Effects of 4-OH Tamoxifen on HEC 1B Endometrial Cancer Cells

1. Effects of 4-OH Tamoxifen on HEC 1B Endometrial Cancer Cells Tracey Einem, Carolina Boet, Dr. Maria Cuevas1, Dr. Maha Zewail-Foote2 1Biology Department, 2 Chemistry Department, Southwestern University, 1001 E. University Ave. Georgetown, TX 78626 einemt@southwestern.edu, boetc@southwestern.edu, cuevasm@southwestern.edu, footezm@southwestern.edu Western Blot Analysis Cell protein extracts were prepared from HEC 1B cells treated with 0, 1μM, and 10μM 4-OH TAM for 24 hours at the incubating conditions previously described. Western blot analysis was performed using standard procedure. ABSTRACT Figure 3: DNA Gel Electrophoresis of extracts from ER positive HEC 1A (lanes 2-4) and ER negative HEC 1B (lanes 9-11) endometrial adenocarcinoma cells treated with 10μM 4-OH TAM and estrogen. Characteristic DNA fragmentation (180-200bp) was not seen following DNA gel electrophoresis in all samples. This suggests apoptosis did not occur in either cell line. Tamoxifen, a well-known drug for breast cancer treatment, has been found to be an estrogen antagonist in the breast, but a partial estrogen agonist in the endometrium. In addition, tamoxifen metabolites have been shown to produce DNA adducts in a variety of tissues. In this study, we investigated the potential of 4-OH tamoxifen (4-OH TAM) to exert proliferative effects via an ER independent pathway. For this purpose, we used the ER negative human endometrial adenocarcinoma cell line, HEC 1B, and compared results with the ER positive human endometrial adenocarcinoma cell line, HEC 1A. We treated ER negative HEC 1B cells with different estrogen and 4-OH TAM concentrations ranging from 0-100M and incubated at 37ºC, 5% CO2 atmosphere. Using a dye-exclusion assay and colorimetric method (MTS Assay) we observed that lower concentrations of 4-OH TAM had little effect on HEC 1B cell proliferation. However, at higher concentrations (10,100M), cell proliferation was inhibited by almost 100% within 24 hours. When HEC 1B cells were treated with different doses of estrogen, we observed an initial proliferative response after 24 hours at low doses, followed by a partial inhibition of growth after 2 to 3 days. However, at the highest dose (100M), we observed a complete inhibition of cell proliferation. In contrast, ER positive HEC 1A cells were refractory to low dose estrogen but, like HEC1B cells, were completely growth inhibited by 100M estrogen. When ER positive HEC 1A cells were treated with higher concentrations of 4-OH TAM, no inhibition was observed at 1M whereas at 10M, a steady decline in cell growth was found. Similar to HEC 1B cells, complete inhibition was observed at 100M within 24 hours. In order to determine if apoptosis is the underlying mechanism of cell death, we incubated HEC 1B cells with 10M 4-OH TAM for 24 hours and assayed for DNA laddering and expression of the pro-apoptotic protein, caspase 8. Preliminary data did not indicate involvement of an apoptotic pathway. These results suggest that 4-OH TAM is promoting cell death via necrosis. RESULTS Figure 4: Western Blot analysis of caspase 8 from 4-OH TAM-treated HEC 1B (ER negative) endometrial adenocarcinoma cells. Protein extracts and western blot analysis were conducted as described in Materials and Methods. 10μL of protein extract from HEC 1B cells treated with 1μM or 10μM 4-OH TAM were fractioned on a 12% polyacrylamide gel, transferred to a PVDF membrane, and immunoblotted with mouse anti-human caspase 8 (BD Biosciences) followed by chemiluminescence. Protein extracts from HEC 1B cells grown in the absence of compound were used as a negative control. The inactive form of caspase 8 (55kDa) is cleaved into smaller activated subunits of 40/36 kDa (doublet) and 23 kDa. A strong band of approximately 55 kDa was detected in all cells, both treated and untreated. A 23kDa band was observed in control and in 1μM HEC 1B cells, whereas a faint band was observed in HEC 1B cells treated with 10μM 4-OH TAM. Thus, the active form of caspase 8 appeared the least expressed at 10μM 4-OH TAM. Figure 1A: Percent Survival for ER negative HEC 1B human endometrial cancer cells treated with different concentrations of 4-OH TAM. HEC 1B cells treated with lower concentrations of 4-OH TAM (1-3 days) resulted in no significant difference in percent survival when compared to untreated cells (red line). Figure 1B: Percent Survival for ER negative HEC 1B human endometrial cancer cells treated with different concentrations of estrogen. HEC 1B cells treated with lower concentrations of estrogen (1-3 days) resulted in no significant difference in percent survival when compared to untreated cells (red line). INTRODUCTION Tamoxifen has been researched for over twenty years as an anti-estrogenic drug used to combat breast cancer. By competing for estrogen receptors, tamoxifen has been proven to reduce the proliferation of breast cancer by 49% through its antagonistic properties (1). On the other hand, in the endometrium, tamoxifen has been shown to be a partial agonist. In addition, the agonistic effect of tamoxifen has been shown to depend on cell type, promoter context, and estrogen receptor subtype ( or ) (2). It has also been shown that tamoxifen and its metabolite, 4-OH TAM, produce DNA adducts in the human, rat, and mouse endometrium as well as in the mouse and rat liver supporting the existence of an alternative ER independent pathway (3,4). By covalently binding to nucleic acids, DNA adducts may disrupt the duplex structure resulting in mutations. If repair mechanisms are not able to remove the mutations upon replication, mutations located within significant genes (such as proto-oncogenes) may initiate an unregulated cell cycle (5). In the end, this uncontrollable cell growth lends way to cancer. In the breast, MCF-7 human adenocarcinoma cells treated with tamoxifen or 4-OH TAM have been demonstrated to undergo a cessation of cell proliferation (6). However, in the endometrium, studies conducted using ER positive HEC 1A human adenocarcinoma cells treated with tamoxifen have exhibited both agonistic and antagonistic tendencies with respect to cell proliferation (7). Furthermore, the effects of tamoxifen and its metabolites on the cell growth of ER negative human endometrial cancer cells (such as HEC 1B) have not been extensively explored. Therefore, this study intended to investigate the effects of 4-OH TAM and estrogen on the proliferation of HEC 1B endometrial cancer cells lacking an ER while using ER positive HEC 1A endometrial cancer cells as a means of comparison. By varying the concentration of 4-OH TAM and estrogen, the antagonistic or agonistic growth effects were analyzed using MTS assay. Tamoxifen-induced DNA adduct formation can result in an agonistic effect due to the mechanisms explained above. However, DNA damage (caused by DNA adducts) can also lead to apoptosis (8). In general, apoptosis is a pathway leading to programmed cell death with visible characteristics such as morphological change, chromatin condensation, and DNA fragmentation (9). If the apoptotic pathway is not utilized, necrosis occurs through the unintentional lysing of the cell. Overall, analyzing the effects of 4-OH TAM and estrogen on ER negative HEC 1B endometrial cancer cells permits a better understanding of the ER independent pathway. DISCUSSION In the present study, we attempted to determine the effects of 4-OH TAM on ER negative HEC 1B endometrial cancer cells while using ER positive HEC 1A endometrial cancer cells as a means of comparison. Through MTS Assay analysis, we discovered that lower concentrations of 4-OH TAM and estrogen had no effect on HEC 1B cell proliferation. However, higher concentrations of 4-OH TAM produced a significant inhibitory effect on the growth of HEC 1A and HEC 1B cell lines when compared to non-treated cells. In addition, HEC 1B and HEC 1A cells were inhibited by higher concentrations of estrogen when compared to controls. Despite the presence of the estrogen receptor within the HEC 1A cells, 100μM estrogen led to a complete inhibition of growth. Similarly, growth of HEC 1B (ER negative) cells was inhibited when exposed to 100μM estrogen. However, a greater inhibition within a shorter amount of time was observed in ER negative HEC 1B cells when compared to ER positive HEC 1A cells. Due to the inhibitory action of these compounds, we investigated the molecular mechanism behind the observed cell death. DNA Gel Electrophoresis and Western blot analysis of the apoptotic protein, caspase 8, did not demonstrate that apoptosis had occurred. From the Western blot analysis, we saw a basal level of expression of caspase 8 in controls and HEC 1B cells treated with 1μM 4-OH TAM (10). On the contrary, an under-expression of active caspase 8 was observed in HEC 1B cells treated with 10μM 4-OH TAM. These results lead us to conclude that necrosis, instead of apoptosis, was the underlying mechanism of cell death. Overall, both estrogen and 4-OH TAM exposure resulted in necrosis in both ER negative HEC 1B and ER positive HEC 1A cells. These results differ from previous studies in which 4-OH TAM was found to be an estrogen agonist in HEC 1A cells using 1μM concentrations (2). However, the fact that HEC 1B cells showed inhibition when exposed to 4-OH TAM and estrogen supports the existence of an ER independent pathway. Figure 1C: Percent Survival for ER negative HEC 1B human endometrial cancer cells treated with higher concentrations of 4-OH TAM. HEC 1B cells treated with higher concentrations of 4-OH TAM (1-3 days) resulted in a significant inhibitory effect (p = 0.0001) at 10μM and 100μM when compared to untreated cells (red line). No significant difference was observed between 1μM and control. Figure 1D: Percent Survival for ER negative HEC 1B human endometrial cancer cells treated with higher concentrations of estrogen. When HEC 1B cells were treated with higher concentrations of estrogen (1-3 days), we observed an initial proliferative response after 24 hours at 1μM and 10μM , followed by a partial inhibition of growth after 2 to 3 days. A significant inhibitory effect (p = 0.0001) was seen at 100μM when compared to untreated cells (red line). OBJECTIVES ACKNOWLEDGEMENTS The objective of this study is to determine the effects of 4-OH TAM on ER negative HEC 1B human endometrial cells in comparison with the effects observed with ER positive HEC 1A human endometrial cells. Specifically, this study aims to: 1. Determine if 4-OH TAM has an agonistic or antagonistic effect on the growth of ER negative HEC 1B endometrial cancer cells. 2. Investigate the molecular mechanism of the agonistic or antagonistic effect on proliferation. We would like to thank Dr. Cheryl Walker for her generous donation of the HEC 1A and HEC 1B endometrial cancer cell lines and her technical advice. Also, we would like to thank Dr. Martín Gonzalez and Dr. Maria Todd for their technical expertise. Finally, a special thanks to Dr. Romi Burks for her statistical guidance. REFERENCES MATERIALS & METHODS • 1. Liu, X., Pisha, E., Tonetti, D.A., Yao, D., Yan, L., Yao, J., Burdette, J.E., Bolton, J.L. (2003) Antiestrogenic and DNA Damaging Effects Induced by Tamoxifen and Toremifene Metabolites. Chem. Res. Toxicol. 16: 832-837. • 2. Castro-Rivera, E. and Safe, S. (2003) 17β-Estradiol- and 4-hydroxytamoxifen-induced transactivation in breast, endometrial and liver cancer cells is dependent on ER-subtype, cell and promoter context. J. Steroid Biochem. Molec. Biol. 84:23-31. • 3. Carthew, P., Edwards, R.E., Nolan, B.M., Tucker, M.J., Smith, L.L. (1999) Compartmentalized Uterotrophic Effects of Tamoxifen, Toremifene, and Estradiol in the Ovariectomized Wister (Han) Rat. Toxicological Sciences. 48:197-205. • 4. Lyman, S.D. and Jordan, V.C. (1985) Metabolism of Tamoxifen and Its Uterotrophic Activity. Biochemical Pharmacology. 34:2787-2794. • 5. Martin, E.A., Brown, K., Gaskell, M., et al., (2003) Tamoxifen DNA Damage Detected in Human Endometrium Using Accelerator Mass Spectrometry. Cancer Research. 63: 8461-8465. • 6. Wang, S., Zhang, B., and Faller, D.V. (2004) BRG1/BRM and prohibitin are required for growth suppression by estrogen antagonists. The EMBO Journal. 23:2293-2303. • 7. Castro-Rivera, E. and Safe, S. (1998) Estrogen-and Antiestrogen-responsiveness of HEC 1A Endometrial Adenocarcinoma Cells in Culture. J. Steroid Biochem. Molec. Biol. 64:287-295. • 8. Wyllie, A.H. (1995) The genetic regulation of apoptosis. Current Opinions in Genetics and Development. 5:97-104. • 9. Dietze, E.C., Caldwell, L.E., Grupin, S.L., Mancini, M., Seewaldt, V.L. (2001) Tamoxifen but Not 4-OH tamoxifen Initiates Apoptosis in p53(-) Normal Human Mammary Epithelial Cells by Inducing Mitochondrial Depolarization. J. Biol. Chem. 276: 5384-5394. • 10. Esposti, M.D., Ferry, G., Masdehors, P., Boutin, J.A., Hickman, J.A., Dive, C. (2003) Post-translational Modification of Bid Has Differential Effects on Its Susceptibility to Cleavage by Caspase 8 or Caspase 3. J. Biol. Chem. 278: 15749-15757. Cell Lines & Tissue Culture Conditions HEC 1B and HEC 1A human endometrial cancer cells were a generous gift from Dr. Cheryl Walker at M.D. Anderson in Smithville. HEC 1A cells were maintained in McCoy’s 5A medium with 1% L-glutamine(Gibco) and supplemented with 10% Fetal Bovine Serum. HEC 1B cells were maintained in Minimum Essential Medium (MEM) (Gibco) supplemented with 10% Fetal Bovine Serum, 2% L-glutamine, and 1% sodium pyruvate. Cells were grown in 75cm2 culture flasks at 37˚C in a 5% CO2 atmosphere. Compounds 4-OH Tamoxifen and Estrogen (Sigma) were dissolved in ethanol, stored, and protected from light in stock solutions of 1mM at -20˚C. MTS Assay HEC 1B (ER negative) and HEC 1A (ER positive) cells were seeded in triplicates into 96-well plates (10,000cells/well). Media was replaced using 5% charcoal stripped bovine serum and cells were allowed to recover for 24 hours. Cells were then incubated with 200μL of various 4-OH TAM and estrogen concentrations (nM to μM range) for 1-3 days. MTS assay was performed using CellTiter 96AQueous One Solution Cell Proliferation Assay (Promega). Absorbance was read at 490nm using a lysis plate reader (BioRad). DNA Gel Electrophoresis Cell DNA extracts were prepared from HEC 1A and HEC 1B cells treated with 10μM 4-OH TAM and estrogen for 24 hours at the incubating conditions previously described. DNA laddering analysis was performed using a DNA Laddering Assay kit (Cayman). Figure 2B: Percent Survival for ER positive HEC 1A human endometrial cancer cells treated with higher concentrations of estrogen. Complete inhibition (p = 0.0001) was observed with HEC 1A cells treated with 100μM estrogen (1-3 days). In contrast, HEC 1A cells treated with 1μM and 10μM estrogen were refractory to low dose estrogen when compared to control (red line). Figure 2A: Percent Survival for ER positive HEC 1A human endometrial cancer cells treated with higher concentrations of 4-OH TAM. A significant inhibition (p = 0.0001) was seen with HEC 1A cells treated with 10μM and 100μM 4-OH TAM (1-3 days) when compared to untreated cells (red line). No inhibition was observed at 1μM.