Artificial intelligence in biology and medicine, and radioprotection research: perspectives from Jerusalem.

While AI is widely used in biomedical research and medical practice, its use is constrained to few specific practical areas, e.g., radiomics. Participants of the workshop on "Artificial Intelligence in Biology and Medicine" (Jerusalem, Feb 14-15, 2023), both researchers and practitioners, aimed to build a holistic picture by exploring AI advancements, challenges and perspectives, as well as to suggest new fields for AI applications. Presentations showcased the potential of large language models (LLMs) in generating molecular structures, predicting protein-ligand interactions, and promoting democratization of AI development. Ethical concerns in medical decision making were also addressed. In biological applications, AI integration of multi-omics and clinical data elucidated the health relevant effects of low doses of ionizing radiation. Bayesian latent modeling identified statistical associations between unobserved variables. Medical applications highlighted liquid biopsy methods for non-invasive diagnostics, routine laboratory tests to identify overlooked illnesses, and AI's role in oral and maxillofacial imaging. Explainable AI and diverse image processing tools improved diagnostics, while text classification detected anorexic behavior in blog posts. The workshop fostered knowledge sharing, discussions, and emphasized the need for further AI development in radioprotection research in support of emerging public health issues. The organizers plan to continue the initiative as an annual event, promoting collaboration and addressing issues and perspectives in AI applications with a focus on low-dose radioprotection research. Researchers involved in radioprotection research and experts in relevant public policy domains are invited to explore the utility of AI in low-dose radiation research at the next workshop.

Fluorescence in situ hybridization analysis of chromosomal aberrations in mouse splenocytes at one- and two-months after total body exposure to iron-56 (Fe) ion particles or X-rays.

High atomic number and energy (HZE) particles such as iron-56 (Fe) ions are a major contributor to health risks in long-term manned space exploration. The aim of this study is to understand radiation-induced differential genotoxic effects between HZE particles and low linear energy transfer (LET) photons. C57BL/6J Jms female mice of 8 weeks old were exposed to total body irradiation of accelerated Fe-particles with a dose ranging from 0.1 to 3.0 Gy or of X-rays with a dose ranging from 0.1 to 5.0 Gy. Chromosomal aberrations (CAs) in splenocytes were examined by fluorescence in situ hybridization at 1- and 2-months after exposure. Clonal expansions of cells with CAs were found to be induced only by X-rays but not by Fe-particles. Dose-dependent increase in the frequencies of stable-type CAs was observed at 1- as well as 2-months after exposure to both radiation types. The frequencies of stable-type CAs in average were much higher in mice exposed to X-rays than those to Fe-particles and did not change significantly between 1- and 2-months after exposure to both radiation types. On the other hand, the frequencies of unstable-type CAs induced by X-rays and Fe-particles were not much different, and they appeared to decrease with time from 1- to 2-months after exposure. These results suggested that larger fraction of stable-type CAs induced by Fe-particles might be non-transmissible than those by X-rays because of some associating lethal alterations on themselves or on other chromosomes in the same cells and that these cells might be removed by 1-month after Fe-TBI. We also demonstrated that exposure to Fe-particles induced insertions at relatively higher frequency to other stable-type CAs than X-rays. Our findings suggest that insertions can be used as indicators of past exposure to high-LET particle radiation.

Enhanced Effects of Chronic Restraint-Induced Psychological Stress on Total Body Fe-Irradiation-Induced Hematopoietic Toxicity in Trp53-Heterozygous Mice.

Humans are exposed to both psychological stress (PS) and radiation in some scenarios such as manned deep-space missions. It is of great concern to verify possible enhanced deleterious effects from such concurrent exposure. Pioneer studies showed that chronic restraint-induced PS (CRIPS) could attenuate Trp53 functions and increase gamma-ray-induced carcinogenesis in Trp53-heterozygous mice while CRIPS did not significantly modify the effects on X-ray-induced hematopoietic toxicity in Trp53 wild-type mice. As high-linear energy transfer (LET) radiation is the most important component of space radiation in causing biological effects, we further investigated the effects of CRIPS on high-LET iron-particle radiation (Fe)-induced hematopoietic toxicity in Trp53-heterozygous mice. The results showed that CRIPS alone could hardly induce significant alteration in hematological parameters (peripheral hemogram and micronucleated erythrocytes in bone marrow) while concurrent exposure caused elevated genotoxicity measured as micronucleus incidence in erythrocytes. Particularly, exposure to either CRISP or Fe-particle radiation at a low dose (0.1 Gy) did not induce a marked increase in the micronucleus incidence; however, concurrent exposure caused a significantly higher increase in the micronucleus incidence. These findings indicated that CRIPS could enhance the deleterious effects of high-LET radiation, particularly at a low dose, on the hematopoietic toxicity in Trp53-heterozygous mice.

Carbon-Ion Beam Irradiation and the miR-200c Mimic Effectively Eradicate Pancreatic Cancer Stem Cells Under in vitro and in vivo Conditions.

PURPOSE: The study investigated the molecular mechanisms that killed pancreatic cancer cells, including cancer stem cells (CSCs), by carbon ion beam irradiation alone or in combination with miRNA-200c under in vitro and in vivo conditions. METHODS: Human pancreatic cancer (PC) cells, PANC1 and PK45, were treated with carbon-ion beam irradiation alone or in combination with microRNA-200c (miR-200c) mimic. Cell viability assay, colony and spheroid formation assay, quantitative real-time PCR analysis of apoptosis-, autophagy-, and angiogenesis-related gene expression, xenograft tumor control and histopathological analyses were performed. RESULTS: The cell viability assay showed that transfection of the miRNA-200c (10 nM) mimic into pancreatic CSC (CD44+/ESA+) and non-CSC (CD44-/ESA-) significantly suppressed proliferation of both types of cell populations described above. Combining carbon-ion beam irradiation with the miRNA-200c mimic significantly reduced the colony as well as spheroid formation abilities compared to that observed with the treatment of carbon-ion beam alone or X-ray irradiation combined with the miRNA-200c mimic. Moreover, the combination of carbon ion beam irradiation and miRNA-200c mimic increased the expression of apoptosis-related gene BAX, autophagy-related genes Beclin-1 and p62, addition of gemcitabine (GEM) further enhanced the expression of these genes. In vivo data showed that carbon-ion beam irradiation in combination with the miRNA-200c mimic effectively suppressed xenograft tumor growth and significantly induced tumor necrosis and cavitation. CONCLUSION: The combination of miRNA-200c mimic and carbon ion beam irradiation may be powerful radiotherapy that significantly kills pancreatic cancer cells containing CSCs and enhances the effect of carbon-ion beam irradiation compared to carbon-ion beam irradiation alone.

Synergistic Effects of Chronic Restraint-Induced Stress and Low-Dose 56Fe-particle Irradiation on Induction of Chromosomal Aberrations in Trp53-Heterozygous Mice.

Astronauts can develop psychological stress (PS) during space flights due to the enclosed environment, microgravity, altered light-dark cycles, and risks of equipment failure or fatal mishaps. At the same time, they are exposed to cosmic rays including high atomic number and energy (HZE) particles such as iron-56 (Fe) ions. Psychological stress or radiation exposure can cause detrimental effects in humans. An earlier published pioneering study showed that chronic restraint-induced psychological stress (CRIPS) could attenuate Trp53 functions and increase carcinogenesis induced by low-linear energy transfer (LET) γ rays in Trp53-heterozygous (Trp53+/-) mice. To elucidate possible modification effects from CRIPS on high-LET HZE particle-induced health consequences, Trp53+/- mice were received both CRIPS and accelerated Fe ion irradiation. Six-week-old Trp53+/- C57BL/6N male mice were restrained 6 h per day for 28 consecutive days. On day 8, they received total-body Fe-particle irradiation (Fe-TBI, 0.1 or 2 Gy). Metaphase chromosome spreads prepared from splenocytes at the end of the 28-day restraint regimen were painted with the fluorescence in situ hybridization (FISH) probes for chromosomes 1 (green), 2 (red) and 3 (yellow). Induction of psychological stress in our experimental model was confirmed by increase in urinary corticosterone level on day 7 of restraint regimen. Regardless of Fe-TBI, CRIPS reduced splenocyte number per spleen at the end of the 28-day restraint regimen. At 2 Gy, Fe-TBI alone induced many aberrant chromosomes and no modifying effect was detected from CRIPS on induction of aberrant chromosomes. Notably, neither Fe-TBI at 0.1 Gy nor CRIPS alone induced any increase in the frequency of aberrant chromosomes, while simultaneous exposure resulted in a significant increase in the frequency of chromosomal exchanges. These findings clearly showed that CRIPS could enhance the frequency of chromosomal exchanges induced by Fe-TBI at a low dose of 0.1 Gy.

Reduced High-Dose Radiation-Induced Residual Genotoxic Damage by Induction of Radioadaptive Response and Prophylactic Mild Dietary Restriction in Mice.

Radioadaptive response (RAR) describes a phenomenon in a variety of in vitro and in vivo systems that a low-dose of priming ionizing radiation (IR) reduces detrimental effects of a subsequent challenge IR at higher doses. Among in vivo investigations, studies using the mouse RAR model (Yonezawa Effect) showed that RAR could significantly extenuate high-dose IR-induced detrimental effects such as decrease of hematopoietic stem cells and progenitor cells, acute radiation hematopoietic syndrome, genotoxicity and genomic instability. Meanwhile, it has been demonstrated that diet intervention has a great impact on health, and dietary restriction shows beneficial effects on numerous diseases in animal models. In this work, by using the mouse RAR model and mild dietary restriction (MDR), we confirmed that combination of RAR and MDR could more efficiently reduce radiogenotoxic damage without significant change of the RAR phenotype. These findings suggested that MDR may share some common pathways with RAR to activate mechanisms consequently resulting in suppression of genotoxicity. As MDR could also increase resistance to chemotherapy and radiotherapy in normal cells, we propose that combination of MDR, RAR, and other cancer treatments (i.e., chemotherapy and radiotherapy) represent a potential strategy to increase the treatment efficacy and prevent IR risk in humans.

Altered Response to Total Body Irradiation of C57BL/6-Tg (CAG-EGFP) Mice.

Application of green fluorescent protein (GFP) in a variety of biosystems as a unique bioindicator or biomarker has revolutionized biological research and made groundbreaking achievements, while increasing evidence has shown alterations in biological properties and physiological functions of the cells and animals overexpressing transgenic GFP. In this work, response to total body irradiation (TBI) was comparatively studied in GFP transgenic C57BL/6-Tg (CAG-EGFP) mice and C57BL/6 N wild type mice. It was demonstrated that GFP transgenic mice were more sensitive to radiation-induced bone marrow death, and no adaptive response could be induced. In the nucleated bone marrow cells of GFP transgenic mice exposed to a middle dose, there was a significant increase in both the percentage of cells expressing pro-apoptotic gene Bax and apoptotic cell death. While in wild type cells, lower expression of pro-apoptotic gene Bax and higher expression of anti-apoptotic gene Bcl-2, and significant lower induction of apoptosis were observed compared to GFP transgenic cells. Results suggest that presence of GFP could alter response to TBI at whole body, cellular and molecular levels in mice. These findings indicate that there could be a major influence on the interpretation of the results obtained in GFP transgenic mice.

Combination of carbon-ion beam and dual tyrosine kinase inhibitor, lapatinib, effectively destroys HER2 positive breast cancer stem-like cells.

To investigate whether carbon-ion beam alone, or in combination with lapatinib, has a beneficial effect in targeting HER2-positive breast cancer stem-like cells (CSCs) compared to that of X-rays, human breast CSCs derived from BT474 and SKBR3 cell lines were treated with a carbon-ion beam or X-rays irradiation alone or in combination with lapatinib, and then cell viability, spheroid formation assays, apoptotic analyses, gene expression analysis of related genes, and immunofluorescent γ-H2AX foci assays were performed. Spheroid formation assays confirmed that ESA+/CD24- cells have CSC properties compared to ESA-/CD24+ cells. CSCs were more highly enriched after X-ray irradiation combined with lapatinib, whereas carbon-ion beam combined with lapatinib significantly decreased the proportion of CSCs. Carbon-ion beam combined with lapatinib significantly suppressed spheroid formation compared to X-rays combined with lapatinib or carbon ion beam alone. Cell cycle analysis showed that carbon ion beam combined with lapatinib predominantly enhanced sub-G1 and G2/M arrested population compared to that of carbon-ion beam, X-ray treatments alone. Carbon-ion beam combined with lapatinib significantly enhanced apoptosis and carbon-ion beam alone dose-dependently increased autophagy-related expression of Beclin1 and in combination with lapatinib greatly enhanced ATG7 expression at protein levels. In addition, a large-sized γH2AX foci in CSCs were induced by carbon ion beam combined with lapatinib treatment in CSCs compared to cells receiving X-rays or carbon-ion beam alone. Altogether, combination of carbon-ion beam irradiation and lapatinib has a high potential to kill HER2-positive breast CSCs, causing severe irreparable DNA damage, enhanced autophagy, and apoptosis.

A multimodal treatment of carbon ions irradiation, miRNA-34 and mTOR inhibitor specifically control high-grade chondrosarcoma cancer stem cells.

BACKGROUND AND PURPOSE: High-grade chondrosarcomas are chemo- and radio-resistant cartilage-forming tumors of bone that often relapse and metastase. Thus, new therapeutic strategies are urgently needed. MATERIAL AND METHODS: Chondrosarcoma cells (CH-2879) were exposed to carbon-ion irradiation, combined with miR-34 mimic and/or rapamycin administration. The effects of treatment on cancer stem cells, stemness-associated phenotype, radioresistance and tumor-initiating properties were evaluated. RESULTS: We show that high-grade chondrosarcoma cells contain a population of radioresistant cancer stem cells that can be targeted by a combination of carbon-ion therapy, miR-34 mimic administration and/or rapamycin treatment that triggers FOXO3 and miR-34 over-expression. mTOR inhibition by rapamycin triggered FOXO3 and miR-34, leading to KLF4 repression. CONCLUSION: Our results show that particle therapy combined with molecular treatments effectively controls cancer stem cells and may overcome treatment resistance of high-grade chondrosarcoma.

Correction: Oh, J.Y., et al. Synergistic Autophagy Effect of miR-212-3p in Zoledronic Acid-Treated In Vitro and Orthotopic In Vivo Models and in Patient-Derived Osteosarcoma Cells, Cancers 2019, 11, 1812.

The authors wish to make the following corrections to this paper [...].

Functionalized mesoporous silica nanoparticles for innovative boron-neutron capture therapy of resistant cancers.

Treatment resistance, relapse and metastasis remain critical issues in some challenging cancers, such as chondrosarcomas. Boron-neutron capture therapy (BNCT) is a targeted radiation therapy modality that relies on the ability of boron atoms to capture low energy neutrons, yielding high linear energy transfer alpha particles. We have developed an innovative boron-delivery system for BNCT, composed of multifunctional fluorescent mesoporous silica nanoparticles (B-MSNs), grafted with an activatable cell penetrating peptide (ACPP) for improved penetration in tumors and with gadolinium for magnetic resonance imaging (MRI) in vivo. Chondrosarcoma cells were exposed in vitro to an epithermal neutron beam after B-MSNs administration. BNCT beam exposure successfully induced DNA damage and cell death, including in radio-resistant ALDH+ cancer stem cells (CSCs), suggesting that BNCT using this system might be a suitable treatment modality for chondrosarcoma or other hard-to-treat cancers.

Carbon-Ion Beam Irradiation Alone or in Combination with Zoledronic acid Effectively Kills Osteosarcoma Cells.

Osteosarcoma (OSA) is the most common malignant bone tumor in children and adolescents. The overall five-year survival rate for all bone cancers is below 70%; however, when the cancer has spread beyond the bone, it is about 15-30%. Herein, we evaluated the effects of carbon-ion beam irradiation alone or in combination with zoledronic acid (ZOL) on OSA cells. Carbon-ion beam irradiation in combination with ZOL significantly inhibited OSA cell proliferation by arresting cell cycle progression and initiating KHOS and U2OS cell apoptosis, compared to treatments with carbon-ion beam irradiation, X-ray irradiation, and ZOL alone. Moreover, we observed that this combination greatly inhibited OSA cell motility and invasion, accompanied by the suppression of the Pi3K/Akt and MAPK signaling pathways, which are related to cell proliferation and survival, compared to individual treatments with carbon-ion beam or X-ray irradiation, or ZOL. Furthermore, ZOL treatment upregulated microRNA (miR)-29b expression; the combination with a miR-29b mimic further decreased OSA cell viability via activation of the caspase 3 pathway. Thus, ZOL-mediated enhancement of carbon-ion beam radiosensitivity may occur via miR-29b upregulation; co-treatment with the miR-29b mimic further decreased OSA cell survival. These findings suggest that the carbon-ion beam irradiation in combination with ZOL has high potential to increase OSA cell death.

Molecular mechanisms underlying the enhancement of carbon ion beam radiosensitivity of osteosarcoma cells by miR-29b.

Carbon ion radiotherapy (CIRT) is more effective than conventional photon beam radiotherapy in treating osteosarcoma (OSA); however, the outcomes of CIRT alone are still unsatisfactory. In this study, we aimed to investigate whether miR-29b acts as a radiosensitizer for CIRT. The OSA cell lines U2OS and KHOS were treated with carbon ion beam alone, γ-ray irradiation alone, or in combination with an miR-29b mimic. OSA cell death as well as invasive and migratory abilities were analyzed through viability, colony formation, Transwell, and apoptosis assays. miR-29 expression was downregulated in OSA tissues compared to that in normal tissues and was associated with metastasis and relapse in patients with OSA. Further, miR-29b was found to directly target the transcription factor Sp1 and suppress the activation of the phosphatase and tensin homolog (PTEN)-AKT pathway. Conversely, Sp1 was found to attenuate the inhibitory effects of miR-29b in OSA cells. When used in combination with miR-29b mimic, carbon ion beam markedly inhibited invasion, migration, and proliferation of OSA cells and promoted apoptosis by inhibiting AKT phosphorylation in a Sp1/PTEN-mediated manner. Taken together, miR-29b mimic improved the radiosensitivity of OSA cells via the PTEN-AKT-Sp1 signaling pathway, presenting a novel strategy for the development of carbon ion beam combination therapy.

Diallyl Disulfide Mitigates DNA Damage and Spleen Tissue Effects After Irradiation.

BACKGROUND Several factors found in foods are beneficial to human health and they may contribute to radiation protection. Taking food factors could be an easy way to reduce the effects of radiation after nuclear accidents, as well as secondary radiation risks after cancer radiotherapy or space missions. Here, diallyl disulfide (DADS), a component of garlic oil, was studied for its ability to mitigate radiation damage. MATERIAL AND METHODS We investigated the effects of DADS on micronucleus (MN) formation and apoptosis in HepG2 cells by use of 4-Gy X-ray irradiation. We also assessed the effects of DADS on radiation damage in vivo by evaluating MN formation in bone marrow cells in mice (BALB/c, 8-week-old females) after oral intake of DADS prior to irradiation with 4 Gy. Several tissue effects were also investigated. RESULTS The presence of DADS inhibited MN formation, whereas DADS had no influence on the radiation-induced inhibition of cell cycle progression in HepG2 cells. An increase in apoptosis in HepG2 cells was induced after irradiation, and this effect was stronger in the presence of DADS than in its absence. In mice, when DADS was administered daily for 3 days prior to irradiation, MN formation in irradiated mice was decreased. The decrease in MN formation in mice was greater with 0.5% DADS compared to 1% DADS. Moreover, an increase in spleen weight observed 3 weeks after irradiation was suppressed in mice administered DADS. CONCLUSIONS DADS is a potential radiation-protective agent that effectively mitigates DNA damage, and its effects in the spleen observed after irradiation may be related to inflammation and carcinogenesis.

Synergistic Autophagy Effect of miR-212-3p in Zoledronic Acid-Treated In Vitro and Orthotopic In Vivo Models and in Patient-Derived Osteosarcoma Cells.

Osteosarcoma (OS) originates from osteoid bone tissues and is prone to metastasis, resulting in a high mortality rate. Although several treatments are available for OS, an effective cure does not exist for most patients with advanced OS. Zoledronic acid (ZOL) is a third-generation bisphosphonate that inhibits osteoclast-mediated bone resorption and has shown efficacy in treating bone metastases in patients with various types of solid tumors. Here, we sought to clarify the mechanisms through which ZOL inhibits OS cell proliferation. ZOL treatment inhibited OS cell proliferation, viability, and colony formation. Autophagy inhibition by RNA interference against Beclin-1 or ATG5 inhibited ZOL-induced OS cell death. ZOL induced autophagy by repressing the protein kinase B/mammalian target of rapamycin/p70S6 kinase pathway and extracellular signal-regulated kinase signaling-dependent autophagy in OS cell lines and patient-derived OS cells. Microarrays of miRNA showed that ZOL increased the levels of miR-212-3p, which is known to play an important role in autophagy, in OS in vitro and in vivo systems. Collectively, our data provided mechanistic insight into how increased miR-212-3p through ZOL treatment induces autophagy synergistically in OS cells, providing a preclinical rationale for conducting a broad-scale clinical evaluation of ZOL + miR-212-3p in treating OS.

High LET Radiation Overcomes In Vitro Resistance to X-Rays of Chondrosarcoma Cell Lines.

Chondrosarcomas are malignant tumors of the cartilage that are chemoresistant and radioresistant to X-rays. This restricts the treatment options essential to surgery. In this study, we investigated the sensitivity of chondrosarcoma to X-rays and C-ions in vitro. The sensitivity of 4 chondrosarcoma cell lines (SW1353, CH2879, OUMS27, and L835) was determined by clonogenic survival assays and cell cycle progression. In addition, biomarkers of DNA damage responses were analyzed in the SW1353 cell line. Chondrosarcoma cells showed a heterogeneous sensitivity toward irradiation. Chondrosarcoma cell lines were more sensitive to C-ions exposure compared to X-rays. Using D10 values, the relative biological effectiveness of C-ions was higher (relative biological effectiveness = 5.5) with cells resistant to X-rays (CH2879) and lower (relative biological effectiveness = 3.7) with sensitive cells (L835). C-ions induced more G2 phase blockage and micronuclei in SW1353 cells as compared to X-rays with the same doses. Persistent unrepaired DNA damage was also higher following C-ions irradiation. These results indicate that chondrosarcoma cell lines displayed a heterogeneous response to conventional radiation treatment; however, treatment with C-ions irradiation was more efficient in killing chondrosarcoma cells, compared to X-rays.

ROS Production and Distribution: A New Paradigm to Explain the Differential Effects of X-ray and Carbon Ion Irradiation on Cancer Stem Cell Migration and Invasion.

Although conventional radiotherapy promotes the migration/invasion of cancer stem cells (CSCs) under normoxia, carbon ion (C-ion) irradiation actually decreases these processes. Unraveling the mechanisms of this discrepancy, particularly under the hypoxic conditions that pertain in niches where CSCs are preferentially localized, would provide a better understanding of the origins of metastases. Invasion/migration, proteins involved in epithelial-to-mesenchymal transition (EMT), and expression of MMP-2 and HIF-1α were quantified in the CSC subpopulations of two head-and-neck squamous cell carcinoma (HNSCC) cell lines irradiated with X-rays or C-ions. X-rays triggered HNSCC-CSC migration/invasion under normoxia, however this effect was significantly attenuated under hypoxia. C-ions induced fewer of these processes in both oxygenation conditions. The differential response to C-ions was associated with a lack of HIF-1α stabilization, MMP-2 expression, or activation of kinases of the main EMT signaling pathways. Furthermore,we demonstrated a major role of reactive oxygen species (ROS) in the triggering of invasion/migration in response to X-rays. Monte-Carlo simulations demonstrated that HO● radicals are quantitatively higher after C-ions than after X-rays, however they are very differently distributed within cells. We postulate that the uniform distribution of ROS after X-rays induces the mechanisms leading to invasion/migration, which ROS concentrated in C-ion tracks are unable to trigger.

Bystander effectors of chondrosarcoma cells irradiated at different LET impair proliferation of chondrocytes.

While the dose-response relationship of radiation-induced bystander effect (RIBE) is controversial at low and high linear energy transfer (LET), mechanisms and effectors of cell-to-cell communication stay unclear and highly dependent of cell type. In the present study, we investigated the capacity of chondrocytes in responding to bystander factors released by chondrosarcoma cells irradiated at different doses (0.05 to 8 Gy) with X-rays and C-ions. Following a medium transfer protocol, cell survival, proliferation and DNA damages were quantified in bystander chondrocytes. The bystander factors secreted by chondrosarcoma cells were characterized. A significant and major RIBE response was observed in chondrocyte cells (T/C-28a2) receiving conditioned medium from chondrosarcoma cells (SW1353) irradiated with 0.1 Gy of X-rays and 0.05 Gy of C-ions, resulting in cell survivals of 36% and 62%, respectively. Micronuclei induction in bystander cells was observed from the same low doses. The cell survival results obtained by clonogenic assays were confirmed using impedancemetry. The bystander activity was vanished after a heat treatment or a dilution of the conditioned media. The cytokines which are well known as bystander factors, TNF-α and IL-6, were increased as a function of doses and LET according to an ELISA multiplex analysis. Together, the results demonstrate that irradiated chondrosarcoma cells can communicate stress factors to non-irradiated chondrocytes, inducing a wide and specific bystander response related to both doses and LET.

Increased Hematopoietic Stem Cells/Hematopoietic Progenitor Cells Measured as Endogenous Spleen Colonies in Radiation-Induced Adaptive Response in Mice (Yonezawa Effect).

The existence of radiation-induced adaptive response (AR) was reported in varied biosystems. In mice, the first in vivo AR model was established using X-rays as both the priming and the challenge doses and rescue of bone marrow death as the end point. The underlying mechanism was due to the priming radiation-induced resistance in the blood-forming tissues. In a series of investigations, we further demonstrated the existence of AR using different types of ionizing radiation (IR) including low linear energy transfer (LET) X-rays and high LET heavy ion. In this article, we validated hematopoietic stem cells/hematopoietic progenitor cells (HSCs/HPCs) measured as endogenous colony-forming units-spleen (CFU-S) under AR inducible and uninducible conditions using combination of different types of IR. We confirmed the consistency of increased CFU-S number change with the AR inducible condition. These findings suggest that AR in mice induced by different types of IR would share at least in part a common underlying mechanism, the priming IR-induced resistance in the blood-forming tissues, which would lead to a protective effect on the HSCs/HPCs and play an important role in rescuing the animals from bone marrow death. These findings provide a new insight into the mechanistic study on AR in vivo.

Effects of carbon ion beam alone or in combination with cisplatin on malignant mesothelioma cells in vitro.

Malignant mesothelioma (MM) is extremely aggressive and a typical refractory cancer. In this study we investigated how effective on killing MM cells by carbon ion beam alone or in combination with cisplatin (CDDP) in vitro. Carbon ion beam (at the center of SOBP with 50 keV/µm of average LET) dose-independently suppressed MM cells MESO-1 and H226 cell viability and in combination with CDDP (25 µM) significantly enhanced its action. Relative biological effectiveness (RBE) values at 73 keV/µm and 13 keV/µm portion of carbon ion beam was estimated as 2.82-2.93 and 1.19-1.22 at D10 level relative to X-ray, respectively by using colony formation assay. Quantitative real time PCR analysis showed that expression of apoptosis-related BAX and autophagy-related Beclin1 and ATG7 was significantly enhanced by carbon ion beam alone or in combination with CDDP. Apoptosis analysis showed that caspase 3/7 activity and the percentage of apoptotic cells was dose-dependently increased after carbon ion beam and it was further increased when combined with CDDP. Spheroid formation ability of cancer stem like CD44+/CD26+ cells was significantly inhibited by carbon ion beam combined with CDDP. Besides, carbon ion beam combined with cisplatin significantly inhibited cell cycle progression (sub-G1 arrest) and induced more large number of γH2AX foci. In conclusion, carbon ion beam combined with CDDP has superior potential to kill MM cells including CSCs with enhanced apoptosis.