Irradiated HeLa Cells Research Articles

Trinuclear ruthenium(II) polypyridyl complexes: Evaluation as photosensitizers for enhanced cervical cancer treatment

Trinuclear ruthenium(II) polypyridyl complexes anchored to benzimidazole-triazine / trisamine scaffolds were investigated as photosensitizers for photodynamic therapy. The trinuclear complexes were noted to produce a significant amount of singlet oxygen in both DMF and aqueous media, are photostable and show appreciable emission quantum yields (ɸem). In our experimental setting, despite the moderate phototoxic activity in the HeLa cervical cancer cell line, the phototoxic indices (PI) of the trinuclear complexes are superior relative to the PIs of a clinically approved photosensitizer, Photofrin®, and the pro-drug 5-aminolevulinic acid (PI: >7 relative to PI: >1 and PI: 4.4 for 5-aminolevulinic acid and Photofrin®, respectively). Furthermore, the ruthenium complexes were noted to show appreciable long-term cytotoxicity upon light irradiation in HeLa cells in a concentration-dependent manner. Consequently, this long-term activity of the ruthenium(II) polypyridyl complexes embodies their ability to reduce the probability of the recurrence of cervical cancer. Taken together, this presents a strong motivation for the development of polymetallic complexes as anticancer agents.

Effect of chemically modified natural porphyrins and copper and silver complexes derived from them on oxidative stress induced by paraquat and radiation

In the present work, chlorophyll [Formula: see text] and deuteroporphyrin IX derivatives and copper (II) and silver (II) porphyrinates based on them were investigated for their ability to prevent oxidative stress in human HeLa cells exposed to paraquat (PQ) using fluorometric microculture cytotoxicity assay (FMCA). Among the compounds studied Ag-methylpyropheophorbide [Formula: see text] and Cu-methylpyropheophorbide [Formula: see text] contributed to a statistically significant increase in the survival of PQ-exposed cells. Additionally, ROS levels in HeLa cells were analyzed by flow cytometry using 2′,7′-dichlorofluorescein diacetate and Ag-methylpyropheophorbide [Formula: see text] and Cu-methylpyropheophorbide [Formula: see text] were shown to arrest PQ-mediated oxidative stress. Furthermore, these two compounds were tested for radioprotective activity in the experiment with irradiation of HeLa cells at doses of 3 Gy and 6 Gy. According to the FMCA results, the addition of Ag-methylpyropheophorbide [Formula: see text] and Cu-methylpyropheophorbide [Formula: see text] to the cell culture had no increase in cell survival compared to the positive control, demonstrating the absence of radioprotective effect of these compounds. It can be assumed that the difference in the effects of these porphyrin derivatives on cell survival is related to different localization of oxidative stress targets in cells exposed to PQ or ionizing radiation.

Copper Nanocluster Enables Simultaneous Photodynamic and Chemo Therapy for Effective Cancer Diagnosis and Treatment in Vitro.

Metal-nanocluster-mediated cancer diagnosis and therapy has drawn considerable attention in recent years due to the unique optical and photophysical properties of metal clusters. This type of material is highly useful for the diagnosis, treatment, and further follow-up of disease. However, a single treatment modality is not sufficient for a complete cure. The use of a multi-therapeutic strategy is among the most promising methods for effective treatment, along with an early-stage diagnosis. To address the multiple therapeutic modalities in a single nanomaterial, a copper nanocluster was synthesized using glutathione, having inherent singlet oxygen generation and emission at 674 nm. A tumor-targeting agent (folic acid) and an anticancer drug (doxorubicin) was conjugated to the copper cluster for cancer diagnosis via targeted imaging and further double therapy (photodynamic and chemotherapy) in vitro. 10.5 μg (18.1 nmol) of drug conjugated copper cluster shows 56 % cell death for 30 second laser irradiation in HeLa cells. Effective cancer cell imaging and therapeutic efficacy are demonstrated in vitro.

Photon Upconversion Hydrogels for 3D Optogenetics

AbstractThe ability to optically induce biological responses in 3D has been dwarfed by the physical limitations of visible light penetration to trigger photochemical processes. However, many biological systems are relatively transparent to low‐energy light, which does not provide sufficient energy to induce photochemistry in 3D. To overcome this challenge, hydrogels that are capable of converting red or near‐IR (NIR) light into blue light within the cell‐laden 3D scaffolds are developed. The upconverted light can then excite optically active proteins in cells to trigger a photochemical response. The hydrogels operate by triplet–triplet annihilation upconversion. As proof‐of‐principle, it is found that the hydrogels trigger an optogenetic response by red/NIR irradiation of HeLa cells that have been engineered to express the blue‐light sensitive protein Cry2olig. While it is remarkable to photoinduce the clustering of Cry2olig with blanket NIR irradiation in 3D, it is also demonstrated how the hydrogels trigger clustering within a single cell with great specificity and spatiotemporal control. In principle, these hydrogels may allow for photochemical control of cell function within 3D scaffolds, which can lead to a wealth of fundamental studies and biochemical applications.

Organic–Inorganic Hybrid Nanoparticles Synthesized with Hypericum perforatum Extract: Potential Agents for Photodynamic Therapy at Ultra-low Power Light

A progressive way in photodynamic therapy is to design photosensitive agents that may operate at lower optical power. Hybrid organic–inorganic silver-, iron-, and silver&iron-contained nanoparticles (NPs) were synthesized using H. perforatum extract and explored as photosensitizers for anticancer and antimicrobial photodynamic therapies at ultra-low power light (2.30 mW/cm2). Hybrid silver- and silver&iron-contained NPs revealed the most promising results for anticancer photodynamic therapy: at a concentration of 25 μg/mL, the difference between viability of non-radiated and irradiated HeLa cells was up to 71%. Importantly, HeLa cancer cells are more assailable for hybrid NP activity than the normal fibroblast MSU-1.1 cell line, which was observed at both “dark” cytotoxicity and DCFH-DA assays. Oxidative stress induced by hybrid iron- and silver&iron-contained NPs in cancer HeLa cells is higher (≥1.7 times) than that in normal fibroblast MSU-1.1 cells. For antimicrobial photodynamic therapy, hybrid silver&iron-contained NPs (at a concentration of 25 μg/mL) were found to be promising photosensitizers being bacteriostatic against E. coli and S. aureus in the “dark” condition and bactericidal after light irradiation. Antimicrobial properties of hybrid silver&iron-contained NPs are the result of synergy of antimicrobial activities of H. perforatum and silver. The highest efficiency against bacteria is achieved at concentrations and conditions at which H. perforatum and silver NPs apart were less or not active. Furthermore, hybrid silver&iron-contained NPs combine photodynamic activities against cancer and bacteria cells with the ability to enhance magnetic resonance imaging (MRI) contrast. This combination makes these NPs promising materials for biomedical application at ultra-low power photodynamic therapy of near-surface cancer or inflammatory disorders, with the possibility of their visualization using MRI.

Titanium Dioxide Nanoparticles Induced HeLa Cell Necrosis under UVA Radiation through the ROS-mPTP Pathway.

Titanium dioxide nanoparticles (nano-TiO2), as a common nanomaterial, are widely used in water purification, paint, skincare and sunscreens. Its safety has always been a concern. Prior studies have shown that ultraviolet A (UVA) can exacerbate the toxicity of nano-TiO2, including inducing cell apoptosis, changing glycosylation levels, arresting cell cycle, inhibiting tumor cell and bacterial growth. However, whether the combination of UVA and nano-TiO2 cause cell necrosis and its mechanism are still rarely reported. In this study, we investigated the cytotoxicity and phototoxicity of mixture crystalline nano-TiO2 (25% rutile and 75% anatase, 21 nm) under UVA irradiation in HeLa cells. Our results showed that the abnormal membrane integrity and the ultrastructure of HeLa cells, together with the decreased viability induced by nano-TiO2 under UVA irradiation, were due to cell necrosis rather than caspase-dependent apoptosis. Furthermore, nano-TiO2 and UVA generated the reactive oxygen species (ROS) and caused the mitochondrial permeability transition pore (mPTP) of HeLa cells to abnormally open. Cell viability was significantly increased after adding vitamin C (VC) or cyclosporin A (CsA) individually to inhibit ROS and mPTP. Clearance of ROS could not only impede the opening of mPTP but also reduce the rate of cell necrosis. The results suggest the possible mechanism of HeLa cell necrosis caused by nano-TiO2 under UVA irradiation through the ROS-mPTP pathway.

Inhibition of Uncoupling Protein 2 Enhances the Radiosensitivity of Cervical Cancer Cells by Promoting the Production of Reactive Oxygen Species.

Objective The mechanism of enhanced radiosensitivity induced by mitochondrial uncoupling protein UCP2 was investigated in HeLa cells to provide a theoretical basis as a novel target for cervical cancer treatment. Methods HeLa cells were irradiated with 4 Gy X-radiation at 1.0 Gy/min. The expression of UCP2 mRNA and protein was assayed by real-time quantitative polymerase chain reaction and western blotting. UCP2 siRNA and negative control siRNA fragments were constructed and transfected into HeLa cells 24 h after irradiation. The effect of UCP2 silencing and irradiation on HeLa cells was determined by colony formation, CCK-8 cell viability, γH2AX immunofluorescence assay of DNA damage, Annexin V-FITC/PI apoptosis assay, and propidium iodide cell cycle assay. The effects on mitochondrial structure and function were investigated with fluorescent probes including dichlorodihydrofluorescein diacetate (DCFH-DA) assay of reactive oxygen species (ROS), rhodamine 123, and MitoTracker Green assay of mitochondrial structure and function. Results Irradiation upregulated UCP2 expression, and UCP2 knockdown decreased the survival of irradiated HeLa cells. UCP2 silencing sensitized HeLa cells to irradiation-induced DNA damage and led to increased apoptosis, cell cycle arrest in G2/M, and increased mitochondrial ROS. Increased radiosensitivity was associated with an activation of P53, decreased Bcl-2, Bcl-xl, cyclin B, CDC2, Ku70, and Rad51 expression, and increased Apaf-1, cytochrome c, caspase-3, and caspase-9 expression. Conclusions UCP2 inhibition augmented the radiosensitivity of cervical cancer cells, and it may be a potential target of radiotherapy of advanced cervical cancer.

The Role of MiR-5094 as a Proliferation Suppressor during Cellular Radiation Response via Downregulating STAT5b.

MicroRNAs (miRNAs) play important roles in the regulation of cellular stress responses. We previously uncovered 10 novel human miRNAs which are induced by X-ray irradiation in HeLa cells using Solexa deep sequencing. The most highly expressed new miRNA, miR-5094, was predicted to target STAT5b. This study wonders whether miR-5094 participates in cellular radiation response via STAT5b. Firstly, direct interaction between miRNA-5094 and the STAT5b 3'-UTR was confirmed by luciferase reporter assay. Then, the radiation responsive expression of miR-5094 and STAT5b were measured in HeLa and Jurkat cells, and the expressions of down-stream genes of STAT5b after ionizing radiation (IR) were detected in HeLa cells. At last, the effects of miR-5094 on survival fraction, cell proliferation, cell cycle arrest and apoptosis induced by IR were investigated in HeLa cells, Jurkat cells and human peripheral blood T cells. It was found that up-regulation of miR-5094 by radiation induction or miRNA mimic transfection suppressed expression of STAT5b, and consequently decreased the transcription of down-stream Igf-1 and Bcl-2. Additionally, over expression of miR-5094 resulted in proliferation suppression and knockdown of miR-5094 by miRNA inhibitor after irradiation partially reversed the proliferation suppression induced by miR-5094 in HeLa cells, Jurkat cells and CD4+ T cells. Collectively, our findings demonstrate that up-regulation of miR-5094 down-regulated the expression of STAT5b, thereby suppressing cell proliferation after X-ray irradiation.

Determination of Endogenous Radioresistance Biomarkers on HeLa Cells

The Genes such as carbonic anhydrase IX (CAIX), hypoxia inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) have been suggested as hypoxic biomarkers in cancer. Indeed, these endogenous biomarkers have been shown to have stronger prognostic value response after treatment by irradiation. However, inconsistent results suggest that factors other than oxygen influence their expression. This present study deciphers the level of expression of different radioresistance biomarkers in both normoxia and hypoxia conditions followed by irradiation of human ovarian tumor cell lines (uterine cervix squamous cell carcinoma (HeLa). HeLa cells were submitted to hypoxia (1% O2) conditions in a Thermo Scientific Heracell i CO2 incubator. The cells were subjected to two doses 4-10 Gy irradiation and re-incubate in their starting conditions for 4 hours, then fixed in 4% paraformaldehyde for 20 min. Protein expressions were assessed by immunocytochemistry staining and fluorescent images were captured by a Axio Imager Z1 fluorescence microscope with oil immersion lens at 63× magnification. In normoxia conditions there was no modification of the level of expression of the CAIX after irradiation. However, an increasing expression level of VEGF was noted. The level of expression of HIF-1 in normoxia was low compared to the other two proteins (CAIX and VEGF). Hypoxia conditions at 2% resulted in a low expression of CAIX and VEGF before and after irradiation at 10 Gy in HeLa cells. HIF-1 had a maximum expression level compared to CAIX and VEGF at 2% oxygen after irradiation in HeLa cells. As tumor hypoxia occurs in a deprived microenvironment, other environmental factors such as irradiation might interact with the effect of low oxygen concentration on gene expression. This study shows that irradiation of HeLa cells has a profound influence on the oxygen dependent induction of certain endogenous hypoxic markers as HIF-1, CAIX, and VEGF.

HLA-DM Mediates Permissiveness of HLA-DPB1 T Cell Epitope Mismatches in Unrelated HCT

Introduction: In 8/8 matched unrelated donor (UD) hematopoietic cell transplantation (HCT), permissive HLA-DPB1 (DP) mismatches within the same functional T Cell Epitope (TCE) group are associated with better outcomes compared to non-permissive mismatches across different TCE groups (Fleischhauer, Blood 2017). This clinical advantage has been shown to be associated with limited in vitro T cell alloreactivity (Meurer, Front Immunol 2019), which in turn is dependent on polymorphic peptide contact amino acids in the DP molecule (Crivello, Biol Blood Marrow Transplant 2015). The HLA class II immunopeptidome is shaped by the peptide editor HLA-DM (DM), and its natural antagonist HLA-DO (DO). Here we investigated the effect of DM/DO activity on the DP immunopeptidome, the breadth of the overall alloresponse to and immunogenicity of permissive and non-permissive DP mismatches, in healthy individuals and in patients after UD-HCT. Methods: HeLa cells expressing single DP alleles in the presence or absence of DM, or in the presence of DM and DO (Rutten, BBMT 2008), were generated for HLA-DPB1*04:02 (DP4) and *10:01 (DP10) as prototypes for 2 distinct TCE groups. The DP immunopeptidomes were analyzed by mass spectrometry. Alloresponses against DP were quantified by CD137 up-regulation assays after co-culture of irradiated HeLa cells with CD4+ responder T cells from 14 healthy blood donors permissive to DP4 and non-permissive to DP10, or from 2 patients referring to the University Hospital Essen, Germany, the latter alive and well >9 months after 8/8 matched UD-HCT with a permissive DP4 or a non-permissive DP10 mismatch, respectively. The breadth of the responding T cell receptor beta (TCRb) repertoire was determined by immunosequencing (Adaptive Biotechnologies, Seattle, USA). The study was performed under informed consent according to the declaration of Helsinki. Results: Reflecting their association with different TCE groups, DP4 and DP10 presented peptidomes with limited (<4%) overlap and different peptide motifs. These features were not changed by the presence or absence of DM. In contrast, the presence of DM resulted in a significant (>50%) shrinking of the peptide repertoire displayed by the same DP antigen in the absence of DM, with approximately 30% peptides shared by the same allele in the two conditions, both for DP4 and for DP10 (Figure 1A). In the presence of DM, the magnitude of the T cell alloresponse to non-permissive DP10 was significantly higher than to permissive DP4, both in healthy individuals (40.7% vs 16.3%, respectively, p<0.0001) and in the informative transplanted patients (Figure 1B). Neither the absence of DM (40.7% vs 45.3%, p=ns) nor the presence of DM with DO (71.6% vs 77.4%, p=ns) altered the magnitude of the non-permissive alloresponse to DP10. Compellingly, both the absence of DM (16.3% vs 39.0%, p<0.001) and the co-expression of DM and DO (21.6% vs 59.5%, p<0.001) significantly increased the response to permissive DP4, again both in healthy individuals and in the informative transplanted patients. The strength of the overall alloresponse was associated with the breadth of the corresponding TCRb repertoire, with significantly higher diversity (1-clonality) in response to non-permissive DP10 (mean 0.68) compared to permissive DP4 (mean 0.48) in the presence of DM, and similar high diversity against both DP antigens in its absence (mean 0.74 vs 0.75 against DP4 and DP10, respectively) in healthy individuals. In the transplanted patients, the permissive alloresponse to DP4 was dominated by a single TCRb that could be retrieved at high frequency also in ex-vivo follow-up samples from the same patient from day +195 and +363, while the non-permissive alloresponse to DP10 was polyclonal (mean 0.62 and 0.61 in the presence and absence of DM, respectively) (Figure 1C). Conclusion: Permissiveness of HLA-DPB1 TCE mismatches is dependent on the peptide editing by DM, and converted into non-permissiveness in its absence or in the presence of its antagonist DO. Permissiveness is associated with the immunopeptidomes of mismatched HLA-DP alloantigens on the MHC side, and with TCRb diversity on the alloreactive T cell side, both in healthy individuals and in patients after UD-HCT. These new mechanistic insights suggest that expression of DM and DO by leukemia or healthy tissues might modulate graft-versus-leukemia and graft-versus-host disease after permissively DP mismatched UD HCT. Disclosures No relevant conflicts of interest to declare.

When Three Isn't a Crowd: A Digyny Concept for Treatment-Resistant, Near-Triploid Human Cancers.

Near-triploid human tumors are frequently resistant to radio/chemotherapy through mechanisms that are unclear. We recently reported a tight association of male tumor triploidy with XXY karyotypes based on a meta-analysis of 15 tumor cohorts extracted from the Mitelman database. Here we provide a conceptual framework of the digyny-like origin of this karyotype based on the germline features of malignant tumors and adaptive capacity of digyny, which supports survival in adverse conditions. Studying how the recombinatorial reproduction via diploidy can be executed in primary cancer samples and HeLa cells after DNA damage, we report the first evidence that diploid and triploid cell sub-populations constitutively coexist and inter-change genomes via endoreduplicated polyploid cells generated through genotoxic challenge. We show that irradiated triploid HeLa cells can enter tripolar mitosis producing three diploid sub-subnuclei by segregation and pairwise fusions of whole genomes. Considering the upregulation of meiotic genes in tumors, we propose that the reconstructed diploid sub-cells can initiate pseudo-meiosis producing two “gametes” (diploid “maternal” and haploid “paternal”) followed by digynic-like reconstitution of a triploid stemline that returns to mitotic cycling. This process ensures tumor survival and growth by (1) DNA repair and genetic variation, (2) protection against recessive lethal mutations using the third genome.

A Porphyrin Dimer-GdDOTA Conjugate as a Theranostic Agent for One- and Two-Photon Photodynamic Therapy and MRI.

A molecular theranostic agent designed for photodynamic therapy (PDT) treatment in the near-infrared and for imaging tissue tumors with magnetic resonance imaging (MRI) is reported. It consists of a linear π-conjugated Zn(II) porphyrin dimer linked at each extremity to a GdDOTA-type complex. This agent has shown very promising potential for PDT applications with good singlet oxygen generation in DMSO and high linear absorption in the near-infrared (λmax = 746 nm, ε ≈ 105 M-1 cm-1). Moreover, this molecule has a propensity for two-photon excited PDT with high two-photon cross sections (∼8000 GM in 880-930 nm range), which should allow for deeper tumor treatments and higher spatial precision as compared to conventional one-photon PDT. Regarding the MRI contrast agent properties, the molecule has shown superior relaxivity (14.4 mM-1 s-1 at 40 MHz, 298 K) in comparison to clinical contrast agents and the ability to be internalized in cells, thanks to its amphiphilic character. Irradiation of HeLa cells using either one-photon (740 nm) or two-photon excitation (910 nm) has led in both cases to important cell death.

Photodynamic Therapy in HeLa Cells Incubated with Riboflavin and Pectin‐coated Silver Nanoparticles

Riboflavin (Rf) is an endogenous photosensitizer, which can participate in Type I and Type II processes. We have recently shown that the yield of the triplet excited states of Rf is enhanced in the presence of pectin-coated silver nanoparticles (Pec@AgNP) due to formation of a complex between Rf and Pec@AgNP (Rf-Pec@AgNP). Consequently, under aerobic conditions, the amounts of singlet molecular oxygen and superoxide radical anion generated are also larger in the presence of the nanoparticles. This result made us suspect that the nanoparticles could have a beneficial effect in Rf-based PDT. To prove this hypothesis, we here compared the photodamage in HeLa cells incubated with Rf in the presence and in the absence of Pec@AgNP applying several optical assays. We used fluorescence imaging of irradiated HeLa cells incubated with Annexin V and propidium iodide to evaluate the occurrence of apoptosis/necrosis, the reduction of the tetrazolium dye MTT to formazan and neutral red uptake to prove cell viability, as well as synchrotron infrared microscopy of single cells to evaluate possible structural changes of DNA and nuclear proteins. The enhanced photodamage observed in the presence of Pec@AgNP seems to indicate that Rf enters into the cells complexed with the nanoparticles.

Abstract 4414: Radiation-induced miR-5094 expression inhibits cell proliferation by targeting STAT5b

Abstract MicroRNAs (miRNAs) are highly conserved small non-coding RNAs that alter protein expression and regulate multiple intracellular processes including cellular stress response. We previously uncovered several novel miRNAs, whose expressions are induced by X-rays irradiation in HeLa cells, using a miRNA array. Here, we examined the function of miR-5094 during cellular radiation response. With software predictions and confirmatory experiments, we identified the signal transduction and activator of transcription 5b (STAT5b), a known regulator of proliferation and survival, as a target of miR-5094. The study also showed that both miR-5094 induction by X-rays irradiation and miR-5094 mimics transfection decreased STAT5b expression, down-regulated STAT5b downstream genes, and consequently, reduced the cell survival and cell viability. Knockdown of miR-5094 by miRNA inhibitor after radiation partially reversed these suppressive effects. As STAT5b plays an important role in lymphocyte proliferation, we further confirmed the proliferation inhibitory effects of miR-5094/STAT5b pathway on Jurkat cells and human peripheral blood CD4+ T cells during cell radiation response. In conclusion, our study provided the first evidence that radiation induces upregulation of miR-5094, which in turn inhibits cell proliferation via targeting STAT5b. Citation Format: Nan Ding, Junrui Hua, Jinpeng He, Wenjun Wei, Hailong Pei, Guangming Zhou, Jufang Wang, Altaf A. Wani, Qianzheng Zhu. Radiation-induced miR-5094 expression inhibits cell proliferation by targeting STAT5b [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4414.

Comprehensive circular RNA expression profile in radiation-treated HeLa cells and analysis of radioresistance-related circRNAs.

BackgroundCervical cancer is one of the most common cancers in women worldwide. Malignant tumors develop resistance mechanisms and are less sensitive to or do not respond to irradiation. With the development of high-throughput sequencing technologies, circular RNA (circRNA) has been identified in an increasing number of diseases, especially cancers. It has been reported that circRNA can compete with microRNAs (miRNAs) to change the stability or translation of target RNAs, thus regulating gene expression at the transcriptional level. However, the role of circRNAs in cervical cancer and the radioresistance mechanisms of HeLa cells are unknown. The objective of this study is to investigate the role of circRNAs in radioresistance in HeLa cells.MethodsHigh-throughput sequencing and bioinformatics analysis of irradiated and sham-irradiated HeLa cells. The reliability of high-throughput RNA sequencing was validated using quantitative real-time polymerase chain reaction. The most significant circRNA functions and pathways were selected by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. A circRNA–miRNA–target gene interaction network was used to find circRNAs associated with radioresistance. Moreover, a protein–protein interaction network was constructed to identify radioresistance-related hub proteins.ResultsHigh-throughput sequencing allowed the identification of 16,893 circRNAs involved in the response of HeLa cells to radiation. Compared with the control group, there were 153 differentially expressed circRNAs, of which 76 were up-regulated and 77 were down-regulated. GO covered three domains: biological process (BP), cellular component (CC) and molecular function (MF). The terms assigned to the BP domain were peptidyl-tyrosine dephosphorylation and regulation of cell migration. The identified CC terms were cell–cell adherens junction, nucleoplasm and cytosol, and the identified MF terms were protein binding and protein tyrosine phosphatase activity. The top five KEGG pathways were MAPK signaling pathway, endocytosis, axon guidance, neurotrophin signaling pathway, and SNARE interactions in vesicular transport. The protein–protein interaction analysis indicated that 19 proteins might be hub proteins.ConclusionsCircRNAs may play a major role in the response to radiation. These findings may improve our understanding of the role of circRNAs in radioresistance in HeLa cells and allow the development of novel therapeutic approaches.

Induction of autophagy and interleukin 6 secretion in bystander cells: metabolic cooperation for radiation-induced rescue effect?

We hypothesized that radiation-induced rescue effect (RIRE) shared similar mechanisms with ‘metabolic cooperation’, in which nutrient-deprived cancer cells prompted normal cells to provide nutrients. Our data demonstrated that X-ray irradiation induced autophagy in HeLa cells, which could last at least 18 h, and proved that the irradiated cells (IRCs) resorted to breaking down their own intracellular components to supply the molecules required for cell-repair enhancement (e.g. to activate the NF-κB pathway) in the absence of support from bystander unirradiated cells (UICs). Furthermore, autophagy accumulation in IRCs was significantly reduced when they were partnered with UICs, and more so with UICs with pre-induced autophagy before partnering (through starvation using Earle’s Balanced Salt Solution), which showed that the autophagy induced in UICs supported the IRCs. Our results also showed that interleukin 6 (IL-6) was secreted by bystander UICs, particularly the UICs with pre-induced autophagy, when they were cultured in the medium having previously conditioned irradiated HeLa cells. It was established that autophagy could activate the signal transducer and activator of transcription 3 (STAT3) that was required for the IL-6 production in the autophagy process. Taken together, the metabolic cooperation of RIRE was likely initiated by the bystander factors released from IRCs, which induced autophagy and activated STAT3 to produce IL-6 in bystander UICs, and was finally manifested in the activation of the NF-κB pathway in IRCs by the IL-6 secreted by the UICs.

Inhibition of EGFR nuclear shuttling decreases irradiation resistance in HeLa cells.

Cervical cancer is a leading cause of mortality in women worldwide. The resistance to irradiation at the advanced stage is the main reason for the poor prognosis and high mortality. This work aims to elucidate the molecular mechanism underlying the radio-resistance. In this study, we determined the pEGFR-T654 and pDNA-PK-T2609 expression level changes in irradiated HeLa cells treated with T654 peptide, a nuclear localization signal (NLS) inhibitor, to inhibit EGFR nuclear transport. Cell viability, cell cycle and migratory capacity were analyzed. Xenograft animal model was used to evaluate the effect of EGFR nuclear transport inhibition on the tumor growth in vivo. The enhanced translocation of nuclear EGFR in the irradiated HeLa cells correlated with the increasing level of pEGFR-T654 and pDNA-PK-T2609. Inhibition of EGFR nuclear translocation by NLS peptide inhibitor attenuated DNA damage repair in the irradiated HeLa cells, decreased cell viability and promoted cell death through arrest at G0 phase. NLS peptide inhibitor impaired the migratory capacity of irradiated HeLa cells, and negatively affected tumorigenesis in xenograft mice. This work puts forward a potential molecular mechanism of the irradiation resistance in cervical cancer cells, providing a promising direction towards an efficient therapy of cervical cancer.

DNA damage response induces structural alterations in histone H3–H4

Synchrotron-radiation circular-dichroism spectroscopy was used to reveal that the DNA damage response induces a decrement of α-helix and an increment of β-strand contents of histone H3–H4 extracted from X-ray–irradiated human HeLa cells. The trend of the structural alteration was qualitatively opposite to that of our previously reported results for histone H2A–H2B. These results strongly suggest that histones share roles in DNA damage responses, particularly in DNA repair processes and chromatin remodeling, via a specific structural alteration of each histone.

Dynamically-enhanced retention of gold nanoclusters in HeLa cells following X-rays exposure: A cell cycle phase-dependent targeting approach

Background and purposeCell cycle phase could affect the cellular uptake of nanoparticles. Based on the fact that ionizing radiation exposure can delay cell cycle progression including inducing G2/M phase arrest, we propose that ionizing radiation exposure is a cell cycle phase-dependent targeting approach for intracellular delivery of nano-agents in tumor cells. Materials and methodsWe synthesized luminescent gold nanoclusters (AuNCs) using a one-pot green synthetic method. Subsequently, we used the as-prepared AuNCs as both “nano-agents” and fluorescent trafficking probes for our study using human cervical carcinoma HeLa cells. Estimating the cellular uptake of AuNCs and cell cycle analysis were performed following X-rays irradiation and cell synchronization. ResultsOur work showed that X-rays irradiation could delay the division of HeLa cells and thereby enhance the retention of AuNCs in HeLa cells, which is a reverse strategy compared with other studies on synergistic nano-radiotherapy. Our results demonstrated that the cell cycle synchronization influenced the cellular uptake processes of AuNCs, suggesting that dynamic cell cycle progression could affect the cellular uptake kinetics of AuNCs. ConclusionWe consider that the radiation-induced cell division delay might provide a possible mechanism underlying the enhanced effect for the cellular uptake of AuNCs in irradiated HeLa cells.

Comparison of the Radiosensitizing Effect of ATR, ATM and DNA-PK Kinase Inhibitors on Cervical Carcinoma Cells

Here, we compared the effects of inhibitors of three phosphatidylinositol-3-kinase-related kinases, ATM, ATR a DNA-PK, on radiosensitization of cervical carcinoma cells. We demonstrated that DNA-PK inhibitor NU7441 enhanced phosphorylation of Chk1 and Chk2 kinases 2 h after irradiation of HeLa cells at a dose of 8 Gy in contrast to ATM kinase inhibitor KU55933, which completely blocked the Chk2 kinase phosphorylation on threonine 68, and ATR kinase inhibitor VE-821, which blocked the Chk1 kinase phosphorylation on serine 345. Most HeLa cells were accumulated in G2 phase of the cell cycle 24 h after irradiation at a high dose of 15 Gy, which was even potentiated after adding the inhibitors NU7441 and KU55933. Compared to all other irradiated groups, inhibitor VE-821 increased the number of cells in S phase and reduced the number of cells in G2 phase 24 h after irradiation at the high dose of 15 Gy. HeLa cells entered the mitotic cycle with unrepaired DNA, which resulted in cell death and the radiosensitizing effect of VE-821. Short-term application of the inhibitors (2 h before and 30 min after the irradiation by the dose of 8 Gy) significantly decreased the colony-forming ability of HeLa cells. Using real-time monitoring of cell proliferation by the xCELLigence system we demonstrated that while the radiosensitizing effect of VE-821 (ATR inhibitor) is manifested early after the irradiation, the radiosensitizing effect of KU55933 (ATM inhibitor) and NU7441 (DNA-PK inhibitor) is only observed as late as 72 h after the irradiation.