Inhibition of Rac1 attenuates radiation-induced lung injury while suppresses lung tumor in mice

Radiation pneumonitis is a major complication of radiation therapy. However, the detailed cellular mechanisms have not been clearly defined. Based on the recognition that basement membrane disruption occurs in acute lung injury and that matrix metalloproteinase (MMP)-2 can degrade type IV collagen, one of the major components of the basement membrane, we hypothesized that ionizing radiation would modulate MMP-2 production in human lung epithelial cells. To evaluate this, the modulation of MMP-2 with irradiation was investigated in normal human bronchial epithelial cells as well as in A549 cells. We measured the activity of MMP-2 in the conditioned medium with zymography and the MMP-2 mRNA level with RT-PCR. Both of these cells constitutively expressed 72-kDa gelatinolytic activity, corresponding to MMP-2, and exposure to radiation increased this activity. Consistent with the data of zymography, ionizing radiation increased the level of MMP-2 mRNA. This radiation-induced increase in MMP-2 expression was mediated via p53 because the p53 antisense oligonucleotide abolished the increase in MMP-2 activity as well as the accumulation of p53 after irradiation in A549 cells. These results indicate that MMP-2 expression by human lung epithelial cells is involved in radiation-induced lung injury.

Abstract Backgrounds: There is still little progress in the effective treatment of radiation-induced lung injury (RILI), a key dose-limiting factor for thoracic radiotherapy. Ras-related C3 botulinum toxin substrate1, Rac1, is a small guanosine triphosphatase involved in various mechanisms of radiation-induced damage and is over-expressed/mutated in various tumors. The gain-of-function mutation of Rac1 mediates tumor cells’ resistance to radiotherapy. Therefore, inhibiting Rac1 has the potential of protecting normal tissues from radiation-induced injury, and at the same time, sensitizing tumor to radiation therapy, which makes it a promising ideal target for radiation protection. To investigate the protective effects and mechanisms of Rac1 inhibition on RILI, and explore the possible mechanisms that mediate the differential effects of Rac1 inhibition on normal lung tissue and tumor cells.Methods: 60Co radioactive source was used for ionizing radiation (IR). RILI mouse model was constructed. Influence of Rac1 inhibition which was achieved by Rac1-specific inhibitor, NSC23766, on RILI were studied by H & E and Masson staining, and immunohistochemical staining of vimentin, TGF-βand γ-H2AX. Normal mouse lung epithelial cell line, MLE-12, and mouse lung cancer cell line, LLC, were used to study the effects of Rac1 inhibition on the cellular level. RNA-seq analysis was used for screening differential gene expression caused by Rac1 knockdown. The molecular mechanisms of Rac1 inhibition were studied at the cellular level. Subcutaneous tumor-bearing nude mouse model and orthotopic lung tumor-bearing mouse model were constructed to verify the bidirectional effects of Rac1 inhibition in vivo. Results: RILI of mouse was alleviated by intraperitoneal injection of NSC23766. Rac1 inhibition/knockdown reduced the radiation-induced damage of MLE-12 while aggravated that of LLC. Rac1 translocated from cytoplasm to nucleus after radiation. Tumor protein p53-inducible nuclear protein 1, Trp53inp1, was down-regulated by Rac1 knockdown. In vivo study further proved the differential effects of Rac1 inhibition. Rac1 was over-expressed and mutated in LLC cells, and the expression level of Trp53inp1 significantly lower, compared with that of MLE-12.Conclusion: Rac1 inhibition reduced the radiation-induced damage of normal lung epithelial cells, thereby alleviating RILI of mouse. These effects were partially mediated by down-regulating the expression of Trp53inp1. However, Rac1 inhibition significantly increased the sensitivity of LLC to radiation damage and inhibited its growth. The over-expression and mutation of Rac1, and the significant low expression of Trp53inp1 in LLC, may be the fundamental reasons mediating the differential effects of Rac1 inhibition.

Radiation-induced damage to normal lung parenchyma remains a dose-limiting factor in thorax-associated radiotherapy (RT). Severe early and late complications with lungs can increase the risk of morbidity in cancer patients after RT. Herein, senescence of lung epithelial cells following RT-induced cellular stress, or more precisely the respective altered secretory profile, the senescence-associated secretory phenotype (SASP), was suggested as a central process for the initiation and progression of pneumonitis and pulmonary fibrosis. We previously reported that abrogation of certain aspects of the secretome of senescent lung cells, in particular, signaling inhibition of the SASP-factor Ccl2/Mcp1 mediated radioprotection especially by limiting endothelial dysfunction. Here, we investigated the therapeutic potential of a combined metformin treatment to protect normal lung tissue from RT-induced senescence and associated lung injury using a preclinical mouse model of radiation-induced pneumopathy. Metformin treatment efficiently limited RT-induced senescence and SASP expression levels, thereby limiting vascular dysfunctions, namely increased vascular permeability associated with increased extravasation of circulating immune and tumor cells early after irradiation (acute effects). Complementary in vitro studies using normal lung epithelial cell lines confirmed the senescence-limiting effect of metformin following RT finally resulting in radioprotection, while fostering RT-induced cellular stress of cultured malignant epithelial cells accounting for radiosensitization. The radioprotective action of metformin for normal lung tissue without simultaneous protection or preferable radiosensitization of tumor tissue might increase tumor control probabilities and survival because higher radiation doses could be used.

: Radiation-induced lung injury is characterized by an acute pneumonia phase followed by a fibrotic phase. At the time of irradiation, a rapid, short-lived burst of reactive oxygen species (ROS) such as hydroxyl radicals (•OH) occurs, but chronic radiation-induced lung injury may occur due to excess ROS such as H2O2 , O2•− , ONOO− , and •OH. Molecular hydrogen (H2 ) is an efficient antioxidant that quickly diffuses cell membranes, reduces ROS such as •OH and ONOO− , and suppresses damage caused by oxidative stress in various organs. In 2011, through the evaluation of electron-spin resonance and fluorescent indicator signals, we had reported that H2 can eliminate •OH and can protect against oxidative stress-related apoptotic damage induced by irradiation of cultured lung epithelial cells. We had explored for the first time the radioprotective effects of H2 treatment on acute and chronic radiation-induced lung damage in mice by inhaled H2 gas (for acute) and imbibed H2 -enriched water (for chronic). Thus, we had proposed that H2 be considered a potential radioprotective agent. Recent publications have shown that H2 directly neutralizes highly reactive oxidants and indirectly reduces oxidative stress by regulating the expression of various genes. By regulating gene expression, H2 functions as an anti-inflammatory and anti-apoptotic molecule and promotes energy metabolism. The increased evidence obtained from cultured cells or animal experiments reveal a putative place for H2 treatment and its radioprotective effect clinically. This review focuses on major scientific advances of in the treatment of H2 as a new class of radioprotective agents.

Radiation-induced lung injury (RILI) occurs in up to 30% of thoracic radiotherapy (RT) cases and is a major limiting factor of dose escalation to achieve tumor control and improve survival. RILI can be separated into two phases: an early inflammatory phase and a late fibrotic phase. Imaging has the potential to provide a helpful understanding of RILI for diagnosis, monitoring and treatment. Current clinical imaging methods rely on anatomical imaging and occasionally incorporate functional imaging. With the advent of molecular imaging, specific targeted probes can be designed to image RILI at every stage of the process. Molecular imaging is still in its infancy and most new RILI imaging techniques are still under development. This chapter summarizes the different imaging methods used clinically for RILI imaging and explores new developments for the future of RILI management.

In our previous study, we demonstrated that cyanidin-3-O-glucoside (C3G)-rich haskap (Lonicera caerulea L.) berry extracts can attenuate the carcinogen-induced DNA damage in normal lung epithelial cells in vitro. Here, the efficacy of lyophilized powder of whole haskap berry (C3G-HB) in lowering tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, (NNK)-induced lung tumorigenesis in A/JCr mice was investigated. Three weeks after daily oral administration of C3G-HB (6 mg of C3G in 0.2 g of C3G-HB/mouse/day), lung tumors were initiated by a single intraperitoneal injection of NNK. Dietary C3G-HB supplementation was continued, and 22 weeks later, mice were euthanized. Lung tumors were visualized through positron emission tomography (PET) and magnetic resonance imaging (MRI) 19 weeks after NNK injection. Dietary supplementation of C3G-HB significantly reduced the NNK-induced lung tumor multiplicity and tumor area but did not affect tumor incidence. Immunohistochemical analysis showed reduced expression of proliferative cell nuclear antigen (PCNA) and Ki-67 in lung tissues. Therefore, C3G-HB has the potential to reduce the lung tumorigenesis, and to be used as a source for developing dietary supplements or nutraceuticals for reducing the risk of lung cancer among high-risk populations.

Glutamine metabolism provides energy to tumor cells and also produces reactive oxygen species (ROS). Excessive accumulation of ROS can damage mitochondria and eventually lead to cell death. xCT (SLC7A11) is responsible for the synthesis of glutathione in order to neutralize ROS. In addition, mitophagy can remove damaged mitochondria to keep the cell alive. Ionizing radiation kills tumor cells by causing the accumulation of ROS, which subsequently induces nuclear DNA damage. With this in mind, we explored the mechanism of intracellular ROS accumulation induced by ionizing radiation and hypothesized new methods to enhance the effect of radiotherapy. We used MCF-7 breast cancer cells and HCT116 colorectal cancer cells in our study. The above-mentioned cells were irradiated with different doses of X-rays or carbon ions. Clone formation assays were used to detect cell proliferation, enzyme-linked immunosorbent assay (ELISA) detected ATP, and glutathione (GSH) production, while the expression of proteins was detected by Western blot and quantitative real-time PCR analysis. The production of ROS was detected by flow cytometry, and immunofluorescence was used to track mitophagy-related processes. Finally, BALB/C tumor-bearing nude mice were irradiated with X-rays in order to further explore the protein expression found in tumors with the use of immunohistochemistry. Ionizing radiation increased the protein expressions of ASCT2, GLS, and GLUD in order to upregulate the glutamine metabolic flux in tumor cells. This caused an increase in ATP secretion. Meanwhile, ionizing radiation inhibited the expression of the xCT (SLC7A11) protein and reduced the generation of glutathione, leading to excessive accumulation of intracellular ROS. The mitophagy inhibitor, or knockdown Parkin gene, is able to enhance the ionizing radiation-induced ROS production and increase nucleus DNA damage. This combined treatment can significantly improve the killing effect of radiation on tumor cells. We concluded that ionizing radiation could upregulate the glutamine metabolic flux and enhance ROS accumulation in mitochondria. Ionizing radiation also decreased the SLC7A11 expression, resulting in reduced GSH generation. Therefore, inhibition of mitophagy can increase ionizing radiation-induced cell death.