Additive antitumor effect of arsenic trioxide with exposure to ionizing radiation to human acute promyelocytic leukemia HL­60 cells.

Arsenic trioxide (ATO) is expected to be a chemical drug with antitumor activity against acute promyelocytic leukemia (APL), a type of acute myeloid leukemia. In Japan, its antitumor effects were confirmed in clinical trials for APL, and it has been approved in various countries around the world. However, there have been no reports on ATO's antitumor effects on radioresistant leukemia cells, which can be developed during radiotherapy and in combination with therapeutic radiation beams. The present study sought to clarify the antitumor effect of ATO on APL cells with radiation resistance and determine its efficacy when combined with ionizing radiation (IR). The radiation­resistant HL60 (Res­HL60) cell line was generated by subjecting the native cells to 4­Gy irradiation every week for 4 weeks. The half­maximal inhibitory concentration (IC50) for cell proliferation by ATO on native cell was 0.87 µM (R2=0.67), while the IC50 for cell proliferation by ATO on Res­HL60 was 2.24 µM (R2=0.91). IR exposure increased the sub­G1 and G2/M phase ratios in both cell lines. The addition of ATO resulted in a higher population of G2/M after 24 h rather than 48 h. When the rate of change in the sub­G1 phase was examined in greater detail, the sub­G1 phase in both control cells without ATO significantly increased by exposure to IR at 24 h, but only under the condition of 2 Gy irradiation, it had continued to increase at 48 h. Res­HL60 supplemented with ATO showed a higher rate of sub­G1 change at 24 h; however, 2 Gy irradiation resulted in a decrease compared with the control. There was a significant increase in the ratio of the G2/M phase in cells after incubation with ATO for 24 h, and exposure to 2 Gy irradiation caused an even greater increase. To determine whether the inhibition of cell proliferation and cell cycle disruptions is related to reactive oxygen species (ROS) activity, intracellular ROS levels were measured with a flow cytometric assay. Although the ROS levels of Res­HL60 were higher than those of native cells in the absence of irradiation, they did not change after 0.5 or 2 Gy irradiation. Furthermore, adding ATO to Res­HL60 reduced intracellular ROS levels. These findings provide important information that radioresistant leukemia cells respond differently to the antitumor effect of ATO and the combined effect of IR.

Prediction of hub genes and key pathways associated with the radiation response of human hematopoietic stem/progenitor cells using integrated bioinformatics methods.

Hematopoietic stem cells (HSCs) are indispensable for the maintenance of the entire blood program through cytokine response. However, HSCs have high radiosensitivity, which is often a problem during radiation therapy and nuclear accidents. Although our previous study has reported that the combination cytokine treatment (interleukin-3, stem cell factor, and thrombopoietin) improves the survival of human hematopoietic stem/progenitor cells (HSPCs) after radiation, the mechanism by which cytokines contribute to the survival of HSPCs is largely unclear. To address this issue, the present study characterized the effect of cytokines on the radiation-induced gene expression profile of human CD34+ HSPCs and explored the hub genes that play key pathways associated with the radiation response using a cDNA microarray, a protein-protein interaction-MCODE module analysis and Cytohubba plugin tool in Cytoscape. This study identified 2,733 differentially expressed genes (DEGs) and five hub genes (TOP2A, EZH2, HSPA8, GART, HDAC1) in response to radiation in only the presence of cytokines. Furthermore, functional enrichment analysis found that hub genes and top DEGs based on fold change were enriched in the chromosome organization and organelle organization. The present findings may help predict the radiation response and improve our understanding of this response of human HSPCs.

Proteomics and secreted lipidomics of mouse-derived bone marrow cells exposed to a lethal level of ionizing radiation.

High doses of ionizing radiation (IR) exposure can lead to the development of severe acute radiation syndrome with bone marrow failure. Defining risk factors that predict adverse events is a critical mission to guide patient selection for personalized treatment protocols. Since non-hematopoietic stem cells act as feeder cells in the niche and their secreted lipids may regulate hematopoietic stem cells, we focused on non-hematopoietic stem cells and aimed to discover biomarkers that can assess radiation exposure from their secreted lipids. Bone marrow stromal cells (BMSCs) and osteoblast differentiation-inducing cells (ODICs) isolated from mouse femurs were exposed to lethal doses of IR and the proteomic differences between BMSC and ODIC cell layers were compared. We observed an increased Nrf2-mediated oxidative stress response and IL6 expression in ODICs and decreased expression of mitochondrial proteins in BMSCs. To elucidate secreted factors, lipidomics of the cultures were profiled; the relevant lipids distinguishing IR-exposed and control groups of BMSC were acyl-acyl phosphatidylcholine (PC aa C34:1 and PC aa C34:4), lysophosphatidylcholine (lyso-PC a C18:0 and lyso PC a C17:0) and sphingomyelin (SM C20:2). These analyses suggest that certain lipids are candidate markers for the toxic effects of IR.

Hepatic gene expression and functional changes associated with nonalcoholic steatohepatitis.

Non­alcoholic steatohepatitis (NASH) is a pathological condition of the liver in which hepatocyte steatosis, invasion of inflammatory cells and hepatic injury occur without alcohol abuse. Despite the known risk of liver cancer and liver fibrosis that may progress to liver cirrhosis that exists with NASH, an understanding of related gene expression and associated functional changes remains insufficient. The present study used a mouse model of NASH induced by a high­fat diet to examine gene expression in the liver and to search for transcripts that could predict early liver fibrosis in the future. Mice fed a high­fat diet for 2 weeks showed typical NASH liver histology by hematoxylin and eosin staining, and increased fibrosis was confirmed by Sirius red staining after 6 weeks. Functional changes associated with liver damage, liver inflammation, liver steatosis and liver fibrosis were predicted by toxicological ontology analysis using Ingenuity Pathways Analysis. Downregulated microRNA (miR)­21 and upregulated collagen type III α1 mRNA in the liver and upregulated exosomal miR­21 in serum of mice fed a high­fat diet for 1 and/or 2 weeks were confirmed by reverse transcription­quantitative PCR, suggesting that these changes occur prior to histological confirmation of fibrosis. Therefore, it may be possible to predict future liver fibrosis by analyzing fibrosis­related genes that shift prior to pathological findings.

Dose-dependent expression of extracellular microRNAs in HCT116 colorectal cancer cells exposed to high-dose-rate ionising radiation.

Biomarkers of tumour response to radiotherapy may help optimise cancer treatment. The aim of the present study was to identify changes in extracellular microRNAs (miRNAs) as a biomarker of radiation-induced damage to human colorectal cancer cells. HCT116 cells were exposed to increasing doses of X-rays, and extracellular miRNAs were analysed by microarray. The results were correlated with the frequency of micronuclei. A total of 59 miRNAs with a positive correlation and 4 with a negative correlation between dose (up to 6 Gy) and extracellular miRNA expression were identified. In addition, for doses between 0 and 10 Gy, 12 miRNAs among those 59 miRNAs with a positive correlation were identified; for these extracellular miRNAs, a significantly positive correlation was observed between their expression and the frequency of micronuclei for doses up to 10 Gy. These results suggest that specific miRNAs may be considered as cell damage markers and may serve as secreted radiotherapy response biomarkers for colorectal cancer; however, the results must be further validated in serum samples collected from patients undergoing radiotherapy.

HER2-positive breast cancer that resists therapeutic drugs and ionizing radiation releases sphingomyelin-based molecules to circulating blood serum.

Breast cancer is the second most common cancer in the world based on incidence, reaching more than 2 million new cases in 2018, while continuing to increase. Invasive ductal carcinoma is the most common type of this cancer, making up approximately 70-80% of all breast cancer diagnoses. In particular, the type of breast cancer overexpressing human epidermal growth factor receptor 2 (HER2) has potential of strong proliferation, migration and invasion and early treatment is necessary. The authors identified and studied a single patient displaying complete therapeutic resistance to monoclonal anti-HER2 antibody therapy, chemotherapy and radiotherapy. A patient who exhibited resistance to postoperative adjuvant therapy after mastectomy was selected from HER2-positive breast cancer, and this patient had the grade of T4bN2aM0, Stage IIIB. The patient samples, blood serum and cancer tissue, were analyzed by metabolome and immunostaining technique, respectively. The characteristics of peripheral blood serum and solid tumor were investigated, aiming to find new serum biomarker(s) using the metabolomics technique. A correlation between the appearance of HER2-positive cancer tissue and serum concentration of the sphingomyelin family was found. In addition, HER2-positive tumor tissue in both the primary and recurrent cancer express the sphingomyelinase. These results suggest that sphingomyelins from this cancer tissue leads to therapy resistance, induction of invasion and strong proliferation.

Changes in miRNA expressions in the injured small intestine of mice following high­dose radiation exposure.

The small intestine is one of the most highly regenerative and radiosensitive tissues in mammals, including humans. Exposure to high doses of ionizing radiation causes serious intestinal damage. Recently, several investigations have been conducted using radioprotective agents to determine ways for reducing intestinal damage caused by radiation exposure. However, a thorough understanding of functional changes occurring in the small intestine of mice exposed to high­dose radiation is necessary for developing novel and more potent radioprotective agents. In this study, we examined changes in microRNA (miRNA/miR) expressions in the small intestine of mice at 72 h after X­ray exposure (10 Gy). We identified seven upregulated miRNAs and six downregulated miRNAs in the small intestine of mice following radiation exposure using miRNA microarray analysis. Particularly, miR­34a­5p was highly expressed, which was confirmed by reverse transcription-quantitative PCR. Forkhead box P1 (Foxp1) was predicted to be a target of the mRNA of miR­34a­5p using OmicsNet. Decreased Foxp1 expression in the small intestine following radiation exposure was confirmed, suggesting that Foxp1 expression recovery may induce the suppression of radiation­induced enteritis. Therefore, miR­34a­5p is a potential target molecule for developing novel radioprotective agents.

Time-related study on external exposure dose of 2-deoxy-2-[F-18]fluoro-D-glucose PET for workers' safety.

Time-course study of individual dose equivalents of 2-deoxy-2-[F-18]fluoro-D-glucose positron emission tomography (18F-FDG PET) was conducted in different hospital workers, and the daily work duties were analyzed. For the measurements, a semiconductor dosimeter was used. The values at intervals of 1 min and 1 h, the monthly cumulative and daily cumulative doses, and trend graphs were acquired with dedicated software and displayed on the reader. The following radiation workers with duties involving maximum external exposure work were included: doctors making diagnoses (4.8 µSv/procedure), nurses removing injection needles (3.1 µSv/procedure), pharmacists performing quality control tests (2.9 µSv/procedure), nuclear medicine technologists assisting patient positioning (6.5 µSv/procedure), and cyclotron engineers performing daily checks (13.4 µSv/procedure). The results of analysis of daily work duties revealed the influencing factors of external exposure dose. To reduce the external exposure dose, investigators should shorten the patient's contact time with the 18F-FDG source or patient tracer.

Understanding the mechanism underlying the acquisition of radioresistance in human prostate cancer cells.

Acquisition of radioresistance (RR) has been reported during cancer treatment with fractionated irradiation. However, RR is poorly understood in the prognosis of radiotherapy. Although radiotherapy is important in the treatment of prostate cancer (PCa), acquisition of RR has been reported in PCa with an increased number of cancer stem cells (CSCs), neuroendocrine differentiation (NED) and epithelial-mesenchymal transition. However, to the best of our knowledge, the mechanism underlying RR acquisition during fractionated irradiation remains unclear. In the present study, human PCa cell lines were subjected to fractionated irradiation according to a fixed schedule as follows: Irradiation (IR)1, 2 Gy/day with a total of 20 Gy; IR2, 4 Gy/day with a total of 20 Gy; and IR3, 4 Gy/day with a total of 56 Gy. The expression of cluster of differentiation (CD)44, a CSC marker, was identified to be increased by fractionated irradiation, particularly in DU145 cells. The expression levels of CD133 and CD138 were increased compared with those in parental cells following a single irradiation or multiple irradiations; however, the expression levels decreased with subsequent irradiation. RR was evidently acquired by exposure to 56 Gy radiation, which resulted in increased expression of the NED markers CD133 and CD138, and increased mRNA expression levels of the pluripotency-associated genes octamer-binding transcription factor 4 and Nanog homeobox. These data indicate that radiation-induced CSCs emerge due to the exposure of cells to fractionated irradiation. In addition, the consequent increase in the expression of NED markers is possibly induced by the increased expression of pluripotency-associated genes. Therefore, it can be suggested that cancer cells acquire RR due to increased expression of pluripotency-associated genes following exposure to fractionated irradiation.

Exosomes released from pancreatic cancer cells enhance angiogenic activities via dynamin-dependent endocytosis in endothelial cells in vitro.

Pancreatic cancer has the lowest 5 year survival rate among all cancers. Several extracellular factors are involved in the development and metastasis of pancreatic cancer to distant organs. Exosomes are lipid-bilayer, membrane-enclosed nanoparticles that are recognised as important mediators of cell-to-cell communications. However, the role of exosomes released from pancreatic cancer cells in tumour micro-environment remains unknown. Here, we show that exosomes released from pancreatic cancer PK-45H cells activate various gene expressions in human umbilical vein endothelial cells (HUVECs) by in vitro analyses. In addition, these exosomes released from PK-45H cells promote phosphorylation of Akt and ERK1/2 signalling pathway molecules and tube formation via dynamin-dependent endocytosis in HUVECs. Our findings suggested that exosomes released from pancreatic cancer cells may act as a novel angiogenesis promoter.

Cell­to­cell communication via extracellular vesicles among human pancreatic cancer cells derived from the same patient.

Despite existing multimodal therapies, pancreatic cancer exhibits high metastatic capability and poor prognosis. Extracellular vesicles (EVs) are nanoparticles comprising lipid bilayers and various other components, such as protein and nucleic acids, derived from secreted cells. Recent research has demonstrated the involvement of EVs released from cancer cells in the metastasis of cancer cells to distant organs. However, the effects of EVs released from pancreatic cancer cells on other pancreatic cancer cells in a tumor microenvironment remain unclear. The present study aimed to elucidate that EVs released from PK­45H pancreatic cancer cells are taken up by PK­45P pancreatic cancer cells derived from the same patient through dynamin­related endocytosis. Additionally, EVs released from PK­45H cells augment the phosphorylation of classical mitogen­activated protein kinase (MAPK) pathways in PK­45P cells. The uptake of EVs released from PK­45H cells by PK­45P cells stimulates cell migration through the classical MAPK­dependent pathway, suggesting that EVs released from one pancreatic cancer cell are taken up by other surrounding pancreatic cancer cells and could be critical inducers of cancer metastasis in the tumor microenvironment.

[Screening for Biological Marker of Dose-optimization in Cancer Radiotherapy].

PURPOSE AND METHODS: External radiotherapy of target regions using high-energy beams leads to excessive exposure along with individual variation in therapeutic and adverse effects. However, high-precision radiotherapy utilizes 3D-multi detector computed tomography to confirm both target position and administer radiation dose. To install the individual bioinformation in the radiotherapy plan (particularly, radiosensitivity into the target region and/or the around normal tissue), the investigation of biomarkers, which are able to estimate their radiosensitivity was performed. The aim of this investigation is to screen for suitable radiosensitivity biomarkers using the human colorectal cancer-derived HCT 116 cell line. RESULTS: We found that cell damage and micronucleus frequency significantly increased dose dependently after exposure to 6 Gy X-irradiation (1 Gy/min). In contrast, total RNA concentration (69.8-85.2 ng/ml) remained stable in the cell culture supernatant despite radiation dose variation. Additionally, 52 specific micro RNAs were detected after exposure to 6 Gy X-irradiation. CONCLUSION: These results suggest that radiosensitivity, including extent of cellular damage in target or normal tissue, can be indirectly estimated by monitoring the expression of micro RNAs.

Protective Effect of the c-mpl Agonist Romiplostim on Megakaryocytopoiesis of Human CD34+ Hematopoietic Progenitor Cells Exposed to Ionizing Radiation.

A thrombopoiesis-stimulating protein, the myeloproliferative leukemia virus protooncogene (Mpl) ligand romiplostim (RP), is currently approved as a therapeutic agent for idiopathic thrombocytopenic purpura in many countries. Although the action of the initial MPL ligand thrombopoietin (TPO) on human megakaryocytic regeneration from irradiated human hematopoietic stem cells has been examined, there are few reports on the action of RP. In the present study, freshly prepared nonirradiated and 2-Gy X-irradiated human CD34 positive (CD34+) cells from placental umbilical cord blood were cultured with a combination of RP and various cytokines. As a result, the effect of RP on cell proliferation of nonirradiated CD34+ cells was found to be comparable to that of TPO. However, the stimulating activity of RP on megakaryocytic progenitor-derived colony formation was markedly lower compared with TPO. Regarding the action of RP with various cytokines, the present results showed that a combination of RP with interleukin-3 (IL-3) or IL-3 plus stem cell factor (SCF) showed a high regenerative effect on cell proliferation, megakaryopoiesis, thrombopoiesis, and megakaryocyte colony formation from X-irradiated CD34+ cells. The present study showed that human recombinant RP has potential effects on human megakaryocytic regeneration from X-irradiated human CD34+ cells and synergistically acts with IL-3 and IL-3 plus SCF, just as observed with TPO.

A radioresistant fraction of acute promyelocytic leukemia cells exhibit CD38 cell-surface antigen and mRNA expression.

In the present study, the cell viability and cluster of differentiation (CD)38 mRNA expression were evaluated in radioresistant (Res)-HL60 acute promyelocytic leukemia (APL) cells. Cell viability in Res-HL60 cells was higher compared with wild-type HL60 cells, but did not differ between high and mid/low CD38 antigen expression groups in Res-HL60 cells. A higher expression of CD38 mRNA in Res-HL60 cells was observed, particularly in the CD38high cell subpopulation. Furthermore, the expression of CD38 mRNA was upregulated following exposure to X-irradiation. In contrast, the characteristic expression of CD45 and CCAAT/enhancer-binding protein α mRNA were not altered. These results suggest that the accumulation of CD38 protein in radioresistant APL cells, resulting from the constant expression of CD38 mRNA induced by X-irradiation, is a characteristic response of the radioresistant-surviving fraction; however, the accumulation of CD38 did not influence the extent of radioresistant behavior.

Serum miR-375-3p increase in mice exposed to a high dose of ionizing radiation.

Exposure to high-doses of ionizing radiation (IR) leads to development of a strong acute radiation syndrome (ARS) in mammals. ARS manifests after a latency period and it is important to develop fast prognostic biomarkers for its early detection and assessment. Analysis of chromosomal aberrations in peripheral blood lymphocytes is the gold standard of biological dosimetry, but it fails after high doses of IR. Therefore, it is important to establish novel biomarkers of exposure that are fast and reliable also in the high dose range. Here, we investigated the applicability of miRNA levels in mouse serum. We found significantly increased levels of miR-375-3p following whole body exposure to 7 Gy of X-rays. In addition, we analyzed their levels in various organs of control mice and found them to be especially abundant in the pancreas and the intestine. Following a dose of 7 Gy, extensive cell death occurred in these tissues and this correlated negatively with the levels of miR-375-3p in the organs. We conclude that high expressing tissues of miR-375-3p may secrete this miRNA in serum following exposure to 7 Gy. Therefore, elevated miR-375-3p in serum may be a predictor of tissue damage induced by exposure to a high radiation dose.

Anti-tumor and anti-invasion effects of a combination of 4-methylumbelliferone and ionizing radiation in human fibrosarcoma cells.

Hyaluronan (HA) is a major component of the extracellular matrix that is synthesized in excess in cancer tissues. 4-methylumbelliferone (MU) inhibits the synthesis of HA and is closely related to the invasion and metastasis of cancer. However, the effects of MU in conjunction with cancer radiotherapy remain unknown. The present study assessed the anti-tumor and anti-invasion effects of the concomitant use of ionizing radiation (IR) and 100 µM MU on human fibrosarcoma HT1080 cells. Cell viability and cellular invasion potency assays were performed. There was a greater decrease in the viability of cells cultured with a combination of 2 Gy IR and MU compared with untreated control cells. In addition, cell cycle distribution analysis demonstrated that a higher proportion of these cells were in the sub-G1 phase and higher fractions of annexin-V positive, propidium iodide positive cells (i.e., apoptotic cells) were observed. HA concentration in the 2 Gy irradiated culture was similar to that in the non-irradiated control culture, however, it significantly decreased following the administration of both MU alone and 2 Gy IR with MU. Furthermore, treatment with 2 Gy IR and MU resulted in a significant decrease in the invasion rate and matrix metalloproteinase (MMP)-2 and MPP-9 expression. Taken together, these results suggest that the administration of MU with 2 Gy IR is effective at reducing HA production, cell invasion and the metastatic potential of cancer cells.

Ascorbic acid does not reduce the anticancer effect of radiotherapy.

The present study hypothesized that the therapeutic use of ascorbic acid (AsA) in combination with radiation may reduce therapy-related side effects and increase the antitumor effects. The aim of the study was to examine the association between the scavenged activity of AsA and the biological anticancer effect of hydroxyl (OH) radicals generated by X-ray irradiation. Cell survival, DNA fragmentation of human leukemia HL60 cells and the amount of OH radicals were investigated following X-ray irradiation and AsA treatment. The number of living cells decreased, and DNA fragmentation increased at AsA concentrations >1 mM. Electron spin resonance spectra revealed that X-ray irradiation generated OH radicals, which were scavenged by AsA at concentrations >75 µM. The AsA concentration inside the cell was 75 µM when cells underwent extracellular treatment with 5 mM AsA, which significantly induced HL60 cell death even without irradiation. No increase in the number of viable HL60 cells was observed following AsA treatment with irradiation when compared to irradiation alone. In conclusion, the disappearance of the radiation anticancer effects with AsA treatment in combination with radiotherapy for cancer treatment is not a cause for concern.

Application of cell sorting for enhancing the performance of the cytokinesis-block micronucleus assay.

Among the numerous methods available to assess genotoxicity, the cytokinesis-block micronucleus (CBMN) assay is very popular due its relative simplicity and power to detect both clastogenic and aneugenic compounds. A problem with the CBMN assay is that all DNA damaging agents also inhibit the ability of cells to progress through mitosis, leading to a low number of binucleated cells (BNCs). One method to resolve this issue is to ensure a sufficient proportion of BNCs in the samples. In the current study, the applicability of a cell sorting system capable of isolating cell fractions containing abundant BNCs was investigated. Furthermore, to investigate the relationship between the cell division delay due to radiation exposure and the generation of BNCs and micronuclei (MN), we assessed a series of lag times between radiation exposure and addition of cytochalasin-B (Cyt-B). Cells from the human chronic myelogenous leukemia cell line K562 were exposed to X-rays (2 Gy and 4 Gy), and Cyt-B was subsequently added at 0, 6 and 12 h following irradiation. After treatment with Cyt-B for 24 h, the percentage of BNCs, the MN frequency and the cell cycle distribution were analyzed. In addition, cells displaying the DNA contents corresponding to BNCs were isolated and analyzed. The results indicate that applying the cell sorter to the CBMN assay increased the percentage of BNCs compared with the standard method. Thus, this technique is a promising way of enhancing the capacity of the CBMN assay.

Genetic network profiles associated with established resistance to ionizing radiation in acute promyelocytic leukemia cells and their extracellular vesicles.

Radiation-resistant acute promyelocytic leukemia (APL) cells present challenges to treatment, and the acquisition of resistance to ionizing radiation (IR) is a matter of clinical concern. However, little information is available on the behavior of radio-resistant APL in terms of gene expression profiles and intercellular communication. In this study, cDNA microarray and RT-PCR were used to analyze the intracellular genetic network and extracellular vesicles (EVs), respectively, in the established radio-resistant HL60 (Res-HL60) cell line. Significant changes in the expression of 7,309 known mRNAs were observed in Res-HL60 relative to control. In addition, 7 mRNAs were determined as targets because significant changes in the expression were observed using Ingenuity analysis software, confirming the quantitative RT-PCR. However, EVs from Res-HL60 cells did not include these target molecules. These results suggest that radio-resistant APL is regulated by the expression and suppression of specific molecules, and these molecules are not transferred between cells by EVs.