Ruochun Huang

Identification of interleukin-8 as estrogen receptor-regulated factor involved in breast cancer invasion and angiogenesis by protein arrays

With the goal of identifying key factors involved in human breast cancer progression, we applied human cytokine antibody arrays we have developed to screen cytokine expression levels in human breast cancer cell lines and identified interleukin (IL)‐8 as a key factor involved in breast cancer invasion and angiogenesis. Elevated expression of IL‐8 in breast cancer cells was associated with breast cancer invasiveness and angiogenesis. Neutralization of antibody against IL‐8 specifically blocked IL‐8‐mediated tumor cell invasion and angiogenesis. Furthermore, IL‐8 levels in human breast cancer cells were closely related to estrogen receptor (ER) status. ER positive cells expressed low levels of IL‐8 whereas ER negative cells expressed high levels of IL‐8. Expression of exogenous ERα substantially inhibited IL‐8 expression. Our findings raise intriguing questions regarding the role of IL‐8 in the development and progression of human breast cancer in association with ER status.

Connexin 43 (cx43) enhances chemotherapy‐induced apoptosis in human glioblastoma cells

Stable re‐expression of connexin 43 (cx43) in human glioblastoma suppresses transformation and tumorigenicity. The present study was designed to examine the role of cx43 in chemotherapy‐induced apoptosis. Expression of cx43 in human glioblastoma cells significantly increased sensitivity to several common chemotherapeutic agents, including etoposide, paclitaxel (Taxol) and doxorubicin, compared with control‐transfected cells. The increased sensitivity to chemotherapeutic agents resulted from apoptosis as evidenced by Hoechst dye staining, TUNEL assay and annexin V assay. These cx43‐mediated effects were coupled with decreased expression of the specific apoptosis inhibitor bcl‐2. Over‐expression of bcl‐2 in cx43‐transfected cells partially confers the resistance to apoptosis induced by etoposide, suggesting that the cx43‐mediated apoptosis to chemotherapeutic agents is regulated in part through the down‐regulation of bcl‐2 expression. Furthermore, the cx43‐mediated apoptosis in response to chemotherapeutic drugs may not be linked to increased gap junctional communication in cx43‐transfected cells. Our results demonstrate a new role of cx43 in the mediation of apoptosis during chemotherapy.

Hydrogen peroxide promotes transformation of rat liver non-neoplastic epithelial cells through activation of epidermal growth factor receptor

Previous studies demonstrated that hydrogen peroxide (H2O2) is a tumor promoter in the rat liver epithelial cell line T51B. We investigated the pathway linking H2O2 to tumor promotion. H2O2 can directly induce tyrosine phosphorylation of epidermal growth factor receptor (EGFR). H2O2 and epidermal growth factor exerted similar effects on the induction of early growth response genes, disruption of gap junction communication, triggering of calcium inflow, and promotion of transformation. Furthermore, the effect of H2O2 on tumor promotion was blocked by abrogation of EGFR activation. Our results suggested that tumor promotion by H2O2 is mediated mainly through activation of EGFR in T51B cells.

Enhanced chemosensitization in multidrug-resistant human breast cancer cells by inhibition of IL-6 and IL-8 production.

Drug resistance remains a major hurdle to successful cancer treatment. Many mechanisms such as overexpression of multidrug-resistance related proteins, increased drug metabolism, decreased apoptosis, and impairment of signal transduction pathway can contribute multidrug resistance (MDR). Recent studies strongly suggest a close link between cytokines and drug resistance. To identify new targets involved in drug resistance, we established a multidrug-resistant human breast cancer cell line MCF-7/R and examined the cytokine profile using cytokine antibody array technology. Among 120 cytokines/chemokines screened, IL-6, IL-8, and 13 other proteins were found to be markedly increased in drug-resistant MCF-7/R cell line as compared to sensitive MCF-7/S cell line, while 7 proteins were specifically reduced in drug-resistant MCF-7/R cells. Neutralizing antibodies against IL-6 and IL-8 partially reversed the drug resistance of MCF-7/R to paclitaxel and doxorubicin, while a neutralizing antibody against MCP-1 had no significant effect. Inhibition of endogenous IL-6 or IL-8 by siRNA technology significantly enhanced drug sensitivity of MCF-7/R cells. Furthermore, overexpression of IL-6 or IL-8 expression by transfection increased the ADM resistance in MCF-7/S cells. Our data suggest that increased expression levels of IL-6 and IL-8 may contribute to MDR in human breast cancer cells.

Detection of multiple cytokines by protein arrays from cell lysate and tissue lysate.

Abstract Previously we demonstrated that multiple cytokines could be simultaneously detected using an antibody-based protein array system with high sensitivity and specificity from conditioned medium and serum. Here, we created a higher density array system to simultaneously detect 35 cytokines from cell lysates and tissue lysates. This assay combines the advantages of the specificity of enzyme-linked immunosorbent assays (ELISA), sensitivity of enhanced chemiluminescence (ECL), and high-throughput of microspot. In this system, capture antibodies dissolved in methanol were spotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were then incubated with tissue lysates or cell lysates. After removing unbound proteins by extensive washing, the membranes were exposed to horseradish peroxidase (HRP)-conjugated antibody(ies). The signals were visualized with an ECL system. High specificity, sensitivity, and accuracy of this approach were demonstrated. This approach can be used in any general laboratory setting without any sophisticated equipment. It should be feasible to extend this concept to develop a high-throughput protein array system. Combining nitrocellulose membrane-based and PVDF membrane-based approaches, the human cytokine array system can be applied to detect multiple cytokine expression from cell lysate, tissue lysate, serum, plasma, and conditioned medium. Future applications of this new approach include direct protein expression profiling, immunological disease diagnostics, and discovery of new biomarkers.

Enhanced apoptosis under low serum conditions in human glioblastoma cells by connexin 43 (Cx43).

Connexin 43 (Cx43), a structural component of gap junctions, is believed to function as a tumor suppressor gene. Previously, we showed that expression of Cx43 suppresses cell proliferation and tumorigenicity of human glioblastoma cells [Huang et al., Cancer Res 58:5089–5096, 1998] and enhances apoptosis in response to chemotherapeutic agents [Huang et al., Int J Cancer 92:130–138, 2001]. In the present study, we demonstrated that expression of Cx43 in human glioblastoma cells potentiated an apoptotic program under low‐serum conditions. The Cx43‐mediated effect was coupled with a decreased expression of the specific apoptosis‐inhibitor bcl‐2. Overexpression of bcl‐2 in Cx43‐transfected cells conferred resistance to apoptosis induced under low‐serum conditions, suggesting that the Cx43‐mediated apoptosis under low‐serum conditions is regulated, in part, through the downregulation of bcl‐2 expression. Furthermore, application of the phosphatidylinositol‐3′‐OH kinase inhibitor LY294002 specifically induced apoptosis in Cx43‐transfected cells. Our results demonstrate a new role of Cx43 in the mediation of apoptosis under low serum conditions.

Enhanced Protein Profiling Arrays with ELISA-Based Amplification for High-Throughput Molecular Changes of Tumor Patients’ Plasma

Purpose: The purpose of this study is to develop a high-throughput approach to detect protein expression from hundreds and thousands of samples and to apply this technology to profile circulating angiogenic factor protein levels in patients with gynecological tumors. Experimental Design: Analytes containing a mixture of protein are immobilized onto antibody-coated surface of support in array format. The presence of protein in analytes is detected with biotin-labeled antibody coupled with an enhanced chemiluminescence or fluorescence detection system. The exact amount of protein can be quantitatively measured. The expression levels of five angiogenic factors (angiogenin, interleukin 8, vascular endothelial growth factor, platelet-derived growth factor, and epidermal growth factor) from 157 samples were quantitatively measured using this novel protein array technology and were statistically analyzed. The expression patterns of angiogenic factors were analyzed using two-way hierarchical cluster analysis approach. Results: A novel protein array technology, which can simultaneously and quantitatively measure few protein levels from hundreds and thousands of samples was developed. Only minute amounts of sample are required for the assay. This approach also features high sensitivity and specificity. Using this novel protein array approach, we analyzed the plasma expression levels of five angiogenic factors in 137 patients diagnosed with a tumor and 20 controls. Statistical analysis reveals different expression levels of angiogenic factors between patients and controls. Cluster analysis suggests a possible classification of normal subjects from patients. Conclusions: Enhanced protein profiling arrays provide a high-throughput and sensitive system to detect one or few protein from hundreds and thousands of samples. Such an approach should have broad application in biomedical discovery.

Profiling of human cytokines in healthy individuals with vitamin E supplementation by antibody array

Previously, we demonstrated that vitamin E supplementation decreases autoantibodies to oxidized lipid-protein complexes (J. Med. Food 1 (2000) 247). Utilizing an in vitro modeling system, we also demonstrated that vitamin E blocks the tumor promotion process in liver epithelial cells (Carcinogenesis 20 (1999) 485 and Mol. Carcinog. 30 (2001) 209). To investigate the molecular mechanisms of vitamin E function, we developed a human cytokine array system that is capable of detecting the expression of 35 cytokines simultaneously. Using this new technology, we analyzed the potential vitamin E-regulated cytokines in vitamin E supplementation individuals. The cytokine arrays showed that expression of several cytokines, particularly monocyte chemoattractant protein-1 (MCP-1), was profoundly reduced in vitamin E supplementation individuals. Moreover, addition of vitamin E to several cultured cells significantly down-regulated the expression of MCP-1. Our results suggested that MCP-1 may be one of the most important targets of antioxidant vitamin E. To the best of our knowledge, this is the first report describing the down-regulation of MCP-1 in vitamin E supplementation in vivo.

The promise of cytokine antibody arrays in the drug discovery process

The introduction of cytokine antibody arrays has added a new approach for investigators to simultaneously measure multiple cytokine levels in biological samples. Several different platforms have been developed. The ability to measure hundreds of cytokine levels with high specificity and sensitivity within a very limited amount of samples is a powerful tool. Many investigators worldwide have applied this novel technology in their biomedical research, particularly in drug discovery. Undoubtedly, the technology will continue to be improved and the application increased in the next several years.

Identification of five serum protein markers for detection of ovarian cancer by antibody arrays.

Background Protein and antibody arrays have emerged as a promising technology to study protein expression and protein function in a high-throughput manner. These arrays also represent a new opportunity to profile protein expression levels in cancer patients’ samples and to identify useful biosignatures for clinical diagnosis, disease classification, prediction, drug development and patient care. We applied antibody arrays to discover a panel of proteins which may serve as biomarkers to distinguish between patients with ovarian cancer and normal controls. Methodology/Principal Findings Using a case-control study design of 34 ovarian cancer patients and 53 age-matched healthy controls, we profiled the expression levels of 174 proteins using antibody array technology and determined the CA125 level using ELISA. The expression levels of those proteins were analyzed using 3 discriminant methods, including artificial neural network, classification tree and split-point score analysis. A panel of 5 serum protein markers (MSP-alpha, TIMP-4, PDGF-R alpha, and OPG and CA125) was identified, which could effectively detect ovarian cancer with high specificity (95%) and high sensitivity (100%), with AUC =0.98, while CA125 alone had an AUC of 0.87. Conclusions/Significance Our pilot study has shown the promising set of 5 serum markers for ovarian cancer detection.