Rethinking the Selection of Pathological T-Classification for Non-Small-Cell Lung Cancer in Varying Degrees of Visceral Pleural Invasion: A SEER-Based Study.

Background: The T classification of non-small-cell lung cancer (NSCLC) was upgraded from T1 to T2 when accompanied by visceral pleural invasion (VPI). However, the association between VPI and prognostic outcomes was obscure in NSCLC patients with ≤3 cm tumor size (TS), which leaded the controversy of selection of T classification. The goal was to evaluate the effect of VPI on the prognosis of NSCLC with ≤ 3cm TS and present a modified T classification. Methods: A total of 14,934 NSCLC patients without distant metastasis were recruited through a retrospective study in the SEER database. The effect of VPI on lung cancer specific survival (LCSS) was evaluated using survival curve and COX regression analysis in NSCLC patients with ≤3 cm TS. Results: Although there was no difference of the LCSS of PL0 and PL1 patients with ≤2 cm TS in patients without lymph node (LN) metastasis, the LCSS was lower in PL2 patients than those in PL0 (T1a: p < 0.001; T1b: p = 0.001). Moreover, the LCSS was decreased in PL1 and PL2 patients with 2-3 cm TS compared with PL0 (T1c: PL1, p < 0.001; PL2, p = 0.009) of patients without LN metastasis. No difference of LCSS was observed in patients with LN metastasis between PL0 with PL1 and PL2. Conclusion: In NSCLC patients without LN metastasis and TS ≤ 2 cm, tumor with PL1 should remain defined as T1, tumor with PL2 should be defined as T2. However, 2-3 cm TS patients with PL1 or PL2 should both defined as T2. Meanwhile, ≤3 cm TS patients with LN metastasis can be regarded as T1, whether NSCLC patients accompanied with PL1 or PL2.

Ceramide induces the apoptosis of non­small cell lung cancer cells through the Txnip/Trx1 complex.

Ceramide is a biologically active sphingomyelin that inhibits cell growth and proliferation. In previous studies, it was demonstrated that the use of lipopolysaccharides induces acid sphingomyelinases to produce ceramide, promoting lung cancer cell apoptosis; however, the specific mechanisms of this action remain unclear. Thioredoxin­interacting protein (Txnip) plays an important role in the signal transmission of redox reactions inside and outside the cell. Thus, it was hypothesized that ceramide induces apoptosis in lung adenocarcinoma cells (A549 and PC9) by modulating the Txnip/Trx1 complex. In the present study, the Cell Counting kit­8 method was used to detect cell activity and the drug concentration. Hoechst 33258 staining and flow cytometry were used to detect cell apoptosis, and the positional association between Txnip and Trx1 upregulated by ceramide was observed by immunofluorescence confocal microscopy. Reverse transcription­quantitative polymerase chain reaction and western blot analysis were used to detect the changes in related gene, mRNA and protein expression levels. The results revealed that ceramide treatment resulted in the upregulation of Txnip and in the reduction of Trx1 activities. However, the Txnip inhibitor, verapamil, reversed these changes. The analysis of mRNA expression further verified the changes observed in the protein expression of Txnip, Trx1 and apoptosis­related proteins. On the whole, the present study demonstrates that ceramide induces the apoptosis of lung cancer cells by regulating the Txnip/Trx1 complex.

Role of ASM/Cer/TXNIP signaling module in the NLRP3 inflammasome activation.

BACKGROUND: This study aimed to explore the effects of ceramide (Cer) on NLRP3 inflammasome activation and their underlying mechanisms. METHODS: Lipopolysaccharide (LPS)/adenosine triphosphate (ATP)-induced NLRP3 inflammasome activation in J774A.1 cells and THP-1 macrophages was used as an in vitro model of inflammation. Western blotting and real-time PCR (RT-PCR) were used to detect the protein and mRNA levels, respectively. IL-1ß and IL-18 levels were measured by ELISA. ASM assay kit and immunofluorescence were used to detect ASM activity and Cer content. RESULTS: Imipramine, a well-known inhibitor of ASM, significantly inhibited LPS/ATP-induced activity of ASM and the consequent accumulation of Cer. Additionally, imipramine suppressed the LPS/ATP-induced expression of thioredoxin interacting protein (TXNIP), NLRP3, caspase-1, IL-1ß, and IL-18 at the protein and mRNA level. Interestingly verapamil, a TXNIP inhibitor, suppressed LPS/ATP-induced activation of TXNIP/NLRP3 inflammasome but did not affect LPS/ATP-induced ASM activation and Cer formation. TXNIP siRNA and verapamil inhibited C2-Cer-induced upregulation of TXNIP and activation of the NLRP3 inflammasome. In addition, the pretreatment of cells with sulfo-N-succinimidyl oleate (SSO), an irreversible inhibitor of the scavenger receptor CD36, blocked Cer-induced upregulation of nuclear factor-κB (NF-κB) activity, TXNIP expression, and NLRP3 inflammasome activation. Inhibition of NF-κB activation by SN50 prevented Cer-induced upregulation of TXNIP and activation of the NLRP3 inflammasome but did not affect CD36 expression. CONCLUSION: This study demonstrated that the ASM/Cer/TXNIP signaling pathway is involved in NLRP3 inflammasome activation. The results documented that the CD36-dependent NF-κB-TXNIP signaling pathway plays an essential role in the Cer-induced activation of NLRP3 inflammasomes in macrophages.

Salvianolic acid B exerts a protective effect in acute liver injury by regulating the Nrf2/HO-1 signaling pathway.

Salvianolic acid B (Sal B) exerts strong antioxidant activity and eliminates the free radical effect. However, how it affects the antioxidant pathway is not very clear. The objective of this study was to investigate the underlying mechanism of Sal B in CCl4-induced acute liver injury, especially its effect on the Nrf2/HO-1 signaling pathway. For the in vivo experiment, an acute liver injury model was induced using CCl4 and treated with Sal B. For the in vitro experiment, an oxidative damage model was established followed by Sal B treatment. Serum biochemical indicators and reactive oxygen species activity were detected using corresponding kits. Oxidant/antioxidant status was determined based on the levels of malondialdehyde, glutathione, and superoxide dismutase. Nrf2 and HO-1 levels were analyzed by Western blotting and immunohistochemical staining. Sal B treatment improved liver histology, decreased the aminotransferase levels, and attenuated oxidative stress in the acute liver injury model. Nrf2 and HO-1 levels were increased both in vivo and in vitro. Sal B suppresses acute liver injury and Nrf2/HO-1 signaling plays a key role in this process.

MiR-541-3p reverses cancer progression by directly targeting TGIF2 in non-small cell lung cancer.

Lung cancer remains a leading cause of cancer-associated mortality worldwide, and non-small lung cancer (NSCLC) is responsible for over 80 % of lung cancer-related deaths. Identifying novel molecular biomarker that can inhibit the progression of lung cancer will facilitate the development of new treatment strategies. Herein, we demonstrated that miR-541-3p is a tumor-suppressor microRNA (miRNA) in NSCLC progression. We found that expression of miR-541-3p was decreased obviously in NSCLC tissues and plasma. Down-regulation of miR-541-3p was associated with TNM stage and postoperative survival. Overexpression of miR-541-3p inhibited the growth and metastasis of NSCLC cells. The TGIF2 was a direct target of miR-541-3p and promoted the growth and metastasis of NSCLC cells. Further study showed that TGIF2 could reverse the inhibitory effect of miR-541-3p on growth and metastasis of NSCLC cells. Taken together, our data highlight the pivotal role of miR-541-3p in the progression of NSCLC. Thus, miR-541-3p may be a potential prognostic marker and of treatment relevance for NSCLC progression intervention.

The association between osteopontin and survival in non-small-cell lung cancer patients: a meta-analysis of 13 cohorts.

In the last decade, osteopontin (OPN) was identified as one of the important proteins that promote the metastasis of tumor. However, the association between OPN overexpression and clinical outcome of non-small-cell lung cancer (NSCLC) was unclear. The purpose of this study is to investigate the role of OPN in NSCLC patients. A total of 13 studies are included to explore the relationship between the OPN elevation and the overall survival (OS) and disease-free survival (DFS) in NSCLC patients. We searched for related articles in PubMed, Web of Science, Google Scholar, and Cochrane Library databases, which were published before January 31, 2015. Hazard ratio (HR), odds ratio (OR), and 95% confidence interval (CI) in the high OPN expression group compared with the low OPN expression group were calculated and analyzed. Primary results were summarized by using a fixed-effects model or a random-effects model. The stratified analyses in subgroups were also performed. Thirteen cohort studies, which involved 1,630 patients, were included. Subgroup analyses were performed by area and test method of OPN. We found that OPN was significantly associated with poor OS (HR =2.20, 95% CI 1.71-2.83, P<0.001) and DFS (HR =2.11, 95% CI 1.62-2.74, P<0.001) in NSCLC patients. OPN overexpression tended to be associated with the presence of advanced tumor TNM stage (III and IV) (OR =2.57, 95% CI 1.61-4.11, P<0.001). The Egger's test suggested that there was no publication bias in OS studies (P=0.062) and DFS studies (P=0.740). These data indicate that OPN seems to have a significant predictive potential in estimating survival in NSCLC.

[Effect of C8-ceramide on apoptosis of alveolar type II epithelial cells].

OBJECTIVE: To investigate the effects of exogenous C8-ceramide on primary alveolar type II epithelial cells (AECII). METHODS: AECII were isolated from 18 day gestational age ICR mice, and cultured in Dulbecco's Modified Eagle Medium with 95% air and 5% CO2at 37 °C. The cells were treated with different concentrations (0, 20, 40 and 80 µmol/L) of C8-ceramide for 24 h and treated with 80 µmol/L of C8-ceramide for different time (3, 6, 12, and 24 h) to observe the viability of cells by MTT assay. Then, the annexin V externalization assay and TUNEL assay were performed to determine apoptosis after the cells were treated with different concentrations (0, 20, 40, and 80 µmol/L) of C8-ceramide for different time (12 and 24 h). RESULTS: The results of MTT assay showed that cell viability decreased in a dose- and time- dependent manner. In Annexin V externalization assay, the cells showed a distribution of (20.63 ± 0.86)%, (22.67 ± 1.72)%, (42.03 ± 1.34)%, (50.40 ± 1.30)% and (20.93 ± 2.51)%, (28.93 ± 3.19)%, (47.00 ± 1.08)%, (57.77 ± 3.04)% cells in apoptosis after treated with 0, 20, 40, 80 µmol/L of C8-ceramide for 12 and 24 h (It is significant difference from untreated cells except for 20 µmol/L at 12 h point, all P<0.05). In TUNEL assay, the cells showed a distribution of (5.51 ± 1.41)%, (13.24 ± 2.62)%, (35.10 ± 4.59)%, (75.07 ± 4.37)% and (5.56 ± 1.36)%, (21.34 ± 2.09)%, (37.12 ± 2.11)%, (91.33 ± 0.72)% cells in apoptosis after treated with 0, 20, 40, 80 µmol/L of C8-ceramide for 12 and 24 h (It is significant difference from untreated cells at the same time point, all P<0.05). CONCLUSION: The results demonstrate that C8-ceramide can decrease the viability of AECII and induce apoptosis.