Membrane-Bound CD40L Promotes Senescence and Initiates Senescence-Associated Secretory Phenotype via NF-κB Activation in Lung Adenocarcinoma.

BACKGROUND/AIMS: Cellular senescence acts as a barrier against tumorigenesis. The CD40L transgene, expressed in some tumor cells, not only becomes visible to antigen-presenting cells but also actively catalyzes its own termination. Here, we evaluated the effect of a membrane-bound mutant form of human CD40L (CD40L-M) on senescence and the senescence-associated secretory phenotype (SASP) in non-small cell lung cancer (NSCLC). METHODS: CD40 expression levels in the NSCLC cell lines A549/TR, A549/DDP and H460 were examined by flow cytometry. Senescent cells and tissues were identified via SA-ß-gal activity. Cell proliferation was visualized by EdU labeling. qRT-PCR, Western blotting, and immunofluorescence staining were conducted to assess mRNA and protein expression levels of CD40L, γ-H2A.X, p65, p-p65, IκBα, p53, p21 and p16. Cytokines secreted from transfected cells were tested by ELISA and cell migration assay. Capsid tyrosine-modified rAAV5-CD40L-M was packaged and carried out in vivo. RESULTS: Overexpression of CD40L-M promoted senescence, inhibited proliferation, increased DNA damage-associated γ-H2A.X, and initiated the SASP in CD40-positive NSCLC cells. NF-κB signaling was activated by CD40L-M overexpression in these cells. Knockdown of NF-κB partially overcame senescence and failed to induce SASP. Furthermore, increased p53 and p21 protein levels induced by CD40L-M were also reduced following NF-κB suppression. CONCLUSIONS: These data showed that the membrane-bound CD40L mutant may promote cellular senescence and initiate the SASP of NSCLC cells in an NF-κB-dependent manner. Therefore, CD40L-M-induced senescence may be a potential approach to protect against lung adenocarcinoma.

Synthesis of novel galactose functionalized gold nanoparticles and its radiosensitizing mechanism.

BACKGROUND: Biocompatible gold nanoparticles (GNPs) are potentially practical and efficient agents in cancer radiotherapy applications. In this study, we demonstrated that GNPs can significantly modulate irradiation response of hepatocellular carcinoma cells in vitro and investigated the underlying mechanisms. We co-grafted galactose (GAL) targeting hepatocyte specific asialoglycoprotein receptor and Polyethylene Glycol (PEG) onto GNPs surfaces to increase GNPs targeting specificity and stability. RESULTS: This novel GAL-PEG-GNPs and bare GNPs show similar appearance and cytotoxicity profiles, while more GAL-PEG-GNPs can be effectively uptaken and could enhance cancer cell killing. CONCLUSION: GAL-PEG-GNPs have better radiosensitization to HepG2. The sensitization mechanism of GAL-PEG-GNPs is related to the apoptotic gene process activated by generation of a large amount of free radicals induced by GNPs.

Evaluation of serum creatinine- and cystatin C-based equations for the estimation of glomerular filtration rate in a Chinese population.

OBJECTIVE: This study aimed to evaluate the applicability of a selection of glomerular filtration rate (GFR) estimating equations based on serum creatinine (SCr) and serum cystatin C in a Chinese population. MATERIAL AND METHODS: Estimated GFR values from 10 equations were compared with reference GFR (rGFR) from the (99m)Tc-DTPA renal dynamic imaging method. The study enrolled 569 Chinese participants (41.5% women, 53.5 ± 16.9 years, range 19-92 years), with mean rGFR 74.80 ± 26.10 (range 9.8-146.8 ml/min/1.73 m(2)). RESULTS: Bland-Altman analysis illustrated that the 95% agreement limits of all the equations surpassed the acceptable tolerance (<60 ml/min/1.73 m(2)), of which the MacIsaac equation was the closest one, reaching 71.7 ml/min/1.73 m(2). Linear regression analysis also demonstrated a consistent result. When assessed in all participants, the accuracy of the six equations reached and exceeded the acceptable level (≥70%), of which the Shanghai and MacIsaac equations gained more accuracy than others. When compared in subgroups, the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI), MacIsaac and Cockcroft-Gault (CG) equations were optimal for rGFR stages ≥ 90 ml/min/1.73 m(2), 30-89 ml/min/1.73 m(2) and < 30 ml/min/1.73 m(2), respectively. CONCLUSION: The results demonstrated that further improvement is needed for the selected 10 equations. Not all the cystatin C equations were superior to SCr equations. They have their own applicability at various GFR levels. At present, the CKD-EPI, MacIsaac and CG equations may be applied to evaluate GFR in normal, mild to moderate and severe kidney function, respectively.

Down-regulation of ALKBH2 increases cisplatin sensitivity in H1299 lung cancer cells.

AIM: To elucidate the combined effect of alkylated DNA repair protein alkB homolog 2 (ALKBH2)-targeting gene therapy and cisplatin (cDDP) chemotherapy on the non-small cell lung cancer (NSCLC) H1299 cell line. METHODS: ALKBH2 was down-regulated in H1299 cells by lentivirus-mediated RNA interference (RNAi). Changes in ALKBH2 expression were determined using real-time RT-PCR and Western blotting. Cell viability was evaluated using MTT assay. DNA synthesis in proliferating cells was determined using BrdU incorporation assay. Cell apoptosis was determined using flow cytometry. RESULTS: Lentivirus-mediated ALKBH2 silencing alone did not induce apoptosis or attenuate the growth potential of H1299 cells within five days post-infection. Combined treatment modalities with lentivirus-mediated ALKBH2 down-regulation and cDDP (333 µmol/L) were significantly more potent in inhibiting cell growth and inducing apoptosis than mono-chemotherapy. CONCLUSION: Combined treatment modalities of ALKBH2 knockdown and cDDP chemotherapy have the potential to improve the efficacy in the treatment of NSCLC.

The CYP1B1 Leu432Val polymorphism contributes to lung cancer risk: evidence from 6501 subjects.

The polymorphism of cytochrome P4501B1 (CYP1B1) codon 432 (rs1056836, CYP1B1*3, or Leu432Val) is thought to have a significant effect on lung cancer risk, but the results are inconsistent. In this meta-analysis, we assessed 9 published studies involving 6501 subjects that investigated the association between the CYP1B1 codon 432 polymorphism and risk of lung cancer. Overall, the CYP1B1 Leu/Val and Val/Val-variant genotypes were associated with a significantly increased risk of lung cancer in different genetic models (heterozygote comparison: OR=1.22; 95% CI=1.02-1.45, P(heterogeneity)=0.068; homozygote comparison: OR=1.41; 95% CI=1.08-1.85, P(heterogeneity)=0.071; dominant model comparison: OR=1.26; 95% CI=1.04-1.51, P(heterogeneity)=0.019; and recessive model comparison: OR=1.17; 95% CI=1.02-1.34, P(heterogeneity)=0.429). In the stratified analysis by ethnicity, significantly increased risks were found among Caucasians for Leu/Val vs Leu/Leu (OR=1.30; 95% CI=1.03-1.64; P(heterogeneity)=0.092), and dominant model (OR=1.35; 95% CI=1.03-1.77; P(heterogeneity)=0.015). However, no significant associations were found in both Europeans and African-Americans for all genetic models. In the subgroup analyses by smoking status, a significantly increased risk of lung cancer was found among smokers (dominant model: OR=1.46; 95% CI=1.08-1.83; P(heterogeneity)=0.175). However, we did not find any statistically significant association by subgroup analyses of pathological type. This meta-analysis suggests that the CYP1B1 Val allele is a low-penetrant risk factor for developing lung cancer.

TRAIL-R1 polymorphisms and cancer susceptibility: an evidence-based meta-analysis.

Published data on the association between tumour necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL-R1 or DR4) polymorphisms rs20575 (C626G), rs2230229 (A1322G) and rs20576 (A683C) and cancer risk are inconclusive. To derive a more precise estimation of the relationship, a meta-analysis was performed. A total of nine studies, among which eight articles including 2941 cases and 3358 controls described C626G genotypes, three articles including 736 cases and 668 controls described A1322G genotypes and three studies totalling 1550 cases and 2257 controls described A683C genotypes were involved in this meta-analysis. Overall, all three polymorphisms were associated with cancer susceptibility. For C626G polymorphism, there was no association between C626G polymorphism and the risk of cancer in all genetic models when all the eligible studies were pooled into the meta-analysis. In the subgroup analysis by source of controls, statistically significantly reduced cancer risks were found among groups with population-based controls for CG versus CC (OR=0.77, 95% CI:0.65-0.91, P(heterogeneity)=0.007) and dominant model (OR=0.84, 95% CI:0.72-0.99, P(heterogeneity)=0.409). For A1322G polymorphism, we found it was associated with a significantly elevated cancer risk of all cancer types in different genetic models (homozygote comparison: OR=2.80, 95% CI:1.16-6.76, P(heterogeneity)=0.905; dominant model comparison: OR=1.57, 95% CI:1.02-2.41, P(heterogeneity)=0.167; and recessive model comparison: OR=1.22, 95% CI:0.94-1.60, P(heterogeneity)=0.535). Similar results were obtained from A683C polymorphism (homozygote comparison: OR=3.21, 95% CI:1.26-8.20, P(heterogeneity)=0.012; dominant model comparison: OR=1.61, 95% CI: 1.09-2.36, P(heterogeneity)=0.000; and recessive model comparison: OR=2.79, 95% CI: 1.17-6.68, P(heterogeneity)=0.025). In summary, this meta-analysis suggests that TRAIL-R1 C626G polymorphism is marginally associated with cancer susceptibility, and both TRAIL-R1 A1322G G allele and A683C C allele are associated with increased risk for cancer.

[Adeno-associated virus-mediated CD40 ligand transfer into human lung cancer cells].

OBJECTIVE: To investigate the transduction efficiency of serotype 1, 2, 5, 6, 7, 8, 9, 10 recombinant adeno-associated viruses (rAAV) in human lung cancer cell line A549 cells and compare the transduction efficiency of conventional AAV vectors with that of self-complementary AAV (scAAV) vectors. Furthermore, the capacity of A549 cells expressing transgenic CD40L to stimulate dendritic cells (DCs) was evaluated. METHODS: Lung cancer A549 cells were infected with 1 x 10(4) particules per cell of AAV encoding the green fluorescent protein (GFP) or human CD40L driven by CMV promotor, and transgene expression was analyzed by flow cytometry and fluorescence microscopy. Stimulation of isolated human dendritic cells by CD40L-expressing tumor cells was quantified by measuring secreted interleukin-12 with immunoassay. RESULTS: Serotype AAV2/5 transduced A549 cells much more efficiently than serotypes AAV2/1, AAV2/2, AAV2/6, AAV2/7, AAV2/8, AAV2/9 and AAV2/10. The transduction efficiency of scAAV2/5 was significantly higher than that of conventional AAV2/5. Furthermore, pre-treatment with carboplatin substantially increased AAV-mediated transgene expression. The scAAV2/5 vectors encoding human CD40L was used to generate CD40L. A549 cells transduce by these vectors were co-cultured with immature human DCs. As a consequence, interleukin-12 was released and measured in the culture supernatant. Specificity of immunostimulatory effect of CD40L was confirmed by blocking with a monoclonal antibody binding to human CD40L. CONCLUSION: scAAV2/5 transduce lung adenocarcinoma A549 cell efficiently, and co-administration of chemotherapeutic agent carboplatin further enhances its transduction efficiency. It is confirmed that lung cancer cells infected with a CD40L-encoding scAAV2/5 construct can activate human DCs to secrete interleukin-12. Our findings provided a basis for future immunotherapeutic approaches including intratumoral transfer of stimulating factors.

Adeno-associated virus mediated gene transfer into lung cancer cells promoting CD40 ligand-based immunotherapy.

Expression of the CD40 ligand (CD40L) on tumors can activate host immune systems and produce antitumor effects against the tumors. To deliver the CD40L gene efficiently, we evaluated the efficiency of transduction of different serotypes of adeno-associated virus (AAV) vectors in lung cancer A549 cells and compared the transduction efficiency of a conventional AAV vector with that of self-complementary AAV (scAAV) vectors as well. We determined that serotype AAV2/5 transduced A549 cells much more efficiently than serotypes AAV2/1, AAV2/2, AAV2/6, AAV2/7, AAV2/8, AAV2/9 and AAV2/10. And the transduction efficiency of scAAV2/5 was significantly higher than conventional AAV2/5. Furthermore, pre-treatment with carboplatin, which is a chemotherapeutic agent used in lung cancer chemotherapy, substantially increased AAV-mediated transgene expression. The scAAV2/5 vectors encoding human CD40L were used to tranduce CD40L into A549 cells, which were then co-cultivated with immature human dendritic cells (DCs). Interleukin 12 (IL-12) that was released was measured in the culture supernatant. Specificity of the immunostimulatory effect of CD40L was confirmed by blocking with a monoclonal antibody binding to human CD40L. The in vivo antitumor activity was evaluated in BALB/c nude mice bearing A549 lung cancer. scAAV2/5-CD40L showed significant inhibitory effects on the growth of transplanted tumor cells as compared with control group. These studies suggest that recombinant AAV2/5-CD40L may provide an effective form of therapy for lung cancer.