GCN5 mediates DNA-PKcs crotonylation for DNA double-strand break repair and determining cancer radiosensitivity.

BACKGROUND: DNA double-strand break (DSB) induction and repair are important events for determining cell survival and the outcome of cancer radiotherapy. The DNA-dependent protein kinase (DNA-PK) complex functions at the apex of DSBs repair, and its assembly and activity are strictly regulated by post-translation modifications (PTMs)-associated interactions. However, the PTMs of the catalytic subunit DNA-PKcs and how they affect DNA-PKcs's functions are not fully understood. METHODS: Mass spectrometry analyses were performed to identify the crotonylation sites of DNA-PKcs in response to γ-ray irradiation. Co-immunoprecipitation (Co-IP), western blotting, in vitro crotonylation assays, laser microirradiation assays, in vitro DNA binding assays, in vitro DNA-PK assembly assays and IF assays were employed to confirm the crotonylation, identify the crotonylase and decrotonylase, and elucidate how crotonylation regulates the activity and function of DNA-PKcs. Subcutaneous xenografts of human HeLa GCN5 WT or HeLa GCN5 siRNA cells in BALB/c nude mice were generated and utilized to assess tumor proliferation in vivo after radiotherapy. RESULTS: Here, we reveal that K525 is an important site of DNA-PKcs for crotonylation, and whose level is sharply increased by irradiation. The histone acetyltransferase GCN5 functions as the crotonylase for K525-Kcr, while HDAC3 serves as its dedicated decrotonylase. K525 crotonylation enhances DNA binding activity of DNA-PKcs, and facilitates assembly of the DNA-PK complex. Furthermore, GCN5-mediated K525 crotonylation is indispensable for DNA-PKcs autophosphorylation and the repair of double-strand breaks in the NHEJ pathway. GCN5 suppression significantly sensitizes xenograft tumors of mice to radiotherapy. CONCLUSIONS: Our study defines K525 crotonylation of DNA-PKcs is important for the DNA-PK complex assembly and DSBs repair activity via NHEJ pathway. Targeting GCN5-mediated K525 Kcr of DNA-PKcs may be a promising therapeutic strategy for improving the outcome of cancer radiotherapy.

PARP1 modulates METTL3 promoter chromatin accessibility and associated LPAR5 RNA m6A methylation to control cancer cell radiosensitivity.

Chromatin remodeling and N6-methyladenosine (m6A) modification are two critical layers in controlling gene expression and DNA damage signaling in most eukaryotic bioprocesses. Here, we report that poly(ADP-ribose) polymerase 1 (PARP1) controls the chromatin accessibility of METTL3 to regulate its transcription and subsequent m6A methylation of poly(A)+ RNA in response to DNA damage induced by radiation. The transcription factors nuclear factor I-C (NFIC) and TATA binding protein (TBP) are dependent on PARP1 to access the METTL3 promoter to activate METTL3 transcription. Upon irradiation or PARP1 inhibitor treatment, PARP1 disassociated from METTL3 promoter chromatin, which resulted in attenuated accessibility of NFIC and TBP and, consequently, suppressed METTL3 expression and RNA m6A methylation. Lysophosphatidic Acid Receptor 5 (LPAR5) mRNA was identified as a target of METTL3, and m6A methylation was located at A1881. The level of m6A methylation of LPAR5 significantly decreased, along with METTL3 depression, in cells after irradiation or PARP1 inhibition. Mutation of the LPAR5 A1881 locus in its 3' UTR results in loss of m6A methylation and, consequently, decreased stability of LPAR5 mRNA. METTL3-targeted small-molecule inhibitors depress murine xenograft tumor growth and exhibit a synergistic effect with radiotherapy in vivo. These findings advance our comprehensive understanding of PARP-related biological roles, which may have implications for developing valuable therapeutic strategies for PARP1 inhibitors in oncology.

Exposure to polystyrene nanoplastics induces abnormal activation of innate immunity via the cGAS-STING pathway.

Endogenous immune defenses provide an intrinsic barrier against external entity invasion. Microplastics in the environment, especially those at the nanoscale (nanoplastics or NPs), may pose latent health risks through direct exposure. While links between nanoplastics and inflammatory processes have been established, detailed insights into how they may perturb the innate immune mechanisms remain uncharted. Employing murine and macrophage (RAW264.7) cellular models subjected to polystyrene nanoplastics (PS-NPs), our investigative approach encompassed an array of techniques: Cell Counting Kit-8 assays, flow cytometric analysis, acridine orange/ethidium bromide (AO/EB) fluorescence staining, cell transfection, cell cycle scrutiny, genetic manipulation, messenger RNA expression profiling via quantitative real-time PCR, and protein expression evaluation through western blotting. The results showed that PS-NPs caused RAW264.7 cell apoptosis, leading to cell cycle arrest, and activated the cGAS-STING pathway. This resulted in NF-κB signaling activation and increased pro-inflammatory mediator expression. Importantly, PS-NPs-induced activation of NF-κB and its downstream inflammatory cascade were markedly diminished after the silencing of the STING gene. Our findings highlight the critical role of the cGAS-STING pathway in the immunotoxic effects induced by PS-NPs. We outline a new mechanism whereby nanoplastics may trigger dysregulated innate immune and inflammatory responses via the cGAS/STING pathway.

Association between per- and polyfluoroalkyl substances (PFAS) and depression in U.S. adults: A cross-sectional study of NHANES from 2005 to 2018.

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) are widespread persistent organic pollutants (POPs) associated with diseases including osteoporosis, altered immune function and cancer. However, few studies have investigated the association between PFAS mixture exposure and Depression in general populations. METHODS: Nationally representative data from the National Health and Nutrition Examination Survey (NHANES) (2005-2018) were used to analyze the association between PFAS and Depression in U.S. adults. Total 12,239 adults aged 20 years or older who had serum PFAS measured and answered Patient Health Questionnaire-9 (PHQ-9) were enrolled in this study. PFAS monomers detected in all 7 investigation cycles were included in the study. Generalized additive model (GAM) was used to fit smooth curves and threshold effect analysis was carried out to find the turning point of smooth curves. Generalized linear model (GLM) was used to describe the non-linear relationship between PFAS and depression and unconditioned logistic regression was used to risk analysis. RESULTS: The median of total serum PFAS concentration was 14.54 ng/mL. The curve fitting results indicated a U-shaped relationship between total serum PFAS and depression: PFAS< 39.66 ng/mL, A negative correlation between PHQ-9 score and serum PFAS concentration was observed (ß 0.047,95%CI -0.059, -0.036). The depression PHQ-9 score decreased with the increase of serum PFAS concentration. PFAS ≥ 39.66 ng/mL, A positive correlation was observed between PFAS and PHQ-9 score (ß 0.010,95% CI 0.003, 0.017). The depression PHQ-9 score increased with the increase of serum PFAS concentration. CONCLUSIONS: Our study provides new clues to the association of PFAS with depression, and large population-based cohort studies that can validate the causal association as well as toxicological mechanism studies are needed for validation.

LPAR5 confers radioresistance to cancer cells associated with EMT activation via the ERK/Snail pathway.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is a critical event contributing to more aggressive phenotypes in cancer cells. EMT is frequently activated in radiation-targeted cells during the course of radiotherapy, which often endows cancers with acquired radioresistance. However, the upstream molecules driving the signaling pathways of radiation-induced EMT have not been fully delineated. METHODS: In this study, RNA-seq-based transcriptome analysis was performed to identify the early responsive genes of HeLa cells to γ-ray irradiation. EMT-associated genes were knocked down by siRNA technology or overexpressed in HeLa cells and A549 cells, and the resulting changes in phenotypes of EMT and radiosensitivity were assessed using qPCR and Western blotting analyses, migration assays, colony-forming ability and apoptosis of flow cytometer assays. RESULTS: Through RNA-seq-based transcriptome analysis, we found that LPAR5 is downregulated in the early response of HeLa cells to γ-ray irradiation. Radiation-induced alterations in LPAR5 expression were further revealed to be a bidirectional dynamic process in HeLa and A549 cells, i.e., the early downregulating phase at 2 ~ 4 h and the late upregulating phase at 24 h post-irradiation. Overexpression of LPAR5 prompts EMT programing and migration of cancer cells. Moreover, increased expression of LPAR5 is significantly associated with IR-induced EMT and confers radioresistance to cancer cells. Knockdown of LPAR5 suppressed IR-induced EMT by attenuating the activation of ERK signaling and downstream Snail, MMP1, and MMP9 expression. CONCLUSIONS: LPAR5 is an important upstream regulator of IR-induced EMT that modulates the ERK/Snail pathway. This study provides further insights into understanding the mechanism of radiation-induced EMT and identifies promising targets for improving the effectiveness of cancer radiation therapy.

Tissue Reactions and Mechanism in Cardiovascular Diseases Induced by Radiation.

The long-term survival rate of cancer patients has been increasing as a result of advances in treatments and precise medical management. The evidence has accumulated that the incidence and mortality of non-cancer diseases have increased along with the increase in survival time and long-term survival rate of cancer patients after radiotherapy. The risk of cardiovascular disease as a radiation late effect of tissue damage reactions is becoming a critical challenge and attracts great concern. Epidemiological research and clinical trials have clearly shown the close association between the development of cardiovascular disease in long-term cancer survivors and radiation exposure. Experimental biological data also strongly supports the above statement. Cardiovascular diseases can occur decades post-irradiation, and from initiation and development to illness, there is a complicated process, including direct and indirect damage of endothelial cells by radiation, acute vasculitis with neutrophil invasion, endothelial dysfunction, altered permeability, tissue reactions, capillary-like network loss, and activation of coagulator mechanisms, fibrosis, and atherosclerosis. We summarize the most recent literature on the tissue reactions and mechanisms that contribute to the development of radiation-induced cardiovascular diseases (RICVD) and provide biological knowledge for building preventative strategies.

The Promising Therapeutic Approaches for Radiation-Induced Pulmonary Fibrosis: Targeting Radiation-Induced Mesenchymal Transition of Alveolar Type II Epithelial Cells.

Radiation-induced pulmonary fibrosis (RIPF) is a common consequence of radiation for thoracic tumors, and is accompanied by gradual and irreversible organ failure. This severely reduces the survival rate of cancer patients, due to the serious side effects and lack of clinically effective drugs and methods. Radiation-induced pulmonary fibrosis is a dynamic process involving many complicated and varied mechanisms, of which alveolar type II epithelial (AT2) cells are one of the primary target cells, and the epithelial-mesenchymal transition (EMT) of AT2 cells is very relevant in the clinical search for effective targets. Therefore, this review summarizes several important signaling pathways that can induce EMT in AT2 cells, and searches for molecular targets with potential effects on RIPF among them, in order to provide effective therapeutic tools for the clinical prevention and treatment of RIPF.

MiRNA-155-5p inhibits epithelium-to-mesenchymal transition (EMT) by targeting GSK-3ß during radiation-induced pulmonary fibrosis.

Radiation-induced pulmonary fibrosis (RIPF) is a major lung complication in using radiotherapy to treat thoracic diseases. MicroRNAs (miRNAs) are reported to be the therapeutic targets for many diseases. However, the miRNAs involved in the pathogenesis of RIPF are rarely studied as potential therapeutic targets. Alveolar epithelial cells participate in RIPF formation by undergoing epithelial-mesenchymal transition (EMT). Here we demonstrated the critical role of miR-155-5p in radiation-induced EMT and RIPF. Using the previously established EMT cell model, we found that miR-155-5p was significantly down-regulated through high-throughput sequencing. Irradiation could decrease the expression of miR-155-5p in intro and in vivo, and it was inversely correlated to RIPF formation. Ectopic miR-155-5p expression inhibited radiation-induced-EMT in vitro and in vivo. Knockdown of glycogen synthase kinase-3ß (GSK-3ß), the functional target of miR-155-5p, reversed the induction of EMT and enhanced the phosphorylation of p65, a subunit of NF-κB, which were mediated by the down-regulation of miR-155-5p. Moreover, our finding demonstrated that ectopic miR-155-5p expression alleviated RIPF in mice by the GSK-3ß/NF-κB pathway. Thus, radiation downregulates miR-155-5p in alveolar epithelial cells that induces EMT, which contributes to RIPF using GSK-3ß/NF-κB pathway. Our observation provides further understanding on the regulation of RIPF and identifies potential therapeutic targets.

Vanillin derivative VND3207 activates DNA-PKcs conferring protection against radiation-induced intestinal epithelial cells injury in vitro and in vivo.

Vanillin is a natural compound endowed with antioxidant and anti-mutagenic properties. We previously identified the vanillin derivative VND3207 with strong radio-protective and antioxidant effects and found that VND3207 confers survival benefit and protection against radiation-induced intestinal injury (RIII) in mice. We also observed that VND3207 treatment enhanced the expression level of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) in human lymphoblastoid cells with or without γ-irradiation. DNA-PKcs is a critical component of DNA double strand break repair pathway and also regulates mitotic progression by stabilizing spindle formation and preventing mitotic catastrophe in response to DNA damage. In the present study, we found that VND3207 protected intestinal epithelial cells in vitro against ionizing radiation by promoting cell proliferation and inhibiting cell apoptosis. In addition, VND3207 promoted DNA-PKcs activity by increasing autophosphorylation at S2056 site. Consistent with this, VND3207 significantly decreased the number of γH2AX foci and mitotic catastrophe after radiation. DNA-PKcs deficiency abolished these VND3207 radio-protective effects, indicating that DNA-PKcs activation is essential for VND3207 activity. In conclusion, VND3207 promoted intestinal repair following radiation injury by regulating the DNA-PKcs pathway.

Modeling and multiscale characterization of the quantitative imaging based fibrosis index reveals pathophysiological, transcriptome and proteomic correlates of lung fibrosis induced by fractionated irradiation.

Pulmonary fibrosis represents a leading cause of morbidity and mortality worldwide. Therapy induced lung fibrosis constitutes a pivotal dose-limiting side effect of radiotherapy and other anticancer agents. We aimed to develop objective criteria for assessment of fibrosis and discover pathophysiological and molecular correlates of lung fibrosis as a function of fractionated whole thoracic irradiation. Dose-response series of fractionated irradiation was utilized to develop a non-invasive and quantitative measure for the degree of fibrosis - the fibrosis index (FI). The correlation of FI with histopathology, blood-gas, transcriptome and proteome responses of the lung tissue was analyzed. Macrophages infiltration and polarization was assessed by immunohistochemistry. Fibrosis development followed a slow kinetic with maximum lung fibrosis levels detected at 24-week post radiation insult. FI favorably correlated with radiation dose and surrogates of lung fibrosis i.e., enhanced pro-inflammatory response, tissue remodeling and extracellular matrix deposition. The loss of lung architecture correlated with decreased epithelial marker, loss of microvascular integrity with decreased endothelial and elevated mesenchymal markers. Lung fibrosis was further attributed to a switch of the inflammatory state toward a macrophage/T-helper cell type 2-like (M2/Th2) polarized phenotype. Together, the multiscale characterization of FI in radiation-induced lung fibrosis (RILF) model identified pathophysiological, transcriptional and proteomic correlates of fibrosis. Pathological immune response and endothelial/epithelial to mesenchymal transition were discovered as critical events governing lung tissue remodeling. FI will be instrumental for deciphering the molecular mechanisms governing lung fibrosis and discovery of novel targets for treatment of this devastating disease with an unmet medical need.

Exosome-packaged miR-1246 contributes to bystander DNA damage by targeting LIG4.

BACKGROUND: An increasing number of studies have recently reported that microRNAs packaged in exosomes contribute to multiple biological processes such as cancer progression; however, little is known about their role in the development of radiation-induced bystander effects. METHODS: The exosomes were isolated from the culture medium of BEP2D cells with or without γ-ray irradiation by ultracentrifugation. To monitor DNA damage and repair efficiency, the DNA double-strand break biomarker 53BP1 foci, comet, micronuclei, expression of DNA repair genes and NHEJ repair activity were detected. The miR-1246 targeting sequence of the DNA ligase 4 (LIG4) mRNA 3'UTR was assessed by luciferase reporter vectors. RESULTS: miR-1246 was increased in exosomes secreted from 2 Gy-irradiated BEP2D cells and inhibited the proliferation of nonirradiated cells. The miR-1246 mimic, exosomes from irradiated cells, and radiation-conditioned cell culture medium increased the yields of 53BP1 foci, comet tail and micronuclei in nonirradiated cells, and decreased NHEJ efficiency. miR-1246 downregulated LIG4 expression by directly targeting its 3'UTR. CONCLUSIONS: Our findings demonstrate that miR-1246 packaged in exosomes could act as a transfer messenger and contribute to DNA damage by directly repressing the LIG4 gene. Exosomal miR-1246 may be a critical predictor of and player in radiation-induced bystander DNA damage.

The vanillin derivative 6-bromine-5-hydroxy-4-methoxybenzaldehyde induces aberrant mitotic progression and enhances radio-sensitivity accompanying suppression the expression of PLK1 in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the most common form of esophageal cancer in China. Since chemotherapy is the standard clinical intervention for advanced ESCC, the development of highly effective and minimal/non-toxic drugs is essential to improve the clinical outcome and prognosis of the patients. A novel derivative of vanillin, 6-bromine-5-hydroxy-4-methoxybenzaldehyde (BVAN08), has been recently reported to activate different cell death pathways in cancer cells. In this study, we demonstrate that BVAN08 exhibits a potent anti-proliferation effect on ESCC cells (TE-1 and ECA-109) by inhibiting the expression of PLK1, an important mitotic kinase. Consistent with this, BVAN08 induces mitotic arrest and chromosomal misalignment in ESCC cells. The disruption of microtubule nucleation around centrosomes is also observed in BVAN08 treated ESCC cells. Furthermore, BVAN08 enhances radio-sensitivity of ESCC cells by prolonging DNA damage repair. These findings underscore the potential value of BVAN08 in cancer therapeutics and demonstrate the underlying mechanism by which BVAN08 induces mitotic catastrophe and enhances radio-sensitivity in ESCC cells.

MLN4924 suppresses the BRCA1 complex and synergizes with PARP inhibition in NSCLC cells.

Like ubiquitination, several studies have demonstrated that neddylation is implicated to be involved in the double strand break repair. BRCA1 is one of the key repair factors in the homologous recombination repair and may play a downstream role of the neddylation. BRCA1 is also a frequently mutated gene in cancers, which serve as the targets for PARP inhibitors. Here we further investigated the correlation between neddylation and BRCA1 complex using neddylation inhibitor MLN4924. MLN4924 efficiently inhibited the recruitment of components of BRCA1 complex to DNA damage sites. Thus MLN4924 may collaborate with PARP inhibitor to suppress tumor. Our results showed that combination MLN4924 and PARP inhibitor Olaparib impaired the DNA repair process in NSCLC cells. Furthermore, MLN4924 and Olaparib significantly inhibited the cancer cell growth. Kaplan-Meier survival analysis from lung cancer patients showed that high expression of NEDD8, BRCA1 and PARPs correlate with worse overall survival. Thus the combination of MLN4924 and PARP inhibitor may serve as a new strategy for NSCLC treatment.

IER5 is involved in DNA Double-Strand Breaks Repair in Association with PAPR1 in Hela Cells.

The immediate early response gene 5 (IER5) is a radiation response gene induced in a dose-independent manner, and has been suggested to be a molecular biomarker for biodosimetry purposes upon radiation exposure. Here, we investigated the function of IER5 in DNA damage response and repair. We found that interference on IER5 expression significantly decreased the efficiency of repair of DNA double-strand breaks induced by ionizing radiations in Hela cells. We found that IER5 participates in the non-homologous end-joining pathway of DNA breaks repair. Additionally, we identified a number of potential IER5-interacting proteins through mass spectrometry-based protein assays. The interaction of IER5 protein with poly(ADP-Ribose) polymerase 1 (PARP1) and Ku70 was further confirmed by immunoprecipitation assays. We also found that Olaparib, a PARP1 inhibitor, affected the stability of IER5. These results indicate that targeting of IER5 may be a novel DNA damage response-related strategy to use during cervical cancer radiotherapy or chemotherapy.

Stabilization of 4E-BP1 by PI3K kinase and its involvement in CHK2 phosphorylation in the cellular response to radiation.

Objectives: 4E-BP1 is a family member of eIF4E binding proteins (4E-BPs) which act as the suppressors of cap-dependent translation of RNA via competitively associating with cap-bound eIF4E. RNA translation regulation is an important manner to control the cellular responses to a series of stress conditions such as ionizing radiation (IR)-induced DNA damage response and cell cycle controlling. This study aimed to determine the mechanism of 4E-BP1 stabilization and its potential downstream target(s) in the response to IR. Methods: PI3Ks kinase inhibitors were used to determine the signaling control of 4E-BP1 phosphorylation and protein stability. shRNA strategy was employed to silence the expression of 4E-BP1 in HeLa and HepG2 cells, and determine its effect on the irradiation-induced CHK2 phosphorylation. The protein degradation/stability was investigated by western blotting on the condition of blocking novel protein synthesis by cycloheximide (CHX). Results: The phosphorylation of 4E-BP1 at Thr37/46 was significantly increased in both HepG2 and HeLa cells by ionizing radiation. Depression of 4E-BP1 by shRNA strategy resulted in an incomplete G2 arrest at the early stage of 2 hours post-irradiation, as well as a higher accumulation of mitotic cells at 10 and 12 hours post-irradiation as compared to the control cells. Consistently, the CHK2 phosphorylation at Thr68 induced by IR was also attenuated by silencing 4E-BP1 expression. Both PI3K and DNA-PKcs kinase inhibitors significantly decreased the protein level of 4E-BP1, which was associated with the accelerated degradation mediated by ubiquitination-proteasome pathway. Conclusion: PI3K kinase activity is necessary for maintaining 4E-BP1 stability. Our results also suggest 4E-BP1 a novel biological role of regulating cell cycle G2 checkpoint in responding to IR stress in association with controlling CHK2 phosphorylation.

The involvement of c-Myc in the DNA double-strand break repair via regulating radiation-induced phosphorylation of ATM and DNA-PKcs activity.

Deregulation of c-Myc often occurs in various human cancers, which not only contributes to the genesis and progression of cancers but also affects the outcomes of cancer radio- or chemotherapy. In this study, we have investigated the function of c-Myc in the repair of DNA double-strand break (DSB) induced by γ-ray irradiation. A c-Myc-silenced Hela-630 cell line was generated from HeLa cells using RNA interference technology. The DNA DSBs were detected by γ-H2AX foci, neutral comet assay and pulsed-field gel electrophoresis. We found that the capability of DNA DSB repair in Hela-630 cells was significantly reduced, and the repair kinetics of DSB was delayed as compared to the control Hela-NC cells. Silence of c-myc sensitized the cellular sensitivity to ionizing radiation. The phosphorylated c-Myc (Thr58/pSer62) formed the consistent co-localisation foci with γ-H2AX as well as the phosphorylated DNA-PKcs/S2056 in the irradiated cells. Moreover, depression of c-Myc largely attenuated the ionizing radiation-induced phosphorylation of the ataxia telangiectasia mutated (ATM) and decreased the in vitro kinase activity of DNA-PKcs. Taken together, our results demonstrated that c-Myc protein functions in the process of DNA double-strand break repair, at least partially, through affecting the ATM phosphorylation and DNA-PKcs kinase activity. The overexpression of c-Myc in tumours can account for the radioresistance of some tumour cell types.

DNA-PKcs associates with PLK1 and is involved in proper chromosome segregation and cytokinesis.

Accurate mitotic regulation is as important as intrinsic DNA repair for maintaining genomic stability. It is believed that these two cellular mechanisms are interconnected with DNA damage. DNA-PKcs is a critical component of the non-homologous end-joining pathway of DNA double-stranded break repair, and it was recently discovered to be involved in mitotic processing. However, the underlying mechanism of DNA-PKcs action in mitotic control is unknown. Here, we demonstrated that depletion of DNA-PKcs led to the dysregulation of mitotic progression in response to DNA damage, which eventually resulted in multiple failures, including failure to segregate sister chromatids and failure to complete cytokinesis, with daughter cells becoming fused again. The depletion of DNA-PKcs resulted in a notable failure of cytokinesis, with a high incidence of multinucleated cells. There were also cytoplasmic bridges containing DNA that continuously connected the daughter cells after DNA damage was induced. Phosphorylated DNA-PKcs (T2609) colocalizes with PLK1 throughout mitosis, including at the centrosomes from prophase to anaphase and at the kinetochores from prometaphase to metaphase, with accumulation at the midbody during cytokinesis. Importantly, DNA-PKcs was found to associate with PLK1 in the mitotic phase, and the depletion of DNA-PKcs resulted in the overexpression of PLK1 due to increased protein stability. However, deficiency in DNA-PKcs attenuated the recruitment of phosphorylated PLK1 to the midbody but not to the kinetochores and centrosomes. Our results demonstrate the functional association of DNA-PKcs with PLK1, especially in chromosomal segregation and cytokinesis control.

Urokinase-type plasminogen activator receptor regulates apoptotic sensitivity of colon cancer HCT116 cell line to TRAIL via JNK-p53 pathway.

The urokinase-type plasminogen activator receptor (uPAR) serves not only as an anchor for urokinase-type plasminogen activator but also participates in intracellular signal transduction events. In this study, we investigated whether uPAR could modulate TRAIL-induced apoptosis in human colon cancer cells HCT116. Using an antisense strategy, we established a stable HCT116 cell line with down-regulated uPAR. The sensitivity to TRAIL-induced apoptosis was evaluated by FACS analysis. Our results show that the inhibition of uPAR could sensitize HCT116 to TRAIL-induced apoptosis. uPAR inhibition changed the expression of mitochondrial apoptotic pathway proteins, including Bcl-2, Bax, Bid and p53, in a pro-apoptotic manner. We also found that the inhibition of uPAR down-regulated the phosphorylation of FAK, ERK and JNK. The inhibition of p53 by RNA interference rescued cells from enhanced apoptosis, thus indicating that p53 is critical for enhancing TRAIL-induced apoptosis. Furthermore, JNK, but not ERK, inhibition involved in the up-regulation of p53. JNK negatively regulated p53 protein level. Overall, our results show that uPAR inhibition can sensitize colon cancer cells HCT116 to TRAIL-induced apoptosis via active p53 and mitochondrial apoptotic pathways that JNK inhibition is involved.

Radiation-targeted immunotherapy: A new perspective in cancer radiotherapy.

In contemporary oncology, radiation therapy and immunotherapy stand as critical treatments, each with distinct mechanisms and outcomes. Radiation therapy, a key player in cancer management, targets cancer cells by damaging their DNA with ionizing radiation. Its effectiveness is heightened when used alongside other treatments like surgery and chemotherapy. Employing varied radiation types like X-rays, gamma rays, and proton beams, this approach aims to minimize damage to healthy tissue. However, it is not without risks, including potential damage to surrounding normal cells and side effects ranging from skin inflammation to serious long-term complications. Conversely, immunotherapy marks a revolutionary step in cancer treatment, leveraging the body's immune system to target and destroy cancer cells. It manipulates the immune system's specificity and memory, offering a versatile approach either alone or in combination with other treatments. Immunotherapy is known for its targeted action, long-lasting responses, and fewer side effects compared to traditional therapies. The interaction between radiation therapy and immunotherapy is intricate, with potential for both synergistic and antagonistic effects. Their combined use can be more effective than either treatment alone, but careful consideration of timing and sequence is essential. This review explores the impact of various radiation therapy regimens on immunotherapy, focusing on changes in the immune microenvironment, immune protein expression, and epigenetic factors, emphasizing the need for personalized treatment strategies and ongoing research to enhance the efficacy of these combined therapies in cancer care.

Voltage-dependent anion channel 1 mediates mitochondrial fission and glucose metabolic reprogramming in response to ionizing radiation.

The ionizing radiation (IR) represents a formidable challenge as an environmental factor to mitochondria, leading to disrupt cellular energy metabolism and posing health risks. Although the deleterious impacts of IR on mitochondrial function are recognized, the specific molecular targets remain incompletely elucidated. In this study, HeLa cells subjected to γ-rays exhibited concomitant oxidative stress, mitochondrial structural alterations, and diminished ATP production capacity. The γ-rays induced a dose-dependent induction of mitochondrial fission, simultaneously manifested by an elevated S616/S637 phosphorylation ratio of the dynamin-related protein 1 (DRP1) and a reduction in the expression of the mitochondrial fusion protein mitofusin 2 (MFN2). Knockdown of DRP1 effectively mitigated γ-rays-induced mitochondrial network damage, implying that DRP1 phosphorylation may act as an effector of radiation-induced mitochondrial damage. The mitochondrial outer membrane protein voltage-dependent anion channel 1 (VDAC1) was identified as a crucial player in IR-induced mitochondrial damage. The VDAC1 inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), counteracts the excessive mitochondrial fission induced by γ-rays, consequently rebalancing the glycolytic and oxidative phosphorylation equilibrium. This metabolic shift was uncovered to enhance glycolytic capacity, thus fortifying cellular resilience and elevating the radiosensitivity of cancer cells. These findings elucidate the intricate regulatory mechanisms governing mitochondrial morphology under radiation response. It is anticipated that the development of targeted drugs directed against VDAC1 may hold promise in augmenting the sensitivity of tumor cells to radiotherapy and chemotherapy.