Acute myeloid leukemia (AML)-derived mesenchymal stem cells induce chemoresistance and epithelial-mesenchymal transition-like program in AML through IL-6/JAK2/STAT3 signaling.

Acute myeloid leukemia (AML) has a high rate of treatment failure due to increased prevalence of therapy resistance. Mesenchymal stem cells (MSCs) in the leukemia microenvironment contribute to chemoresistance in AML, but the specific mechanism remains unclear. The critical role of the epithelial-mesenchymal transition (EMT)-like profile in AML chemoresistance has been gradually recognized. However, there is no research to suggest that the AML-derived bone marrow mesenchymal stem cells (AML-MSCs) induce the EMT program in AML thus far. We isolated AML-MSCs and cocultured them with AML cells. We found that AML-MSCs induced a significant mesenchymal-like morphology in drug-resistant AML cells, but it was scarce in parental AML cells. The AML-MSCs promoted growth of AML cells in the presence or absence of chemotherapeutics in vitro and in vivo. Acute myeloid leukemia MSCs also induced EMT marker expression in AML cells, especially in chemoresistant AML cells. Mechanistically, AML-MSCs secreted abundant interleukin-6 (IL-6) and upregulated IL-6 expression in AML cells. Acute myeloid leukemia cells upregulated IL-6 expression in AML-MSCs in turn. Meanwhile, AML-MSCs activated the JAK2/STAT3 pathway in AML cells. Two JAK/STAT pathway inhibitors counteracted the AML-MSCs induced morphology change and EMT marker expression in AML cells. In conclusion, AML-MSCs not only promote the emergence of chemoresistance but also enhance it once AML acquires chemoresistance. AML-MSCs induce EMT-like features in AML cells; this phenotypic change could be related to chemoresistance progression. AML-MSCs induce the EMT-like program in AML cells through IL-6/JAK2/STAT3 signaling, which provides a therapeutic target to reverse chemoresistance in AML.

Identification of novel candidate genes and small molecule drugs in ovarian cancer by bioinformatics strategy.

Background: Ovarian cancer (OC) is the most lethal type of malignancies in the female reproductive system. This study aimed to identify novel biomarkers and potential small molecule drugs in OC by integrating two expression profile datasets. Methods: GSE18520 and GSE14407 from the Gene Expression Omnibus (GEO) database were selected and the overlapped differentially expressed genes (DEGs) were detected. The Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analysis were performed to establish the protein-protein interaction (PPI) network of DEGs and identified the hub genes. Gene Expression Profiling Interactive Analysis (GEPIA), Oncomine database and The Human Protein Atlas (HPA) were used to validate the expression of the identified hub genes. The prognostic value of these hub genes were evaluated by the Kaplan Meier plotter online tool. The expression of NCAPG was further explored by immunohistochemistry in our OC tissues. Moreover, CMap database was used to look for prospective small compounds with therapeutic efficacy based on OC RNA-seq. Results: A total of 433 DEGs were identified. The DEGs were mainly enriched in negative regulation of transcription and pathways in cancer. A PPI network was constructed with 344 nodes and 1,596 interactions. The top ten module genes were chosen as hub genes. Among which, survival analysis showed that patients with high expression of CCNB1, TOP2A, NUSAP1, NCAPG, KIF20A and DLGAP5 had poorer survival results than those with low expression. These six genes were all overexpressed in OC tissue by means of bioinformatics analysis. In our clinical patients, the expression rate of NCAPG in OC tissues was significantly higher than that in benign serous ovarian cystadenoma and borderline serous ovarian cystadenoma tissues. Meanwhile, several small molecules with potential therapeutic efficacy against OC were identified in our study. Conclusions: By means of bioinformatics analysis, we identified six real hub genes and indicated a group of candidate small molecule drugs as adjunctive agents for OC. They could be the potential novel biomarkers for the diagnosis and promising therapeutic targets of OC.

Immunofluorescence analysis of cytokeratin 8/18 staining is a sensitive assay for the detection of cell apoptosis.

Apoptosis is one of the major types of programmed cell death. During this process, cells experience a series of morphological and biochemical changes. Flow cytometric analysis of Annexin V staining of cell surface phosphatidylserine, in combination with a DNA-staining dye to probe the permeability of the cell membrane, is an established method for detecting apoptosis. The present study aimed to show that the immunofluorescence analysis of cytokeratin (CK) 8/18 staining may provide a new and sensitive assay for the detection of apoptotic cells. Tumor cells were treated with 20 µM cisplatin to induce apoptosis. Following 12 and 24 h of cisplatin treatment, cells were collected and stained with 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI) and fluorescein-labeled anti-CK8/18 antibody. The apoptotic cells were subsequently examined by fluorescence microscopy. Annexin V-fluorescein isothiocyanate/propidium iodide staining followed by flow cytometric analysis confirmed that cisplatin was able to induce apoptosis in tumor cells. Immunofluorescence analysis demonstrated that apoptotic cells had a distinct CK8/18 staining pattern. In living cells, CK8/18 was uniformly distributed in the cytoplasm and cytosol; however in the apoptotic cells with a condensed and/or fragmented apoptotic nucleus (as identified by DAPI staining), fluorescein-labeled anti-CK8/18 antibody exhibited unusual punctate and/or bubbly staining in the cytosol. In the apoptotic cells that could not be identified by DAPI staining, fluorescein-labeled CK8/18 displayed polarized aggregated staining in the cytosol. These results indicate that fluorescein-conjugated CK8/18 may be a useful and sensitive indicator of cell apoptosis.

Correlation between p65 and TNF-α in patients with acute myelocytic leukemia.

The correlation between the expression levels of p65 and TNF-α in patients with acute myelocytic leukemia (AML) and AML cell lines were investigated. The bone marrow samples of 30 AML patients and 10 non-leukemia controls were studied. The mRNA expression levels of p65 and TNF-α were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Pearson's Correlation test was used to demonstrate the correlation between TNF-α and p65 expression levels in AML specimens. Receiver operating characteristic (ROC) curves were plotted to determine whether TNF-α and p65 expression levels could be used to differentiate AML samples from non-leukemia samples. MG132 and anti-TNF-α antibody were used to inhibit the expression of p65 and TNF-α in the AML cell line, HL-60. The expression of p65 and TNF-α were detected by RT-qPCR and western blot analysis. The mRNA expression levels of p65 and TNF-α were significantly increased in AML patients compared with non-leukemia control bone marrow samples by RT-qPCR, and the two molecules expression pattern's exhibited sufficient predictive power to distinguish AML patients from non-leukemia control samples. Pearson's correlation analysis demonstrated that TNF-α expression was strongly correlated with p65 expression in AML bone marrow samples. In HL-60 cells, inhibition of TNF-α reduced the expression of p65; in addition, inhibition of p65 reduced the expression of TNF-α as assessed by RT-qPCR and western blot analysis. p65 and TNF-α were highly expressed in AML patients, and these 2 molecules were strongly correlated. The present study indicates that p65 and TNF-α have potential as molecular markers to distinguish AML patients from non-leukemia control samples, and that these 2 molecules may be useful prognostic factor for patients with AML.

Inhibition of tumor necrosis factor-α enhances apoptosis induced by nuclear factor-κB inhibition in leukemia cells.

Inhibition of nuclear factor-κB (NF-κB) results in antitumor activity in leukemia cells, and may be a potential therapeutic strategy for the treatment of leukemia. However, a significant limitation of NF-κB inhibition in the treatment of leukemia is the low efficiency of this technique. NF-κB inhibitor treatment induces apoptosis in leukemia cells; however, it additionally causes inflammatory molecules to induce increased sensitivity of healthy hematopoietic cells to cell death signals, therefore limiting its clinical applications. Tumor necrosis factor-α (TNF-α) is a key regulator of inflammation, and induces a variety of actions in leukemic and healthy hematopoietic cells. TNF-α induces NF-κB-dependent and -independent survival signals, promoting the proliferation of leukemia cells. However, in healthy hematopoietic cells, TNF-α induces death signaling, an effect which is enhanced by the inhibition of NF-κB. Based on these observations, the present study hypothesized that inhibition of TNF-α signaling may be able to protect healthy hematopoietic cells and other tissue cells, while increasing the anti-leukemia effects of NF-κB inhibition on leukemia cells. The role and underlying molecular mechanisms of TNF-α inhibition in the regulation of NF-κB inhibition-induced apoptosis in leukemia cells was therefore investigated in the present study. The results indicated that inhibition of TNF-α enhanced NF-κB inhibition-induced apoptosis in leukemia cells. It was also revealed that protein kinase B was significant in the regulation of TNF-α and NF-κB inhibition-induced apoptosis. During this process, intrinsic apoptotic pathways were activated. A combination of NF-κB and TNF-α inhibition may be a potential specific and effective novel therapeutic strategy for the treatment of leukemia.

Clinical significance of serum expression of GROß in esophageal squamous cell carcinoma.

AIM: To determine the association between serum levels of growth-related gene product ß (GROß) and clinical parameters in esophageal squamous cell carcinoma (ESCC). METHODS: Using enzyme-linked immunosorbent assay, serum GROß levels were measured in ESCC patients (n = 72) and healthy volunteers (n = 83). The association between serum levels of GROß and clinical parameters of ESCC was analyzed statistically. RESULTS: The serum GROß levels were much higher in ESCC patients than in healthy controls (median: 645 ng/L vs 269 ng/L, P < 0.05). Serum GROß levels were correlated positively with tumor size, lymph node metastasis, and tumor-node-metastasis (TNM) staging, but not with gender or the histological grade of tumors in ESCC patients. The sensitivity and specificity of the assay for serum GROß were 73.61% and 56.63%, respectively. CONCLUSION: GROß may function as an oncogene product and contribute to tumorigenesis and metastasis of ESCC.

GROß and its downstream effector EGR1 regulate cisplatin-induced apoptosis in WHCO1 cells.

Cisplatin is one of the most widely used chemotherapeutic agents employed for treatment of a wide variety of solid tumors, including human esophageal squamous cell carcinoma (ESCC). However, a major limitation of cisplatin-based chemotherapy of ESCC is the rather low-effective rate. Understanding the molecular events of limited efficacy of cisplatin-based chemotherapy of ESCC could lead to strategies resulting in improved therapeutic benefits. The CXC chemokine family has been reported to be related to inflammatory reaction, injure recovery, cell proliferation, apoptosis and even to be involved in the regulation of chemotherapeutic agent-induced apoptosis. CXCL2 chemokine, also known as GROß (growth-related gene product ß), belongs to the CXC chemokine group. The known functions of GROß are related to attracting neutrophils to sites of inflammation, modulation of the neurotransmitter release, cell proliferation and apoptosis. However, little is known about the relationship between GROß and chemotherapeutic agent-induced apoptosis. This study was designed to provide insights into the possible role of GROß in the regulation of cisplatin-induced apoptosis in ESCCs. We report here that inhibition of expression of GROß can decrease cisplatin-induced apoptosis in WHCO1 cells. EGR1 is a downstream factor regulated by GROß. Silencing expression of EGR1 can also decrease cisplatin-induced apoptosis in WHCO1 cells. The activation of caspase 9 was delayed in cells in which GROß and EGR1 were knocked down after cisplatin treatment. All these results indicate that GROß and its downstream factor EGR1 are involved in regulating cisplatin-induced apoptosis in WHCO1 cells, and during this process the intrinsic apoptotic pathway is activated. It may be useful to examine the expression levels of GROß and EGR1 in ESCC patients to select those likely to respond well to cisplatin.