Cynthia Cunningham

Antitumor effect of β-elemene in non-small-cell lung cancer cells is mediated via induction of cell cycle arrest and apoptotic cell death

Abstract.β-Elemene is a novel anticancer drug, which was extracted from the ginger plant. However, the mechanism of action of β-elemene in non-small-cell lung cancer (NSCLC) remains unknown. Here we show that β-elemene had differential inhibitory effects on cell growth between NSCLC cell lines and lung fibroblast and bronchial epithelial cell lines. In addition, β-elemene was found to arrest NSCLC cells at G2-M phase, the arrest being accompanied by decreases in the levels of cyclin B1 and phospho-Cdc2 (Thr-161) and increases in the levels of p27kip1 and phospho-Cdc2 (Tyr-15). Moreover, β-elemene reduced the expression of Cdc25C, which dephosphorylates/activates Cdc2, but enhanced the expression of the checkpoint kinase, Chk2, which phosphorylates/ inactivates Cdc25C. These findings suggest that the effect of β-elemene on G2-M arrest in NSCLC cells is mediated partly by a Chk2-dependent mechanism. We also demonstrate that β-elemene triggered apoptosis in NSCLC cells. Our results clearly show that β-elemene induced caspase-3, −7 and −9 activities, decreased Bcl-2 expression, caused cytochrome c release and increased the levels of cleaved caspase-9 and poly(ADP-ribose) polymerase in NSCLC cells. These data indicate that the effect of β-elemene on lung cancer cell death may be through a mitochondrial release of the cytochrome c-mediated apoptotic pathway.

Antiproliferative effect of β-elemene in chemoresistant ovarian carcinoma cells is mediated through arrest of the cell cycle at the G2-M phase

Abstract.Elemene is a natural antitumor plant drug. However, the effect of elemene on cell growth in ovarian cancer is unknown. In this study, we show that β-elemene inhibited the proliferation of cisplatin-resistant human ovarian cancer cells and their parental cells, but had only a marginal effect in human ovary cells, indicating differential inhibitory effects on cell growth between ovarian cancer cells and normal ovary cells. We also demonstrated for the first time that β-elemene markedly enhanced cisplatin-induced growth inhibition in resistant cells compared to sensitive cells. In addition, cell cycle analysis revealed a synergistic effect of β-elemene and cisplatin on the induction of cell cycle G2-M arrest in our resistant ovarian carcinoma cells. Furthermore, we showed that treatment of these cells with both drugs downregulated cyclin B1 and Cdc2 expression, but elevated the levels of p53, p21waf1/cip1, p27kip1 and Gadd45. Finally, the combination of β-elemene and cisplatin was found to increase the phosphorylation of Cdc2 and Cdc25C, which leads to a reduction in Cdc2-cyclin B1 activity. These novel findings suggest that β-elemene sensitizes chemoresistant ovarian carcinoma cells to cisplatin-induced growth suppression partly through modulating the cell cycle G2 checkpoint and inducing cell cycle G2-M arrest, which lead to blockade of cell cycle progression.

Influence of the Route of Infection on Development of T-Cell Receptor β-Chain Repertoires of Reovirus-Specific Cytotoxic T Lymphocytes

ABSTRACT It is well established that the route of infection affects the nature of the adaptive immune response. However, little is known about the effects of the route of exposure on development of cytotoxic T-lymphocyte (CTL) responses. Alternative antigen-presenting cell populations, tissue-restricted expression of class I major histocompatibility complex-encoded molecules, and unique T-cell receptor (TCR)-bearing cells in mucosal tissues could influence the selection and expansion of responder T cells. This study addresses the question of whether the route of virus infection affects the selection and expansion of subpopulations of virus-specific CTLs. Mice were infected orally or in the hind footpads with reovirus, and the repertoires of TCR β-chains expressed on virus-specific CD8+ T cells in Peyers patches or lymph nodes and spleens were examined. CD8+ cells expressing the variable gene segment of the TCR β-chain 6 (Vβ6) expanded in the spleens of mice infected by either route and in CTL lines established from the spleens and draining lymphoid tissues. Adoptively transferred Vβ6+ CD8+ T cells from orally or parenterally infected donors expanded in reovirus-infected severe combined immunodeficient recipient mice and mediated cytotoxicity ex vivo. Furthermore, recovered Vβ6+ cells were enriched for clones utilizing uniform complementarity-determining region 3 (CDR3) lengths. However, sequencing of CDR3β regions from Vβ6+ CD8+ cells indicated that Jβ gene segment usage is significantly more restricted in CTLs from orally infected mice, suggesting that the route of infection affects selection and/or subsequent expansion of virus-specific CTLs.

MAPK and PI3K kinase inhibition reduces proliferation of Barrett’s adenocarcinoma in vitro

Objectives. Esophageal adenocarcinoma usually arises from Barrett’s esophagus. Mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) play a critical role in cell survival. We hypothesized that their inhibition in Barrett’s cancer cells would decrease cellular proliferation and alter apoptosis in vitro.Methods. Two Barrett’s-associated adenocarcinoma cell lines, SEG-1 (wild type p53) and BIC-1 (mutant p53), were treated with MAPK (U0126) and PI3K (LY294002) inhibitors at 20 μM concentrations. After 24 and 72 h, cell viability was measured by MTT assay. Apoptosis and necrosis was evaluated by the Annexin V FITC assay. Statistical analysis was performed by ANOVA. Results. LY 294002 and U0126 treatment produced significant reductions (range 15.7 to 62.0%, P < 0.001) in cellular proliferation at both 24 and 72 h in the SEG-1 cells. BIC-1 cell viability was reduced (39.3 to 56.4%, P < 0.001) at 72 h. Both early and late apoptotic activity was significantly increased (P < 0.05) in the SEG-1 cells. Necrosis was significantly reduced (P < 0.01) using both inhibitors. No changes in either early or late apoptosis or necrosis were observed in the BIC-1 cells. Conclusions. Herein, we report the first demonstration of significant anti-proliferative effects against Barrett’s adenocarcinoma by MAPK and PI3 kinase inhibition in vitro. Pro-apoptotic mechanisms prevail in the wild-type p53 cells. Further investigation is warranted to advance the clinical treatment of this devastating disease.

Immunology of the Gastrointestinal Tract

The gastrointestinal system is essentially a long muscular tube, the functional surface of which is a thin, mucus-coated layer approx 1 mm thick, that is joined at both ends with the external integument and, thus, is a contact surface with the external environment (1). The surface area of the adult human intestine is estimated to be approx 300 M2 (2). This surface is constantly exposed to antigens, which, proximally, is mostly of dietary origin and, distally, tends to be bacterial products derived from colonic flora. Providing a protective barrier at this external surface is complicated by the need to selectively absorb nutrients. To prevent the colonization and/or invasion of the intestinal mucosa by foreign organisms, the intestine makes use of a number of innate and adaptive defense factors. This chapter provides a broad overview of immune responses in the intestine.