Normal Lung Fibroblasts Research Articles

Warfarin-Capped Gold Nanoparticles: Synthesis, Cytotoxicity, and Cellular Uptake.

Currently, research studies on nanoparticle cytotoxicity, uptake or internalization into the body’s cells are of great interest for the improvement of diagnostic and therapeutic applications. We report here the synthesis and characterization of very stable novel warfarin-capped gold nanoparticles with an average diameter of 54 ± 10 nm which were prepared using sodium warfarin as a reducing agent. The nanoparticles were tested in terms of cytotoxicity and cellular internalization in vitro on two cell lines: normal lung fibroblast HFL-1 and human retinal pigment epithelial D407 cells. Our results showed that the normal lung fibroblast HFL-1 cells were more sensitive to the nanoparticle treatment compared to the human retinal pigment epithelial D407 cells. Moreover, any signs of potential cytotoxicity occurred during the first 24 h of treatment, the cellular viability remaining largely unchanged for longer exposure times. Transmission electron microscopy and dark field hyperspectral imaging revealed that the nanoparticles were effectively delivered and released to the HFL-1 and D407 cells’ cytoplasm. Our results provide valuable information to further investigate sodium warfarin-capped gold nanoparticles for possible biological applications.

Miniature Non-thermal Plasma Induced Cell Cycle Arrest and Apoptosis in Lung Carcinoma Cells

Non-thermal plasma has been a promising new cancer treatment modality in plasma oncology field. It generates extracellular and intracellular reactive species which are key factors for the treatment of cancer cells. In this study, we investigated the differential effect of non-thermal plasma on both A549 lung adenocarcinoma and MRC-5 lung fibroblast cells. Extracellular generation of reactive species in both A549 lung cancer and MRC-5 normal lung fibroblast cells were similar, whereas intracellular penetration of reactive species generated by plasma in A549 cancer cells were almost fourfold higher than the normal cells. Interestingly, A549 cancer cells treated for shorter (15 and 30 s) and longer durations (60 and 120 s) get arrested in S-phase (19%) and G2/M phase (28%) respectively. In a healthy MRC-5 cells, few cells arrested in S and G2/M phase in relative to A549 cells for all treatment time. Finally, we evaluated the expression of apoptosis-related genes, H2AX, BAX, P53, Caspase-8, and ATM on normal and cancer cells. There was a higher expression of BAX gene for 120 s plasma treated A549 cell samples at day 1 relative to MRC-5 cells. These findings demonstrate that non-thermal plasma generated reactive species creates intracellular stress, that arrests cell cycle and induces apoptosis in cancer cells. This study suggests that non-thermal plasma could be a potential therapy for lung cancer treatment.

Ferulic acid enhances the radiation sensitivity of lung and liver carcinoma cells by collapsing redox homeostasis: mechanistic involvement of Akt/p38 MAPK signalling pathway

The major drawback of anticancer therapy is the development of resistance against drugs and radiation at the later phase of treatment which may lead to recurrences of the disease. Therefore, strategy was taken to enhance radiation sensitivity of lung (A549) and liver (HepG2) carcinoma cells by treatment with ferulic acid (FA) prior to irradiation. FA pre-treatment initially decreased reactive oxygen species (ROS) level in carcinoma cells which induced reductive stress and cytostasis. To overcome this stress, cellular mechanism increased the Keap1 level to down-regulate nuclear localisation of Nrf2 and its dependent antioxidant system. The antioxidant system reached the lowest level after 3 and 6 h of FA treatment in A549 and HepG2 cells respectively. As endogenous ROS were still being generated at same rate, ROS level was clearly higher than control which changed the reductive stress to oxidative stress. Exposure to γ-radiation in this condition further increased ROS level and caused radio-sensitisation of carcinoma cells. Combination of irradiation (IR) and FA activated mitochondrial apoptotic pathway and concomitantly inhibited the cell cycle progression and survival pathway over the IR group. Moreover, the combination treatment showed significant tumour regression, caspase 3 activation and nuclear fragmentation in tumour tissue compared to radiation alone. In contrast, FA pre-treatment protected peripheral blood mononuclear cells (PBMC) and normal lung fibroblast WI38 cells from radiation damage. Together, combination treatment offers effective strategy of killing cancer cells and demonstrates its potential for increasing the efficacy of radio-therapy.

39P - The regulation of INK4 locus by long non-coding RNAs

BackgroundINK4 locus is located on human chromosome 9p21 region and encodes three tumor suppressor genes, p15, p16 and ARF. So far, we revealed that a long non-coding RNA (lncRNA), ANRIL, transcribed from INK4 locus represses the transcription of p15 and p16 genes. ANRIL associates with polycomb protein complexes and recruits them on INK4 locus, leading to the transcriptional repression. The depletion of ANRIL inhibits the proliferation of cancer cells such as non-small cell lung cancer and colorectal cancer, indicating that ANRIL functions to promote cancer cells proliferation. Recently, we found a novel lncRNA induced by oncogenic Ras signal (named LION: lncRNA induced by oncogenic Ras signal). In this study, we showed that LION is involved in the transcriptional regulation of INK4 locus and cell proliferation. MethodsHuman non-small cell lung cancer cell H1299 and colorectal cancer cell HCT116 were transfected with siRNA oligonucleotides against LION. The expression analysis of INK4 locus genes was performed by quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR). Cell cycle analysis was performed by using Muse cell analyzer. ResultsRT-PCR analysis showed that LION is highly expressed in several cancer cells such as non-small cell lung cancer, cervical cancer and colorectal cancer compared with normal lung fibroblasts. Silencing LION by siRNA oligonucleotides inhibits the proliferation of H1299, HCT116 and HeLa cells. Q-RT-PCR analysis showed that silencing LION increases the p15 and p16 mRNA, suggesting that LION is involved in the transcriptional repression of INK4 locus. Cell cycle analysis showed that silencing LION causes G2/M phase arrest in cell cycle, suggesting that LION functions to promote G2/M transition. ConclusionsLION is involved in the promotion of cancer cells proliferation such as H1299 and HCT116 cells via regulating p15, p16 and other genes related to G2/M phase control. Legal entity responsible for the studyThe authors. FundingJSPS KAKENHI. DisclosureAll authors have declared no conflicts of interest.

Design and synthesis of substituted dihydropyrimidinone derivatives as cytotoxic and tubulin polymerization inhibitors

An operationally simple Biginelli protocol was employed for the synthesis of new C6-carbon based aryl α-haloacrylamide-linked dihydropyrimidinone derivatives. The synthesized compounds were appraised for their in vitro antiproliferative potential against a selected panel of human cancer cell lines especially MCF-7 (human breast cancer), MDA-MB-231 (human breast cancer), HCT-116 (human colon cancer), HCT-15 (human colorectal adenocarcinoma), HT-29 (human colon adenocarcinoma) and DU145 (human prostate cancer) along with normal lung fibroblasts (HFL-1). Preferably, compounds containing α-haloacrylamide (10a-g) functionality were found to exhibit most significant cytotoxicity (IC50 value 0.54 ± 0.12 to 8.35 ± 0.82 µM) against the listed cancer cell lines, particularly towards breast cancer cell lines MCF-7 and MDA-MB-231 (IC50 value 0.54 ± 0.12 to 3.70 ± 0.24 µM). In the seam of synthesized compounds, compound 10f exhibited potent antiproliferative activity against breast cancer cell lines namely MCF-7 (IC50 value 0.54 ± 0.12 µM) and MDA-MB-231 (IC50 value 1.18 ± 0.32 µM). Further to understand the underlying apoptosis mechanisms, different staining techniques such as AO/EB, DCFDA, and DAPI staining were performed. To know the extent of apoptosis and loss of mitochondrial membrane potential in MCF-7 cell lines, annexin V-FITC/PI and JC-1 were performed. Cell cycle analysis revealed that compound 10f arrested the cells at G2/M phase in a dose-dependent manner. The compound 10f also found to exhibit significant inhibition of tubulin polymerization (IC50 of 6.91 ± 0.43 μM) with microtubule destabilizing properties. Molecular docking studies also revealed that compound 10f efficiently interacted with critical catalytically active residues Ser178, Val238, and Val318 of the α/β-tubulin by a hydrogen bond.

In vitro screening for compounds from Hypericum longistylum with anti-pulmonary fibrosis activity

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a poor prognosis and limited therapies, and transforming growth factor-β1 (TGF-β1) plays a central role in the pathogenesis of IPF. Here, we aimed to investigate the chemical constituents and biological activities of Hypericum longistylum and detect whether the isolated compounds inhibit the TGF-β1/Smad3 signaling pathway to identify candidate compounds for the treatment of pulmonary fibrosis. Fifteen compounds (1-15) were isolated from H. longistylum and their structures were elucidated on the basis of spectroscopic analyses. An in vitro MTT assay was used to test the effect of these fifteen compounds on fibroblast cytotoxicity and vitality. Furthermore, their bioactivities were screened using a TGF-β1/Smad3 pathway luciferase reporter in vitro. MTT screening found that compounds 1-15 had no deleterious effects on normal mouse lung fibroblasts and no significant inhibition of vitality. Luciferase assay showed that compounds 14 and 15 could significantly inhibit the TGF-β1/Smad3 pathway with the inhibition rates of 67.92% and 93.10%, respectively. Both compounds can be used as lead compounds for structural modification and optimization to obtain more drug candidates for the treatment of pulmonary fibrosis.

Interferon-\u03b3 enhances the antifibrotic effects of pirfenidone by attenuating IPF lung fibroblast activation and differentiation

BackgroundIdiopathic pulmonary fibrosis (IPF) pathogenesis involves multiple pathways, and combined antifibrotic therapy is needed for future IPF therapy. Inhaled interferon-γ (IFN-γ) was recently shown to be safe and without systemic effects in patients with IPF.AimTo examine the in vitro effects of individual and combined treatment with IFN-γ and pirfenidone (PFD) on normal and IPF fibroblast activation and extracellular matrix remodeling after TGF-β1 and PDGF-BB stimulation.MethodsIPF and normal human lung fibroblasts (NHLF) were treated with IFN-γ, PFD or a combination of both drugs in the presence of either TGF-β1 or PDGF-BB. The effects of TGF-β1 and PDGF-BB treatment on cell viability, proliferation, differentiation and migration were examined. The expression of collagen 1, matrix metalloproteinases (MMPs) and tissue inhibitors of MMP (TIMPs) was analyzed using qPCR, Western blotting and gelatin zymography. Total collagen content in conditioned media was also measured using a Sircol assay.ResultsCompared to that of PFD, the effect of IFN-γ in downregulating normal and IPF lung fibroblast differentiation to myofibroblasts in response to TGF-β1 was more potent. Importantly, the combination of IFN-γ and PFD had a possibly synergistic/additive effect in inhibiting the TGF-β1- and PDGF-BB-induced proliferation, migration and differentiation of normal and IPF lung fibroblasts. Furthermore, both drugs reversed TGF-β1-induced effects on MMP-1, − 2, − 3, − 7, and − 9, while only PFD promoted TIMP-1 and-2 expression and release.ConclusionsOur findings demonstrate that the antifibrotic effects of IFN-γ and PFD on normal and IPF lung fibroblasts are different and complementary. Combination therapy with inhaled IFN-γ and PFD in IPF is promising and should be further explored in IPF clinical trials.

Арония черноплодная (Aronia melanocarpa) как источник соединений с потенциальной геропротекторной активностью

Арония черноплодная (Aronia melanocarpa (Micht.) Elliot) известна как ценная плодовая культура. В статье представлен обзор эффектов экстрактов стеблей, листьев и ягод A. melanocarpa на продолжительность жизни модельных организмов, а также потенциальных последствий применения экстрактов аронии для организма в целом. Анализ литературы показывает, что экстракт аронии черноплодной способствует увеличению продолжительности жизни и является потенциальным геропротектором.

Conditioned medium from asbestos-exposed fibroblasts affects proliferation and invasion of lung cancer cell lines.

The importance of the role of fibroblasts in cancer microenvironment is well-recognized. However, the relationship between fibroblasts and asbestos-induced lung cancer remains underexplored. To investigate the effect of the asbestos-related microenvironment on lung cancer progression, lung cancer cells (NCI-H358, Calu-3, and A549) were cultured in media derived from IMR-90 lung fibroblasts exposed to 50 mg/L asbestos (chrysotile, amosite, and crocidolite) for 24 h. The kinetics and migration of lung cancer cells in the presence of asbestos-exposed lung fibroblast media were monitored using a real-time cell analysis system. Proliferation and migration of A549 cells increased in the presence of media derived from asbestos-exposed lung fibroblasts than in the presence of media derived from normal lung fibroblasts. We observed no increase in proliferation and migration in lung cancer cells cultured in asbestos-exposed lung cancer cell medium. In contrast, increased proliferation and migration in lung cancer cells exposed to media from asbestos-exposed lung fibroblasts was observed for all types of asbestos. Media derived from lung fibroblasts exposed to other stressors, such as hydrogen peroxide and UV radiation didn’t show as similar effect as asbestos exposure. An enzyme-linked immunosorbent assay (ELISA)-based cytokine array identified interleukin (IL)-6 and IL-8, which show pleiotropic regulatory effects on lung cancer cells, to be specifically produced in higher amounts by the three types of asbestos-exposed lung fibroblasts than normal lung fibroblasts. Thus, the present study demonstrated that interaction of lung fibroblasts with asbestos may support the growth and metastasis of lung cancer cells and that chrysotile exposure can lead to lung cancer similar to that caused by amphibole asbestos (amosite and crocidolite).

Potentiation of in vitro apoptotic effects of δ-Tocotrienol and Jerantinine A on human lung adenocarcinoma cells

Introduction: The apoptotic effects of single-compound and combined sub-effective concentrations of δ-tocotrienol and jerantinine A on human lung adenocarcinoma (A549) cells were investigated. Methods: Assays including cell viability, histochemical and immunofluorescence staining techniques, flow cytometry and enzyme activity were used. Results: The combination of δ-tocotrienol with jerantinine A at sub-effective concentrations induced a synergistic effect and improved selective toxicity towards cancerous A549 cells over normal lung fibroblast (MRC5) cells compared to the single-compound jerantinine. Morphological features of apoptosis were evident on treated A549 cells. Combined sub-effective concentrations of δ-tocotrienol with jerantinine A induced a predominantly G2/M cell cycle arrest and characterised by a disruption of microtubular networks mediated via caspase 8, 9 and 3 enzymatic activities. Conclusion: These findings demonstrated improved potency in vitro and reduced dose-related toxicity of jerantinine A to normal cells through prospective combined treatment between low-concentration δ-tocotrienol and jerantinine A for lung cancer.

Curcumin relieves paraquat‑induced lung injury through inhibiting the thioredoxin interacting protein/NLR pyrin domain containing 3‑mediated inflammatory pathway.

When paraquat (PQ) enters the human body, it increases oxidative stress and inflammation, ultimately resulting in acute lung injury (ALI). Curcumin, a naturally occurring compound, has been reported to ameliorate PQ-induced ALI; however, the underlying molecular mechanisms remain unclear. In the present study, normal lung fibroblasts (WI-38VA13) were treated with 10 µmol/l PQ for 48 h, followed by a further 48 h incubation with 300 µmol/l curcumin. Cells were then harvested to determine their viability. Flow cytometry was performed to analyze the levels of reactive oxygen species (ROS) and the rate of apoptosis. The levels of apoptotic proteins and activation of the thioredoxin interacting protein/NLR pyrin domain containing 3 (TXNIP/NLRP3) axis were measured via reverse transcription-quantitative polymerase chain reaction and western blot analyses. Proinflammatory cytokine levels were examined using enzyme-linked immunosorbent assays. Finally, the expression levels of Notch1, extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylated-ERK1/2 were evaluated via western blotting. Following treatment with curcumin, PQ-induced increases in ROS levels and apoptosis were significantly attenuated, and Bcl-2 expression levels were upregulated, whereas those of Bax were downregulated. It was also observed that curcumin treatment downregulated the expression levels of TXNIP, NLRP3, interleukin (IL)-1β and IL-18, and downstream caspase-1 compared with PQ treatment alone. Curcumin significantly attenuated the upregulation of Notch1 without affecting ERK1/2 phosphorylation. The present findings suggested that the inhibitory effects of curcumin on TXINP1 may inhibit activation of the NLRP3 inflammasome, subsequently suppressing the upregulation of proinflammatory cytokines and ultimately improving PQ-induced ALI.

Plumbagin induces paraptosis in cancer cells by disrupting the sulfhydryl homeostasis and proteasomal function

Plumbagin (PLB) is an active secondary metabolite extracted from the roots of Plumbago rosea. In this study, we report that plumbagin effectively induces paraptosis by triggering extensive cytoplasmic vacuolation followed by cell death in triple negative breast cancer cells (MDA-MB-231), cervical cancer cells (HeLa) and non-small lung cancer cells (A549) but not in normal lung fibroblast cells (WI-38). The vacuoles originated from the dilation of the endoplasmic reticulum (ER) and were found to be empty. The cell death induced by plumbagin was neither apoptotic nor autophagic. Plumbagin induced ER stress mainly by inhibiting the chymotrypsin-like activity of 26S proteasome as also evident from the accumulation of polyubiquitinated proteins. The vacuolation and cell death were found to be independent of reactive oxygen species generation but was effectively inhibited by thiol antioxidant suggesting that plumbagin could modify the sulfur homeostasis in the cellular milieu. Plumbagin also resulted in a decrease in mitochondrial membrane potential eventually decreasing the ATP production. This is the first study to show that Plumbagin induces paraptosis through proteasome inhibition and disruption of sulfhydryl homeostasis and thus further opens up the lead molecule to potential therapeutic strategies for apoptosis-resistant cancers.

Synthesis and biochemical studies of novel organic selenides with increased selectivity for hepatocellular carcinoma and breast adenocarcinoma

Nineteen organoselenides were synthesized and tested for their intrinsic cytotoxicity in hepatocellular carcinoma (HepG2) and breast adenocarcinoma (MCF-7) cell lines and their corresponding selective cytotoxicity (SI) was estimated using normal lung fibroblast (WI-38) cells. Most of the organic selenides exhibited good anticancer activity, and this was more pronounced in HepG2 cells. Interestingly, the naphthoquinone- (5), thiazol- (12), and the azo-based (13) organic selenides demonstrated promising SI (up to 76). Furthermore, the amine 4c, naphthoquinone 5, and azo-based 13 and 15 organic selenides were able to down-regulate the expression of Bcl-2 and up-regulate the expression levels of IL-2, IL-6 and CD40 in HepG2 cells compared to untreated cells. Moreover, most of the synthesized candidates manifested good free radical-scavenging and GPx-like activities comparable to vitamin C and ebselen. The obtained results suggested that some of the presented organoselenium candidates have promising anti-HepG2 and antioxidant activities.

Hsa_circ_0001946 Inhibits Lung Cancer Progression and Mediates Cisplatin Sensitivity in Non-small Cell Lung Cancer via the Nucleotide Excision Repair Signaling Pathway.

Background: Despite great advances in the diagnosis and treatment of non-small cell lung cancer (NSCLC), early diagnosis remains a challenge because patients usually have advanced lung cancer at the time they are diagnosed. The limited efficacy of conventional chemotherapy is another major problem in the treatment of NSCLC. Based on a published set of sequencing data, we find that hsa_circ_0001946 is a circRNA molecule with a significantly different expression level in three cell lines (human normal lung fibroblasts cell line MRC-5, human NSCLC cell line A549, cisplatin-resistant cell line A549/DDP), NSCLC tissues and paired adjacent normal tissues. We believe that hsa_circ_0001946 may have an effect on the progression of NSCLC and its sensitivity to cisplatin.Methods: We focused on investigating the circular RNA, hsa_circ_0001946. RNA interference of hsa_circ_0001946 was carried out in A549 cell lines to determine the effect of reduced hsa_circ_0001946 expression on lung cancer progression and was analyzed by Cell Counting Kit-8 (CCK-8), 5-ethynyl-20-deoxyuridine, clone formation, Hoechst, wound healing, and transwell assays. The nucleotide excision repair (NER) signaling pathway was identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Moreover, cellular responses to cisplatin were assessed through CCK-8 and flow cytometry assays. Western blot analysis and host-cell reactivation assay were used to determine the effect of hsa_circ_0001946 on NER signaling.Results: In this study, we found that the reduced expression of hsa_circ_0001946 promoted the viability, proliferation, migration, and invasion of NSCLC cells, as well as inhibition of cell apoptosis. Our findings suggest that hsa_circ_0001946 can affect the sensitivity of NSCLC cells to the chemotherapeutic drug cisplatin via modulation of the NER signaling pathway.Conclusions: Our study demonstrated the role of hsa_circ_0001946 in NSCLC pathogenesis, development, and chemosensitivity, and suggests that hsa_circ_0001946 may serve as a novel biomarker for the diagnosis and prediction of platinum-based chemosensitivity in patients with NSCLC.

Pirfenidone attenuates lung fibrotic fibroblast responses to transforming growth factor-\u03b21

BackgroundPirfenidone, an antifibrotic agent used for the treatment of idiopathic pulmonary fibrosis (IPF), functions by inhibiting myofibroblast differentiation, which is involved in transforming growth factor (TGF)-β1-induced IPF pathogenesis. However, unlike normal lung fibroblasts, the relationship between pirfenidone responses of TGF-β1-induced human fibrotic lung fibroblasts and lung fibrosis has not been elucidated.MethodsThe effects of pirfenidone were evaluated in lung fibroblasts isolated from fibrotic human lung tissues after TGF-β1 exposure. The ability of two new pharmacological targets of pirfenidone, collagen triple helix repeat containing protein 1(CTHRC1) and four-and-a-half LIM domain protein 2 (FHL2), to mediate contraction of collagen gels and migration toward fibronectin were assessed in vitro.ResultsCompared to control lung fibroblasts, pirfenidone significantly restored TGF-β1-stimulated fibroblast-mediated collagen gel contraction, migration, and CTHRC1 release in lung fibrotic fibroblasts. Furthermore, pirfenidone attenuated TGF-β1- and CTHRC1-induced fibroblast activity, upregulation of bone morphogenic protein-4(BMP-4)/Gremlin1, and downregulation of α-smooth muscle actin, fibronectin, and FHL2, similar to that observed post-CTHRC1 inhibition. In contrast, FHL2 inhibition suppressed migration and fibronectin expression, but did not downregulate CTHRC1.ConclusionsOverall, pirfenidone suppressed fibrotic fibroblast-mediated fibrotic processes via inverse regulation of CTHRC1-induced lung fibroblast activity. Thus, CTHRC1 can be used for predicting pirfenidone response and developing new therapeutic targets for lung fibrosis.

Subcellular compartmentalization of PKM2 identifies anti-PKM2 therapy response in vitro and in vivo mouse model of human non-small-cell lung cancer.

Pyruvate kinase M2 (PKM2) is an alternatively spliced variant, which mediates the conversion of glucose to lactate in cancer cells under normoxic conditions, known as the Warburg effect. Previously, we demonstrated that PKM2 is one of 97 genes that are overexpressed in non-small-cell lung cancer (NSCLC) cell lines. Herein, we demonstrate a novel role of subcellular PKM2 expression as a biomarker of therapeutic response after targeting this gene by shRNA or small molecule inhibitor (SMI) of PKM2 enzyme activity in vitro and in vivo. We examined two established lung cancer cell lines, nine patients derived NSCLC and three normal lung fibroblast cell lines for PKM2 mRNA, protein and enzyme activity by RT-qPCR, immunocytochemistry (ICC), and Western blot analysis. All eleven NSCLC cell lines showed upregulated PKM2 enzymatic activity and protein expression mainly in their cytoplasm. Targeting PKM2 by shRNA or SMI, NSCLC cells showed significantly reduced mRNA, enzyme activity, cell viability, and colony formation, which also downregulated cytosolic PKM2 and upregulated nuclear enzyme activities. Normal lung fibroblast cell lines did not express PKM2, which served as negative controls. PKM2 targeting by SMI slowed tumor growth while gene-silencing significantly reduced growth of human NSCLC xenografts. Tumor sections from responding mice showed >70% reduction in cytoplasmic PKM2 with low or undetectable nuclear staining by immunohistochemistry (IHC). In sharp contrast, non-responding tumors showed a >38% increase in PKM2 nuclear staining with low or undetectable cytoplasmic staining. In conclusion, these results confirmed PKM2 as a target for cancer therapy and an unique function of subcellular PKM2, which may characterize therapeutic response to anti-PKM2 therapy in NSCLC.

Expression of tesmin (MTL5) in non‑small cell lung cancer: A preliminary study.

Lung cancer is the most commonly diagnosed cancer and the most frequent cause of death worldwide. Tesmin (testis‑specific metallothionein‑like protein; MTL‑5) is a 60‑kDa protein which has cysteine‑rich motifs (CXC domain), characteristic of metallothioneins (MTs). Tesmin expression has been observed in germ cells during spermatogenesis, oogenesis and also in various cell nuclei after exposure to heavy metal ions. Yet, the role of tesmin in carcinogenesis is unknown. The aim of the present study was to evaluate the localization and intensity of tesmin expression in non‑small cell lung cancer (NSCLC) and its association with the clinicopathological data of patients. A total of 121 cases of NSCLC and 20 cases of non‑cancerous tissue samples from the surgical margin (control) were used for immunohistochemistry (IHC). In addition, 20 cases of frozen NSCLC tissues and 20 cases of control were used for the in vivo study. Normal lung fibroblasts (IMR‑90) and lung cancer cell lines NCI‑H1703 (lung squamous cell carcinoma), NCI‑H522 and A549 (both adenocarcinomas of the lung) were used for western blot analysis (WB) and RT‑PCR studies. Positive cytoplasmic tesmin expression was observed in 88.42% of the examined cases of NSCLC. Statistical analysis showed increased IHC tesmin expression in cancer cells compared to that noted in the controls. In addition, MTL5 mRNA and WB tesmin protein expression were also higher in cancer cases compared to the controls. A positive correlation between tesmin and Ki‑67 IHC expression was demonstrated (r=0.32; P<0.001). Higher WB tesmin expression was also associated with shorter overall survival (P<0.05, Mantel‑Cox test). The in vitro study revealed higher tesmin protein (WB) and MTL5 (qPCR) in lung cancer cell lines compared to the lung fibroblast control cell line. Higher tesmin expression in cancer cells compared to control cells may suggest a role of tesmin in NSCLC carcinogenesis. A positive correlation between tesmin and Ki‑67 could indicate a possible role of tesmin in the proliferation of NSCLC.

Potential role of myeloid-derived suppressor cells in pulmonary fibrosis

Abstract Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective curative therapy. Recruitment of bone marrow-derived myeloid cells is implicated in lung fibrosis by promoting fibrosis via paracrine mechanisms. Recent evidence shows increased circulating myeloid-derived suppressor cells (MDSC) in IPF patients. More recently, the importance of MDSC is suggested by the observation of MDSC accumulation in IPF lungs. The objective of this study was to assess the accumulation/expansion of MDSC in bleomycin-induced mouse model of pulmonary fibrosis and human IPF, and investigate their potential role in lung myofibroblast differentiation. Flow cytometry analysis showed that MDSCs in lung were increased in a mouse model of lung fibrosis, as well as in peripheral blood samples from IPF patients. The increase of the monocytic subtype of MDSC (M-MDSC) was greater than that for the granulocytic subtype (G-MDSC) in the peripheral blood samples from patients with IPF. In vitro co-culture of normal mouse lung fibroblasts with sorted bone marrow (BM)-derived MDSCs using trans-well inserts revealed paracrine activation of lung fibroblasts by activated M-MDSC, but not GMDSC, as manifested by significantly elevated mRNA levels of α-smooth muscle actin and TGFβ mRNAs. Both quiescent M-MDSC and G-MDSC failed to stimulate fibroblast activation or myofibroblast differentiation. Interestingly, the level of TGFβ mRNA in M-MDSC was higher than G-MDSC. These findings indicated that in pulmonary fibrosis, BM-derived MDSCs were mobilized and recruited to the lung, and the recruited M-MDSC subpopulation subsequently played a paracrine role by activating resident lung fibroblasts and myofibroblast differentiation.

Leptin enhances cytokine/chemokine production by normal lung fibroblasts by binding to leptin receptor

BackgroundObesity is a known risk and exacerbation factor for bronchial asthma. Leptin is an adipokine secreted by adipocytes and enhances energy consumption. Earlier studies have shown that leptin also activates inflammatory cells and structural cells, including airway epithelial cells, thereby exacerbating inflammation. However, little is known about leptin's effect on normal human lung fibroblasts (NHLFs), which are deeply involved in airway remodeling in asthma. This study aimed to elucidate the direct effect of leptin on NHLFs. MethodsNHLFs were co-cultured with leptin, and production of cytokines/chemokines was analyzed with real-time PCR and cytometric bead arrays (CBA). Expression of alpha smooth muscle actin (α-SMA) in the lysate of NHLFs stimulated with leptin was assessed by western blotting. Expression of leptin receptor (Ob-R) was analyzed by real-time PCR and flow cytometry. NHLFs were transfected with Ob-R small interference ribonucleic acid (siRNA) by electroporation and used for experiments. ResultsLeptin enhanced production of CCL11/Eotaxin, monocyte chemoattractant protein-1 (CCL2/MCP-1), CXCL8/IL-8, interferon gamma-induced protein 10 (CXCL10/IP-10) and IL-6 by NHLFs at both the protein and messenger ribonucleic acid (mRNA) levels. Leptin also slightly, but significantly, elevated expression of α-SMA. We found robust Ob-R expression on cell surfaces, and transfection with Ob-R siRNA suppressed the enhanced production of CCL11/Eotaxin, CXCL10/IP-10 and IL-6 by leptin, although not completely. ConclusionsThese findings indicate that leptin may contribute to worsening of asthma in obese patients by enhancing production of inflammatory mediators by binding to Ob-R and accelerating myofibroblast differentiation.

The Nutritional Cytokine Leptin Promotes NSCLC by Activating the PI3K/AKT and MAPK/ERK Pathways in NSCLC Cells in a Paracrine Manner

Purpose Leptin is a nutritional cytokine encoded by the obesity gene whose concentration in the tumor microenvironment is closely related to the occurrence and progression of cancer. However, previous evidence has suggested that there is no clear relationship between serum leptin concentrations and lung cancer progression. Cancer-associated fibroblasts (CAFs), the most abundant component of the tumor microenvironment in a variety of solid tumors, were recently reported to produce leptin. Therefore, it was inferred that leptin is most likely to affect non-small-cell lung cancer (NSCLC) through an autocrine and paracrine mechanism. In the current study, we investigated the paracrine effect and mechanism of leptin produced by CAFs on NSCLC by establishing a novel in vitro cell coculture system. Methods A noncontact coculture device was designed and made by 3D printing. CAFs and paired normal lung fibroblasts (NLFs) from 5 patients were successfully isolated and cocultured with two NSCLC cell lines in a coculture system. The background expression of leptin was detected by western blot. The in situ expression of leptin and its receptor (Ob-R) in NSCLC tissues and paired normal lung tissues was analyzed by immunohistochemistry. Furthermore, we downregulated the expression of leptin in CAFs and assessed changes in its promotion on NSCLC cells in the coculture system. Finally, changes in the phosphorylation of ERK1/2 and AKT were examined to investigate the molecular mechanisms responsible for the paracrine promotion of NSCLC cells by leptin. Results Leptin was overexpressed in nearly all five primary CAF lines compared with its expression in paired NLFs. IHC staining showed that the expression of leptin was high in NSCLC cells, slightly lower in CAF, and negative in normal lung tissue. Ob-R was strongly expressed in NSCLC cells. The ability of A549 and H1299 cells to proliferate and migrate was enhanced by high leptin levels in both the cocultured fibroblasts and the culture medium. Furthermore, western blot assays suggested that the MAPK/ERK1/2 and PI3K/AKT signaling pathways were activated by leptin produced by CAFs, which demonstrated that the functions of paracrine leptin in NSCLC are as those of the serum leptin to other cancers. Conclusion Leptin produced by CAF promotes proliferation and migration of NSCLC cells probably via PI3K/AKT and MAPK/ERK1/2 signaling pathways in a paracrine manner.