53BP1 loss suppresses the radiosensitizing effect of icotinib hydrochloride in colorectal cancer cells

Background: This study aimed to investigate the influence of the expression of P53-binding protein 1 (53BP1), a key component in DNA damage repair pathways, on the radiosensitizing effect of icotinib hydrochloride in colorectal cancer and to elucidate the mechanisms underlying this influence.

Materials and methods: Real-time RT-PCR and Western blotting were performed to verify the gene-knockout effect of 53BP1 small hairpin RNA (ShRNA), and colony formation assay was employed to investigate the influence of 53BP1 downregulation on the radiosensitizing effect of icotinib hydrochloride in HCT116 cells. Cell apoptosis, cell cycle distributions, and histone H2AX (γ-H2AX) fluorescence foci after 53BP1 knockdown were evaluated. Relative protein expression in the ataxia telangiectasia mutated kinase (ATM)-checkpoint kinase-2 (CHK2)-P53 pathway was measured by Western blot analysis to unravel the molecular mechanisms linking the pathway to the above phenomena.

Results: Icotinib hydrochloride increased the radiosensitivity of HCT116 cells; however, this effect was suppressed by the downregulation of 53BP1 expression, a change that inhibited cell apoptosis, increased the percentage of HCT116 cells arrested in S-phase and inhibited the protein expression of key molecules in the ATM-CHK2-P53 apoptotic pathway.

Conclusion: Our studies confirmed that the loss of 53BP1 serves as a negative regulator of the radiosensitizing effect of icotinib in part by suppressing the ATM-CHK2-P53 apoptotic pathway.     

Estimation of low-dose radiation-responsive proteins in the absence of genomic instability in normal human fibroblast cells

Purpose: Low-dose radiation has various biological effects such as adaptive responses, low-dose hypersensitivity, as well as beneficial effects. However, little is known about the particular proteins involved in these effects. Here, we sought to identify low-dose radiation-responsive phosphoproteins in normal fibroblast cells.

Materials and methods: We assessed genomic instability and proliferation of fibroblast cells after γ-irradiation by γ-H2AX foci and micronucleus formation analyses and BrdU incorporation assay, respectively. We screened fibroblast cells 8?h after low-dose (0.05?Gy) γ-irradiation using Phospho Explorer Antibody Microarray and validated two differentially expressed phosphoproteins using Western blotting.

Results: Cell proliferation proceeded normally in the absence of genomic instability after low-dose γ-irradiation. Phospho antibody microarray analysis and Western blotting revealed increased expression of two phosphoproteins, phospho-NFκB (Ser536) and phospho-P70S6K (Ser418), 8?h after low-dose radiation.

Conclusions: Our findings suggest that low-dose radiation of normal fibroblast cells activates the expression of phospho-NFκB (Ser536) and phospho-P70S6K (Ser418) in the absence of genomic instability. Therefore, these proteins may be involved in DNA damage repair processes.     

Potentiality of syringetin for preferential radiosensitization to cancer cells

Purpose: To examine the enhancing effects of syringetin on the radiosensitivity of normal and cancer cells, and the related mechanism.

Materials and methods: We used normal human lung and mouse fibroblasts as well as human lung and mouse cancer cells derived from the above normal fibroblasts. Cell radiosensitivity was measured using a colony formation assay. Apoptosis was analyzed with DAPI staining and Western blots. DNA lesions were analyzed with γH2AX immunofluorescent staining.

Results: The colony formation assay showed that syringetin enhanced radiosensitivity more effectively in cancer cells (H1299 and C3H/MCA clone 15) compared with normal cells (HFL-III and C3H/10T1/2). The radiosensitizing effect of syringetin was observed in mutated p53 and wild-type p53-transfected H1299 cells regardless of p53 status. Apoptosis was more frequently observed in X-ray-irradiated H1299 cells combined with syringetin compared with X-ray-only-treated cells. Enhanced apoptosis by syringetin was not observed in HFL-III cells. Western blot analysis showed that X-ray-induced Caspase-3 activation was enhanced by syringetin in H1299 cells. The number of X-ray-induced DNA double-strand breaks (DSB) measured by quantitative analysis of γH2AX foci was the same for H1299 cells treated with X-rays with or without syringetin.

Conclusions: This study supports the hypothesis that syringetin enhances radiosensitivity more effectively in cancer cells than in normal cells through enhancement of the Caspase-3-mediated apoptosis pathway. Syringetin could be useful in the development of novel efficacious radiosensitizers.     

Association of ribosomal protein S6 kinase 1 with cellular radiosensitivity of non-small lung cancer

Purpose: Ribosomal S6 kinase 1 (S6K1) plays an important role in cell proliferation, protein translation and cell survival. This study investigated the possibility of using S6K1 as a new target in the radiotherapy of non-small cell lung cancer (NSCLC) and its potential mechanism.

Materials and methods: shRNA interference technology was applied to silence the expression of S6K1 in A549 and H460, and clonogenic assay was performed to measure the radiosensitizing effects. DNA repair was monitored by γH2AX foci formation. Cell cycle and apoptosis were measured by flow cytometry assays. Protein expression was analyzed by Western blotting. S6K1 and lentivirus containing programmed cell death 4 (PDCD4), a substrate of S6K1, were co-transfected into cells and radiosensitivity was also detected.

Results: S6K1 knockdown significantly enhanced the radiosensitivity of NSCLC cells. The notable findings in response to this combined treatment were prolonged delay in radiation-induced DNA DSB repair, robust G2/M checkpoint arrest, increased apoptosis and upregulation of PDCD4. Moreover, PDCD4 knockdown reversed the radiosensitizing effects of S6K1 inhibition in NSCLC.

Conclusions: S6K1 is a promising tumor-specific target for the enhancement of NSCLC radiosensitivity and its effects may be mediated by increased expression of PDCD4.     

The drill wobble effect: oversizing the femoral tunnel during ACL reconstruction

Objectives: In ACL reconstruction, autologous tendon graft can be attached to the femur, within a boney tunnel, using an Endobutton device. The ultimate aim being to achieve biological fixation and incorporation into the bone. Accurate bone tunnel diameter to match the tendon graft is vital to biologic incorporation and strength. The common technique of in sequence passing a guide wire, a cannulated 4.5 mm Endobutton drill, then a cannulated femoral socket drill causes the guidewire to lose cortical fixation and stability before the femoral socket drill is passed.

The Objective of this study is to analyze this common technique of femoral socket creation and determine if it results in unintentionally oversizing the femoral socket due to femoral socket drill-wobble over a destabilised guide wire.

Methods: 12 cadaveric femoral pairs equally divided between two groups underwent femoral socket creation in one of the two following sequences.

Group 1: Guidewire, 4.5 mm endobutton drill, 8 mm femoral socket drill. Group 2: Guidewire, 8 mm femoral socket drill, 4.5 mm endobutton drill. The created femoral tunnels apertures and calibres were measured and then compared for accuracy between the two groups.

Results: Passing the 4.5 mm drill before the 8 mm socket drill results in oversized tunnel apertures and calibres when compared to passing an 8 mm socket drill after the 4.5 mm drill has been passed (p<0.0001).

Conclusion: To most precisely create an 8 mm femoral socket in ACL reconstruction, the 8 mm femoral socket reamer followed by the 4.5 mm should be passed over the guide wire to prevent guide wire destabilization and drill-wobble.     

Effect of cell phone-like electromagnetic radiation on primary human thyroid cells

Purpose To evaluate the potential carcinogenic effects of radiofrequency energy (RFE) emitted by cell phones on human thyroid primary cells.

Materials and methods Primary thyroid cell culture was prepared from normal thyroid tissue obtained from patients who underwent surgery at our department. Subconfluent thyroid cells were irradiated under different conditions inside a cell incubator using a device that simulates cell phone-RFE. Proliferation of control and irradiated cells was assessed by the immunohistochemical staining of antigen Kiel clone-67 (Ki-67) and tumor suppressor p53 (p53) expression. DNA ploidy and the stress biomarkers heat shock protein 70 (HSP70) and reactive oxygen species (ROS) was evaluated by fluorescence-activated cell sorting (FACS).

Results Our cells highly expressed thyroglobulin (Tg) and sodium-iodide symporter (NIS) confirming the origin of the tissue. None of the irradiation conditions evaluated here had an effect neither on the proliferation marker Ki-67 nor on p53 expression. DNA ploidy was also not affected by RFE, as well as the expression of the biomarkers HSP70 and ROS.

Conclusion Our conditions of RFE exposure seem to have no potential carcinogenic effect on human thyroid cells. Moreover, common biomarkers usually associated to environmental stress also remained unchanged. We failed to find an association between cell phone-RFE and thyroid cancer. Additional studies are recommended.     

Reduction of anterior glenohumeral dislocations: a new closed reduction technique

Objective: A new closed reduction technique for anterior glenohumeral dislocations and tuberosity fracture dislocations is introduced.

Methods: Forty-one consecutive patients with an acute anterior glenohumeral dislocation or tuberosity fracture dislocation underwent closed reduction by an orthopaedic surgeon employing this new method.

Results: Closed reduction was successful in 88% of patients using the reduction maneuver. Associated fracture with glenohumeral dislocation did not influence the success rate of the reduction maneuver. An assistant was needed in 15% of cases. No complications related to the reduction maneuver were noted amongst the cohort.

Conclusion: This novel reduction technique is safe demonstrating excellent success rates both for anterior shoulder dislocations and tuberosity fracture-dislocations.     

Photon-induced Auger effect in biological systems: a review

Purpose: In this paper, we review interesting findings reported in the studies of the biological effects induced by inner-shell ionization with the aim of interpreting them from a mechanistic viewpoint, in particular, of the Auger effect of atoms on different biological systems.

Materials and methods: More than 70 published papers are cited on the Auger effects ranging from DNA-related elements (carbon, nitrogen, oxygen and phosphorus) to mammalian cells. Externally administrated bromine, iodine, and platinum have also been cited for the present endeavor. Those significant works all needed a highly monochromatized X-rays from brilliant synchrotron light sources.

Results: We have assembled a coherent view on the inner-shell effects of the Auger process that contrasts to the overall effects with the outer-shell ionization processes.

Conclusion: Some of these studies have reported that the Auger effect significantly enhances biological effects as compared with irradiation at below K-ionization energy. The Auger-specific molecular degradation mode of DNA, involving extensive fragmentation of the deoxypentose moiety, has also been revealed. We conclude that the selectively localized effect on the specified atoms through inner-shell ionization followed by the Auger process should have a definite impact on the current radiation effect studies, which are largely based on non-selective outer-shell ionizations.     

Screening of biomarkers for liver adenoma in low-dose-rate γ-ray-irradiated mice

Purpose: Chronic low-dose-rate (20 mGy/day) γ-irradiation increases the incidence of hepatocellular adenomas (HCA) in female B6C3F1 mice. The purpose of this study is to identify potential serum biomarkers for these HCAs by a new approach.

Material and methods: Microarray analysis were performed to compare the gene expression profiles of HCAs from mice exposed to low-dose-rate γ-rays with those of normal livers from non-irradiated mice. From the differentially expressed genes, those for possibly secretory proteins were selected. Then, the levels of the proteins in sera were analysed by ELISA.

Results: Microarray analysis identified 4181 genes differentially expressed in HCAs (>2.0-fold). From these genes, those for α-fetoprotein (Afp), α-1B-glycoprotein (A1bg) and serine peptidase inhibitor Kazal type-3 (Spink3) were selected as the genes for candidate proteins. ELISA revealed that the levels of Afp and A1bg proteins in sera significantly increased and decreased, respectively, in low-dose-rate irradiated mice with HCAs and also same tendency was observed in human patients with hepatocellular carcinomas.

Conclusion: These results indicate that A1bg could be a new serum biomarker for liver tumor. This new approach of using microarray to select genes for secretory proteins is useful for prediction of novel tumor markers in sera.     

Radiobiological effects of tritiated water short-term exposure on V79 clonogenic cell survival

Purpose: We set out to improve the accuracy of absorbed dose calculations for in vitro measurements of the relative biological effectiveness (RBE) of tritiated water (HTO) for the clonogenic cell survival assay, also considering the influence of the end-of-track linear energy transfer (LET) of low-energy electrons.

Materials and methods: The COmputation Of Local Electron Release (COOLER) program was adopted to investigate the cell geometry and the tritium full beta-decay spectrum impact on the S-values and subsequently on the RBE of HTO for clonogenic cell survival at similar high dose rates (HDR).

Results: S-values for cells growing in suspension are usually comparable to those for adherent cells. RBEs calculated at the 10% survival fraction through the use of the average energy are almost similar to those obtained with the beta-spectrum. For adherent cells, an RBE of 1.6 was found when HTO cell survival curves were compared to acute γ-ray exposures. Irrespective of the geometrical configuration, the RBE was 2.0 when the comparison was made with similar dose rates.

Conclusions: These results underline the importance of irradiating at equal dose rates and cell culture conditions when measuring in vitro RBE-values.     

Heart rate recovery time in metabolically healthy and metabolically unhealthy obese children

Background: Autonomic dysfunction is an emerging non-traditional cardiovascular risk factor that correlates with obesity, components of metabolic syndrome, as well as cardiorespiratory fitness. As a simple and validated index of autonomic balance, heart rate recovery (HRR) has been reported as a useful biomarker for predicting cardiovascular morbidity and mortality.

Objectives: The aim of this study was to compare HRR in metabolically healthy vs. metabolically unhealthy obese children.

Methods: A total of 56 obese children of whom 31 had metabolic syndrome were examined. All the participants underwent the multistage submaximal cycle ergometer test and HRR was determined one minute after the test.

Results: The HRR was significantly lower (18.9 ± 3.7) in a group of metabolically unhealthy obese children compared to metabolically healthy obese children (24 ± 4.1) p < 0.001. Logistic regression analysis showed that reduction in HRR was also influenced by higher BMI.

Conclusion: Our findings implicate the presence of the autonomic dysfunction as reflected by impaired heart rate recovery (HRR) in obese children with metabolic syndrome.     

Synergistic effect of Ebselen and gamma radiation on breast cancer cells

Purpose: To explore the synergistic effect of a seleno-organic compound Ebselen (Ebs) and/or γ-radiation to exert antitumor effects on human breast cancer (MCF-7) cell line in vitro.

Materials and methods: Ebs cytotoxicity at various concentrations (10, 25, 50 and 75 μg), cell proliferation and clonogenic assay of Ebs and/or γ-radiation (at 1, 3 and 6?Gy), expression of p-IκBα and NF-κB, inflammatory cytokines levels (TNF-α, IL-2, INF-γ, IL-10 and TGF-β), apoptotic factors (Caspase-3, Granzyme-B and TRAIL) and angiogenic factor (VEGF) were investigated.

Results: The results showed that the effective dosage of this combination was observed at 25 μg/ml of Ebs with γ-radiation at 6?Gy. Data displayed a significant reduction in NF-κB mRNA along with an elevation in granzyme-B mRNA and TRAIL mRNA expression. Furthermore, protein expression of caspase-3 was elevated, whereas p-IκBα and p-NF-κB(p65) protein expression was reduced significantly. Also, a significant decline in TNF-α, IL-2, INF-γ, TGF-β with a significant increase in IL-10 levels were revealed. Meanwhile, a significant decrease in VEGF level and proliferation capacity were observed.

Conclusions: We conclude that a combination of Ebs with radiotherapy has a major antitumor efficiency in inducing apoptosis and inhibiting cancer cell progression, due to the synergistic effect in regulating gene and protein expression, and in a modulating response of pro-and anti-inflammatory cytokines.     

Inducing mutations through γ-irradiation in seeds of Mucuna pruriens for developing high L-DOPA-yielding genotypes

Purpose: To develop elite genotypes in Mucuna pruriens (L.) DC with high L-DOPA (L-3, 4 dihydroxyphenylalanine) yields, with non-itching characteristics and better adaptability by applying γ-irradiation. Molecular and chemical analysis was performed for screening based on specific characteristics desired for developing suitable genotypes.

Materials and methods: Developed, mutant populations were analyzed for L-DOPA % in seeds through TLC (thin layer chromatography), and the results obtained were validated with the HPLC (High performance liquid chromatography). The DNA (Deoxyribonucleic acid) was isolated from the leaf at the initial stage and used for DNA polymorphism. RNA (Ribonucleic acid) was isolated from the leaf during maturity and used for expression analysis.

Results: The selected mutant T-I-7 showed 5.7% L-DOPA content compared to 3.18% of parent CIM-Ajar. The total polymorphism obtained was 57% with the molecular marker analysis. The gene expression analysis showed higher fold change expression of the dopadecarboxylase gene (DDC) in control compared to selected mutants (T-I-7, T-II-23, T-IV-9, T-VI-1).

Conclusion: DNA polymorphism was used for the screening of mutants for efficient screening at an early stage. TLC was found suitable for the large-scale comparative chemical analysis of L-DOPA. The expression profile of DDC clearly demonstrated the higher yields of L-DOPA in selected mutants developed by γ-irradiation in the seeds of the control.     

Nano alterations of membrane structure on both γ-irradiated and stored human erythrocytes

Purpose: Storage and ionizing radiation of human red blood cells (RBC) produce alterations on RBC membranes and modify their normal shape and functionality. We investigated early morphological and biochemical changes in RBC due to those stressing agents at the nanoscale level and their impact on blood quality.

Materials and methods: Whole blood samples from healthy donors were γ-irradiated with 15, 25, 35, and 50?Gy. Non-irradiated and non-stored RBC were used as control samples. Irradiated blood samples were stored separately at 4?°C and analyzed immediately and after 5 and 13 d. Atomic force microscopy (AFM), osmotic fragility and Raman spectroscopy were used to detect morphological and biochemical changes.

Results: RBC function is challenged by both irradiation and storage. The storage procedure caused nanometric variations over the surface of RBC membrane for both irradiated and non-irradiated cells. The membrane of RBC became more fragile, while the biochemical fingerprint of hemoglobin (Hb) remained unaltered.

Conclusions: Our work shows that the irradiation procedure leads to an increase in the number and size of nanovesicles along with the dose. The functionality of RBC can be affected from changes in the roughness, becoming more fragile and susceptible to breakage.     

A systematic literature review of synovial chondromatosis and pigmented villonodular synovitis of the hip

Objectives: Benign synovial diseases of the hip including Synovial Chondromatosis (SC) and Pigmented Villonodular Synovitis (PVNS) are devastating diseases. Initially, patients present with hip pain unrelieved by conservative measures. The diagnosis of PVNS and SC are often delayed, leading to progression of joint damage.

The purpose of this review is to present the latest on the diagnosis, management, and prognosis of SC and PVNS of the hip.

Methods: An extensive systematic search of MEDLINE and PUBMED Databases was performed. Data parameters were set from 2005 to present day with set inclusion criteria. Systematic reviews were excluded.

Results: 427 abstracts were identified, with 12 articles meeting all inclusion criteria. Eight studies focused on SC, and 5 on PVNS. 233 patients with SC of the hip and 98 patients with PVNS of the hip were identified, a total of 331 patients.

Discussion: Benign Synovial disorders of the hip are rare. In patients with chronic hip pain secondary to benign synovial disorders, early diagnosis and surgical intervention demonstrate good outcomes, and patients may benefit due to prevention of morbidity from further joint destruction. There is no clear consensus between higher successes through open versus arthroscopic surgical debridement. In the final phase of benign synovial disorders of the hip, THA of different types based on the patient’s age should be considered.     

Effects of gamma rays on the regeneration of murine hair follicles in the natural hair cycle

Purpose: This review evaluates the effects of γ-rays on the regeneration of murine hair follicles in the natural hair cycle. A series of studies were performed to investigate this issue, in which the whole bodies of C57BL/10JHir mice in the 1st telogen phase of the hair cycle were irradiated with γ-rays.

Results: The dermis of the irradiated skin showed a decrease in hair follicle density and induction of curved hair follicles along with the presence of white hairs and hypopigmented hair bulbs in the 2nd and 3rd anagen phases. An increased frequency of hypopigmented hair bulbs was still observed in the later hair cycle at postnatal day 200. There was no significant difference in the number of stem cells in the hair bulge region between control and irradiated skin.

Conclusions: These results show that the effects of γ-rays on the pigmentation of murine hair follicles are persistently carried over to later hair cycles, although those on the number and structure of hair follicles appear to be hidden by the effects of aging. Our findings may be important for understanding the mechanisms of the actions of stem cells on hair regeneration in connection with age-related phenotypes.     

Cardiovascular sequel of neck irradiation in head and neck cancer patients

Purpose: The baroreflex is an important afferent mechanism controlling autonomic functions. As afferent nerves course through the neck, they are susceptible to damage by neck irradiation in head and neck cancer patients. With increased survival of head and neck cancer patients because of improved therapy, the cardiovascular morbidity and mortality in them have become apparent and this is of clinical concern. There are few case reports of baroreflex failure as a chronic sequel to neck irradiation.

Objectives: The present study evaluated the changes in cardio-autonomic tone and postural cardiovascular reflex in neck-irradiated patients.

Methods: Head and neck cancer patients who had received neck irradiation (n?=?15) and healthy controls (n?=?15) were evaluated for heart rate variability with time domain analysis of 5?min ECG recording. Postural cardiovascular reflexes were studied with changes in blood pressure and heart rate in the lying to standing test.

Results: Our results suggest that there is a reduction in overall time domain measures of heart rate variability and weakened postural reflexes in neck-irradiated patients.

Conclusion: Decreased heart rate variability in neck-irradiated patients reflects an independent risk of cardiovascular morbidity. The early detection of cardiovascular impairment in such patients may help healthcare professionals in providing better care. Furthermore, the dose delivered to the carotid sinus should be monitored and restricted.     

DNA repair pathway choice at various conditions immediately post irradiation

Purpose: To clarify which DNA double-strand break repair pathway, non-homologous end-joining (NHEJ), homologous recombination repair (HRR) or both, plays a key role in potentially lethal damage repair (PLDR).

Methods and materials: Combining published data and our new potentially lethal damage repair (PLDR) data, we explain whether similar to sublethal damage repair (SLDR), PLDR also mainly depends on NHEJ versus HRR. The PLDR data used the same cell lines: wild type, HRR or NHEJ-deficient fibroblast cells, as those SLDR data published by our laboratory previously. The PLDR condition that we used was as commonly described by many other groups: the cells were collected immediately or overnight post ionizing radiation for colony formation after cultured to a plateau phase with a low concentration of serum medium.

Results: Enough data from other groups and our lab showed that wild type or HRR-deficient cells had efficient PLDR, but NHEJ deficient cells did not.

Conclusion: NHEJ contributes more to PLDR than HRR in mammalian cells, which is similar to SLDR. Since both SLDR and PLDR are relevant to clinical tumor status while undergoing radiotherapy, such clarification may benefit radiotherapy in the near future.     

Increasing levels of saliva alpha amylase in electrohypersensitive (EHS) patients

Purpose: To assess the level of various salivary and urinary markers of patients with electromagnetic hypersensitivity (EHS) and to compare them with those of a healthy control group.

Materials and methods: We analyzed samples from 30 EHS individuals and a matched control group of 25 individuals (non-EHS) aged between 22 and 66. We quantified cortisol both in saliva and urine, alpha amylase (sAA), immunoglobulin A and C Reactive Protein levels in saliva and neopterin in urine (uNeopterin).

Results: sAA was found to be significantly higher (p?<?0.005) in the EHS group. uNeopterin and sAA analysis showed a significant difference based on the duration of EHS.

Conclusion: Higher levels of sAA in EHS participants may suggest that the sympathetic adrenal medullar system is activated. However, most of the analyzed markers of the immune system, sympathetic activity and circadian rhythm did not vary significantly in the EHS group. There is a trend to the higher levels of some variables in subgroups according to the EHS duration.