Chunxu Yang

Redistribution of DR4 and DR5 in lipid rafts accounts for the sensitivity to TRAIL in NSCLC cells.

The selective toxicity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) against tumor cells makes it a potential targeted drug for treating non-small cell lung carcinomas (NSCLC). However, the majority of established human NSCLC cell lines are either partially or completely resistant to TRAIL, resulting in the limitation for clinical use of rhTRAIL and its agonistic antibodies. In this study, compared to TRAIL-sensitive H460 cell line, TRAIL-resistant A549 cell line showed a similar expression level of DR5 and a higer expression level of DR4. It indicates that there is no positive correlation between the expression levels of death receptors and sensitivity to TRAIL. However, tests on A549 cells with DR4 siRNA transfection revealed that DR4-competitive binding to TRAIL could not affect the capacity of TRAIL in inducing apoptosis. Instead, further studies found that the aggregation of DR4 and DR5 in lipid rafts only occured in H460 cells with TRAIL pretreatment. It suggested that the TRAIL-induced redistribution of DR4 and DR5 in lipid rafts contributed to the sensitivity to TRAIL in TRAIL-sensitive NSCLC H460 cell line, which was also confirmed by intervention tests of the cholesterol-sequestering agent nystatin.

The miR-15 Family Enhances the Radiosensitivity of Breast Cancer Cells by Targeting G2 Checkpoints

Enhancing radiosensitivity is an important area of investigation for improving breast cancer therapy outcomes. The aim of this study was to assess the role of the miR-15 family in the radiosensitivity of breast cancer cells. MicroRNAs (miRNAs) encoded by the miR-15 cluster are known to induce G1 arrest and apoptosis by targeting G1 checkpoints and the anti-apoptotic B cell lymphoma 2 (BCL-2) gene. However, the effect of the miR-15 family on G2/M arrest and radiosensitivity remains poorly understood. In the current study, cells transfected with miR-15a/15b/16 mimic or inhibitor were irradiated and examined by: clonogenic assays, phosphorylated H2AX assay, flow cytometry, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), real-time PCR and Western blot. Real-time PCR was also used to monitor time-dependent changes of miR-15a/15b/16 expression after irradiation. A putative target site for miR-15a/15b/16 within the Chk1 and Wee1 3′ UTRs was confirmed using luciferase reporter assays. Additionally, siRNA was used to validate the effect of Chk1 and Wee1 on radiosensitivity in breast cancer cells. In our study, we investigated the effects of radiation on the miR-15 family and found a time-dependent change in the expression of miR-15a/15b/16 in breast cancer cells postirradiation, as well as an increase in miR-15 family-mediated sensitization of breast cancer cells to radiation. The increase in radiosensitivity induced by the miR-15 family was associated with persistent unrepaired DNA damage, abrogation of radiation-induced G2 arrest and suppressed cell proliferation, and appear to involve both the checkpoint kinase 1 (Chk1) and Wee1. In addition, we found that inhibition of the miR-15 family could not induce cell resistance to radiation. These findings suggest that the expression of the miR-15 family contributes to increased radiosensitivity of breast cancer cells by influencing G2/M checkpoint proteins.

Concomitant overexpression of EGFR and CXCR4 is associated with worse prognosis in a new molecular subtype of non-small cell lung cancer.

Although the relationships between CXCR4 and EGFR expression and survival in non‑small cell lung cancer (NSCLC) have been studied independently, dual CXCR4/EGFR tumor status and its relationship with survival has not been previously investigated. In the present study, we examined the relationship between CXCR4 expression, EGFR expression and dual CXCR4/EGFR expression and survival in patients with NSCLC (n=125) using immunohistochemical techniques. Overall survival was estimated using Kaplan-Meier and Cox proportional hazards models adjusting for patient age, tumor stage and type of treatments. Patients with CXCR4-positive tumors were significantly associated with distant metastasis and tended to have poorer prognosis compared to patients with CXCR4-negative tumors (HR=2.172, 95% CI=1.229‑3.839). No significant association between EGFR expression and survival was found; however co-expression of CXCR4/EGFR was a significant prognostic factor of worse overall survival (HR=2.741, 95% CI=1.330‑5.741). Furthermore, we showed that EGF enhanced the expression of CXCR4 in NSCLC cells through the PI-3K pathway, and treatment of NSCLC cells with EGFR phosphorylation inhibitor, AG1478, resulted in downregulation of the expression of CXCR4. These results suggest an important interaction between CXCR4 and EGFR intra-cellular pathways that may activate signals of tumor progression and may provide a valid explanation for the poor overall survival rate of patients whose co-expression of CXCR4 and EGFR is detected in tissue sections. Based on EGFR and CXCR4 expression, new molecular subtypes of NSCLC established in the present study can be used for customization of NSCLC treatment. Our results also showed that EGFR and CXCR4 are potential therapeutic targets for NSCLC and that simultaneous inhibition of EGFR and CXCR4 in NSCLC patients with concomitant expression of both CXCR4 and EGFR may be an effective treatment strategy.

Absence of death receptor translocation into lipid rafts in acquired TRAIL-resistant NSCLC cells.

Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a major limitation for its clinical use. The mechanisms of TRAIL resistance have been mostly studied in the context of cell lines that are intrinsically resistant to TRAIL. However, little is known about the molecular alterations that contribute to the development of acquired resistance during treatment with TRAIL. In this study, we established H460R, an isogenic cell line with acquired TRAIL resistance, from the TRAIL‑sensitive human lung cancer cell line H460 to investigate the mechanisms of acquired resistance. The acquired TRAIL‑resistant H460R cells remained sensitive to cisplatin. The mRNA and protein expression levels of death receptor 4 (DR4) and death receptor 5 (DR5) were not altered in either of the TRAIL-treated cell lines. Nevertheless, tests in which the DR4 or DR5 gene was overexpressed or silenced suggest that death receptor expression is necessary but not sufficient for TRAIL‑induced apoptosis. Compared with parental TRAIL-sensitive H460 cells, H460R cells showed a decreased TRAIL-induced translocation of DR4/DR5 into lipid rafts. Further studies showed that nystatin partially prevented lipid raft aggregation and DR4 and DR5 clustering and reduced apoptosis in H460 cells again. Analysis of apoptotic molecules showed that more pro-caspase-8, FADD, caspase-3 and Bid, but less cFLIP in H460 cells than in H460R cells. Our findings suggest that the lack of death receptor redistribution negatively impacts DISC assembly in lipid rafts, which at least partially leads to the development of acquired resistance to TRAIL in H460R cells.

Antisense Oligonucleotide Targeting Matrix Metalloproteinase-7 (MMP-7) Changes the Ultrastructure of Human A549 Lung Adenocarcinoma Cells

Antisense oligonucleotide (ASODN) targeting specific gene can be capable of potently downregulating proliferation and invasion in human cancer cells. However, the underlying mechanisms are less well defined. Here the authors show that matrix metalloproteinase-7 (MMP-7) ASODN changes the ultrastructure of human A549 lung adenocarcinoma cells. Transfection of MMP-7 ASODN significantly lowered the expression of MMP-7 protein in A549 cells. Decreased microvilli, endoplasmic reticulum dilation, swelling of mitochondria, and formation of apoptotic bodies were observed by transmission electron microscope. Collectively, the findings identified the morphological mechanism that MMP-7 ASODN inhibited proliferation and invasion in A549 cells.

The interaction between different types of activated RAW 264.7 cells and macrophage inflammatory protein-1 alpha

BackgroundTwo major ways of macrophage (MΦ) activation can occur in radiation-induced pulmonary injury (RPI): classical and alternative MΦ activation, which play important roles in the pathogenesis of RPI. MΦ can produce chemokine MΦ inflammatory protein-1α (MIP-1α), while MIP-1α can recruit MΦ. The difference in the chemotactic ability of MIP-1α toward distinct activated MΦ is unclear. We speculated that there has been important interaction of MIP-1α with different activated MΦ, which might contribute to the pathogenesis of RPI.MethodsClassically and alternatively activated MΦ were produced by stimulating murine MΦ cell line RAW 264.7 cells with three different stimuli (LPS, IL-4 and IL-13); Then we used recombinant MIP-1α to attract two types of activated MΦ. In addition, we measured the ability of two types of activated MΦ to produce MIP-1α at the protein or mRNA level.ResultsChemotactic ability of recombinant MIP-1α toward IL-13-treated MΦ was the strongest, was moderate for IL-4-treated MΦ, and was weakest for LPS-stimulated MΦ (p < 0.01). The ability of LPS-stimulated MΦ to secrete MIP-1α was significantly stronger than that of IL-4-treated or IL-13-treated MΦ (p < 0.01). The ability of LPS-stimulated MΦ to express MIP-1α mRNA also was stronger than that of IL-4- or IL-13-stimulated MΦ (p < 0.01).ConclusionsThe chemotactic ability of MIP-1α toward alternatively activated MΦ (M2) was significantly greater than that for classically activated MΦ (M1). Meanwhile, both at the mRNA and protein level, the capacity of M1 to produce MIP-1α is better than that of M2. Thus, chemokine MIP-1α may play an important role in modulating the transition from radiation pneumonitis to pulmonary fibrosis in vivo, through the different chemotactic affinity for M1 and M2.

Deubiquitinase USP9X deubiquitinates β-catenin and promotes high grade glioma cell growth

β-catenin is a crucial signal transduction molecule in the Wnt/β-catenin signal pathway, and increased β-catenin expression has consistently been found in high grade gliomas. However, the mechanisms responsible for β-catenin overexpression have remained elusive. Here we show that the deubiquitinase USP9X stabilizes β-catenin and thereby promotes high grade glioma cell growth. USP9X binds β-catenin and removes the Lys 48-linked polyubiquitin chains that normally mark β-catenin for proteasomal degradation. Increased USP9X expression correlates with increased β-catenin protein in high grade glioma tissues. Moreover, patients with high grade glioma overexpressing USP9X have a poor prognosis. Knockdown of USP9X suppresses cell proliferation, inhibits G1/S phase conversion, and induces apoptosis in U251 and A172 cells. Interestingly, c-Myc and cyclinD1, which are important downstream target genes in the Wnt/β-catenin signal pathway, also show decreased expression in cells with siRNA-mediated down-regulation of USP9X. Down-regulation of USP9X also consistently inhibits the tumorigenicity of primary glioma cells in vivo. In summary, these results indicate that USP9X stabilizes β-catenin and activates Wnt/β-catenin signal pathway to promote glioma cell proliferation and survival. USP9X could also potentially be a novel therapeutic target for high grade gliomas.

The ultrastructural difference between CD133-positive U251 glioma stem cells and normal U251 glioma cells.

Glioma stem cells (GSC) have higher tumorigenic potential and stronger chemoresistance and radioresistance than normal glioma cells. The mechanisms behind these phenomena have remained elusive. The authors have isolated CD133-positive U251 GSCs from U251 glioma cells and detected the expression of stem cell markers (CD133 and nestin) of U251 GSCs by immunofluorescence staining. Then the ultrastructures of U251 GSCs and normal U251 glioma cells were observed by transmission electron microscopy and the ultrastructural differences between them were compared. Increased cell nucleus atypia, rougher endoplasmic reticulum, and more microvilli were observed in CD133-positive U251 GSCs than in normal U251 glioma cells. In summary, these ultrastructural differences support the hypothesis that GSCs have stronger tumorigenic ability and resistance to chemotherapy and radiotherapy.

Elevated expression of USP9X correlates with poor prognosis in human non-small cell lung cancer.

BACKGROUND The aim of this study was to investigate the expression of ubiquitin-specific peptidase 9, X-linked (USP9X) in non-small cell lung cancer (NSCLC) patients and to evaluate the relevance of USP9X expression to tumor prognosis. METHODS Ninety-five patients who underwent surgical resection for clinical stage I-IIIA NSCLC between July 2008 and July 2011 were included in this study. Immunohistochemical analysis of USP9X expression was performed on 95 NSCLC tissues and 32 adjacent normal lung parenchymal tissues from these patients. The Chi-squared test was used to compare the clinicopathological characteristics between different groups. Kaplan-Meier analysis and a Cox regression model were used to determine the independent prognostic factors. A P value <0.05 was considered to be significant. RESULTS The expression of USP9X was found to be significantly higher in NSCLC tissue (44.2%) than in adjacent normal lung parenchymal tissue (6.3%) (P<0.001). High USP9X expression was significantly associated with positive lymph node metastasis (P<0.001), clinical stage (P<0.001) and a reduced overall survival rate (P=0.001) in patients with NSCLC. Based on the multivariate analysis, the elevated expression of the USP9X protein was a significant predictor of poor prognosis for NSCLC patients (HR =2.244, P=0.028). CONCLUSIONS The current study demonstrated that the expression of USP9X in NSCLC tissue was significantly higher than that in normal lung tissue and that this elevated expression level of USP9X was associated with poor prognosis among NSCLC patients, suggesting that USP9X might serve as a prognostic biomarker for NSCLC.

Involvement of Cdc25c in Cell Cycle Alteration of a Radioresistant Lung Cancer Cell Line Established with Fractionated Ionizing Radiation

Cancer patients often suffer from local tumor recurrence after radiation therapy. Cell cycling, an intricate sequence of events which guarantees high genomic fidelity, has been suggested to affect DNA damage responses and eventual radioresistant characteristics of cancer cells. Here, we established a radioresistant lung cancer cell line, A549R , by exposing the parental A549 cells to repeated γ-ray irradiation with a total dose of 60 Gy. The radiosensitivity of A549 and A549R was confirmed using colony formation assays. We then focused on examination of the cell cycle distribution between A549 and A549R and found that the proportion of cells in the radioresistant S phase increased, whereas that in the radiosensitive G1 phase decreased. When A549 and A549R cells were exposed to 4 Gy irradiation the total differences in cell cycle redistribution suggested that G2-M cell cycle arrest plays a predominant role in mediating radioresistance. In order to further explore the possible mechanisms behind the cell cycle related radioresistance, we examined the expression of Cdc25 proteins which orchestrate cell cycle transitions. The results showed that expression of Cdc25c increased accompanied by the decrease of Cdc25a and we proposed that the quantity of Cdc25c, rather than activated Cdc25c or Cdc25a, determines the radioresistance of cells.