Ya Guang Liu

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.

Induction of Aromatase Expression in Cervical Carcinomas: Effects of Endogenous Estrogen on Cervical Cancer Cell Proliferation

Epidemiologic studies have implicated estrogenic exposure as well as human papilloma virus (HPV) infection in cervical carcinogenesis, and some studies have suggested that estrogen and HPV may play synergistic roles in cervical tumorigenesis. In this study, we report a novel finding that approximately 35% of cervical carcinomas tested (n = 19) express aromatase, the enzyme responsible for converting androgen to estrogen, the rate-limiting and final step in estrogen biosynthesis. On the other hand, no aromatase expression was detected in precancerous (n = 42) or normal cervical (n = 17) tissue samples. Increased aromatase was associated with increases in estrogen receptors (ER-alpha and ER-beta) and a decrease in progesterone receptor levels, suggesting that in situ estrogen signaling via ER may be involved in tumor growth. Stable overexpression of aromatase in HPV+ cervical cancer cells resulted in increased cellular proliferation, anchorage-independent growth, and ER expression and activity. In contrast, little change in ER was observed in HPV- cells. Steroid hormone receptor expression observed in vitro paralleled that seen in cervical carcinomas expressing aromatase. Aromatase overexpression also induced the expression of cyclin D1, proliferating cell nuclear antigen, and the HPV oncogenes, E6 and E7. Furthermore, the data underscores the importance of steroid receptor (estrogen and progesterone receptors) regulation in cervical carcinogenesis. To our knowledge, this is the first report demonstrating the induction of aromatase expression in cervical carcinomas, and opens the possibility that aromatase inhibitors may be potential therapeutic agents in cervical carcinomas expressing aromatase.

Elevated Expression of the Oncogene c-fms and Its Ligand, the Macrophage Colony-Stimulating Factor-1, in Cervical Cancer and the Role of Transforming Growth Factor-β1 in Inducing c-fms Expression

Cervical cancer is the third most common gynecologic cancer in the United States. The presence and possible involvement of several cytokines have been studied in cervical cancer; however, very little data, if any, are available on whether cervical tumors are responsive to stimulation by the macrophage colony-stimulating factor-1 (CSF-1). Given the involvement of c-fms and its ligand CSF-1 in gynecologic cancers, such as that of the uterus and the ovaries, we have examined the expression of c-fms and CSF-1 in cervical tumor (n = 17) and normal cervix (n = 8) samples. The data show that c-fms and its ligand are significantly higher in cervical carcinomas compared with normal samples. Immunohistochemistry not only showed that tumor cells expressed significantly higher levels of c-fms but also c-fms levels were markedly higher in tumor cells than tumor-associated stromal cells. Blocking c-fms activity in cervical cancer cells, which express CSF-1 and c-fms, resulted in increased apoptosis and decreased motility compared with control, suggesting that CSF-1/c-fms signaling may be involved in enhanced survival and possibly invasion by cervical cancer cells via an autocrine mechanism. Combined, the data show for the first time the induction of CSF-1 and c-fms in cervical carcinomas and suggest that c-fms activation may play a role in cervical carcinogenesis. Additionally, our data suggest that transforming growth factor-beta1 may be a factor in inducing the expression of c-fms in cervical cancer cells. The data suggest that c-fms may be a valuable therapeutic target in cervical cancer.

Overexpression of the Colony-Stimulating Factor (CSF-1) and/or Its Receptor c-fms in Mammary Glands of Transgenic Mice Results in Hyperplasia and Tumor Formation

A number of recent studies have suggested that the colony-stimulating factor (CSF-1) and its receptor c-fms may be involved in the development of mammary glands during lactation and breast cancer. To study the role of CSF-1 or its receptor in initiation of mammary tumorigenesis, we have generated two independent lines of transgenic mice that overexpress either CSF-1 or c-fms under the control of the mouse mammary tumor virus promoter. Mammary glands of the virgin CSF-1 transgenic mice show increased ductal branching, hyperplasia, dysplasia, and other preneoplastic changes, which are indicative of increased cellular proliferation. Similar changes were also evident in the mammary glands of the c-fms transgenic mice. These changes became more prominent with age and resulted in mammary tumor formation. Moreover, secondary events like dimethylbenz(a)anthracene treatment accelerated mammary tumor formation in these mice. Although the expression of estrogen receptor α was not significantly changed in either of the transgenic mouse strains, progesterone receptor levels was higher in both transgenic lines as compared with the nontransgenic littermates. Expression of G1 cyclins was prominently increased in the mammary glands of both the CSF-1 and c-fms transgenic lines, suggesting increased cell cycle progression in these strains. In addition, the proliferation marker proliferating cell nuclear antigen (PCNA) and the mitogen-responsive transcription factor c-jun were also increased as compared with the nontransgenic controls. These findings, along with the histological data, support the hypothesis that CSF-1 and its receptor are involved in the etiology of breast cancer.

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.

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.

Cytotoxic Activities, Cellular Uptake, Gene Regulation, and Optical Imaging of Novel Platinum(II) Complexes

A new class of platinum(II) coordination complexes and their dye tagged conjugates has been synthesized from N-substituted diaminocyclohexane ligands. The in vitro anticancer activities of the platinum compounds have been validated against the breast cancer cell-line MCF-7 and the normal cell-line MCF-10A via sulforhodamine B and colony formation assay. The platinum compounds and the corresponding metal-free ligands exhibited higher drug efficiencies than cisplatin and oxaliplatin against MCF-7 cells. Cellular uptake and DNA-bound Pt were demonstrated by atomic absorption spectroscopy. The platinum complexes displayed increased cellular accumulation and DNA binding as compared with cisplatin. Real-time reverse transcription polymerase chain reaction assay was employed to investigate drug effects on mRNA expression in MCF-7 cells. The results indicated that the study compounds are effective in regulating cyclin D1, Bcl-2, and p53 genes; yet, oxaliplatin is less effective in manipulating those genes. The luminescent probe that was integrated into the platinum complexes made it possible to monitor cellular drug distribution using optical imaging. Targeting of tumor cell nuclei by the study compounds was confirmed by confocal microscopy. Taken together, these new platinum(II)-based antitumor agents are different from marketed platinum drugs in several critical aspects and could have potential in cancer therapy.

Estrogen receptor alpha is required for mammary development and the induction of mammary hyperplasia and epigenetic alterations in the aromatase transgenic mice.

Aromatase transgenic mice exhibit hyperplastic and dysplastic changes, attesting to the importance of local estrogen in breast carcinogenesis. These mice also show increased levels of the estrogen receptor alpha and beta (ERalpha, ERbeta) suggesting that this receptor may play an important role in the initiation of estrogen-mediated mammary hyperplasia observed in these mice. To address the specific role of ERalpha in the mammary development and in the induction of estrogen-mediated hyperplasia in aromatase transgenic mice, we have generated MMTV-aromatase x ERalpha knockout cross (referred as aromatase/ERKO). Even though ERbeta is expressed in aromatase/ERKO mice, lack of ERalpha leads to impaired mammary growth in these mice. The data suggest that ERalpha plays an important role in the mammary gland development as well as in the induction of mammary hyperplasia in aromatase transgenic mice. Lack of ERalpha expression in the aromatase/ERKO mice resulted in a decrease in the expression of Cyclin D1, PCNA and TGFbeta relative to the aromatase parental strain. The studies involving aromatase/ERKO mice show that lack of ERalpha results in impaired mammary development even in the presence of continuous tissue estrogen, suggesting estrogen/ERalpha-mediated actions are critical for mammary development and carcinogenesis.

Chiral salicyl diamines: potent anticancer molecules.

A set of 12 enantiomeric diamine‐based small molecules was synthesized and screened for anticancer activity against five human cancer cell lines: NCI‐H460, A549, MCF‐7, SK‐BR‐3, and T‐47D. The salicyl diamino compounds (1–6) were found to induce inhibition of the growth of cancer cells at submicromolar concentrations. The lead compound, N,N′‐bis‐salicyl‐(1R,2R)‐diaminocyclohexane (1) displayed single‐reagent anticancer activity with an IC50 value equal to or less than 2.0 μM in H460 and A‐549 cancer cells. SRB and colony formation assays indicated that compound 1 shows greater cytotoxic activity toward MCF‐7 cells than MCF‐10A cells. Real‐time RT‐PCR analysis demonstrated that compound 1, is an extremely efficient regulator of antiapoptotic genes, Bcl‐xL, Bcl‐2, and the cell cycle related gene, cyclin D1. This study provides a new insight into the development of novel small molecules in the treatment of human breast cancers.

Artificial zinc(II) complexes regulate cell cycle and apoptosis-related genes in tumor cell lines

Various proteins involved in transcriptional regulation possess highly selective DNA‐binding domains, known as zinc fingers. However, little is known about small‐molecule zinc(II) complexes in the regulation of gene expression and programmed cell death. A new family of zinc(II) complexes is reported, which might be useful against human cancer cells. By using template synthesis and in vitro cell‐line screening, a set of zinc(II) complexes has been found to induce apoptosis of cancer cells and display single‐reagent in vitro cytotoxicity. The method used to synthesize the molecules resulted in “built‐in” luminescent behavior. Confocal optical imaging clearly demonstrated penetration through the cell membrane by these metal complexes. We have discovered that C3, the meso‐zinc(II) complex is an extremely efficient regulator of the cell cycle and anti‐apoptosis genes bcl‐2 and bcl‐xL. This study provides a new insight into the development of zinc(II) complexes as potential drugs.