Background: SIRT1, which belongs to the Sirtuin family of NAD-dependent enzymes, plays diverse roles in aging, metabolism, and disease biology. It could regulate cell survival and has been shown to be a protective factor in heart function. Hence, we verified the mechanism by which SIRT1 regulates doxorubicin induced cardiomyocyte injury in vivo and in vitro. Methods: We analyzed SIRT1 expression in doxorubicin-induced neonatal rat cardiomyocyte injury model and adult mouse heart failure model. SIRT1 was over-expressed in cultured neonatal rat cardiomyocyte by adenovirus mediated gene transfer. SIRT1 agonist resveratrol was used to treat the doxorubicin-induced heart failure mouse model. Echocardiography, reactive oxygen species (ROS) production, TUNEL, qRT-PCR, and Western blotting were performed to analyze cell survival, oxidative stress, and inflammatory signal pathways in cardiomyocytes. Results: SIRT1 expression was down-regulated in doxorubicin induced cardiomocyte injury, accompanied by elevated oxidative stress and cell apoptosis. SIRT1 over-expression reduced doxorubicin induced cardiomyocyte apoptosis with the attenuated ROS production. SIRT1 also reduced cell apoptosis by inhibition of p38MAPK phosphorylation and caspase-3 activation. The SIRT1 agonist resveratrol was able to prevent doxorubicin-induced heart function loss. Moreover, the SIRT1 inhibitor niacinamide could reverse SIRT1's protective effect in cultured neonatal rat cardiomyocytes. Conclusions: These results support the role of SIRT1 as an important regulator of cardiomyocyte apoptosis during doxorubicin-induced heart injury, which may represent a potential therapeutic target for doxorubicin-induced cardiomyopathy.

Pulmonary exposure to multi-walled carbon nanotubes (MWCNT) induces an inflammatory and rapid fibrotic response, although the long-term signaling mechanisms are unknown. The aim of this study was to examine the effects of 1, 10, 40, or 80 μg MWCNT administered by pharyngeal aspiration on bronchoalveolar lavage (BAL) fluid for polymorphonuclear cell (PMN) infiltration and lactate dehydrogenase (LDH) activity and lung histopathology for inflammatory and fibrotic responses in mouse lungs 1 month, 6 months, and 1 year post-exposure. As MWCNT are often structurally compared to asbestos, a 120 μg crocidolite asbestos group was incorporated as a positive control for comparative purposes. Results showed that MWCNT increased BAL fluid LDH activity and PMN infiltration in a dose-dependent manner at all 3 post-exposure times. Asbestos exposure elevated LDH activity at all 3 post-exposure times and PMN infiltration at 1- and 6 months post-exposure. Pathological changes in the lung, the presence of MWCNT or asbestos, and fibrosis were noted at 40 and 80 μg MWCNT and in asbestos-exposed mice at 1 year post-exposure. To determine potential signaling pathways involved with MWCNT-associated pathological changes in comparison to asbestos, we determined up- and down-regulated gene expression in lung tissue at 1 year post-exposure. Exposure to MWCNT tended to favor those pathways involved in immune responses, specifically T-cell responses, whereas exposure to asbestos tended to favor pathways involved in oxygen species production, electron transport, and cancer. Data indicate that MWCNT are biopersistent in the lung and induce inflammatory and fibrotic pathological changes similar to crocidolite asbestos, but may reach these endpoints by different mechanisms.

Multiwalled carbon nanotubes (MWCNT) are one of the most commonly produced nanomaterials, and pulmonary exposure during production, use, and disposal is a concern for the developing nanotechnology field. The airway epithelium is the first line of defense against inhaled particles. In a mouse model, MWCNT were reported to reach the alveolar space of the lung after in vivo exposure, penetrate the epithelial lining, and result in inflammation and progressive fibrosis. This study sought to determine the cellular and gene expression changes in small airway epithelial cells (SAEC) after in vitro exposure to MWCNT in an effort to elucidate potential toxicity mechanisms and signaling pathways. A direct interaction between SAEC and MWCNT was confirmed by both internalization of MWCNT and interaction at the cell periphery. Following exposure, SAEC showed time-dependent increases in reactive oxygen species production, total protein phosphotyrosine and phosphothreonine levels, and migratory behavior. Analysis of gene and protein expression suggested altered regulation of multiple biomarkers of lung damage, carcinogenesis, and tumor progression, as well as genes involved in related signaling pathways. These results demonstrate that MWCNT exposure resulted in the activation of SAEC. Gene expression data derived from MWCNT exposure provide information that may be used to elucidate the underlying mode of action of MWCNT in the small airway and suggest potential prognostic gene signatures for risk assessment.

PurposeThis study aims to develop a multi-gene assay predictive of the clinical benefits of chemotherapy in non-small cell lung cancer (NSCLC) patients, and substantiate their protein expression as potential therapeutic targets.Patients and methodsThe mRNA expression of 160 genes identified from microarray was analyzed in qRT-PCR assays of independent 337 snap-frozen NSCLC tumors to develop a predictive signature. A clinical trial JBR.10 was included in the validation. Hazard ratio was used to select genes, and decision-trees were used to construct the predictive model. Protein expression was quantified with AQUA in 500 FFPE NSCLC samples.ResultsA 7-gene signature was identified from training cohort (n = 83) with accurate patient stratification (P = 0.0043) and was validated in independent patient cohorts (n = 248, P < 0.0001) in Kaplan-Meier analyses. In the predicted benefit group, there was a significantly better disease-specific survival in patients receiving adjuvant chemotherapy in both training (P = 0.035) and validation (P = 0.0049) sets. In the predicted non-benefit group, there was no survival benefit in patients receiving chemotherapy in either set. The protein expression of ZNF71 quantified with AQUA scores produced robust patient stratification in separate training (P = 0.021) and validation (P = 0.047) NSCLC cohorts. The protein expression of CD27 quantified with ELISA had a strong correlation with its mRNA expression in NSCLC tumors (Spearman coefficient = 0.494, P < 0.0088). Multiple signature genes had concordant DNA copy number variation, mRNA and protein expression in NSCLC progression.ConclusionsThis study presents a predictive multi-gene assay and prognostic protein biomarkers clinically applicable for improving NSCLC treatment, with important implications in lung cancer chemotherapy and immunotherapy.

Inhalation exposure to multi-walled carbon nanotubes (MWCNT) in mice results in inflammation, fibrosis, and the promotion of lung adenocarcinoma; however, the molecular basis behind these pathologies is unknown. This study determined global mRNA and miRNA profiles in whole blood from mice exposed by inhalation to MWCNT that correlated with the presence of lung hyperplasia, fibrosis, and bronchiolo-alveolar adenoma and adenocarcinoma. Six-week-old, male, B6C3F1 mice received a single intraperitoneal injection of either the DNA-damaging agent methylcholanthrene (MCA, 10 μg/g body weight) or vehicle (corn oil). One week after injections, mice were exposed by inhalation to MWCNT (5 mg/m³, 5 hours/day, 5 days/week) or filtered air (control) for a total of 15 days. At 17 months post-exposure, mice were euthanized and examined for the development of pathological changes in the lung, and whole blood was collected and analyzed using microarray analysis for global mRNA and miRNA expression. Numerous mRNAs and miRNAs in the blood were significantly up- or down-regulated in animals developing pathological changes in the lung after MCA/corn oil administration followed by MWCNT/air inhalation, including fcrl5 and miR-122-5p in the presence of hyperplasia, mthfd2 and miR-206-3p in the presence of fibrosis, fam178a and miR-130a-3p in the presence of bronchiolo-alveolar adenoma, and il7r and miR-210-3p in the presence of bronchiolo-alveolar adenocarcinoma, among others. The changes in miRNA and mRNA expression, and their respective regulatory networks, identified in this study may potentially serve as blood biomarkers for MWCNT-induced lung pathological changes.