Crosstalk between autophagy and epithelial-mesenchymal transition and its application in cancer therapy.

Autophagy is a highly conserved catabolic process that mediates degradation of pernicious or dysfunctional cellular components, such as invasive pathogens, senescent proteins, and organelles. It can promote or suppress tumor development, so it is a "double-edged sword" in tumors that depends on the cell and tissue types and the stages of tumor. The epithelial-mesenchymal transition (EMT) is a complex biological trans-differentiation process that allows epithelial cells to transiently obtain mesenchymal features, including motility and metastatic potential. EMT is considered as an important contributor to the invasion and metastasis of cancers. Thus, clarifying the crosstalk between autophagy and EMT will provide novel targets for cancer therapy. It was reported that EMT-related signal pathways have an impact on autophagy; conversely, autophagy activation can suppress or strengthen EMT by regulating various signaling pathways. On one hand, autophagy activation provides energy and basic nutrients for EMT during metastatic spreading, which assists cells to survive in stressful environmental and intracellular conditions. On the other hand, autophagy, acting as a cancer-suppressive function, is inclined to hinder metastasis by selectively down-regulating critical transcription factors of EMT in the early phases. Therefore, the inhibition of EMT by autophagy inhibitors or activators might be a novel strategy that provides thought and enlightenment for the treatment of cancer. In this article, we discuss in detail the role of autophagy and EMT in the development of cancers, the regulatory mechanisms between autophagy and EMT, the effects of autophagy inhibition or activation on EMT, and the potential applications in anticancer therapy.

The relationship between autophagy and the immune system and its applications for tumor immunotherapy.

Autophagy is a genetically well-controlled cellular process that is tightly controlled by a set of core genes, including the family of autophagy-related genes (ATG). Autophagy is a "double-edged sword" in tumors. It can promote or suppress tumor development, which depends on the cell and tissue types and the stages of tumor. At present, tumor immunotherapy is a promising treatment strategy against tumors. Recent studies have shown that autophagy significantly controls immune responses by modulating the functions of immune cells and the production of cytokines. Conversely, some cytokines and immune cells have a great effect on the function of autophagy. Therapies aiming at autophagy to enhance the immune responses and anti-tumor effects of immunotherapy have become the prospective strategy, with enhanced antigen presentation and higher sensitivity to CTLs. However, the induction of autophagy may also benefit tumor cells escape from immune surveillance and result in intrinsic resistance against anti-tumor immunotherapy. Increasing studies have proven the optimal use of either ATG inducers or inhibitors can restrain tumor growth and progression by enhancing anti-tumor immune responses and overcoming the anti-tumor immune resistance in combination with several immunotherapeutic strategies, indicating that induction or inhibition of autophagy might show us a prospective therapeutic strategy when combined with immunotherapy. In this article, the possible mechanisms of autophagy regulating immune system, and the potential applications of autophagy in tumor immunotherapy will be discussed.

Anesthetic agent etiomidate induces apoptosis in N2a brain tumor cell line.

The present study identified the cytotoxic effects of etomidate on the N2a neuroblastoma cell line. Etomidate induced apoptosis in N2a cells in a concentration­dependent manner, which was confirmed by western blotting and flow cytometry. Phase contrast microscopy was used to analyze the effect of etomidate on morphological characteristics. The number of the apoptotic cells was increased and confirmed by DAPI and PI staining, which served as a characteristic hallmark of apoptosis. Additionally, etomidate led to loss of mitochondrial membrane potential and resulted in the generation of reactive oxygen species in N2a cells. The western blot analysis revealed that N2a cells treated with etomidate had a significant modulation of pro­apoptotic proteins, includingpoly ADP­ribose polymerase (PARP), cleaved PARP, caspase­9 and procaspase­3. In conclusion, the present study determined that etomidate induced cytotoxic and apoptotic effects in N2a brain tumor cells in vitro.

HOTAIR, a long non-coding RNA driver of malignancy whose expression is activated by FOXC1, negatively regulates miRNA-1 in hepatocellular carcinoma.

Evidence is rapidly accumulating that long non-coding RNAs (lncRNAs) are involved in human tumorigenesis and are dysregulated in multiple cancers, including hepatocellular carcinoma (HCC). lncRNAs can regulate essential pathways that contribute to tumor initiation and progression with tissue specificity, which suggests that lncRNAs may be valuable biomarkers and therapeutic targets. HOX transcript antisense intergenic RNA (HOTAIR) has previously been demonstrated to be an oncogene and a negative prognostic factor in a variety of cancers; however, the factors that contribute to the upregulation of HOTAIR and the interaction between HOTAIR and microRNAs (miRNAs or miRs) are largely unknown. In the present study, the expression levels of HOTAIR, forkhead box C1 (FOXC1) and miRNA-1 were examined in 50 matched pairs of HCC and HCC cells. The effects of HOTAIR on HCC cell proliferation were tested using trypan blue exclusion assay. The effect of HOTAIR on HCC growth in vivo was determined in a (nu/nu) mouse model. A computational screening of HOTAIR promoter was conducted to search for transcription factor-binding sites. FOXC1 binding to the promoter region of HOTAIR was confirmed using a chromatin immunoprecipitation assay. A search for miRNAs that had complementary base paring with HOTAIR was performed utilizing an online software program. The interaction between miR-1 and HOTAIR was examined using a luciferase reporter assay. Gain and loss of function approaches were used to determine the changes of HOTAIR or miR-1 expression. The relative levels of FOXC1 and HOTAIR expression in HCC tissues and HepG2 cells were significantly higher than those in normal liver LO2 cells and adjacent carcinoma tissues; the relative expression of miR-1 exhibited the opposite pattern. Overexpression of HOTAIR promoted HCC cell proliferation and progression of tumor xenografts. The present authors have demonstrated that FOXC1 binds to the upstream region of HOTAIR in HCC cells and that FOXC1 activates lncRNA HOTAIR expression in HCC HepG2 cells, which suggests that HOTAIR harbors a miRNA-1 binding site. The present data revealed that this binding site is vital for the regulation of miRNA-1 by HOTAIR. Furthermore, HOTAIR negatively regulated the expression of miRNA-1 in HepG2 cells. Additionally, the present study demonstrated that the oncogenic activity of HOTAIR is in part based on the negative regulation of miR-1. Taken together, these results suggest that HOTAIR is a FOXC1-activated driver of malignancy, which acts in part through the repression of miR-1.

Correlations of microRNA-124a and microRNA-30d with clinicopathological features of breast cancer patients with type 2 diabetes mellitus.

This study intends to investigate the correlations of miR-124a and miR-30d with clinicopathological features of breast cancer (BC) patients with type 2 diabetes mellitus (T2DM). A total of 72 BC patients with T2DM (diabetic group) and 144 BC patients without T2DM (non-diabetic group) were enrolled in this study. Blood glucose was detected by glucose oxidase methods. Glycosylated hemoglobin (HbA1c) was measured by high performance liquid chromatography. Fasting insulin (FIns) was measured by chemiluminescent microparticle immunoassay. Automatic biochemical analyzer was used to detect triglyceride, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C). Estradiol (E2) was detected by radioimmunoassay. Homeostasis model assessment was applied to assess the insulin resistance (HOMA-IR) and ß-cell insulin secretion (HOMA-IS). The expressions of miR124a and miR-30d were measured by quantitative real-time polymerase chain reaction (qRT-PCR). There were significant differences in age, the ratio of menopause, body mass index (BMI), HDL-C, TC, 2-h plasma glucose (2hPG), FIns, HbA1c, HOMA-IS and HOMA-IR between the diabetic and non-diabetic groups. The diabetic group had higher incidence of lymph node metastasis than non-diabetic group. The miR-124a expression was down-regulated while the miR-30d expression was up-regulated in BC patients with T2DM. The correlation analysis showed that miR-124a expression was positively correlated with HDL-C, while it was negatively correlated with age, HbA1c, LDL-C and E2. However, the miR-30d expression was negatively correlated with HDL-C but positively correlated with age, HbA1c, LDL-C and E2. In conclusion, miR-124a and miR-30d may be correlated with clinicopathological features of BC patients with T2DM. The miR-124a and miR-30d could serve as novel biomarkers for early diagnosis of BC in patients with T2DM.

[The rheology properties of common hydrophilic gel excipients].

To investigate theological properties of common hydrophilic gel excipients such as Carbopol based on viscosity, the viscosity was determined by rotation method and falling-ball method. Linear regression was made between ln(eta) and concentration, the slope of which was used to explore the relation between viscosity and concentration of different excipients. The viscosity flow active energy (E(eta)) was calculated according to Arrhenius equation and was used to investigate the relation between viscosity and temperature of different excipients. The results showed that viscosities measured by two methods were consistent. Concentration of guargum (GG) and hydroxypropylmethyl cellulose (HPMC) solution had a great influence on the viscosity, k > 5; while concentration of polyvinylpyrrolidone-K30 (PVP-K30) and polyethylene glycol 6000 (PEG6000) exerted a less effect on viscosity, k < 0.2; viscosity flow active energy of different excipients were close, which ranged from 30 to 40 kJ x mol(-1). Therefore, theological properties study could provide the basis for application of excipients and establish a foundation for the research of relation between excipients structure, property and function.

[Expression and clinical significance of epidermal growth factor receptor and protein kinase B in gastric carcinoma].

OBJECTIVE: To evaluate the association between survival and expressions of epidermal growth factor receptor(EGFR) and protein kinase B(AKT) in gastric carcinoma specimens. METHODS: EGFR and AKT were detected by immunohistochemistry in 84 patients with gastric cancer and 15 controls. Association between their expressions and clinical characteristics as well as prognosis were analyzed. RESULTS: In patients with gastric cancer,expression rates were significantly higher than those in 15 normal gastric tissues for EGFR(55.9% vs. 20%, P<0.05) and AKT(64.3% vs. 6.7%, P<0.01). Expression of EGFR was associated with late staging(P<0.01), while expression of AKT was associated with lymph node metastasis(P<0.01). Overall 5-year survival rate was significantly higher in patients with EGFR negative expression (49.0% vs. 22.0%, P<0.05). Overall 5-year survival rate was significantly higher in patients with AKT expression(45.7% vs. 30.2%, P<0.05). CONCLUSION: Expression of EGFR and AKT negative may be used to evaluate the degree of differentiation and prognosis in patients with gastric carcinoma.

[Rituximab-mediated sensitization of B-NHL cell lines to apoptosis induced by gemcitabine and Navelbine in vitro].

This study was purposed to explore the Rituximab (RTX)-mediated sensitization of B-NHL cell lines to apoptosis induced by Gemcitabine or Navelbine and its possible mechanism. The inhibitory rate of B-NHL cell proliferation was detected by XTT method, the IC50 from Gemcitabine or Navelbine and combination of Gemcitabine or Navelbine with RTX was compared. The expression level of antiapoptotic protein BCL-2 in Daudi, Ramos, Raji and Namalwa cells treated with RTX of 20 µg/ml for 24 hours was detected by Western blot. The results showed that the RTX as a single agent could weakly induce the apoptosis of Daudi, Namalwa, Raji and Ramos lymphoma cell lines, the inhibiting rate of cell proliferation ranged from 3% to 10%; RTX could sensitize significantly the cytotoxity of Gemcitabine or Navelbine in Daudi, Namalwa and Raji cell lines; The expression of BCL-2 in Raji and Namalwa cell lines treated with RTX of 20 microg/ml for 24 hours was down-regulated. It is concluded that RTX can sensitize the cytotoxicity of Gemcitabine or Navelbine to the human lymphoma cell lines which displayed the synergistic effect on Daudi, Namalwa and Raji cell lines. The BCL-2 expression level in Raji and Namalwa cell lines treated by RTX is down-regulated which may be one of the mechanisms sensitizing the cytotoxicity of Gemcitabine or Navelbine.

[Preclinical study of apoptosis of B-NHL cell lines induced by anti-CD20 monoclonal antibody].

The aim of this study was to investigate the effect of anti-CD20 monoclonal antibody (McAb) on induction of apoptosis in malignant B cell lines in vitro and to explore its possible mechanism. The human Burkitt's lymphoma cell lines (Daudi, Namalwa, Raji and Ramos cells) were cultured in vitro. The inhibitory rate of cell proliferation was detected by XTT assay, the apoptosis of cells was determined by flow cytometry. The expression of BCL-2 in human Burkitt's lymphoma cell lines (Daudi, Namalwa, Raji and Ramos cells) treated with rituximab (20 microg/ml) for 24 hours was analyzed by Western blot. The results showed that the anti-CD20 McAb had a slight anti-proliferation effect on the Daudi, Namalwa, Raji cell lines and no effect on the Ramos cell line. There is no correlation between the effect and the concentration of anti-CD20 McAb. Anti-CD20 McAb as a single agent could weakly induce the apoptosis of four cell lines. The inhibitory rate of cell proliferation ranged from 3% to 10%. Expression of BCL-2 protein was down-regulated after treated by anti-CD20 McAb for 24 hours in Raji and Namalwa cell lines. It is concluded that the anti-CD20 McAb as a monomer can slightly inhibit the proliferation of Daudi, Namalwa and Raji cell lines, the inhibition does not dependent on the treating time and the concentrations of anti-CD20 McAb. Anti-CD20 McAb as a monomer can weakly induce the apoptosis of four cell lines. Expression of BCL-2 in Raji and Namalwa cell lines is down-regulated after the cells were treated by anti-CD20 McAb for 24 hours. Down-regulation of BCL-2 expression may be one of the mechanisms enhancing the cytotoxicity of cytotoxic drugs.

Sustained low-level expression of interferon-gamma promotes tumor development: potential insights in tumor prevention and tumor immunotherapy.

Although the proinflammatory cytokine interferon-gamma (IFN-gamma) has been generally thought to enhance antitumor immune responses and be involved in antitumor mechanisms of many other immunotherapy molecules, it has also been reported that IFN-gamma could promote tumor immune evasion. In this report, by using an ideal mouse model that expresses IFN-gamma locally in muscle, we demonstrate that sustained low-level expression of IFN-gamma promotes the development of several types of tumor including H22 hepatoma, MA782/5S mammary adenocarcinoma and B16 melanoma. However, transitory expression of IFN-gamma does not have such an effect. On the other hand, sustained high-level expression of IFN-gamma mediates significant antitumor effect on H22 hepatoma. Low level of IFN-gamma upregulates expression of PD-L1, PD-L2, CTLA-4 and Foxp3, which may partly account for the tumor immune evasion promoted by IFN-gamma. Furthermore, blockade of PD-L inhibits IFN-gamma's tumor-promoting effect. Our findings provide a mechanistic link between chronic inflammation and cancer and would have potential implications for cancer prevention and also for the design of cytokine-based cancer immunotherapy.

[HLA genotyping by automatic semi-quantitative polymerase chain reaction-reverse sequence specific oligonucleotide].

OBJECTIVE: To explore a HLA genotyping method that can be used for organ transplantation tissue typing, and especially for establishing hemopoietic stem cell bank and the cord blood stem cell bank by analyzing the PCR-DNA. METHODS: Modified automatic method based on semi-quantitative amplification system of HLA-I, I allele genotyping was established using reverse sequence-specific oligonucleotide with polymerase chain reaction (PCR-RSSO), and was compared with PCR (using sequence specific primer, PCR-SSR) and manual PCR-RSSO in terms of the accuracy, resolution, and the quantity of DNA consumption. A total of 635 blood samples were genotyped with HLA-A, B, C, DR and DQ alleles using auto semi-quantitative PCR-RSSO, 166 of which were also examined using PCR-SSP and manual PCR-RSSO simultaneously. RDSULTS: The success rate of automatic semi-quantitative PCR-RSSO, PCR-SSP and manual PCR-RSSO was 98.4% (3 124/3 175), 98.8% (656/664) and 88.3% (732/830) respectively, with no significant difference between the former 2 as indicated by X2 test (P>0.05). Auto semi-quantitative PCR-RSSO, however, yielded significantly higher success rate than manual PCR-RSSO (P<0.05). CONCLUSION: PCR-RSSO is capable of identifying 706 alleles of HLA-I, II antigens which amounts to 75.43% of the 936 alleles published by WHO in 2000, with intermediate to high resolution, even in the case of the homozygote. The hybridization results documenting the original data can be conveniently preserved. This method therefore possesses the merits of low expenses, low labor intensity, and rapid processing of large number of samples.