induced autophagy is not cytoprotective in function in these experimental models. Furthermore, secretome analysis (nLC-MS/MS) of p53 +/+ H460 cells showed no significant association with radioresistance-related proteins in comparison with p53 -/- H460 cells. The rate and extent of apoptosis was quite low and similar in the two cells lines, essentially ruling out apoptotic cell death as the basis for differential radiation sensitivity. Finally, Senescence was more pronounced in the p53 +/+ cells compared to the p53 -/- cells, which may contribute to the greater radiation sensitivity in the p53 +/+ cells. However, the most relevant finding in this work was that when autophagy is the nonprotective form, it does not confer inherent radiation resistance in tumor cells. Citation Format: Jingwen Xu, Emmanuel Kenneth Cudjoe, Tareq Saleh, Nipa Patel, Moureq Alotaibi, Yingliang Wu, Santiago Lima, David Gewirtz. Nonprotective autophagy fails to confer resistance to radiation [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 1326.

Patterns of local-regional failure after intensity-modulated radiotherapy for nasopharyngeal carcinoma

Objective To investigate the dosimetric patterns of local-regional after intensity-modulated radiotherapy (IMRT) for nasopharyngeal carcinoma (NPC), and to provide a clinical basis for further improvement in the therapeutic effect for NPC. Methods The patterns of local-regional in 364 previously untreated NPC patients without distant metastasis who were admitted to our hospital from December 2007 to April 2012 were analyzed. The failures were classified as failure if Vrecur within the 95% isodose curve (V95%) was ≥95%, failure if V95% was less than 95% and not less than 20%, or failure if V95% was<20%. The Kaplan-Meier method was used to calculate survival rates. Results The median follow-up was 40 months (range 3-84 months). The 3-year local failure-free survival, regional failure-free survival, distant metastasis-free survival, overall survival, and progression-free survival were 94.1%, 95.4%, 86.5%, 90.9%, and 78.7%, respectively. A total of 35 patients experienced local-regional recurrence, and in-field recurrence, marginal recurrence, and out-field accounted for 81.1%, 8.1%, and 10.8%, respectively. Conclusions In-field failures are the main dosimetric patterns of local-regional recurrence after IMRT for NPC, suggesting that radiation resistance of tumor cells is an important reason for local-regional failure. Key words: Nasopharyngeal neoplasms/intensity-modulated radiotherapy; Recurrence; Patterns of local-regional failure; Dosimetric analysis

Effects of HERV-R env Knockdown in Combination with Ionizing Radiation on Apoptosis-Related Gene Expression in A549 Lung Cancer Cells

Radiotherapy has played a key role in the management of non–small-cell lung cancer (NSCLC). However, the use of radiotherapy in treating NSCLC is limited because of the intrinsic radiation resistance of tumor cells and injury to adjacent normal tissues. Many oncogenes are reported to be involved in radioresistance. Thus, novel moleculartargeting approaches to enhance the radiosensitivity of NSCLC cells are required to improve the therapeutic efficiency of radiotherapy. In this study, we report that expression of the human endogenous retrovirus-R (HERV-R) env gene is greatly elevated in γ-irradiation resistant A549 cells compared with radiation sensitive H460 cells. In addition, the HERV-R env gene was significantly increased in A549 cells after treatment with γ-irradiation. HERV-R env knockdown by siRNA in irradiated A549 cells led to overexpression of TP53 mRNA, followed by significant elevation in the levels of CDKN1A mRNA. Moreover, the expression of the apoptosis-related FAS-1 gene was increased, whereas the expression levels of the anti-apoptotic gene BCL2 were significantly decreased in the A549 cells in which the HERV-R env was suppressed by γ-irradiation. These results suggest that knockdown of HERV-R env with γ-irradiation causes cell cycle disturbances, which in turn induces apoptosis. In conclusion, the combination of HERV-R env knockdown and γ-irradiation has the potential to improve the therapeutic efficiency of radiotherapy for NSCLC.

Targeting Notch to overcome radiation resistance.

Radiotherapy represents an important therapeutic strategy in the treatment of cancer cells. However, it often fails to eliminate all tumor cells because of the intrinsic or acquired treatment resistance, which is the most common cause of tumor recurrence. Emerging evidences suggest that the Notch signaling pathway is an important pathway mediating radiation resistance in tumor cells. Successful targeting of Notch signaling requires a thorough understanding of Notch regulation and the context-dependent interactions between Notch and other therapeutically relevant pathways. Understanding these interactions will increase our ability to design rational combination regimens that are more likely to be safe and effective. Here we summarize the role of Notch in mediating resistance to radiotherapy, the different strategies to block Notch in cancer cells and how treatment scheduling can improve tumor response. Finally, we discuss a need for reliable Notch related biomarkers in specific tumors to measure pathway activity and to allow identification of a subset of patients who are likely to benefit from Notch targeted therapies.

DNA Double-Strand Break Repair as Determinant of Cellular Radiosensitivity to Killing and Target in Radiation Therapy

Radiation therapy plays an important role in the management of a wide range of cancers. Besides innovations in the physical application of radiation dose, radiation therapy is likely to benefit from novel approaches exploiting differences in radiation response between normal and tumor cells. While ionizing radiation induces a variety of DNA lesions, including base damages and single-strand breaks, the DNA double-strand break (DSB) is widely considered as the lesion responsible not only for the aimed cell killing of tumor cells, but also for the general genomic instability that leads to the development of secondary cancers among normal cells. Homologous recombination repair (HRR), non-homologous end-joining (NHEJ), and alternative NHEJ, operating as a backup, are the major pathways utilized by cells for the processing of DSBs. Therefore, their function represents a major mechanism of radiation resistance in tumor cells. HRR is also required to overcome replication stress – a potent contributor to genomic instability that fuels cancer development. HRR and alternative NHEJ show strong cell-cycle dependency and are likely to benefit from radiation therapy mediated redistribution of tumor cells throughout the cell-cycle. Moreover, the synthetic lethality phenotype documented between HRR deficiency and PARP inhibition has opened new avenues for targeted therapies. These observations make HRR a particularly intriguing target for treatments aiming to improve the efficacy of radiation therapy. Here, we briefly describe the major pathways of DSB repair and review their possible contribution to cancer cell radioresistance. Finally, we discuss promising alternatives for targeting DSB repair to improve radiation therapy and cancer treatment.

γ-radiation induces cellular sensitivity and aberrant methylation in human tumor cell lines

Purpose: Ionizing radiation induces cellular damage through both direct and indirect mechanisms, which may include effects from epigenetic changes. The purpose of this study was to determine the effect of ionizing radiation on DNA methylation patterns that may be associated with altered gene expression.Materials and methods: Sixteen human tumor cell lines originating from various cancers were initially tested for radiation sensitivity by irradiating them with γ-radiation in vitro and subsequently, radiation sensitive and resistant cell lines were treated with different doses of a demethylating agent, 5-Aza-2′-Deoxycytidine (5-aza-dC) and a chromatin modifier, Trichostatin-A (TSA). Survival of these cell lines was measured using 3-(4, 5-Dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium (MTT) and clonogenic assays. The effect of radiation on global DNA methylation was measured using reverse phase high performance liquid chromatography (RP-HPLC). The transcription response of methylated gene promoters, from cyclin-dependent kinase inhibitor 2A (p16INK4a) and ataxia telangiectasia mutated (ATM) genes, to radiation was measured using a luciferase reporter assay.Results: γ-radiation resistant (SiHa and MDAMB453) and sensitive (SaOS2 and WM115) tumor cell lines were examined for the relationship between radiation sensitivity and DNA methylation. Treatment of cells with 5-aza-dC and TSA prior to irradiation enhanced DNA strand breaks, G2/M phase arrest, apoptosis and cell death. Exposure to γ-radiation led to global demethylation in a time-dependent manner in tumor cells in relation to resistance and sensitivity to radiation with concomitant activation of p16INK4a and ATM gene promoters.Conclusion: These results provide important information on alterations in DNA methylation as one of the determinants of radiation effects, which may be associated with altered gene expression. Our results may help in delineating the mechanisms of radiation resistance in tumor cells, which can influence diagnosis, prognosis and eventually therapy for human cancers.

Interference with Activator Protein-2 transcription factors leads to induction of apoptosis and an increase in chemo- and radiation-sensitivity in breast cancer cells

BackgroundActivator Protein-2 (AP-2) transcription factors are critically involved in a variety of fundamental cellular processes such as proliferation, differentiation and apoptosis and have also been implicated in carcinogenesis. Expression of the family members AP-2α and AP-2γ is particularly well documented in malignancies of the female breast. Despite increasing evaluation of single AP-2 isoforms in mammary tumors the functional role of concerted expression of multiple AP-2 isoforms in breast cancer remains to be elucidated. AP-2 proteins can form homo- or heterodimers, and there is growing evidence that the net effect whether a cell will proliferate, undergo apoptosis or differentiate is partly dependent on the balance between different AP-2 isoforms.MethodsWe simultaneously interfered with all AP-2 isoforms expressed in ErbB-2-positive murine N202.1A breast cancer cells by conditionally over-expressing a dominant-negative AP-2 mutant.ResultsWe show that interference with AP-2 protein function lead to reduced cell number, induced apoptosis and increased chemo- and radiation-sensitivity. Analysis of global gene expression changes upon interference with AP-2 proteins identified 139 modulated genes (90 up-regulated, 49 down-regulated) compared with control cells. Gene Ontology (GO) investigations for these genes revealed Cell Death and Cell Adhesion and Migration as the main functional categories including 25 and 12 genes, respectively. By using information obtained from Ingenuity Pathway Analysis Systems we were able to present proven or potential connections between AP-2 regulated genes involved in cell death and response to chemo- and radiation therapy, (i.e. Ctgf, Nrp1, Tnfaip3, Gsta3) and AP-2 and other main apoptosis players and to create a unique network.ConclusionsExpression of AP-2 transcription factors in breast cancer cells supports proliferation and contributes to chemo- and radiation-resistance of tumor cells by impairing the ability to induce apoptosis. Therefore, interference with AP-2 function could increase the sensitivity of tumor cells towards therapeutic intervention.

The Suppression of Hypoxia-inducible Factor and Vascular Endothelial Growth Factor by siRNA Does not Affect the Radiation Sensitivity of Multicellular Tumor Spheroids

The hypoxic microenvironment is closely associated with the radiation resistance of tumor cells. Hypoxia induces several genes such as hypoxia-inducible factor (HIF-1) and vascular endothelial growth factor (VEGF) to promote tumor cell growth and survival. The up-regulated expression levels of HIF-1 and VEGF in tumor cells also correlate with their resistance to radiation, suggesting that these genes are potential therapeutic targets for strategies designed to enhance radiation effects. To further investigate this possibility, we investigated the effects of suppressing these genes upon the radiation sensitivity of cancer cells. We conducted these experiments using multicellular spheroids as a three-dimensional in vitro tumor model and RNA interference as the method of gene suppression. SQ5 human lung carcinoma cells were treated with HIF-1/VEGF siRNA and/or radiation. Reversed transfection methods were employed for the spheroids. Gene expression was analyzed using quantitative RT-PCR and western blotting. Cell toxicity was qualified by colony formation assay. Compared with monolayer cells, spheroids showed up-regulated expression of HIF-1 and increased radiation resistance. Hypoxic conditions elevated the expression of HIF-1 and VEGF and enhanced the surviving fraction of spheroids after exposure to radiation. However, when the expression of HIF-1 and VEGF was down-regulated by transfection of targeting siRNA, this did not influence the cytotoxic effects of the radiation under either normoxic or hypoxic conditions. We have established a method to transfect siRNA into spheroid cells. Our current data indicate that the functions of HIF-1 or VEGF are independent of radiation sensitivity in spheroids under either normoxic or hypoxic conditions.

HSV-1 amplicon-mediated post-transcriptional inhibition of Rad51 sensitizes human glioma cells to ionizing radiation

Standard treatment for glioblastoma multiforme and other brain tumors consists of surgical resection followed by combined radio-/chemotherapy. However, radiation resistance of tumor cells limits the success of this treatment, and the tumors invariably recur. Therefore, the selective inhibition of molecular mediators of radiation resistance may provide therapeutic benefit to the patient. One of these targets is the Rad51 protein, which is a key component of the homologous recombinational repair of DNA double-strand breaks. Here, we investigated whether post-transcriptional silencing of Rad51 by herpes simplex virus-type 1 (HSV-1) amplicon vector-mediated short interfering RNA expression can enhance the antitumor effect of radiation therapy. We demonstrate that these vectors specifically and efficiently inhibited the radiation-induced recruitment of Rad51 into nuclear foci in human glioma cells. The combination of vector-mediated silencing of Rad51 expression and treatment with ionizing radiation resulted in a pronounced reduction of the survival of human glioma cells in culture. In athymyc mice, a single intratumoral injection of Rad51-specific HSV-1 amplicon vector followed by a single radiation treatment resulted in a significant decrease in tumor size. In control animals, including mice that received an intratumoral injection of Rad51-specific amplicon vector but no radiation treatment, the tumor sizes increased.