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1.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810266

RESUMO

The conformational variation of the viral capsid structure plays an essential role both for the environmental resistance and acid nuclear release during cellular infection. The aim of this study was to evaluate how capsid rearrangement in engineered phages of M13 protects viral DNA and peptide bonds from damage induced by UV-C radiation. From in silico 3D modelling analysis, two M13 engineered phage clones, namely P9b and 12III1, were chosen for (i) chemical features of amino acids sequences, (ii) rearrangements in the secondary structure of their pVIII proteins and (iii) in turn the interactions involved in phage capsid. Then, their resistance to UV-C radiation and hydrogen peroxide (H2O2) was compared to M13 wild-type vector (pC89) without peptide insert. Results showed that both the phage clones acquired an advantage against direct radiation damage, due to a reorganization of interactions in the capsid for an increase of H-bond and steric interactions. However, only P9b had an increase in resistance against H2O2. These results could help to understand the molecular mechanisms involved in the stability of new virus variants, also providing quick and necessary information to develop effective protocols in the virus inactivation for human activities, such as safety foods and animal-derived materials.


Assuntos
Bacteriófago M13/efeitos da radiação , Proteínas do Capsídeo/química , Tolerância a Radiação , Raios Ultravioleta , Bacteriófago M13/química , Bacteriófago M13/efeitos dos fármacos , Farmacorresistência Viral , Peróxido de Hidrogênio/toxicidade , Domínios Proteicos
2.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806361

RESUMO

Oral cancers constitute the majority of head and neck tumors, with a relatively high incidence and poor survival rate in developing countries. While the five-year survival rates of the oral cancer patients have increased to 65%, the overall survival for advanced stages has been at 27% for the past ten years, emphasizing the necessity for further understanding the etiology of the disease, diagnosis, and formulating possible novel treatment regimens. MicroRNAs (miRNAs), a family of small non-coding RNA, have emerged as master modulators of gene expression in various cellular and biological process. Aberrant expression of these dynamic molecules has been associated with many human diseases, including oral cancers. The deregulated miRNAs have been shown to control various oncogenic processes, including sustaining proliferative signaling, evading growth suppressors, resisting cell death activating invasion and metastasis, and inducing angiogenesis. Hence, the aberrant expression of miRNAs associated with oral cancers, makes them potential candidates for the investigation of functional markers, which will aid in the differential diagnosis, prognosis, and development of novel therapeutic regimens. This review presents a holistic insight into our understanding of the role of miRNAs in regulating various hallmarks of oral tumorigenesis.


Assuntos
MicroRNAs/genética , Neoplasias Bucais/etiologia , Neoplasias Bucais/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/etiologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Apoptose/genética , Biomarcadores Tumorais/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/biossíntese , Neoplasias Bucais/patologia , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Oncogenes , Tolerância a Radiação/genética , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia
3.
World J Surg Oncol ; 19(1): 131, 2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33882945

RESUMO

BACKGROUND: Radiotherapy is a main therapeutic method for cancers, including colon cancer. In the current study, we aim to explore the effects of circular RNA (circRNA) circ_0055625 in the progression and radiosensitivity of colon cancer and the underlying mechanism. METHODS: The expression of circ_0055625 and musashi homolog 1 (MSI1) mRNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR). MSI1 protein expression was determined by Western blot. Cell proliferation was assessed by cell counting kit-8 (CCK-8) and colony formation assays. Cell survival fraction, apoptosis, and invasion were investigated by colony formation assay, flow cytometry analysis, and transwell invasion assay, respectively. Cell migration was detected by wound-healing and transwell migration assays. The binding relationship between microRNA-338-3p (miR-338-3p) and circ_0055625 or MSI1 was predicted by online databases and identified by Dual-Luciferase Reporter Assay. The effects of circ_0055625 silencing on the tumor formation and radiosensitivity of colon cancer in vivo were explored by in vivo tumor formation assay. RESULTS: Circ_0055625 and MSI1 were upregulated in colon cancer tissues and cells relative to control groups. Radiation treatment apparently increased the expression of circ_0055625 and MSI1 in colon cancer cells. Circ_0055625 knockdown or MSI1 silencing repressed cell proliferation, migration, and invasion and promoted cell apoptosis and radiosensitivity in colon cancer. Also, circ_0055625 silencing-mediated effects were attenuated by MSI1 overexpression. Additionally, circ_0055625 silencing reduced MSI1 expression, which could be attenuated by miR-338-3p inhibitor. Mechanically, circ_0055625 acted as a sponge for miR-338-3p to regulate MSI1. Furthermore, circ_0055625 knockdown hindered tumor growth and improved radiosensitivity in vivo. CONCLUSION: Circ_0055625 repression inhibited the progression and radioresistance of colon cancer by downregulating MSI1 through sponging miR-338-3p. This result might provide a theoretical basis for improving the therapy of colon cancer with radiation.


Assuntos
Neoplasias do Colo , MicroRNAs/genética , Proteínas do Tecido Nervoso/genética , RNA Circular/genética , Proteínas de Ligação a RNA/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/radioterapia , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Inativação de Genes , Inativação Gênica , Humanos , Proteínas do Tecido Nervoso/biossíntese , Prognóstico , Proteínas de Ligação a RNA/biossíntese , Tolerância a Radiação/genética , Tolerância a Radiação/efeitos da radiação , Transfecção
4.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33804169

RESUMO

Glioblastoma multiforme (GBM) is a malignant primary brain tumor with poor patient prognosis. Although the standard treatment of GBM is surgery followed by chemotherapy and radiotherapy, often a small portion of surviving tumor cells acquire therapeutic resistance and become more aggressive. Recently, altered kinase expression and activity have been shown to determine metabolic flux in tumor cells and metabolic reprogramming has emerged as a tumor progression regulatory mechanism. Here we investigated novel kinase-mediated metabolic alterations that lead to acquired GBM radioresistance and malignancy. We utilized transcriptomic analyses within a radioresistant GBM orthotopic xenograft mouse model that overexpresses the dual specificity tyrosine-phosphorylation-regulated kinase 3 (DYRK3). We find that within GBM cells, radiation exposure induces DYRK3 expression and DYRK3 regulates mammalian target of rapamycin complex 1 (mTORC1) activity through phosphorylation of proline-rich AKT1 substrate 1 (PRAS40). We also find that DYRK3 knockdown inhibits dynamin-related protein 1 (DRP1)-mediated mitochondrial fission, leading to increased oxidative phosphorylation (OXPHOS) and reduced glycolysis. Importantly, enforced DYRK3 downregulation following irradiation significantly impaired GBM cell migration and invasion. Collectively, we suggest DYRK3 suppression may be a novel strategy for preventing GBM malignancy through regulating mitochondrial metabolism.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Dinaminas/genética , Glioblastoma/radioterapia , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Camundongos , Mitocôndrias/genética , Mitocôndrias/patologia , Mitocôndrias/efeitos da radiação , Fosforilação Oxidativa/efeitos da radiação , Proteínas Proto-Oncogênicas c-akt/genética , Tolerância a Radiação/genética , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 46(2): 113-120, 2021 Feb 28.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-33678646

RESUMO

OBJECTIVES: Radiotherapy is one of the main therapies for colorectal cancer, but radioresistance often leads to radiotherapy failure. To improve the radioresistance, we explore the effect of oligomycin A, the H+-ATP synthase inhibitor, on the sensitivity of HT29 colorectal cancer cells to irradiation and its underlying mechanisms. METHODS: The effects of different concentrations of oligomycin A on the survival rate and glycolysis of HT29 colorectal cancer cells at different time points were investigated via MTT and glycolysis assay. siRNA-PFK1 was synthesized in vitro and transfected into HT29 cells. The effects of oligomycin A on radiosensitivity of HT29 colorectal cancer cells were measured via MTT and colony formation assay. Western blotting was used to detect the effect of oligomycin A on the expression of glycolytic enzyme PFK1. We compared difference between the effects of siRNA-PFK1 group and oligomycin A combined with siRNA-PFK1 group on cell survival and glycolysis. After 4 Gy X-ray irradiation, the effects of cell survival and glycolysis between the siRNA-PFK1 group and the oligomycin A combined with siRNA-PFK1 group were compared. RESULTS: Compared with the 0 µmol/L oligomycin A group, the cell survival rate of HT29 cells treated with 4 µmol/L oligomycin A was significantly increased (P<0.05), and the glucose uptake, the lactic acid, and the ATP production were also significantly increased (all P<0.01). After X-ray irradiation at different doses (0, 2, 4, 6, and 8 Gy), the colony formation rate and cell survival rate of the 4 µmol/L oligomycin A treated group were significantly higher than those in the 0 µmol/L oligomycin A group (both P<0.01). The sensitization enhancement ratio of oligomycin A on HT29 colorectal cancer cells was 0.4886. The expression of PFK1 in the 4 µmol/L oligomycin A group was significantly higher than that in the 0 µmol/L oligomycin A group (P<0.001). The glycolysis level, colony formation rate, and cell survival rate of the siRNA-PFK1 HT29 cells group were significantly lower than those in the 0 µmol/L oligomycin A group (all P<0.05), while the results in the 4 µmol/L oligomycin A combined with siRNA-PFK1 group were significantly higher than those in the siRNA-PFK1 group (all P<0.001). After 4 Gy X-ray irradiation, the colony formation rate and cell survival rate in the siRNA-PFK1 group were decreased compared with those in the irradiation group (P<0.01 or P<0.001), while the results of the 4 µmol/L oligomycin A combined with siRNA-PFK1 group were significantly higher than those in the siRNA-PFK1 group (both P<0.001). CONCLUSIONS: Oligomycin A can promote the radioresistance of HT29 colorectal cancer cells, which may be related to up-regulation of the PFK1 expression and increase of cell glycolysis.


Assuntos
Neoplasias Colorretais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Células HT29 , Humanos , Oligomicinas/farmacologia , Tolerância a Radiação
6.
Adv Clin Exp Med ; 30(2): 173-181, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33650331

RESUMO

BACKGROUND: Radioresistance is a huge obstacle in radiotherapy of non-small cell lung cancer (NSCLC) and how to raise radiosensitivity is an urgent issue. OBJECTIVES: In this study, we investigated the role and molecular mechanism of sodium new houttuyfonate (SNH) in regulation of radiosensitivity of NSCLC cells. MATERIAL AND METHODS: The Cell Counting Kit-8 (CCK-8) was used to measure cell viabilities of NSCLC cell lines A549 and HCC827 after a treatment with SNH (0 mM, 0.1 mM and 0.3 mM). It examined cytotoxicity induced by X-ray (0 Gy, 1 Gy, 2 Gy, 4 Gy, 6 Gy, and 8 Gy) after SNH (0.3 mM) treatment, while flow cytometry was used for apoptosis detection use. Expression of miR-147a or signal transducer and activator of transcription (STAT3) in selected cell lines was assessed through real-time quantitative polymerase chain reaction (RT-qPCR). The CCK-8 was then applied to measure cytotoxicity in cells with miR-147a upregulation or STAT3 suppression under irradiation apoptosis changes were detected with flow cytometry. Thereafter, binding conditions between miR-147a and STAT3 were checked using luciferase reporter assays. Expressions of STAT3 in A549 transfected by siNC, siSTAT3, and by siSTAT3 and miR-147a mimics were checked using RT-qPCR and the phosphorylation of STAT3 was observed using enzyme-linked immunosorbent assay (ELISA). RESULTS: The SNH treatment significantly suppressed cell viabilities and increased apoptosis of lung cancer cells. Cytotoxicity and apoptosis in A549 cells were both enhanced after SNH treatment and raised as the dosages of X-ray grew. MiR-147a presented lower expression in lung cancer cells and was upregulated by SNH, which further contributed to higher cell apoptosis after irradiation. STAT3 directly bound miR-147a and was more expressed in NSCLC cells. Inhibited phosphorylation of STAT3 promoted apoptosis in A549 cells after X-ray exposure. Overexpressed miR-147a inactivated STAT3 signaling, adding to cell apoptosis after irradiation. CONCLUSIONS: SNH-induced miR-147a increased radiosensitivity of A549 cells through inactivation of STAT3 pathway.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , MicroRNAs , Apoptose , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/radioterapia , MicroRNAs/genética , Tolerância a Radiação , Fator de Transcrição STAT3 , Ácidos Sulfônicos
7.
Int J Mol Sci ; 22(5)2021 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-33673647

RESUMO

Small RNAs are essential to coordinate many cellular processes, including the regulation of gene expression patterns, the prevention of genomic instability, and the suppression of the mutagenic transposon activity. These processes determine the aging, longevity, and sensitivity of cells and an organism to stress factors (particularly, ionizing radiation). The biogenesis and activity of small RNAs are provided by proteins of the Argonaute family. These proteins participate in the processing of small RNA precursors and the formation of an RNA-induced silencing complex. However, the role of Argonaute proteins in regulating lifespan and radioresistance remains poorly explored. We studied the effect of knockdown of Argonaute genes (AGO1, AGO2, AGO3, piwi) in various tissues on the Drosophila melanogaster lifespan and survival after the γ-irradiation at a dose of 700 Gy. In most cases, these parameters are reduced or did not change significantly in flies with tissue-specific RNA interference. Surprisingly, piwi knockdown in both the fat body and the nervous system causes a lifespan increase. But changes in radioresistance depend on the tissue in which the gene was knocked out. In addition, analysis of changes in retrotransposon levels and expression of stress response genes allow us to determine associated molecular mechanisms.


Assuntos
Proteínas Argonauta/antagonistas & inibidores , Proteínas de Drosophila/antagonistas & inibidores , Drosophila melanogaster/crescimento & desenvolvimento , Longevidade/genética , RNA Interferente Pequeno/genética , Tolerância a Radiação/genética , Animais , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/efeitos da radiação , Feminino , Raios gama , Masculino , Especificidade de Órgãos , Interferência de RNA
8.
Int J Mol Sci ; 22(5)2021 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-33673439

RESUMO

Ionizing radiation (IR) is used for patients diagnosed with unresectable non-small cell lung cancer (NSCLC). However, radiotherapy remains largely palliative due to the survival of specific cell subpopulations. In the present study, the sublines of NSCLC cells, A549IR (p53wt) and H1299IR (p53null) survived multifraction X-ray radiation exposure (MFR) at a total dose of 60 Gy were investigated three weeks after the MFR course. We compared radiosensitivity (colony formation), expression of epithelial-mesenchymal transition (EMT) markers, migration activity, autophagy, and HR-dependent DNA double-strand break (DSB) repair in the bulk and entire CD44high/CD166high CSC-like populations of both parental and MFR survived NSCLC cells. We demonstrated that the p53 status affected: the pattern of expression of N-cadherin, E-cadherin, Vimentin, witnessing the appearance of EMT-like phenotype of MFR-surviving sublines; 1D confined migratory behavior (wound healing); the capability of an irradiated cell to continue to divide and form a colony of NSCLC cells before and after MFR; influencing the CD44/CD166 expression level in MFR-surviving NSCLC cells after additional single irradiation. Our data further emphasize the impact of p53 status on the decay of γH2AX foci and the associated efficacy of the DSB repair in NSCLC cells survived after MFR. We revealed that Rad51 protein might play a principal role in MFR-surviving of p53 null NSCLC cells promoting DNA DSB repair by homologous recombination (HR) pathway. The proportion of Rad51 + cells elevated in CD44high/CD166high population in MFR-surviving p53wt and p53null sublines and their parental cells. The p53wt ensures DNA-PK-mediated DSB repair for both parental and MFR-surviving cells irrespectively of a subsequent additional single irradiation. Whereas in the absence of p53, a dose-dependent increase of DNA-PK-mediated non-homologous end joining (NHEJ) occurred as an early post-irradiation response is more intensive in the CSC-like population MFR-surviving H1299IR, compared to their parental H1299 cells. Our study strictly observed a significantly higher content of LC3 + cells in the CD44high/CD166high populations of p53wt MFR-surviving cells, which enriched the CSC-like cells in contrast to their p53null counterparts. The additional 2 Gy and 5 Gy X-ray exposure leads to the dose-dependent increase in the proportion of LC3 + cells in CD44high/CD166high population of both parental p53wt and p53null, but not MFR-surviving NSCLC sublines. Our data indicated that autophagy is not necessarily associated with CSC-like cells' radiosensitivity, emphasizing that careful assessment of other milestone processes (such as senescence and autophagy-p53-Zeb1 axis) of primary radiation responses may provide new potential targets modulated for therapeutic benefit through radiosensitizing cancer cells while rescuing normal tissue. Our findings also shed light on the intricate crosstalk between autophagy and the p53-related EMT, by which MFR-surviving cells might obtain an invasive phenotype and metastatic potential.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/radioterapia , Quebras de DNA de Cadeia Dupla , Neoplasias Pulmonares/radioterapia , Tolerância a Radiação , Reparo de DNA por Recombinação , Proteína Supressora de Tumor p53/metabolismo , Células A549 , Autofagia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Linhagem Celular Tumoral , Movimento Celular , DNA/metabolismo , Reparo do DNA por Junção de Extremidades , Transição Epitelial-Mesenquimal , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/fisiopatologia , Rad51 Recombinase/metabolismo , Raios X
9.
Int J Nanomedicine ; 16: 2071-2085, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33727814

RESUMO

Background: Radiation therapy remains an important treatment modality in cancer therapy, however, resistance is a major problem for treatment failure. Elevated expression of glutathione is known to associate with radiation resistance. We used glutathione overexpressing small cell lung cancer cell lines, SR3A-13 and SR3A-14, established by transfection with γ-glutamylcysteine synthetase (γ-GCS) cDNA, as a model for investigating strategies of overcoming radiation resistance. These radiation-resistant cells exhibit upregulated human copper transporter 1 (hCtr1), which also transports cisplatin. This study was initiated to investigate the effect and the underlying mechanism of iron-platinum nanoparticles (FePt NPs) on radiation sensitization in cancer cells. Materials and Methods: Uptakes of FePt NPs in these cells were studied by plasma optical emission spectrometry and transmission electron microscopy. Effects of the combination of FePt NPs and ionizing radiation were investigated by colony formation assay and animal experiment. Intracellular reactive oxygen species (ROS) were assessed by using fluorescent probes and imaged by a fluorescence-activated-cell-sorting caliber flow cytometer. Oxygen consumption rate (OCR) in mitochondria after FePt NP and IR treatment was investigated by a Seahorse XF24 cell energy metabolism analyzer. Results: These hCtr1-overexpressing cells exhibited elevated resistance to IR and the resistance could be overcome by FePt NPs via enhanced uptake of FePt NPs. Overexpression of hCtr1 was responsible for the increased uptake/transport of FePt NPs as demonstrated by using hCtr1-transfected parental SR3A (SR3A-hCtr1-WT) cells. Increased ROS and drastic mitochondrial damages with substantial reduction of oxygen consumption rate were observed in FePt NPs and IR-treated cells, indicating that structural and functional insults of mitochondria are the lethal mechanism of FePt NPs. Furthermore, FePt NPs also increased the efficacy of radiotherapy in mice bearing SR3A-hCtr1-WT-xenograft tumors. Conclusion: These results suggest that FePt NPs can potentially be a novel strategy to improve radiotherapeutic efficacy in hCtr1-overexpressing cancer cells via enhanced uptake and mitochondria targeting.


Assuntos
Ligas/farmacologia , Transportador de Cobre 1/metabolismo , Ferro/farmacologia , Nanopartículas Metálicas/química , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Platina/farmacologia , Tolerância a Radiação , Aerobiose , Animais , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Nanopartículas Metálicas/ultraestrutura , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Modelos Biológicos , Tolerância a Radiação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Resultado do Tratamento , Raios X
10.
Methods Mol Biol ; 2269: 37-47, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33687670

RESUMO

Ionizing radiation is a critical component of glioblastoma (GBM) therapy. Recent data have implicated glioblastoma stem-like cells (GSCs) as determinants of GBM development, maintenance, and treatment response. Understanding the response of GSCs to radiation should thus provide insight into the development of improved GBM treatment strategies. Towards this end, in vitro techniques for the analysis of GSC radiosensitivity are an essential starting point. One such method, the clonogenic survival assay has been adapted to assessing the intrinsic radiosensitivity of GSCs and is described here. As an alternative method, the limiting dilution assay is presented for defining the radiosensitivity of GSC lines that do not form colonies or only grow as neurospheres. In addition to these cellular strategies, we describe γH2AX foci analysis, which provides a surrogate marker for radiosensitivity at the molecular level. Taken together, the in vitro methods presented here provide tools for defining intrinsic radiosensitivity of GSCs and for testing agents that may enhance GBM radioresponse.


Assuntos
Biomarcadores Tumorais , Loci Gênicos , Glioblastoma , Histonas , Proteínas de Neoplasias , Células-Tronco Neoplásicas , Tolerância a Radiação , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/radioterapia , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
11.
J Vis Exp ; (169)2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33779614

RESUMO

Radiation dosimetry is critical in the accurate delivery and reproducibility of radiation schemes in preclinical models for high translational relevance. Prior to performing any in vitro or in vivo experiments, the specific dose output for the irradiator and individual experimental designs must be assessed. Using an ionization chamber, electrometer, and solid water setup, the dose output of wide fields at isocenter can be determined. Using a similar setup with radiochromic films in the place of the ionization chamber, dose rates for smaller fields at different depths can also be determined. In vitro clonogenic survival assays of cancer cells in response to radiation treatment are inexpensive experiments that provide a measure of inherent radio-sensitivity of cell lines by fitting these data with the traditional linear-quadratic model. Model parameters estimated from these assays, combined with the principles of biologic effective doses, allows one to develop varying fractionation schedules for radiation treatment that provide equivalent effective doses in tumor-bearing animal experiments. This is an important factor to consider and correct for in comparing in vivo radiation therapy schedules to eliminate potential confounding of results due to variance in the delivered effective doses. Taken together, this article provides a general method for dose output verification preclinical animal and cabinet irradiators, in vitro assessment of radio-sensitivity, and verification of radiation delivery in small living organisms.


Assuntos
Neoplasias da Mama/radioterapia , Radiometria/instrumentação , Animais , Neoplasias da Mama/patologia , Proliferação de Células , Fracionamento da Dose de Radiação , Feminino , Humanos , Modelos Lineares , Camundongos , Tolerância a Radiação , Radiometria/métodos , Eficiência Biológica Relativa , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
12.
J Cancer Res Clin Oncol ; 147(5): 1275-1286, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33687564

RESUMO

Esophageal squamous cell carcinoma (ESCC) is one of most lethal malignancies with high aggressive potential in the world. Radiotherapy is used as one curative treatment modality for ESCC patients. Due to radioresistance, the 5-year survival rates of patients after radiotherapy is less than 20%. Tumor radioresistance is very complex and heterogeneous. Cancer-associated fibroblasts (CAFs), as one major component of tumor microenvironment (TME), play critical roles in regulating tumor radioresponse through multiple mechanisms and are increasingly considered as important anti-cancer targets. Cancer stemness, which renders cancer cells to be extremely resistant to conventional therapies, is involved in ESCC radioresistance due to the activation of Wnt/ß-catenin, Notch, Hedgehog and Hippo (HH) pathways, or the induction of epithelial-mesenchymal transition (EMT), hypoxia and autophagy. Non-protein-coding RNAs (ncRNAs), which account for more than 90% of the genome, are involved in esophageal cancer initiation and progression through regulating the activation or inactivation of downstream signaling pathways and the expressions of target genes. Herein, we mainly reviewed the role of CAFs, cancer stemness, non-coding RNAs as well as others in the development of radioresistance and clarify the involved mechanisms. Furthermore, we summarized the potential strategies which were reported to reverse radioresistance in ESCC. Together, this review gives a systematic coverage of radioresistance mechanisms and reversal strategies and contributes to better understanding of tumor radioresistance for the exploitation of novel intervention strategies in ESCC.


Assuntos
Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Tolerância a Radiação/fisiologia , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Transição Epitelial-Mesenquimal/fisiologia , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/metabolismo , Humanos , RNA não Traduzido/metabolismo , Transdução de Sinais/fisiologia
13.
Adv Clin Exp Med ; 30(1): 55-65, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33529508

RESUMO

BACKGROUND: The radiosensitivity of colon cancer cells can be regulated by noncoding RNAs. OBJECTIVES: In this study, the lncRNA antisense non-coding RNA in the INK4 locus (ANRIL) was selected to analyze its regulatory role in chitooligosaccharides (COS)-related radiosensitivity in colon cancer cells. MATERIAL AND METHODS: The ANRIL expression in colon cancer cell lines was examined using real-time quantitative polymerase chain reaction (RT-qPCR), based on which we selected the cell line that presented the highest expression of ANRIL for radiosensitivity research. The cells were exposed to X-rays (0 Gy, 2 Gy, 4 Gy, and 6 Gy) and evaluated for changes in ANRIL and miR-181a-5p expression using RT-qPCR. Cell viability was evaluated using the CCK8 method, while apoptosis was detected with flow cytometry assays. Dual luciferase assays validated the binding between ANRIL and miR-181a-5p. The cell survival rates after differential COS treatments (0 mg/mL, 1.0 mg/mL, 2.0 mg/mL, 3.0 mg/mL, 4.0 mg/mL, and 5.0 mg/mL) were rated using CCK8 assay. The cells with the strongest dosage of COS (5.0 mg/mL) were selected to further investigate the role of ANRIL/miR-181a-5p in modulating the radiosensitivity observed with CCK-8 and flow cytometry assays. RESULTS: The ANRIL was highly expressed in colon cancer cells lines, especially in the SW480 cell line. Irradiation significantly decreased cell viability and ANRIL expression in a dose-dependent manner. The overexpression of ANRIL reduced the cell apoptosis rate after irradiation. MiR-181a-5p directly bound to ANRIL and was upregulated by irradiation in a dose-dependent manner. The suppression of miR-181a-5p decreased cell apoptosis. The COS treatment notably downregulated cell survival and promoted apoptosis in cells exposed to irradiation. The overexpression of ANRIL partially reversed COS-induced apoptosis and the inhibition rate; the upregulation of miR-181a-5p could counteract the impact of ANRIL regulation in cells. CONCLUSIONS: The ANRIL negatively regulated radiosensitivity induced by COS in colon cancer cells by sponging miR-181a-5p.


Assuntos
Neoplasias do Colo , MicroRNAs/genética , RNA Longo não Codificante/genética , Linhagem Celular Tumoral , Quitina/análogos & derivados , Neoplasias do Colo/genética , Humanos , Tolerância a Radiação
14.
Nat Commun ; 12(1): 898, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33563973

RESUMO

Radiation sensitivity varies greatly between tissues. The transcription factor p53 mediates the response to radiation; however, the abundance of p53 protein does not correlate well with the extent of radiosensitivity across tissues. Given recent studies showing that the temporal dynamics of p53 influence the fate of cultured cells in response to irradiation, we set out to determine the dynamic behavior of p53 and its impact on radiation sensitivity in vivo. We find that radiosensitive tissues show prolonged p53 signaling after radiation, while more resistant tissues show transient p53 activation. Sustaining p53 using a small molecule (NMI801) that inhibits Mdm2, a negative regulator of p53, reduced viability in cell culture and suppressed tumor growth. Our work proposes a mechanism for the control of radiation sensitivity and suggests tools to alter the dynamics of p53 to enhance tumor clearance. Similar approaches can be used to enhance killing of cancer cells or reduce toxicity in normal tissues following genotoxic therapies.


Assuntos
Tolerância a Radiação , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Tolerância a Radiação/efeitos dos fármacos , Distribuição Tecidual/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Proteína Supressora de Tumor p53/efeitos da radiação , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Mutat Res ; 861-862: 503301, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33551102

RESUMO

Ataxia-telangiectasia (AT) is a rare inherited recessive disorder which is caused by a mutated Ataxia-telangiectasia mutated (ATM) gene. Hallmarks include chromosomal instability, cancer predisposition and increased sensitivity to ionizing radiation. The ATM protein plays an important role in signaling of DNA double-strand breaks (DSB), thereby phosphorylating the histone H2A.X. Non-functional ATM protein leads to defects in DNA damage response, unresolved DSBs and genomic instability. The aim of this study was to evaluate chromosomal aberrations and γH2A.X foci as potential radiation sensitivity biomarkers in AT patients. For this purpose, lymphocytes of 8 AT patients and 10 healthy controls were irradiated and induced DNA damage and DNA repair capacity were detected by the accumulation of γH2A.X foci. The results were heterogeneous among AT patients. Evaluation revealed 2 AT patients with similar γH2A.X foci numbers as controls after 1 h while 3 patients showed a lower induction. In regard to DNA repair, 3 of 5 AT patients showed poor damage repair. Therefore, DNA damage induction and DNA repair as detected by H2A.X phosphorylation revealed individual differences, seems to depend on the underlying individual mutation and thus appears not well suited as a biomarker for radiation sensitivity. In addition, chromosomal aberrations were analyzed by mFISH. An increased frequency of spontaneous chromosomal breakage was characteristic for AT cells. After irradiation, significantly increased rates for non-exchange aberrations, translocations, complex aberrations and dicentric chromosomes were observed in AT patients compared to controls. The results of this study suggested, that complex aberrations and dicentric chromosomes might be a reliable biomarker for radiation sensitivity in AT patients, while non-exchange aberrations and translocations identified both, spontaneous and radiation-induced chromosomal instability.


Assuntos
Ataxia Telangiectasia/genética , Aberrações Cromossômicas , Histonas/genética , Tolerância a Radiação , Adolescente , Adulto , Ataxia Telangiectasia/patologia , Ataxia Telangiectasia/radioterapia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Reparo do DNA , Feminino , Humanos , Masculino , Fosforilação , Radiação Ionizante , Adulto Jovem
16.
Radiat Res ; 195(4): 378-384, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33543294

RESUMO

Radiotherapy plays an important role in the treatment of hepatocellular carcinoma (HCC). Cyclin G1 is a novel member of the cyclin family, and it is abnormally expressed in HCC. In this study we investigated the role of cyclin G1 in the radiotherapy of HCC cells. The expression of cyclin G1 was silenced by transfection of cyclin G1-siRNA into HepG2 cells and Huh7 cells, and the expression of cyclin G1 mRNA and protein was measured by qRT-PCR and Western blot analysis. The proliferation was analyzed using MTT assay, and the radiosensitivity of HCC cells was detected using colony formation assay and a xenograft tumor model. The expression of apoptosis-related proteins (Bcl-2 and Bax) was detected by Western blot analysis, and caspase-3 was detected using fluorimetry. The expression of cyclin G1 mRNA and protein in HepG2/Huh7-cyclin G1-siRNA cells was found to be significantly decreased compared to that in HepG2/Huh7 cells. Silencing the expression of cyclin G1 inhibited the proliferation of HCC cells and enhanced radiosensitivity in HCC cells in vitro and in vivo. Knockdown of cyclin G1 expression significantly decreased Bcl-2 expression, and increased Bax expression and caspase-3 activity in HCC cells. Silencing of cyclin G1 expression enhances the radiosensitivity of HCC cells in vitro and in vivo. The mechanism for this may be related to the regulation of apoptosis-related proteins.


Assuntos
Carcinoma Hepatocelular/radioterapia , Ciclina G1/genética , Neoplasias Hepáticas/radioterapia , Tolerância a Radiação/genética , Animais , Apoptose/efeitos da radiação , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Caspase 3/genética , Linhagem Celular Tumoral , Ciclina G1/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Xenoenxertos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína X Associada a bcl-2/genética
17.
Radiat Res ; 195(4): 307-323, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33577641

RESUMO

Medical countermeasures (MCMs) for hematopoietic acute radiation syndrome (H-ARS) should be evaluated in well-characterized animal models, with consideration of at-risk populations such as pediatrics. We have developed pediatric mouse models of H-ARS and delayed effects of acute radiation exposure (DEARE) for efficacy testing of MCMs against radiation. Male and female C57BL/6J mice aged 3, 4, 5, 6, 7 and 8 weeks old (±1 day) were characterized for baseline hematopoietic and gastrointestinal parameters, radiation response, efficacy of a known MCM, and DEARE at six and 12 months after total-body irradiation (TBI). Weanlings (age 3 weeks) were the most radiosensitive age group with an estimated LD50/30 of 712 cGy, while mice aged 4 to 8 weeks were more radioresistant with an estimated LD50/30 of 767-787 cGy. Female weanlings were more radiosensitive than males at 3 and 4 weeks old but became significantly more radioresistant after the pubertal age of 5 weeks. The most dramatic increase in body weight, RBC counts and intestinal circumference length occurred from 3 to 5 weeks of age. The established radiomitigator Neulasta® (pegfilgrastim) significantly increased 30-day survival in all age groups, validating these models for MCM efficacy testing. Analyses of DEARE among pediatric survivors revealed depressed weight gain in males six months post-TBI, and increased blood urea nitrogen at 12 months post-TBI which was more severe in females. Hematopoietic DEARE at six months post-TBI appeared to be less severe in survivors from the 3- and 4-week-old groups but was equally severe in all age groups by 12 months of age. Similar to our other acute radiation mouse models, there was no appreciable effect of Neulasta used as an H-ARS MCM on the severity of DEARE. In summary, these data characterize a pediatric mouse model useful for assessing the efficacy of MCMs against ARS and DEARE in children.


Assuntos
Síndrome Aguda da Radiação/tratamento farmacológico , Filgrastim/farmacologia , Sistema Hematopoético/efeitos dos fármacos , Polietilenoglicóis/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Síndrome Aguda da Radiação/etiologia , Síndrome Aguda da Radiação/fisiopatologia , Animais , Modelos Animais de Doenças , Sistema Hematopoético/fisiopatologia , Sistema Hematopoético/efeitos da radiação , Humanos , Camundongos , Pediatria , Tolerância a Radiação/efeitos da radiação , Irradiação Corporal Total/efeitos adversos
18.
Radiat Res ; 195(4): 324-333, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33577642

RESUMO

Long non-coding RNAs (lncRNAs) are involved in diverse biological processes, including DNA damage repair, and are of interest as potential biomarkers of radiosensitivity. We investigated whether lncRNA radiosensitivity signatures could be derived for use in cancer patients treated with radiotherapy. Signature development involved radiosensitivity measurements for cell lines and primary tumor samples, and patient outcome after radiotherapy. A 10-lncRNA signature trained on radiosensitivity measurements in bladder cell lines showed a trend towards independent validation. In multivariable analyses, patients with tumors classified as radioresistant by the lncRNA signature had poorer local relapse-free survival (P = 0.065) in 151 patients with muscle-invasive bladder cancer who underwent radiotherapy. An mRNA-based radiosensitivity index signature performed similarly to the lncRNA bladder signature for local relapse-free survival (P = 0.055). Pathway analysis showed the lncRNA signature associated with molecular processes involved in radiation responses. Knockdown of one of the lncRNAs in the signature showed a modest increase in radiosensitivity in one cell line. An alternative approach involved training on primary cervical tumor radiosensitivity or local control after radiotherapy. Both approaches failed to generate a cervix lncRNA radiosensitivity signature, which was attributed to the age of samples in our cohorts. Our work highlights challenges in validating lncRNA signatures as biomarkers in archival tissue from radiotherapy cohorts, but supports continued investigation of lncRNAs for a role in radiosensitivity.


Assuntos
Biomarcadores Tumorais/genética , RNA Longo não Codificante/genética , Tolerância a Radiação/genética , Neoplasias da Bexiga Urinária/radioterapia , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/genética , Transcriptoma/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia
19.
J Biol Regul Homeost Agents ; 35(1): 117-129, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33593046

RESUMO

Peptidyl arginine deiminase 4 (PADI4), an enzyme that converts arginine residues to citrulline residues in the presence of calcium ions, affects the biochemical activities of proteins. The biological function of PADI4 as well as its mechanism in nasopharyngeal carcinoma (NPC) necessitates further investigation. PADI4 expression in NPC tissues and cells was detected using Western blot. qRT-PCR was used to determine the expression of miR-335-5p and PADI4 mRNA in NPC tissues and cells. BrdU assay and CCK-8 assay were employed to detect cell proliferation. Cell migration and invasion were evaluated using Transwell assay. NPC cells were exposed to different doses of radiation in vitro, and then colony formation assays were used to detect colony survival. The target relationship between miR-335-5p and PADI4 was verified using Western blot, qRT-PCR, and dual-luciferase reporter gene assays. Compared with normal mucosal epithelial tissues and cell lines, the expression level of PADI4 in NPC tissues and cells was significantly up-regulated. PADI4 overexpression promoted the proliferation, migration, and invasion of NPC cells. Under radiation, NPC cell survival was significantly promoted by the up-regulation of PADI4. Conversely, knock-down of PADI4 suppressed the above-mentioned malignant phenotypes. MiR-335-5p could bind with the 3' UTR of PADI4 mRNA, and suppressed the expression of PADI4. PADI4 down-regulated the expression of p21 and activated the mTOR signaling pathway. PADI4, which is negatively regulated by miR-335-5p, promotes the proliferation, migration, invasion and radioresistance of NPC cells by regulating the p21 and mTOR signaling pathways.


Assuntos
MicroRNAs/provisão & distribução , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/radioterapia , Proteína-Arginina Desiminase do Tipo 4 , Tolerância a Radiação/genética
20.
Radiat Res ; 195(1): 93-100, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33429432

RESUMO

Cold inducible RNA binding protein (CIRP), also named A18 hnRNP or CIRBP, is a cold-shock RNA-binding protein which can be induced upon various cellular stresses. Its expression level is induced in various cancer tissues relative to adjacent normal tissues; this is believed to play a critical role in cancer development and progression. In this study, we investigated the role of CIRP in cells exposed to ionizing radiation. Our data show that CIRP reduction causes cell colony formation and cell viability reduction after irradiation. In addition, CIRP knockdown cells demonstrated a higher DNA damage rate but less cell cycle arrest after irradiation. As a result, the induced DNA damage with less DNA repair processes led to an increased cell apoptosis rate in CIRP knockdown cells postirradiation. These findings suggest that CIRP is a critical protein in irradiated cells and can be used as a potential target for sensitizing cancer cells to radiation therapy.


Assuntos
Neoplasias/radioterapia , Proteínas de Ligação a RNA/genética , Tolerância a Radiação/genética , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Técnicas de Silenciamento de Genes , Humanos , Neoplasias/genética , Neoplasias/patologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Radiação Ionizante
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