Radiosensitivity Of Cell Lines Research Articles

BIOM-41. HMGB2 IS A CANDIDATE PROGNOSTIC BIOMARKER IN BOTH ADULT AND PEDIATRIC DIFFUSE GLIOMAS

Abstract Few prognostic molecular markers are common to pediatric- and adult diffuse gliomas (PDG and ADG). HMGB2 is a protein expressed in human brain development and in de-differentiated high-grade gliomas (HGG), which we have recently described as a putative prognostic and predictive biomarker in ADG, associated with proliferative activity and EIF2 signaling pathway upregulation. Additionally, we showed that HMGB2 shRNA knockdown (HMGB2-KD) reduced cell proliferation and colony formation, and radio-sensitized ADG cell lines. Aims: To assess the prognostic potential of HMGB2 in PDG and its association with stemness. METHODS 1) We singled out HMGB2 from proteomic profiles of an independent PDG cohort previously associated with EIF2 and MYCN signaling upregulation for survival analysis. 2) PDG cells SF188 were de-differentiated with substitution of DMEM by a neurobasal (NB) medium devoid of serum for stemness association studies. 3) We generated HMGB2-KD clones of PDG cell lines SF188 (grown in DMEM and NB) and KNS42 with 3 different shRNAs. RESULTS HMGB2 expression was significantly related to histological grade in PDG (grade 3 vs. grade 2 and grade 4 vs. grade 2: fold change= 8.16 and 9.39; p=0.0045 and <0.001; FDR= 0.04 and 0.0006, respectively) and survival (p=0.004, FDR=0.142 for PFS and p=0.03, FDR=0.291 for OS), in a Cox model including age and grade as covariables. Compared with cells growing in DMEM, SF188 cells growing in NB medium had a morphological change from stellate cells to round cells growing in spheres, with 2-fold increase in C-MYC and HMGB2 expression by Western analysis. HMGB2-KD resulted in >90% reduction of HMGB2 expression in SF188 and >70% in KNS42. CONCLUSION Collectively, our preliminary results suggest that HMGB2 is associated with stemness, has similar roles in PDG and ADG, and may be validated as prognostic/predictive biomarker in both adult and pediatric populations. Mechanistic studies of radiation response are underway.

Significant Impact of let-7d MicroRNA on Breast Cancer Cell Lines Post-Radiation Treatment

Introduction: Radiotherapy is a common treatment for breast cancer treatment, that induces DNA damage. These DNA damages are addressed through various repair pathways, which regulate the DNA repair systems and confer radio-resistance. Non-coding RNAs are a big proportion of genome transcripts without the potential to encode proteins. Related studies demonstrated that radiation affects the expression of non-coding RNAs. Let-7d, a tumor suppressor in numerous cancers, has some target genes that play a role in the DNA repair system. Materials and Methods: Human breast cancer cell lines MDA-MB-231 and MCF-7 were cultured in a Dulbecco's Modified Eagle Medium. The exponentially growing cells were treated with some doses of X-rays. After radiation treatment and cell harvesting, RNA was extracted, and cDNA synthesis was done. The let-7d miRNA expression changes were calculated with real-time quantitative reverse transcription polymerase chain reaction. Results: The results implied that radiation caused increased let-7d expression in breast cancer cell lines after radiation treatment. In addition, the results showed that 24 h after radiation, the expression of let-7d in the radioresistant cell line was higher than the radiosensitive one; 48 h after radiation, the expression of let-7d in the radiosensitive cell line was higher than the other one. Conclusions: The results demonstrated that radiation treatment increased let-7d miRNA expression in both radiosensitive and radio-resistant breast cancer cell lines. Therefore, let-7d might be involved in the radiosensitivity of breast cancer.

Differential biological effect of low doses of ionizing radiation depending on the radiosensitivity in a cell line model

Purpose Exposure to low doses (LD) of ionizing radiation (IR), such as the ones employed in computed tomography (CT) examination, can be associated with cancer risk. However, cancer development could depend on individual radiosensitivity. In the present study, we evaluated the differences in the response to a CT-scan radiation dose of 20 mGy in two lymphoblastoid cell lines with different radiosensitivity. Materials and Methods Several parameters were studied: gene expression, DNA damage, and its repair, as well as cell viability, proliferation, and death. Results were compared with those after a medium dose of 500 mGy. Results After 20 mGy of IR, the radiosensitive (RS) cell line showed an increase in DNA damage, and higher cell proliferation and apoptosis, whereas the radioresistant (RR) cell line was insensitive to this LD. Interestingly, the RR cell line showed a higher expression of an antioxidant gene, which could be used by the cells as a protective mechanism. After a dose of 500 mGy, both cell lines were affected by IR but with significant differences. The RS cells presented an increase in DNA damage and apoptosis, but a decrease in cell proliferation and cell viability, as well as less antioxidant response. Conclusions A differential biological effect was observed between two cell lines with different radiosensitivity, and these differences are especially interesting after a CT scan dose. If this is confirmed by further studies, one could think that individuals with radiosensitivity-related genetic variants may be more vulnerable to long-term effects of IR, potentially increasing cancer risk after LD exposure.

Identification of the unfolded protein response pathway as target for radiosensitization in pancreatic cancer

Background and purposeDue to the high intrinsic radioresistance of pancreatic ductal adenocarcinoma (PDAC), radiotherapy (RT) is only beneficial in 30% of patients. Therefore, this study aimed to identify targets to improve the efficacy of RT in PDAC. Materials and methodsAlamar Blue proliferation and colony formation assay (CFA) were used to determine the radioresponse of a cohort of 38 murine PDAC cell lines. A gene set enrichment analysis was performed to reveal differentially expressed pathways. CFA, cell cycle distribution, γH2AX FACS analysis, and Caspase 3/7 SYTOX assay were used to examine the effect of a combination treatment using KIRA8 as an IRE1α-inhibitor and Ceapin-A7 as an inhibitor against ATF6. ResultsThe unfolded protein response (UPR) was identified as a pathway highly expressed in radioresistant cell lines. Using the IRE1α-inhibitor KIRA8 or the ATF6-inhibitor Ceapin-A7 in combination with radiation, a radiosensitizing effect was observed in radioresistant cell lines, but no substantial alteration of the radioresponse in radiosensitive cell lines. Mechanistically, increased apoptosis by KIRA8 in combination with radiation and a cell cycle arrest in the G1 phase after ATF6 inhibition and radiation have been observed in radioresistant cell lines. ConclusionSo, our data show evidence that the UPR is involved in radioresistance of PDAC. Increased apoptosis and a G1 cell cycle arrest seem to be responsible for the radiosensitizing effect of UPR inhibition. These findings are supportive for developing novel combination treatment concepts in PDAC to overcome radioresistance.

The impact of DNA double-strand break repair pathways throughout the carbon ion spread-out Bragg peak beam.

Following carbon ion beam irradiation in mammalian cells, such as used in carbon ion radiotherapy (CIRT), it has been suggested that the balance between whether nonhomologous end joining (NHEJ) or homologous recombination (HR) is utilized depends on the DNA double-strand break (DSB) complexity. Here, we quantified DSB distribution and identified the importance of each DSB repair pathway at increasing depths within the carbon ion spread-out Bragg peak (SOBP) beam range. Chinese hamster ovary (CHO) cell lines were irradiated in a single biological system capable of incorporating the full carbon ion SOBP beam range. Cytotoxicity and DSB distribution/repair kinetics were examined at increasing beam depths using cell survival as an endpoint and γ-H2AX as a surrogate marker for DSBs. We observed that proximal SOBP had the highest number of total foci/cell and lowest survival, while distal SOBP had the most dense tracks. Both NHEJ- and HR-deficient CHO cells portrayed an increase in radiosensitivity throughout the full carbon beam range, although NHEJ-deficient cells were the most radiosensitive cell line from beam entrance up to proximal SOBP and demonstrated a dose-dependent decrease in ability to repair DSBs. In contrast, HR-deficient cells had the greatest ratio of survival fraction at entrance depth to the lowest survival fraction within the SOBP and demonstrated a linear energy transfer (LET)-dependent decrease in ability to repair DSBs. Collectively, our results provide insight into treatment planning and potential targets to inhibit, as HR was a more beneficial pathway to inhibit than NHEJ to enhance the cell killing effect of CIRT in targeted tumor cells within the SOBP while maintaining limited unwanted damage to surrounding healthy cells.

Integrin β1 regulates the perineural invasion and radioresistance of oral squamous carcinoma cells by modulating cancer cell stemness

Perineural invasion and radioresistance are the main determinants of treatment outcomes in oral squamous cell carcinoma (OSCC), but the exact mechanism is still unknown. We conducted an in vitro experiment to evaluate the role of integrin β1 (ITGB1) in the perineural invasion, radioresistance, and tumor aggressiveness of OSCC. Two OSCC cell lines (SCC25, SCC15) and radiation-induced radioresistant OSCC cell lines were used in this study. The expression of ITGB1 was compared between control radiosensitive and radioresistant OSCC cell lines. ITGB1 was inhibited by small hairpin RNA, and then the adhesion to neuronal cells, responsiveness to radiation, and aggressiveness of both OSCC cell lines were evaluated. Expression of ITGB1 and adhesion to neuronal cells were increased in radioresistant OSCC compared with control radiosensitive OSCC, and increased ITGB1 expression was more prominent in cancer stem cell-like cells. When the expression of ITGB1 was inhibited, the adhesion to neuronal cells, resistance to radiation, and invasion and migration of radioresistant OSCC were significantly reduced. Moreover, the expression of cancer stem cell markers and size of spheroid formations were also significantly attenuated by inhibiting ITGB1. These findings suggest that ITGB1 may be a significant contributor to perineural invasion and the maintenance of radioresistance in OSCC cells, and is associated with cancer stem cell-like cells. Furthermore, our results suggest a possible relationship between perineural invasion and radioresistance of OSCC. More detailed research is warranted to evaluate the role of ITGB1 as a novel emerging therapeutic target for radioresistant OSCC.

Characterization of Intrinsic Radiation Sensitivity in a Diverse Panel of Normal, Cancerous and CRISPR-Modified Cell Lines.

Intrinsic radiosensitivity is a major determinant of radiation response. Despite the extensive amount of radiobiological data available, variability among different studies makes it very difficult to produce high-quality radiosensitivity biomarkers or predictive models. Here, we characterize a panel of 27 human cell lines, including those derived from lung cancer, prostate cancer, and normal tissues. In addition, we used CRISPR-Cas9 to generate a panel of lines with known DNA repair defects. These cells were characterised by measuring a range of biological features, including the induction and repair of DNA double-strand breaks (DSBs), cell cycle distribution, ploidy, and clonogenic survival following X-ray irradiation. These results offer a robust dataset without inter-experimental variabilities for model development. In addition, we used these results to explore correlations between potential determinants of radiosensitivity. There was a wide variation in the intrinsic radiosensitivity of cell lines, with cell line Mean Inactivation Doses (MID) ranging from 1.3 to 3.4 Gy for cell lines, and as low as 0.65 Gy in Lig4-/- cells. Similar substantial variability was seen in the other parameters, including baseline DNA damage, plating efficiency, and ploidy. In the CRISPR-modified cell lines, residual DSBs were good predictors of cell survival (R2 = 0.78, p = 0.009), as were induced levels of DSBs (R2 = 0.61, p = 0.01). However, amongst the normal and cancerous cells, none of the measured parameters correlated strongly with MID (R2 < 0.45), and the only metrics with statistically significant associations are plating efficiency (R2 = 0.31, p = 0.01) and percentage of cell in S phase (R2 = 0.37, p = 0.005). While these data provide a valuable dataset for the modelling of radiobiological responses, the differences in the predictive power of residual DSBs between CRISPR-modified and other subgroups suggest that genetic alterations in other pathways, such as proliferation and metabolism, may have a greater impact on cellular radiation response. These pathways are often neglected in response modelling and should be considered in the future.

Molecular Influence of the ATM Protein in the Treatment of Human Cells with Different Radioprotective Drugs: Comparisons between Antioxidative and Pro-Episkevic Strategies

The radiation protection strategy with chemical agents has long been based on an antioxidative approach consisting in reducing the number of radical oxygen and nitrogen species responsible for the formation of the radiation-induced (RI) DNA damage, notably the DNA double-strand breaks (DSB), whose subset participates in the RI lethal effect as unrepairable damage. Conversely, a DSB repair-stimulating strategy that may be called the "pro-episkevic" approach (from the ancient Greek episkeve, meaning repair) can be proposed. The pro-episkevic approach directly derives from a mechanistic model based on the RI nucleoshuttling of the ATM protein (RIANS) and contributes to increase the number of DSB managed by NHEJ, the most predominant DSB repair and signaling pathway in mammalians. Here, three radioresistant and three radiosensitive human fibroblast cell lines were pretreated with antioxidative agents (N-acetylcysteine or amifostine) or to two pro-episkevic agents (zoledronate or pravastatin or both (ZOPRA)) before X-ray irradiation. The fate of the RI DSB was analyzed by using γH2AX and pATM immunofluorescence. While amifostine pretreatment appeared to be the most efficient antioxidative process, ZOPRA shows the most powerful radiation protection, suggesting that the pro-episkevic strategy may be an alternative to the antioxidative one. Additional investigations are needed to develop some new drugs that may elicit both antioxidative and pro-episkevic properties and to quantify the radiation protection action of both types of drugs applied concomitantly.

Insights into the characteristics of primary radioresistant cervical cancer using single-cell transcriptomics

Radioresistance is a major cause of radiotherapy failure among patients with cervical cancer (CC), the fourth most common cause of cancer mortality in women worldwide. Traditional CC cell lines lose intra-tumoral heterogeneity, posing a challenge for radioresistance research. Meanwhile, conditional reprogramming (CR) maintains intra-tumoral heterogeneity and complexity, as well as the genomic and clinical characteristics of original cells and tissues. Three radioresistant and two radiosensitive primary CC cell lines were developed under CR conditions from patient specimens, and their characteristics were verified via immunofluorescence, growth kinetics, clone forming assay, xenografting, and immunohistochemistry. The CR cell lines had homogenous characteristics with original tumor tissues and maintained radiosensitivity in vitro and in vivo, while also maintaining intra-tumoral heterogeneity according to single-cell RNA sequencing analysis. Upon further investigation, 20.83% of cells in radioresistant CR cell lines aggregated in the G2/M cell cycle phase, which is sensitive to radiation, compared to 38.1% of cells in radiosensitive CR cell lines. This study established three radioresistant and two radiosensitive CC cell lines through CR, which will benefit further research investigating radiosensitivity in CC. Our present study may provide an ideal model for research on development of radioresistance and potential therapeutic targets in CC.

EXPRESSION OF H/ACA snoRNA IN CELL LINES WITH CHROMOSOMAL ABNORMALITIES AFTER IRRADIATION

The H/ACA snoRNA family is involved in pseudouridine biogenesis. It prevents genetic changes in cells and makes them more stable due to ribosomal RNA characteristics. Therefore, the study of H/ACA snoRNA expression in cell lines with chromosomal disorders after irradiation is of particular interest.&#x0D; The purpose of the study is to analyze the effect of chromosomal disorders on H/ACA snoRNA expression in radioresistant K562 and radiosensitive HL-60 cell lines after radiation exposure.&#x0D; Materials and Methods. K562 and HL-60 cell lines were exposed to radiation (4 Gy). H/ACA snoRNA expression was analyzed by NGS sequencing (1, 4, and 24 hours after irradiation).&#x0D; Results. The authors revealed differences in H/ACA snoRNA expression by chromosomes in the studied cell lines, as well as the impact of chromosomal abnormalities on H/ACA snoRNA expression after radiation exposure. Changes in the copy number of normal chromosomes lead to minor changes in H/ACA snoRNA expression. Marker chromosomes disrupt H/ACA snoRNA expression. Thus, is becomes impossible to use H/ACA snoRNAs located in abnormal chromosomes as radioresistance markers. Moreover, marker chromosomes decrease the number of H/ACA snoRNAs expressed in K562, despite the greater amount of genetic material.

Alternating Radiation Fractionations Overcome Radioresistance in Patient-Derived Glioblastoma Cells

<h3>Purpose/Objective(s)</h3> Historically, radiotherapy fractions for Glioblastoma (GBM) are delivered in equal doses over a given number of days, though this regimen has not changed in the past century and does not accommodate for the development of radioresistance. Using mathematical oncological simulations, we propose that ramping up dose can overcome radioresistance in patient-derived xenograft (PDX) GBM cells more sufficiently than the same dose delivered in equal fractions. <h3>Materials/Methods</h3> We generated a radiation-sensitive (JX14P) and -resistant (JX14P-RT) PDX GBM cell line by implanting primary tumors into the flank of athymic nude mice. To achieve radioresistance, we treated these mice with 6 fractions of 2 Gy of radiation over 14 days. In silico simulations utilizing the radiosensitivity parameters based on the linear-quadratic model and JX14P and JX14P-RT doubling time, suggested a total regimen of 16 Gy, with an average of 4 Gy/fx, delivered in fractions of 1 Gy, 3 Gy, 5 Gy, or 7 Gy (Ramp-Up, "RU") would result in a smaller surviving fraction than the equivalent total dose of 4 Gy, 4 Gy, 4 Gy, 4 Gy (Equal Dosing, "ED") in JX14P and JX14PRT. The equivalent opposite regimen of 7 Gy, 5 Gy, 3 Gy, and 1 Gy (Ramp-Down, "RD") was also investigated. 5000 cells were plated in DMEM-F12 media with B27 supplement and 20 ng/ml EGF and FGF and irradiated every three days. Viability was determined using CellTiterGlo, nine days after the final dose of radiation. This experiment was done in triplicates. Statistical analysis of viability between the different regimens was assessed using a two-way ANOVA with a multiple comparisons Tukey test. <h3>Results</h3> Our preliminary results indicate that delivering a total dose of 16 Gy, alternating radiation fractionations every three days of 1 Gy, 3 Gy, 5 Gy, or 7 Gy, enhances cytotoxicity in both radiation-sensitive and radiation-resistant PDX models when compared to no treatment and fractions of equal dose (4 Gy) delivered in the same manner (Cell line vs luminescence, <i>P</i><0.0001). <h3>Conclusion</h3> This data suggests RU and RD dosing is superior to ED in the radiosensitive cell lines ED (<i>P</i><0.0001). However, in the radioresistant cell lines, only RU was superior to ED (<i>P</i><0.0001). We are also exploring the effects of this novel treatment regimen in our radiation-sensitive and -resistant PDX GBM cell lines when co-cultured with different starting proportions. This is the first known experiment where two regimens with the same average dose per fraction and total dose results in vastly different cytotoxicity.

Disruption of mitochondrial function augments the radiosensitivity of prostate cancer cell lines.

Ionizing radiation is one of the most widely used therapeutic methods in the treatment of prostate cancer, but the problem is developing radioresistance of the tumour. There is evidence that metabolic reprogramming in cancer is one of the major contributors to radioresistance and mitochondria play a crucial role in this process. The aim of the study was to assess the influence of oxidative phosphorylation uncoupling to radiosensitivity of prostate cancer cells differing in metabolic phenotype. LNCaP, PC-3 and DU-145 cells were exposed to X-rays and simultaneously treated with 2,4-dinitrophenol (2,4-DNP). The radiosensitive of cell lines was determined by cell clonogenic assay and cell cycle analysis. The cytotoxic effect was evaluated with MTT and CVS (Crystal violet staining) assay, apoptosis detection and cell cycle analysis. The phenotype of the cells was determined by glucose uptake and lactate release, ATP level measurement as well as basal reactive oxygen species level and mRNA expression of genes related to oxidative stress defence. The synergistic effect of 2,4-dinitrophenol and X-ray was observed only in the case of the LNCaP cell line. Phenotypic analysis indicates that this may be due to the highest dependence of these cells on oxidative phosphorylation and sensitivity to disruption of their redox status.

The effect of chromosome abnormalities on expression of SnoRNA in radioresistant and radiosensitive cell lines after irradiation.

In this paper, we have studied the role of chromosomal abnormalities in the expression of small nucleolar RNAs (snoRNAs) of radioresistant (K562) and radiosensitive (HL-60) leukemia cell line. Cells were exposed to an X-ray dose of 4 Gy. SnoRNA expression was investigated using NGS sequencing. The distribution of expressed snoRNAs on chromosomes has been found to be different for two cell lines. The most significant differences in the expression of snoRNAs were found in the K562 cell line based on the analysis of the dynamics of log2fc values. The type of clustering, the number and type of snoRNAs slightly differed in the chromosomes with trisomy and monosomy and had a pronounced difference in pairs with marker chromosomes in both cell lines. In this study, we have demonstrated that chromosomal abnormalities alter the expression of snoRNA after irradiation. Trisomies and monosomies do not have such a noticeable effect on the expression of snoRNAs as the presence of marker chromosomes.

COX4-1 promotes mitochondrial supercomplex assembly and limits reactive oxide species production in radioresistant GBM

Glioblastoma (GBM) is a fatal disease with recurrences often associated with radioresistance. Although often effective at treating newly diagnosed GBM, increasing evidence suggests that radiotherapy-induced alterations in tumor metabolism promote GBM recurrence and aggressiveness. Using isogenic radiosensitive and radioresistant GBM cell lines and patient-derived xenolines, we found that acquired radioresistance is associated with a shift from a glycolytic metabolism to a more oxidative metabolism marked by a substantial increase in the activity of the mitochondrial respiratory chain complex cytochrome c oxidase (CcO). This elevated CcO activity was associated with a switch in the isoform expression of the CcO regulatory subunit COX4, from COX4-2 to COX4-1, assembly of CcO-containing mitochondrial supercomplexes (SCs), and reduced superoxide (O2•-) production. Overexpression of COX4-1 in the radiosensitive cells was sufficient to promote the switch from glycolytic to oxidative metabolism and the incorporation of CcO into SCs, with a concomitant reduction in O2•- production. Conversely, silencing of COX4-1 expression in normally radioresistant cells reduced CcO activity, promoted the disassembly of mitochondrial SCs, and increased O2•- production. Additionally, gain or loss of COX4-1 expression was sufficient to induce the radioresistant or radiosensitive phenotype, respectively. Our results demonstrate that COX4-1 promotes SC assembly in GBM cells, and SC assembly may in turn regulate the production of reactive oxygen species and thus the acquisition of radioresistance in GBM.

Uncertainties Associated with Clonogenic Assays using a Cs-137 Irradiator and Ir-192 Afterloader: A Comprehensive Compilation for Radiation Researchers.

Clonogenic assays are the gold standard for measuring cell clonogenic survival and enable quantification of a cell line's radiosensitivity through the calculation of the surviving fraction, the ratio of cell clusters (colonies) formed after radiation exposure compared to the number formed without exposure. Such studies regularly utilize Cs-137 irradiators. While uncertainties for specific procedural aspects have been described previously, a comprehensive review has not been completed. We therefore quantified uncertainties associated with clonogenic assays performed using a Cs-137 Shepherd irradiator, and a recently established brachytherapy afterloader in vitro radiation delivery apparatus (BAIRDA), through a series of experiments and a literature review. The clonogenic assay is subject to uncertainties that affect the determination of the surviving fraction (e.g., accuracy of the number of cells seeded, potential effects of hypothermia, and the threshold number of cells for a cluster to be identified as a colony). Furthermore, dose delivery uncertainties related to both the Cs-137 irradiator and BAIRDA were also quantified. The combined standard (k = 1) uncertainty was ± 6.0% in the surviving fraction for the Cs-137 irradiator (±6.3% for BAIRDA), up to ± 2.2% in the dose delivered by the Cs-137 irradiator, and up to ± 4.3% in the dose delivered by BAIRDA. The largest individual uncertainties were associated with the number of cells seeded on a plate (3.4%) and inter-observer variability in counting (4.1%), suggesting that effective reduction of uncertainties in the conduct of the clonogenic assay may provide the greatest relief on the uncertainty budget. Finally, measurable impact on experimental findings was assessed by applying this uncertainty to clonogenic assays of SW756 cells using either a Cs-137 irradiator or BAIRDA, introducing a maximum shift in the reported radiobiological parameters α/β and T1/2 of 0.3 Gy and 0.4 h, respectively, while the 95% confidence interval increased by 0.5 Gy and decreased by 0.4 h, respectively. Though the overall impact on radiobiological parameter estimation was small, the individual uncertainties could have a significant influence in other applications of in vitro experiments in radiation biology. Hence, better understanding of the uncertainties associated with both clonogenic assays and the radiation source used can improve the accuracy of experimental analysis and reproducibility of the results.

Radiation-induced DNA damage and altered expression of p21, cyclin D1 and Mre11 genes in human fibroblast cell lines with different radiosensitivity.

Radiotherapy plays a pivotal role in the treatment of cancer. One of the main challenges in this treatment modality is radiation-induced complications in some patients affected by high radiosensitivity (RS). The differences in RS are determined mainly by genetic factors. Therefore, identifying the genes and mechanisms that affect RS in different cells is essential for evaluating radiotherapy outcomes. In the present study, the ability to repair DNA double-stranded breaks (DSB) is evaluated, followed by examining the expression levels of CDKN1A (p21), cyclinD1, and Mre11 genes in human fibroblasts with different RSs. Cellular RS was measured by survival fraction at 2 Gy (SF2). The γ-H2AX assay was used for assessing DNA repair capacity. Eventually, gene expression levels from each cell line 4 and 24 h after irradiation (at 2, 4, and 8 Gy) were measured by real-time PCR. The SF2 values for the cell lines ranged from 0.286 to 0.641, and RS differences of fibroblast cells were identified. Among the studied genes, the expression of Mre11 was the most important. Analysis of the real-time PCR data showed that changes in Mre11 gene expression (4 h after 8 Gy irradiation) were directly correlated with the RS (R2 = 0.905). The difference in the expression of the p21 gene (4 h after 4 Gy irradiation) was also promising. Finally, the flow cytometry analysis showed that the radioresistant cell lines quickly repaired DBS damages. However, the repair process was slow in the radiosensitive cell line, and the residual damage is significantly higher than other cell lines (P < 0.01). This study indicates that changes in the expression of p21 and Mre11 genes play an important role in cell response to radiation and thus these genes can be introduced as biomarkers to predict RS in normal cell lines.

Gemcitabine-Based Chemoradiotherapy Enhanced by a PARP Inhibitor in Pancreatic Cancer Cell Lines.

Pancreatic ductal adenocarcinoma is a devastating disease with a 5-year overall survival of 9% for all stages. Gemcitabine-based chemoradiotherapy for locally advanced pancreatic cancer is highly toxic. We conducted an in vitro study to determine whether poly(ADP-ribose) polymerase-1 inhibition radiosensitized gemcitabine-based chemotherapy. Human pancreatic cancer cell lines, MIA PaCa-2, AsPC-1, BxPC-3 and PANC-1 were treated with gemcitabine (10 nM) and/or olaparib (1 µM). Low-LET gamma single dose of 2, 5 and 10 Gy radiations were carried out. Clonogenic assay, PAR immunoblotting, cell cycle distribution, γH2Ax, necrotic and autophagic cell death quantifications were performed. Treatment with olaparib alone was not cytotoxic, but highly radiosensitized cell lines, particularly at high dose per fraction A non-cytotoxic concentration of gemcitabine radiosensitized cells, but less than olaparib. Interestingly, olaparib significantly enhanced gemcitabine-based radiosensitization in PDAC cell lines with synergistic effect in BxPC-3 cell line. All cell lines were radiosensitized by the combination of gemcitabine and olaparib, through an increase of unrepaired double-strand, a G2 phase block and cell death. Radiosensitization was increased with high dose of radiation. The combination of olaparib with gemcitabine-based chemoradiotherapy could lead to an enhancement of local control in vivo and an improvement in disease-free survival.

ALG3 contributes to stemness and radioresistance through regulating glycosylation of TGF-\u03b2 receptor II in breast cancer

BackgroundRadiotherapy is a conventional and effective local treatment for breast cancer. However, residual or recurrent tumors appears frequently because of radioresistance. Novel predictive marker and the potential therapeutic targets of breast cancer radioresistance needs to be investigated.MethodsIn this study, we screened all 10 asparagine-linked glycosylation (ALG) members in breast cancer patients’ samples by RT-PCR. Cell viability after irradiation (IR) was determined by CCK-8 assay and flow cytometry. The radiosensitivity of cell lines with different ALG3 expression was determined with the colony formation assay by fitting the multi-target single hit model to the surviving fractions. Cancer stem-like traits were assessed by RT-PCR, Western blot, and flow cytometry. The mechanisms of ALG3 influencing radiosensitivity was detected by Western blot and immunoprecipitation. And the effect of ALG3 on tumor growth after IR was verified in an orthotopic xenograft tumor models. The association of ALG3 with prognosis of breast cancer patients was confirmed by immunohistochemistry.ResultsALG3 was the most significantly overexpressing gene among ALG family in radioresistant breast cancer tissue. Overexpression of ALG3 predicted poor clinicopathological characteristics and overall survival (OS), and early local recurrence-free survival (LRFS) in breast cancer patients. Upregulating ALG3 enhanced radioresistance and cancer stemness in vitro and in vivo. Conversely, silencing ALG3 increased the radiosensitivity and repressed cancer stemness in vitro, and more importantly inhibition of ALG3 effectively increased the radiosensitivity of breast cancer cells in vivo. Mechanistically, our results further revealed ALG3 promoted radioresistance and cancer stemness by inducing glycosylation of TGF-β receptor II (TGFBR2). Importantly, both attenuation of glycosylation using tunicamycin and inhibition of TGFBR2 using LY2109761 differentially abrogated the stimulatory effect of ALG3 overexpression on cancer stemness and radioresistance. Finally, our findings showed that radiation played an important role in preventing early recurrence in breast cancer patients with low ALG3 levels, but it had limited efficacy in ALG3-overexpressing breast cancer patients.ConclusionOur results suggest that ALG3 may serve as a potential radiosensitive marker, and an effective target to decrease radioresistance by regulating glycosylation of TGFBR2 in breast cancer. For patients with low ALG3 levels, radiation remains an effective mainstay therapy to prevent early recurrence in breast cancer.

Oncolytic virotherapy promotes radiosensitivity in soft tissue sarcoma by suppressing anti-apoptotic MCL1 expression.

Soft tissue sarcoma (STS) is a rare cancer that develops from soft tissues in any part of the body. Despite major advances in the treatment of STS, patients are often refractory to conventional radiotherapy, leading to poor prognosis. Enhancement of sensitivity to radiotherapy would therefore improve the clinical outcome of STS patients. We previously revealed that the tumor-specific, replication-competent oncolytic adenovirus OBP-301 kills human sarcoma cells. In this study, we investigated the radiosensitizing effect of OBP-301 in human STS cells. The in vitro antitumor effect of OBP-301 and ionizing radiation in monotherapy or combination therapy was assessed using highly radiosensitive (RD-ES and SK-ES-1) and moderately radiosensitive (HT1080 and NMS-2) STS cell lines. The expression of markers for apoptosis and DNA damage were evaluated in STS cells after treatment. The therapeutic potential of combination therapy was further analyzed using SK-ES-1 and HT1080 cells in subcutaneous xenograft tumor models. The combination of OBP-301 and ionizing radiation showed a synergistic antitumor effect in all human STS cell lines tested, including those that show different radiosensitivity. OBP-301 was found to enhance irradiation-induced apoptosis and DNA damage via suppression of anti-apoptotic myeloid cell leukemia 1 (MCL1), which was expressed at higher levels in moderately radiosensitive cell lines. The combination of OBP-301 and ionizing radiation showed a more profound antitumor effect compared to monotherapy in SK-ES-1 (highly radiosensitive) and HT1080 (moderately radiosensitive) subcutaneous xenograft tumors. OBP-301 is a promising antitumor reagent to improve the therapeutic potential of radiotherapy by increasing radiation-induced apoptosis in STS.

Impact of DNA repair and reactive oxygen species levels on radioresistance in pancreatic cancer

PurposeRadioresistance in pancreatic cancer patients remains a critical obstacle to overcome. Understanding the molecular mechanisms underlying radioresistance may achieve better response to radiotherapy and thereby improving the poor treatment outcome. The aim of the present study was to elucidate the mechanisms leading to radioresistance by detailed characterization of isogenic radioresistant and radiosensitive cell lines. MethodsThe human pancreatic cancer cell lines, Panc-1 and MIA PaCa-2 were repeatedly exposed to radiation to generate radioresistant (RR) isogenic cell lines. The surviving cells were expanded, and their radiosensitivity was measured using colony formation assay. Tumor growth delay after irradiation was determined in a mouse pancreatic cancer xenograft model. Gene and protein expression were analyzed using RNA sequencing and Western blot, respectively. Cell cycle distribution and apoptosis (Caspase 3/7) were measured by FACS analysis. Reactive oxygen species generation and DNA damage were analyzed by detection of CM-H2DCFDA and γH2AX staining, respectively. Transwell chamber assays were used to investigate cell migration and invasion. ResultsThe acquired radioresistance of RR cell lines was demonstrated in vitro and validated in vivo. Ingenuity pathway analysis of RNA sequencing data predicted activation of cell viability in both RR cell lines. RR cancer cell lines demonstrated greater DNA repair efficiency and lower basal and radiation-induced reactive oxygen species levels. Migration and invasion were differentially affected in RR cell lines. ConclusionsOur data indicate that repeated exposure to irradiation increases the expression of genes involved in cell viability and thereby leads to radioresistance. Mechanistically, increased DNA repair capacity and reduced oxidative stress might contribute to the radioresistant phenotype.