MCT-1 oncogene contributes to increased in vivo tumorigenicity of MCF7 cells by promotion of angiogenesis and inhibition of apoptosis.

Overexpression of a novel oncogene MCT-1 (multiple copies in a T cell malignancy) causes malignant transformation of murine fibroblasts. To establish its role in the pathogenesis of breast cancer in humans, we generated stable transfectants of MCF7 breast cancer cells negative for endogenous MCT-1 (MCF7-MCT-1). Overexpression of MCT-1 in these cells resulted in a slight elevation of estrogen receptor-alpha, and higher rates of DNA synthesis and growth in response to estradiol compared with the empty vector control (MCF7-EV). The pure antiestrogen fulvestrant inhibited the estradiol-stimulated proliferation of MCF7-MCT-1 cells. The MCF7-MCT-1 clones showed increased invasiveness in the presence of 50% serum compared with the MCF7-EV. In a tumor xenograft model, MCT-1-overexpressing cells showed higher take rates and formed significantly larger tumors than MCF7-EV controls. When we examined angiogenic phenotype and molecular mediators of angiogenesis in MCF7-MCT-1 tumors in vivo, we found greater microvascular density and lower apoptosis in the MCF7-MCT-1 tumors compared with MCF7-EV controls accompanied by a dramatic decline in the levels of angiogenesis inhibitor, thrombospondin-1 (TSP1). In vitro, blocking TSP1 in the medium conditioned by MCT-1-negative cells restored its angiogenic potential to that of the MCF7-MCT-1 cells. Conversely, despite an increase in mRNA encoding vascular endothelial growth factor upon MCT-1 overexpression, vascular endothelial growth factor protein levels have not been notably altered. Taken together, our results suggest that MCT-1 may contribute to the pathogenesis and progression of human breast cancer via at least two routes: promotion of angiogenesis through the decline of TSP1 and inhibition of apoptosis.

Gene expression profiles with activation of the estrogen receptor alpha-selective estrogen receptor modulator complex in breast cancer cells expressing wild-type estrogen receptor.

Selective Estrogen Receptor Modulators (SERMs) are a new class of drugsthat bind to estrogen receptor (ER) and elicit agonistic or antagonistic responses, depending on the target tissue. We have developed an in vitro system in which some SERMs (4-hydroxytamoxifen and resveratrol) demonstrate estrogenic response through wild-type (wt) ER, whereas others (raloxifene and GW7604) remain antiestrogenic. This system mimics the tamoxifen-resistant phenotype in clinic, when resistant tumors contain wtER. We used Atlas cDNA arrays to study gene expression profiles after ER activation by different SERMs in MDA-MB-231 human breast cancer cells stably transfected with wtER. Cells were treated with estradiol, four different SERMs, and the pure antiestrogen ICI 182,780. The obtained expression data were analyzed using GeneSpring software. Real-time reverse transcription-PCR was used to verify the array data. Our results showed that treatment with various compounds altered the expression of a diverse group of genes, revealing sets of overlapping genes that may represent a complex network of genes of interrelated signal transduction pathways. Sets of "agonistic" and "antagonistic" genes were identified on the basis of the known response to different SERMs. Further analysis of selected sets of genes revealed functionally related group of genes in each set, encoding proteins that were related to cell proliferation, survival, and apoptosis. Flow cytometry data indicated an antiapoptotic activity in cells treated with agonists versus apoptotic activity in cells treated with antagonists. A model for estradiol-like (survival) and antiestrogen-like (apoptosis) activities of SERMs on the basis of their gene expression profiles is suggested.

Resveratrol acts as an estrogen receptor (ER) agonist in breast cancer cells stably transfected with ER alpha.

Resveratrol (Res) is a phytoestrogen found in grapes and present in red wine. Res has been shown to function as an estrogen receptor (ER) agonist, but it remains unclear whether it may also exert antagonist activity. Our aim was to study the effects of Res at both the molecular (TGFalpha gene activation) and the cellular (cell growth) levels in breast cancer cells stably transfected with wild-type (wt) ER(D351) and mutant (mut) ER (D351Y). TGFalpha mRNA induction was used as a specific marker of estradiol (E(2)) responsiveness. Res caused a concentration-dependent (10(-8)-10(-4) M) stimulation of TGFalpha mRNA, indicating that it acts as an estrogen agonist in these cell lines. The pure antiestrogen ICI 182,780 (ICI) blocked Res-induced activation of TGFalpha, consistent with action through an ER-mediated pathway. Further studies that combined treatments with E(2) and Res showed that Res does not act as an antagonist in the presence of various (10(-11)-10(-8) M) concentrations of E(2). To determine whether Res can be classified as a type I or type II estrogen (Jordan et al., Cancer Res 2001;61:6619-23,), we examined Res with the D351G ER in the TGFalpha assay and found that Res belongs to the type I estrogens. Both Res and E(2) had concentration-dependent growth inhibitory effects in cells expressing wtER and D351Y ER. Although the pure antiestrogen ICI blocked the growth inhibitory effects of E(2), it did not block the inhibitory effects of Res, suggesting that the antiproliferative effects of Res also involve ER-independent pathways. Interestingly, Res differentially affected the levels of ER protein in these 2 cell lines: Res down-regulated wtER levels while significantly up-regulating the amount of mutD351Y ER. Co-treatment with ICI resulted in strongly reduced ER levels in both cell lines. Gene array studies revealed Res-induced up-regulation of more than 80 genes, among them a profound activation of p21(CIP1)/WAF1, a gene associated with growth arrest. The p21(CIP1)/WAF1 protein levels measured by Western blotting confirmed Res-induced significant up-regulation of this protein in both cell lines. In summary, Res acts as an ER agonist at low doses but also activates ER-independent pathways, some of which inhibit cell growth.

Optical forces for noninvasive cellular analysis.

A novel, noninvasive measurement technique for quantitative cellular analysis is presented that utilizes the forces generated by an optical beam to evaluate the physical properties of live cells in suspension. In this analysis, a focused, near-infrared laser line with a high cross-sectional intensity gradient is rapidly scanned across a field of cells, and the interaction of those cells with the beam is monitored. The response of each cell to the laser depends on its size, structure, morphology, composition, and surface membrane properties; therefore, with this technique, cell populations of different type, treatment, or biological state can be compared. To demonstrate the utility of this cell analysis platform, we evaluated the early stages of apoptosis induced in the U937 cancer cell line by the drug camptothecin and compared the results with established reference assays. Measurements on our platform show detection of cellular changes earlier than either of the fluorescence-based Annexin V or caspase assays. Because no labeling or additional cell processing is required and because accurate assays can be performed with a small number of cells, this measurement technique may find suitable applications in cell research, medical diagnostics, and drug discovery.