Radiation-induced Apoptosis Research Articles

Curcumin Enhanced Ionizing Radiation-Induced Immunogenic Cell Death in Glioma Cells through Endoplasmic Reticulum Stress Signaling Pathways.

Objective Local radiotherapy may cause distant tumor regression via inducing immunogenic cell death (ICD). Here, we investigated the effect of curcumin on ionizing radiation-induced immunogenic cell death in normoxic or hypoxic glioma cells and its mechanism in vitro and vivo. Methods Hypoxic or normoxic glioma cell apoptosis and the cell surface exposure of calreticulin (CRT) were detected by flow cytometry. Extracellular ATP and HSP70 were measured by chemiluminescence assay and ELISA, respectively. Endoplasmic reticulum (ER) stress protein levels were detected by western blot. Moreover, the induction of bona fide ICD was detected by vaccination assays in mice bearing glioma model. Spleen lymphocytes and tumor-infiltrating lymphocyte subsets were analyzed by flow cytometry and immunohistochemistry. Results Curcumin incubation before X-ray irradiation significantly increased radiation-induced apoptosis rate in normoxic or hypoxic glioma cells. Curcumin enhanced radiation-induced CRT exposure, release of HSP70 and ATP, and ER stress signaling activity. After treatment with ER stress pathway inhibitors, cell apoptosis and CRT exposure induced by the combination treatment of curcumin and X-ray were reduced. In vaccination experiments, glioma cells irradiated by X-ray produced a strong immunogenic response rejecting tumor formation in 70% mice. In comparison, cells treated by curcumin and X-ray produced a stronger immune response rejecting tumor formation in 90% mice. The combination treatment increased the percentage of tumor-infiltrating CD4+, CD8+ T lymphocytes, and CD11c+ dendritic cells compared to X-ray irradiation alone. Conclusion Ionizing radiation-induced normoxic or hypoxic glioma immunogenic cell death could be further enhanced by curcumin through activating the ER stress PERK-eIF2α and IRE1α-XBP1 signaling pathways.

PRSS1 Mutations Affect Pancreatic Ductal Adenocarcinoma Radiosensitivity via AKT and Extracellular Regulated Protein Kinases Pathways

Radioresistance is the leading cause of failed radiation therapy for pancreatic ductal cancer (PDAC). The relevance of the cationic trypsinogen gene (PRSS1) in PDAC radioresistance is unknown, despite its association with tumor responses to therapy in numerous malignancies. Here we established two PRSS1 point mutation PDAC cell lines: c. 338 T > G and c.410 C > T. Compared to their parental cells, elevated AKT and ERK phosphorylation concentrations were observed in Panc-1 and MIA PaCa-2 c. 338 T > G and c.410 C > T cells with point mutations. The PRSS1 mutation restored the sensitivity of radioresistant cells to radiation through increased ionizing radiation-induced apoptosis by down regulating p-AKT and p-ERK. Based on these results, we hypothesized that a PRSS1 mutation in PDAC increased cell radiosensitivity by decreasing p-AKT and p-ERK. Our findings provide a molecular basis for optimizing radiation in patients with PDAC.

Radioprotection of deinococcal exopolysaccharide BRD125 by regenerating hematopoietic stem cells.

There is a substantial need for the development of biomaterials for protecting hematopoietic stem cells and enhancing hematopoiesis after radiation damage. Bacterial exopolysaccharide (EPS) has been shown to be very attractive to researchers as a radioprotectant owing to its high antioxidant, anti-cancer, and limited adverse effects. In the present study, we isolated EPS from a novel strain, Deinococcus radiodurans BRD125, which produces EPS in high abundance, and investigated its applicability as a radioprotective biomaterial. We found that EPS isolated from EPS-rich D. radiodurans BRD125 (DeinoPol-BRD125) had an excellent free-radical scavenging effect and reduced irradiation-induced apoptosis. In addition, bone-marrow and spleen-cell apoptosis in irradiated mice were significantly reduced by DeinoPol-BRD125 administration. DeinoPol-BRD125 enhanced the expression of hematopoiesis-related cytokines such as GM-CSF, G-GSF, M-CSF, and SCF, thereby enhancing hematopoietic stem cells protection and regeneration. Taken together, our findings are the first to report the immunological mechanism of a novel radioprotectant, DeinoPol-BRD125, which might constitute an ideal radioprotective and radiation mitigating agent as a supplement drug during radiotherapy.

Protective effects of an aqueous extract of Protaetia brevitarsis seulensis larvae against radiation-induced testicular injury in mice.

The larvae of Protaetia brevitarsis seulensis have been used as a food ingredient and are known for their nutritional value and anti-inflammatory properties. However, whether P.brevitarsis seulensis larvae demonstrate protective effects against radiation-induced testicular injury has not been investigated. In this study, the protective effects of an aqueous extract of P.brevitarsis seulensis larvae (PBE) against radiation-induced testicular injury were tested. Male C57BL/6 mice were administered PBE (5 or 10mg/kg) orally for 14 days before exposure to focal pelvic irradiation. Histopathological examinations were conducted at 8h and 30 d after radiation exposure. PBE pretreatment reduced the radiation-induced apoptosis of germ cells at 8h after irradiation and significantly increased testis and epididymis weights relative to those of the irradiated control mice at 30 days. PBE protected against histopathological damage and decreased the radiation-induced effects on the epithelium height and seminiferous tubule diameter. Furthermore, the extract ameliorated the radiation-induced morphological abnormalities of sperm cells and improved their motility. It also prevented a decrease in the epididymal sperm count caused by irradiation. Moreover, the extract alleviated the generation of reactive oxygen species, and its antioxidative activity increased in a dose-dependent manner. Among the six major compounds isolated from PBE, benzoic acid and uridine showed the highest antioxidant activities. These results suggest that PBE protects against radiation-induced testicular injury via its antioxidative properties. Thus, it has potential clinical applicability as a neoadjuvant therapy for the prevention of testicular damage caused by cancer radiotherapy.

Knockdown of SHMT2 enhances the sensitivity of gastric cancer cells to radiotherapy through the Wnt/β-catenin pathway.

Gastric cancer (GC) is one of the most common malignant tumors. The mechanism of GC radioresistance and new radiosensitizers must be revealed and developed to treat GC. Serine hydroxymethyltransferase 2 (SHMT2) is responsible for encoding the mitochondrial form of the pyridoxal phosphate-dependent enzyme. SHMT2 plays a critical role in several types of cancers, while its possible effect on the radiological resistance in GC is still unclear. In this study, we investigated the role of SHMT2 in the radiological resistance of GC. Our data confirmed that SHMT2 was highly expressed in radiation-resistant GC cells. SHMT2 reduced the radiosensitivity of GC cells. In addition, SHMT2 is involved in radiation-induced GC cell apoptosis. Further, SHMT2 regulated the Wnt/β-catenin pathway, therefore reducing the radiosensitivity of GC cells in vivo. In conclusion, we revealed that depletion of SHMT2 enhanced the sensitivity of GC cells to interventional radiotherapy through the Wnt/β-catenin pathway.

SMPDL3b modulates radiation-induced DNA damage response in renal podocytes.

The kidneys are radiosensitive and dose-limiting organs for radiotherapy (RT) targeting abdominal and paraspinal tumors. Excessive radiation doses to the kidneys ultimately lead to radiation nephropathy. Our prior work unmasked a novel role for the lipid-modifying enzyme, sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b), in regulating the response of renal podocytes to radiation injury. In this study, we investigated the role of SMPDL3b in DNA double-strand breaks (DSBs) repair in vitro and in vivo. We assessed the kinetics of DSBs recognition and repair along with the ATM pathway and nuclear sphingolipid metabolism in wild-type (WT) and SMPDL3b overexpressing (OE) human podocytes. We also assessed the extent of DNA damage repair in SMPDL3b knock-down (KD) human podocytes, and C57BL6 WT and podocyte-specific SMPDL3b-knock out (KO) mice after radiation injury. We found that SMPDL3b overexpression enhanced DSBs recognition and repair through modulating ATM nuclear shuttling. OE podocytes were protected against radiation-induced apoptosis by increasing the phosphorylation of p53 at serine 15 and attenuating subsequent caspase-3 cleavage. SMPDL3b overexpression prevented radiation-induced alterations in nuclear ceramide-1-phosphate (C1P) and ceramide levels. Interestingly, exogenous C1P pretreatment radiosensitized OE podocytes by delaying ATM nuclear foci formation and DSBs repair. On the other hand, SMPDL3b knock-down, in vitro and in vivo, induced a significant delay in DSBs repair. Additionally, increased activation of apoptosis was induced in podocytes of SMPDL3b-KO mice compared to WT mice at 24 h post-irradiation. Together, our results unravel a novel role for SMPDL3b in radiation-induced DNA damage response. The current work suggests that SMPDL3b modulates nuclear sphingolipid metabolism, ATM nuclear shuttling, and DSBs repair.

Nicotinamide mitigates radiation injury in submandibular gland by protecting mitochondrial structure and functions.

Radiation damage to salivary gland is inevitable in head and neck cancer patients receiving radiotherapy. Safe and effective treatments for protecting salivary glands from radiation are still unavailable. Mitochondrial damage is a critical mechanism in irradiated salivary gland; however, treatment targeting mitochondria has not received much attention. Nicotinamide is a key component of the mitochondrial metabolism. Here, we investigated the effects and underlying mechanisms of nicotinamide on protecting irradiated submandibular gland. Submandibular gland cells and tissues were randomly divided into four groups: control, nicotinamide alone, radiation alone, and radiation with nicotinamide pretreatment. Cell viability was detected by PrestoBlue cell viability reagent. Histopathological alterations were observed with HE staining. Pilocarpine-stimulated saliva was measured from Wharton's duct. Cell apoptosis was determined by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Nicotinamide phosphoribosyl transferase was examined with immunofluorescence. The levels of nicotinamide adenine dinucleotide, mitochondrial membrane potential, and adenosine triphosphate were measured with the relevant kits. The mitochondrial ultrastructure was observed under transmission electron microscopy. Nicotinamide significantly mitigated radiation damage both in vitro and in vivo. Also, nicotinamide improved saliva secretion and reduced radiation-induced apoptosis in irradiated submandibular glands. Moreover, nicotinamide improved nicotinamide phosphoribosyl transferase and the levels of nicotinamide adenine dinucleotide/adenosine triphosphate and mitochondrial membrane potential, all of which were decreased by radiation in submandibular gland cells. Importantly, nicotinamide protected the mitochondrial ultrastructure from radiation. These findings demonstrate that nicotinamide alleviates radiation damage in submandibular gland by replenishing nicotinamide adenine dinucleotide and maintaining mitochondrial function and ultrastructure, suggesting that nicotinamide could be used as a prospective radioprotectant for preventing radiation sialadenitis.

Tanshinone IIA Accomplished Protection against Radiation-Induced Cardiomyocyte Injury by Regulating the p38/p53 Pathway.

Background Radiotherapy is one of the major strategies for treating tumors, and it inevitably causes damage to relevant tissues and organs during treatment. Radiation-induced heart disease (RIHD) refers to radiation-induced cardiovascular adverse effects caused by thoracic radiotherapy. Currently, there is no uniform standard in the treatment of RIHD. Methods In our group study, by administering a dose of 4 Gy radiation, we established a radiation injured cardiomyocyte model and explored the regulatory relationship between tanshinone IIA and p38 MAPK in cardiomyocyte injury. We assessed cell damage and proliferation using clonogenic assay and lactate dehydrogenase (LDH) release assay. The measures of antioxidant activity and oxidative stress were conducted using superoxide dismutase (SOD) and reactive oxygen species (ROS). The apoptosis rate and the relative expression of apoptotic proteins were conducted using flow cytometry and western blot. To assess p38 and p53 expressions and phosphorylation levels, western blot was performed. Results Experimental results suggested that tanshinone IIA restored cell proliferation in radiation-induced cardiomyocyte injury (∗∗P < 0.01), and the level of LDH release decreased (∗P < 0.05). Meanwhile, tanshinone IIA could decrease the ROS generation induced by radiation (∗∗P < 0.01) and upregulate the SOD level (∗∗P < 0.01). Again, tanshinone IIA reduced radiation-induced cardiomyocyte apoptosis (∗∗P < 0.01). Finally, tanshinone IIA downregulated radiation-induced p38/p53 overexpression (∗∗∗P < 0.001). Conclusions The treatment effects of tanshinone IIA against radiation-induced myocardial injury may be through the regulation of the p38/p53 pathway.

Multifunctional PEGylated Niosomal Nanoparticle-Loaded Herbal Drugs as a Novel Nano-Radiosensitizer and Stimuli-Sensitive Nanocarrier for Synergistic Cancer Therapy.

Nowadays, radiotherapy is one of the most effective treatments for breast cancer. In order to overcome the radioresistance of cancer cells, radio-sensitizing agents can be used combined with irradiation to increase the therapeutic efficiency. Curcumin can enhance the radiosensitivity of cancer cells and decrease their viability by the accumulation of these cells in the G2 phase. The encapsulation of curcumin in a nanoniosomal delivery system increases aqueous solubility and bioavailability, resulting in increased radio sensitivity. The present study aimed to enhance the radio-sensitizing effect of the curcumin-containing nanoniosome (Cur-Nio) when combined with irradiation. Thus, curcumin (0.5 mg ml−1) was loaded on a PEGylated nanoniosome containing Tween 60, cholesterol, DOTAP, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG) (at ratios of 70:30:10:5, respectively) by the thin-film hydration method. The particle size, zeta potential, entrapment efficiency, and drug-release rate of formulated nanoniosomes were determined. In order to assess cytotoxicity and apoptosis, different doses of irradiation along with various concentrations of free curcumin and Cur-Nio (single or in combination with irradiation) were treated with breast cancer cells. The particle size and zeta potential of Cur-Nio were reported to be 117.5 nm and −15.1 mV, respectively. The entrapment efficiency (EE%) and loading capacities were 72.3% and 6.68%, respectively. The drug-release rate during 6 h was 65.9%. Cell survival in the presence of curcumin at doses of 1 and 3 Gy showed a significant reduction compared with cells irradiated at 48 h and 72 h (p < 0.000). Also, the rate of cytotoxicity and apoptosis was significantly higher in cells treated with the combination of curcumin-containing nanoniosomes and irradiation in comparison with those treated with free curcumin. These findings indicate that the efficacy of pre-treatment with Cur-Nio as a radiosensitizer during radiotherapy enhances irradiation-induced breast cancer cell apoptosis and is a useful strategy to increase the effectiveness of breast cancer therapy.

Chlorogenic Acid, the Main Antioxidant in Coffee, Reduces Radiation-Induced Apoptosis and DNA Damage via NF-E2-Related Factor 2 (Nrf2) Activation in Hepatocellular Carcinoma.

Radiotherapy produces excessive reactive oxygen species (ROS), which can lead to DNA damage and apoptosis in tumor cells, thereby killing malignant cells. Chlorogenic acid (CGA) is a well-known antioxidant in coffee due to its strong ability to remove ROS. However, the effect of CGA on radiotherapeutic efficacy remains unclear. In this study, we showed that CGA could hinder the therapeutic effect of radiotherapy by inhibiting radiation-induced apoptosis and DNA damage via scavenging excessive ROS and activating the NF-E2-related factor 2 (Nrf2) antioxidant system in hepatocellular carcinoma (HCC) cells and a murine model. The knockdown of Nrf2 reversed CGA-mediated radiation resistance in HCC cells. In conclusion, CGA might be a potential tumor-protective compound upon irradiation and reduce the efficacy of radiotherapy via ROS scavenging and Nrf2 activation.

The Efficacy of Radiation is Enhanced by Metformin and Hyperthermia Alone or Combined Against FSaII Fibrosarcoma in C3H Mice.

The effects of mild-temperature hyperthermia (MTH) and metformin, alone or in combination, on the efficacy of high-dose hypofractionated radiation against experimental tumors were investigated. FSaII fibrosarcoma grown subcutaneously in the hind legs of C3H mice was irradiated with a single 15 Gy dose using a 60Co irradiator. The radio frequency capacitive method was used to heat the tumors at 41.0°C for 30 min. Metformin was intraperitoneally (i.p.) administered daily to tumor-bearing mice at a dose of 150 mg/kg. The expression levels of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and programmed cell death-ligand 1 (PD-L1) were determined by immunohistochemical staining of the excised tumor tissues. The apoptosis of tumor cells in vivo was quantified by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and cleaved caspase-3 staining of the excised tumor tissues. Irradiation of tumors markedly increased the expression of HIF-1α, VEGF, and PD-L1, and MTH and metformin used either alone or in combination significantly abrogated the radiation-induced upregulation of these proteins. MTH and metformin alone or combined increased the radiation-induced apoptosis in tumor cells and enhanced the radiation-induced suppression of tumor growth. The findings indicated that the increased tumor response to 15 Gy irradiation by MTH and metformin alone or in combination was due, in part, to the abrogation of the radiation-induced upregulation of HIF-1α and its downstream targets VEGF and PD-L1.

Human Mesenchymal Stromal Cells Do Not Cause Radioprotection of Head-and-Neck Squamous Cell Carcinoma.

Radiotherapy of head-and-neck squamous cell carcinoma (HNSCC) can cause considerable normal tissue injuries, and mesenchymal stromal cells (MSCs) have been shown to aid regeneration of irradiation-damaged normal tissues. However, utilization of MSC-based treatments for HNSCC patients undergoing radiotherapy is hampered by concerns regarding potential radioprotective effects. We therefore investigated the influence of MSCs on the radiosensitivity of HNSCCs. Several human papillomavirus (HPV)-negative and HPV-positive HNSCCs were co-cultured with human bone marrow-derived MSCs using two-dimensional and three-dimensional assays. Clonogenic survival, proliferation, and viability of HNSCCs after radiotherapy were assessed depending on MSC co-culture. Flow cytometry analyses were conducted to examine the influence of MSCs on irradiation-induced cell cycle distribution and apoptosis induction in HNSCCs. Immunofluorescence stainings of γH2AX were conducted to determine the levels of residual irradiation-induced DNA double-strand breaks. Levels of connective tissue growth factor (CTGF), a multifunctional pro-tumorigenic cytokine, were analyzed using enzyme-linked immunosorbent assays. Neither direct MSC co-culture nor MSC-conditioned medium exerted radioprotective effects on HNSCCs as determined by clonogenic survival, proliferation, and viability assays. Consistently, three-dimensional microwell arrays revealed no radioprotective effects of MSCs. Irradiation resulted in a G2/M arrest of HNSCCs at 96 h independently of MSC co-culture. HNSCCs’ apoptosis rates were increased by irradiation irrespective of MSCs. Numbers of residual γH2AX foci after irradiation with 2 or 8 Gy were comparable between mono- and co-cultures. MSC mono-cultures and HNSCC-MSC co-cultures exhibited comparable CTGF levels. We did not detect radioprotective effects of human MSCs on HNSCCs. Our results suggest that the usage of MSC-based therapies for radiotherapy-related toxicities in HNSCC patients may be safe in the context of absent radioprotection.

Fenofibrate Attenuates Radiation-Induced Oxidative Damage to the Skin through Fatty Acid Binding Protein 4 (FABP4).

Radiation facilities and radioactive materials have been widely used in military, industry, medicine, science and nuclear facilities, which has significantly increased the potential of large-scale, uncontrolled exposure to radiation. The skin is one of the radiosensitive organ systems and radiation-induced skin injury remains a serious concern after ionizing radiation exposure. Our previous report indicates the involvement of the peroxisome proliferator-activated receptor pathway in the response of skin tissues to ionizing radiation. PPARα is a member of the PPAR nuclear hormone receptor superfamily, which can be activated by fibrate ligands. However, the protection of fenofibrate against ionizing radiation in skin keratinocytes and fibroblasts has not been described. The PPARα mRNA levels in irradiated and nonirradiated skin tissues of rats were determined by real-time assay. The expression of PPARα, and FABP4 were evaluated by western blot and IHC assay. The cell proliferation was detected by colony formation. The γH2AX foci and ROS levels in irradiated WS1 cells with FABP4 overexpression than in control cells were performed by Immunofluorescence assay. We found that PPARα expression was lower in the irradiated skin tissues of mouse, rat, monkey, and human patients than in their nonirradiated counterparts. PPARα fenofibrate significantly decreased radiation-induced ROS and apoptosis in a dose-dependent manner in human keratinocyte HaCaT and skin fibroblast WS1 cells. Moreover, fenofibrate significantly decreased radiation-induced ROS and malondialdehyde (MDA) levels in electron beam irradiated skin tissues of rats. Mechanistically, the proximal promoter of fatty acid binding protein 4 (FABP4) harbored three binding sites of PPARα and fenofibrate stimulated the transcription of FABP4 in skin cells. FABP4 overexpression decreased radiation-induced ROS and γH2AX foci. FABP4 inhibitor BMS309403 abrogated the ROS-eliminating activity as well as the lipid-accumulating role of fenofibrate, indicating that FABP4 mediates the radioprotective role of fenofibrate. In addition, FABP4 overexpression significantly decreased radiation-induced oxidative damage in vivo. These results confirm that fenofibrate attenuated radiation-induced oxidative damage to the skin by stimulating FABP4.

Mitigation of radiation injury to reproductive system of male mice by Trichostatin A

Trichostatin A (TSA), derived from the bacteria Streptomyces hygroscopicus, is a hydroxamic acid having various biological properties such as histone deacetylase inhibition, anticancer and radiomitigative action. However the mitigative activity of TSA against radiation-induced damages in the mouse reproductive system has not yet been elucidated. The present study unraveled the effects of 2 Gy whole body irradiation (60Co γ- radiation) on C57BL/6 mice male reproductive system including structural damages to testes, increase in apoptosis and reduction in germ cell viability, reduced fertility as well as increased genomic instability in the next generation. Moreover, hematological study and micronuclei assay were used to record chances of radiation-induced hematologic cancer and disruption of genomic integrity in F1 generation. Interestingly, TSA administration 1 and 24 h post-irradiation attenuated radiation-induced morphological damage and cellular apoptosis in testes. In male mice, TSA restored hematological parameters and micronuclei frequency to normal levels, restored sperm viability, and helped them overcome radiation-induced temporary sterility 5 weeks after the irradiation. Thus our results showed that TSA reduced the probability of radiation-induced hematologic cancers as well as genotoxicity and restored genomic integrity in the progenies of paternally exposed mice by reducing radiation-induced apoptosis in spermatogenic cells and restoring cell proliferation. This study suggested that TSA could be used as potential radiomitigator for male reproductive system.

Dimethyl Sulfoxide Attenuates Radiation-Induced Testicular Injury through Facilitating DNA Double-Strand Break Repair.

The testis is susceptible to ionizing radiation, and male infertility and sexual dysfunction are prevalent problems after whole-body or local radiation exposure. Currently, there is no approved agent for the prevention or treatment of radiation-induced testicular injury. Herein, we investigated the radioprotective effect of dimethyl sulfoxide (DMSO), an organosulfur compound that acts as a free radical scavenger, on testicular injury. Treatment of mice with a single dose of DMSO prior to 5 Gy irradiation restored sex hormones and attenuated the reduction in testis weight. Histological analyses revealed that DMSO alleviated the distorted architecture of seminiferous tubules and promoted seminiferous epithelium regeneration following irradiation. Moreover, DMSO provided quantitative and qualitative protection for sperm and preserved spermatogenesis and fertility in male mice. Mechanistically, DMSO treatment enhanced GFRα-1+ spermatogonial stem cell and c-Kit+ spermatogonial survival and regeneration after radiation. DMSO also alleviated radiation-induced oxidative stress and suppressed radiation-induced germ cell apoptosis in vivo and in vitro. Additionally, DMSO efficiently reduced DNA damage accumulation and induced the expression of phosph-BRCA1, BRCA1, and RAD51 proteins, indicating that DMSO facilitates DNA damage repair with a bias toward homologous recombination. In summary, our findings demonstrate the radioprotective efficacy of DMSO on the male reproductive system, which warrants further studies for future application in the preservation of male fertility during conventional radiotherapy and nuclear accidents.

Cell Senescence-Related Pathways Are Enriched in Breast Cancer Patients With Late Toxicity After Radiotherapy and Low Radiation-Induced Lymphocyte Apoptosis.

BackgroundRadiation-induced late effects are a common cause of morbidity among cancer survivors. The biomarker with the best evidence as a predictive test of late reactions is the radiation-induced lymphocyte apoptosis (RILA) assay. We aimed to investigate the molecular basis underlying the distinctive RILA levels by using gene expression analysis in patients with and without late effects and in whom we had also first identified differences in RILA levels.Patients and MethodsPeripheral blood mononuclear cells of 10 patients with late severe skin complications and 10 patients without symptoms, selected from those receiving radiotherapy from 1993 to 2007, were mock-irradiated or irradiated with 8 Gy. The 48-h response was analyzed in parallel by RILA assay and gene expression profiling with Affymetrix microarrays. Irradiated and non-irradiated gene expression profiles were compared between both groups. Gene set enrichment analysis was performed to identify differentially expressed biological processes.ResultsAlthough differentially expressed mRNAs did not reach a significant adjusted p-value between patients suffering and not suffering clinical toxicity, the enriched pathways indicated significant differences between the two groups, either in irradiated or non-irradiated cells. In basal conditions, the main differentially expressed pathways between the toxicity and non-toxicity groups were the transport of small molecules, interferon signaling, and transcription. After 8 Gy, the differences lay in pathways highly related to cell senescence like cell cycle/NF-κB, G-protein-coupled receptors, and interferon signaling.ConclusionPatients at risk of developing late toxicity have a distinctive pathway signature driven by deregulation of immune and cell cycle pathways related to senescence, which in turn may underlie their low RILA phenotype.

RNF216 Alleviates Radiation-Induced Apoptosis and DNA Damage Through Regulating Ubiquitination-Mediated Degradation of p53 in Glioblastoma.

Glioblastoma (GBM) is the most common and lethal subtype of glioma, characterized by uncontrolled cancer cell proliferation, extensive infiltration, and therapeutic resistance. Ring finger protein 216 (RNF216) is a RING-type E3 ubiquitin ligase aberrantly expressed in multiple human cancers. Tumor protein 53 (p53) is a transcription factor that acts as a tumor suppressor. This study aimed to compare the RNF216 expression in GBM tissues and normal peritumoral tissues and to examine the effects of RNF216 overexpression/knockdown on tumorigenesis, radioresistance, and the p53 pathway in GBM. The results showed that RNF216 was overexpressed in GBM tissues and cell lines, and high RNF216 expression was related to a poor prognosis. RNF216 overexpression promoted GBM cell growth and inhibited apoptosis, while RNF216 knockdown impaired GBM cell growth and enhanced cell death. RNF216 was also highly expressed in recurrent GBM tissues compared with paired primary tumors. The upregulation of RNF216 not only facilitated GBM cell growth but also protected cells against X-ray irradiation-induced apoptosis and DNA damage, while RNF216 knockdown exerted opposite effects. Moreover, the implantation of GBM cells with RNF216 silencing suppressed tumorigenesis and increased radiosensitivity of mice bearing GBM xenografts. Further analysis revealed that RNF216 overexpression reduced the stability of p53 protein via ubiquitination and negatively regulated the p53 pathway, while RNF216 knockdown preserved the p53 protein. In conclusion, RNF216 effectively attenuated radiation-induced apoptosis and DNA damage in GBM via inducing ubiquitination-mediated degradation of p53. These findings suggest the potential therapeutic use of RNF216 inhibition for tumorigenesis and therapeutic resistance in GBM.

Hax-1 Regulates Radiation-Induced Mitochondrial-Dependent Apoptosis of Uveal Melanoma Cells through PI3K/AKT/eNOS Pathway.

Uveal melanoma is an aggressive skin cancer that remains insurmountable and is accompanied by inferior prognostic results. The proliferative and survival mechanisms of uveal melanoma cells need to be further investigated to improve the treatment of uveal melanoma. According to reports, HAX-1 is an antiapoptotic protein vital for multiple malignancies. Nevertheless, the role and causal link of HAX-1 in uveal melanoma are still elusive. The survival diversity of uveal melanoma sufferers with diverse haX-1 expressing levels was studied by TCGA database. Patients in the riskhigh group exhibited greater levels of HAX-1 in contrast to the risklow group, and individuals with higher HAX-1 levels displayed inferior survival times. The outcomes of CCK-8 and clonogenesis revealed that the proliferative rate of haX-1 knockout cells was slower. The result of scratch experiment shows that the ability of scratch recovery after HAX-1 is reduced. Transwell migration and tumor cell pelletization experiments showed that siHAX-1 significantly reduced cell migration and tumor cell pelletization. After haX-1 was knocked out, the loss of MMP was decreased, the transfer of CyT C was elevated, and the protein expression of Bax, Caspase 3, and Bcl2 was elevated, suggesting that mitochondria-induced apoptosis was increased. Sihax-1 treatment remarkably decreased the phosphonation of phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR)/endothelial NO synthase (eNOS) in mum-2B and C918. Pretreatment with LY294002 significantly restored iHAX-1-induced decline in PI3K/AKT/mTOR/eNOS phosphorylation. Therefore, our results suggest that haX-1 induces radiation-dependent apoptosis of UM cells via the PI3K/AKT/eNOS signal path.

Autophagic State Confers Facultative Stem Cell Capacity in The Intestinal Epithelium

BackgroundThe intestinal epithelium is a highly proliferative tissue that undergoes complete turnover in 3 to 5 days. The presence of intestinal stem cells expressing the canonical Wnt target gene Lgr5, known as active intestinal stem cells (a‐ISCs), are responsible for generating daughter cells that differentiate into specialized epithelial cells to carryout various functions in the intestine. The proliferative nature of the intestinal epithelium renders it susceptible to DNA damage‐inducing injury, such as high‐dose irradiation and chemotherapy. Although a‐ISCs and early daughter cells are killed by these insults, a radio‐resistant population of facultative intestinal stem cells (f‐ISCs) can regenerate lost a‐ISCs to restore homeostasis following injury. The functional characteristics of f‐ISCs are becoming increasingly understood, however, whether any epithelial cell can act as an f‐ISC is less clear. One reason for this ambiguity is a heavy reliance on Cre‐driven reporter mouse models with low recombination efficiency. Our objective was to find a functional marker to identify f‐ISCs based on cellular state rather than gene expression. The autophagy pathway has been shown to protect against DNA damage and irradiation‐induced apoptosis in the intestinal epithelium. Furthermore, recent studies demonstrate a requirement for the autophagy pathway during cellular de‐differentiation in both gastric chief and pancreatic acinar cells following metaplasia‐inducing injury. Given that autophagy plays roles in both radio‐resistance and cellular plasticity, two integral features of f‐ISCs, we hypothesized that autophagic activity could serve to identify cells with f‐ISC capacity.Methods and ResultsUsing the autophagic vesicle tracer dye CytoID combined with fluorescence‐activated cell sorting, we demonstrate that intestinal epithelial cells with high levels of CytoID exhibit increased organoid‐formation efficiency (OFE) compared to CytoID ‘low’ cells. Single cell sequencing reveals that the CytoID high population is largely composed of secretory cells including Paneth, Goblet, Tuft, and Enteroendocrine cells, whereas the CytoID low population consisted mainly of a‐ISCs and absorptive Enterocytes. Using reporter mice and antibodies against cell surface receptors, we observe that CytoID can identify cells with high OFE within Enteroendocrine, Paneth, and Goblet cell lineages. Finally, we demonstrate that autophagy is required for the enhanced organoid‐formation observed in CytoID high cells by plating these cells in the presence of the lysosomal inhibitor Bafilomycin A1.ConclusionsOur new data support the notion that epithelial cells in a high autophagic state are biased to secretory lineages and that autophagy status functions as a lineage agnostic marker of f‐ISCs. Furthermore, our data suggests that autophagy is required for organoid formation in these lineages.

SOCS1 counteracts ROS-mediated survival signals and promotes apoptosis by modulating cell cycle to increase radiosensitivity of colorectal cancer cells

As negative regulators of cytokine signaling pathways, suppressors of cytokine signaling (SOCS) proteins have been reported to possess both pro-tumor and anti-tumor functions. Our recent studies have demonstrated suppressive effects of SOCS1 on epithelial to mesenchymal signaling in colorectal cancer cells in response to fractionated ionizing radiation or oxidative stress. The objective of the present study was to determine the radiosensitizing action of SOCS1 as an anti-tumor mechanism in color-ectal cancer cell model. In HCT116 cells exposed to ionizing radiation, SOCS1 over-expression shifted cell cycle arrest from G2/M to G1 and promoted radiation-induced apoptosis in a p53-dependent manner with down-regulation of cyclin B and up-regulation of p21. On the other hand, SOCS1 knock-down resulted in a reduced apoptosis with a decrease in G1 arrest. The regulatory action of SOCS1 on the radiation response was mediated by inhibition of radiation-induced Jak3/STAT3 and Erk activities, thereby blocking G1 to S transition. Radiation-induced early ROS signal was responsible for the activation of Jak3/Erk/STAT3 that led to cell survival response. Our data col-lectively indicate that SOCS1 can promote radiosensitivity of colorectal cancer cells by counteracting ROS-mediated survival signal, thereby blocking cell cycle progression from G1 to S. The resulting increase in G1 arrest with p53 activation then contributes to the promotion of apoptotic response upon radiation. Thus, induction of SOCS1 expression may increase therapeutic efficacy of radiation in tumors with low SOCS1 levels.