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Hormonal Effect on Occludin Expression in the Endometrial Adenocarcinoma HEC-1A and HEC-1B cells. Morgan R. Gallo, Kelsey A. Rice, Taylor D. Vickers and Maria E. Cuevas Biology Department, Southwestern University Methods Objectives Results According to the American Cancer Society 1 , endometrial cancer is the most common female reproductive cancer in the United States with an incidence of 1 in 37 women. Alterations of tight junction (TJs) proteins have been reported in a number of human cancers. Occludin is one of several TJ proteins responsible for the proper structure and functions of TJs, including restriction of paracellular transport and maintenance of cell polarity 2 . While disruption of TJs has been associated with tumorigenesis, very few studies have investigated the role of occludin in the development and progression of endometrial cancer. In this study we evaluated the possible effects of estradiol and 4-hydroxytamoxifen (4- OHT) on occludin expression and the invasive capability of HEC-1A and HEC-1B adenocarcinoma cell lines. Results show that HEC-1A cells overexpressed two low molecular weight (46, 58 kDa) and the expected 65 kDa isoforms of occludin, whereas HEC-1B only expressed the 46 kDa isoform. After treatment with 0-100 nM estradiol (E 2 ), we observed a biphasic effect on occludin expression on both cell lines. In contrast, when cells were treated with 4-OHT a dose-dependant inhibition on occludin expression was observed. In addition, we observed a decrease on the invasive capability of HEC-1A and HEC-1B with increased E 2 concentration. Our data suggest that at low concentrations, E 2 promotes invasion of the HEC-1A and HEC-1B cells by increasing the expression levels of the two low molecular weight isoforms of occludin. Cell Lines and Tissue Culture Conditions: Cells were cultured in their respective medium (HEC-1A in McCoys 5A and HEC-1B in MEM) supplemented with 10% fetal bovine serum (FBS),1% penicillin and streptomycin and 2mM L-glutamine (PSG). Cells were maintained in a 5%CO 2 atmosphere at 37°C. Western Blot Analysis. Protein extracts were run on a pre-cast 10% SDS-PAGE gel for occludin protein analysis. Polyacrylamide gels were then transferred to Immobilon-P PVDF membranes. The membranes were probed for 1 h at RT with 1 μg/ml mouse-anti occludin primary antibody (Life Technologies) in 5% milk/PBS solution. Membranes were then probed at RT for 1 h with a 1:3000 dilution of goat-anti mouse HRP- conjugated secondary antibody (BioRad Laboratories). For signal detection the enhanced chemiluminescence (ECL) kit (Amersham) was used according to manufacturer’s instructions. The specific objectives of the present study were to determine: 1) Occludin expression levels in a panel of female cancer cell lines. 2) The possible effect of estradiol (E 2 ) and 4-hydroxytamoxifen (4-OHT) on occludin expression in the endometrial adenocarcinoma HEC-1A and HEC-1B cell lines. 3) The possible effect of E 2 and 4-OHT on the invasive capability of HEC-1A and HEC-1B. HEC-1A 46 Actin 46 58 65 kDa A. E 2 (nM) B. 4-OHT (nM) 0 10 50 100 FBS 0 10 50 100 FBS Actin 46 58 65 kDa Figure 1: Levels of occludin expression in a panel of female cancer cell lines. Extracts were prepared from log phase cultures of the following cell lines: human normal mammary epithelial (HMEC); breast (MCF-7, MDA-MB-231), cervical (HeLa); ovarian (SK-OV-3); and endometrial (RL95-2, HEC-1A and HEC-1B). Equal amounts of each protein extract were subjected to SDS-PAGE and transferred onto PVDF membrane. Following incubation with occludin antibody we observed strong signals corresponding to 65, 58 and 46 kDa in HEC-1A and MCF-7. Notably, HEC-1B only expressed the lower molecular weight isoform. Actin 46 Actin 0 10 50 100 FBS Figure 2: Biphasic effect of estradiol (A) and 4-OHT dose-dependent inhibition (B) on occludin expression in the HEC-1A cell line. Cells were plated (2 x 10 5 cells/well) onto 6- well plates and cultured in their respective media, supplemented with 10%FBS/1%PSG for 48 h. Prior to hormone exposure cells were washed twice with PBS, serum starved for 24 h followed by addition of media containing charcoal stripped fetal bovine serum (CSFBS) containing 0-100 nM E 2 or 4- OHT. Conc (nM) 65 kDa 58 kDa 46 kDa 58 /65 kDa 46/65 kDa 0 1.0 1.0 1.0 1.0 1.0 10 1.60 ± 1.0 2.8 ± 1.85 1.20 ± 0.14 1.70 0.70 50 0.98 ± 0.3 0.44 ± 0.18 0.80 ± 0.22 0.45 0.80 100 2.44 ± 1.9 1.40 ± 0.35 1.30 ± 0.9 0.60 0.54 FBS 13.6 ± 15.6 2.00 ± 0.23 0.70 ± 0.3 0.14 0.50 Table 1: Densitometric analysis of the biphasic E 2 on HEC-1A A. E 2 (nM) 46 Actin 0 10 50 100 FBS B. 4-OHT (nM) Figure 3: Biphasic effect of estradiol (A) and 4-OHT dose-dependent (B) expression in the HEC-1B cell line. The experiment was conducted as described in figure 2. Table 3: Densitometric analysis the effects of E 2 and 4-OHT on HEC-1B Conc (nM) 46 kDa 0 1.0 10 2.0 ± 0.6 50 1.54 ± 0.7 100 1.63 ± 0.5 FBS 2.4 ± 1.5 Table 2: Densitometric analysis of 4-OHT effect on HEC-1A Conc (nM) E 2 4-OHT 46 kDa 0 1.0 1.0 10 1.0 1.0 50 0.96 0.80 100 1.190 0.74 FBS 1.39 1.1 MAIN RESULTS: HEC-1A Experiment: We observed an increase in the lower molecular weight (58 and 46 kDa) occludin isoforms at 10 nM E 2 . The same concentration also increased the ratio of 58/65 kDa by 11-fold and the 46/65 kDa by 14-fold. MAIN RESULTS: Notably, we only observed the 46 kDa isoform of occludin. However, we again observed the estrogen biphasic and 4-OHT dose-dependent inhibition of occludin expression. kDa 0 10 50 100 Estradiol concentration (nM) ND 0.1 0.2 0.3 0.4 0.5 Absorbance (560 nm) HEC-1A HEC-1B Figure 4: E 2 dose-dependent inhibition on HEC-1A and HEC-1B invasion capabilities. Invasive potential was tested using a Boyden Chamber with 8 μm pore size polycarbonate membrane coated with ECMatrix (EMD, Inc.). Cells were serum starved for 48 h in phenol free medium prior to plating. Cell suspension containing 3 x 10 5 cells/300 μl was loaded on the upper chamber and FBS containing medium was added to the lower chamber as the chemoattractant. E 2 and 4-OHT (0-100 nM) were diluted in medium before plating. Cells were incubated for 24 h at 37 o C in 5%CO 2 atmosphere. Inserts were removed and the cells on the lower chamber were stained and collected following the manufacturer’s instructions. MAIN RESULTS: We observed a dose-dependent inhibition on the invasive capabilities in both cell lines . At 10 nM E 2 concentration, HEC-1B appeared to exhibit the highest invasive potential. Furthermore, the inhibitory effect seems to be more pronounced in the HEC-1B cell line. Abstract Western blots were quantified by computer-aided determination of OD using ImageJ (NIH) and normalized to actin. Means (± SD) of three experiments in each point of changes in densitometry of occludin 65, 58 and 46 kDa isoforms in response to treatments with E 2 . Means (± SD) of two experiments in each point of changes in densitometry of occludin 46 kDa isoform in response to 4- OHT. References 1. American Cancer Society (online). Available: http://www.cancer.org/cancer/endometrialcancer/detailedguide/endometrial-uterine- cancer-key-statistics (last updated 02/03/2014) 2. Schneeberger EE and Lynch RD. The tight junction: a multifunctional complex. Am J Physiol Cell Phsysiol 2004; 286:1213-1228. Acknowledgements We would like to thank the Howard Hughes Medical Institute Grant (HHMI) and Southwestern University for their support of the SCOPE program.
