Aurora-A/NF-ĸB Signaling Is Associated With Radio-resistance in Human Lung Adenocarcinoma.

BACKGROUND/AIM: This study aimed to discuss the effect and possible molecular mechanisms of Aurora-A/NF-ĸB signaling on the radiotherapy resistance of human docetaxel-resistant lung adenocarcinoma cells. MATERIALS AND METHODS: The human lung adenocarcinoma SPC-A1 and SPC-A1/DTX cell lines were utilized in the present study. The MTT assay measured the sensitivity of cells to radiotherapy. The tumor-initiating ability of the cells was detected in vitro by cloning assays. Apoptosis was quantified by flow cytometry. Real-time quantitative PCR and western blotting were used to detect the mRNA and protein expression of the Aurora-A/NF-ĸB, respectively. Tumors transplanted subcutaneously into nude mice were used to test the effect of Aurora-A on the in vivo sensitivity of the tumors to radiotherapy. RESULTS: The SPC-A1/DTX docetaxel-resistant lung adenocarcinoma cells were radio-resistant compared with the parental SPC-A1 cells. Up-regulated aurora-A was responsible for the in vitro radio-resistance of docetaxel-resistant SPC-A1/DTX cells. Nuclear transcription factor NF-ĸB was identified as a downstream target gene of Aurora-A in SPC-A1/DTX cells, and NF-ĸB also participated in the radio-resistance of SPC-A1/DTX cells regulated by Aurora-A. CONCLUSION: The Aurora-A/NF-ĸB pathway is association with radio-resistance of human lung adenocarcinoma docetaxel-resistant cells.

Chemo-resistant Gastric Cancer Associated Gene Expression Signature: Bioinformatics Analysis Based on Gene Expression Omnibus.

BACKGROUND/AIM: This study aimed to identify biomarkers for predicting the prognosis of advanced gastric cancer patients who received docetaxel, cisplatin, and S-1 (DCS). MATERIALS AND METHODS: Gene expression profiles were obtained from the Gene Expression Omnibus database (GSE31811). Gene-Ontology-enrichment and KEGG-pathway analysis were used for evaluating the biological functions of differentially-expressed genes. Protein-protein interaction (PPI) network and Kaplan-Meier survival analyses were employed to assess the prognostic values of hub genes. RESULTS: A total of 1,486 differentially expressed genes (DEGs) were identified, including 13 up-regulated and 1,473 down-regulated genes. KEGG pathways such as metabolic pathways, cell adhesion molecules (CAMs), PI3K-Akt signaling pathway and pathways in cancer were significantly represented. In the PPI network, the top ten hub genes ranked by degree were GNG7, PLCB1, CALML5, FGFR4, GRB2, JAK3, ADCY7, ADCY9, GNAS and KDR. Five DEGs, including ANTXR1, EFNA5, GAMT, E2F2 and NRCAM, were associated with relapse-free survival and overall survival. CONCLUSION: ANTXR1, EFNA5, GAMT, E2F2 and NRCAM are potential biomarkers and therapeutic targets for DCS treatment in GC.

The Anticancer Effects of Atractylenolide III Associate With the Downregulation of Jak3/Stat3-Dependent IDO Expression.

Objective: Indoleamin-2,3-dioxygenase-1 (IDO) has been identified as a checkpoint protein involved in generating the immunosuppressive tumor microenvironment that supports tumor growth. It has been reported that atractylenolide III (ATLIII) has anticancer and immune modulatory effects. This study is to determine the anticancer effects of ATLIII with the Jak3/Stat3-dependent IDO inactivation. Methods: We assessed the cytotoxicity of ATLIII and IFN-γ on lung cancer cells by MTT. We determined the efficacy of ATLIII on IFN-γ-induced IDO expression by RT-PCR and Western blot. We also determined the efficacy of ATLIII on Jak3/Stat3 pathway expression induced by IFN-γ and Jak3/Stat3-dependent IDO activation. Further molecular docking assay predicted the binding activity and site of ATLIII to Jak3 protein. Additional immunofluorescence staining was used to measure the Stat3 intracellular localization. Finally, we performed mouse animal experiments to observe changes in the expression of IDO, p-Jak3, p-Stat3, and tryptophan/kynurenine after ATLIII administration. Results: ATLIII showed no cytotoxicity at a wide of dosage range. ATLIII reduced the phosphorylation level of Jak3 and Stat3 in response to IFN-γ stimulation, then remarkably reduced the nuclear translocation of p-Stat3 by IFN-γ. Lastly, ATLIII significantly downregulated the expression level of IDO at a wide dosage range. Molecular docking assay showed that the oxygen atom on the five-membered ring of ATLIII was capable of forming a hydrogen bond with Leu905-NH2 site of Jak3 protein. Further evidence showed that though IFN-γ had normal capacity to trigger Stat3 phosphorylation, nuclear translocation, and promoter luciferase activity, ATLIII failed to trigger efficacy on reducing these changes under forced Jak3-Leu905 mutant expression condition. Finally, we confirmed this view in in vivo experiments. Conclusion: ATLIII has shown significant efficacy to inhibit IFN-γ-triggered Jak3/Stat3 pathway-dependent IDO activation, and do so through a direct binding to Jak3 protein. This study elucidated a new mechanism for the anticancer effect of ATLIII, which may provide a feasible target for the clinical immunotherapy of malignant tumors.

Autophagy: novel applications of nonsteroidal anti-inflammatory drugs for primary cancer.

In eukaryotic cells, autophagy is a process associated with programmed cell death. During this process, cytoplasmic proteins and organelles are engulfed by double-membrane autophagosomes, which then fuse with lysosomes to form autolysosomes. These autolysosomes then degrade their contents to recycle the cellular components. Autophagy has been implicated in a wide variety of physiological and pathological processes that are closely related to tumorigenesis. In recent years, an increasing number of studies have indicated that nonsteroidal anti-inflammatory drugs, such as celecoxib, meloxicam, sulindac, aspirin, sildenafil, rofecoxib, and sodium salicylate, have diverse effects in cancer that are mediated by the autophagy pathway. These nonsteroidal anti-inflammatory drugs can modulate tumor autophagy through the PI3K/Akt/mTOR, MAPK/ERK1/2, P53/DRAM, AMPK/mTOR, Bip/GRP78, CHOP/ GADD153, and HGF/MET signaling pathways and inhibit lysosome function, leading to p53-dependent G1 cell-cycle arrest. In this review, we summarize the research progress in autophagy induced by nonsteroidal anti-inflammatory drugs and the molecular mechanisms of autophagy in cancer cells to provide a reference for the potential benefits of nonsteroidal anti-inflammatory drugs in cancer chemotherapy.

A microRNA-4516 inhibitor sensitizes chemo-resistant gastric cancer cells to chemotherapy by upregulating ING4.

Objective MicroRNA (miRNA) is an important factor in the regulation of gene transcription. This study was aimed at investigating the role of miR-4516 in the chemoresistance of gastric cancer cells. Methods miR-4516 expression levels were measured in gastric cancer cell line SGC7901 and in 5-fluorouracil (5-FU)-resistant SGC7901 cells (SGC7901/5-FU) via microarray analysis and RT-PCR. A miR-4516 inhibitor and negative controls were transfected into SGC7901/5-FU cells. A miR-4516 mimic and negative controls were transfected into SGC7901 cells. CCK8 and flow-cytometric assays were performed to evaluate the sensitivity of SGC7901/5-FU cells to 5-FU. Western blot experiments detected the expression of Bcl-2, Bax, Caspase-3, P-gp and ING4 protein. Results Additionally, ING4 was demonstrated to be downregulated in SGC7901/5-FU cells and inversely correlated with miR-4516 expression. Rescue experiments revealed that overexpression of ING4 attenuated the inhibitory effects of miR-4516 on the proliferation of gastric cancer cells. ING4 was predicted to be a potential target of miR-4516. Synergism of the inhibitory effects correlated with a reduction in the expression of the anti-apoptotic protein Bcl-2 and the drug resistance-related protein P-gp as well as strong expression of apoptosis-related proteins (Bax, Caspase-3). Thus, a miR-4516 inhibitor sensitized gastric cancer SGC7901/5-FU cells to 5-FU by enhancing apoptosis. We then corroborated these results with in vivo experiments. Conclusion We found that miR-4516 might be a potential therapeutic target in chemo-resistant gastric cancer.

Correction: A microRNA-4516 inhibitor sensitizes chemo-resistant gastric cancer cells to chemotherapy by upregulating ING4.

[This corrects the article DOI: 10.1039/C8RA06419A.].

Sequential treatment with betulinic acid followed by 5-fluorouracil shows synergistic cytotoxic activity in ovarian cancer cells.

Betulinic acid selectively inhibits the growth of ovarian carcinoma cell lines without affecting the normal cells. In the present study, the effect of 5-fluorouracil (5-FU) and betulinic acid (BA) combination on ovarian carcinoma cells was studied. The results demonstrated that ovarian carcinoma cells on concurrent or 5-FU followed by BA treatment show increased Sub-G1 cell population, increased rate of cell apoptosis and morphological changes in mitochondrial membrane. In OVCAR 432 cells treatment with sequential combination of 5-FU and BA increased the Sub-G1 cell population to 51.3% and growth inhibition rate of > 72%. However, exposure to BA before 5-FU treatment caused a decrease in rate of inhibition to < 35%. Treatment with combination of 5 µM of 5-FU and 1 µM of BA for 48 h, led to an induction of apoptosis in 79.7% and induced morphological changes in OVCAR 432 cells. The Western blot results showed high concentration of cytochrome c in the cell cytosol after 24 h of 5-FU and BA combination treatment. Treatment of BA-responsive RMS-13 cells with 5-FU and BA combination resulted in inhibition of GLI1, GLI2, PTCH1, and IGF2 genes. In addition, we found a significant reduction in hedgehog activity of RMS-13 cells after 5-FU and BA combination treatment by means of a hedgehog-responsive reporter assay. Therefore, 5-FU and BA combination can be a promising regimen for the treatment of ovarian carcinoma.

Gene microarray assessment of multiple genes and signal pathways involved in androgen-dependent prostate cancer becoming androgen independent.

To study the gene expression change and possible signal pathway during androgen-dependent prostate cancer (ADPC) becoming androgen-independent prostate cancer (AIPC), an LNCaP cell model of AIPC was established using flutamide in combination with androgen-free environment inducement, and differential expression genes were screened by microarray. Then the biological process, molecular function and KEGG pathway of differential expression genes are analyzed by Molecule Annotation System (MAS). By comparison of 12,207 expression genes, 347 expression genes were acquired, of which 156 were up-ragulated and 191 down-regulated. After analyzing the biological process and molecule function of differential expression genes, these genes are found to play crucial roles in cell proliferation, differntiation, cell cycle control, protein metabolism and modification and other biological process, serve as signal molecules, enzymes, peptide hormones, cytokines, cytoskeletal proteins and adhesion molecules. The analysis of KEGG show that the relevant genes of AIPC transformation participate in glutathione metabolism, cell cycle, P53 signal pathway, cytochrome P450 metabolism, Hedgehog signal pathway, MAPK signal pathway, adipocytokines signal pathway, PPAR signal pathway, TGF-ß signal pathway and JAK-STAT signal pathway. In conclusion, during the process of ADPC becoming AIPC, it is not only one specific gene or pathway, but multiple genes and pathways that change. The findings above lay the foundation for study of AIPC mechanism and development of AIPC targeting drugs.

Preliminary study on the mechanism of oridonin-induced apoptosis in human squamous cell oesophageal carcinoma cell line EC9706.

OBJECTIVE: To study the apoptosis-inducing effect of the Chinese medicine oridonin in the human oesophageal squamous cell carcinoma cell line EC9706, in vitro. METHODS: The effect of oridonin on cell proliferation was studied using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Effects on the cell cycle and apoptosis were studied by flow cytometry; effects on intracellular Ca(2+) concentration were studied by measuring the fluorescence intensity of a fluorescent Ca(2+) probe by laser scanning confocal microscopy. RESULTS: The EC9706 cell-proliferation rate decreased with time and oridonin concentration (10-40 µmol/l). The number of cells in G0 and G1 phases increased significantly following exposure to oridonin for 48 and 72 h respectively, and oridonin was shown to be most effective at inducing apoptosis in EC9706 cells at 40 µmol/l. Compared with the control group, all concentrations of oridonin tested (10-40 µmol/l) significantly increased the Ca(2+) fluorescence intensity of EC9706 cells. CONCLUSIONS: Oridonin was shown to inhibit proliferation and induce apoptosis in the human oesophageal squamous cell carcinoma cell line EC9706, in vitro. These data provide preliminary experimental evidence for the anticancer effects of oridonin, which is as a traditional Chinese medicine used to treat various cancers, including oesophageal squamous cell carcinoma. Further studies are required to elucidate the mode of action.

Identification of candidate genes related to rice grain weight under high-temperature stress.

The rise of global warming presents a problem for all living organisms, including rice and other staple plants. High temperatures impair rice grain weight by inhibiting the filling of the caryopses during the milky stage. The molecular mechanism behind this process, however, is poorly understood. Identifying candidate genes involved in responses to high-temperature stress may provide a basis for the improvement of heat tolerance in rice. Using paired, genetically similar heat-tolerant and heat-sensitive rice lines as plant materials, cDNA-AFLP analysis revealed a total of 54 transcript derived fragments (TDFs), mainly from the heat-tolerant lines. This clearly indicated variations in gene expression between the two rice lines. BLAST results showed that 28 of the 54 TDFs were homologous sequences. These homologous genes were found to encode proteins involved in signal transduction, oxidation, transcriptional regulation, transport, and metabolism. The functions and differential expression patterns of some important genes are further discussed. High temperature stress may trigger a wide range of changes in gene expression in rice caryopses, in turn affecting functions ranging from signal transduction to cellular metabolism. Forty-five of the 54 TDFs were mapped to rice chromosomes. The genes identified in the present study would make good candidates for further study into the molecular mechanisms underlying rice adaptation to high-temperature stress.

Antisense interleukin-5 reduces eosinophil infiltration and hyperresponsiveness in an allergic asthma model.

Interleukin-5 (IL-5) involves in the development of airway inflammation and hyperresponsiveness through activation of eosinophils. Thus, inhibition of IL-5 expression seems to be an attractive approach for asthma therapy. In this study, an antisense IL-5 gene transferred by recombinant adeno-associated virus (asIL-5) was constructed to transfect murine allergic asthma model. Our results showed that asIL-5 efficiently inhibited the IL-5 mRNA expression and significantly attenuated the inflammation in lung tissues. Significant decreasing of eosinophils and inflammatory cells were found in peripheral blood and bronchoalveolar lavage fluid (BALF). In addition, significant inhibition of airway hyperresponsiveness (AHR) was also found in the mice treated with asIL-5. These observations demonstrate that antisense oligonucleotid against IL-5 delivered by adeno-associated virus system is possibly an efficacious therapeutic strategy for allergic asthma and other eosinophil-related disorders.

RNA interference against interleukin-5 attenuates airway inflammation and hyperresponsiveness in an asthma model.

Interleukin-5 (IL-5) accompanies the development of airway inflammation and hyperresponsiveness through the activation of eosinophils. Therefore, interference of IL-5 expression in lung tissue seems to be an accepted approach in asthma therapy. In this study, we designed a small interfering RNA (siRNA) to inhibit the expression of IL-5. The siRNAs against IL-5 were constructed in a lentivirus expressing system, and 1.5x10(6) IFU (inclusion-forming unit) lentiviruses were administered intratracheally to ovalbumin (OVA)-sensitized murine asthmatic models. Our results show that lentivirus-delivered siRNA against IL-5 efficiently inhibited the IL-5 messenger ribonucleic acid (mRNA) expression and significantly attenuated the inflammation in lung tissue. Significant decrease of eosinophils and inflammatory cells were found in peripheral blood, bronchoalveolar lavage fluid (BALF), and lung tissue. In addition, significant inhibition of airway hyperresponsiveness (AHR) was found in the mice treated with siRNA against IL-5. These observations demonstrate that siRNA delivered by means of the lentivirus system is possibly an efficacious therapeutic approach for asthma.

Differentiation of Mouse Embryonic Stem Cells into Neural Cells on Silk Fibroin Scaffolds / 中国生物工程杂志

Induced the mouse embryonic stem(ES)cells into neural cells on silk fibroin via the improved 4-/4+ RA method to explore the effect of the silk fibroin to the ES-derived neurons' growth,adherence and differentiation.Suspended the ES cells into EBs and then transferred them to three different substrates-coated 35 mm dishes including gelatin,Bombyx mori silk fibroin(SF) and Tussah silk fibroin(TSF) to identify the adherence and proportion of ES cells-derived neurons under these three substrates.The results showed that the EBs adhered to the gelatin and TSF are faster than to the SF.The average adhesive rate on gelatin and TSF are 90.3% and 84.4% respectively,and only 38.5% on SF,all the proportion of ?-Ⅲ-Tubulin positive cells is approximately 40%.It may provide important experimental information for tissue engineering,in which ES cells-derived neuron cells and silk fibroin materials are scaffolds,and also offer a source for cell therapy research of neurodegenerative disease.