Preclinical Evaluation of the ATR Inhibitor BAY 1895344 as a Radiosensitizer for Head and Neck Squamous Cell Carcinoma.

PURPOSE: Despite aggressive multimodal treatment that typically includes definitive or adjuvant radiation therapy (RT), locoregional recurrence rates approach 50% for patients with locally advanced human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC). Thus, more effective therapeutics are needed to improve patient outcomes. We evaluated the radiosensitizing effects of ataxia telangiectasia and RAD3-related (ATR) inhibitor (ATRi) BAY 1895344 in preclinical models of HNSCC. METHODS AND MATERIALS: Murine and human HPV-negative HNSCC cells (MOC2, MOC1, JHU-012) were treated with vehicle or ATRi with or without 4 Gy. Checkpoint kinase 1 phosphorylation and DNA damage (γH2AX) were evaluated by Western blot, and ATRi half-maximal inhibitory concentration was determined by MTT assay for HNSCC cells and immortalized murine oral keratinocytes. In vitro radiosensitization was tested by clonogenic assay. Cell cycle distribution and mitotic catastrophe were evaluated by flow cytometry. Mitotic aberrations were quantified by fluorescent microscopy. Tumor growth delay and survival were assessed in mice bearing MOC2 or JHU-012 transplant tumors treated with vehicle, ATRi, RT (10 Gy × 1 or 8 Gy × 3), or combined ATRi + RT. RESULTS: ATRi caused dose-dependent reduction in checkpoint kinase 1 phosphorylation at 1 hour post-RT (4 Gy) and dose-dependent increase in γH2AX at 18 hours post-RT. Addition of RT to ATRi led to decreased BAY 1895344 half-maximal inhibitory concentration in HNSCC cell lines but not in normal tissue surrogate immortalized murine oral keratinocytes. Clonogenic assays demonstrated radiosensitization in the HNSCC cell lines. ATRi abrogated the RT-induced G2/M checkpoint, leading to mitosis with unrepaired DNA damage and increased mitotic aberrations (multinucleated cells, micronuclei, nuclear buds, nucleoplasmic bridges). ATRi and RT significantly delayed tumor growth in MOC2 and JHU-012 in vivo models, with improved overall survival in the MOC2 model. CONCLUSIONS: These findings demonstrated that BAY 1895344 increased in vitro and in vivo radiosensitivity in HPV-negative HNSCC preclinical models, suggesting therapeutic potential warranting evaluation in clinical trials for patients with locally advanced or recurrent HPV-negative HNSCC.

WEE1 kinase inhibitor MK-1775 sensitizes oral tongue squamous cell carcinoma cells to radiation irrespective of TP53 status.

OBJECTIVE: Oral tongue squamous cell carcinoma (OTSCC) frequently harbors non-functional p53 and depends on G2/M checkpoint mediated by WEE1. WEE1 suppression has been identified as a promising anti-tumor strategy. This study investigated the capacity of WEE1 kinase inhibitor (MK-1775) and its underlying mechanisms in enhancing radiation responses of OTSCC cells in vitro. MATERIALS AND METHODS: WEE1 kinase expression and its downstream target (CDK1) were investigated in OTSCC versus normal oral tissue. A synergistic combination of MK-1775 with radiation on OTSCC cell lines with different p53 statuses was assessed by viability assay. The radio-sensitizing effects of MK-1775 on apoptosis, cell cycle, DNA damage, and mitotic entry were also determined. RESULTS: Irradiation enhanced CDK1 expression in all tested cell lines, though the effect was far more pronounced in p53 mutated cell lines. MK-1775 exhibited inhibitory effects against the survival of all cell lines and enhanced their response to the radiation. These effects were strongly elicited by induction of apoptosis and lethal mitosis, but less likely by abrogation of radiation-induced G2 arrest. CONCLUSION: These results demonstrate the efficacy of MK-1775 in enhancing the radiation effect on OTSCC in vitro associated with a significant apoptotic death rate, identifying WEE1 inhibitor as a potent radiosensitizer in OTSCC irrespective of p53 mutational status.

USP44 regulates irradiation-induced DNA double-strand break repair and suppresses tumorigenesis in nasopharyngeal carcinoma.

Radiotherapy is the primary treatment for patients with nasopharyngeal carcinoma (NPC), and approximately 20% of patients experience treatment failure due to tumour radioresistance. However, the exact regulatory mechanism remains poorly understood. Here, we show that the deubiquitinase USP44 is hypermethylated in NPC, which results in its downregulation. USP44 enhances the sensitivity of NPC cells to radiotherapy in vitro and in vivo. USP44 recruits and stabilizes the E3 ubiquitin ligase TRIM25 by removing its K48-linked polyubiquitin chains at Lys439, which further facilitates the degradation of Ku80 and inhibits its recruitment to DNA double-strand breaks (DSBs), thus enhancing DNA damage and inhibiting DNA repair via non-homologous end joining (NHEJ). Knockout of TRIM25 reverses the radiotherapy sensitization effect of USP44. Clinically, low expression of USP44 indicates a poor prognosis and facilitates tumour relapse in NPC patients. This study suggests the USP44-TRIM25-Ku80 axis provides potential therapeutic targets for NPC patients.

Oncogenic KRAS drives radioresistance through upregulation of NRF2-53BP1-mediated non-homologous end-joining repair.

KRAS-activating mutations are oncogenic drivers and are correlated with radioresistance of multiple cancers, including colorectal cancer, but the underlying precise molecular mechanisms remain elusive. Herein we model the radiosensitivity of isogenic HCT116 and SW48 colorectal cancer cell lines bearing wild-type or various mutant KRAS isoforms. We demonstrate that KRAS mutations indeed lead to radioresistance accompanied by reduced radiotherapy-induced mitotic catastrophe and an accelerated release from G2/M arrest. Moreover, KRAS mutations result in increased DNA damage response and upregulation of 53BP1 with associated increased non-homologous end-joining (NHEJ) repair. Remarkably, KRAS mutations lead to activation of NRF2 antioxidant signaling to increase 53BP1 gene transcription. Furthermore, genetic silencing or pharmacological inhibition of KRAS, NRF2 or 53BP1 attenuates KRAS mutation-induced radioresistance, especially in G1 phase cells. These findings reveal an important role for a KRAS-induced NRF2-53BP1 axis in the DNA repair and survival of KRAS-mutant tumor cells after radiotherapy, and indicate that targeting NRF2, 53BP1 or NHEJ may represent novel strategies to selectively abrogate KRAS mutation-mediated radioresistance.

Nafamostat mesilate, a nuclear factor kappa B inhibitor, enhances the antitumor action of radiotherapy on gallbladder cancer cells.

Nuclear factor kappa B (NF-κB) is a transcriptional factor that can be activated by radiotherapy and chemotherapy. The synthetic protease inhibitor nafamostat mesilate (NM) inhibits NF-κB activity and exerts antitumor actions in various types of cancer. In the present study, we hypothesized that NM might enhance the antitumor action of radiotherapy on gallbladder cancer (GBC) cells by inhibiting radiation-induced NF-κB activity. Thus, we investigated the correlation between radiotherapy and NF-κB activity in GBC cells. We assessed the in vitro effects of radiotherapy with or without NM on NF-κB activity, apoptosis of GBC cells (NOZ and OCUG-1), induction of apoptotic cascade, cell cycle progression, and viability of GBC cells using four treatment groups: 1) radiation (5 Gy) alone; 2) NM (80 µg/mL and 40 µg/mL, respectively) alone; 3) combination (radiation and NM); and 4) vehicle (control). The same experiments were performed in vivo using a xenograft GBC mouse model. In vitro, NM inhibited radiation-induced NF-κB activity. Combination treatment significantly attenuated cell viability and increased cell apoptosis and G2/M phase cell cycle arrest compared with those in the other groups for NOZ and OCUG-1 cells. Moreover, combination treatment upregulated the expression of apoptotic proteins compared with that after the other treatments. In vivo, NM improved the antitumor action of radiation and increased the population of Ki-67-positive cells. Overall, NM enhanced the antitumor action of radiotherapy on GBC cells by suppressing radiation-induced NF-κB activity. Thus, the combination of radiotherapy and NM may be useful for the treatment of locally advanced unresectable GBC.

Coralyne Radiosensitizes A549 Cells by Upregulation of CDKN1A Expression to Attenuate Radiation Induced G2/M Block of the Cell Cycle.

Coralyne is a synthetic analog of berberine related to protoberberine-isoquinoline alkaloids. Isoquinoline derivatives and analogs are renowned as potent radiosensitizers with potential medical application. In the present study, we investigated the effect of coralyne on the cell death, cytoskeletal changes and cell cycle progression of irradiated A549 cells. A clonogenic assay revealed that coralyne pretreatment decreased the viability of A549 cells in a time- and dose-dependent manner. Moreover, exposure to coralyne and ionizing radiation (IR) markedly altered the filamentous actin cytoskeletal architecture and integrin-ß binding sites of A549 cells. Treatment with 1-25 µM coralyne in combination with 2 Gy of IR significantly reduced the percentage of cells in G2/M phase compared with 2 Gy IR alone. These results indicate that coralyne is a potent radiosensitizing agent that may find an application in medicine.

Pharmacological Inhibition of HSP90 Radiosensitizes Head and Neck Squamous Cell Carcinoma Xenograft by Inhibition of DNA Damage Repair, Nucleotide Metabolism, and Radiation-Induced Tumor Vasculogenesis.

PURPOSE: Recent preclinical studies suggest combining the HSP90 inhibitor AT13387 (Onalespib) with radiation (IR) against colon cancer and head and neck squamous cell carcinoma (HNSCC). These studies emphasized that AT13387 downregulates HSP90 client proteins involved in oncogenic signaling and DNA repair mechanisms as major drivers of enhanced radiosensitivity. Given the large array of client proteins HSP90 directs, we hypothesized that other key proteins or signaling pathways may be inhibited by AT13387 and contribute to enhanced radiosensitivity. Metabolomic analysis of HSP90 inhibition by AT13387 was conducted to identify metabolic biomarkers of radiosensitization and whether modulations of key proteins were involved in IR-induced tumor vasculogenesis, a process involved in tumor recurrence. METHODS AND MATERIALS: HNSCC and non-small cell lung cancer cell lines were used to evaluate the AT13387 radiosensitization effect in vitro and in vivo. Flow cytometry, immunofluorescence, and immunoblot analysis were used to evaluate cell cycle changes and HSP90 client protein's role in DNA damage repair. Metabolic analysis was performed using liquid chromatography-Mass spectrometry. Immunohistochemical examination of resected tumors post-AT13387 and IR treatment were conducted to identify biomarkers of IR-induced tumor vasculogenesis. RESULTS: In agreement with recent studies, AT13387 treatment combined with IR resulted in a G2/M cell cycle arrest and inhibited DNA repair. Metabolomic profiling indicated a decrease in key metabolites in glycolysis and tricarboxylic acid cycle by AT13387, a reduction in Adenosine 5'-triphosphate levels, and rate-limiting metabolites in nucleotide metabolism, namely phosphoribosyl diphosphate and aspartate. HNSCC xenografts treated with the combination exhibited increased tumor regrowth delay, decreased tumor infiltration of CD45 and CD11b+ bone marrow-derived cells, and inhibition of HIF-1 and SDF-1 expression, thereby inhibiting IR-induced vasculogenesis. CONCLUSIONS: AT13387 treatment resulted in pharmacologic inhibition of cancer cell metabolism that was linked to DNA damage repair. AT13387 combined with IR inhibited IR-induced vasculogenesis, a process involved in tumor recurrence postradiotherapy. Combining AT13387 with IR warrants consideration of clinical trial assessment.

Inhibition of SOCS6 confers radioresistance in esophageal squamous cell carcinoma.

Esophageal cancer is one of the most common cancer of the digestive system and radiotherapy is widely applied in advanced esophageal cancer treatment, however radioresistance (RR) is one of the major reasons for radiotherapy failure. There is limited knowledge on the mechanisms that cause RR, here we identify suppressors of cytokine signaling 6 (SOCS6) is a negative regulator of radioresistance in ESCC cells. SOCS6 deficiency in ESCC cells conferred radioresistance in vitro and in vivo by increasing radiation-induced G2/M arrest, DNA damage repair and inhibiting radiation-induced apoptosis. Moreover, the transcriptome sequencing analysis demonstrates that the transcription of SOCS6 was partially p53-dependent. Importantly we found that highly correlated SOCS6 and P53 express lower in RR esophageal cancer tissues compare with radiosensitive ones. Collectedly our study uncovers that SOCS6, as a downstream effector of p53, is a key regulator involved in the radioresistance of ESCC.

TTK inhibitor promotes radiosensitivity of liver cancer cells through p21.

The monopolar spindle 1 ((hMps1/TTK) is a serine/threonine kinase that plays an important role in spindle assembly checkpoint signaling. To explore the possible relationship between TTK inhibition and radiosensitivity, we examined whether TTK inhibition influences cellular susceptibility of radiation. And we further revealed its mechanisms. We found that the expression of TTK was obviously higher in liver cancer tissues compared to the normal liver tissues. Kaplan-Meier Plotter demonstrated that patients with low TTK expression levels had a longer overall survival than patients with high TTK expression levels. TTK inhibitor AZ3146 could simulated liver cancer cells to accumulate in the G2/M phase, which ultimately enhances DNA damage with more γ-H2AX foci and more apoptosis and necrosis induced by radiation, which prompted that TTK inhibition sensitized liver cancer cells to radiation. In addition, TTK inhibition altered cell-cycle progression and exacerbated centrosome abnormalities, resulting in enhanced mitotic catastrophe (MC) induced by radiation in a p21-mediated manner. In this study, we present evidences that the TTK inhibitor promotes the radiosensitivity of liver cancer cells through regulating cell cycle in p21-mediated manner in vitro, indicating that TTK inhibitor may be an attractive radiosensitizer for the patients with liver cancer.

Targeting Aurora B kinase with Tanshinone IIA suppresses tumor growth and overcomes radioresistance.

Aurora B kinase is aberrantly overexpressed in various tumors and shown to be a promising target for anti-cancer therapy. In human oral squamous cell carcinoma (OSCC), the high protein level of Aurora B is required for maintaining of malignant phenotypes, including in vitro cell growth, colony formation, and in vivo tumor development. By molecular modeling screening of 74 commercially available natural products, we identified that Tanshinone IIA (Tan IIA), as a potential Aurora B kinase inhibitor. The in silico docking study indicates that Tan IIA docks into the ATP-binding pocket of Aurora B, which is further confirmed by in vitro kinase assay, ex vivo pull-down, and ATP competitive binding assay. Tan IIA exhibited a significant anti-tumor effect on OSCC cells both in vitro and in vivo, including reduction of Aurora B and histone H3 phosphorylation, induction of G2/M cell cycle arrest, increase the population of polyploid cells, and promotion of apoptosis. The in vivo mouse model revealed that Tan IIA delayed tumor growth of OSCC cells. Tan IIA alone or in combination with radiation overcame radioresistance in OSCC xenograft tumors. Taken together, our data indicate that Tan IIA is an Aurora B kinase inhibitor with therapeutic potentials for cancer treatment.

SPRTN protease-cleaved MRE11 decreases DNA repair and radiosensitises cancer cells.

The human MRE11/RAD50/NBS1 (MRN) complex plays a crucial role in sensing and repairing DNA DSB. MRE11 possesses dual 3'-5' exonuclease and endonuclease activity and forms the core of the multifunctional MRN complex. We previously identified a C-terminally truncated form of MRE11 (TR-MRE11) associated with post-translational MRE11 degradation. Here we identified SPRTN as the essential protease for the formation of TR-MRE11 and characterised the role of this MRE11 form in its DNA damage response (DDR). Using tandem mass spectrometry and site-directed mutagenesis, the SPRTN-dependent cleavage site for MRE11 was identified between 559 and 580 amino acids. Despite the intact interaction of TR-MRE11 with its constitutive core complex proteins RAD50 and NBS1, both nuclease activities of truncated MRE11 were dramatically reduced due to its deficient binding to DNA. Furthermore, lack of the MRE11 C-terminal decreased HR repair efficiency, very likely due to abolished recruitment of TR-MRE11 to the sites of DNA damage, which consequently led to increased cellular radiosensitivity. The presence of this DNA repair-defective TR-MRE11 could explain our previous finding that the high MRE11 protein expression by immunohistochemistry correlates with improved survival following radical radiotherapy in bladder cancer patients.

Centipedegrass extract enhances radiosensitivity in melanoma cells by inducing G2/M cell cycle phase arrest.

Melanoma is aggressive, highly metastatic, and potentially fatal. In the case of patients with advanced melanoma, it is difficult to expect a good prognosis, since this cancer has low sensitivity to chemotherapy and radiation therapy. The use of natural ingredients may enhance existing therapies. Centipedegrass extract (CGE) which contains phenolic structures and C-glycosyl flavones, has been shown to have anti-inflammatory effects and anti-cancer effects. The purpose of this study was to evaluate the radio sensitizing effects of CGE in combination with ionizing radiation (IR). Two melanoma cell lines were exposed to IR after treatment with CGE at concentrations that were not toxic alone. The effects of CGE + IR on cell survival, cell cycle, and apoptotic cell death were examined using MTT and Muse® Cell Analyzer, and fluorescence microscopy. Molecular signaling mechanisms were explored by western blots. Our findings showed that co-treatment of CGE + IR reduced the survival of melanoma cells more than IR alone. Also, cell cycle arrest in CGE-treated cells was enhanced and these cells became more radiosensitive. CGE + IR increased apoptotic cell death more than IR alone. Western blot results showed that the effect of CGE + IR involved MAPKs (ERK1/2, p38, and JNK) pathway. Our study suggests that CGE + IR treatment enhanced radio-sensitization and cell death of melanoma cells via cell cycle arrest and the MAPKs pathway.

In vitro effects of Trastuzumab Emtansine (T-DM1) and concurrent irradiation on HER2-positive breast cancer cells.

BACKGROUND: To determine the effects of concurrent irradiation and T-DM1 on HER2-positive breast cancer cell lines. METHODS: Five human breast cancer cell lines (in vitro study) presenting various levels of HER2 expression were used to determine the potential therapeutic effect of T-DM1 combined with radiation. The toxicity of T-DM1 was assessed using viability assay and cell cycle analysis was performed by flow cytometry after BrdU incorporation. HER2 cells were irradiated at different dose levels after exposure to T-DM1. Survival curves were determined by cell survival assays (after 5 population doubling times). RESULTS: The results revealed that T-DM1 induced significant lethality due to the intracellular action of DM1 on the cell cycle with significant G2/M phase blocking. Even after a short time incubation, the potency of T-DM1 was maintained and even enhanced over time, with a higher rate of cell death. After irradiation alone, the D10 (dose required to achieve 10% cell survival) was significantly higher for high HER2-expressing cell lines than for low HER2-expressing cells, with a linearly increasing relationship. In combination with irradiation, using conditions that allow cell survival, T-DM1 does not induce a radiosensitivity. CONCLUSIONS: Although there is a linear correlation between intrinsic HER2 expression and radioresistance, the results indicated that T-DM1 is not a radiation-sensitizer under the experimental conditions of this study that allowed cell survival. However, further investigations are needed, in particular in vivo studies before reaching a final conclusion.

Synergistic Tumoricidal Effects of Alpha-Lipoic Acid and Radiotherapy on Human Breast Cancer Cells via HMGB1.

PURPOSE: Radiotherapy (RT) is one of main strategies of cancer treatment. However, some cancer cells are resistant to radiation-induced cell death, including apoptosis. Therefore, alternative approaches targeting different anti-tumor mechanisms such as cell senescence are required. This study aimed to investigate the synergistic effect of alpha-lipoic acid (ALA) on radiation-induced cell death and senescence in MDA-MB-231 human breast cancer cells. MATERIALS AND METHODS: The cells were divided into four groups depending on the cell treatment (control, ALA, RT, and ALA+RT). Cells were analyzed for morphology, apoptotic cell death, mitochondrial reactive oxygen species, membrane potential, cellular senescence, and cell cycle. RESULTS: Our data showed that ALA significantly promoted apoptotic cell death when combined with RT, as reflected by Annexin V staining, expression of apoptosis-related factors, mitochondrial damages as well as cell morphological changes and reduction of cell numbers. In addition, ALA significantly enhanced radiation-induced cellular senescence, which was shown by increased HMGB1 expression in the cytosol fraction compared to the control, increased p53 expression compared to the control, activation of p38 as well as nuclear factor кB, and G2/M cell cycle arrest. CONCLUSION: The current study is the first report showing a new mode of action (senescence induction) of ALA beyond apoptotic cell death in MDA-MB-231 cancer cells known to be resistant to RT.

Radiosensitizing effects of c­myc gene knockdown­induced G2/M phase arrest by intrinsic stimuli via the mitochondrial signaling pathway.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents and its long­term survival rate has stagnated in the past decades. Previous studies have shown that tumors in the G2/M phase are more sensitive to radiotherapy. The proto­oncogene c­myc is a transformed member of the myc family and c­myc­interacting zinc finger protein­1 (Miz­1) is a poly­Cys2His2 zinc finger (ZF) activator of cell cycle regulator genes, such as the cyclin­dependent kinase inhibitor p21. C­myc can repress the expression of p21 by binding to Miz­1 and abolishing the interaction between Miz­1 and its co­activators, which induces G2/M phase arrest. Therefore, the present study investigated the radiosensitizing effects of the c­myc gene and the sensitizing apoptosis pathway, aiming to identify a more effective combination radiotherapy treatment for osteosarcoma. The present study demonstrated that the c­myc gene was overexpressed in osteosarcoma cells compared to osteoblasts. Following inhibition of c­myc gene expression in osteosarcoma cells, tumor proliferation was significantly hindered after inducing G2/M phase arrest via regulating G2/M phase­associated proteins. Additionally, it was revealed that inhibiting c­myc gene expression combined with radiotherapy could significantly increase the apoptosis rate of osteosarcoma cells via the mitochondrial signaling pathway. In summary, the present study verified the radiosensitizing effects of c­myc gene knockdown­induced G2/M phase arrest, which was achieved by intrinsic stimuli through the mitochondrial signaling pathway.

Curcumin from Jianghuang (Rhizoma Curcumae Longae) protects against exposure to ultraviolet B by antioxidation and attenuating mitochondrion-dependent apoptosis.

OBJECTIVE: To investigate the protective effect of curcumin extracted from Jianghuang (Rhizoma Curcumae Longae) against ultraviolet B (UVB) and the possible mechanism. METHODS: Effects of curcumin were detected in vivo and in vitro. Morphological changes of white guinea pig skin were assessed by hematoxylin and eosin staining. HaCaT cell proliferation was measured by 3-[4,5-dimethylthylthiazol-2-yl]-2,5 diphenyltetrazolium broide (MTT) assays. The cell cycle distribution, apoptotic rate, level of reactive oxygen species (ROS), mitochondrial membrane potential, and intracellullar calcium ion concentration of HaCaT cells were detected by flow cytometry. Antioxidant levels in skin tissues and HaCat cells were measured by biochemical methods. RESULTS: UVB inhibited in vitro cell proliferation by inducing G2/M arrest, increasing ROS, apoptosis, and necrosis, and decreasing B-cell lymphoma-2, and increasing Bax, cytochrome c, and caspase-3 levels. CONCLUSION: Curcumin blocks the effects of UVB by reducing ROS and apoptosis, and reversing UVB-induced changes in the expression of apoptotic proteins. The mitochondrial pathway is involved in curcumin-regulated apoptosis.

Cyclin K interacts with ß-catenin to induce Cyclin D1 expression and facilitates tumorigenesis and radioresistance in lung cancer.

Rationale: Radioresistance remains the major cause of local relapse and distant metastasis in lung cancer. However, the underlying molecular mechanisms remain poorly defined. This study aimed to investigate the role and regulatory mechanism of Cyclin K in lung cancer radioresistance. Methods: Expression levels of Cyclin K were measured by immunohistochemistry in human lung cancer tissues and adjacent normal lung tissues. Cell growth and proliferation, neutral comet and foci formation assays, G2/M checkpoint and a xenograft mouse model were used for functional analyses. Gene expression was examined by RNA sequencing and quantitative real-time PCR. Protein-protein interaction was assessed by immunoprecipitation and GST pull-down assays. Results: We report that Cyclin K is frequently overexpressed and correlates with poor prognosis in lung cancer patients. Functionally, we demonstrate that Cyclin K depletion results in reduced proliferation, defective G2/M checkpoint and enhanced radiosensitivity in lung cancer. Mechanistically, we reveal that Cyclin K interacts with and promotes the stabilization of ß-catenin protein, thereby upregulating the expression of Cyclin D1. More importantly, we show that Cyclin D1 is the major effector that mediates the biological functions of Cyclin K in lung cancer. Conclusions: These findings suggest that Cyclin K positively modulates the ß-catenin/Cyclin D1 axis to promote tumorigenesis and radioresistance in lung cancer, indicating that Cyclin K may represent a novel attractive biomarker for lung cancer radiotherapy.

Radiosensitization of NSCLC cells to X-rays and carbon ions by the CHK1/CHK2 inhibitor AZD7762, Honokiol and Tunicamycin.

Although radiotherapy, especially carbon-ion radiotherapy, is an effective treatment modality against non-small-cell lung cancer (NSCLC), studies using radiation combined with sensitizer for improving the efficacy of radiotherapy are still needed. In this work, we aimed to investigate in NSCLC A549 and H1299 cell lines the effects of different linear energy transfer (LET) radiations combined with diverse sensitizing compounds. Cells pretreated with the CHK1/CHK2 inhibitor AZD7762, Honokiol or Tunicamycin were irradiated with low-LET X-rays and high-LET carbon ions. Cell survival was assessed using the clonogenic cell survival assay. Cell cycle distribution and apoptosis were measured with flow cytometry, and DNA double strand break (DSB) and repair were detected using γ-H2AX immunofluorescence staining. Our results revealed that AZD7762, Honokiol and Tunicamycin demonstrated low cytotoxicity to NSCLC cells and a pronounced radiosensitizing effect on NSCLC cells exposed to carbon ions than X-rays. Unrepaired DNA DSB damages, the abrogation of G2/M arrest induced by irradiation, and finally apoptotic cell death were the main causes of the radiosensitizing effect. Thus, our data suggest that high-LET carbon ion combined with these compounds may be a potentially effective therapeutic strategy for locally advanced NSCLC.

Berberine, a natural alkaloid sensitizes human hepatocarcinoma to ionizing radiation by blocking autophagy and cell cycle arrest resulting in senescence.

OBJECTIVE: To study the radiosensitizing potential of Berberine and the underlying mechanism in human hepatocarcinoma (HepG2) cells. METHODS: HepG2 cells were challenged with X-rays in combination with Berberine treatment and several in vitro assays were performed. Alteration in cell viability was determined by MTT assay. Changes in intracellular ROS levels, mitochondrial membrane potential/mass, intracellular acidic vesicular organelles as well as cell cycle arrest and apoptotic cell death were analysed by flow cytometry. Induction of autophagy was assessed by staining the cells with Monodansylcadaverine/Lysotracker red dyes and immunoblotting for LC3I/II and p62 proteins. Phase-contrast/fluorescence microscopy was employed to study mitotic catastrophe and senescence. Cellular senescence was confirmed by immunoblotting for p21 levels and ELISA for Interleukin-6. KEY FINDINGS: X-rays + Berberine had a synergistic effect in reducing cell proliferation accompanied by a robust G2/M arrest. Berberine-mediated radiosensitization was associated with elevated levels of LC3II and p62 suggesting blocked autophagy that was followed by mitotic catastrophe and senescence. Treatment of cells with X-rays + Berberine resulted in increased oxidative stress, hyperpolarized mitochondria with increased mitochondrial mass and reduced ATP levels. CONCLUSIONS: The study expands the understanding of the pharmacological properties of Berberine and its applicability as a radiosensitizer towards treating liver cancer.

Different Mechanisms Underlie the Metabolic Response of GBM Stem-Like Cells to Ionizing Radiation: Biological and MRS Studies on Effects of Photons and Carbon Ions.

Glioblastoma multiforme (GBM) is a malignant primary brain tumor with very poor prognosis, high recurrence rate, and failure of chemo-radiotherapy, mainly due to a small fraction of cells with stem-like properties (GSCs). To study the mechanisms of GSCs resistance to radiation, two GSC lines, named line #1 and line #83, with different metabolic patterns and clinical outcome, were irradiated with photon beams and carbon ions and assessed by 1H Magnetic Resonance Spectroscopy (MRS). Both irradiation modalities induced early cytotoxic effects in line #1 with small effects on cell cycle, whereas a proliferative G2/M cytostatic block was observed in line #83. MR spectroscopy signals from mobile lipids (ML) increased in spectra of line #1 after photon and C-ion irradiation with effects on lipid unsaturation level, whereas no effects were detected in line #83 spectra. Gamma-Aminobutyric Acid (GABA), glutamic acid (glu) and Phosphocreatine (pCr) signals showed a significant variation only for line #1 after carbon ion irradiation. Glucose (glc) level and lactate (Lac) extrusion behaved differently in the two lines. Our findings suggest that the differences in irradiation response of GSCs #1 and #83 lines are likely attributable to their different metabolic fingerprint rather than to the different radiation types.