Cellular Radiosensitivity Research Articles

Downregulation of CTRP1 Reduces Radio-resistance in Glioblastoma Cells by Inhibiting the Expression of CD133 after X-ray and Carbon Ion Irradiation

Glioblastomas (GBMs), the most prevalent primary malignant brain tumors, present significant challenges due to their invasive nature, high recurrence rates, and limited treatment options. Radiotherapy is a cornerstone in the management of GBMs; however, resistance to treatment poses a substantial obstacle. This study investigates the role of adipokine C1q/TNF-related protein 1 (CTRP1) in the radio-sensitivity of GBMs, utilizing both X-ray and carbon ion irradiation. Expression analyses revealed elevated CTRP1 and CD133 levels in GBMs tissues, which were associated with poor patient survival. Carbon ion irradiation demonstrated superior growth inhibition compared to X-ray, particularly in U87 (high CD133) cells. Moreover, CTRP1 expression increased following radiation exposure, especially after X-ray treatment. Knockdown of CTRP1 enhanced radio-sensitivity by reducing cell proliferation and increasing apoptosis, while exacerbating oxidative stress. Bioinformatics analysis revealed CTRP1's involvement in DNA damage repair pathways. Our findings establish a novel connection between CTRP1 and cellular radio-sensitivity. Targeting CTRP1, especially in U87 (high CD133) cells, enhances GBMs radio-sensitivity, offering potential therapeutic avenues.

Downregulation of circSTX6 suppresses tumor progression while facilitating radiosensitivity in cervical squamous cell carcinoma

Backgroundcervical squamous cell carcinoma (CSCC) is the second gynecological tumors that seriously threaten women’s life quality. Circular RNA (circRNA) is related with cervical cancer carcinogenesis and radiosensitivity. AimTo investigate the performance of hsa_circ_0007905 (circSTX6) on regulating cellular activities and radiosensitivity in CSCC. MethodsThe relative expression of circSTX6 in different tissue samples was detected by RT-qPCR. The cellular activity influence of circSTX6 in cervical cancer cells was measured by CCK-8 and Transwell assays. The survival fractions of cancer cells were detected after the radiation treatment to explore the relationship between circSTX6 and radiosensitivity of cervical cancer. The downstream miRNAs were predicted and analyzed. Rescue experiments confirmed their targeting relationship. Bioinformatic analysis was performed to identify the potential targets of miR-203a-3p. ResultscircSTX6 was increased and miR-203a-3p was decreased in cervical cancer tissues and radio-resistant tissues. CircSTX6 expression was related to the patient’s survival rates. CircSTX6 absence decreased cervical cancer cell proliferation and invasion while enhancing the sensitivity of cervical cancer cells to radiotherapy by regulating miR-203a-3p. RAB27B may be a target of miR-203a-3p. ConclusioncircSTX6 may be a clinical prognostic biomarker in CSCC. The absence of circSTX6 inhibits cellular behaviors and increases the sensitivity of cervical cancer cells to radiation by modulating miR-203a-3p/RAB27B axis.

Discovery of Novel PROTAC SIRT6 Degraders with Potent Efficacy against Hepatocellular Carcinoma.

Sirtuin 6 (SIRT6), a member of the SIRT family, plays essential roles in the regulation of metabolism, inflammation, aging, DNA repair, and cancer development, making it a promising anticancer drug target. Herein, we present our use of proteolysis-targeting chimera (PROTAC) technology to formulate a series of highly potent and selective SIRT6 degraders. One of the degraders, SZU-B6, induced the near-complete degradation of SIRT6 in both SK-HEP-1 and Huh-7 cell lines and more potently inhibited hepatocellular carcinoma (HCC) cell proliferation than the parental inhibitors. In preliminary mechanistic studies, SZU-B6 hampered DNA damage repair, promoting the cellular radiosensitization of cancer cells. Our SIRT6 degrader SZU-B6 displayed promising antitumor activity, particularly when combined with the well-known kinase inhibitor sorafenib or irradiation in an SK-HEP-1 xenograft mouse model. Our results suggest that these PROTACs might constitute a potent therapeutic strategy for HCC.

Artemis deficiency: A large cohort including a novel variant with increased radiosensitivity.

Artemis deficiency is an autosomal recessive disorder characterized by a combined immunodeficiency with increased cellular radiosensitivity. In this review, the clinical and genetic characteristics of 15 patients with DCLRE1C variants are presented. The demographic, clinical, immunologic, and genetic characteristics of patients with confirmed DCLRE1C variants diagnosed between 2013 and 2023 were collected retrospectively. Three patients were evaluated for radiosensitivity by the Comet assay, compared with age- and sex-matched healthy control. Seven patients who had severe infections in the first 6 months of life were diagnosed with T-B-NK+ SCID (severe combined immunodeficiency). Among them, four individuals underwent transplantation, and one of those died due to post-transplant complications in early life. Eight patients had hypomorphic variants. Half of them were awaiting a suitable donor, while the other half had already undergone transplantation. The majority of patients were born into a consanguineous family (93.3%). Most patients had recurrent sinopulmonary infections (73.3%), and one patient had no other infection than an acute respiratory infection before diagnosis. Two patients (13.3%) had autoimmunity in the form of autoimmune hemolytic anemia. Growth retardation was observed in only one patient (6.6%), and no malignancy was detected in the surviving 11 patients during the median (IQR) of 21.5 (12-45) months of follow-up. Three patients who had novel variants exhibited increased radiosensitivity and compromised DNA repair, providing a potential vulnerability to malignant transformation. Early diagnosis, radiation avoidance, and careful preparation for transplantation contribute to minimizing complications, enhancing life expectancy, and improving the patient's quality of life.

Seventy Years of Dose-response Models: From the Target Theory to the Use of Big Databases Involving Cell Survival and DNA Repair.

Radiobiological data, whether obtained at the clinical, biological or molecular level has significantly contributed to a better description and prediction of the individual dose-response to ionizing radiation and a better estimation of the radiation-induced risks. Particularly, over the last seventy years, the amount of radiobiological data has considerably increased, and permitted the mathematical formulas describing dose-response to become less empirical. A better understanding of the basic radiobiological mechanisms has also contributed to establish quantitative inter-correlations between clinical, biological and molecular biomarkers, refining again the mathematical models of description. Today, big data approaches and, more recently, artificial intelligence may finally complete and secure this long process of thinking from the multi-scale description of radiation-induced events to their prediction. Here, we reviewed the major dose-response models applied in radiobiology for quantifying molecular and cellular radiosensitivity and aimed to explain their evolution: Specifically, we highlighted the advances concerning the target theory with the cell survival models and the progressive introduction of the DNA repair process in the mathematical models. Furthermore, we described how the technological advances have changed the description of DNA double-strand break (DSB) repair kinetics by introducing the important notion of DSB recognition, independent of that of DSB repair. Initially developed separately, target theory on one hand and, DSB recognition and repair, on the other hand may be now fused into a unified model involving the cascade of phosphorylations mediated by the ATM kinase in response to any genotoxic stress.

Biological dose optimization incorporating intra-tumoural cellular radiosensitivity heterogeneity in ion-beam therapy treatment planning

Objective. Treatment plans of ion-beam therapy have been made under an assumption that all cancer cells within a tumour equally respond to a given radiation dose. However, an intra-tumoural cellular radiosensitivity heterogeneity clearly exists, and it may lead to an overestimation of therapeutic effects of the radiation. The purpose of this study is to develop a biological model that can incorporate the radiosensitivity heterogeneity into biological optimization for ion-beam therapy treatment planning. Approach. The radiosensitivity heterogeneity was modeled as the variability of a cell-line specific parameter in the microdosimetric kinetic model following the gamma distribution. To validate the developed intra-tumoural-radiosensitivity-heterogeneity-incorporated microdosimetric kinetic (HMK) model, a treatment plan with H-ion beams was made for a chordoma case, assuming a radiosensitivity heterogeneous region within the tumour. To investigate the effects of the radiosensitivity heterogeneity on the biological effectiveness of H-, He-, C-, O-, and Ne-ion beams, the relative biological effectiveness (RBE)-weighted dose distributions were planned for a cuboid target with the stated ion beams without considering the heterogeneity. The planned dose distributions were then recalculated by taking the heterogeneity into account. Main results. The cell survival fraction and corresponding RBE-weighted dose were formulated based on the HMK model. The first derivative of the RBE-weighted dose distribution was also derived, which is needed for fast biological optimization. For the patient plan, the biological optimization increased the dose to the radiosensitivity heterogeneous region to compensate for the heterogeneity-induced reduction in biological effectiveness of the H-ion beams. The reduction in biological effectiveness due to the heterogeneity was pronounced for low linear energy transfer (LET) beams but moderate for high-LET beams. The RBE-weighted dose in the cuboid target decreased by 7.6% for the H-ion beam, while it decreased by just 1.4% for the Ne-ion beam. Significance. Optimal treatment plans that consider intra-tumoural cellular radiosensitivity heterogeneity can be devised using the HMK model.

Effect of Raltitrexed on ECA109 Cellular Radiosensitivity and its Mechanism in Esophageal Cancer.

To investigate the effect of raltitrexed + X-ray irradiation on esophageal cancer ECA109 cells and analyze the potential action mechanism. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to analyze the inhibitory effect of raltitrexed on cell proliferation. The effect of raltitrexed on radiosensitivity was studied through a clone-forming experiment. The scratch assay and invasion test were performed to understand the cell migration and invasion abilities. The apoptosis rate change was measured using a flow cytometer, and Western Blotting was used to determine the expression of B cell lymphoma-2 (Bcl-2) and Bcl2-associated X protein (Bax) in each group. Raltitrexed significantly inhibited ECA109 proliferation in a time-dose-dependent manner; there were significant differences among different concentrations and times of action. The results of the clone-forming experiment showed a sensitization enhancement ratio of 1.65, and this demonstrated a radiosensitization effect. After the combination of raltitrexed with X-ray, the cell migration distance was shortened, and the number of cells penetrating the membrane was reduced. Raltitrexed can inhibit the growth of esophageal cancer ECA109 cells and has a radiosensitization effect.

Mechanism of Musashi2 affecting radiosensitivity of lung cancer by modulating DNA damage repair.

Identifying new targets for overcoming radioresistance is crucial for improving the efficacy of lung cancer radiotherapy, given that tumor cell resistance is a leading cause of treatment failure. Recent research has spotlighted the significance of Musashi2 (MSI2) in cancer biology. In this study, we first demonstrated that MSI2 plays a key function in regulating the radiosensitivity of lung cancer. The expression of MSI2 is negatively correlated with overall survival in cancer patients, and the knockdown of MSI2 inhibits tumorigenesis and increases radiosensitivity of lung cancer cells. Cellular radiosensitivity, which is closely linked to DNA damage, is influenced by MSI2 interaction with ataxia telangiectasia mutated and Rad3-related kinase (ATR) and checkpoint kinase 1 (CHK1) post-irradiation; moreover, knockdown of MSI2 inhibits the ATR-mediated DNA damage response pathway. RNA-binding motif protein 17 (RBM17), which is implicated in DNA damage repair, exhibits increased interaction with MSI2 post-irradiation. We found that knockdown of RBM17 disrupted the interaction between MSI2 and ATR post-irradiation and increased the radiosensitivity of lung cancer cells. Furthermore, we revealed the potential mechanism of MSI2 recruitment into the nucleus with the assistance of RBM17 to activate ATR to promote radioresistance. This study provides novel insights into the potential application of MSI2 as a new target in lung cancer radiotherapy.

Live Imaging to Quantify Cellular Radiosensitivity in Patient-Derived Tumor Organoids.

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, often (but not always) because of differences in the ability of malignant cells to repair oxidative DNA damage as elicited by ionizing rays. Clonogenic assays have been employed for decades to assess the sensitivity of cultured cancer cells to ionizing irradiation, largely because irradiated cancer cells often die in a delayed manner that is difficult to quantify with short-term flow cytometry- or microscopy-assisted techniques. Unfortunately, clonogenic assays cannot be employed as such for more complex tumor models, such as patient-derived tumor organoids (PDTOs). Indeed, irradiating established PDTOs may not necessarily abrogate their growth as multicellular units, unless their stem-like compartment is completely eradicated. Moreover, irradiating PDTO-derived single-cell suspensions may not properly recapitulate the sensitivity of malignant cells to RT in the context of established PDTOs. Here, we detail an adaptation of conventional clonogenic assays that involves exposure of established PDTOs to ionizing radiation, followed by single-cell dissociation, replating in suitable culture conditions and live imaging. Non-irradiated (control) PDTO-derived stem-like cells reform growing PDTOs with a PDTO-specific efficiency, which is negatively influenced by irradiation in a dose-dependent manner. In these conditions, PDTO-forming efficiency and growth rate can be quantified as a measure of radiosensitivity on time-lapse images collected until control PDTOs achieve a predefined space occupancy.

TAB182 regulates glycolytic metabolism by controlling LDHA transcription to impact tumor radiosensitivity

Metabolic reprogramming, a hallmark of cancer, is closely associated with tumor development and progression. Changes in glycolysis play a crucial role in conferring radiation resistance to tumor cells. How radiation changes the glycolysis status of cancer cells is still unclear. Here we revealed the role of TAB182 in regulating glycolysis and lactate production in cellular response to ionizing radiation. Irradiation can significantly stimulate the production of TAB182 protein, and inhibiting TAB182 increases cellular radiosensitivity. Proteomic analysis indicated that TAB182 influences several vital biological processes, including multiple metabolic pathways. Knockdown of TAB182 results in decreased lactate production and increased pyruvate and ATP levels in cancer cells. Moreover, knocking down TAB182 reverses radiation-induced metabolic changes, such as radioresistant-related lactate production. TAB182 is necessary for activating LDHA transcription by affecting transcription factors SP1 and c-MYC; its knockdown attenuates the upregulation of LDHA by radiation, subsequently suppressing lactate production. Targeted suppression of TAB182 significantly enhances the sensitivity of murine xenograft tumors to radiotherapy. These findings advance our understanding of glycolytic metabolism regulation in response to ionizing radiation, which may offer significant implications for developing new strategies to overcome tumor radioresistance.

Statin-Sensitive Akt1/Src/Caveolin-1 Signaling Enhances Oxidative Stress Resistance in Rhabdomyosarcoma.

Identifying the molecular mechanisms underlying radioresistance is a priority for the treatment of RMS, a myogenic tumor accounting for approximately 50% of all pediatric soft tissue sarcomas. We found that irradiation (IR) transiently increased phosphorylation of Akt1, Src, and Cav1 in human RD and RH30 lines. Synthetic inhibition of Akt1 and Src phosphorylation increased ROS levels in all RMS lines, promoting cellular radiosensitization. Accordingly, the elevated activation of the Akt1/Src/Cav1 pathway, as detected in two RD lines characterized by overexpression of a myristoylated Akt1 form (myrAkt1) or Cav1 (RDCav1), was correlated with reduced levels of ROS, higher expression of catalase, and increased radioresistance. We found that treatment with cholesterol-lowering drugs such as lovastatin and simvastatin promoted cell apoptosis in all RMS lines by reducing Akt1 and Cav1 levels and increasing intracellular ROS levels. Combining statins with IR significantly increased DNA damage and cell apoptosis as assessed by γ histone 2AX (γH2AX) staining and FACS analysis. Furthermore, in combination with the chemotherapeutic agent actinomycin D, statins were effective in reducing cell survival through increased apoptosis. Taken together, our findings suggest that the molecularly linked signature formed by Akt1, Src, Cav1, and catalase may represent a prognostic determinant for identifying subgroups of RMS patients with higher probability of recurrence after radiotherapy. Furthermore, statin-induced oxidative stress could represent a treatment option to improve the success of radiotherapy.

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.

YAP/Aurora A-mediated ciliogenesis regulates ionizing radiation-induced senescence via Hedgehog pathway in tumor cells

Primary cilia are antenna-like organelles that play critical roles in sensing and responding to various signals. Nevertheless, the function of primary cilia in cellular response to ionizing radiation (IR) in tumor cells remains unclear. Here, we show that primary cilia are frequently expressed in tumor cells and tissues. Notably, IR promotes cilia formation and elongation in time- and dose-dependent manners. Mechanistic study shows that the suppression of YAP/Aurora A pathway contributes to IR-induced ciliogenesis, which is diminished by Aurora A overexpression. The ciliated tumor cells undergo senescence but not apoptosis in response to IR and the abrogation of cilia formation is sufficient to elevate the lethal effect of IR. Furthermore, we show that IR-induced ciliogenesis leads to the activation of Hedgehog signaling pathway to drive senescence and resist apoptosis, and its blockage enhances cellular radiosensitivity by switching senescence to apoptosis. In summary, this work shows evidence of primary cilia in coordinating cellular response to IR in tumor cells, which may help to supply a novel sensitizing target to improve the outcome of radiotherapy.

Effect of bisphosphonates and statins on the in vitro radiosensitivity of breast cancer cell lines.

Early-stage breast cancer is usually treated with breast-conserving surgery followed by adjuvant radiation therapy. Acute skin toxicity is a common radiation-induced side effect experienced by many patients. Recently, a combination of bisphosphonates (zoledronic acid) and statins (pravastatin), or ZOPRA, was shown to radio-protect normal tissues by enhancing DNA double-strand breaks (DSB) repair mechanism. However, there are no studies assessing the effect of ZOPRA on cancerous cells. The purpose of this study is to characterize the in vitro effect of the zoledronic acid (ZO), pravastatin (PRA), and ZOPRA treatment on the molecular and cellular radiosensitivity of breast cancer cell lines. Two breast cancer cell lines, MDA MB 231 and MCF-7, were tested. Cells were treated with different concentrations of pravastatin (PRA), zoledronate (ZO), as well as their ZOPRA combination, before irradiation. Anti-γH2AX and anti-pATM immunofluorescence were performed to study DNA DSB repair kinetics. MTT assay was performed to assess cell proliferation and viability, and flow cytometry was performed to analyze the effect of the drugs on the cell cycle distribution. The clonogenic assay was used to assess cell survival. ZO, PRA, and ZOPRA treatments were shown to increase the residual number of γH2AX foci for both cell lines. ZOPRA treatment was also shown to reduce the activity of the ATM kinase in MCF-7. ZOPRA induced a significant decrease in cell survival for both cell lines. Our findings show that pretreatment with ZOPRA can decrease the radioresistance of breast cancer cells at the molecular and cellular levels. The fact that ZOPRA was previously shown to radioprotect normal tissues, makes it a good candidate to become a therapeutic window-widening drug.

Nanodiamond Effects on Cancer Cell Radiosensitivity: The Interplay between Their Chemical/Physical Characteristics and the Irradiation Energy

Nanoparticles are being increasingly studied to enhance radiation effects. Among them, nanodiamonds (NDs) are taken into great consideration due to their low toxicity, inertness, chemical stability, and the possibility of surface functionalization. The objective of this study is to explore the influence of the chemical/physical properties of NDs on cellular radiosensitivity to combined treatments with radiation beams of different energies. DAOY, a human radioresistant medulloblastoma cell line was treated with NDs—differing for surface modifications [hydrogenated (H-NDs) and oxidized (OX-NDs)], size, and concentration—and analysed for (i) ND internalization and intracellular localization, (ii) clonogenic survival after combined treatment with different radiation beam energies and (iii) DNA damage and apoptosis, to explore the nature of ND–radiation biological interactions. Results show that chemical/physical characteristics of NDs are crucial in determining cell toxicity, with hydrogenated NDs (H-NDs) decreasing either cellular viability when administered alone, or cell survival when combined with radiation, depending on ND size and concentration, while OX-NDs do not. Also, irradiation at high energy (γ-rays at 1.25 MeV), in combination with H-NDs, is more efficient in eliciting radiosensitisation when compared to irradiation at lower energy (X-rays at 250 kVp). Finally, the molecular mechanisms of ND radiosensitisation was addressed, demonstrating that cell killing is mediated by the induction of Caspase-3-dependent apoptosis that is independent to DNA damage. Identifying the optimal combination of ND characteristics and radiation energy has the potential to offer a promising therapeutic strategy for tackling radioresistant cancers using H-NDs in conjunction with high-energy radiation.

Heme Biosynthesis is Crucial for Cell Survival and Mitochondrial OXPHOS after X Irradiation.

Heme is an essential component of the hemoproteins involved in the mitochondrial electron transport chain (ETC). Cancer cells have been reported to display high heme levels and increased activity of heme-containing proteins. Consistently, inhibition of heme biosynthesis by the ALAD inhibitor succinylacetone (SA) has been shown to reduce tumor cell survival. These observations indicate that heme biosynthesis is essential for cancer cell proliferation. X irradiation has been shown to increase mitochondrial mass, membrane potential, oxygen consumption, reactive oxygen species (ROS) production, and ATP synthesis. This finding suggests that radiation activates mitochondrial oxidative phosphorylation (OXPHOS). However, although heme is an essential component of the mitochondrial ETC, whether radiation influences heme biosynthesis remains unclear. In this study, we evaluated heme biosynthesis activity after X irradiation and examined the effects of heme biosynthesis inhibition by SA on cellular radiosensitivity and mitochondrial OXPHOS function. We demonstrated that X irradiation significantly increased ALAS1 mRNA levels and cellular heme content. Inhibition of heme biosynthesis by SA significantly decreased cellular heme content and sensitized cancer cells to radiation. We also showed that SA reduced cellular ATP levels, mitochondrial membrane potential, and mitochondrial ROS production, suggesting mitochondrial OXPHOS dysfunction. SA decreased the expression of mitochondrial heme-related proteins COX2 and cytochrome c but did not influence COX1 and VDAC expression. These results indicate that inhibition of heme biosynthesis decreased mitochondrial ETC protein expression and OXPHOS activity, which triggered cellular ATP depletion and radiosensitization after X irradiation. In summary, heme biosynthesis is upregulated by X irradiation and is essential for mitochondrial OXPHOS and cell survival.

Mean global DNA methylation serves as independent prognostic marker in IDH-wildtype glioblastoma.

The IDH-wildtype glioblastoma (GBM) patients have a devastating prognosis. Here, we analyzed the potential prognostic value of global DNA methylation of the tumors. DNA methylation of 492 primary samples and 31 relapsed samples, each treated with combination therapy, and of 148 primary samples treated with radiation alone were compared with patient survival. We determined the mean methylation values and estimated the immune cell infiltration from the methylation data. Moreover, the mean global DNA methylation of 23 GBM cell lines was profiled and correlated to their cellular radiosensitivity as measured by colony formation assay. High mean DNA methylation levels correlated with improved survival, which was independent from known risk factors (MGMT promoter methylation, age, extent of resection; P = 0.009) and methylation subgroups. Notably, this correlation was also independent of immune cell infiltration, as higher number of immune cells indeed was associated with significantly better OS but lower mean methylation. Radiosensitive GBM cell lines had a significantly higher mean methylation than resistant lines (P = 0.007), and improved OS of patients treated with radiotherapy alone was also associated with higher DNA methylation (P = 0.002). Furthermore, specimens of relapsed GBM revealed a significantly lower mean DNA methylation compared to the matching primary tumor samples (P = 0.041). Our results indicate that mean global DNA methylation is independently associated with outcome in glioblastoma. The data also suggest that a higher DNA methylation is associated with better radiotherapy response and less aggressive phenotype, both of which presumably contribute to the observed correlation with OS.

Role of The circ-HIPK3, circ-PVT1, miR-25, and miR-149 in Response of Breast Cancer Cells to Ionizing Radiation.

Determining cellular radiosensitivity of breast cancer (BC) patients through molecular markers before radiation therapy (RT) allows accurate prediction of individual's response to radiation. The aim of this study was therefore to investigate the potential role of epigenetic biomarkers in breast cancer cellular radiosensitivity. In this experimental study, we treated two BC cell lines, MDA-MB 231 and MCF-7, with doses of 2, 4, and 8Gy of irradiation for 24 and 48 hours. Expression levels of circ-HIPK3, circ-PVT1, miR-25, and miR- 149 were quantified using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Significance of the observations was statistically verified using one-way ANOVA with a significance level of P<0.05. Annexin V-FITC/PI binding assay was utilized to measure cellular apoptosis. The rate of cell apoptosis was significantly higher in MCF-7 cells compared to MDA-MB-231 cells at doses of 4Gy and 8Gy (P=0.013 and P=0.004, respectively). RNA expression analysis showed that circ-HIPK3 was increased in the MDA-MB-231 cell line compared to the MCF-7 cell line after exposure to 8Gy for 48 hours. Expression of circ-PVT1 was found to be higher in MDA-MB-231 cells compared to MCF-7 cells after exposure to 8Gy for 24 hours, likewise after exposure to 4Gy and 8Gy for 48 hours. After exposing 8Gy, expression of miR-25 was increased in MDA-MB-231 cells compared to MCF-7 cells at 24 and 48 hours. After exposing 8Gy dose, expression of miR-149 was increased in MCF-7 cells compared to MDA-MB-231 cells at 24 and 48 hours. circ-HIPK3, circ-PVT1, and miR-25 played crucial roles in the mechanisms of radioresistance in breast cancer. Additionally, miR-149 was involved in regulating cellular radiosensitivity. Therefore, these factors provided predictive information about a tumor's radiosensitivity or its response to treatment, which could be valuable in personalizing radiation dosage.

Sodium-Glucose Cotransporter 2 (SGLT2) Inhibition Induces DNA Damage and Radiosensitization in Glioblastoma Cell Lines

Glioblastomas (GBM) are highly dependent on glucose for energy to promote cell proliferation and tumor growth. Preclinical studies have shown that sodium-glucose cotransporter 2 (SGLT2) inhibitors, drugs that are FDA approved for diabetes, reduce the viability and invasiveness of GBM cells. We hypothesize that SGLT2 inhibitors decrease GBM cell growth and viability by promoting DNA damage. The SGLT2 inhibitors canagliflozin, dapagliflozin and empagliflozin were used and cell studies were performed under low glucose (0.6 mM) media conditions. In the cell proliferation assays, cells were counted in triplicate after 24-, 48- and 72-hour treatment with SGLT2 inhibitors. Glucose transport assays were performed using a 6-fluorescent iodinated glucose analog (6FIGA) which is selectively transported by SGLTs and not by glucose transporters (GLUTs). Immunoblots were used to measure protein expression of SGLT2 and markers of DNA damage (phospho-ATM and gamma-H2AX). Cellular radiosensitization was evaluated by clonogenic survival assays. SGLT2 inhibition with canagliflozin, dapagliflozin and empagliflozin decreased cell proliferation in LN229, U118MG and LN18 GBM cells lines. In LN229 cells, 72-hour treatment with canagliflozin (30 µM) decreased cell proliferation by 60% compared to vehicle control cells (p<0.0001) whereas dapagliflozin (30 µM) and empagliflozin (30 µM) decreased cell proliferation compared to vehicle control cells by 17% (p<0.0001) and 19% (p<0.001), respectively. Glucose transport assays show that 6FIGA uptake is reduced by canagliflozin and dapagliflozin in LN229 and U87 GBM cells. Canagliflozin treatment (50 µM) for 24 hours in LN229 cells results in increased phosphorylated ATM protein and increased gamma-H2AX protein, markers of DNA damage, by immunoblot. SGLT2 protein expression was not affected by canagliflozin. Treatment with canagliflozin (50 µM) 48 hours prior to 2, 4 and 6 Gy of ionizing radiation reduced clonogenic survival of LN229, LN18 and U118MG GBM cells. SGLT2 inhibitors canagliflozin, dapagliflozin and empagliflozin decrease GBM cell proliferation, with the highest inhibitory effect seen with canagliflozin. Canagliflozin treatment alone increases DNA damage signaling in GBM cells. Canagliflozin radiosensitizes GBM cells. This work provides preliminary preclinical rationale for canagliflozin as a radiosensitizer in GBM.

Induction of epithelial-mesenchymal transition in thyroid follicular cells is associated with cell adhesion alterations and low-dose hyper-radiosensitivity.

Epithelial-mesenchymal transition (EMT) is associated with altered cellular adhesion. We previously demonstrated that cellular adhesion influences Low-dose Hyper-Radiosensitivity (HRS) in a variety of tumor cells. However, the relationship of low-dose HRS with the phenotypic plasticity incurred by EMT during the neoplastic transformation remains to be elucidated. To investigate whether acquisition of EMT phenotype during progressive neoplastic transformation may affect low-dose radiation sensitivity. Primary thyroid cells obtained from a human cystic thyroid nodule were first subjected to nutritional stress. This yielded immortalized INM-Thy1 cell strain, which was further treated with either multiple γ-radiation fractions (1.5 Gy each) or repetitive cycles of 3-methylcholanthrene and phorbol-12-myristate-13-acetate, yielding two progressive transformants, viz., INM-Thy1R and INM-Thy1C. Morphological alterations, chromosomal double-minutes, cell adhesion proteins, anchorage dependency, tumorigenicity in nude mice and cellular radiosensitivity were studied in these strains. Both transformants (INM-Thy1R, INM-Thy1C) displayed progressive tumorigenic features, viz., soft agar colony growth and solid tumor growth in nude mice, coupled with features of epithelial-mesenchymal transition and activated Wnt pathway. Incidentally, the chemical-induced transformant (INM-Thy1C) displayed a prominent HRS (αs/αr=29.35) which remained unaffected at high cell density. However, the parental (INM-Thy1) cell line as well as radiation-induced transformant (INM-Thy1R) failed to show this hypersensitivity. The study shows that induction of EMT in thyroid follicular cells may accompany increased susceptibility to low-dose ionizing radiation, which was attenuated by adaptive resistance acquired during radiation-induced transformation.