Surfactant protein D inhibits activation of non-small cell lung cancer-associated mutant EGFR and affects clinical outcomes of patients.

Tyrosine kinase inhibitor (TKI)-sensitive and TKI-resistant mutations of epidermal growth factor receptor (EGFR) are associated with lung adenocarcinoma. EGFR mutants were previously shown to exhibit ligand-independent activation. We have previously demonstrated that pulmonary surfactant protein D (SP-D, SFTPD) suppressed wild-type EGFR signaling by blocking ligand binding to EGFR. We herein demonstrate that SFTPD downregulates ligand-independent signaling in cells harboring EGFR mutations such as TKI-sensitive exon 19 deletion (Ex19del) and L858R mutation as well as TKI-resistant T790M mutation, subsequently suppressing cellular growth and motility. Lectin blotting and ligand blotting in lung cancer cell lines suggested that EGFR mutants express oligomannose-type N-glycans and interact with SFTPD directly. Cross-linking assay indicated that SFTPD inhibits ligand-independent dimerization of EGFR mutants. We also demonstrated that SFTPD reduced dimerization-independent phosphorylation of Ex19del and T790M EGFR mutants using point mutations that disrupted the asymmetric dimer interface. It was confirmed that SFTPD augmented the viability-suppressing effects of EGFR-TKIs. Furthermore, retrospective analysis of 121 patients with lung adenocarcinoma to examine associations between serum SFTPD levels and clinical outcome indicated that in TKI-treated patients with lung cancer harboring EGFR mutations, including Ex19del or L858R, high serum SFTPD levels correlated with a lower number of distant metastases and prolonged overall survival and progression-free survival. These findings suggest that SFTPD downregulates both TKI-sensitive and -resistant EGFR mutant signaling, and SFTPD level is correlated with clinical outcome. These findings illustrate the use of serum SFTPD level as a potential marker to estimate the efficacy of EGFR-TKIs.

Surfactant protein D suppresses lung cancer progression by downregulation of epidermal growth factor signaling.

Surfactant protein D (SP-D) is a member of the collectin family that has an important role in maintaining pulmonary homeostasis. In this study, we demonstrated that SP-D inhibited the proliferation, migration and invasion of A549 human lung adenocarcinoma cells. We found that SP-D suppressed epidermal growth factor (EGF) signaling in A549 cells, H441 human lung adenocarcinoma cells and human EGF receptor (EGFR) stable expression CHO-K1 cells. A binding study using (125)I-EGF demonstrated that SP-D downregulated the binding of EGF to EGFR. A ligand blot indicated that SP-D bound to EGFR, and a lectin blot suggested that EGFR in A549 cells had both high-mannose type and complex type N-glycans. We purified the recombinant extracellular domain of EGFR (soluble EGFR=soluble EGFR (sEGFR)), and demonstrated that SP-D directly bound to sEGFR in a Ca(2+)-dependent manner. The binding of SP-D to sEGFR was suppressed by EDTA, mannose or N-glycopeptidase F treatment. Mass spectrometric analysis indicated that N-glycans in domain III of EGFR were of a high-mannose type. These data suggest that SP-D reduces EGF binding to EGFR through the interaction between the carbohydrate recognition domain of SP-D and N-glycans of EGFR, and downregulates EGF signaling. Our finding suggests the novel type of regulation system of EGF signaling involving lectin-to-carbohydrate interaction and downregulation of ligand binding.

Annexin V decreases PS-mediated macrophage efferocytosis and deteriorates elastase-induced pulmonary emphysema in mice.

Efferocytosis is believed to be a key regulator for lung inflammation in chronic obstructive pulmonary disease. In this study we pharmacologically inhibited efferocytosis with annexin V and attempted to determine its impact on the progression of pulmonary emphysema in mouse. We first demonstrated in vitro and in vivo efferocytosis experiments using annexin V, an inhibitor for phosphatidylserine-mediated efferocytosis. We then inhibited efferocytosis in porcine pancreatic elastase (PPE)-treated mice. PPE-treated mice were instilled annexin V intranasally starting from day 8 until day 20. Mean linear intercept (Lm) was measured, and cell apoptosis was assessed in lung specimen obtained on day 21. Cell profile, apoptosis, and mRNA expression of matrix metalloproteinases (MMPs) and growth factors were evaluated in bronchoalveolar lavage (BAL) cells on day 15. Annexin V attenuated macrophage efferocytosis both in vitro and in vivo. PPE-treated mice had a significant higher Lm, and annexin V further increased that by 32%. More number of macrophages was found in BAL fluid in this group. Interestingly, cell apoptosis was not increased by annexin V treatment both in lung specimens and BAL fluid, but macrophages from mice treated with both PPE and annexin V expressed higher MMP-2 mRNA levels and had a trend for higher MMP-12 mRNA expression. mRNA expression of keratinocyte growth factor tended to be downregulated. We showed that inhibited efferocytosis with annexin V worsened elastase-induced pulmonary emphysema in mice, which was, at least partly, attributed to a lack of phenotypic change in macrophages toward anti-inflammatory one.

Treatment results of carcinoma in situ of the glottis: an analysis of 82 cases.

OBJECTIVES: To evaluate the results of different treatment modalities for carcinoma in situ of the glottis, and to identify important prognostic factors for outcome. DESIGN: Review of 82 cases treated definitively for glottic carcinoma in situ between 1958 and 1998. The median follow-up for all patients was 112 months, and 90% had more than 2 years of follow-up. SETTING: Academic tertiary care referral centers. INTERVENTION: Fifteen patients were treated with vocal cord stripping (group 1), 13 with more extensive surgery (group 2) including endoscopic laser resection (11 patients) and hemilaryngectomy (2 patients), and 54 with radiotherapy (group 3). Thirty patients had anterior commissure involvement and 9 had bilateral vocal cord involvement. Radiotherapy was delivered via opposed lateral fields at 1.5 to 2.4 Gy per fraction per day (median fraction size, 2 Gy), 5 days per week. The median total dose was 64 Gy, and the median overall time was 47 days. MAIN OUTCOME MEASURES: Initial locoregional control (LRC), ultimate LRC, and larynx preservation. RESULTS: The 10-year initial LRC rates were 56% for group 1, 71% for group 2, and 79% for group 3. Of those who failed, the median time to relapse was 11 months for group 1, 17 months for group 2, and 41 months for group 3. Univariate analysis showed that the difference in initial LRC rates between groups 1 and 3 was statistically significant (P =.02), although it was not statistically significant on multivariate analysis (P =.07). Anterior commissure involvement was an important prognostic factor for LRC on both univariate (P =.03) and multivariate (P =.04; hazard ratio, 1.6) analysis, and its influence appeared to be mainly confined to the surgically treated patients (groups 1 and 2). The 10-year larynx preservation rates were 92% for group 1, 70% for group 2, and 85% for group 3. Anterior commissure involvement was the only important prognostic factor for larynx preservation (P =. 01) on univariate analysis. All but 2 patients in whom treatment failed underwent successful salvage surgery. Voice quality was deemed good to excellent in 73% of the patients in group 1, 40% in group 2, and 68% in group 3. CONCLUSIONS: Treatment of carcinoma in situ of the glottis with vocal cord stripping or more extensive surgery or radiotherapy provided excellent ultimate LRC and comparable larynx preservation rates. Anterior commissure involvement was associated with poorer initial LRC and larynx preservation, particularly in the surgically treated patients. The choice of initial treatment should be individualized, depending on patient age, reliability, and tumor extent. Pretreatment and posttreatment objective evaluation of voice quality should be helpful in determining the best therapy for these patients.

Carbon monoxide overproduced by heme oxygenase-1 causes a reduction of vascular resistance in perfused rat liver.

This study aimed to examine whether livers overexpressing heme oxygenase (HO)-1 could alter the vascular resistance through the vasorelaxing action of carbon monoxide (CO). The relationship among HO-1 expression, CO generation, and the vascular resistance was assessed in perfused rat livers pretreated with hemin, an inducer of HO-1. At 18 h after the hemin treatment, livers displayed marked increases in HO-1 expression in hepatocytes and venous CO flux and a reduction of the basal resistance. The reduction of the resistance in hemin-treated livers was canceled by administration of oxyhemoglobin, a reagent trapping both CO and nitric oxide (NO), but not by methemoglobin, which captures NO but not CO. Liposome-encapsulated oxyhemoglobin, which cannot access the space of Disse, did not cause vasoconstriction. Furthermore, these livers became less sensitive to endothelin-1, a vasoconstrictive peptide, than the untreated controls through mechanisms involving CO. On the other hand, at 12 or 24 h after the treatment when the HO-1 induction was not accompanied by CO overproduction, neither a decrease in the basal resistance nor vascular hyporeactivity to endothelin-1 was observed. These results suggest that CO overproduced in the extrasinusoidal compartment is a determinant of the HO-1-mediated vasorelaxation in the liver.

Induction of heme oxygenase-1 suppresses venular leukocyte adhesion elicited by oxidative stress: role of bilirubin generated by the enzyme.

This study aimed to examine whether an elevated activity of heme oxygenase (HO)-1 in the tissue attenuates endothelial cell-leukocyte interactions microvessels in vivo. When rats were pretreated with an intraperitoneal injection of hemin, an HO-1 inducer, mesenteric tissues, including their microvessels, displayed a marked induction of HO-1 concurrent with an increase in plasma concentrations of bilirubin-IXalpha, the product of HO-catalyzed degradation of protoheme IX. In these rats, oxidative stress such as superfusion with H(2)O(2) and ischemia-reperfusion of the tissues neither induced rolling nor exhibited adherent responses of leukocytes in venules. In contrast, the oxidative stresses evoked marked rolling and adhesion of leukocytes in the control rats without HO-1 induction. The HO-1 induction also downregulated leukocyte adhesion elicited by other pro-oxidant stimuli such as N(omega)-nitro-L-arginine methyl ester. The decreases in the oxidant-elicited leukocyte adhesive responses under HO-1-inducing conditions were restored by perfusion with zinc protoporphyrin-IX, an HO inhibitor, but not with copper protoporphyrin-IX, which did not inhibit the enzyme. Furthermore, the effects of zinc protoporphyrin-IX were repressed by superfusion with bilirubin or biliverdin at the micromolar level, but not by the same concentration of carbon monoxide, another product of HO. These results indicate that induction of the HO-1 activity serves as a potential stratagem to prevent oxidant-induced microvascular leukocyte adhesion through the action of bilirubin, a product of HO reaction.