Microarray Analysis Reveals Overexpression of both Integral Membrane and Cytosolic Tight Junction Genes in Endometrial Cancer Cell Lines.

Deregulation of tight junction (TJ) proteins and the associated disruption of TJ function has been demonstrated to play a role in the development of endometrial cancer. In the current study, we have shown overexpression of claudin-3 and -4 mRNA (by RT-PCR) and protein (by immunoblotting) in a panel of 9 human endometrial cancer cell lines. To further expand our understanding of the complex role of TJ deregulation in endometrial cancer, we also investigated the expression of 84 TJ and TJ-associated genes (encoding the array of proteins that function within the TJ network from the membrane to nuclear signaling pathways) by microarray analysis. Consistent with the claudin-3 and -4 RT-PCR and immunoblot findings described above, we observed overexpression of the claudin-3 and -4 genes by microarray analysis. Further, we observed overexpression of an additional three genes in 8 of the 9 endometrial cancer cell lines: OCLN (occludin), F11R (JAM-A) and TJP3 (ZO-3). OCLN and F11R encode integral membrane proteins whereas TJP3 encodes a cytosolic scaffolding protein that indirectly links membrane TJ proteins to the actin cytoskeleton and cell signaling pathways. Our data suggest that the structural disruption of TJs coupled with the downstream deregulation of signaling pathways involved in cellular proliferation and migration may contribute to the development of endometrial cancer.

Comparison of transepithelial resistance measurement techniques: Chopsticks vs. Endohm.

BACKGROUND: TER measurements across confluent cellular monolayers provide a useful indication of TJ strength between epithelial and endothelial cells in culture. Having a reliable and accurate method of measuring cell-to-cell adhesion is critical to studies in pathophysiology and cancer metastasis. However, the use of different technical approaches to measure TER has reportedly yielded inconsistent measurements within the same cell lines. METHODS: In the current study, we compared the peak TER values for the MDCK (canine kidney) and MCF-7 (human breast cancer) epithelial cell lines using two common approaches (Chopstick and Endohm) and two types of polymer inserts (PC and PET). RESULTS: Both cell lines demonstrated a statistically significant difference in the peak TERs obtained using the two different approaches. Further, the MDCK (but not the MCF-7) cells demonstrated a statistically significant difference between the peak TERs when using the same approach but different inserts. CONCLUSION: Our study indicates the importance of using a single approach when seeking to measure and compare the TER values of cultured cell lines.

Overexpression and delocalization of claudin-3 protein in MCF-7 and MDA-MB-415 breast cancer cell lines.

Tumor-specific deregulated expression of claudins, integral membrane proteins found in tight junctions (TJs), has indicated a possible role for TJ disruption in cancer progression. The current study demonstrates the marked overexpression of claudin-3 protein in two breast cancer cell lines of metastatic origin (MCF-7 and MDA-MB-415). Immunofluorescence and differential detergent fractionation analyses revealed that, although claudin-3 was primarily localized at cell junctions, it was also detected intracellularly. Similarly, the siRNA-mediated suppression of claudin-3 did not considerably affect its pattern of subcellular distribution relative to mock-transfected cells. However, there appeared to be a preferential loss of claudin-3 signal in the cytoskeletal fraction. Wound-healing assays were conducted to assess the effect of endogenous overexpression versus siRNA-mediated suppression of claudin-3 on cellular motility in MCF-7 cells. Suppression of claudin-3 protein levels resulted in a marked decrease in the rate of cellular motility relative to mock-transfected cells. These findings suggest that overexpression of claudin-3 may be important in disrupting TJ integrity and thus contribute to enhanced cellular motility, a key component of tumor progression.

Estrogen-dependent expression and subcellular localization of the tight junction protein claudin-4 in HEC-1A endometrial cancer cells.

Endometrial cancer is the most common female reproductive cancer in the United States and is associated with deregulated tight junction protein expression. Given the highly estrogen-responsive nature of this tissue, we investigated the effects of estrogen and its agonist, 4-OH TAM, on the expression and subcellular localization of the tight junction protein claudin-4 (CLDN-4), in HEC-1A endometrial cancer cells. In untreated HEC-1A cells, we observed dramatic overexpression of claudin-4 protein. In addition, differential detergent extraction analysis indicated that claudin-4 was localized primarily in the membrane but also found in the cytosolic, nuclear and cytoskeletal fractions. Upon exposure of HEC-1A to estradiol (E2), we observed a biphasic effect both on the overall expression of claudin-4 protein and on its cytosolic and cytoskeletal presence as demonstrated by immunoblot analysis. Immunofluorescence analysis also revealed a biphasic effect of E2 on claudin-4 expression. In contrast, we observed no changes in expression levels nor in the subcellular distribution patterns of claudin-4 in HEC-1A cells treated with different concentrations of 4-OH TAM. The intracellular presence of CLDN-4 coupled with the biphasic effects of E2 on CLDN-4 expression in the cytoskeleton suggest that this protein may be involved in cell signaling to and from TJs.

In vitro cytotoxicity of 4'-OH-tamoxifen and estradiol in human endometrial adenocarcinoma cells HEC-1A and HEC-1B.

Tamoxifen (TAM), used to treat estrogen receptor (ER)-positive breast cancer, is a well known estrogen antagonist in the breast, but a partial estrogen agonist in the endometrium. In addition, TAM metabolites, such as 4'-hydroxy-tamoxifen (4-OH-TAM), have been shown to be more potent than the parent compound. The objective of this study was to determine the effects of 4-OH-TAM and estradiol (E2) on two human endometrial adenocarcinoma cell lines, HEC-1B and HEC-1A. When HEC-1B cells were treated with lower concentrations (10-1,000 nM) of 4-OH-TAM or E2 for 1-3 days, no significant difference in the percentage of cell survival was observed among the varying concentrations. At higher 4-OH-TAM or E2 concentrations (1-100 µM), HEC-1B and HEC-1A cells responded similarly with an obvious decrease in cell growth noted at 10 and 100 µM 4-OH-TAM and 100 µM E2. In order to address the observed cell death, DNA laddering was performed at various time intervals with 4-OH-TAM (10 µM) or E2 (10 or 50 µM). DNA gel electrophoresis failed to show the typical laddering pattern (180-200 bp) observed in apoptosis. Furthermore, western blot analysis of caspase-8 and -3 failed to demonstrate caspase activity. These results suggest that apoptosis was not the underlying cellular mechanism of cell death. Due to the lack of apoptotic markers, a cytotoxic (cell death) effect was differentiated from a cytostatic (growth inhibition) effect confirming that cell death had occurred. In summary, micromolar concentrations of 4-OH-TAM induced a non-apoptotic cytotoxic effect in the endometrium; however further studies are needed to elucidate the cytotoxic pathway being utilized.

In vitro cytotoxic activity of anthrapyrazole analogues in human prostate DU-145 and testicular NTERA-2 carcinoma cells.

Anthrapyrazoles are potent cytotoxic agents that intercalate into DNA, causing DNA strand breaks, inhibition of DNA synthesis and topoisomerase II. In this study, we investigated the in vitro cytotoxic activity of two anthrapyrazole analogues (AP-10 and AP-11) in human prostate (DU-145) and testicular (NTERA-2) carcinoma cells. The cytotoxic activity of these analogues was determined using the MTS cell growth inhibition assay. The IC50 of AP-10 on NTERA-2 and DU-145 cells was found to be 0.2 and 0.4 microM, respectively. AP-11 inhibited cell growth with an IC50 value of 1.2 microM (NTERA-2) and 3.2 microM (DU-145). Using trypan blue dye exclusion assay, we were able to confirm the cytotoxic effect of AP-10 and AP-11 on DU-145 cells, thereby distinguishing it from the cytostatic effect. To determine whether cells were able to recover after exposure to the anthrapyrazole analogues, DU-145 and NTERA-2 cells were exposed to the IC50 concentration of AP-10 and AP-11. After a 1-h exposure, fresh media containing either testosterone or dihydrotestosterone were added daily for five days and the cell growth rate was compared to the control. Although cells exposed to AP-10 and AP-11 were able to recover, they never attained the growth rate observed in the control cultures. The DNA fragmentation assay did not provide evidence of apoptosis. In conclusion, our results demonstrated that AP-10 had a higher cytotoxic activity than AP-11, and apoptosis appeared not to be involved in the biological activity of these compounds.