Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder manifesting with ectopic connective tissue mineralization, caused by mutations in the ABCC6 gene, with ∼35% of all mutations being ...premature termination mutations. In this study, we investigated the therapeutic potential of the nonsense codon read-through-inducing drug, PTC124, in treating PXE. The ability of this drug to facilitate read-through of nonsense mutations was examined in HEK293 cells transfected with human ABCC6 expression constructs harboring seven different PXE-associated nonsense mutations, and was evaluated by immunofluorescence and In-Cell ELISA. Our data demonstrated that PTC124 did not exhibit cytotoxicity in concentrations up to 20μgml−1, and the facilitated read-through varied not only with dose but also with sequence context. Considering the redundancy of the genetic code, it was postulated that in case of the most common recurrent nonsense mutation, p.R1141X, the read-through may result in substitution of the arginine 1,141 by glycine, tryptophan, or cysteine. Their potential pathogenicity was tested in a recently developed zebrafish messenger RNA (mRNA) rescue assay, and demonstrated that all three mRNA transcripts were able to rescue abcc6a morpholino-induced phenotype of zebrafish. Thus, our results suggest that read-through of nonsense mutations in ABCC6 by PTC124 may have potential for pharmacologic treatment of PXE.
Objectives
The effectiveness and costs of new treatments should be assessed in relation to existing practice. We describe treatments, survival and costs for advanced or metastatic small cell lung ...cancer (SCLC) patients receiving systemic therapy in the period preceding the introduction of immunotherapies.
Materials and Methods
This was a retrospective cohort study of patients aged ≥65 years, identified using linked Surveillance, Epidemiology, and End Results and Medicare databases. Individuals with a new primary diagnosis of SCLC between January 2007 and December 2013 were followed until December 2014. Chemotherapy treatments, health care visits and costs (in 2016 USD), and survival were determined by line of therapy.
Results
A total of 11 812 patients were identified with SCLC. First‐line (1L) chemotherapy was received by 6509 (55.1%) patients, most (93.2%) with carboplatin‐ (71.0%) or cisplatin‐ (22.2%) based therapies, typically combined with etoposide (79.2%). Second‐ (2L) and third‐ (3L) line chemotherapies were received by 2238 (18.9%) and 679 (5.7%) patients, of which 48.4% and 30.9%, respectively, were platinum‐based. The median durations of 1L, 2L, and 3L carboplatin‐based therapies were 5.9, 4.8, and 5.4 months, respectively, and the corresponding durations of cisplatin‐based therapies were 5.3, 4.2, and 5.3 months. During 1L, 2L, and 3L chemotherapies, patients averaged 8.2, 7.4, and 7.3 health care visits per month, respectively, and incurred total mean health care costs of $60 223, $42 636, and $35 903 per patient, respectively. Median survival from the start of 1L, 2L, and 3L chemotherapy was 9.2, 6.0, and 5.7 months, respectively.
Conclusion
First‐line chemotherapy was primarily platinum‐based, and a plethora of different regimens was used for 2L and 3L chemotherapies. Median survival from the start of 1L chemotherapy was 9 months, with an associated health care cost of $60 000. These data highlight an unmet medical need among SCLC patients receiving systemic therapy.
The objective of this manuscript was to describe the existing practice of small cell lung cancer (SCLC) care as a real‐world benchmark with which to assess the performance of the anticipated new immunotherapies. First‐line chemotherapy was largely platinum‐based, consistent with US guidelines while a variety of regimens were used for second‐ and third‐line chemotherapies, reflecting the lack of an accepted standard of care for these patients. Median survival from the start of each line of chemotherapy was <12 months. The data presented in this manuscript provide a historical benchmark with which to assess the costs of the anticipated new immunotherapies and their effects on survival, as well as on the burden of SCLC on patients and on the health care system.
Staphylococcus aureus is an important human pathogen whose virulence relies on the secretion of many different proteins. In general, the secretion of most proteins in S. aureus, as well as other ...bacteria, is dependent on the type I signal peptidase (SPase)-mediated cleavage of the N-terminal signal peptide that targets a protein to the general secretory pathway. The arylomycins are a class of natural product antibiotics that inhibit SPase, suggesting that they may be useful chemical biology tools for characterizing the secretome. While wild-type S. aureus (NCTC 8325) is naturally resistant to the arylomycins, sensitivity is conferred via a point mutation in its SPase. Here, we use a synthetic arylomycin along with a sensitized strain of S. aureus and multidimensional protein identification technology (MudPIT) mass spectrometry to identify 46 proteins whose extracellular accumulation requires SPase activity. Forty-four possess identifiable Sec-type signal peptides and thus are likely canonically secreted proteins, while four also appear to possess cell wall retention signals. We also identified the soluble C-terminal domains of two transmembrane proteins, lipoteichoic acid synthase, LtaS, and O-acyteltransferase, OatA, both of which appear to have noncanonical, internal SPase cleavage sites. Lastly, we identified three proteins, HtrA, PrsA, and SAOUHSC_01761, whose secretion is induced by arylomycin treatment. In addition to elucidating fundamental aspects of the physiology and pathology of S. aureus, the data suggest that an arylomycin-based therapeutic would reduce virulence while simultaneously eradicating an infection.
Introduction
Hypoxia‐inducible factor‐2α (HIF‐2α) modulates the hypoxic response pathway in tumors; however, mutations in pathways (including SDHA, SDHB, SDHC, SDHD, FH, and VHL genes) that are ...suspected to activate HIF‐2α are poorly understood, with limited understanding of the prevalence and clinical prognosis.
Methods
This retrospective observational study used a de‐identified nationwide (US‐based) clinico‐genomic database (CGDB) across 15 available tumor types.
Results
Among the 9467 adult patients with advanced/metastatic solid tumors included in the analysis, any mutation at the above‐mentioned six genes was observed in 1.8% (95% CI: 1.5–2.1) of patients. The mutation prevalence ranged from 0.05% of SDHD to 0.93% of VHL. When further stratified by tumor type, the prevalence of gene mutation in each tumor type was well below 1%, except for VHL with 44% in renal cell carcinomas (RCC). Excluding RCC, the prevalence of any HIF‐2α gene mutations in the study population was 0.9% (95% CI: 0.8–1.2). The median overall survival (OS) from 1 and 2 L therapy among patients with any HIF‐2α gene mutation was 14.5 (95% CI: 11.5–24.2) and 9.3 (95% CI: 6.0–18.1) months, respectively, compared with 13.4 (95% CI: 12.9–13.9) and 9.8 (95% CI: 9.3–10.4) months among patients without HIF‐2α gene mutations.
Discussion and Conclusions
The prevalence of HIF‐2α related gene mutations was generally low (<1%) across the 15 solid tumor types, except for VHL in RCC. No significant association between HIF‐2α gene mutation status and OS was identified among patients evaluated in this study.
The development of novel targeted therapies has become an important research focus for lung cancer treatment. Our previous study has shown leptomycin B (LMB) significantly inhibited proliferation of ...lung cancer cells; however, p53 wild type lung cancer cells were resistant to LMB. Therefore, the objective of this study was to develop and evaluate a novel therapeutic strategy to sensitize LMB-resistant lung cancer cells by combining LMB and doxorubicin (DOX). Among the different treatment regimens, pretreatment with DOX (pre-DOX) and subsequent treatment with LMB to A549 cells significantly decreased the 50% inhibitory concentration (IC50) as compared to that of LMB alone (4.4 nM vs. 10.6 nM, P<0.05). Analysis of cell cycle and apoptosis by flow cytometry further confirmed the cytotoxic data. To investigate molecular mechanisms for this drug combination effects, p53 pathways were analyzed by Western blot, and nuclear proteome was evaluated by two dimensional-difference gel electrophoresis (2D-DIGE) and mass spectrometry. In comparison with control groups, the levels of p53, phospho-p53 (ser15), and p21 proteins were significantly increased while phospho-p53 (Thr55) and survivin were significantly decreased after treatments of pre-DOX and LMB (P<0.05). The 2D-DIGE/MS analysis identified that sequestosome 1 (SQSTM1/p62) had a significant increase in pre-DOX and LMB-treated cells (P<0.05). In conclusion, our results suggest that drug-resistant lung cancer cells with p53 wild type could be sensitized to cell death by scheduled combination treatment of DOX and LMB through activating and restoring p53 as well as potentially other signaling pathway(s) involving sequestosome 1.
Abstract Objective Our recent study demonstrated the protective action of green tea polyphenols (GTPs) against bone loss in ovariectomized (OVX) rats through their antioxidant capacities to scavenge ...reactive oxygen species. The objective of the present study was to evaluate the alterations of liver protein profiles in estrogen-deficient middle-aged rats after GTP treatment by a gel-based proteomic approach. This may lead to understanding the mechanisms of GTPs in promoting bone health. Methods Liver samples were obtained from 14-mo-old female OVX rats treated with no GTPs (OVX) or 0.5% (w/v) GTPs (OVX + GTP) in drinking water for 16 wk ( n = 10/group). Two-dimensional difference gel electrophoresis combined with mass spectrometry was used to compare the liver protein profiles of pooled samples from the OVX and OVX + GTP groups. Liver proteins were labeled in duplicate by reversing the fluorescent dyes. Results Approximately 800 protein spots were detected. The expression levels of superoxide dismutase-1 and adenosine triphosphate synthase were 2.0-fold and 1.5-fold higher in the OVX + GTP group versus the OVX group, respectively, whereas the expression level of catechol-O-methyltransferase was 1.5-fold lower in the OVX + GTP group versus the OVX group. The changes of superoxide dismutase-1 and catechol-O-methyltransferase in individual liver samples were confirmed by western blots. Conclusion Our data provide further evidence for the antioxidant role of GTPs by increasing superoxide dismutase-1 and adenosine triphosphate synthase and the estrogen-associated effect of GTPs by decreasing catechol-O-methyltransferase.
The p53 tumor suppressor gene plays an essential role in tumorigenesis, and the chromosomal region maintenance 1 (CRM1) has been suggested to export p53 protein from the nucleus to the cytoplasm. The ...objectives of the present study were to evaluate p53 expression and subcellular localization as well as CRM1 expression using immunohistochemistry in our established bitransgenic mouse lung tumor model. In this model, expression of the mutant human Ki-rasG12C allele was regulated in a doxycycline (DOX)-inducible, lung-specific manner. Following treatment with curcumin, we found that although overall p53 expression levels were not significantly changed among the three groups, lung lesions in mice treated with DOX alone had the highest proportion of N>C (nucleus predominant) p53 staining (46±7%), followed by lung lesions in mice co-treated with DOX and curcumin (31±12%) and controls (17±4%). CRM1 expression was dramatically inhibited in lung lesions in mice treated with DOX (0±0) as compared to controls (90±17, P=0.001), and could be partially reversed after curcumin treatment (47±21, P=0.028, DOX vs. DOX+curcumin). Collectively, the results from this study demonstrated that p53 accumulated in the nucleus in lung lesions in mice expressing the mutant Ki-rasG12C transgene as a result of down-regulation of CRM1. Furthermore, these alterations could be partially reversed by curcumin treatment. p53 subcellular localization resulting from CRM1 alterations may play an important role in lung tumorigenesis.
Curcumin, a polyphenolic compound, is the active component of Curcuma longa and has been extensively investigated as an anticancer drug that modulates multiple pathways. Eukaryotic initiation factors ...(eIFs) have been known to play important roles in translation initiation, which controls cell growth and proliferation. Little is known about the effects of curcumin on eIFs in lung cancer. The objective of this study was to exam the curcumin cytotoxic effect and modulation of two major rate-limiting translation initiation factors, including eIF2α and eIF4E protein expression levels in lung adenocarcinoma epithelial cell line A549. Cytotoxicity was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and protein changes were determined by Western blot. A549 cells were treated with 0-240 μM curcumin for 4-96 h. The inhibitory effects of curcumin on cytotoxicity were dose- and time-dependent (P < 0.001). The 50% inhibitory curcumin concentrations (IC50s) at 24, 48, 72, and 96 h were 93, 65, 40, and 24 μM, respectively. Protein expressions of eIF2α, eIF4E, Phospho-4E-BP1 were down-regulated, while Phospho-eIF2α and Phospho-eIF4E were up-regulated after A549 cells were treated with 20 and 40 μM curcumin for 24 h. In addition, the effects of curcumin on these protein expression changes followed a significant dose-response (P < 0.05, trend test). These findings suggest that curcumin could reduce cell viability through prohibiting the initiation of protein synthesis by modulating eIF2α and eIF4E.
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