Human esophageal cancer cell death mediated by apoptosis-inducing nucleosides from CD57+HLA-DRbright natural suppressor cell line.

We approached the induction of apoptosis in human esophageal cancer cells (T.Tn) by apoptosis-inducing nucleosides (AINs) from CD57+HLA-DRbright-natural suppressor (57.DR-NS) cell line originated in decidua. The 57.DR-NS cells generated apoptosis in T.Tn cells by AINs released into the cultures. We isolated a series of AINs by high performance liquid chromatography (HPLC) from 57.DR-NS cell culture fluids. Each AIN and its mixture induced apoptotic cell death in T.Tn cells, detected by DNA fragmentation in agarose gels, terminal deoxynucleotide transferase mediated dUTP-nick end labeling (TUNEL) method and accumulation of sub-G1 DNA content with flow cytometry. Furthermore, we found the occurrence of DNA strand breaks followed by the activation of caspase-3 during AINs-induced apoptosis in T.Tn cells. Thus, we validated that AINs could induce apoptosis in T.Tn cells mediated through DNA strand breaks and activation of caspase-3.     

Apoptotic cell death of human gastric tumor induced by nucleosides from CD57+HLA-DRbright natural suppressor cell line

CD57+HLA-DRbright natural suppressor (57.DR-NS) cell line derived from human decidual tissue generated apoptosis in a human gastric carcinoma cell line (GCIY) but not in a human diploid normal cell line (WI-38). The factors released from 57.DR-NS cells were purified by thin-layer-chromatography (TLC) and separated by high performance liquid chromatography (HPLC) into six components. The physicochemical structures of the six components were determined as a series of nucleosides and their modified forms collectively termed in a previous study. They could generate apoptotic cell death of GCIY malignant target cells, but not disturb the viability of WI-38 normal target cells. The administration of AINs into GCIY tumor bearing SCID mice resulted in drastic suppression of tumor growth followed by the decrease in tumor size due to the occurrence of apoptosis in tumor tissues.     

Effects of apoptosis-inducing nucleosides released from CD57+HLA-DRbright natural suppressor cell line on human breast cancer cell death and growth

The CD57+HLA-DRbright-natural suppressor (57.DR-NS) cell line induced apoptosis in estrogen-non-responsive human breast carcinoma MDA-MB-435 cells by apoptosis-inducing nucleosides (AINs) released into the cultures. We obtained six active AINs isolated by high performance liquid chromatography (HPLC) from 57.DR-NS cell cultures. Each AIN isolated from 57.DR-NS cell cultures induced apoptosis in MDA-MB-435 cells. We found the occurrence of DNA strand breaks followed by the activation of caspase-3 during AIN-induced apoptosis in MDA-MB-435 cells. The data obtained here indicated that 57.DR-NS cells could induce apoptosis in MDA-MB-435 cells mediated by AINs through DNA strand breaks and activation of caspase-3. Furthermore, the administration of AINs into MDA-MB-435 tumor-bearing SCID mice culminated in strong suppression of tumor growth with no change of body weight of experimental mice suggesting no side effects of AINs.     

Human T cell leukemia cell death by apoptosis-inducing nucleosides from CD57(+) HLA-DR(bright) natural suppressor cell line.

Apoptosis-inducing nucleosides (AINs) released from CD57( +) HLA-DR(bright) natural suppressor (57.DR-NS) cell line, derived from human decidual tissue, were isolated from 57.DR-NS cell culture supernatant by the combination of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Apoptotic cell death was strongly induced in human T cell leukemia Molt4 cells treated with AINs, absolutely depending on DNA strand breaks, with activation of the caspase cascade, especially caspase-3. The administration of AINs to Molt4 tumor-bearing severe combined immunodeficiency (SCID) mice resulted in drastic suppression of tumor growth, with a decrease of tumor size and the appearance of apoptotic signals in tumor tissue. Thus, AINs are candidates for development as anticancer agents.     

Induction of apoptosis by piceatannol in human leukemic U937 cells through down-regulation of Bcl-2 and activation of caspases

Piceatannol is a polyphenol that is found in abundant quantities in grapes and wine. Although recent experimental data revealed the proapoptotic potency of piceatannol, the molecular mechanisms underlying the anti-leukemic activity have not yet been studied in detail. This study examined the effects of piceatannol on the growth of the human leukemia cell line U937. The results showed that piceatannol inhibits the viability of U937 cells by inducing apoptosis, as evidenced by the formation of apoptotic bodies, DNA fragmentation and the accumulation of the sub-G1 phase. RT-PCR and immunoblotting data showed that treating the cells with piceatannol caused the down-regulation of anti-apoptotic Bcl-2 and cIAP-2 expression. Piceatannol-induced apoptosis was also associated with the proteolytic activation of caspase-3, and the degradation/cleavage of poly (ADP-ribose) polymerase protein. z-DEVD-fmk, a caspase-3-specific inhibitor, blocked the activation of caspase-3 and increased the survival of the piceatannol-treated U937 cells, suggesting that caspase-3 activation is essential for piceatannol-induced apoptosis.     

Human T Cell Leukemia Cell Death by Apoptosis-inducing Nucleosides from CD57+ HLA-DRbright Natural Suppressor Cell Line

Apoptosis-inducing nucleosides (AINs) released from CD57⁺HLA-DR^bright natural suppressor (57.DR-NS) cell line, derived from human decidual tissue, were isolated from 57.DR-NS cell culture supernatant by the combination of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Apoptotic cell death was strongly induced in human T cell leukemia Molt4 cells treated with AINs, absolutely depending on DNA strand breaks, with activation of the caspase cascade, especially caspase-3. The administration of AINs to Molt4 tumor-bearing severe combined immunodeficiency (SCID) mice resulted in drastic suppression of tumor growth, with a decrease of tumor size and the appearance of apoptotic signals in tumor tissue. Thus, AINs are candidates for development as anticancer agents.     

Therapeutic effects of novel anti-tumor reagent, apoptosis inducing nucleosides from CD57+HLA-DRbright natural suppressor cell line on human gastric carcinoma-bearing SCID mice

To further provide scientific evidence before clinical application, the anti-tumor effects of apoptosis inducing nucleosides (AINs) released from CD57+HLA-DRbright natural suppressor (CD57.DR-NS) cell line on human gastric carcinoma (GCIY)-bearing severe combined immunodeficiency (SCID) mice were examined by monitoring tumor cell growth and change of body weight of mice. The results obtained evidenced that AINs strongly induced apoptosis in the tumor tissues in SCID mice with decrease of tumor size and without loss of body weight. We found that peak 5 and peak 6 (P5 and P6) components among six components (AINs) isolated from CD57.DR-NS cell cultures by high performance liquid chromatography (HPLC) are the most effective. The anti-tumor effective dosage of P5, P6 and their mixture, P5+P6, were obtained in dose-dependent manner. Thus, the most effective method of administration of AINs for tumor regression without exhaustion was established in the present study. Corresponding to the previous study that AINs could generate apoptosis in malignant cells while lacking the toxicity in normal cells, the results obtained in the present preclinical experiments suggested anti-tumor efficacy of AINs with possible refrainment from side-effects in clinical trials.     

Amrubicin induces apoptosis in human tumor cells mediated by the activation of caspase-3/7 preceding a loss of mitochondrial membrane potential

Amrubicin, a completely synthetic 9-aminoanthracycline derivative, inhibits cell growth by stabilizing a topoisomerase II-DNA complex. This study was designed to examine the apoptosis induced in human leukemia U937 cells by amrubicin and its active metabolite amrubicinol. Amrubicin, amrubicinol and other antitumor agents, such as daunorubicin and etoposide, induced typical apoptosis with characteristic nuclear morphological change and DNA fragmentation. Measuring the population of sub-G(1) phase cells, it was found that under conditions where cell growth was inhibited by either amrubicin or amrubicinol, U937 cells underwent apoptotic cell death in a dose-dependent manner accompanied by an arrest of the cell cycle at G(2)/M. Furthermore, amrubicin- and amrubicinol-induced apoptosis was mediated by the activation of caspase-3/7, but not caspase-1, preceding a loss of mitochondrial membrane potential. These results indicate that both a reduction in mitochondrial membrane potential and the activation of caspase-3/7 are key events in the apoptosis induced by amrubicin and amrubicinol as well as the other antitumor agents. In addition, studies with oligomycin suggested that the apoptosis induced by amrubicin and amrubicinol involved substantially different pathways from that triggered by daunorubicin and etoposide. Oligomycin blocked the etoposide-induced increase in the number of sub-G(1) phase cells without preventing the activation of caspase-3/7, and had no inhibitory effect on the expansion of the sub-G(1) population in daunorubicin-treated cells, whereas apoptosis-related changes caused by amrubicin and amrubicinol were suppressed in the presence of oligomycin.     

Caspase-3 mediated cleavage of BRCA1 during UV-induced apoptosis

The breast cancer suppressor protein, BRCA1 plays an important role in mediating cell cycle arrest, apoptosis and DNA responses to DNA damage signals. In this study, we show that BRCA1 level is downregulated during UV-induced apoptosis by caspase-3 mediated cleavage. Cleavage of BRCA1 by caspase-3 produced a fragment that contained the C-terminal of the molecule. Accordingly, treatment of cells with caspase-3 inhibitor or mutation of a specific caspase-3 cleavage site (DLLD) at amino acid 1151-1154 of BRCA1 abolished cleavage and consequential accumulation of the BRCA1 C-terminal fragment. Whereas expression of the non-cleavable BRCA1 (D/A 1154) mutant conferred the resistance phenotype to UV-induced cell death, expression of the cleaved BRCA1 C-terminus induced cell death in the absence of UV. Examination of the mechanism of C-terminus-induced cell death revealed that the cleaved fragment triggers the apoptotic response through activation of BRCA1 downstream effectors, GADD45 and JNK. Altogether, results of our study demonstrate a functional role for caspase-3 mediated cleavage of BRCA1 during UV-induced apoptosis.     

Induction of apoptosis by isothiocyanate sulforaphane in human cervical carcinoma HeLa and hepatocarcinoma HepG2 cells through activation of caspase-3

Sulforaphane (SFN) is an isothiocyanate that is found in abundant quantities in many cruciferous vegetables including broccoli and cauliflower. Its inhibitory effects on tumor cell growth in vitro and in vivo, which is dependent on the direct effect on cancer cells, has attracted considerable attention. This study examined the effects of SFN on the growth of human cervical carcinoma HeLa and hepatocarcinoma HepG2 cells. The results showed that SFN inhibits the viability of both HeLa and HepG2 cells by inducing apoptosis, as evidenced by the formation of apoptotic bodies and the accumulation of the sub-G1 phase. RT-PCR and immunoblotting showed that treating the cells with SFN caused the down-regulation of anti-apoptotic Bcl-2 and Bcl-XL, and the up-regulation of pro-apoptotic Bax expression. SFN-induced apoptosis was associated with the proteolytic activation of caspase-3, and the degradation/cleavage of poly (ADP-ribose) polymerase and the beta-catenin protein. z-DEVD-fmk, a caspase-3 specific inhibitor, blocked the activation of caspase-3 and increased the survival of the SFN-treated HeLa and HepG3 cells, suggesting that caspase-3 activation is essential for SFN-induced apoptosis.     

Piplartine induces caspase-mediated apoptosis in PC-3 human prostate cancer cells

The present study examined the anti-proliferative effects of piplartine on the human prostate cancer cell line PC-3. This is the first report demonstrating the piplartine anti-cancer activity toward prostate cancer cell lines, although its precise mechanism of action is still not completely defined. In MTT assays, it preferentially inhibited growth of androgen-independent PC-3 cells in a dose-dependent (3-30 microM) and time-dependent (12-48 h) manner. In PC-3 cells, it showed an IC50 of 15 microM after 24 h of treatment. After a 24-30 microM treatment for 24 h, there were some reduction of cell volume, cell vacuolization, chromatin condensation and increased number of apoptotic cells visible by light and fluorescence microscopy. Agarose gel electrophoresis revealed that cells treated with piplartine exhibited DNA fragmentation. In addition, growth inhibition of PC-3 cells was associated with G2/M arrest and sub-G1 accumulation. Higher concentrations (24-30 microM) of piplartine modulated apoptosis-related protein expression by down-regulating cdc-2 expression and up-regulating PARP/procaspase-3 cleavage. Also, PC-3 cells treated with piplartine demonstrated caspase-3 activation, as observed with an in vitro caspase-3 colorimetric assay kit. Taken together, these results demonstrated that high concentrations of piplartine exhibited anti-proliferative and anti-cancer effects on PC-3 cells and that caspase-3-mediated PARP cleavage and cell cycle arrest at G2/M phase are involved in the underlying cellular mechanism of the apoptosis process.     

Ionizing radiation but not anticancer drugs causes cell cycle arrest and failure to activate the mitochondrial death pathway in MCF-7 breast carcinoma cells

There is considerable evidence that ionizing radiation (IR) and chemotherapeutic drugs mediate apoptosis through the intrinsic death pathway via the release of mitochondrial cytochrome c and activation of caspases -9 and -3. Here we show that MCF-7 cells that lack caspase-3 undergo a caspase-dependent apoptotic cell death in the absence of DNA fragmentation and alpha-fodrin cleavage following treatment with etoposide or doxorubicin, but not after exposure to IR. Re-expression of caspase-3 restored DNA fragmentation and alpha-fodrin cleavage following drug treatment, but it did not alter the radiation-resistant phenotype of these cells. In contrast to the anticancer drugs, IR failed to induce the intrinsic death pathway in MCF-7/casp-3 cells, an event readily observed in IR-induced apoptosis of HeLa cells. Although IR-induced DNA double-strand breaks were repaired with similar efficiencies in all cell lines, cell cycle analyses revealed a persistent G2/M arrest in the two MCF-7 cell lines, but not in HeLa cells. Together, our data demonstrate that caspase-3 is required for DNA fragmentation and alpha-fodrin cleavage in drug-induced apoptosis and that the intrinsic death pathway is fully functional in MCF-7 cells. Furthermore, they show that the radiation-resistant phenotype of MCF-7 cells is not due to the lack of caspase-3, but is caused by the failure of IR to activate the intrinsic death pathway. We propose (1) different signaling pathways are induced by anticancer drugs and IR, and (2) IR-induced G2/M arrest prevents the generation of an apoptotic signal required for the activation of the intrinsic death pathway.     

The cytotoxicity of Scytosiphon lomentaria against HL-60 promyelocytic leukemia cells

This study examined the cytotoxicity of Scytosiphon lomentaria, using various cancer cell lines. The ethyl acetate (EtOAc) fraction of this alga showed the cytotoxicity to leukemia cells, including HL-60. When HL-60 cells were treated with its EtOAc fraction, several apoptotic characteristics, such as DNA fragmentation, chromatin condensation, and an increase of the population of sub-G1 hypodiploid cells, were observed. Moreover, the EtOAc fraction decreased c-Myc expression in a dose-dependent manner. In order to understand the mechanism of apoptosis induction by S. lomentaria, we examined the changes of Bcl-2 and Bax protein expression levels. The EtOAc fraction reduced Bcl-2, an antiapoptotic protein, but increased Bax, a proapoptotic protein, in a dose-dependent manner. When we examined the activation of caspase-3, an effector of apoptosis, the expression of the active form (19 kDa) of caspase-3 increased, and the increase of their activities was demonstrated by the cleavage of poly (ADP-ribose) polymerase, a substrate of caspase-3, to 85 kDa. The results suggest that the inhibitory effect of S. lomentaria on the growth of HL-60 appears to arise from the induction of apoptosis by way of the down-regulation of Bcl-2 and the activation of caspase.     

Sulforaphane sensitizes tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis through downregulation of ERK and Akt in lung adenocarcinoma A549 cells

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in some cancer cells, including A549 lung adenocarcinoma cells. However, treatment with TRAIL in combination with subtoxic concentrations of sulforaphane (SFN) sensitizes TRAIL-resistant A549 cells to TRAIL-mediated apoptosis. Combined treatment with SFN and TRAIL induced chromatin condensation, DNA fragmentation, annexin V staining and sub-G(1) phase DNA content. These indicators of apoptosis correlate with the induction of caspase-3 activity that results in the cleavage of poly(ADP-ribose) polymerase and the release of lactate dehydrogenase. Both the cytotoxic effect and apoptotic characteristics induced by combined treatment were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, demonstrating the important role of caspase-3 in the observed cytotoxic effect. Combined treatment also triggered the activation of p38 MAPK and JNK, and downregulation of ERK and Akt. Inhibitors of ERK (PD98059) or Akt (LY294002), but not p38 MAPK, resulted in significantly decreased cell viability. Although the activation of JNK was increased in response to combined treatment, inhibition of the JNK pathway significantly attenuated cell viability. These results indicate that caspase-3 is a key regulator of apoptosis in response to combined SFN and TRAIL in human lung adenocarcinoma A549 cells through downregulation of ERK and Akt.     

Antimycin A induces apoptosis in As4.1 juxtaglomerular cells

Antimycin A, an inhibitor of electron transport in mitochondria, has been used as reactive oxygen species (ROS) generator in the biological system. Here, we investigated the in vitro effect of antimycin A on apoptosis in As4.1 juxtaglomerular cells. Antimycin A efficiently induced apoptosis in As4.1 cells as evidenced by flow cytometric detection of sub-G(1) DNA content, annexin V binding assay and DAPI staining. This apoptotic process was accompanied by loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bcl-2 decrease, caspase-3 activation and PARP cleavage. All of caspase inhibitors tested in this experiment failed to rescue As4.1 cells from antimycin A-induced cell death at the time of 48 h in view of sub-G(1) cells and annexin V positive staining cells. However, with regard to the mitochondrial membrane potential (DeltaPsi(m)), pan caspase inhibitor (Z-VAD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK) at the concentration of 25 microM noticeably decreased the loss of mitochondrial membrane potential (DeltaPsi(m)) in antimycin A-treated cells. Taken together, we have demonstrated that antimycin A as an inhibitor of electron transport in mitochondria potently induces apoptosis in As4.1 juxtaglomerular cells.     

Genistein sensitizes TRAIL-resistant human gastric adenocarcinoma AGS cells through activation of caspase-3

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in some cancer cells, including AGS gastric adenocarcinoma cells. However, treatment with TRAIL in combination with subtoxic concentrations of genistein sensitizes TRAIL-resistant AGS cells to TRAIL-mediated apoptosis. Combined treatment with genistein and TRAIL-induced chromatin condensation and sub-G1 phase DNA content. These indicators of apoptosis are correlated with the activation of death receptors (DR5) and induction of caspase-3 activity, which results in the cleavage of poly(ADP-ribose)polymerase. Both the cytotoxic effect and apoptotic characteristics induced by combined treatment were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, which demonstrates the important role of caspase-3 in the observed cytotoxic effect. These results indicate that caspase-3 is a key regulator of apoptosis in response to combined genistein and TRAIL in human gastric adenocarcinoma AGS cells through the activation of DR5 and mitochondrial dysfunction.     

Inhibition of cell growth and induction of apoptosis in human prostate cancer cell lines by 6-aminoquinolone WM13

Fluoroquinolones affect the proliferation and apoptotic cell death of several human malignancies. Therefore, we investigated whether new 6-aminoquinolone derivatives, initially synthesized as anti-HIV agents, could affect the proliferation and apoptotic cell death of human prostate cancer cell lines. PC3 and LNCaP cell lines were used as models of androgen-resistant and androgen-responsive prostate cancer, and proliferation of PC3 and LNCaP cells was strongly inhibited by 6-aminoquinolone WM13. Cytotoxicity, which was more pronounced in LNCaP, was accompanied by morphological changes, DNA damage, arrest at the S/G(2)/M phase of the cell cycle, and an increase of the sub-G(1) population. Molecular mechanism underlying WM13-induced cell death involved caspase-8 and -3 and modulation of the expression of apoptotic genes, as well as cleavage of poly-ADP ribose polymerase. Cell death following the treatment of human prostate cancer cell lines with WM13 can be attributed to apoptosis which, depending on the cell line, proceeds through different pathways.     

Activation of caspases-3/7 is dispensable for idarubicin-induced apoptotic DNA fragmentation in human leukemia cells

Idarubicin (IDA) is a 4-demethoxy-anthracycline analogue of daunorubicin (DNR). IDA has been recognized as a potent anti-leukemic agent. However, the molecular mechanism of IDA-induced cell death remains unclear. In the present study, we investigated the activity of IDA to induce apoptosis in human leukemia HL-60 and Jurkat cells, and studied its relationship with activation of caspases-3/7. IDA induced apoptotic DNA fragmentation in a time- and dose-dependent manner. The kinetics of apoptotic DNA fragmentation induced by IDA was well correlated with that of caspase-3/7 activation. We examined the effect of caspase-3/7 inhibitor Ac-DEVD-CHO on IDA-induced apoptosis in HL-60 and Jurkat cells. Ac-DEVD-CHO abolished IDA-induced caspases-3/7 activation in both cell lines. We have also found that L-carnitine can inhibit recombinant caspase-3 activity in vitro. L-carnitine treatment prevented IDA-induced caspases-3/7 activation in both cell lines in a dose-dependent manner. However, neither Ac-DEVD-CHO nor L-carnitine inhibited IDA-induced apoptotic internucleosomal DNA fragmentation in HL-60 or Jurkat cells. These data suggest that caspase-3/7 may be dispensable for idarubicin-induced internucleosomal DNA cleavage during apoptosis in human leukemia cells.     

A superoxide anion generator, pyrogallol, inhibits the growth of HeLa cells via cell cycle arrest and apoptosis

We investigated the in vitro effects of pyrogallol on cell growth, cell cycle regulation, and apoptosis in HeLa cells. Pyrogallol inhibited the growth of HeLa cells with an IC(50) of approximately 45 microM. Pyrogallol induced arrest during all phases of the cell cycle and also very efficiently resulted in apoptosis in HeLa cells, as evidenced by flow cytometric detection of sub-G1 DNA content, annexin V binding assay, and DAPI staining. This apoptotic process was accompanied by the loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bcl-2 decrease, caspase-3 activation, and PARP cleavage. Pan-caspase inhibitor (Z-VAD) could rescue some HeLa cells from pyrogallol-induced cell death, while caspase-8 and -9 inhibitors unexpectedly enhanced the apoptosis. When we examined the changes of the ROS, H(2)O(2) or O(2)(*-) in pyrogallol-treated cells, H(2)O(2) was slightly increased and O(2)(*-) significantly was increased. In addition, we detected a decreased GSH content in pyrogallol-treated cells. Only pan-caspase inhibitor showing recovery of GSH depletion and reduced intracellular O(2)(*-) level decreased PI staining in pyrogallol-treated HeLa cells, which indicates dead cells. In summary, we have demonstrated that pyrogallol as a generator of ROS, especially O(2) (*-), potently inhibited the growth of HeLa cells through arrests during all phases of the cell cycle and apoptosis.     

Se-methylselenocysteine induces apoptosis through caspase activation in HL-60 cells

Apoptosis, a programmed process of cell suicide, has been proposed as the most plausible mechanism for the chemopreventive activities of selenocompounds. In our study, we found that Se-methylselenocysteine (MSC) induced apoptosis through caspase activation in human promyelocytic leukemia (HL-60) cells. Measurements of cytotoxicity, DNA fragmentation and apoptotic morphology revealed that MSC was more efficient at inducing apoptosis than selenite, but was less toxic. Moreover, MSC increased both the apoptotic cleavage of poly(ADP-ribose) polymerase (PARP) and caspase-3 activity, whereas selenite did not. We next examined whether caspases and serine proteases are required for the apoptotic induction by MSC. A general caspase inhibitor, z-VAD-fmk, dramatically decreased cytotoxicity in MSC-treated HL-60 cells and several other apoptotic features, such as, caspase-3 activation, the apoptotic DNA ladder, TUNEL-positive staining and the DNA double-strand break. Interestingly, a general serine protease inhibitor, AAPV-cmk, also effectively inhibited MSC-mediated cytotoxicity and apoptosis. These results demonstrate that MSC is a selenocompound that efficiently induces apoptosis in leukemia cells and that proteolytic machinery, in particular caspase-3, is necessary for MSC-induced apoptosis. On the other hand, selenite-induced cell death could be derived from necrosis rather than apoptosis, since selenite did not significantly induce several apoptotic phenomena, including the activation of caspase-3.