Radiation Sensitivity Of Cells Research Articles

PS02.060: EXPLORATION OF RADIOSENSITIVITY-RELATED LNCRNA IN ESOPHAGEAL CANCER STEM CELL

Abstract Background Esophageal cancer stem cells are the most radiation-resistant cells, to study radiosensitivity with esophageal cancer stem cells is a better method, Previously study was only the relevant microRNA research, so we explore radiosensitivity-related LncRNA in esophageal cancer stem cell. Methods The Eca109 cells were divided into tumor sphere cells group and adherent cells group by using suspended and adherent culture. Differences in stem cell characteristics of two groups were compared by CD44 positive expression rate, CCK8 cell proliferation and plate cloning formation. Using Arraystar LncRNA microarray analysis the gene expression differences of CD44+ esophageal cancer stem cells and CD44− esophageal cancer cells sorting out by flow separation technology. Bioinformatics analysis and real-time PCR were applied to verified the specific LncRNA related to radiation sensitivity of esophageal cancer stem cells. Results 1.The result of CCK8 cell proliferation experience showed that the absorbance value of the tumor sphere cells group was significantly higher than and adherent cells group after 24h, 48h and 72h (P < 0.05). The cell clone formation rate of the tumor sphere cells group was higher than adherent cells group (P < 0.05). 2. The plate cloning formation showed that the values of D0, Dq, N and SF2 were significantly higher in the tumor sphere cells group than adherent cells group, and the radiation sensitization ratio was 1.556. 3. Arraystar LncRNA chip analysis showed following the criteria that DEGseq at |log2Ratio| ≥ 1和q < 0.05, 4961 DEGs including 2517 up-regualted genes and 2444 down-regulated genes were identified. 4. We verified the top 10 difference LncRNA genes by realtime PCR. The results showed that MSTRG. 73,085 was down-regulated and its expression level on average was 20.7 times (chip was 49.3 times), CH17–360D5.2, RP11–439E19.10, MSTRG.424, MSTRG.64087, MSTRG.15903 was up-regulated and their expression level on average were 2.7, 8.7, 3.7, 5, 7 (chip were 8.2, 5.2, 78.9, 3, 4.7 times). Conclusion We verified the different LncRNA genes by arraystar LncRNA chip and realtime PCR, The results showed that MSTRG. 73,085 was down-regulated and CH17–360D5.2, RP11–439E19.10, MSTRG.424, MSTRG.64087, MSTRG.15903 was up-regulated. These LncRNA genes may be related to the radiosensitivity of esophageal cancer stem cells and could be a potential new target for esophageal squamous carcinoma. Disclosure All authors have declared no conflicts of interest.

Salvianolic acid B renders glioma cells more sensitive to radiation via Fis-1-mediated mitochondrial dysfunction

Glioma remains the leading cause of brain tumor-related death worldwide, and radiation is a standard adjuvant therapy with proven efficacy. Salvianolic acid B (SalB), a bioactive compound isolated from Radix Salviae, has been shown to exert anti-cancer effects in many cancer cell lines, including glioma. This study aimed to investigate whether SalB could affect response to radiation in human glioma cells. We found that SalB decreased cell viability of U87 cells in a-dose-dependent manner. A subthreshold dose of SalB at 0.5 μM, which had no effect on cell viability and apoptosis, significantly increased radiation sensitivity of U87 cells in a dose- and time-dependent manner, but had no effect on sensitivity to temozolomide (TMZ). Similar results were also observed in human glioma U373 cells. In addition, SalB aggravated the radiation-induced apoptosis and mitochondrial dysfunction, as measured by mitochondrial Ca2+ buffering capacity and mitochondrial swelling. SalB treatment markedly promoted mitochondrial fission and differently regulated the expression of fission proteins. Furthermore, downregulation of the fission protein Fis-1 using siRNA was found to partially reversed the SalB-induced effects on cell viability, apoptosis and mitochondrial fission in U87 cells. In conclusion, our results suggest that a subthreshold dose of SalB renders glioma cells more sensitive to radiation via Fis-1-mediated mitochondrial dysfunction, and radiotherapy combined with SalB might be a novel treatment for glioma patients.

Silencing the expression of MTDH increases the radiation sensitivity of SKOV3 ovarian cancer cells and reduces their proliferation and metastasis.

Ovarian cancer has a high mortality rate among women worldwide. Radiotherapy is considered an effective method of ovarian cancer treatment, however, radioresistance presents a challenge. It is necessary to develop techniques that can increase radiosensitivity in ovarian cancer, and gene therapy is a promising option. The aim of the present study was to investigate the effects of metadherin (MTDH) silencing on the radiosensitivity of ovarian cancer. Ovarian cancer tissues (n=273) and normal ovarian tissues (n=277) were used, as were SKOV3 ovarian cancer cells and the immortalized human ovarian epidermal HOSEpiC cell line. MTT, Transwell and wound-healing assays were performed to assess the proliferation, invasion and migration abilities of the SKOV3 cells. Colony-forming assays and flow cytometry were applied to detect the radiosensitivity and apoptosis of the SKOV3 cells. Nude mouse xenograft models were established to evaluate the effect of MTDH gene silencing on tumor growth and the efficacy of radiotherapy. Ovarian cancer, in tissues and cells, was demonstrated to have a high level of MTDH. Additionally, MTDH silencing was found to significantly inhibit proliferation, migration and invasion, and induce apoptosis in SKOV3 cells, and it was suggested that MTDH depletion significantly increased the sensitivity of the SKOV3 cells to X-ray radiation. MTDH silencing enhanced radiosensitivity and delayed tumor growth in the nude mouse xenograft model. Collectively, the results obtained in the present study suggest the potential role of MTDH silencing as a technique for ameliorating radioresistance in ovarian cancer. The present study provides a promising experimental basis for the improvement of ovarian cancer radiotherapy treatment.

Differential Radiation Sensitivity in p53 Wild-Type and p53-Deficient Tumor Cells Associated with Senescence but not Apoptosis or (Nonprotective) Autophagy.

Studies of radiation interaction with tumor cells often focus on apoptosis as an end point; however, clinically relevant doses of radiation also promote autophagy and senescence. Moreover, functional p53 has frequently been implicated in contributing to radiation sensitivity through the facilitation of apoptosis. To address the involvement of apoptosis, autophagy, senescence and p53 status in the response to radiation, the current studies utilized isogenic H460 non-small cell lung cancer cells that were either p53-wild type (H460wt) or null (H460crp53). As anticipated, radiosensitivity was higher in the H460wt cells than in the H460crp53 cell line; however, this differential radiation sensitivity did not appear to be a consequence of apoptosis. Furthermore, radiosensitivity did not appear to be reduced in association with the promotion of autophagy, as autophagy was markedly higher in the H460wt cells. Despite radiosensitization by chloroquine in the H460wt cells, the radiation-induced autophagy proved to be essentially nonprotective, as inhibition of autophagy via 3-methyl adenine (3-MA), bafilomycin A1 or ATG5 silencing failed to alter radiation sensitivity or promote apoptosis in either the H460wt or H460crp53 cells. Radiosensitivity appeared to be most closely associated with senescence, which occurred earlier and to a greater extent in the H460wt cells. This finding is consistent with the in-depth proteomics analysis on the secretomes from the H460wt and H460crp53 cells (with or without radiation exposure) that showed no significant association with radioresistance-related proteins, whereas several senescence-associated secretory phenotype (SASP) factors were upregulated in H460wt cells relative to H460crp53 cells. Taken together, these findings indicate that senescence, rather than apoptosis, plays a central role in determination of radiosensitivity; furthermore, autophagy is likely to have minimal influence on radiosensitivity under conditions where autophagy takes the nonprotective form.

Palbociclib enhances radiosensitivity of hepatocellular carcinoma and cholangiocarcinoma via inhibiting ataxia telangiectasia–mutated kinase–mediated DNA damage response

AimPalbociclib is an oral cyclin-dependent kinase 4/6 inhibitor, which is efficacious in treating breast cancer. Currently, there are numerous active clinical trials testing palbociclib alone or in combination with other medications for treating various types of malignancies. Here, we evaluated the anti-cancer effect of palbociclib in combination with radiation therapy (RT) for treating human hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) and addressed the molecular mechanism behind the combination therapy. MethodsImmunofluorescence staining of γH2AX or 53BP1 was used to determine the effect of palbociclib on double-strand break (DSB) repair. Clonogenic assays, sphere formation and cell death ELISA were performed to study the sensitising effect of palbociclib on radiation-induced cytotoxicity. Signal alteration in DSB repair pathways was examined by Western blot analysis. Finally, we evaluated the in vivo anti-cancer activity and the associated molecular events of the combination therapy in a preclinical HCC xenograft model. ResultsPalbociclib affected the kinetics of DNA repair and enhanced the radiation sensitivity of HCC and CCA cells. Importantly, we found that palbociclib inhibits ataxia telangiectasia–mutated (ATM) kinase, the key upstream kinase responding to RT-induced DSBs. Furthermore, we showed that the inhibitory effect of palbociclib on RT-induced ATM kinase activation is mediated by protein phosphatase 5 (PP5). Both in vitro and in vivo investigations revealed that the inhibition of the PP5-ATM axis by palbociclib after DNA damage is responsible for the synergism between palbociclib and RT. ConclusionOur findings provide a novel combination strategy against liver cancer cells. Clinical trials using palbociclib as an adjuvant in RT are warranted.

Methyl jasmonate enhances the radiation sensitivity of esophageal carcinoma cells by inhibiting the 11-ketoprostaglandin reductase activity of AKR1C3.

PurposeIn our previous study, we found that AKR1C3 was a radioresistance gene in KY170R cells. Downregulating the expression of AKR1C3 could enhance the radiosensitivity of esophageal carcinoma cells. In this study, we investigated whether methyl jasmonate (MeJ), an inhibitor of Aldo-keto reductase family1 member C3 (AKR1C3), could overcome radiation resistance in AKR1C3 highly expressed cells.Patients and methodsWe used clone formation assays to detect radiosensitivity effects. Flow cytometry assays were used to detect reactive oxygen species (ROS) accumulation and apoptosis. Enzyme linked immunosorbent assays (ELISAs) were used to detect the concentrations of prostaglandin F2 (PGF2) and prostaglandin D2 (PGD2) in the cells after incubation with MeJ. Western blotting was used to detect AKR1C3 and peroxisome proliferator-activated receptor gamma (PPARγ) expression.ResultsWe found that AKR1C3 was highly expressed in radioresistant esophageal carcinoma cells. MeJ inhibited the expression of AKR1C3 and enhanced the radiation sensitivity of esophageal carcinoma cells expressing high levels of AKR1C3 (P<0.05). MeJ could inhibit the 11-ketoprostaglandin reductase activity of AKR1C3 in a dose-dependent manner in KY170R cells. Incubation of KY170R cells with 200 µmol/L of MeJ for 24 h reduced the expression of PGF2 by roughly 30% (P<0.05). The PPAR pathway inhibitor GW9662 prevented the radiation sensitivity enhancement imparted by MeJ. After adding GW9662, there were no significant differences between the radiation sensitivities of MeJ-treated and -untreated KY170R cells (P>0.05). The radiation sensitivity effect of MeJ also depended upon the generation of ROS in KY170R cells; 48 h after irradiation, ROS levels in the MeJ group was twofold higher than in the untreated KY170R cells (P<0.05). The ROS scavenger, N-acetyl cysteine, could reverse the radiosensitivity effects of MeJ (P>0.05).ConclusionOur results indicate that MeJ can increase the radiation sensitivity of AKR1C3-overexpressing KY170R cells by inhibiting the 11-ketoprostaglandin reductase activity of AKR1C3 and increasing cellular ROS levels.

Radiation Sensitivity of Adipose-Derived Stem Cells Isolated from Breast Tissue.

Within their niche, adipose-derived stem cells (ADSCs) are essential for homeostasis as well as for regeneration. Therefore, the interest of physicians is to use ADSCs as a tool for radiation oncology and regenerative medicine. To investigate related risks, this study analyses the radiation response of adult stem cells isolated from the adipose tissue of the female breast. To avoid donor-specific effects, ADSCs isolated from breast reduction mammoplasties of 10 donors were pooled and used for the radiobiological analysis. The clonogenic survival fraction assay was used to classify the radiation sensitivity in comparison to a more radiation-sensitive (ZR-75-1), moderately sensitive (MCF-7), and resistant (MCF10A) cell lines. Afterwards, cytotoxicity and genotoxicity of irradiation on ADSCs were investigated. On the basis of clonogenic cell survival rates of ADSCs after irradiation, we assign ADSCs an intermediate radiation sensitivity. Furthermore, a high repair capacity of double-strand breaks is related to an altered cell cycle arrest and increased expression of cyclin-dependent kinase (CDK) inhibitor p21. ADSCs isolated from breast tissue exhibit intermediate radiation sensitivity, caused by functional repair mechanisms. Therefore, we propose ADSCs to be a promising tool in radiation oncology.

PO-133 Inhibition of HIF-1α enhances radiation sensitivity in tumour cells and mediates tumour growth decrease in mice

IntroductionPoor prognosis of many solid tumours is often associated with hypoxic regions and an increased level of hypoxia-inducible-factor-1α (HIF-1α). Previous findings indicate that HIF-1α expression is relevant for radiation resistance. It was shown that HIF-1α is connected to molecules involved in DNA damage repair and checkpoint control. HIF-1α has an enhanced activity in tumours after radiation treatment (IR) and also mediates low radiation sensitivity.Material and methodsIn vitro cultured Lewis Lung Carcinoma (LLC) cells with doxycycline (Dox)-inducible knockdown (KD) of HIF-1α were used. Cells were incubated under hypoxic conditions (Hx, 1%) and irradiated with 5 Gy. Cell cycle distribution analysis was performed by PI staining and FACS. Colony formation assay, caspase-3 assay, PARP-1 cleavage and Annexin/PI staining were used to quantify apoptosis and long term survival. DNA double strand break (DSB) repair was investigated by pulse field gel electrophoresis, Western blot and immunofluorescent staining with γH2AX, 53 BP1, RAD51, BRCA1 antibodies. In vivo the induction of HIF-1α KD was performed by tamoxifen administration to HIF-1αfl/flCre-ERki/+ C57BL/6 mice. The LLC cells with constitutive HIF-1α targeting (shHIF) or scrambled (shscr) shRNA-expressing vectors were inoculated into the flank of the mice. IR was delivered once at day 10 with a dose of 15 Gy using an X-ray source.Results and discussionsWe demonstrated an inhibition of decrement of phosphorylated H2AX after Hx and IR in HIF-1α deficient cells. Fast repair kinetics of the cells remained unchanged, whereas the long term survival of the cells with reduced HIF-1α was decreased. Moreover, these cells displayed an increased rate of apoptosis after IR. We also observed persisted RAD51 foci after 24 hour in HIF-1α deficient cells which indicates an alteration in homologous recombination repair (HRR) of the cells. Furthermore, in vivo experiments with a HIF-1α deficient stromal cells in the mice and injected HIF-1α deficient tumours cells indicate a decrease of tumour growth.ConclusionThese results imply that inactivation of HIF-1α disrupts DSB repair, in particular HRR. Furthermore, they highlight the importance of the interaction of tumour and microenvironment with respect to radiation sensitivity. It was shown that inhibition of HIF-1α enhances radiation sensitivity of tumour cells which is potentially helpful for the development of novel tumour therapies.

Decreased expression level and DNA-binding activity of specificity protein 1 via cyclooxygenase-2 inhibition antagonizes radiation resistance, cell migration and invasion in radiation-resistant lung cancer cells.

Radiation is able to inhibit tumor growth, promote tumor cell apoptosis and prolong patient survival. However, radiation resistance remains a major impediment to radiotherapy. Local and metastatic recurrences following radiation are still large impediments to overall survival. Although cyclooxygenase-2 (COX-2) inhibitors may induce radiation sensitivity in cancer cells, the underlying mechanisms are not fully understood. The present study demonstrated high potential for cell proliferation, migration and invasion in radiation-resistant lung cancer cell lines. The present study observed the overexpression of specificity protein 1 (Sp1) in these cells, and the overexpression of Sp1 induced upregulation of matrix metalloproteinase (MMP)-2, MMP-9, B cell lymphoma-2, in addition to a high potential for radiation resistance, migration and invasion in these cells. The present study revealed that the COX-2 selective inhibitor, celecoxib, enhanced radiation sensitivity and inhibited migration and invasion in these cells by inhibiting the expression and DNA-binding activity of Sp1. Furthermore, celecoxib downregulated Sp1 by inhibiting c-Jun N-terminal kinase (JNK). Taken together, the present study demonstrated that Sp1 overexpression in radiation-resistant cancer cells and COX-2 inhibitors enhanced radiation sensitivity and inhibited the migration and invasion of cancer cells, at least partially, via inactivation of the JNK/Sp1 signaling pathway and a decrease in Sp1 DNA-binding activity.

Centrosome Clustering Is a Tumor-selective Target for the Improvement of Radiotherapy in Breast Cancer Cells

Owing to the frequent observation of centrosome amplification in human cancers, cancer cells have a unique mechanism to suppress detrimental multipolar division by clustering multiple centrosomes into two functional spindle poles, known as centrosome clustering. This study investigated whether inhibition of centrosome clustering enhances the radiation sensitivity of breast cancer cells. In this study, inhibition of centrosome clustering was examined by using various centrosome-declustering agents and KIFC1 siRNA in three breast cancer cell lines and two normal fibroblast cell lines. The combination effect of radiation and centrosome declustering was evaluated by cell viability, clonogenic, immunofluorescence assay. This study showed that targeting centrosome clustering enhanced the efficacy of radiotherapy of breast cancer cells with less damage to normal cells. Ionizing radiation induced centrosome amplification in breast cancer cells, but not in normal fibroblast cells. Notably, we showed that centrosome declustering efficiently radiosensitized the centrosome-amplified breast cancer cells through induction of multipolar spindles but did not affect the viability of normal fibroblasts in response to irradiation. Furthermore, KIFC1 mediated the radiosensitivity of the centrosome-amplified breast cancer cells. Our data provided the first evidence that centrosome clustering is a tumor-selective target for the improvement of radiotherapy in breast cancer cells.

Role of the NRP-1-mediated VEGFR2-independent pathway on radiation sensitivity of non-small cell lung cancer cells.

To determine if inhibiting neuropilin-1 (NRP-1) affects the radiosensitivity of NSCLC cells through a vascular endothelial growth factor receptor 2 (VEGFR2)-independent pathway, and to assess the underlying mechanisms. The expression of VEGFR2, NRP-1, related signaling molecules, abelson murine leukemia viral oncogene homolog 1 (ABL-1), and RAD51 were determined by RT-PCR and Western blotting, respectively. Radiosensitivity was assessed using the colony-forming assay, and the cell apoptosis were analyzed by flow cytometry. We selected two cell lines with high expression levels of VEGFR2, including Calu-1 cells that have high NRP-1 expression, and H358 cells that have low NRP-1 expression. Upon inhibition of p-VEGFR2 by apatinib in Calu-1 cells, the expression of NRP-1 protein and other related proteins in the pathway was still high. Upon NRP-1 siRNA treatment, the expression of both NRP-1 and RAD51 decreased (p < 0.01; p < 0.05). Upon ABL-1 siRNA treatment, the expression of NRP-1 was increased and the expression of RAD51 was unchanged. Calu-1 cells treated with NRP-1 siRNA exhibited significantly higher apoptosis and radiation sensitivity in radiation therapy compared to Calu-1 cells treated with apatinib alone (p < 0.01; p < 0.01). The apoptosis and radiation sensitivity in H358 cells with NRP-1 overexpression was similar to the control group regardless of VEGFR2 inhibition. We demonstrated that when VEGFR2 was inhibited, NRP-1 appeared to regulate RAD51 expression through the VEGFR2-independent ABL-1 pathway, consequently regulating radiation sensitivity. In addition, the combined inhibition of VEGFR2 and NRP-1 appears to sensitize cancer cells to radiation.

Research of radiation resistance delivery through paracrine effects on non-small cell lung cancer

Objective To investigate the role and possible mechanisms of paracrine in radiation tolerance of non-small cell lung cancer (NSCLC) by observing the effects of radiation-resistant NSCLC H460R cell secretion on the radiosensitivity of parental H460 cells. Methods H460 cells were inoculated and cultured, and then divided into control group, conditional culturing group, irradiation group and irradiation combined conditional culturing group. One-time irradiation with 137Cs γ-rays was conducted with a dose rate of 1.02 Gy/min. Effect of H460R conditioned media on the radiation sensitivity of H460 cells was observed by clonogenic assay. The percentage of phospho-histone histone H2AX (γH2AX) positive cells was detected by immunofluorescence staining. The cyclical changes of the cells were detected by flow cytometry. The expression of DNA damage-associated proteins γH2AX and Rad51 were detected by Western Blot. Results The results showed that compared with the single γ-rays irradiation, the number of cell clones was significantly increased in the H460R cells after γ-rays irradiation combined with conditional culturing treatment at different doses of 2, 4, and 6 Gy, and the differences were statistically significant (2,4 Gy: all P<0.05; 6 Gy: P<0.01). The results of immunofluorescence staining showed that the percentage of γH2AX positive cells in the conditional culturing group was higher than that in the control group [(39.40±2.51)% vs. (25.21±2.05)%], and the difference was statistically significant (P<0.01). This value in the irradiation combined conditional culturing group was lower than that in irradiated group [(60.48±2.79)% vs. (80.65±2.05)%], and the difference was statistically significant (P<0.001). The flow cytometry analysis showed that the percentage of cells in G2/M phase for the irradiation (4 Gy) combined conditional culturing group was higher than that of irradiated group [(26.83±1.42)% vs. (15.73±1.29)%], and the difference was statistically significant (P<0.001). The Western Blot results showed that γH2AX and Rad51 protein expression in the irradiation (4 Gy) combined conditional culturing group respectively was lower and higher than that in irradiated group. Conclusion In the tumor microenvironment, radiation-resistant H460R cells can enhance the radioresistance of H460 cells by paracrine, which may be related to the promotion of DNA repair ability after irradiation Key words: Non-small cell lung cancer; Paracrine; Radioresistance; DNA repair

Glutathione-Depleting Gold Nanoclusters for Enhanced Cancer Radiotherapy through Synergistic External and Internal Regulations.

The therapeutic performance of cancer radiotherapy is often limited by the overexpression of glutathione (GSH) in tumors and low radiation sensitivity of cancerous cells. To address these issues, the facilely prepared histidine-capped gold nanoclusters (Au NCs@His) were adopted as a radiosensitizer with a high sensitization enhancement ratio of ∼1.54. On one hand, Au NCs@His can inherit the local radiation enhancement property of gold-based materials (external regulation); on the other hand, Au NCs@His can decrease the intracellular GSH level, thus preventing the generated reactive oxygen species (ROS) from being consumed by GSH, and arrest the cells at the radiosensitive G2/M phase (internal regulation).

AZD9291 Increases Sensitivity to Radiation in PC-9-IR Cells by Delaying DNA Damage Repair after Irradiation and Inducing Apoptosis.

AZD9291 is a novel, irreversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), which is administered orally. It has been proven effective in non-small cell lung cancer (NSCLC) patients, with both EGFR-sensitizing and EGFR T790M mutations in preclinical models. However, the potential therapeutic effects of AZD9291 combined with other modalities, including ionizing radiation, are not well understood. The presence of AZD9291 significantly increases the cell-killing effects of radiation in PC-9-IR cells with a secondary EGFR mutation (T790M), which was developed from NSCLC PC-9 cells (human lung adenocarcinoma cell with EGFR 19 exon 15 bp deletion) after chronic exposure to increasing doses of gefitinib, and in H1975 cells (human lung adenocarcinoma cell with EGFR exon 20 T790M mutation de novo), but not in PC-9 cells or in H460 cells (human lung adenocarcinoma cell with wild-type EGFR). In PC-9-IR cells, AZD9291 remarkably decreases phosphorylation levels of EGFR, extracellular regulated protein kinase (ERK), and protein kinase B (AKT). AZD9291 increases sensitivity to radiation in PC-9-IR cells by delaying deoxyribonucleic acid (DNA) damage repair after irradiation and inducing apoptosis, and enhances tumor growth inhibition when combined with radiation in PC-9-IR xenografts. Our findings suggest a potential therapeutic effect of AZD9291 as a radiation sensitizer in lung cancer cells with an acquired EGFR T790M mutation, providing a rationale for a clinical trial using the combination of AZD9291 and radiation in NSCLCs harboring acquired T790M mutation.

EEF-2 Kinase-targeted miR-449b confers radiation sensitivity to cancer cells

The roles of microRNA in regulation of various biological processes and in modulation of therapeutic effects have been widely appreciated. In this study, we found a positive correlation between miR-449 b expression and radiation sensitivity in cancer cells and in tumor specimens from patients. We showed that eEF-2 kinase, a negative regulator of global protein synthesis, is a target of miR-449 b. Introducing a miR-449 b mimic into cancer cells led to suppression of eEF-2 kinase expression, leading to increases of protein synthesis and depletion of cellular ATP. Further, we demonstrated that the miR-449 b mimic rendered the cancer cells more sensitive to ionizing radiation both in vitro (cell culture) and in vivo (animal xenograft model). Moreover, the radiation sensitivity conferred by miR-449 b could be blunted by cycloheximide, an inhibitor of protein synthesis, or by direct delivery of ATP liposome, supporting eEF-2 kinase as a mediator of the radio-sensitizing effects of miR-449 b. These results indicate that miR-449 b, which is frequently down-regulated in radio-resistant cancers, may represent a new critical determinant of radio-sensitivity.

Overexpression of β-Catenin Decreases the Radiosensitivity of Human Nasopharyngeal Carcinoma CNE-2 Cells

Background/Aims: Nasopharyngeal carcinoma (NPC) is rare worldwide but remains highly prevalent in endemic regions, notably in southern China. Radiotherapy remains the treatment of choice for NPC, but radioresistance has been identified as a major cause of therapeutic failure. The Wnt/β-catenin signaling has been found to be involved in NPC radioresistance; however, the effect of β-catenin overexpression on radioresistance remains unknown in NPC until now. This study aimed to examine the impact of β-catenin overexpression on the radiosensitivity of human NPC CNE-2 cells. Methods: Immunohistochemistry was performed to detect the β-catenin expression in normal nasopharyngeal specimens and NPC specimens. The human NPC CNE-2 cell line overexpressing β-catenin was modeled by transfection with the pcDNA3.1/Hygro(+)/β-catenin recombinant vector (transfection group), while cells transfected with the pcDNA3.1/Hygro(+) vector served as negative controls and non-transfected cells served as blank controls. The expression of key molecules of the Wnt/β-catenin signaling pathway was determined using Western blotting and qPCR assays, and the changes of radiation sensitivity were measured with a colony-formation assay. Cell viability was measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide) assay. In addition, the cell cycle and apoptosis was detected using flow cytometry and the TCF/LEF transcriptional activity was measured with a Dual Luciferase Reporter Assay System. Results: Immunohistochemical staining showed high β-catenin expression in radioresistant NPC specimens, and low expression in radiosensitive NPC specimens and normal nasopharyngeal specimens. Western blotting and qPCR assays detected higher β-catenin expression in the transfection group than in the negative and blank controls (P < 0.01). Down-regulation of GSK-3β expression (P < 0.05) and up-regulation of Cyclin D1 expression (P < 0.01) was detected in β-catenin overexpressing NPC cells exposed to X-ray radiation relative to negative and blank controls. Colony-formation assay revealed higher D₀, D_q and SF in the transfection group than in the negative and blank control groups post-radiation, and the SER in the transfection group was 0.75-fold and 0.68-fold greater than that in the blank and negative control groups, respectively. MTT assay revealed that the viability of CNE-2 cells was significantly higher in the transfection group (96% ± 8.72%) than in the negative control group (74.67 ± 7.05%) and the blank control group (75.33% ± 7.02%) 24 h post-exposure to 6 Gy X-ray radiation (P < 0.05). X-ray radiation led to a lower proportion of CNE-2 cells at the G2/M phase and a lower apoptotic rate in the transfection group than in the negative and blank control groups (P < 0.05). In addition, the TCF/LEF transcriptional activity was higher in the transfection group than in the negative and blank control groups (P < 0.01), and 6 Gy X-ray radiation elevated the TCF/LEF transcriptional activity relative to 0 Gy radiation in the transfection group (P < 0.01). Conclusion: β-catenin overexpression may decrease the radiation sensitivity in NPC CNE-2 cells through activating the downstream transcriptional factors of β-catenin, and reducing G2/M arrest and cell apoptosis.

Targeting Allostery with Avatars to Design Inhibitors Assessed by Cell Activity: Dissecting MRE11 Endo- and Exonuclease Activities.

For inhibitor design, as in most research, the best system is question dependent. We suggest structurally defined allostery to design specific inhibitors that target regions beyond active sites. We choose systems allowing efficient quality structures with conformational changes as optimal for structure-based design to optimize inhibitors. We maintain that evolutionarily related targets logically provide molecular avatars, where this Sanskrit term for descent includes ideas of functional relationships and of being a physical embodiment of the target's essential features without requiring high sequence identity. Appropriate biochemical and cell assays provide quantitative measurements, and for biomedical impacts, any inhibitor's activity should be validated in human cells. Specificity is effectively shown empirically by testing if mutations blocking target activity remove cellular inhibitor impact. We propose this approach to be superior to experiments testing for lack of cross-reactivity among possible related enzymes, which is a challenging negative experiment. As an exemplary avatar system for protein and DNA allosteric conformational controls, we focus here on developing separation-of-function inhibitors for meiotic recombination 11 nuclease activities. This was achieved not by targeting the active site but rather by geometrically impacting loop motifs analogously to ribosome antibiotics. These loops are neighboring the dimer interface and active site act in sculpting dsDNA and ssDNA into catalytically competent complexes. One of our design constraints is to preserve DNA substrate binding to geometrically block competing enzymes and pathways from the damaged site. We validate our allosteric approach to controlling outcomes in human cells by reversing the radiation sensitivity and genomic instability in BRCA mutant cells.

I-Kappa-B Kinase-epsilon activates nuclear factor-kappa B and STAT5B and supports glioblastoma growth but amlexanox shows little therapeutic potential in these tumors

Aim: This study aims to analyze the role of I-kappa-B kinase (IKK)-epsilon in glioblastoma (GBM). Methods: A series of in vitro, in vivo, microarray, and immunohistochemical assessments were performed to evaluate the biological effects of IKK-epsilon on cell signaling, radiation sensitivity, and patient survival in GBM condition. Results: IKK-epsilon was strongly expressed in 75% of 195 primary GBM samples but did not correlate with patient survival. No correlation was established between the copy number, messenger RNA (mRNA) expression, and protein expression in 38 fresh tumor samples, nor between IKK-epsilon mRNA expression and survival in 543 GBM of the TCGA repository. In vitro, IKK-epsilon contributed to the growth and migration of glioma cells, independent of their EGFRVIII status. IKK-epsilon activated nuclear factor (NF)-κB and STAT5B in vitro, confirming the observed correlation surgical GBM samples. IKK-epsilon silencing did not alter the sensitivity of GBM cells to ionizing radiation. Amlexanox, inhibitor of IKK-epsilon and TBK1, poorly (IC50 > 100 μM) decreased cell growth and increased NF-κB activity in GBM cells, in vitro, notably due to TBK1 inhibition. In vivo, oral amlexanox failed to inhibit the growth of intracerebral U87 GBM xenografts in nude mice. Conclusion: The results confirm a moderate pro-oncogenic role of IKK-epsilon in GBM, but question the potential of amlexanox as a therapeutic drug.

Fractionation enhances acute oligodendrocyte progenitor cell radiation sensitivity and leads to long term depletion.

Ionizing radiation (IR) is commonly used to treat central nervous system (CNS) cancers and metastases. While IR promotes remission, frequent side effects including impaired cognition and white matter loss occur following treatment. Fractionation is used to minimize these CNS late side effects, as it reduces IR effects in differentiated normal tissue, but not rapidly proliferating normal or tumor tissue. However, side effects occur even with the use of fractionated paradigms. Oligodendrocyte progenitor cells (OPCs) are a proliferative population within the CNS affected by radiation. We hypothesized that fractionated radiation would lead to OPC loss, which could contribute to the delayed white matter loss seen after radiation exposure. We found that fractionated IR induced a greater early loss of OPCs than an equivalent single dose exposure. Furthermore, OPC recovery was impaired following fractionated IR. Finally, reduced OPC differentiation and mature oligodendrocyte numbers occurred in single dose and fractionated IR paradigms. This work demonstrates that fractionation does not spare normal brain tissue and, importantly, highlights the sensitivity of OPCs to fractionated IR, suggesting that fractionated schedules may promote white matter dysfunction, a point that should be considered in radiotherapy.

Knockdown of AMPKα decreases ATM expression and increases radiosensitivity under hypoxia and nutrient starvation in an SV40-transformed human fibroblast cell line, LM217

BackgroundPresence of unperfused regions containing cells under hypoxia and nutrient starvation contributes to radioresistance in solid human tumors. It is well known that hypoxia causes cellular radioresistance, but little is known about the effects of nutrient starvation on radiosensitivity. We have reported that nutrient starvation induced decrease of mTORC1 activity and decrease of radiosensitivity in an SV40-transformed human fibroblast cell line, LM217, and that nutrient starvation induced increase of mTORC1 activity and increase of radiosensitivity in human liver cancer cell lines, HepG2 and HuH6 (Murata et al., BBRC 2015). Knockdown of mTOR using small interfering RNA (siRNA) for mTOR suppressed radiosensitivity under nutrient starvation alone in HepG2 cells, which suggests that mTORC1 pathway regulates radiosensitivity under nutrient starvation alone. In the present study, effects of hypoxia and nutrient starvation on radiosensitivity were investigated using the same cell lines. MethodsLM217 and HepG2 cells were used to examine the effects of hypoxia and nutrient starvation on cellular radiosensitivity, mTORC1 pathway including AMPK, ATM, and HIF-1α, which are known as regulators of mTORC1 activity, and glycogen storage, which is induced by HIF-1 and HIF-2 under hypoxia and promotes cell survival. ResultsUnder hypoxia and nutrient starvation, AMPK activity and ATM expression were increased in LM217 cells and decreased in HepG2 cells compared with AMPK activity under nutrient starvation alone or ATM expression under hypoxia alone. Under hypoxia and nutrient starvation, radiosensitivity was decreased in LM217 cells and increased in HepG2 cells compared with radiosensitivity under hypoxia alone. Under hypoxia and nutrient starvation, knockdown of AMPK decreased ATM activity and increased radiation sensitivity in LM217 cells. In both cell lines, mTORC1 activity was decreased under hypoxia and nutrient starvation. Under hypoxia alone, knockdown of mTOR slightly increased ATM expression but did not affect radiosensitivity in LM217. Under hypoxia and nutrient starvation, HIF-1α expression was suppressed and glycogen storage was reduced. ConclusionOur data suggest that AMPK regulates ATM expression and partially regulates radiosensitivity under hypoxia and nutrient starvation. The molecular mechanism underlying the induction of ATM expression by AMPK remains to be elucidated.