Single neonatal irradiation induces long-term gene expression changes in the thyroid gland, which may be involved in the tumorigenesis

Exposure to ionizing radiation in childhood has been recognized as a risk factor for thyroid cancer. We previously demonstrated that neonatal X-irradiation induced specific deformation of the thyroid follicles. Here, we further analyzed this model to understand the possible relationship with thyroid carcinogenesis. Wistar rats were subjected to cervical X-irradiation at different ages of 1–8 weeks old and at different doses of 1.5–12 Gy. For tumor promotion, rats were fed with an iodine-deficient diet (IDD). In cervically X-irradiated neonatal rats, the size of thyroid follicles decreased, accompanied by an increase in the number of TUNEL-positive cells. Fas and Lgals3 mRNA levels increased, while Mct8 and Lat4 expressions decreased. The co-administration of IDD induced the proliferation and the upregulation in Lgals3 expression, resulting in thyroid adenoma development at 28 weeks post-exposure. Our data demonstrated that single neonatal X-irradiation induced continuous apoptotic activity in the thyroid with the long-term alternation in Fas, Mct8, Lat4, and Lgals3 mRNA expressions. Some of these changes were similar to those induced by IDD, suggesting that neonatal X-irradiation may partially act as a thyroid tumor promoter. These radiation-induced thyroidal changes may be enhanced by the combined treatment with IDD, resulting in the early development of thyroid adenoma.

RPRM negatively regulates ATM levels involving its phosphorylation mediated by CDK4/CDK6

Sensitizing cancer cells to radio- and chemotherapy remains a hot topic in cancer treatment. Here it is identified that Protein Reprimo (RPRM) negatively regulates the levels of ataxia-telangiectasia mutated (ATM) protein kinase, a master regulator of DNA damage response (DDR) in the presence of DNA double-strand breaks (DSBs), resulting in impaired DNA repair efficiency and enhanced cellular sensitivity to genotoxic agents. Mechanistically, although RPRM is primarily located in cytoplasm, it rapidly translocates to nucleus shortly after induced by X-irradiation, interacts with ATM and promotes the nuclear export and proteasomal degradation of ATM. The nuclear translocation of RPRM is associated with its phosphorylation at serine 98, which is mediated by cyclin-dependent kinases 4/6 (CDK4/6). Inhibition of CDK4/6 stabilizes RPRM and promotes its nuclear import, in turn enhances the nuclear export of ATM and the reduction of ATM levels. As a result, RPRM overexpression and its phosphorylation inhibition sensitize cells to genotoxic agents. Moreover, RPRM deficiency significantly increases resistance to radiation-induced damage both in vitro and in vivo. These findings establish a crucial regulatory mechanism in which ATM is negatively modulated by RPRM, suggesting that RPRM may serve as a novel target for both cancer therapy and radiation protection.

Structural and functional state of Heren carcinoma after local fractional X-irradiation and irradiation combined with meloxicam

Background. One of the most important problems of oncology is the overcoming of therapeutic resistance of tumors, which occurs in particular due to increased levels of the enzyme cyclooxygenase 2 (COX-2). It is known that the growth of COX-2 and the product of its activity, prostaglandin-E2 in cancer, promotes such processes in the body as tumor growth, stimulation of proliferation, induction of cancer stem cells, inhibition of apoptosis, activation of angiogenesis, invasion, metastasis and development of chemoresistance. The use of COX-2 inhibitors, which are nonsteroidal anti-inflammatory drugs (NSAIDs), significantly limits these processes and improves survival and mortality in cancer patients, and in combination with chemotherapeutics eliminates the resistance they cause. Purpose – study of the structural and functional state of Guerin’s carcinoma cells after the combined use of nonsteroidal anti-inflammatory drug meloxicam and local X-irradiation in total doses of 1.0 and 10 Gy. Materials and methods. On 33 rats with inoculated Guerin’s carcinoma, the ultrastructure of tumor cells (TC) was studied using standard methods of electron microscopy 24 hours after the combined use of the meloxicam drug at a dose of 0.2 mg per 1 kg of body weight one day before the first and 2 hours before the second session fractional local X-irradiation in total doses of 1 and 10 Gy (twice daily at 0.5 and 5.0 Gy, respectively. The mitotic index (the number of cells in the state of mitosis per 100 TC,%), the apoptosis index (the number of cells in the state of apoptotic death per 100 TC,%) and the frequency of TC with small nuclei (%). Results. It was found that irradiation of Guerin’s tumor in a total dose of 10 Gy causes disturbances in the ultrastructure associated with damage to the nuclear apparatus of the TC. Pleiomorphism of the nuclei, the appearance of binucleated cells and micronuclei, a significant decrease in mitotic activity and a slight increase in the apoptosis index are observed. Stimulation of the functional activity of macrophages is also noted. Under irradiation in a total dose of 1 Gy, such effects are less pronounced or completely absent, such as, for example, the processes of phagocytosis. The frequency index of TC with small nuclei is equally reliably increased at both radiation doses. The administration of the drug meloxicam leads to a significant decrease in mitotic activity and an increase in the frequency of small cells, while the ultrastructural picture of the tumor remains almost unchanged. With the combined action of the drug and radiation in both doses, violations of the fine structure of the OC are identical to those found during irradiation. At the same time, the mitotic index in the group with the combined effect of the drug and radiation at a dose of 10 Gy is significantly lower than with only irradiation.In addition, at both doses, the frequency of small forms of PC significantly increases in comparison with the indicators of both the intact control group and the corresponding irradiation groups. Only in combination with radiation does meloxicam reliably stimulate apoptosis, while in other groups its index remains at the level of control values. The relationship was confirmed, which was constantly revealed in all experimental groups, between a decrease in the level of the mitotic index and an increase in the frequency of TC with small nuclei in Guerin’s carcinoma. An inverse correlation was found between these indicators (r = 0.80, P = 0.05). Conclusions. The combined action of the drug and irradiation significantly increases the effectiveness of both therapeutic factors due to the property of meloxicam to reliably inhibit proliferative activity and promote post-radiation development of apoptosis in tumor tissue. The presence of a correlation between the mitotic index and the frequency of cells with small nuclei in Guerin’s tumor may indicate the relationship between cell growth and division. Under the combined action of both investigated factors, changes in the tumor ultrastructure are mainly caused by irradiation. The administration of meloxicam increases the efficiency of the combined use of both therapeutic agents due to its ability to reliably inhibit proliferative activity and promote post-radiation development of apoptosis in tumor tissue. The presence of a correlation dependence between the mitotic index and the frequency of cells with small nuclei in Guerin’s tumor may indicate the relationship between the processes of cell growth and division.

Assessment of Mutations Induced by Cold Atmospheric Plasma Jet Treatment Relative to Known Mutagens in Escherichia coli

The main bactericidal components of cold atmospheric plasma (CAP) are thought to be reactive oxygen and nitrogen species (RONS) and UV radiation, both of which have the capacity to cause DNA damage and mutations. Here, the mutagenic effects of CAP on Escherichia coli were assessed in comparison to X- and UV-irradiation. DNA damage and mutagenesis were screened for using a diffusion-based DNA fragmentation assay and modified Ames test respectively. Mutant colonies obtained from the latter were quantitated and sequenced. CAP was found to elicit a similar mutation spectrum to X-irradiation, that did not resemble that for UV implying that CAP produced RONS are more likely the mutagenic component of CAP. CAP treatment was also shown to promote resistance to the antibiotic ciprofloxacin. Our data suggest that CAP treatment has mutagenic effects that may have important phenotypic consequences.

Altered Induction of Reactive Oxygen Species by X-rays in Hematopoietic Cells of C57BL/6-Tg (CAG-EGFP) Mice

Previous work pointed to a critical role of excessive production of reactive oxygen species (ROS) in increased radiation hematopoietic death in GFP mice. Meanwhile, enhanced antioxidant capability was not demonstrated in the mouse model of radio-induced adaptive response (RAR) using rescue of radiation hematopoietic death as the endpoint. ROS induction by ex vivo X-irradiation at a dose ranging from 0.1 to 7.5 Gy in the nucleated bone marrow cells was comparatively studied using GFP and wild type (WT) mice. ROS induction was also investigated in the cells collected from mice receiving a priming dose (0.5 Gy) efficient for RAR induction in WT mice. Significantly elevated background and increased induction of ROS in the cells from GFP mice were observed compared to those from WT mice. Markedly lower background and decreased induction of ROS were observed in the cells collected from WT mice but not GFP mice, both receiving the priming dose. GFP overexpression could alter background and induction of ROS by X-irradiation in hematopoietic cells. The results provide a reasonable explanation to the previous study on the fate of cells and mice after X-irradiation and confirm enhanced antioxidant capability in RAR. Investigations involving GFP overexpression should be carefully interpreted.

Ultrafiltration Extract of Radix Angelica Sinensis and Radix Hedysari Attenuates Risk of Low-Dose X-Ray Radiation-Induced Myocardial Fibrosis In Vitro

The risk of radiation-induced heart damage (RIHD) is a growing concern since recent advances in radiation therapy (RT) for cancer treatments have significantly improved the number of survivors. Radiation-induced myocardial fibrosis (RIMF) is the final pathological condition of RIHD and main change leading to serious cardiovascular complications following RT. The aim of this study was to investigate the effect of ultrafiltration extract of Radix Angelica Sinensis and Radix Hedysari (RAS-RH) on the proliferation, apoptosis, and reactive oxygen species (ROS) of cardiac fibroblasts after X-irradiation in vitro. The RAS-RH extract was from the Danggui Buxue decoction (DBD) in TCM. Primary cardiac fibroblasts were irradiated with 1 Gy X-ray to evaluate the effect of RAS-RH on the expression levels of cell proliferation, apoptosis, ROS, and fibrotic molecules. Our data demonstrated that X-irradiation at 1 Gy resulted in the proliferation of cardiac fibroblasts; RAS-RH attenuated the myocardial fibrosis. Furthermore, X-ray radiation reduced the apoptosis of cardiac fibroblasts; RAS-RH accelerated the apoptosis of these cells after irradiation. In addition, the damage driven by ROS in primary cardiac fibroblasts after irradiation was weakened by RAS-RH and the expression of TGF-β1, Col1, and α-SMA increased after irradiation; RAS-RH decreased the expression of these makers. Overall, these data indicate that low-dose X-ray irradiation boosts myocardial fibrosis, and the effect of RAS-RH protects against fibrosis via attenuating the proliferation and accelerating the apoptosis of myocardial fibroblasts after X-irradiation.

Proof of Concept Study for Increasing Tenascin-C-Targeted Drug Delivery to Tumors Previously Subjected to Therapy: X-Irradiation Increases Tumor Uptake

In treatment-refractory cancers, tumor tissues damaged by therapy initiate the repair response; therefore, tumor tissues must be exposed to an additional burden before successful repair. We hypothesized that an agent recognizing a molecule that responds to anticancer treatment-induced tissue injury could deliver an additional antitumor agent including a radionuclide to damaged cancer tissues during repair. We selected the extracellular matrix glycoprotein tenascin-C (TNC) as such a molecule, and three antibodies recognizing human and murine TNC were employed to evaluate X-irradiation-induced changes in TNC uptake by subcutaneous tumors. TNC expression was assessed by immunohistochemical staining of BxPC-3 tumors treated with or without X-irradiation (30 Gy) for 7 days. Antibodies against TNC (3–6, 12–2–7, TDEAR) and a control antibody were radiolabeled with 111In and injected into nude mice having BxPC-3 tumors 7 days after X-irradiation, and temporal uptake was monitored for an additional 4 days by biodistribution and single-photon emission computed tomography with computed tomography (SPECT/CT) studies. Intratumoral distribution was analyzed by autoradiography. The immunohistochemical signal for TNC expression was faint in nontreated tumors but increased and expanded with time until day 7 after X-irradiation. Biodistribution studies revealed increased tumor uptake of all three 111In-labeled antibodies and the control antibody. However, a statistically significant increase in uptake was evident only for 111In-labeled 3–6 (35% injected dose (ID)/g for 30 Gy vs. 15% ID/g for 0 Gy at day 1, p < 0.01), whereas limited changes in 111In-labeled TDEAR2, 12–2–27, and control antibody were observed (several % ID/g for 0 and 30 Gy). Serial SPECT/CT imaging with 111In-labeled 3–6 or control antibody provided consistent results. Autoradiography revealed noticeably stronger signals in irradiated tumors injected with 111In-labeled 3–6 compared with each of the nonirradiated tumors and the control antibody. The signals were observed in TNC-expressing stroma. Markedly increased uptake of 111In-labeled 3–6 in irradiated tumors supports our concept that an agent, such as an antibody, that recognizes a molecule involved in tissue injury repair, such as TNC, could enhance drug delivery to tumor tissues that have undergone therapy. The combination of antibody 3–6 coupled to a tumoricidal drug and conventional therapy has the potential to achieve better outcomes for patients with refractory cancer.