The miR-15b-Smurf2-HSP27 axis promotes pulmonary fibrosis.

BACKGROUND: Heat shock protein 27 (HSP27) is overexpressed during pulmonary fibrosis (PF) and exacerbates PF; however, the upregulation of HSP27 during PF and the therapeutic strategy of HSP27 inhibition is not well elucidated. METHODS: We have developed a mouse model simulating clinical stereotactic body radiotherapy (SBRT) with focal irradiation and validated the induction of RIPF. HSP25 (murine form of HSP27) transgenic (TG) and LLC1-derived orthotropic lung tumor models were also used. Lung tissues of patients with RIPF and idiopathic pulmonary fibrosis, and lung tissues from various fibrotic mouse models, as well as appropriated cell line systems were used. Public available gene expression datasets were used for therapeutic response rate analysis. A synthetic small molecule HSP27 inhibitor, J2 was also used. RESULTS: HSP27 expression with its phosphorylated form (pHSP27) increased during PF. Decreased mRNA expression of SMAD-specific E3 ubiquitin-protein ligase 2 (Smurf2), which is involved in ubiquitin degradation of HSP27, was responsible for the increased expression of pHSP27. In addition, increased expression of miRNA15b was identified with decreased expression of Smurf2 mRNA in PF models. Inverse correlation between pHSP27 and Smurf2 was observed in the lung tissues of PF animals, an irradiated orthotropic lung cancer models, and PF tissues from patients. Moreover, a HSP27 inhibitor cross-linked with HSP27 protein to ameliorate PF, which was more effective when targeting the epithelial to mesenchymal transition (EMT) stage of PF. CONCLUSIONS: Our findings identify upregulation mechanisms of HSP27 during PF and provide a therapeutic strategy for HSP27 inhibition for overcoming PF.

Design, synthesis, and biological evaluation of novel HSP27 inhibitors to sensitize lung cancer cells to clinically available anticancer agents.

Expression of heat shock protein (HSP) correlates with the oncogenic status of malignant cells and plays an important role in tumorigenesis. HSP27 is constitutively expressed at specific stages of cancer development, and several clinical trials have reported correlations between HSP27 expression and tumor progression, metastasis, and chemoresistance in various types of cancer cells. These findings indicate that HSP27 is a major drug target, particularly in chemo-resistant cancers. As part of our ongoing efforts to improve the previously identified J2, a HSP27 cross-linker, we, in this study, report the identification of NK16 as a novel inducer of abnormal HSP27 dimers that did not affect the expression of HSP90 in an NCI-H460 lung cancer cell model. When NCI-H460 cells were treated with NK16 in combination with the anticancer drug cisplatin or paclitaxel, cleavage of PARP and caspase-3 was increased compared to administration of cisplatin or paclitaxel alone. Similar results were obtained in an NCI-H460-xenografted mouse model, in which tumor growth was suppressed more by co-administration of NK16 and paclitaxel than by paclitaxel alone. We propose NK16 as a meaningful strategy to improve the anticancer efficacy of cisplatin and paclitaxel.

Sodium dichloroisocyanurate toxicity in rats during a 90-day inhalation toxicity study.

Sodium dichloroisocyanurate-96% (NaDCC) is commonly used to treat drinking water, industrial water, and wastewater. However, exposure to NaDCC by inhalation can have toxic pulmonary effects in humans. In the present study, we evaluated the potential toxicity of NaDCC following a 90-day inhalation toxicity study in Sprague-Dawley/Crl:CD (SD) rats. The animals were exposed to 0.4, 2.0, or 10.0 mg/m3 NaDCC for 90 days. In addition, male and female rats from the 10.0 mg/m3 group were set up as the recovery group for 14 days. The bronchoalveolar lavage fluid showed a concentration-dependent increase in the total cell count, with a significant increase in neutrophils in both the sexes in the 10.0 mg/m3 group compared to the negative control group. In the 10.0 mg/m3 group, lung organ weight was significantly increased among the female rats. Histopathological examination showed eosinophilic droplets in the olfactory/respiratory epithelium, mucous cell hyperplasia, atrophy/degeneration of the tracheal branches, and wall thickening of the alveolar ducts in the nasal cavity of both sexes in the 10.0 mg/m3 group. The adverse effects of NaDCC exposure were observed to decrease during the 14-day recovery period in both sexes. Based on pathological observations, the "no observed adverse effect concentration (NOAEC)" of inhaled NaDCC was 2.0 mg/m3 for both sexes. These results are expected to provide a scientific basis for inhalation toxicity data of NaDCC.

Nicotinamide adenine dinucleotide phosphate oxidase 2 deletion attenuates polyhexamethylene guanidine-induced lung injury in mice.

Inhalation of polyhexamethylene guanidine phosphate (PHMG) can cause pulmonary fibrosis. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox) are enzymes that produce reactive oxygen species, which may be involved in tissue damage in various lung diseases. To investigate whether the Nox2 isoform of Nox is involved in the progression of PHMG-induced lung damage, we studied the contribution of Nox2 in PHMG-induced lung injury in Nox2-deficient mice. We treated wild-type (WT) and Nox2 knockout mice with a single intratracheal instillation of 1.1 mg/kg PHMG and sacrificed them after 14 days. We analyzed lung histopathology and the number of total and differential cells in the bronchoalveolar lavage fluid. In addition, the expressions of cytokines, chemokines, and profibrogenic genes were analyzed in the lung tissues. Based on our results, Nox2-deficient mice showed less PHMG-induced pulmonary damage than WT mice, as indicated by parameters such as body weight, lung weight, total cell count, cytokine and chemokine levels, fibrogenic mediator expression, and histopathological findings. These findings suggest that Nox2 may have the potential to contribute to PHMG-induced lung injury and serves as an essential signaling molecule in the development of PHMG-induced pulmonary fibrosis by regulating the expression of profibrogenic genes.

Drug like HSP27 cross linkers with chromenone structure ameliorates pulmonary fibrosis.

Background: Pulmonary fibrosis (PF) is a progressive lung disease characterized by fibroblast accumulation and collagen deposition, resulting in lung scarring and impaired gas exchange. Current treatments for idiopathic pulmonary fibrosis (IPF) have limited efficacy and significant side effects. Heat shock protein 27 (HSP27) has emerged as a potential therapeutic target for PF due to its involvement in fibrotic processes. However, effective HSP27 inhibitors for PF treatment are still lacking. Methods: To assess the anti-fibrotic effects of NA49, we utilized murine PF models induced by radiation (IR) or bleomycin (BLM). We administered NA49 to the PF mice and evaluated its impact on lung fibrosis progression. We also investigated the molecular mechanisms underlying NA49's effects, focusing on its inhibition of EMT-related signaling pathways. Results: In our study, we evaluated the potential of a novel HSP27 inhibitor, NA49, in preclinical models of PF. NA49 effectively suppressed PF development in radiation and bleomycin-induced PF models. It reduced fibrosis, inhibited NFkB signaling, and downregulated EMT-related molecules. Importantly, we evaluated the safety profile of NA49 by assessing its impact on DNA strand breakage. Compared to previous HSP27 inhibitors, NA49 showed lower levels of DNA damage in human lung epithelial cells, and suggests that NA49 may have reduced toxicity compared to other HSP27 inhibitors. Overall, our results demonstrate that NA49 effectively inhibits PF development in preclinical models. It reduces lung fibrosis, inhibits EMT-related signaling pathways, and exhibits improved safety profiles. These findings highlight the potential of NA49 as a promising candidate for the treatment of PF. Conclusion: NA49 exhibited significant anti-fibrotic effects, inhibiting fibrosis development and EMT-related signaling pathways. Moreover, NA49 showed improved safety profiles compared to previous HSP27 inhibitors.

HSPB1 inhibitor J2 attenuates lung inflammation through direct modulation of Ym1 production and paracrine signaling.

Heat shock protein beta-1 (HSPB1) is a multifaceted protein that controls cellular stress, modulates cell differentiation and development, and inhibits apoptosis of cancer cells. Increased HSPB1 expression is highly associated with poor outcomes in lung cancer by enhancing cell migration and invasion; therefore, targeting HSPB1 may be a promising therapeutic for lung cancer and fibrosis. Although the HSPB1 inhibitor J2 has been reported to exhibit potent antifibrotic effects, it remains unclear whether and how J2 directly modulates inflammatory immune responses in pulmonary fibrosis. In this study, we found that J2 potently attenuated irradiation or bleomycin-induced pulmonary fibrosis by significantly inhibiting the infiltration and activation of T cells and macrophages. J2 inhibited T-cell proliferation and subsequently suppressed T helper cell development. Although there was no significant effect of J2 on cell proliferation of M1 and M2 macrophages, J2 specifically increased the expression of Ym1 in M2 macrophages without affecting the expression of other M2 markers. Interestingly, J2 increased lysosomal degradation of HSPB1 and inhibited HSPB1-induced repression of signal transducer and activator of transcription 6 (STAT6), which simultaneously increased STAT6 and Ym1 expression. Ym1 production and secretion by J2-treated M2 macrophages substantially decreased IL-8 production by airway epithelial cells in vitro and in vivo, resulting in attenuation of airway inflammation. Taken together, we suggest that J2 has potential as a therapeutic agent for pulmonary fibrosis with increased HSPB1 expression through direct immune suppression by Ym1 production by M2 macrophages as well as T-cell suppression.

Lung-targeted delivery of TGF-ß antisense oligonucleotides to treat pulmonary fibrosis.

Pulmonary fibrosis is a serious respiratory disease, with limited therapeutic options. Since TGF-ß is a critical factor in the fibrotic process, downregulation of this cytokine has been considered a potential approach for disease treatment. Herein, we designed a new lung-targeted delivery technology based on the complexation of polymeric antisense oligonucleotides (pASO) and dimeric human ß-defensin 23 (DhBD23). Antisense oligonucleotides targeting TGF-ß mRNA were polymerized by rolling circle amplification and complexed with DhBD23. After complexation with DhBD23, pASO showed improved serum stability and enhanced uptake by fibroblasts in vitro and lung-specific accumulation upon intravenous injection in vivo. The pASO/DhBD23 complex delivered into the lung downregulated target mRNA, and subsequently alleviated lung fibrosis in mice, as demonstrated by western blotting, quantitative reverse-transcriptase PCR (qRT-PCR), immunohistochemistry, and immunofluorescence imaging. Moreover, as the complex was prepared only with highly biocompatible materials such as DNA and human-derived peptides, no systemic toxicity was observed in major organs. Therefore, the pASO/DhBD23 complex is a promising gene therapy platform with lung-targeting ability to treat various pulmonary diseases, including pulmonary fibrosis, with low side effects.

Identification of molecular signatures involved in radiation-induced lung fibrosis.

In radiotherapy, radiation (IR)-induced lung fibrosis has severe and dose-limiting side effects. To elucidate the molecular effects of IR fibrosis, we examined the fibrosis process in irradiated mouse lung tissues. High focal IR (90 Gy) was exposed to a 3-mm volume of the left lung in C57BL6 mice. In the diffused irradiation, 20 Gy dose delivered with a 7-mm collimator almost covered the entire left lung. Histological examination for lung tissues of both irradiated and neighboring regions was done for 4 weeks after irradiation. Long-term effects (12 months) of 20Gy IR were compared on a diffuse region of the left lung and non-irradiated right lung. Fibrosis was initiated as early as 2 weeks after IR in the irradiated lung region and neighboring region. Upregulation of gtse1 in both 90Gy-irradiated and neighboring regions was observed. Upregulation of fgl1 in both 20Gy diffused irradiated and non-irradiated lungs was identified. When gtse1 or flg1 was knock-downed, TGFß or IR-induced epithelial-mesenchymal transition was inhibited, accompanied with the inhibition of cellular migration, suggesting fibrosis responsible genes. Immunofluorescence analysis using mouse fibrotic lung tissues suggested that fibrotic regions showed increased expressions of Gtse1 and Fgl1, indicating novel molecular signatures of gtse1and fgl1 for IR-induced lung fibrosis. Even though their molecular mechanisms and IR doses or irradiated volumes for lung fibrosis may be different, these genes may be novel targets for understanding IR-induced lung fibrosis and in treatment strategies. KEY MESSAGES: Upregulation of gtse1 by 90Gy focal irradiation and upregulation of fgl1 by 20Gy diffused irradiation are identified in mouse lung fibrosis model. Gtse1 and Fgl1 are involved in radiation or TGFß-induced epithelial-mesenchymal transition. Radiation-induced fibrotic regions of mouse lungs showed increased expressions of Gtse1 and Fgl1. Gtse1 and Fgl1 are suggested to be novel targets for radiation-induced lung fibrosis.

The Hsp27-Mediated IkBα-NFκB Signaling Axis Promotes Radiation-Induced Lung Fibrosis.

PURPOSE: Lung fibrosis is a major side effect experienced by patients after lung cancer radiotherapy. However, effective protection strategies and underlying treatment targets remain unclear. In an effort to improve clinical outcomes, pharmacologic treatment of fibrosis is becoming increasingly popular; however, no ideal therapeutic strategy is yet available. EXPERIMENTAL DESIGN: We used a mouse model to irradiate high focal (90 or 75 Gy) to 3-mm volume of the left lung. Lung tissues of mice were subjected to microarray, mRNA expression, and immunohistochemical analysis. Correlations of radiation (IR)-induced epithelial-mesenchymal transition (EMT) were validated in lung cell lines using appropriate treatments to activate or inhibit selected pathways. RESULTS: The expression of Hsp27 was increased during IR-induced lung fibrosis in a mouse model. Inhibition of functional Hsp27 using shRNA and a synthetic small molecule inhibitor (J2) in lung cells alleviated IR-mediated EMT. The activation of NFkB pathways via direct interaction between Hsp27 and IkBα resulted in increased expressions of Twist, IL-1ß, and IL-6 and facilitated IR-mediated EMT, which was identified as an underlying mechanism of Hsp27-mediated fibrosis after IR. J2 also inhibited IR-induced lung fibrosis in an orthotopic lung cancer model, and IR-induced lung fibrotic tissues from patients showed higher expression of Hsp27 than unirradiated lungs. CONCLUSIONS: Collectively, IkBα-NFkB signaling activation by Hsp27, which resulted in the facilitation of Twist, IL1ß, and IL6 expression, is involved in the EMT process that is tightly connected to the development of IR-induced lung fibrosis. Our findings also suggest that inhibition of Hsp27 has the potential to become a valuable therapeutic strategy for IR-induced lung fibrosis.

Identification of radiation response genes and proteins from mouse pulmonary tissues after high-dose per fraction irradiation of limited lung volumes.

PURPOSE: The molecular effects of focal exposure of limited lung volumes to high-dose per fraction irradiation (HDFR) such as stereotactic body radiotherapy (SBRT) have not been fully characterized. In this study, we used such an irradiation system and identified the genes and proteins after HDFR to mouse lung, similar to those associated with human therapy. METHODS AND MATERIALS: High focal radiation (90 Gy) was applied to a 3-mm volume of the left lung of C57BL6 mice using a small-animal stereotactic irradiator. As well as histological examination for lungs, a cDNA micro array using irradiated lung tissues and a protein array of sera were performed until 4 weeks after irradiation, and radiation-responsive genes and proteins were identified. For comparison, the long-term effects (12 months) of 20 Gy radiation wide-field dose to the left lung were also investigated. RESULTS: The genes ermap, epb4.2, cd200r3 (up regulation) and krt15, hoxc4, gdf2, cst9, cidec, and bnc1 (down-regulation) and the proteins of AIF, laminin, bNOS, HSP27, ß-amyloid (upregulation), and calponin (downregulation) were identified as being responsive to 90 Gy HDFR. The gdf2, cst9, and cidec genes also responded to 20 Gy, suggesting that they are universal responsive genes in irradiated lungs. No universal proteins were identified in both 90 Gy and 20 Gy. Calponin, which was downregulated in protein antibody array analysis, showed a similar pattern in microarray data, suggesting a possible HDFR responsive serum biomarker that reflects gene alteration of irradiated lung tissue. These genes and proteins also responded to the lower doses of 20 Gy and 50 Gy HDFR. CONCLUSIONS: These results suggest that identified candidate genes and proteins are HDFR-specifically expressed in lung damage induced by HDFR relevant to SBRT in humans.