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[beta]-estradiol induces cytotoxic effects to human T-lymphoma (Jurkat) cells through oxidative stress.

INTRODUCTION

Estradiol is a form of estrogen, and is a female sex hormone produced by the ovaries. Estrogen is necessary for many processes in the body. In vitro studies indicated that the addition of estradiol to cultures of human lymphocytes decreases in CD4+/CD8+ T-cell-subset ratios (Athreya et al., 1993) and enhances immunoglobulin secretion (Kanda and Tamaki, 1999). Studies indicated that estrogens can affect cells of the immune system and may play a role in modulating lymphocyte development and function (Smithson et al., 1998, Grimaldi et al., 2002). Published reports suggest that estrogens may have important actions at other steps in the atherogenic process (Nathan and Chaudhuri, 1997). However, the mechanisms by which [beta]-estradiol-induces cytotoxic effects on cancer cells are not yet completely understood. In the present study, we use the human Jurkat T-lymphoma cell line as a test model to evaluate whether [beta]-estradiol-induced cytotoxicity is associated with oxidative stress.

MATERIALS AND METHODS

Chemicals and Test Media

Growth medium RPMI 1640 containing 1 mmol/L L-glutamine was purchased from Gibco BRL products (Grand Island, NY). Fetal bovine serum (FBS) and phosphate buffered saline (PBS) were obtained from Sigma Chemical Company (St. Louis, MO).

Tissue Culture

Human T-lymphoma (Jurkat) cells were obtained from American Type Culture Collection (Manassas, VA, USA). In the laboratory, cells were stored in the liquid nitrogen until use. They were next thawed by gentle agitation of their containers (vials) for 2 minutes in a water bath at 37[degrees]C. After thawing, the contents of each vial was transferred to a 25 [cm.sup.2] tissue culture flask, diluted with up to 10 mL of RPMI 1640 containing 1 mmol/L L-glutamine (GIBCO/BRL, Gaithersburg, MD) and supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (w/v) penicillin/streptomycin. The 25 [cm.sup.2] culture flasks, each containing 2 x [10.sup.6] viable cells, were observed under the microscope, and then placed in a humidified 5 % C[O.sub.2] incubator at 37[degrees]C. Three times a week, the cells were diluted under same conditions to maintain a density of 5 x 105/mL, and harvested in the exponential phase of growth.

Cell treatment and biochemical test for cell viability

The in vitro cytotoxicity of [beta]-estradiol against Jurkat T-cells was determined by MTT assay as previously described (Yedjou and Tchounwou, 2007). Briefly, to 180 [micro]L aliquots in six replicates of the cell suspension (5 x [10.sup.5] cells/mL) seeded to 96-well polystyrene tissue culture plates, 20 [micro]L aliquots of stock solutions were added to each well using distilled water as solvent to make-up final [beta]-estradiol concentrations of 1, 2, 4, 8, and 16 [micro]M. Control cells received 20 pL of distilled water. Cells were placed in a humidified 5% C[O.sub.2] incubator for 12 hr at 37[degrees]C. After incubation, 20 pL aliquots of MTT solution (5 mg/mL in PBS) were added to each well and re-incubated for 4 h at 37[degrees]C followed by low centrifugation at 800 rpm for 5 min. Then, the 200 [micro]L of supernatant culture medium were carefully aspirated and 200 [micro]L aliquots of dimethylsulfoxide (DMSO) were added to each well to dissolve the formazan crystals after incubation for 10 min to dissolve air bubbles, the culture plate was placed on a Biotek micro-plate reader and the absorbance was measured at 550 nm. All assays were performed in six replicates for each [beta]-estradiol dose. Statistical analysis was done to determine the means [+ or -] SDs of cell viability. Cell viability rate was calculated as the percentage of MTT absorption as follows: % survival = (mean experimental absorbance/mean control absorbance x 100).

Biochemical test for oxidative stress by lipid peroxidation assay

Lipid peroxidation is traditionally quantified to measure malondialdehyde (MDA) and 4-hydroxynonenal. The extraction procedure and measurement of the extracted MDA was performed according to the manufacturer's instructions (Calbiochem-Novabiochem, San Diego, CA). Malondialdehydes (MDA) are formed during lipid peroxidation (Halliwell and Gutteridge 1984). Briefly, 2 x [10.sup.6] Jurkat T-cells/mL untreated as a control and cells treated with [beta]-estradiol were incubated in a total volume of 10 ml growth medium for 48 hours. After the incubation period, cells were collected in 15 mL tubes, followed by low-speed centrifugation. The cell pellets were resuspended in 0.5 ml of Tris-HCl, pH 7.4, and lysed using a sonicator (W-220; Ultrasonic, Farmingdale, NY) under the conditions of duty cycle-25% and output control-40% for 5 sec on ice. A 200[micro]l aliquot of the culture medium was assayed for MDA according to the lipid peroxidation assay kit protocol. The absorbance of the sample was read at 586 nm, and the concentration of MDA was determined from a standard curve.

Statistical analysis

Experiments were performed at least in triplicates. Data were represented as means [+ or -] SD. Where appropriate, one-way ANOVA test or paired t-test was performed using SAS Software available in the Bio-statistics Core Laboratory at Jackson State University.

RESULTS

[beta]-estradiol Inhibits Cellular viability of Jurkat Tlymphoma Cells

We used the MTT [3-(4, 5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide] assay to examine the cytotoxic effect of [beta]-estradiol on Jurkat T-cells at 24 and 48 hours, respectively (Figure 2). Data generated from these studies clearly showed that [beta]-estradiol exposure significantly reduces the viability of these cell lines. After 24 and 48 hours of exposure, [beta]-estradiol exerted a significant cytotoxic effect on Jurkat T-cells in dose- and time-dependent, showing a relationship with increasing doses of [beta]-estradiol. We observed a biphasic response where physiological (low) doses of [beta]-estradiol induce cell growth and cellular proliferation of Jurkat T -cells, whereas higher doses inhibit cell growth and induce cell death.

[beta]-estradiol Induces Oxidative Stress in Jurkat T-lymphoma Cells

The standard curve generated from lipid peroxidation assay is presented in Figure 3, and the effect of [beta]-estradiol on malondialdehyde (MDA) production in Jurkat T-cells is presented in Figure 4. MDA is an end product of lipid peroxidation, which is a key determinant of cellular injury. As seen on figure 4, there is a significant increase in MDA level at all doses tested as result of oxidative stress, a biomarker of cellular injury. A maximum level (21.5 [micro]M) of malondialdehyde (MDA) production was observed at the highest dose tested compared to (4.95 [micro]M) in the control. This increase demonstrates that [beta]-estradiol augments MDA production in Jurkat T-cells.

DISCUSSION

Natural estrogens such as estradiol, estrone, and estriol are produced from natural sources such as the urine of pregnant mares and women, the adrenals and testes of stallions, and the human placenta and amniotic fluid (Roberts, 1986). To prove the cytotoxic efficacy of P estradiol in Jurkat T-cell model, cell viability was determined by the MTT assay. As shown in Figure 2, [beta]-estradiol treatment increased the percentage of dead cells in a dose- and time-dependent manner. Consistent with our results, in vitro studies indicated that the addition of estradiol to cultures of human lymphocytes causes a decrease in CD4+/CD8+ T-cell-subset ratios (Athreya et al., 1993) and enhances immunoglobulin secretion (Kanda and Tamaki, 1999). Several scientific reports indicated that estrogen toxicity resulted in the death of the patient (Suess et al., 1992; Sanpera et al., 2002) or in a long recovery period (Hall, 1992; Acke et al., 2003).

To test the hypothesis that oxidative stress may be involved in [beta]-estradiol induced anti-tumor activity in Jurkat T-cells, we performed lipid peroxidation assay. Our result obtained from this assay showed a slight significant (p < 0.05) increase in malondialdehydes (a by-product of lipid peroxidation and biomarker of oxidative stress) production in [beta]-estradiol-treated cells compared to the control. Similarly, a recent study in our laboratory indicated that garlic compounds generate MDA production in HL-60 cells leading to phosphatidylserine externalization, caspase-3 activation, nucleosomal DNA fragmentation, and cell death (Yedjou and Tchounwou, 2012). High level of MDA production detected in the Jurkat T-cells may be indicative of the therapeutic mechanisms of action of [beta]-estradiol in human T-lymphoma (Jurkat) cells.

CONCLUSIONS

Results from this study indicate at the cellular level that [beta]-estradiol significantly reduced the viability of human T-lymphoma (Jurkat) cells in a dose and time-dependent manner. At the molecular level, [beta]-estradiol treatment resulted in a significant increase in lipid peroxidation generation in Jurkat T-cells. Findings from this study indicate that [beta]-estradiol is highly cytotoxic to Jurkat T-cells. This cytotoxicity is found to be mediated through oxidative stress, which is a biomarker of cellular injury.

ACKNOWLEDGEMENTS

This work was supported by the Mississippi INBRE, funded by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103476. We also acknowledge the National Institutes of Health (NIH-NIMHD Grant No. G12MD07581) through the RCMI Center for Environmental Health at Jackson State University (Jackson, Mississippi, USA).

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Clement Yedjou, Joseph Cameron, Ariane T. Mbemi, and Paul Tchounwou

Natural Chemotherapeutics Research Laboratory, NIH-Center for Environmental Health, College of Science, Engineering and Technology, Jackson State University, 1400 Lynch Street, P.O. Box 18540, Jackson, Mississippi, USA.
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Author:Yedjou, Clement; Cameron, Joseph; Mbemi, Ariane T.; Tchounwou, Paul
Publication:Journal of the Mississippi Academy of Sciences
Article Type:Report
Date:Apr 1, 2015
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