Synergism of non-thermal plasma and low concentration RSL3 triggers ferroptosis via promoting xCT lysosomal degradation through ROS/AMPK/mTOR axis in lung cancer cells.

BACKGROUND: Though (1S, 3R)-RSL3 has been used widely in basic research as a small molecular inducer of ferroptosis, the toxicity on normal cells and poor pharmacokinetic properties of RSL3 limited its clinical application. Here, we investigated the synergism of non-thermal plasma (NTP) and low-concentration RSL3 and attempted to rise the sensitivity of NSCLC cells on RSL3. METHODS: CCK-8 assay was employed to detect the change of cell viability. Microscopy and flowcytometry were applied to identify lipid peroxidation, cell death and reactive oxygen species (ROS) level respectively. The molecular mechanism was inspected with western blot and RT-qPCR. A xenograft mice model was adopted to investigate the effect of NTP and RSL3. RESULTS: We found the synergism of NTP and low-concentration RSL3 triggered severe mitochondria damage, more cell death and rapid ferroptosis occurrence in vitro and in vivo. NTP and RSL3 synergistically induced xCT lysosomal degradation through ROS/AMPK/mTOR signaling. Furthermore, we revealed mitochondrial ROS was the main executor for ferroptosis induced by the combined treatment. CONCLUSION: Our research shows NTP treatment promoted the toxic effect of RSL3 by inducing more ferroptosis rapidly and provided possibility of RSL3 clinical application.

Exogenous carbon monoxide promotes GPX4-dependent ferroptosis through ROS/GSK3ß axis in non-small cell lung cancer.

The gas therapy is drawing increasing attention in the treatment of many diseases including cancer. As one of gas signaling molecules, carbon monoxide (CO) has been proved to exert anti-cancer effects via triggering multiple cell death types, such as autophagy, apoptosis and necrosis. Here, we showed that low concentration CO delivered from CO-releasing molecule 3 (CORM-3) effectively induced ferroptosis, known as a novel proinflammatory programmed cell death, in vitro and in vivo. Mechanistically, we found that CO triggered ferroptosis by modulating the ROS/GSK3ß/GPX4 signaling pathway, resulting in the accumulation of lipid hydroperoxides and the occurrence of ferroptosis. We think our findings provide novel insights into the anti-cancer mechanisms of CO, and suggest that CO could potentially be exploited as a novel ferroptosis inducer for cancer treatment in the future.

Ferroptosis triggered by STAT1- IRF1-ACSL4 pathway was involved in radiation-induced intestinal injury.

Radiation-induced intestinal injury (RIII), a common gastrointestinal complication caused by radiotherapy on pelvic, abdominal and retroperitoneal tumors, seriously affects the life quality of patients and may result in termination of radiotherapy. At present, the pathogenesis of RIII has not been fully understood. Herein, we demonstrated that ferroptosis played a critical role in RIII occurrence. The RNA sequencing analysis strongly hinted ferroptosis was involved in RIII mice. In line with this, the levels of 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), markers of lipid peroxidation, remarkably increased in RIII mice. And the ferroptosis inhibitor, Ferrostatin-1 (Fer-1), improved the mice survival and alleviated intestinal fibrosis in vivo. Moreover, our results revealed that arachidonic acid (AA) enhanced ferroptosis in cultured intestinal epithelial cells (IECs) and organoids in vitro after irradiation, and AA gavage aggravated RIII in mice. Mechanistic studies revealed the level of ACSL4 protein significantly increased in mouse jejunums and IECs after irradiation. Radiation-induced ferroptosis in IECs was also prevented following ACSL4 knockdown or with the function inhibitor of ACSL4. Furthermore, we found that transcription of ACSL4 induced by irradiation was regulated by STAT1/IRF1 axis, and AMPK activation triggered by AA negatively regulated radiation-induced ferroptosis. Taken together, our results suggest that ferroptosis mediates RIII and reducing dietary AA intake as well as targeting the STAT1-IRF1-ACSL4 axis or AMPK may be the potential approaches to alleviate RIII.

Ionizing Radiation Triggers the Antitumor Immunity by Inducing Gasdermin E-Mediated Pyroptosis in Tumor Cells.

PURPOSE: To understand pyroptosis induced by ionizing radiation and its implications for radiation therapy, we explored the involved factors, possible mechanisms of radiation-induced pyroptosis and consequent antitumor immunity. METHODS AND MATERIALS: The occurrence of pyroptosis was assessed by cell morphology, lactate dehydrogenase release, Annexin V/PI staining and the cleavage of Gasdermin E (GSDME). Cell radiosensitivity was tested with MTT and colony survival assays. Xenograft tumor volume, Ki-67, CD8+ lymphocytes, and ELISA were used to evaluate the effect of GSDME on tumor suppression after irradiation. RESULTS: Irradiation induced pyroptosis in GSDME high-expressing tumor cell lines covering lung, liver, breast, and glioma cancers. Cleavage of GSDME occurred in a dose- and time-dependent manner after irradiation, and pyroptosis could be induced by various kinds of irradiation. The combination of chemotherapy drugs for DNA damage (cisplatin or etoposide) or demethylation (decitabine or azacytidine) and irradiation significantly enhanced the occurrence of pyroptosis. Moreover, we revealed that the Caspase 9/Caspase 3/GSDME pathway was involved in irradiation-induced pyroptosis. Notably, enhanced tumor suppression was observed in Balb/c mice bearing GSDME-overexpressing 4T1 tumors compared with those bearing vector tumors for the promotion of antitumor immunity, which was manifested as distinctly elevated levels of cytotoxic T lymphocytes and release of the related cytokines rather than the direct effect of pyroptosis on tumor cell radiosensitivity. CONCLUSIONS: As an immunogenic cell death caused by radiation, pyroptosis promotes antitumor immunity after irradiation. Our findings may provide new insights to improve the efficacy of tumor radiation therapy.

Impact of COVID-19 on the sleep-wake patterns of preschool children.

OBJECTIVE: To explore the impact of the COVID-19 pandemic on the sleep-wake patterns of preschool children. METHODS: A cohort of preschoolers established before the COVID-19 pandemic was invited to participate in this study. Data including children's demographics, their own and parental sleep-wake patterns, physical activities, and screen time were collected through an online questionnaire from August to September 2020. A comparison was made on the collected data from the same cohort of children before and during the pandemic. RESULTS: The cohort which was established before the pandemic consisted of 3720 preschoolers. For this current study, 642 (17%) participated, and 497 (13%) children who fulfilled the eligibility criteria were included in the final analysis. They showed a delay in their bedtime and wake time on both weekdays and weekends with a 15-30 min increase in nocturnal sleep duration. However, with a reduction in nap time, the average daily sleep duration was shortened by 16.3 ± 64.3 min (p < 0.001) and 27.5 ± 72.9 min (p < 0.001) during weekdays and weekends, respectively. Screen time was increased while outdoor activity duration was decreased. Parental sleep/wake times were also delayed with an increase in sleep duration. Children's sleep habits were associated with screen time and parental sleep/wake patterns. CONCLUSION: Despite school suspension during the COVID-19 pandemic, preschoolers were not sleeping longer. Screen time and parental sleep/wake patterns were the major factors driving the preschoolers' sleep habits. Health education is required to control screen time in children and to promote sleep hygiene among all family members.

Golgi Phosphoprotein 3 Confers Radioresistance via Stabilizing EGFR in Lung Adenocarcinoma.

PURPOSE: Radioresistance is a major cause of treatment failure in tumor radiation therapy, and the underlying mechanisms of radioresistance are still elusive. Golgi phosphoprotein 3 (GOLPH3) has been reported to associate tightly with cancer progression and chemoresistance. Herein, we explored whether GOLPH3 mediated radioresistance of lung adenocarcinoma (LUAD) and whether targeted suppression of GOLPH3 sensitized LUAD to radiation therapy. METHODS AND MATERIALS: The aberrant expression of GOLPH3 was evaluated by immunohistochemistry in LUAD clinical samples. To evaluate the association between GOLPH3 and radioresistance, colony formation and apoptosis were assessed in control and GOLPH3 knockdown cells. γ-H2AX foci and level determination and micronucleus test were used to analyze DNA damage production and repair. The rescue of GOLPH3 knockdown was then performed by exogenous expression of small interfering RNA-resistant mutant GOLPH3 to confirm the role of GOLPH3 in DNA damage repair. Mechanistically, the effect of GOLPH3 on regulating stability and nuclear accumulation of epidermal growth factor receptor (EGFR) and the activation of DNA-dependent protein kinase (DNA-PK) were investigated by quantitative real-time polymerase chain reaction, western blot, immunofluorescence, and coimmunoprecipitation. The role of GOLPH3 in vivo in radioresistance was determined in a xenograft model. RESULTS: In tumor tissues of 33 patients with LUAD, the expression of GOLPH3 showed significant increases compared with those in matched normal tissues. Knocking down GOLPH3 reduced the clonogenic capacity, impaired double-strand break (DSB) repair, and enhanced apoptosis after irradiation. In contrast, reversal of GOLPH3 depletion rescued the impaired repair of radiation-induced DSBs. Mechanistically, loss of GOLPH3 accelerated the degradation of EGFR in lysosome, causing the reduction in EGFR levels, thereby weakening nuclear accumulation of EGFR and attenuating the activation of DNA-PK. Furthermore, adenovirus-mediated GOLPH3 knockdown could enhance the ionizing radiation response in the LUAD xenograft model. CONCLUSIONS: GOLPH3 conferred resistance of LUAD to ionizing radiation via stabilizing EGFR, and targeted suppression of GOLPH3 might be considered as a potential therapeutic strategy for sensitizing LUAD to radiation therapy.

Hemin enhances radiosensitivity of lung cancer cells through ferroptosis.

The principle underlying radiotherapy is to kill cancer cells while minimizing the harmful effects on non-cancer cells, which has still remained as a major challenge. In relation, ferroptosis has recently been proposed as a novel mechanism of radiation-induced cell death. In this study, we investigated and demonstrated the role of Hemin as an iron overloading agent in the generation of reactive oxygen species (ROS) induced by ionizing radiation in lung cancer and non-cancer cells. It was found that the presence of Hemin in irradiated lung cancer cells enhanced the productivity of initial ROS, resulting in lipid peroxidation and subsequent ferroptosis. We observed that application of Hemin as a co-treatment increased the activity of GPx4 degradation in both cancer and normal lung cells. Furthermore, Hemin protected normal lung cells against radiation-induced cell death, in that it suppressed ROS after radiation, and boosted the production of bilirubin which was a lipophilic ROS antioxidant. In addition, we demonstrated significant FTH1 expression in normal lung cells when compared to lung cancer cells, which prevented iron from playing a role in increasing IR-induced cell death. Our findings demonstrated that Hemin had a dual function in enhancing the radiosensitivity of ferroptosis in lung cancer cells while promoting cell survival in normal lung cells.

Micheliolide Enhances Radiosensitivities of p53-Deficient Non-Small-Cell Lung Cancer via Promoting HIF-1α Degradation.

Micheliolide (MCL) has shown promising anti-inflammatory and anti-tumor efficacy. However, whether and how MCL enhances the sensitivity of non-small-cell lung cancer (NSCLC) to radiotherapy are still unknown. In the present paper, we found that MCL exerted a tumor cell killing effect on NSCLC cells in a dose-dependent manner, and MCL strongly sensitized p53-deficient NSCLC cells, but not the cells with wild-type p53 to irradiation (IR). Meanwhile, MCL markedly inhibited the expression of hypoxia-inducible factor-1α (HIF-1α) after IR and hypoxic exposure in H1299 and Calu-1 cells rather than in H460 cells. Consistently, radiation- or hypoxia-induced expression of vascular endothelial growth factor (VEGF) was also significantly inhibited by MCL in H1299 and Calu-1 cells, but not in H460 cells. Therefore, inhibition of the HIF-1α pathway might, at least in part, contribute to the radiosensitizing effect of MCL. Further study showed that MCL could accelerate the degradation of HIF-1α through the ubiquitin-proteosome system. In addition, the transfection of wild-type p53 into p53-null cells (H1299) attenuated the effect of MCL on inhibiting HIF-1α expression. These results suggest MCL effectively sensitizes p53-deficient NSCLC cells to IR in a manner of inhibiting the HIF-1α pathway via promoting HIF-1α degradation, and p53 played a negative role in MCL-induced HIF-1α degradation.

Multiple Stressor Effects of Radon and Phthalates in Children: Background Information and Future Research.

The present paper reviews available background information for studying multiple stressor effects of radon (222Rn) and phthalates in children and provides insights on future directions. In realistic situations, living organisms are collectively subjected to many environmental stressors, with the resultant effects being referred to as multiple stressor effects. Radon is a naturally occurring radioactive gas that can lead to lung cancers. On the other hand, phthalates are semi-volatile organic compounds widely applied as plasticizers to provide flexibility to plastic in consumer products. Links of phthalates to various health effects have been reported, including allergy and asthma. In the present review, the focus on indoor contaminants was due to their higher concentrations and to the higher indoor occupancy factor, while the focus on the pediatric population was due to their inherent sensitivity and their spending more time close to the floor. Two main future directions in studying multiple stressor effects of radon and phthalates in children were proposed. The first one was on computational modeling and micro-dosimetric studies, and the second one was on biological studies. In particular, dose-response relationship and effect-specific models for combined exposures to radon and phthalates would be necessary. The ideas and methodology behind such proposed research work are also applicable to studies on multiple stressor effects of collective exposures to other significant airborne contaminants, and to population groups other than children.

Targeting Nrf2-mediated heme oxygenase-1 enhances non-thermal plasma-induced cell death in non-small-cell lung cancer A549 cells.

Non-thermal plasma (NTP) treatment has been proposed as a potential approach for cancer therapy for killing cancer cells via generation of reactive oxygen species (ROS). As an antioxidant protein, Heme oxygenase-1 (HO-1) has been known to protect cells against oxidative stress. In this paper, we investigated the role of HO-1 activation in NTP-induced apoptosis in A549 cells. Distinctly increased ROS production and apoptosis were observed after NTP exposure. NTP exposure induced HO-1 expression in a dose- and time-dependent manner via activating the translocation of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) from cytoplasm to nucleus. Furthermore, inhibiting HO-1 activation with its specific inhibitor, ZnPP, increased "killing" effect of NTP. Knocking down HO-1 or Nrf2 with the special siRNA also led to elevated ROS level and enhanced NTP-induced cell death. In addition, the c-JUN N-terminal kinase (JNK) signaling pathway was shown to be involved in NTP-induced HO-1 expression. Interestingly, a higher resistance to NTP exposure of A549 cell compared to H1299 and H322 cells was found to be linked to its higher basal level of HO-1 expression. These findings revealed that HO-1 could be considered as a potential target to improve the effect of NTP in cancer therapy.

Monte Carlo studies on photon interactions in radiobiological experiments.

X-ray and γ-ray photons have been widely used for studying radiobiological effects of ionizing radiations. Photons are indirectly ionizing radiations so they need to set in motion electrons (which are a directly ionizing radiation) to perform the ionizations. When the photon dose decreases to below a certain limit, the number of electrons set in motion will become so small that not all cells in an "exposed" cell population can get at least one electron hit. When some cells in a cell population are not hit by a directly ionizing radiation (in other words not irradiated), there will be rescue effect between the irradiated cells and non-irradiated cells, and the resultant radiobiological effect observed for the "exposed" cell population will be different. In the present paper, the mechanisms underlying photon interactions in radiobiological experiments were studied using our developed NRUphoton computer code, which was benchmarked against the MCNP5 code by comparing the photon dose delivered to the cell layer underneath the water medium. The following conclusions were reached: (1) The interaction fractions decreased in the following order: 16O > 12C > 14N > 1H. Bulges in the interaction fractions (versus water medium thickness) were observed, which reflected changes in the energies of the propagating photons due to traversals of different amount of water medium as well as changes in the energy-dependent photon interaction cross-sections. (2) Photoelectric interaction and incoherent scattering dominated for lower-energy (10 keV) and high-energy (100 keV and 1 MeV) incident photons. (3) The fractions of electron ejection from different nuclei were mainly governed by the photoelectric effect cross-sections, and the fractions from the 1s subshell were the largest. (4) The penetration fractions in general decreased with increasing medium thickness, and increased with increasing incident photon energy, the latter being explained by the corresponding reduction in interaction cross-sections. (5) The areas under the angular distribution curves of photons exiting the medium layer and subsequently undergoing interactions within the cell layer became smaller for larger incident photon energies. (6) The number of cells suffering at least one electron hit increased with the administered dose. For larger incident photon energies, the numbers of cells suffering at least one electron hit became smaller, which was attributed to the reduction in the photon interaction cross-section. These results highlighted the importance of the administered dose in radiobiological experiments. In particular, the threshold administered doses at which all cells in the exposed cell array suffered at least one electron hit might provide hints on explaining the intriguing observation that radiation-induced cancers can be statistically detected only above the threshold value of ~100 mSv, and thus on reconciling controversies over the linear no-threshold model.

Non-thermal plasma induces mitochondria-mediated apoptotic signaling pathway via ROS generation in HeLa cells.

Non-thermal plasma (NTP) has been proposed as a novel therapeutic method for anticancer treatment. Although increasing evidence suggests that NTP selectively induces apoptosis in some types of tumor cells, the molecular mechanisms underlying this phenomenon remain unclear. In this study, we further investigated possible molecular mechanisms for NTP-induced apoptosis of HeLa cells. The results showed that NTP exposure significantly inhibited the growth and viability of HeLa cells. Morphological observation and flow cytometry analysis demonstrated that NTP exposure induced HeLa cell apoptosis. NTP exposure also activated caspase-9 and caspase-3, which subsequently cleaved poly (ADP- ribose) polymerase. Furthermore, NTP exposure suppressed Bcl-2 expression, enhanced Bax expression and translocation to mitochondria, activated mitochondria-mediated apoptotic pathway, followed by the release of cytochrome c. Further studies showed that NTP treatment led to ROS generation, whereas blockade of ROS generation by N-acetyl-l-cysteine (NAC, ROS scavengers) significantly prevented NTP-induced mitochondrial alteration and subsequent apoptosis of HeLa cells via suppressing Bax translocation, cytochrome c and caspase-3 activation. Taken together, our results indicated that NTP exposure induced mitochondria-mediated intrinsic apoptosis of HeLa cells was activated by ROS generation. These findings provide insights to the therapeutic potential and clinical research of NTP as a novel tool in cervical cancer treatment.

Monte Carlo studies on neutron interactions in radiobiological experiments.

Monte Carlo method was used to study the characteristics of neutron interactions with cells underneath a water medium layer with varying thickness. The following results were obtained. (1) The fractions of neutron interaction with 1H, 12C, 14N and 16O nuclei in the cell layer were studied. The fraction with 1H increased with increasing medium thickness, while decreased for 12C, 14N and 16O nuclei. The bulges in the interaction fractions with 12C, 14N and 16O nuclei were explained by the resonance spikes in the interaction cross-section data. The interaction fraction decreased in the order: 1H > 16O > 12C > 14N. (2) In general, as the medium thickness increased, the number of "interacting neutrons" which exited the medium and then further interacted with the cell layer increased. (3) The area under the angular distributions for "interacting neutrons" decreased with increasing incident neutron energy. Such results would be useful for deciphering the reasons behind discrepancies among existing results in the literature.

Conversion coefficients for determination of dispersed photon dose during radiotherapy: NRUrad input code for MCNP.

Radiotherapy is a common cancer treatment module, where a certain amount of dose will be delivered to the targeted organ. This is achieved usually by photons generated by linear accelerator units. However, radiation scattering within the patient's body and the surrounding environment will lead to dose dispersion to healthy tissues which are not targets of the primary radiation. Determination of the dispersed dose would be important for assessing the risk and biological consequences in different organs or tissues. In the present work, the concept of conversion coefficient (F) of the dispersed dose was developed, in which F = (Dd/Dt), where Dd was the dispersed dose in a non-targeted tissue and Dt is the absorbed dose in the targeted tissue. To quantify Dd and Dt, a comprehensive model was developed using the Monte Carlo N-Particle (MCNP) package to simulate the linear accelerator head, the human phantom, the treatment couch and the radiotherapy treatment room. The present work also demonstrated the feasibility and power of parallel computing through the use of the Message Passing Interface (MPI) version of MCNP5.

Akt/mTOR mediated induction of bystander effect signaling in a nucleus independent manner in irradiated human lung adenocarcinoma epithelial cells.

Cytoplasm is an important target for the radiation-induced bystander effect (RIBE). In the present work, the critical role of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway in the generation of RIBE signaling after X-ray irradiation and the rapid phosphorylation of Akt and mTOR was observed in the cytoplasm of irradiated human lung adenocarcinoma epithelial (A549) cells. Targeting A549 cytoplasts with individual protons from a microbeam showed that RIBE signal(s) mediated by the Akt/mTOR pathway were generated even in the absence of a cell nucleus. These results provide a new insight into the mechanisms driving the cytoplasmic response to irradiation and their impact on the production of RIBE signal(s).

Low Concentration of Exogenous Carbon Monoxide Modulates Radiation-Induced Bystander Effect in Mammalian Cell Cluster Model.

During radiotherapy procedures, radiation-induced bystander effect (RIBE) can potentially lead to genetic hazards to normal tissues surrounding the targeted regions. Previous studies showed that RIBE intensities in cell cluster models were much higher than those in monolayer cultured cell models. On the other hand, low-concentration carbon monoxide (CO) was previously shown to exert biological functions via binding to the heme domain of proteins and then modulating various signaling pathways. In relation, our previous studies showed that exogenous CO generated by the CO releasing molecule, tricarbonyldichlororuthenium (CORM-2), at a relatively low concentration (20 µM), effectively attenuated the formation of RIBE-induced DNA double-strand breaks (DSB) and micronucleus (MN). In the present work, we further investigated the capability of a low concentration of exogenous CO (CORM-2) of attenuating or inhibiting RIBE in a mixed-cell cluster model. Our results showed that CO (CORM-2) with a low concentration of 30 µM could effectively suppress RIBE-induced DSB (p53 binding protein 1, p53BP1), MN formation and cell proliferation in bystander cells but not irradiated cells via modulating the inducible nitric oxide synthase (iNOS) andcyclooxygenase-2 (COX-2). The results can help mitigate RIBE-induced hazards during radiotherapy procedures.

Exogenous Nitric Oxide Suppresses in Vivo X-ray-Induced Targeted and Non-Targeted Effects in Zebrafish Embryos.

The present paper studied the X-ray-induced targeted effect in irradiated zebrafish embryos (Danio rerio), as well as a non-targeted effect in bystander naïve embryos partnered with irradiated embryos, and examined the influence of exogenous nitric oxide (NO) on these targeted and non-targeted effects. The exogenous NO was generated using an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). The targeted and non-targeted effects, as well as the toxicity of the SNAP, were assessed using the number of apoptotic events in the zebrafish embryos at 24 h post fertilization (hpf) revealed through acridine orange (AO) staining. SNAP with concentrations of 20 and 100 µM were first confirmed to have no significant toxicity on zebrafish embryos. The targeted effect was mitigated in zebrafish embryos if they were pretreated with 100 µM SNAP prior to irradiation with an X-ray dose of 75 mGy but was not alleviated in zebrafish embryos if they were pretreated with 20 µM SNAP. On the other hand, the non-targeted effect was eliminated in the bystander naïve zebrafish embryos if they were pretreated with 20 or 100 µM SNAP prior to partnering with zebrafish embryos having been subjected to irradiation with an X-ray dose of 75 mGy. These findings revealed the importance of NO in the protection against damages induced by ionizing radiations or by radiation-induced bystander signals, and could have important impacts on development of advanced cancer treatment strategies.

Characteristics of Protons Exiting from a Polyethylene Converter Irradiated by Neutrons with Energies between 1 keV and 10 MeV.

Monte Carlo method has been used to determine the efficiency for proton production and to study the energy and angular distributions of the generated protons. The ENDF library of cross sections is used to simulate the interactions between the neutrons and the atoms in a polyethylene (PE) layer, while the ranges of protons with different energies in PE are determined using the Stopping and Range of Ions in Matter (SRIM) computer code. The efficiency of proton production increases with the PE layer thickness. However the proton escaping from a certain polyethylene volume is highly dependent on the neutron energy and target thickness, except for a very thin PE layer. The energy and angular distributions of protons are also estimated in the present paper, showing that, for the range of energy and thickness considered, the proton flux escaping is dependent on the PE layer thickness, with the presence of an optimal thickness for a fixed primary neutron energy.