Ruo-Pan Huang

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.

Tumor-induced perturbations of cytokines and immune cell networks.

Until recently, the intrinsically high level of cross-talk between immune cells, the complexity of immune cell development, and the pleiotropic nature of cytokine signaling have hampered progress in understanding the mechanisms of immunosuppression by which tumor cells circumvent native and adaptive immune responses. One technology that has helped to shed light on this complex signaling network is the cytokine antibody array, which facilitates simultaneous screening of dozens to hundreds of secreted signal proteins in complex biological samples. The combined applications of traditional methods of molecular and cell biology with the high-content, high-throughput screening capabilities of cytokine antibody arrays and other multiplexed immunoassays have revealed a complex mechanism that involves multiple cytokine signals contributed not just by tumor cells but by stromal cells and a wide spectrum of immune cell types. This review will summarize the interactions among cancerous and immune cell types, as well as the key cytokine signals that are required for tumors to survive immunoediting in a dormant state or to grow and spread by escaping it. Additionally, it will present examples of how probing secreted cell-cell signal networks in the tumor microenvironment (TME) with cytokine screens have contributed to our current understanding of these processes and discuss the implications of this understanding to antitumor therapies.

Reduced Connexin43 expression in high-grade human brain glioma cells

Connexin43 (cx43), a gap junction protein, is implicated in the suppression of tumor cell growth. Numerous cancer cells show a reduction or loss of cx43 expression compared to their normal counterparts. Our previous studies suggest that cx43 expression is decreased in a variety of human brain tumor cell lines. To further investigate the role of cx43 in the development of human gliomas, we performed the present study on human glioma grades I–IV.

p53 and Egr-1 additively suppress transformed growth in HT1080 cells but Egr-1 counteracts p53-dependent apoptosis

The human fibrosarcoma cell line, HT1080, clone H4, was used to determine if the transformation suppressive functions of p53 and Egr-1 have the same underlying mechanism. This cell line expresses only mutant p53 and no detectable Egr-1. H4 clones stably expressing Egr-1 are less transformed in proportion to the level of Egr-1 expressed, acting through the induction of the TGFβ1 gene. Here, H4 cells and the highest Egr-1 expressing clone were transfected with a vector expressing normal human p53 to derive stable clones expressing p53. The expression of p53 in H4 cells inhibited transformed growth and reduced tumorigenicity. The effect of co-expression of both p53 and Egr-1 was additive, producing cell lines with 30% of normal growth rate and sevenfold reduced tumorigenicity compared with control lines. These results indicated that each factor may act independently by different pathways, although each additively increased the level of p21WAF1 cell cycle inhibitor. However, exposure of the H4-derived cells to UV-C irradiation produced contrasting effects. Cell cycle analyses showed that the presence of p53 was associated with loss of the G1 and S cells to apoptosis after irradiation. In contrast, the expression of Egr-1 increased entry into S/G2 phase of the cell cycle with little apoptosis via a mechanism involving elevated FAK and low caspase activities. Apoptosis was observed only in the cell lines that expressed no Egr-1, especially those expressing wt-p53, and was preceded by high caspase activity. In summary, Egr-1 suppressed transformation and counteracted apoptosis by the coordinated activation of TGFβ1, FN, p21 and FAK, leading to enhanced cell attachment and reduced caspase activity. In the doubly expressing cell line, the survival effect of Egr-1 was dominant over the apoptotic effect of p53.

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.

Suppression of human fibrosarcoma cell growth by transcription factor, Egr-1, involves down-regulation of Bcl-2

Previously, we showed that the transcription factor Egr‐1 suppressed the proliferation of v‐sis transformed NIH3T3 cells and also a number of human tumor cells. Here, we investigate the possible mechanisms responsible for this function. We show that transfected Egr‐1 in human fibrosarcoma cells HT1080 leads to down‐regulation of Bcl‐2. Transient CAT transfection assays reveal that expression of Egr‐1 suppresses Bcl‐2 promoter activity in a dose‐dependent manner. Furthermore, overexpression of Bcl‐2 in Egr‐1‐expressing HT1080 cells enhanced cell proliferation in monolayer culture and increased anchorage‐independent growth. Our results suggest that suppression of tumor cell proliferation by Egr‐1 may be at least partially mediated through the down‐regulation of Bcl‐2. Int. J. Cancer 77:880‐886, 1998.© 1998 Wiley‐Liss, Inc.

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.