Preliminary characterization of IL32 in basal-like/triple negative compared to other types of breast cell lines and tissues

BACKGROUND: Triple negative breast cancer (TNBC) and often basal-like cancers are defined as negative for estrogen receptor, progesterone receptor and Her2 gene expression. Over the past few years an incredible amount of data has been generated defining the molecular characteristics of both cancers. The aim of these studies is to better understand the cancers and identify genes and molecular pathways that might be useful as targeted therapies. In an attempt to contribute to the understanding of basal-like/TNBC, we examined the Gene Expression Omnibus (GEO) public datasets in search of genes that might define basal-like/TNBC. The Il32 gene was identified as a candidate. FINDINGS: Analysis of several GEO datasets showed differential expression of IL32 in patient samples previously designated as basal and/or TNBC compared to normal and luminal breast samples. As validation of the GEO results, RNA and protein expression levels were examined using MCF7 and MDA MB231 cell lines and tissue microarrays (TMAs). IL32 gene expression levels were higher in MDA MB231 compared to MCF7. Analysis of TMAs showed 42% of TNBC tissues and 25% of the non-TNBC were positive for IL32, while non-malignant patient samples and all but one hyperplastic tissue sample demonstrated lower levels of IL32 protein expression. CONCLUSION: Data obtained from several publically available GEO datasets showed overexpression of IL32 gene in basal-like/TNBC samples compared to normal and luminal samples. In support of these data, analysis of TMA clinical samples demonstrated a particular pattern of IL32 differential expression. Considered together, these data suggest IL32 is a candidate suitable for further study

Knockdown of autophagy-related protein 5, ATG5, decreases oxidative stress and has an opposing effect on camptothecin-induced cytotoxicity in osteosarcoma cells

BACKGROUND: Autophagy induction can increase or decrease anticancer drug efficacy. Anticancer drug-induced autophagy induction is poorly characterized in osteosarcoma (OS). In this study, we investigated the impact of autophagy inhibition on camptothecin (CPT)-induced cytotoxicity in OS. METHODS: Autophagy-inhibited DLM8 and K7M3 metastatic murine OS cell lines were generated by infection with lentiviral shRNA directed against the essential autophagy protein ATG5. Knockdown of ATG5 protein expression and inhibition of autophagy was confirmed by immunoblot of ATG5 and LC3II proteins, respectively. Metabolic activity was determined by MTT assay and cell viability was determined by trypan blue exclusion. Acridine orange staining and immunoblotting for LC3II protein expression were used to determine autophagy induction. Oxidative stress was assessed by staining cells with HE and DCFH-DA followed by flow cytometry analysis. Mitochondrial membrane potential was determined by staining cells with TMRE followed by flow cytometry analysis. Immunoblotting was used to detect caspase activation, Parp cleavage and p53 phosphorylation. RESULTS: Autophagy inhibition caused a greater deficit in metabolic activity and cell growth in K7M3 cells compared to DLM8 cells. K7M3 cells exhibited higher basal autophagy levels than DLM8 cells and non-transformed murine MCT3 osteoblasts. Autophagy inhibition did not affect CPT-induced DNA damage. Autophagy inhibition decreased CPT-induced cell death in DLM8 cells while increasing CPT-induced cell death in K7M3 cells. Autophagy inhibition reduced CPT-induced mitochondrial damage and CPT-induced caspase activation in DLM8 cells. Buthionine sulfoximine (BSO)-induced cell death was greater in autophagy-competent DLM8 cells and was reversed by antioxidant pretreatment. Camptothecin-induced and BSO-induced autophagy induction was also reversed by antioxidant pretreatment. Significantly, autophagy inhibition not only reduced CPT-induced oxidative stress but also reduced basal oxidative stress. CONCLUSIONS: The results of this study indicate that autophagy inhibition can have an opposing effect on CPT-induced cytotoxicity within OS. The cytoprotective mechanism of autophagy inhibition observed in DLM8 cells involves reduced CPT-induced oxidative stress and not reduced DNA damage. Our results also reveal the novel finding that knockdown of ATG5 protein reduces both basal oxidative stress and drug-induced oxidative stress

Acute Neurotoxicity and Assessment of a Biomarker of Effect (GFAP) In Male Sprague-Dawley Rats After a Single Exposure to 1,2-Dichloroethane

DCE is a clear synthetic liquid. Its most common use today is to make vinyl chloride and several substances that dissolve grease, glue, and dirt. The purpose ofthis study was to investigate the effects of DCE on the central nervous system by measuring GFAP and GSH brain levels and SMA. Brain regions studied were CTX, BS, CRE and HP. 49-day male Sprague-Dawley rats were given a single dose of DCE and sacrificed 7 and 14 days later to measure GFAP levels. Dose groups for the 7 -day study were Sml/kg saline and O. 7mllkg DCE. Dose groups for the 14-day study were Sml/kg saline, O.3Sml/kg [L-dose] and O.7ml/kg [H-dose] DCE. SMA for the same 1 2 dose groups as used for the 14-day study was measured on days 1,2,7 and 14 using digiscan computerized cages. Brain GSH levels were measured 2 hours following exposure to 5m1Jk:g saline, O.3Sml/kg and O.SOmllkg DCE. The saline group served as the control group in all cases. In the 7 -day study, GFAP levels were significantly (

Knock down of Fas-Associated Protein with Death Domain (FADD) Sensitizes Osteosarcoma to TNFα-induced Cell Death

Fas-associated protein with death domain (FADD) was first identified for its role in linking death receptors to the apoptotic signaling pathway with subsequent cell death. Later studies reported non-apoptotic functions for FADD in normal cells and cancer cells. Non-apoptotic functions for FADD in osteosarcoma (OS) have not been reported. In this study, FADD protein expression was knocked down in human CCHOSD, LM7, and SaOS2 OS cell lines followed by assessment of sensitivity to TNFα- or TRAIL-induced cell death. Knock down of FADD significantly increased TNFα-induced cell death in LM7 and SaOS2 cell lines. The mode of TNFα-induced cell death was apoptosis and not necroptosis. Inhibition of nuclear factor kappa B (NFκB) in wildtype cells increased TNFα-induced cell death to similar levels observed in FADD knockdown cells, suggesting a role for FADD in NFκB pro-survival cell signaling. In addition, knock down of FADD increased SMAC mimetic-mediated TNFα-induced cell death in all cell lines studied. The results of this study indicate that FADD has a pro-survival function in OS following TNFα treatment that involves NFκB signaling. The results also indicate that the pro-survival function of FADD is associated with XIAP activity

Phosphorylated heat shock protein 27 as a potential biomarker to predict the role of chemotherapy-induced autophagy in osteosarcoma response to therapy

Autophagy is a catabolic process involved in cellular homeostasis. Autophagy is increased above homeostatic levels by chemotherapy, and this can either promote or inhibit tumor growth. We previously demonstrated that aerosol gemcitabine (GCB) has a therapeutic effect against osteosarcoma (OS) lung metastases. However, some tumor cells failed to respond to the treatment and persisted as isolated lung metastasis. Here, we examined the mechanisms underlying the dual role of chemotherapy-induced autophagy in OS and sought to identify biomarkers to predict OS response to treatment. In this study, we demonstrate that treatment of various OS cells with GCB induced autophagy. We also showed that GCB reduces the phosphorylation of AKT, mTOR and p70S6K and that GCB-induced autophagy in OS can lead to either cell survival or cell death. Blocking autophagy enhanced the sensitivity of LM7 OS cells and decreased the sensitivity of CCH-OS-D and K7M3 OS cells to GCB. Using a kinase array, we also demonstrated that differences in the phosphorylated heat shock protein 27 (p-HSP27) expression in the various OS cell lines after treatment with GCB, correlates to whether chemotherapy-induced autophagy will lead to increase or decrease OS cells sensitivity to therapy. Increased p-HSP27 was associated with increased sensitivity to anticancer drug treatment when autophagy is inhibited. The results of this study reveal a dual role of autophagy in OS cells sensitivity to chemotherapy and suggest that p-HSP27 could represent a predictive biomarker of whether combination therapy with autophagy modulators and chemotherapeutic drugs will be beneficial for OS patients

Analysis of hsp27 and the autophagy marker LC3B \u3csup\u3eþ\u3c/sup\u3e puncta following preoperative chemotherapy identifies high-risk osteosarcoma patients

Chemotherapy-induced autophagy is a proposed mechanism of chemoresistance and potential therapeutic target in osteosarcoma. We evaluated heat shock protein 27 (HSP27) and autophagy-related proteins as predictors of pathologic treatment response and prognostic markers among osteosarcoma patients who received standard chemotherapy. We analyzed 394 tumor specimens (pre-treatment, post-treatment, and metastases) from 260 osteosarcoma patients by immunohistochemistry for cytoplasmic light chain 3B (LC3B)-positive puncta, sequestosome 1 (SQSTM1), high mobility group box 1 (HMGB1), and HSP27 expression. The staining percentage and intensity for each marker were scored and the extent to which marker expression was correlated with pathologic response, relapse-free survival (RFS), and overall survival (OS) was assessed. LCB3 þ puncta in post-treatment primary tumors (50%) and metastases (67%) was significantly higher than in pre-treatment biopsy specimens (30%; P ¼ 0.023 and \u3c0.001). Among 215 patients with localized osteosarcoma, both pretreatment [multivariate hazard ratio (HR), 26.7; 95% confidence interval (CI), 1.47–484; P ¼ 0.026] and post-treatment HSP27 expression (multivariate HR, 1.85; 95% CI, 1.03–3.33; P ¼ 0.039) were associated with worse OS. Lack of LC3B þ puncta at resection was an independent poor prognostic marker in both univariate (HR, 1.78; 95% CI, 1.05–3.03; P ¼ 0.034) and multivariate models (HR, 1.75; 95% CI, 1.01–3.04; P ¼ 0.045). Patients with LC3B þ / HSP27 tumors at resection had the best 10-year OS (75%) whereas patients with LC3B/HSP27 þ tumors had the worst 10-year survival (25%). Neither HSP27 expression nor the presence of LCB3 þ puncta was correlated with pathologic treatment response. Our findings establish HSP27 expression and LC3B þ puncta as independent prognostic markers in osteosarcoma patients receiving standard chemotherapy and support further investigation into strategies targeting HSP27 or modulating autophagy in osteosarcoma treatment