Synthesis and Characterization of Titanium Nitride-Carbon Composites and Their Use in Lithium-Ion Batteries.

This work focuses on the synthesis of titanium nitride-carbon (TiN-carbon) composites by the thermal decomposition of a titanyl phthalocyanine (TiN(TD)) precursor into TiN. The synthesis of TiN was also performed using the sol-gel method (TiN(SG)) of an alkoxide/urea. The structure and morphology of the TiN-carbon and its precursors were characterized by XRD, FTIR, SEM, TEM, EDS, and XPS. The FTIR results confirmed the presence of the titanium phthalocyanine (TiOPC) complex, while the XRD data corroborated the decomposition of TiOPC into TiN. The resultant TiN exhibited a cubic structure with the FM3-M lattice, aligning with the crystal system of the synthesized TiN via the alkoxide route. The XPS results indicated that the particles synthesized from the thermal decomposition of TiOPC resulted in the formation of TiN-carbon composites. The TiN particles were present as clusters of small spherical particles within the carbon matrix, displaying a porous sponge-like morphology. The proposed thermal decomposition method resulted in the formation of metal nitride composites with high carbon content, which were used as anodes for Li-ion half cells. The TiN-carbon composite anode showed a good specific capacity after 100 cycles at a current density of 100 mAg-1.

Targeting OGG1 and PARG radiosensitises head and neck cancer cells to high-LET protons through complex DNA damage persistence.

Complex DNA damage (CDD), containing two or more DNA lesions within one or two DNA helical turns, is a signature of ionising radiation (IR) and contributes significantly to the therapeutic effect through cell killing. The levels and complexity of CDD increases with linear energy transfer (LET), however, the specific cellular response to this type of DNA damage and the critical proteins essential for repair of CDD is currently unclear. We performed an siRNA screen of ~240 DNA damage response proteins to identify those specifically involved in controlling cell survival in response to high-LET protons at the Bragg peak, compared to low-LET entrance dose protons which differ in the amount of CDD produced. From this, we subsequently validated that depletion of 8-oxoguanine DNA glycosylase (OGG1) and poly(ADP-ribose) glycohydrolase (PARG) in HeLa and head and neck cancer cells leads to significantly increased cellular radiosensitivity specifically following high-LET protons, whilst no effect was observed after low-LET protons and X-rays. We subsequently confirmed that OGG1 and PARG are both required for efficient CDD repair post-irradiation with high-LET protons. Importantly, these results were also recapitulated using specific inhibitors for OGG1 (TH5487) and PARG (PDD00017273). Our results suggest OGG1 and PARG play a fundamental role in the cellular response to CDD and indicate that targeting these enzymes could represent a promising therapeutic strategy for the treatment of head and neck cancers following high-LET radiation.

KH-like Domains in PARP9/DTX3L and PARP14 Coordinate Protein-Protein Interactions to Promote Cancer Cell Survival.

Certain members of the ADP-ribosyltransferase superfamily (ARTD or PARP enzymes) catalyse ADP-ribosylation in response to cellular stress, DNA damage and viral infection and are upregulated in various tumours. PARP9, its binding partner DTX3L and PARP14 protein levels are significantly correlated in head and neck squamous cell carcinoma (HNSCC) and other tumour types though a mechanism where PARP9/DTX3L regulates PARP14 post-transcriptionally. Depleting PARP9, DTX3L or PARP14 expression in HNSCC or HeLa cell lines decreases cell survival through a reduction of proliferation and an increase in apoptosis. A partial rescue of survival was achieved by expressing a PARP14 truncation containing a predicted eukaryotic type I KH domain. KH-like domains were also found in PARP9 and in DTX3L and contributed to protein-protein interactions between PARP9-DTX3L and PARP14-DTX3L. Homodimerization of DTX3L was also coordinated by a KH-like domain and was disrupted by site-specific mutation. Although, cell survival promoted by PARP14 did not require ADP-ribosyltransferase activity, interaction of DTX3L in vitro suppressed PARP14 auto-ADP-ribosylation and promoted trans-ADP-ribosylation of PARP9 and DTX3L. In summary, we characterised PARP9-DTX3L-PARP14 interactions important to pro-survival signalling in HNSCC cells, albeit in PARP14 catalytically independent fashion.

Most DNA repair defects do not modify the relationship between relative biological effectiveness and linear energy transfer in CRISPR-edited cells.

BACKGROUND: Cancer is a highly heterogeneous disease, driven by frequent genetic alterations which have significant effects on radiosensitivity. However, radiotherapy for a given cancer type is typically given with a standard dose determined from population-level trials. As a result, a proportion of patients are under- or over-dosed, reducing the clinical benefit of radiotherapy. Biological optimization would not only allow individual dose prescription but also a more efficient allocation of limited resources, such as proton and carbon ion therapy. Proton and ion radiotherapy offer an advantage over photons due to their elevated Relative Biological Effectiveness (RBE) resulting from their elevated Linear Energy Transfer (LET). Despite significant interest in optimizing LET by tailoring radiotherapy plans, RBE's genetic dependence remains unclear. PURPOSE: The aim of this study is to better define the RBE/LET relationship in a panel of cell lines with different defects in DSB repair pathways, but otherwise identical biological features and genetic background to isolate these effects. METHODS: Normal human cells (RPE1), genetically modified to introduce defects in DNA double-strand break (DSB) repair genes, ATM, BRCA1, DCLRE1C, LIG4, PRKDC and TP53, were used to map the RBE-LET relationship. Cell survival was measured with clonogenic assays after exposure to photons, protons (LET 1 and 12 keV/µm) and alpha particles (129 keV/µm). Gene knockout sensitizer enhancement ratio (SER) values were calculated as the ratio of the mean inactivation dose (MID) of wild-type cells to repair-deficient cells, and RBE values were calculated as the ratio of the MID of X-ray and particle irradiated cells. 53BP1 foci were used to quantify radiation-induced DSBs and their repair following irradiation. RESULTS: Deletion of NHEJ genes had the greatest impact on photon sensitivity (ATM-/- SER = 2.0 and Lig4-/- SER = 1.8), with genes associated with HR having smaller effects (BRCA1-/- SER = 1.2). Wild-type cells showed RBEs of 1.1, 1.3, 5.0 for low- and high-LET protons and alpha particles respectively. SERs for different genes were independent of LET, apart from NHEJ knockouts which proved to be markedly hypersensitive across all tested LETs. Due to this hypersensitivity, the impact of high LET was reduced in cell models lacking the NHEJ repair pathway. HR-defective cells had moderately increased sensitivity across all tested LETs, but, notably, the contribution of HR pathway to survival appeared independent of LET. Analysis of 53BP1 foci shows that NHEJ-defective cells had the least DSB repair capacity after low LET exposure, and no visible repair after high LET exposure. HR-defective cells also had slower repair kinetics, but the impact of HR defects is not as severe as NHEJ defects. CONCLUSIONS: DSB repair defects, particularly in NHEJ, conferred significant radiosensitivity across all LETs. This sensitization appeared independent of LET, suggesting that the contribution of different DNA repair pathways to survival does not depend on radiation quality.

Enhancing radiotherapy outcomes in rectal cancer: A systematic review of targeting hypoxia-induced radioresistance.

Introduction: Neoadjuvant radiotherapy is successfully used in rectal cancer to improve overall survival. However, treatment response is both unpredictable and variable. There is strong evidence to show that the phenomenon of tumour hypoxia is associated with radioresistance, however the mechanism(s) behind this are poorly understood. Consequently, there have only been a small number of studies evaluating methods targeting hypoxia-induced radioresistance. The purpose of this systematic review is to evaluate the potential effectiveness of targeting hypoxia-induced radioresistance in rectal cancer and provide recommendations for future research in this area. Methods: A comprehensive literature search was performed following the PRISMA guidelines. This study was registered on the Prospero database (CRD42023441983). Results: Eight articles met the inclusion criteria. All studies identified were in vitro or in vivo studies, there were no clinical trials. Of the 8 studies identified, 5 assessed the efficacy of drugs which directly or indirectly targeted hypoxia and three that identified potential targets. There was conflicting in vivo evidence for the use of metformin to overcome hypoxia induced radioresistance. Vorinostat, atovaquone, and evofosfamide showed promising preclinical evidence that they can overcome hypoxia-induced radioresistance. Discussion: The importance of investigating hypoxia-induced radioresistance in rectal cancer is crucial. However, to date, only a small number of preclinical studies exist evaluating this phenomenon. This systematic review highlights the importance of further research to fully understand the mechanism behind this radioresistance. There are promising targets identified in this systematic review however, substantially more pre-clinical and clinical research as a priority for future research is needed.

The role of autophagy in hypoxia-induced radioresistance.

Radiotherapy is a widely used treatment modality against cancer, and although survival rates are increasing, radioresistant properties of tumours remain a significant barrier for curative treatment. Tumour hypoxia is one of the main contributors to radioresistance and is common in most solid tumours. Hypoxia is responsible for many molecular changes within the cell which helps tumours to survive under such challenging conditions. These hypoxia-induced molecular changes are predominantly coordinated by the hypoxia inducible factor (HIF) and have been linked with the ability to confer resistance to radiation-induced cell death. To overcome this obstacle research has been directed towards autophagy, a cellular process involved in self degradation and recycling of macromolecules, as HIF plays a large role in its coordination under hypoxic conditions. The role that autophagy has following radiotherapy treatment is conflicted with evidence of both cytoprotective and cytotoxic effects. This literature review aims to explore the intricate relationship between radiotherapy, hypoxia, and autophagy in the context of cancer treatment. It provides valuable insights into the potential of targeting autophagy as a therapeutic strategy to improve the response of hypoxic tumours to radiotherapy.

DNA damage and repair dependencies of ionising radiation modalities.

Radiotherapy is utilised in the treatment of ∼50% of all human cancers, which predominantly employs photon radiation. However, particle radiotherapy elicits significant benefits over conventional photons due to more precise dose deposition and increased linear energy transfer (LET) that generates an enhanced therapeutic response. Specifically, proton beam therapy (PBT) and carbon ion radiotherapy (CIRT) are characterised by a Bragg peak, which generates a low entrance radiation dose, with the majority of the energy deposition being defined within a small region which can be specifically targeted to the tumour, followed by a low exit dose. PBT is deemed relatively low-LET whereas CIRT is more densely ionising and therefore high LET. Despite the radiotherapy type, tumour cell killing relies heavily on the introduction of DNA damage that overwhelms the repair capacity of the tumour cells. It is known that DNA damage complexity increases with LET that leads to enhanced biological effectiveness, although the specific DNA repair pathways that are activated following the different radiation sources is unclear. This knowledge is required to determine whether specific proteins and enzymes within these pathways can be targeted to further increase the efficacy of the radiation. In this review, we provide an overview of the different radiation modalities and the DNA repair pathways that are responsive to these. We also provide up-to-date knowledge of studies examining the impact of LET and DNA damage complexity on DNA repair pathway choice, followed by evidence on how enzymes within these pathways could potentially be therapeutically exploited to further increase tumour radiosensitivity, and therefore radiotherapy efficacy.

The Cellular Response to Complex DNA Damage Induced by Ionising Radiation.

Radiotherapy (ionising radiation; IR) is utilised in the treatment of ~50% of all human cancers, and where the therapeutic effect is largely achieved through DNA damage induction. In particular, complex DNA damage (CDD) containing two or more lesions within one to two helical turns of the DNA is a signature of IR and contributes significantly to the cell killing effects due to the difficult nature of its repair by the cellular DNA repair machinery. The levels and complexity of CDD increase with increasing ionisation density (linear energy transfer, LET) of the IR, such that photon (X-ray) radiotherapy is deemed low-LET whereas some particle ions (such as carbon ions) are high-LET radiotherapy. Despite this knowledge, there are challenges in the detection and quantitative measurement of IR-induced CDD in cells and tissues. Furthermore, there are biological uncertainties with the specific DNA repair proteins and pathways, including components of DNA single and double strand break mechanisms, that are engaged in CDD repair, which very much depends on the radiation type and associated LET. However, there are promising signs that advancements are being made in these areas and which will enhance our understanding of the cellular response to CDD induced by IR. There is also evidence that targeting CDD repair, particularly through inhibitors against selected DNA repair enzymes, can exacerbate the impact of higher LET, which could be explored further in a translational context.

ATM Regulates Differentiation of Myofibroblastic Cancer-Associated Fibroblasts and Can Be Targeted to Overcome Immunotherapy Resistance.

Myofibroblastic cancer-associated fibroblast (myoCAF)-rich tumors generally contain few T cells and respond poorly to immune-checkpoint blockade. Although myoCAFs are associated with poor outcome in most solid tumors, the molecular mechanisms regulating myoCAF accumulation remain unclear, limiting the potential for therapeutic intervention. Here, we identify ataxia-telangiectasia mutated (ATM) as a central regulator of the myoCAF phenotype. Differentiating myofibroblasts in vitro and myoCAFs cultured ex vivo display activated ATM signaling, and targeting ATM genetically or pharmacologically could suppress and reverse differentiation. ATM activation was regulated by the reactive oxygen species-producing enzyme NOX4, both through DNA damage and increased oxidative stress. Targeting fibroblast ATM in vivo suppressed myoCAF-rich tumor growth, promoted intratumoral CD8 T-cell infiltration, and potentiated the response to anti-PD-1 blockade and antitumor vaccination. This work identifies a novel pathway regulating myoCAF differentiation and provides a rationale for using ATM inhibitors to overcome CAF-mediated immunotherapy resistance. SIGNIFICANCE: ATM signaling supports the differentiation of myoCAFs to suppress T-cell infiltration and antitumor immunity, supporting the potential clinical use of ATM inhibitors in combination with checkpoint inhibition in myoCAF-rich, immune-cold tumors.

Factors affecting the radiation response in glioblastoma.

Glioblastoma (GBM) is a highly invasive primary brain tumor in adults with a 5-year survival rate of less than 10%. Conventional radiotherapy with photons, along with concurrent and adjuvant temozolomide, is the mainstay for treatment of GBM although no significant improvement in survival rates has been observed over the last 20 years. Inherent factors such as tumor hypoxia, radioresistant GBM stem cells, and upregulated DNA damage response mechanisms are well established as contributing to treatment resistance and tumor recurrence. While it is understandable that efforts have focused on targeting these factors to overcome this phenotype, there have also been striking advances in precision radiotherapy techniques, including proton beam therapy and carbon ion radiotherapy (CIRT). These enable higher doses of radiation to be delivered precisely to the tumor, while minimizing doses to surrounding normal tissues and organs at risk. These alternative radiotherapy techniques also benefit from increased biological effectiveness, particularly in the case of CIRT. Although not researched extensively to date, combining these new radiation modalities with radio-enhancing agents may be particularly effective in improving outcomes for patients with GBM.

TRIM26 Maintains Cell Survival in Response to Oxidative Stress through Regulating DNA Glycosylase Stability.

Oxidative DNA base lesions in DNA are repaired through the base excision repair (BER) pathway, which consequently plays a vital role in the maintenance of genome integrity and in suppressing mutagenesis. 8-oxoguanine DNA glycosylase (OGG1), endonuclease III-like protein 1 (NTH1), and the endonuclease VIII-like proteins 1-3 (NEIL1-3) are the key enzymes that initiate repair through the excision of the oxidized base. We have previously identified that the E3 ubiquitin ligase tripartite motif 26 (TRIM26) controls the cellular response to oxidative stress through regulating both NEIL1 and NTH1, although its potential, broader role in BER is unclear. We now show that TRIM26 is a central player in determining the response to different forms of oxidative stress. Using siRNA-mediated knockdowns, we demonstrate that the resistance of cells to X-ray radiation and hydrogen peroxide generated as a consequence of trim26 depletion can be reversed through suppression of selective DNA glycosylases. In particular, a knockdown of neil1 or ogg1 can enhance sensitivity and DNA repair rates in response to X-rays, whereas a knockdown of neil1 or neil3 can produce the same effect in response to hydrogen peroxide. Our study, therefore, highlights the importance of TRIM26 in balancing cellular DNA glycosylase levels required for an efficient BER response.

Targeting Metal Impurities for the Detection and Quantification of Carbon Black Particles in Water via spICP-MS.

Carbon black (CB) is a nanomaterial with numerous industrial applications and high potential for integration into nano-enabled water treatment devices. However, few analytical techniques are capable of measuring CB in water at environmentally relevant concentrations. Therefore, we intended to establish a quantification method for CB with lower detection limits through utilization of trace metal impurities as analytical tracers. Various metal impurities were investigated in six commercial CB materials, and the Monarch 1000 CB was chosen as a model for further testing. The La impurity was chosen as a tracer for spICP-MS analysis based on measured concentration, low detection limits, and lack of polyatomic interferences. CB stability in water and adhesion to the spICP-MS introduction system presented a challenge that was mitigated by the addition of a nonionic surfactant to the matrix. Following optimization, the limit of detection (64 µg/L) and quantification (122 µg/L) for Monarch 1000 CB demonstrated the applicability of this approach to samples expected to contain trace amounts of CB. When compared against gravimetric analysis and UV-visible absorption spectroscopy, spICP-MS quantification exhibited similar sensitivity but with the ability to detect concentrations an order of magnitude lower. Method detection and sensitivity was unaffected when dissolved La was spiked into CB samples at environmentally relevant concentrations. Additionally, a more complex synthetic matrix representative of drinking water caused no appreciable impact to CB quantification. In comparison to existing quantification techniques, this method has achieved competitive sensitivity, a wide working range for quantification, and high selectivity for tracing possible release of CB materials with known metal contents.

Overcoming the Impact of Hypoxia in Driving Radiotherapy Resistance in Head and Neck Squamous Cell Carcinoma.

Hypoxia is very common in most solid tumours and is a driving force for malignant progression as well as radiotherapy and chemotherapy resistance. Incidences of head and neck squamous cell carcinoma (HNSCC) have increased in the last decade and radiotherapy is a major therapeutic technique utilised in the treatment of the tumours. However, effectiveness of radiotherapy is hindered by resistance mechanisms and most notably by hypoxia, leading to poor patient prognosis of HNSCC patients. The phenomenon of hypoxia-induced radioresistance was identified nearly half a century ago, yet despite this, little progress has been made in overcoming the physical lack of oxygen. Therefore, a more detailed understanding of the molecular mechanisms of hypoxia and the underpinning radiobiological response of tumours to this phenotype is much needed. In this review, we will provide an up-to-date overview of how hypoxia alters molecular and cellular processes contributing to radioresistance, particularly in the context of HNSCC, and what strategies have and could be explored to overcome hypoxia-induced radioresistance.

Effectiveness of PARP inhibition in enhancing the radiosensitivity of 3D spheroids of head and neck squamous cell carcinoma.

A critical risk factor for head and neck squamous cell carcinoma (HNSCC), particularly of the oropharynx, and the response to radiotherapy is human papillomavirus (HPV) type-16/18 infection. Specifically, HPV-positive HNSCC display increased radiosensitivity and improved outcomes, which has been linked with defective signalling and repair of DNA double-strand breaks (DSBs). This differential response to radiotherapy has been recapitulated in vitro using cell lines, although studies utilising appropriate 3D models that are more reflective of the original tumour are scarce. Furthermore, strategies to enhance the sensitivity of relatively radioresistant HPV-negative HNSCC to radiotherapy are still required. We have analysed the comparative response of in vitro 3D spheroid models of oropharyngeal squamous cell carcinoma to x-ray (photon) irradiation and provide further evidence that HPV-positive cells, in this case now grown as spheroids, show greater inherent radiosensitivity compared to HPV-negative spheroids due to defective DSB repair. We subsequently analysed these and an expanded number of spheroid models, with a particular focus on relatively radioresistant HPV-negative HNSCC, for impact of poly(ADP-ribose) polymerase (PARP) inhibitors (olaparib and talazoparib) in significantly inhibiting spheroid growth in response to photons but also proton beam therapy. We demonstrate that in general, PARP inhibition can further radiosensitise particularly HPV-negative HNSCC spheroids to photons and protons leading to significant growth suppression. The degree of enhanced radiosensitivity was observed to be dependent on the model and on the tumour site (oropharynx, larynx, salivary gland, or hypopharynx) from which the cells were derived. We also provide evidence suggesting that PARP inhibitor effectiveness relates to homologous recombination repair proficiency. Interestingly though, we observed significantly enhanced effectiveness of talazoparib versus olaparib specifically in response to proton irradiation. Nevertheless, our data generally support that PARP inhibition in combination with radiotherapy (photons and protons) should be considered further as an effective treatment for HNSCC, particularly for relatively radioresistant HPV-negative tumours.

Proton beam therapy in rectal cancer: A systematic review and meta-analysis.

INTRODUCTION: Locally advanced rectal cancer is often treated with neoadjuvant chemoradiotherapy and surgery. Radiotherapy carries significant risk of toxicity to organs at risk (OAR). Proton beam therapy (PBT) has demonstrated to be effective in other cancers, delivering equivalent dosimetric radiation but with the benefit of improved sparing of OAR. This review compares dosimetric irradiation of OAR and oncological outcomes for PBT versus conventional photon-based radiotherapy in locally advanced rectal cancer. METHODS: An electronic literature search was performed for studies with comparative cohorts receiving proton beam therapy and photon-based radiotherapy for rectal cancer. RESULTS: Eight articles with a total of 127 patients met the inclusion criteria. There was significantly less irradiated small bowel with PBT compared to three-dimensional conformal radiation therapy (3DCRT) and intensity-modulated radiation therapy (IMRT) (MD -17.01, CI [-24.06, -9.96], p < 0.00001 and MD -6.96, CI [-12.99, -0.94], p = 0.02, respectively). Similar dosimetric results were observed for bladder and pelvic bone marrow. Three studies reported clinical and oncological results for PBT in recurrent rectal cancer with overall survival reported as 43 %, 68 % and 77.2 %, and one study in primary rectal cancer with 100 % disease free survival. CONCLUSION: PBT treatment plans revealed significantly less irradiation of OAR for rectal cancer compared to conventional photon-based radiotherapy. Trials for recurrent rectal cancer and PBT have shown promising results. There are currently no ongoing clinical trials for primary rectal cancer and PBT. More research is required to validate its potential role in dose escalation, higher complete response rate and organ preservation without increasing toxicity.

USP9X Is Required to Maintain Cell Survival in Response to High-LET Radiation.

Ionizing radiation (IR) principally acts through induction of DNA damage that promotes cell death, although the biological effects of IR are more broad ranging. In fact, the impact of IR of higher-linear energy transfer (LET) on cell biology is generally not well understood. Critically, therefore, the cellular enzymes and mechanisms responsible for enhancing cell survival following high-LET IR are unclear. To this effect, we have recently performed siRNA screening to identify deubiquitylating enzymes that control cell survival specifically in response to high-LET α-particles and protons, in comparison to low-LET X-rays and protons. From this screening, we have now thoroughly validated that depletion of the ubiquitin-specific protease 9X (USP9X) in HeLa and oropharyngeal squamous cell carcinoma (UMSCC74A) cells using small interfering RNA (siRNA), leads to significantly decreased survival of cells after high-LET radiation. We consequently investigated the mechanism through which this occurs, and demonstrate that an absence of USP9X has no impact on DNA damage repair post-irradiation nor on apoptosis, autophagy, or senescence. We discovered that USP9X is required to stabilize key proteins (CEP55 and CEP131) involved in centrosome and cilia formation and plays an important role in controlling pericentrin-rich foci, particularly in response to high-LET protons. This was also confirmed directly by demonstrating that depletion of CEP55/CEP131 led to both enhanced radiosensitivity of cells to high-LET protons and amplification of pericentrin-rich foci. Our evidence supports the importance of USP9X in maintaining centrosome function and biogenesis and which is crucial particularly in the cellular response to high-LET radiation.

The Enzyme-Modified Neutral Comet (EMNC) Assay for Complex DNA Damage Detection.

The comet assay is a versatile, simple, and sensitive gel electrophoresis-based method that can be used to measure and accurately quantify DNA damage, particularly single and double DNA strand breaks, in single cells. While generally this is used to measure variation in DNA strand break levels and repair capacity within a population of cells, the technique has more recently been adapted and evolved into more complex analysis and detection of specific DNA lesions, such as oxidized purines and pyrimidines, achieved through the utilization of damage-specific DNA repair enzymes following cell lysis. Here, we detail a version of the enzyme-modified neutral comet (EMNC) assay for the specific detection of complex DNA damage (CDD), defined as two or more DNA damage lesions within 1-2 helical turns of the DNA. CDD induction is specifically relevant to ionizing radiation (IR), particularly of increasing linear energy transfer (LET), and is known to contribute to the cell-killing effects of IR due to the difficult nature of its repair. Consequently, the EMNC assay reveals important details regarding the extent and complexity of DNA damage induced by IR, but also has potential for the study of other genotoxic agents that may induce CDD.

Targeting Acid Ceramidase to Improve the Radiosensitivity of Rectal Cancer.

Previous work utilizing proteomic and immunohistochemical analyses has identified that high levels of acid ceramidase (AC) expression confers a poorer response to neoadjuvant treatment in locally advanced rectal cancer. We aimed to assess the radiosensitising effect of biological and pharmacological manipulation of AC and elucidate the underlying mechanism. AC manipulation in three colorectal cancer cell lines (HT29, HCT116 and LIM1215) was achieved using siRNA and plasmid overexpression. Carmofur and a novel small molecular inhibitor (LCL521) were used as pharmacological AC inhibitors. Using clonogenic assays, we demonstrate that an siRNA knockdown of AC enhanced X-ray radiosensitivity across all colorectal cancer cell lines compared to a non-targeting control siRNA, and conversely, AC protein overexpression increased radioresistance. Using CRISPR gene editing, we also generated AC knockout HCT116 cells that were significantly more radiosensitive compared to AC-expressing cells. Similarly, two patient-derived organoid models containing relatively low AC expression were found to be comparatively more radiosensitive than three other models containing higher levels of AC. Additionally, AC inhibition using carmofur and LCL521 in three colorectal cancer cell lines increased cellular radiosensitivity. Decreased AC protein led to significant poly-ADP ribose polymerase-1 (PARP-1) cleavage and apoptosis post-irradiation, which was shown to be executed through a p53-dependent process. Our study demonstrates that expression of AC within colorectal cancer cell lines modulates the cellular response to radiation, and particularly that AC inhibition leads to significantly enhanced radiosensitivity through an elevation in apoptosis. This work further solidifies AC as a target for improving radiotherapy treatment of locally advanced rectal cancer.

The E3 Ubiquitin Ligase NEDD4L Targets OGG1 for Ubiquitylation and Modulates the Cellular DNA Damage Response.

8-Oxoguanine DNA glycosylase (OGG1) is the major cellular enzyme required for the excision of 8-oxoguanine DNA base lesions in DNA through the base excision repair (BER) pathway, and therefore plays a major role in suppressing mutagenesis and in controlling genome stability. However, the mechanism of regulation of cellular OGG1 protein, particularly in response to oxidative stress, is unclear. We have purified the major E3 ubiquitin ligase responsible for OGG1 ubiquitylation from human cell extracts, and identify this as E3 ubiquitin-protein ligase NEDD4-like (NEDD4L). We demonstrate that recombinant NEDD4L stimulates ubiquitylation of OGG1 in vitro, particularly on lysine 341, and that NEDD4L and OGG1 interact in U2OS cells. Depletion of NEDD4L in U2OS cells has no impact on the stability and steady-state protein levels of OGG1, however, OGG1 stability is enhanced in response to oxidative stress induced by ionizing radiation. Furthermore, ubiquitylation of OGG1 by NEDD4L in vitro inhibits its DNA glycosylase/lyase activity. As a consequence of prolonged OGG1 stability and increased excision activity in the absence of NEDD4L, cells display increased DNA repair capacity but conversely that this decreases cell survival post-irradiation. This effect can be reproduced following OGG1 overexpression, suggesting that dysregulation of OGG1 increases the formation of lethal intermediate DNA lesions. Our study therefore highlights the importance of balancing OGG1 protein levels and BER capacity in maintaining genome stability.

FLASH Radiotherapy: Current Knowledge and Future Insights Using Proton-Beam Therapy.

FLASH radiotherapy is the delivery of ultra-high dose rate radiation several orders of magnitude higher than what is currently used in conventional clinical radiotherapy, and has the potential to revolutionize the future of cancer treatment. FLASH radiotherapy induces a phenomenon known as the FLASH effect, whereby the ultra-high dose rate radiation reduces the normal tissue toxicities commonly associated with conventional radiotherapy, while still maintaining local tumor control. The underlying mechanism(s) responsible for the FLASH effect are yet to be fully elucidated, but a prominent role for oxygen tension and reactive oxygen species production is the most current valid hypothesis. The FLASH effect has been confirmed in many studies in recent years, both in vitro and in vivo, with even the first patient with T-cell cutaneous lymphoma being treated using FLASH radiotherapy. However, most of the studies into FLASH radiotherapy have used electron beams that have low tissue penetration, which presents a limitation for translation into clinical practice. A promising alternate FLASH delivery method is via proton beam therapy, as the dose can be deposited deeper within the tissue. However, studies into FLASH protons are currently sparse. This review will summarize FLASH radiotherapy research conducted to date and the current theories explaining the FLASH effect, with an emphasis on the future potential for FLASH proton beam therapy.