Effects of Low Dose Radiation on Tumor Apoptosis, Cell Cycle and Apoptosis-Related Protein Bcl-2 in Tumor-Bearing Mice

To study the effects of low dose radiation (LDR) on tumor apoptosis, cellcycle progression and changes of apoptosis-related protein Bcl-2 in tumor-bearing mice. Methods:Male mice of Kunming strain were implanted subcutaneously with S180 sarcoma cells in the left inguenas an in situ experimental animal model. Seven days later, the mice were subjected to 75 mGywhole-body γ-irradiation. At 24 and 48 h after the irradiation, all mice were sacrificed. The tumorsizes were measured, and tumor cell apoptosis and cell cycle progression were analyzed by flowcytometry. The expression of apoptosis-related protein Bcl-2 and the apoptotic rate of tumor cellswere observed by immunohistochemistry and electron microscopy. Results: Tumors grew significantlyslower after LDR (P 【 0.05). The tumor cells were arrested in G1 phrase and the expression of Bcl-2protein decreased at 24 h. Apoptotic rate of tumor cells was increased significantly at 48 h afterLDR (P 【 0.01). Conclusion: LDR could cause a G1-phase arrest and increase the apoptosis of tumorcells through the low level of apoptosis-related protein bcl-2 in the tumor-bearing mice. Theorganized immune function and anti-tumor ability are markedly increased after LDR. Our studyprovides practical evidence of clinical application to cancer treatment.

Involvement of Endoplasmic Reticulum Stress in Apoptosis of Testicular Cells Induced by Low-dose Radiation

Summary： The study examined the role of endoplasmic reticulum stress （ERS） and signaling pathways of inositol-requiring enzyme-1 （IRE1）, RNA-activated protein kinase-like ER kinase （PERK） and activating transcription factor-6 （ATF6） in apoptosis of mouse testicular cells treated with low-dose radiation （LDR）. In the dose-dependent experiment, the mice were treated with whole-body X-ray irradiation at different doses （25, 50, 75, 100 or 200 mGy） and sacrificed 12 h later. In the time-dependent experiment, the mice were exposed to 75 mGy X-ray irradiation and killed at different time points （3, 6, 12, 18 or 24 h）. Testicular cells were harvested for experiments. H202 and NO concentrations, and Ca2＋-ATPase activity were detected by biochemical assays, the calcium ion concentration （[Ca2＋]i） by flow cytometry using fluo-3 probe, and GRP78 mRNA and protein expressions by quantitative real-time RT-PCR （qRT-PCR） and Western blotting, respectively. The mRNA expressions of S-XBP1, JNK, caspase-12 and CHOP were measured by qRT-PCR, and the protein expressions of IREla, S-XBP1, p-PERK, p-elF2a, ATF6 p50, p-JNK, pro-caspase-12, cleaved caspase-12 and CHOP by Western blot- ting. The results showed that the concentrations of H202 and NO, the mR_NA expressions of GRP78, S-XBP1, JNK, caspase-12 and CHOP, and the protein expressions of GRP78, S-XBP1, IREla, p-PERK, p-elF2a, ATF6 p50, p-JNK, pro-caspase-12, cleaved caspase-12 and CHOP were significantly increased in a time- and dose-dependent manner after LDR. But the [Ca2]i and Ca2-ATPase activities were sig nificantly decreased in a time and dose-dependent manner. It was concluded that the ERS, regulated by IRE 1, PERK and ATF6 pathways, is involved in the apoptosis of testicular cells in LDR mice, which is associated with ERS-apoptotic signaling molecules of JNK, caspase-12 and CHOP.

INVOLVEMENT OF THE Ca^(2+) PROTEIN KINASE C AND ADENYLATE CYCLACE SIGNAL PATHWAYS IN THE ACTIVATION OF THYMOCYTES IN RESPONSE TO WHOLE BODY IRRADIATION WITH LOW DOSE X RAYS

WT5”BZ] To study the molecular mechanism of the stimulatory effect of low dose radiation(LDR) on T cell activation. [WT5”BX]Methods.[WT5”BZ] Thymocytes from Kunming mice exposed to whole body irradiation(WBI) with different doses of X rays were analyzed for the changes in signal molecules of the phospholipase C phosphatidylinositol biphosphate(PLC IP2) and G protein adenylate cyclase(AC) pathways. [WT5”BX]Results.[WT5”BZ]It was found that[Ca 2+ ] i increased in response to doses within 0 2 Gy which was most marked after 0 075 Gy and the increase was accentuated in the presence of Con A. The changes in CD3 and calcineurin(CN) expression of the thymocytes followed the same pattern as the alterations in [Ca 2+ ] i after LDR. The expression of α,β1 and β2 isoforms of protein kinase C(PKC) was all up regulated after 0 075 Gy with the increase in PKC β1 expression being most marked. The cAMP/cGMP ratio and PKA activity of the thymocytes was lowered after low dose radiation and increased after doses above 0 5 Gy in a dose dependent manner, thus giving rise to J shaped dose response curves. The Ca antagonist TMB 8 and cAMP stimulant cholera toxin suppressed the augmented thymocyte proliferation induced by LDR. [WT5”BX]Conclusion.[WT5”BZ]Data presented in the present paper suggest that activation of the PLC PIP2 signal pathway and suppression of the AC cAMP signal pathway are involved in the stimulation of the thymocytes following WBI with low dose X rays.

Increased Levels of p53 and PARP-1 in EL-4 Cells Probably Related with the Immune Adaptive Response Induced by Low Dose Ionizing Radiation in vitro

Objective This paper is to explore the DNA repair mechanism of immune adaptive response （AR） induced by low dose radiation （LDR）, the changes of mRNA levels and protein expressions of p53, ATM, DNA-PK catalytic subunit （DNA-PKcs） and PARP-1 genes in the LDR-induced AR in EL-4 cells. Methods The apoptosis and cell cycle progression of EL-4 cells were detected by flow cytometry in 12 h after the cells received the pre-exposure of 0.075 Gy X-rays （inductive dose, D 1） and the succeeding high dose irradiation （challenge dose, D2; 1.0, 1.5, and 2.0 Gy X-rays, respectively） with or without wortmannin （inhibitor of ATM and DNA-PK） and 3-aminobenzamid （inhibitor of PARP-1）. And the protein expressions and mRNA levels related to these genes were detected with flow cytometry and reverse transcription-polymerase chain reaction in 12 h after irradiation with D2. Results The mRNA and protein expressions of p53 and PARP-1 in EL-4 cells in the D1 ＋ D2 groups were much lower than those in the D2 groups, and those of PARP-1 in the 3-AB ＋ D2 and the 3-AB ＋ D1 ＋ D2 groups were much lower than those in the D2 and the D1 ＋ D2 groups. The percentage of apoptotic EL-4 cells in the 3-AB ＋ D1 ＋ D2 groups was much higher than that in the D1 ＋ D2 groups, that in the G0/G1 and the G2 ＋ M phases was much higher, and that in the S phase were much lower. Although the ATM and DNA-PKcs mRNA and protein expressions in wortmannin ＋ D1 ＋ D2 groups were much lower than those in the D1 ＋ D2 groups, there were no significant changes in the apoptosis and cell cycle progression between the wortmannin ＋ D1 ＋ D2 and the D1 ＋ D2 groups. Conclusion PARP-1 and p53 might play important roles in AR induced by LDR.

Effect of Cycloheximide on the Adaptive Response Induced by Low Dose Radiation

Human lymphocytes pre-exposed to 10 mGy or 50 mGy of X-rays become less sensitive to subsequent large dose irradiation, exhibited lower rate of chromosome aberration than expected. This adaptive response could be inhibited by cycloheximide, a protein synthesis inhibitor for successive 2 h period ranging from 0.5h before to 4h after the low dose exposure, indicating that the adaptive response was directly related with the protein synthesis.

Effect of Low Dose Radiation on Intracellular Calcium and Protein Kinase C in Lymphocytes

It is first reported in the present paper that whole-body irradiation (WBI) with low dose X-rays could increase intracellular calcium ions ([Ca2+]i) and stimulate protein kinase C (PKC) activity of mouse lymphocytes. Following WBI of male Kunming micc With 75 mGy X-rays at a dose rate of 12.5 mGy/min the mobilization of [Ca2+]i with Con A in CD4+ and CD8+ Cells in the thymus and spleen was potentiated and the amplitude of [Ca2+], mobilization in thymocytes in response to anti-CD3 monoclonal antibody increased with time from 4 to 24 h following low dose radiation. The PKC activity in the homogenate of spleen was markedly stimulated 12 h after WBl with 75 mGy, reaching its peak value at 24-48 h and coming down to lower than normal on day 7. However, the PKC activity in the separated T lymphocytes reached its peak value at 12 h and that in the B lymphocytes reached its peak value on day 4, both coming down to below control on day 7. The implications of this facilitation of signal transduction in T lymphocytes in the mechanism of immunoenhancement after low dose radiation were discussed

Modulation of MMP-2 and TIMP-2 by low dose radiation in mice bearing S180 sarcoma

Objective To investigate the inhibition of low dose radiation （LDR） on S180 sarcomas and its modulation of MMP-2 and TIMP-2 in mice. Methods $180 subcutaneously implanted tumor model mice were randomly divided into two groups： control （N） and low dose radiation （LDR） groups. N mice were sacrificed after 12 h, whereas LDR mice were sacrificed after 12 （LDR-12 h）, 24 （LDR-24 h）, 48 （LDR-48 h）, and 72 （LDR-72 h） h. Thereafter, we measured the tumor volumes. Histopathology was performed, and P-V immunohistochemistry was applied to assess MMP-2 and TIMP-2 expression. Results Compared with the control group, the tumor growth was significantly inhibited in the LDR groups （P 〈 0.05）. MMP-2 expression was considerably reduced in LDR-24h （P 〈 0.05） and LDR-48h （P 〈 0.05）, whereas the change of TIMP-2 was not obvious in the LDR groups （P 〉 0.05） in contrast to that of the control group. Conclusion LDR can effectively suppress the growth of S180 implanted tumors by reducing MMP-2, which is associated with invasion and metastasis.

NK ACTIVITY OF LYMPHOCYTE SUBSETS AND THE EFFECTS OF LOW DOSE RADIATION

This work was supported by the National Natural Science Foundation of China (No. 3870902). Objective: To determine the NK activity of lymphocyte subsets and the effects of low dose radiation. Methods: Lymphocyte subsets were separated by monoclonal antibodies. The NK activity of each subset on tumor cells was detected by radioactive release method. Results: The results showed that besides NK cells, CD 4, CD 8 and B cells alone can kill tumor cells. But the cellkilling activity of NK cells appeared to be strongest. There was synergistic effect between CD 4 and NK cells. The activity of mixed lymphocytes was more than that of only one subset. The effect of low dose radiation (LDR) on NK activity of panlymphocytes or NK cells was different. Conclusion: This paper demonstrated that NK activity of mononuclear cells was called “NK activity of lymphocytes”, but it is not true. Only when NK cells were separated by monoclonal antibodies, its killer activity can be called “activity of NK cells”.

The effect of low dose radiation on cytokine correlated to injury of lungs induced by Bleomycin A5

Objective： The aim of this study was to explore the effect of low dose radiation on cytokine excreted by mice inbreathing of atomization of PYM. Methods： Kunming male mice were randomly divided into three groups： blank group, PYM group （P group）, low dose radiation ＋ PYM group （P ＋ L group）. Mice of P ＋ L group were given whole body low dose radiation 75 mGY, dose rate were 12.5 mGY/min. After 6 h, mice in both P ＋ L group and P group were given inbreathe of atomization of PYM, concentration was 2 mg/mL. Mice were sacrificed after the dl, d7, d14, d21 and d28, IL-6 were detected in alveolar irrigating solution. The tissue samples of mice lung were fixed in 10% formalin, TNF-α and TGF-β were analyzed by immuno- histochemistry. Results： Compared with P group, IL-6 in low dose radiation ＋ PYM group were lower, near to blank group, the difference had notable statistical significance on the dl and the d7, while on the d14, d21, d28, the difference had not statisti- cal significance. It suggested that low dose radiation could reduce the resection of IL-6 at the begin of lung injure induced by low dose radiation. The expression of TGF-β and TNF-α in P ＋ L group were lower than that in P group, but the difference in the two groups had statistical significance by gray analysis P 〈 0.05. Conclusion： In the early stage of lung injure caused by PYM in mice, low dose radiation of 75 mGY can reduce the secretion of IL-6, decrease the production of TGF-β and TNF-α.

The effect of low-dose total body irradiation on tumor-inhibition and signal transduction in tumor tissues of mice bearing S180 sarcoma

Objective： By studying the influence of low-dose total body irradiation to proliferating cell nuclear antigens （PCNA）, epidermal growth factor receptor （EGFR）, erythropoietin （EPO） and vascular endothelial growth factor receptor （VEGFR） of tumor tissues in mice bearing S180 sarcoma, to further explore the mechanism of low doses radiation. Methods：S180 sarcoma cells were implanted subcutaneously into 58 male Kunming mice. Randomly these mice were divided into sham-irradiation （S） group and low-dose radiation （LDR） group. 12 days after implantation, the mice in LDR group were once delivered 75 mGy total-body ^60Co y-ray irradiation, while the mice in S group were left without irradiation. Then the mice in LDR group were executed at 6 h （LDR-6h group）, 12 h （LDR-12 h group）, 24 h （LDR-24 h group）, 48 h （LDR-48 h group） and 72 h （LDR-72h group） after irradiation. Tumor tissues were weighed and histological observed. Immunohistochemical staining was used to detect the expression of PCNA, VEGF, EPO and VEGFR of tumor tissues. Results： Though there was no significant difference between LDR group and S group in tumor weight, after irradiation the expression of PCNA and EPO of tumor tissues in LDR group decreased with time. LDR-24h, LDR-48h and LDR-72h groups were all statistically significantly different from S group. The expression of EGFR and VEGFR also decreased, and LDR-24h group was the lowest （P 〈 0.05）. Conclusion： Seventy-two h after low-dose total body irradiation, there was no significant change in tumor size of mice bearing S180 sarcoma. Low-dose total body radiation decreased the expression of PCNA inhibiting tumor growth; reduced the expression of EGFR in tumor tissue impacting the signal transduction of tumor cells. The study also indicated that low-dose total body irradiation, within a certain period of time, can decrease the expression of hypoxia factor EPO and VEGFR, which may improve the situation of tumor hypoxia and radiosensitivity of tumor itself.

Combination therapy using low dose radiation,plasma exchange,and a robust antiviral may induce synergic beneficial interactions for mitigating severe COVID‐19

Recent studies confirm that current antiviral drugs are not fully effective on coronaviruses.Our research team introduced the idea of radiotherapy at low doses for coronavirus disease 2019(COVID-19)in March 2020.This issue later received much attention from many scientists around the world.Low dose radiation therapy exerts modulatory effects in acute inflammation,potentiate the anti-viral immune responses,and can be localized to the organ affected by inflammatory reactions,such as lungs.The findings of the first clinical trial show that low dose radiation therapy is safe,and reveal early promise of efficacy.Moreover,plasma exchange therapy may be a rescue therapy in severe COVID-19 patients,as it can quickly eliminate the cytokines and chemokines from the circulation.Remdesivir was introduced as an anti-virus agent that prevents viral RNA synthesis and coronavirus replication and reduces the viral load.If antiviral therapy is poor,the virus has ample opportunity to mutate and increase its fitness through adaptive mutations.Given this consideration,instead a single treatment,we preferentially recommend a combination therapy using low dose radiation therapy plasma exchange and a robust antiviral agent(not yet available).This review provides a viewpoint concerning the potential synergistic effects of low dose radiation therapy,plasma exchange therapy and robust antiviral agent in severe acute respiratory syndrome coronavirus type 2(SARS-CoV-2)-infected patients as a more effective strategy for possible treatment of severe COVID-19 patients.

EPIGENETIC NATURE OF RADIATION CARCINOGENESIS AT LOW DOSES

The present paradigm of radiation-induced carcinogenesis has been based on somatic mutation theory initiating a mutation in somatic cells derived from DNA damage by ionizing radiation. A non-threshold linear model has been assumed from high doses to low doses. As an alternative to this, we are proposing a new model that epigenetic changes induced by low dose irradiation may be a first step of carcinogenesis. Our preliminary data suggesting the epigenetic nature and reports on various stress response to ionizing radiation are presented.

Effects of low-dose radiation on tumor growth, erythrocyte immune function and SOD activity in tumor-bearing mice

Background Activating on mammalian and human body LDR is thought to induce adaptive response, enhance immune function and increase anti-tumor ability. This study was designed to assess the effect of low-dose radiation on tumor growth and on erythrocyte immune function and superoxide dismutase (SOD) activity in tumor-bearing mice. Methods Male Kunming mice were subcutaneously implanted with S 180 sarcoma cells in the right inguen to create an experimental in situ animal model. Six hours before implantation, the mice were given 75 mGy X-ray radiation, over the body. Tumor size was observed 5 days later while tumor volume was calculated every other day, allowing for the creation of a graph depicting tumor growth. Fifteen days after implantation, the mice were killed to measure tumor weight and observe the necrotic areas and the location of tumor-infiltrating lymphocytes (TILs). Erythrocyte immune function and SOD activity were also determined. Results Mice pre-exposed to low-dose radiation had a lower tumor formation rate than did those receiving no radiation (P<0.05). Tumor growth was significantly lower in the mice pre-exposed to low-dose radiation; after 15 days, the average tumor weight in the mice pre-exposed to low-dose radiation was also lower (P<0.05). Areas of tumor necrosis and infiltration of TILs were larger in the low-dose radiation group than in the non-radiation group. Erythrocyte immune function and SOD activity were higher in the low-dose radiation group than in the non-radiation group (P<0.05). Conclusion Low-dose radiation can markedly increase the anti-tumor ability of an organism and improve erythrocyte immune function and red blood cell SOD activity as well, suggesting that low-dose radiation might be useful in the clinical treatment of cancer.

SIGNAL TRANSDUCTION IN LYMPHOCYTES AFTER LOW DOSE RADIATION

Mice were given whole-body irradiation (WBI) with 75 mGy X-rays which had previously been found to stimulate immunologic functions. This low dose radiation (LDR) potentiated |Ca2+|i mobilization in thymic and splenic lymphocytes in response to Con A and anti-CD3 McAb and activated protein kinase C in T and B lymphocytes of the spleen. The expression of TCR / CD3 molecules on the thymocytes was en-chanced after LDR indicating an expedited maturation and differentiation process in the thymus. The changes in TCR / CD3 expression and |Ca2-|i mobilization in response to McAb-CD3 after LDR was found to be highly correlated. Meanwhile the transcription of c-fos and c-jun genes was up-regulated beginning 3 hours after LDR. The expression of IL2R in actived thymocytes was potentiated 24 hours after LDR which coincided with the previous finding of increased secretion of IL2 by splenocytes after WBI with 75 mGy X-rays. It is first reported in the present paper that LDR could stimulate immunologic functions through facilitation of the signal transduction process in the lymphocytes.

COVID-19 and low-dose radiation therapy

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the causative pathogen of the coronavirus disease 2019(COVID-19),has caused more than 179 million infections and 3.8 million deaths worldwide.Global health authorities working on the COVID-19 outbreak continue to explore methods to reduce the rate of its transmission to healthy individuals.Treatment protocols thus far have focused on social distancing and masking,treatment with antivirals early in infection,and steroids to reduce the inflammatory response.An alternative approach is therapy with low dose radiation(LDR),which has several advantages compared to the current drugs and medicines.To date more than 10 case reports and pilot clinical trial preliminary outcome are available from different countries.These reports cover a wide range of patient conditions and LDR treatment strategies.Although one report showed the failure to observe the improvement of COVID-19 patients after LDR therapy,the majority showed some clinical improvement,and demonstrated the safety of LDR for COVID-19 patients,particularly with 0.5 Gy.This review aims to summarize the potential rationales and mechanisms of LDR therapy for COVID-19 patients,and its current clinical status and potential use.