Biochemical and Morphological Characteristics of Selenite-Induced Apoptosis in Human Hepatoma Hep G2 Cells

Biochemical and MorphologicalCharacteristics of Selenite-Induced

Apoptosis in Human Hepatoma Hep G2 Cells

HANDAN AK CELIK,*,1 HIKMET HAKAN AYDIN,1

REMZIYE DEVECI,5 ENDER TERZIOGLU,2 SABIRE KARACALI,5

GURAY SAYDAM,3 ULUS AKARCA,4 AND YUCEL BATUR4

Departments of 1Biochemistry, 2Immunology, 3Hematology, and4Gastroenterology, and Science Faculty, 5Department of Biology,

Molecular and Cell Biology Section, Ege University School ofMedicine, Bornova, TR-35100 Izmir, Turkey

Received May 22, 2003; Revised September 24, 2003; Accepted October 24, 2003

ABSTRACT

Selenium is a cellular growth inhibitor in many mammary tumorcells. To comprehend the mechanism for the selenium-induced cell death,we examined the effects of sodium selenite, which has been one of the mostextensively investigated selenium compounds, in human hepatoma HepG2 cells.

Cell viability gradually decreased after treatment with sodium selen-ite within the concentration range of 10–50 µM. Low (10 µM) selenite hasshown a high-percentage laddering pattern compared to the high (25 µM)cytotoxic selenium concentration in agarose gel electrophoresis. G2/M-phase enrichment was also concentration dependent. The most consistenttransmission electron microscopic finding was the existence of large lyso-somes.

Based on these data, we hypothesize that sodium selenite predomi-nantly shows its apoptotic effect over hydrogen selenite accumulation.

Index Entries: Sodium selenite; apoptosis; Hep G2 cells.

Biological Trace Element Research 27 Vol. 99, 2004

© Copyright 2004 by Humana Press Inc.All rights of any nature, whatsoever, reserved.0163-4984/04/99(1–3)–0027 $25.00

* Author to whom all correspondence and reprint requests should be addressed.

INTRODUCTION

Inorganic and organic selenium compounds have been shown toinhibit cell growth in a variety of experimental animal models and cell cul-tures in vivo, and a number of mechanisms have been implicated inaccounting for these effects. Inhibition of cell proliferation and inductionof cell death are induced by all forms of selenium (1). However, sodiumselenite and sodium selenide, which are measured predominantly tohydrogen selenide, caused rapid induction of DNA single-strand breaks asan early event that preceded growth inhibition (1). Sodium selenite is acommonly used inorganic selenium in these studies. There were manyreports showing that selenite, at concentrations in the micromole range,suppressed cell proliferation in culture and induced cytotoxicity, as docu-mented by standard cell viability assays (2). Furthermore, selenite is welldocumented as an inhibitor of cell growth and inducer of DNA fragmen-tation and apoptosis in vitro in many cell lines (3–6).

In the present study, we investigated the effect of selenite, which hasbeen one of the most extensively investigated inorganic selenium com-pound in human hepatoma Hep G2 cell death, and we focused on the bio-chemical and morphological characteristics of apoptosis to comprehendthe mechanism for the selenite-induced cell death.

MATERIALS AND METHODS

Chemicals

3-[4,5-Dimethylthiazol-2-yl-2-yl]-2,5-diphenyltetrazolium bromide(MTT) assay and apoptotic DNA ladder kits were obtained from RocheBiochemicals (Mannheim, Germany); propidium iodide (PI) and Hoechst33342 were obtained from Molecular Probes Europe BV (Leiden, TheNetherlands). All other chemical reagents and cell culture media were pur-chased from Sigma (Deisenhofen, Germany) unless otherwise indicated.

Cells

Human hepatoma Hep G2 cells were obtained from ECACC (Salis-bury, UK). Cells were cultured as a monolayer in RPMI-1640 medium con-taining 10% (v/v) heat-inactivated fetal calf serum (FCS), 100 U ofpenicillin/mL, 100 µg of streptomycin/mL, 2 mM L-glutamine at 37°C ina 75-cm2 culture flask in humidified air containing 5% CO2. The cells wereseeded at 5 × 106 cells/mL of culture medium and passaged once a week.

Experimental Procedures

The cells were washed and then covered with cell culture medium atthe beginning of the experiments. For all experiments, sodium selenite

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Biological Trace Element Research Vol. 99, 2004

(Na2SeO3, FW 172.94) was prepared at 100 µM stock solution and thenadded to the cell culture medium directly to obtain different final concen-trations (2–100 µM selenite). The cells were further incubated at 37°C for24-, 48-, and 72-h periods.

Cell Counting

Cell counting was performed using trypan blue with a hemocytome-ter under an inverted phase-contrast microscope (Olympus, Tokyo, Japan).

MTT Assay

The viability of the cells after treatment with increasing concentra-tions of selenium was measured using the MTT assay. The assay is basedon the cleavage of the yellow tetrazolium salt MTT to purple formozancrystals by metabolic active cells (7). Cells were inoculated onto a 96-wellmicrotiter plate at an initial density of 1 × 105 cells/mL and exposed tovarying concentrations of selenium for 24 h. At the end of the exposure,the medium was removed and the monolayer was washed with phos-phate-buffered saline (PBS) and then given 2 mg/mL MTT in PBS (RocheBiochemicals, Germany). After 4 h incubation at 37°C, the supernatantwas removed and the precipitated formazan crystals were dissolved indimethyl sulfoxide (DMSO). The absorbance proportional to the degree ofcell viability was determined by an enzyme-linked immunoabsorbentassay (ELISA) reader (Bio-Rad, Coda, Hercules, CA, USA) at 595 nm. Thenegative control well was used for zeroing absorbance. The percentage ofcytotoxicity was calculated using the background-corrected absorbanceas follows:

(1–A of experiment well)% Cytotoxicity = ×100

A of positive control well

Apoptotic DNA Ladder

Adherent cells were pelleted and lysed with lysis solution. After lysisof cultured cells in binding buffer, the lysate was applied to a filter tubewith glass fiber fleece and passaged through the glass fiber fleece by cen-trifugation. Residual impurities were removed by a wash step and, subse-quently, DNA was eluted in elution buffer from the column according tothe manufacturer’s instructions. Samples were separated by electrophore-sis on a 1% agarose/Tris-acetate-EDTA (TAE) gel. After electrophoresis,visualization of DNA band was performed by staining with ethidium bro-mide and viewing on an ultraviolet transilluminator (Bio-Rad, Hercules,CA, USA). The gel was photographed under ultraviolet light withPolaroid film.

Selenite-Induced Apoptosis in Hepatoma Cells 29


Flow Cytometric Measurement of Nucleic Acid Staining

Cell permeability was determined after staining with the phenan-threne dye PI (λ excitation max: 540 nm; λ emission max: 625 nm). PI bindsto DNA by intercalating between the bases with little or no sequence andwith a stoichiometry of one dye per 4–5 basepairs of DNA. PI is membraneimpermeable and generally excluded from viable cells. Harvested cellswere washed in cold PBS and adjusted to a cell density of approx 1 × 106

cells/mL in PBS. PI was added to the cell suspension to give a final con-centration of 1 µg/mL for staining and then cells were incubated on ice for30 min. Cell cycle analysis was determined and quantified by the FACScanflow cytometer (Becton Dickinson, Mountain View, CA, USA).

Transmission Electron Microscopy

Morphologic alterations in cultured hepatoma cells treated withsodium selenite was observed by transmission electron microscopy. Cellswere cultured on 50-cm2 culture dishes and exposed to experimental con-ditions. At 24 h, cells were washed with PBS and fixed overnight (16 h)with glutaraldehyde (2% in PBS). After fixation, cells were scraped off witha rubber policeman and centrifuged, and the pellet was resuspended inPBS and treated with osmium tetroxide (1%) overnight (16 h). Thereafter,cells were embedded in Epon 812 resin according to the manufacturer’sinstructions (Fluka, Neu-Ulm, Germany). Ultrathin sections (60 nm) werecontrasted serially with 1% uranyl acetate and 0.66% lead acetate for 10min each. Electron microscopic examination was performed using a Jeol100C (Jeol, Peabody, MA, USA) operated at 80 kV.

RESULTS

Effect of Selenium Concentration on Selenite-InducedCytotoxicity and Proliferation

Selenium-induced cytotoxicity was measured by MTT assay. Theresults from the MTT assays using Hep G2 cells showed that cell viabilitygradually decreased after treatment with selenite within the concentrationrange of 10–50 µM (see Fig. 1). Although the cells maintained exponentialgrowth rates at doses lower than 10 µM of selenite, treatment with higherconcentrations (≥ 10 µM) of selenite significantly enhanced the inhibitoryeffect of Se on cell proliferation. Selenite (≥ 10 µM) treatment often causessheets of cells to detach and float in the medium.

DNA Fragmentation Assay on Agarose Gel

The DNA alterations were studied by the DNA ladder assay. A com-parison of migration patterns of the DNA demonstrated that high-

30 Celik et al.


molecular-weight (HMW) DNA such as that found in the normal control(see Fig. 2, lane 1) is degraded into oligonucleotide fragments followingselenite administration. The administration of sodium selenite (10 µMand 25 µM) after 24 h appeared to be effective at inducing apoptoticresponse when compared to control cells. The dose–response data of theDNA laddering pattern induced by selenite were shown to have adose–dependent reverse relationship between apoptosis and cytotoxic-ity. Low dose (10 µM) of selenite has shown a high-percentage ladderingpattern compared to a high cytotoxic selenium dose (25 µM) (see Fig. 2,lanes 2 and 3). When Hep G2 cells incubated up to 48 h with a minimumselenite concentration (2 µM) consistently, apoptosis was not found.

Determination of Cell Permeability with PI

Nuclear morphology was evaluated with DNA-binding fluorochromePI by flow cytometry (see Fig. 3). Cell suspensions were prepared after cul-ture with sodium selenite for 24 h; subsequently, cells were assayed for cellcycle analysis. Compared with the control group, treatment of seleniteresulted in apoptosis, which was accompanied by G2/M cell accumula-tion. G2/M-phase enrichment was also performed in a concentration-dependent manner in Hep G2 cells. The proportion of Hep G2 cells in theG2/M phase was significantly increased in cells exposed to 10 µM and 25µM selenite from 91.78% of cells in the G0/G1 phase, 2.01% in the S phase,and 5.52% in the G2/M phase for control cells to 77.73%, 4.81%, and 17.01%and 69.43%, 5.73%, and 24.17%, respectively (see Fig. 3d).



Fig. 1. The cytotoxic effect of selenite on Hep G2 cells. Data were obtained byusing the MTT assay. Hep G2 cells (1×105 cells/mL) were cultured with differentdoses of sodium selenite (2, 5, 10, 25, and 50 µM) and the results were comparedwith the control Hep G2 cells. Results of three independent experiments werecombined; mean ± SD values are illustrated.


Fig. 2. Photograph of ultraviolet-illuminated DNA from harvested cellsafter selenite treatment for 24 h. Sampleswere resolved on 1% agarose gels byelectrophoresis and stained with ethid-ium bromide. Lane 1, control cells; lanes2, cells at 24 h after sodium selenite (10µmol/L) addition; lane 3, cells at 48 hafter sodium selenite (25 µmol/L) addi-tion. Lane 4 is the marker.

Fig. 3. Comparison of DNA content histograms of controls (A) and sodiumselenite-treated cells (B). Histograms showing selenite-induced changes in the cellcycle pattern of Hep G2 cells were measured by PI staining and analyzed by flowcytometry. Flow cytometric analysis of the stained cells was carried out with anexcitation at 488 nm on a Becton Dickinson FACS Vantage cytometer. A 24-h cul-ture indicated the percentage of cells with reduced PI fluorescence intensity.Results are representative sets from three independent experiments. Data repre-sent the percentage of cell numbers (mean ± SD) in the cell cycle (C).

Evaluation of Selenite-Induced Morphological Alterations ofHep G2 Cells with Transmission Electron Microscopy

When cultured Hep G2 cells were incubated in cell culture mediumwith selenite for 24 h, electron microscopic observations of selenite-treatedcells indicate that the cells exhibited several characteristics of apoptosis(see Fig. 4B). Selenite-treated Hep G2 cells have had electron-dense cyto-plasm. Free ribosomes as monosomes and polysomes, round, oval, long,



Fig. 4. Morphological alterations in cultured Hep G2 cells exposed to selen-ite (10 µmol/L) as observed by transmission electron microscopy (magnification:20,750×): (A) control and (B) selenium-treated cells. Selenite-treated Hep G2 cellshave an electron-dense cytoplasm and large lipid droplets (I). Granulated endo-plasmic reticulum channels (g) were enlarged as large vacuoles. The most consis-tent finding in the cytoplasm for apoptosis was the existence of large lysosomes(L). Nuclei (N) were irregularly shaped or branched and small sections of thenuclear projections were seen as a distinct structure in the cytoplasm. Moreover, alarge pericellular space appeared around the cells (*).

and enlarged mitochondria, and several lipid droplets in different sizeswere found in the cytoplasm. Granulated endoplasmic reticulum channelswere enlarged as large vacuoli. In addition, several Golgi areas were seen,but there were no formed materials. The most consistent finding in thecytoplasm for apoptosis was the existence of large lysosomes. They con-tain mostly electron-dense residues of lysosomic action. The other charac-teristics for apoptosis were seen in the nuclei. Nuclei were irregularlyshaped or branched, and small sections of the nuclear projections wereseen as a distinct structure in the cytoplasm. Moreover, a large perinuclearspace appeared around the nuclei. Accumulation of heterochromatin,which is another characteristic of apoptosis, was found. Morphologicalcharacteristics of apoptosis in Hep G2 cells are summarized in Table 1.

DISCUSSION

The antineoplastic properties of selenium compounds can be accom-plished by either a decrease in cell proliferation or an increase in apopto-sis or both, which determines tumor growth kinetics. Therefore, apoptosisis an important cellular mechanism for growth regulation and apoptosis isa carefully regulated cell suicide process that occurs as a normal part ofdevelopment (8). Apoptosis is distinguished from necrosis, or accidentalcell death, by characteristic morphological and biochemical features thatinclude nuclear membrane breakdown, chromatin condensation and frag-mentation, cell membrane blebbing, shrinkage of the cytoplasm, and theformation of apoptotic bodies (9).

Selenium is essential in the nutrition of animals and humans. It is anessential component of a number of enzymes. Selenium compounds havealso been found to inhibit tumorigenesis in both human and animal models(10,11). The mechanism of the inverse relationship between selenium andcancer-preventive activity remain to be elucidated (12). The induction ofDNA strand breaks and commitment to apoptosis might explain the selen-ite-mediated growth inhibition and loss of long-term proliferative potential.

Selenium has been shown to be cytotoxic in vitro in multiple-cell mod-els, including Ehrlich ascites and human mammary tumor cells (13,14).Selenium supplementation in laboratory animals decreases tumor forma-tion in several tumor models, including the skin, liver, colon, and pancreas(15). Dietary supplementation with selenite and selenate had only a limitedeffect on selenium concentration in blood compared to supplementation oforganic selenium compounds (16). Adequate dietary selenium supplied asselenite did not accumulate in tissues or increase glutathione peroxidaseactivity. Thus, selenite could be metabolized in a manner that diverts muchof the selenium into pool that provides protection against cancer (17).

Selenium-induced oxidative stress mediates the induction of apopto-sis, the mechanism related to the anticarcinogenic and chemopreventiveeffects of selenium. Cytotoxicity induced by a higher concentration of sele-

34 Celik et al.


nium might be partially correlated with apoptosis (18). Differentiation andapoptosis induced by oxidative stress was significantly attenuated bysuperoxide dismutase, catalase, and deferoxamine (4). Another possiblemechanism could also be related to the lipid peroxidation of cell membrane



Table 1Comparison of Morphological Differences in Hep G2 Cells

(19). Several enzymes that contain essential reactive sulfhydryl groups havebeen shown to be especially sensitive to selenite treatment (20–24).

Selenite is well documented as an inhibitor of cell growth in vitro inmany cell lines. Induction of DNA fragmentation and apoptosis selenite atsimilar doses has also been reported in multiple cell lines (3,5,6). However,this report is the first biochemical and morphological demonstration thatselenite-induced cell cycle block and apoptosis in single-cell-line Hep G2human hepatoma cells (5).

Selenite is an inorganic form of selenium metabolized to H2Se via selen-odiglutathione and glutathione selenopersulfide. Hydrogen selenide is gen-erally regarded as the precursor for supplying selenium in an active form forthe synthesis of selenoproteins. Depending on the cellular requirements forselenium, hydrogen selenide is either used for selenoprotein synthesis or tobe further metabolized via methylation by S-adenosylmethionine to themethylselenol, dimethylselenide, and trimethylselenonium ion in theprocess of elimination from the cell (25–27).

We have demonstrated by MTT assay that selenite induces cellularproliferation at low (<10 µM) concentrations and induced apoptosis athigher concentrations (≥ 10 µM). In general, there is a relation betweenselenite concentration and apoptosis. We have found that a low (10 µM)selenite concentration has a higher apoptotic pattern than the high (25 µM)selenite concentration. Accumulation of excessive hydrogen selenite mightexplain the mechanism. The formation of hydrogen selenite might be a“bottleneck” of selenium metabolism in the cells. Selenite is metabolizedto hydrogen selenite by several glutathione/NADPH reductions. Thus,selenite accumulation might block further selenite metabolism as a resultof the methylation pathway, and the hydrogen selenite concentrationmight increase by augmented oxidant stress by reductive metabolism andreduced NADPH pool as a result of decreased pethose phosphate pathwaybecause of mitochondrial damage in the cancer cells.

We have also demonstrated by transmission electron microscopy thatselenite causes extensive cellular damage. Most consistent findings werelarge lysosomes and large and enlarged mitochondria. Morphologicalfindings also supported the damage caused by oxidative stress in the cells.Based on these data, we suggest that selenite arrests cellular proliferationpossibility by switching metabolic pathways of hydrogen selenite from thedetoxifying methylation pathway to oxidative metabolism. As a result ofincreased hydrogen selenite and its precursors, cells might undergo futilemitochondrial damage/decreased NADPH pool–increased oxidant stresscycle. Thus, this might explain that why high selenite concentrations havepromoted the cell necrosis instead of apoptotic death. Figure 5 shows thathydrogen selenite is a regulatory point of selenium metabolism.

Cell cycle checkpoint controls at the G1 to S transition and the G2 to Mtransition prevent the cell cycle progressing when DNA is damaged. TheG2/M DNA damage checkpoint prevents the cell from entering mitosis(the M-phase). The cdc2-cyclin B kinase is pivotal in regulating this transi-

36 Celik et al.




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tion. The second cascade, the genes that are turned on by p53, constituteeffectors cascade (28). Selenite also inhibits caspace-3-like protease activitythrough a redox mechanism (29). The increased rate of apoptosis caninhibit cancer induction during the promotional phase. Cell cycle proteinkinase cdk2 and protein kinase C are strongly inhibited by various formsof selenium. The decrease in cdk2 kinase activity is accompanied by pro-longed arrest in the S-phase (30).

The inhibitory effect of selenite on the cell cycle resulted in cell cyclearrest at the G2/M boundary, which, at selenite concentrations above 10 µM,was followed by apoptosis. Our experiments shown that the percentage ofG0/G1-phase cells of selenite groups were notably lower than that of thecontrol group. The results point out that selenite could increase the trans-formation of the cell from the G0/G2 phase to the S phase and enrichment inthe G2/M-phase in a concentration-dependent manner. These findings sug-gest that selenite can modulate cellular response through some commonpathways involving both apoptotic and cell cycle regulatory pathways.

To further emphasize our hypothesis that forms of selenium are metab-olized predominantly to hydrogen selenite, which is the rate-limiting step ofselenium metabolism in the induction of apoptosis. Studies suggest that theformulation of the selenium-containing compound is critical for biologicalactivities (26). Therefore, more detailed studies on different selenium com-pounds that induced cellular activity and cell cycle regulation can providesignificantly to better understand the selenium metabolism that participatesin the function of cell survival and death in malignant cells.

ACKNOWLEDGMENTS

We thank Dr. Serdar B. Omay of the Department of Haematology, EgeUniversity School of Medicine for his support to conduct some part of theresearch in his research laboratory.

REFERENCES

1. J. Lu, C. Jiang, M. Kaeck, et al., Dissociation of the genotoxic and growth inhibitioneffects of selenium, Biochem. Pharmacol. 50, 213–219 (1995).

2. D. Medina and D. G. Morrison, Current ideas on selenium as a chemoprevent agent,Pathol. Immunopathol. Res. 7, 187–199 (1988).

3. J. Lu, M. Kaeck, C. Jiang, et al., Selenite induction of DNA strand breaks and apoptosisin mouse leukemic L1210 cells, Biochem. Pharmacol. 47, 1531–1535 (1994).

4. H. M. Shen, C. F. Yang, and C. N. Ong, Sodium selenite-induced oxidative stress andapoptosis in human hepatoma HepG2 cells, Int. J. Cancer 81, 820–828 (1999).

5. M. S. Stewart, R. L. Davis, L. P. Walsh, et al., Induction of differentiation and apoptosis bysodium selenite in human colonic carcinoma cells (HT29), Cancer Lett. 117, 35–40 (1997).

6. H. J. Thompson, A. Wilson, J. Lu, et al., Comparison of the effects of an organic and aninorganic form of selenium on a mammary carcinoma cell line, Carcinogenesis 15,183–186 (1994).

38 Celik et al.


7. D. T. Vistica, P. Skehan, D. Scudiero, et al., Tetrazolium-based assays for cellular viabil-ity: a critical examination of selected parameters affecting formazan production, CancerRes. 51, 2515–2520 (1991).

8. M. F. McCartly, Selenium, calcium channel blockers, and cancer-risk—the yin and yangof apoptosis, Med. Hypotheses 50, 423–433 (1998).

9. Z. Darzynkiewicz, G. Juan, X. Li, et al., Cytometry in cell necrobiology: analysis ofapoptosis and accidental cell death (necrosis), Cytometry 27, 1–20 (1997).

10. C. Jiang, W. Jiang, C. Ip, et al., Selenium-induced inhibition of angiogenesis in mam-mary cancer at chemopreventive levels of intake, Mol. Carcinogen. 26, 213–225 (1999).

11. S. Y. Yu, P. Ao, L. M. Wang, et al., Biochemical and cellular aspects of the anticanceractivity of selenium, Biol. Trace Element Res. 15, 243–255 (1988).

12. G. F. Combs, Jr, Chemopreventive mechanism of selenium, Med. Klin. 15, 94(Suppl. 3),18–24 (1999).

13. C. Ip, Lessons from basic research in selenium and cancer prevention, J. Nutr. 128,1845–1854 (1998).

14. K. A. Poirer and J. A. Milner, The effect of various seleno-compounds on Erlich ascitestumor cells, Biol. Trace Element Res. 1, 25–34 (1979).

15. C. Ip, C. Hayes, R. Budnick, et al., Chemical form of selenium, critical metabolites andcancer prevention, Cancer Res. 51, 595–600 (1991).

16. K. Ortman and B. Pehrson, Effect of selenate as a feed supplement to dairy cows incomparison to selenite and selenium yeast, J. Anim. Sci. 77, 3365–3370 (1999).

17. J. W. Finley, C. D. Davis, and Y. Feng, Selenium from high selenium broccoli protectsrats from colon cancer, J. Nutr. 130, 2384–2389 (2000).

18. D. Y. Cho, U. Jung, and A. S. Chung, Induction of apoptosis by selenite and selen-odiglutathione in HL-60 cells: correlation with cytotoxicity, Biochem. Mol. Biol. Int. 47,781–793 (1999).

19. J. G. Ren, R. L. Zheng, Y. M. Shi, et al., The possible mechanism of the differentiationand apoptosis induced by oxidative stress may be related to the lipid peroxidation ofcell membrane. Cell. Biol. Int. 22, 41–49 (1998).

20. H. E. Ganther and C. Corcoran, Selenotrisulfides. II. Cross-linking of reduced pancre-atic ribonuclease with selenium, Biochemistry 8, 2557–2563 (1969).

21. F. Islam, Y. Watanabe, H. Morii, et al., Inhibition of rat brain prostaglandin D synthaseby inorganic selenocompounds, Arch. Biochem. Biophys. 289, 161–166 (1991).

22. S. Lopez, Y. Miyashita, and S. S. Simons, Jr, Structurally based, selective interaction ofarsenite with steroid receptors, J. Biol. Chem. 265, 16,039–16,042 (1990).

23. G. D. Frenkel, A. Walcott, and C. Middleton, Inhibition of RNA and DNA polymerasesby the product of the reaction of selenite with sulfhydryl compounds, Mol. Pharmacol.31, 112–116 (1987).

24. L. N. Vernie, J. G. Collard, A. P. Eker, et al., Studies on the inhibition of protein synthe-sis by selenodiglutathione, Biochem. J. 180, 213–218 (1979).

25. H. E. Gantner, Selenium metabolism, selenoproteins and mechanisms of cancer pre-vention: complexities with thioredoxin reductase, Carcinogenesis 20, 1647–1666 (1999).

26. H. E. Ganther, Pathways of selenium metabolism including respiratory excretory prod-ucts, J. Am. Coll. Toxicol. 5, 1–5 (1986).

27. R. A. Sunde, Molecular biology of selenoproteins, Annu. Rev. Nutr. 10, 451–474 (1990).28. C. J. Sherr, Cancer cell cycles, Science 274, 1672–1677 (1996).29. H. S. Park, S. H. Huh, Y. Kim, et al., Selenite negatively regulates caspase-3 through a

redox mechanism, J. Biol. Chem. 275, 8487–8491 (2000).30. R. Sinha and D. Medina, Inhibition of cdk2 kinase activity by methylselenocysteine in

synchronized mouse mammary epithelial tumor cells, Carcinogenesis 18, 1541–1547(1997).



Biochemical and Morphological Characteristics of Selenite-Induced Apoptosis in Human Hepatoma Hep G2 Cells

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