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Etoposide is a potent anticancer agent that is used to treat various tumors. We have investigated the dose-dependent effect of etoposide on apoptosis using chronic myeloid leukemia K562 cells treated with low (5 microM) or high (100 microM) concentrations of the drug. At a low concentration, etoposide induced little apoptosis at 24 h, while about 20% of the cells showed apoptosis morphologically at a high concentration. Processing of caspase-3 was slightly detected from 12 h and became obvious at 24 h with 100 microM etoposide. Caspase-3-like protease activity was detected at 24 h with a high concentration. Moreover, these changes were accompanied by cleavage of poly ADP ribose polymerase (PARP). Changes of the mRNA levels of most apoptosis-regulating genes were not prominent at both concentrations, except for the rapid induction of c-IAP-2/HIAP-1 and the down-regulation of Bcl-X(L) by 100 microM etoposide. The downregulation of Bcl-X(L) protein occurred from 6 h, while Bax protein conversely showed a slight increase from 6 h. Taken together, the present findings show that the dose-dependent apoptotic effect of etoposide is based on a change in the balance between Bcl-X(L) and Bax, which precedes the activation of caspase-3.
Mol Cell Biochem 2000 Mar
PMID:Differential responses of Bcl-2 family genes to etoposide in chronic myeloid leukemia K562 cells. 1083 93

Etoposide, a clinically useful anticancer drug, is a potent inhibitor of topoisomerase II. The DNA strand breaks caused by this epipodophyllotoxin lead to apoptotic death of tumor cells. Flow cytometry was used to investigate the relationship between the effects of the drug on the cell cycle of human leukemia HL-60 cells and the variations of the mitochondrial transmembrane potential (DeltaPsi(mt)). Three cationic fluorescent probes, DiOC(6), JC-1, and TMRM, were used to measure drug-induced changes of DeltaPsi(mt). In all three cases, we found that the arrest in the G2/M phase of the cells treated with 0.5 microM etoposide is associated with an increase in the potential of mitochondrial membranes whereas treatment with a tenfold higher drug concentration trigger massive apoptosis and a collapse of DeltaPsi(mt). DNA fragmentation (TUNEL assay) and externalization of phosphatidylserine residues in the outer leaflet of the plasma membrane (annexin V binding) were measured to characterize the apoptotic cell population.
Mol Cell Biol Res Commun 2000 Jul
PMID:Relationship between cell cycle changes and variations of the mitochondrial membrane potential induced by etoposide. 1115 26

Staurosporine, a protein kinase and etoposide, a topoisomerase II inhibitor, are known to enhance apoptosis. The differential effects of these agents on T98G glioblastoma and SK-N-SH neuroblastoma, cell lines both derived from human tumors, have not been determined. We assessed cellular viability, DNA fragmentation and laddering, chromatin condensation, and Poly(ADP-ribose) polymerase (PARP) cleavage induced by these agents at a series of concentrations and times. In addition, to gain an understanding of the mechanism by which these agents work, we measured Protein Kinase C (PKC) activity. Staurosporine induced significant alterations in all apoptotic parameters tested in both cell lines. Etoposide induced apoptotic alterations similar to those caused by staurosporine in neuroblastoma but produced no detectable apoptotic changes in glioblastoma cells. Etoposide induced membrane but not cytosolic PKC activity in neuroblastoma but had no effect on PKC activity in glioblastoma. Our results show that the induction of apoptosis is cell type dependent. PKC activity appears to be crucial in the initiation of apoptosis.
Brain Res Mol Brain Res 2001 Jul 13
PMID:Differential responses of human neuroblastoma and glioblastoma to apoptosis. 1145 93

Protein kinase Cdelta (PKCdelta) is involved in the apoptosis of various cells in response to diverse stimuli. In this study, we characterized the role of PKCdelta in the apoptosis of C6 glioma cells in response to etoposide. We found that etoposide induced apoptosis in the C6 cells within 24 to 48 h and arrested the cells in the G(1)/S phase of the cell cycle. Overexpression of PKCdelta increased the apoptotic effect induced by etoposide, whereas the PKCdelta selective inhibitor rottlerin and the PKCdelta dominant-negative mutant K376R reduced this effect compared to control cells. Etoposide-induced tyrosine phosphorylation of PKCdelta and its translocation to the nucleus within 3 h was followed by caspase-dependent cleavage of the enzyme. Using PKC chimeras, we found that both the regulatory and catalytic domains of PKCdelta were necessary for its apoptotic effect. The role of tyrosine phosphorylation of PKCdelta in the effects of etoposide was examined using cells overexpressing a PKCdelta mutant in which five tyrosine residues were mutated to phenylalanine (PKCdelta5). These cells exhibited decreased apoptosis in response to etoposide compared to cells overexpressing PKCdelta. Likewise, activation of caspase 3 and the cleavage of the PKCdelta5 mutant were significantly lower in cells overexpressing PKCdelta5. Using mutants of PKCdelta altered at individual tyrosine residues, we identified tyrosine 64 and tyrosine 187 as important phosphorylation sites in the apoptotic effect induced by etoposide. Our results suggest a role of PKCdelta in the apoptosis induced by etoposide and implicate tyrosine phosphorylation of PKCdelta as an important regulator of this effect.
Mol Cell Biol 2002 Jan
PMID:Tyrosine phosphorylation of protein kinase Cdelta is essential for its apoptotic effect in response to etoposide. 1173 33

The nuclear architecture of selected chromosomes in apoptotic nuclei of human leukemic cells K-562 and HL-60 was investigated. Etoposide and prolonged confluence were used for the induction of apoptosis. DAPI as well as TUNEL labeling of apoptotic nuclear bodies was combined with visualization of chromosomal territories by the FISH technique. Simultaneous vital staining by annexin V, propidium iodide, and Hoechst 33342 was applied to distinguish apoptotic, necrotic, and intact cell fraction of tested populations. Our FISH analyses revealed that the three-dimensional (3D) structure of apoptotic nuclei as well as the 3D structure of apoptotic bodies is preserved in formaldehyde-fixed cells. High-molecular-weight DNA fragmentation was determined in apoptotic K-562 cells in contrast to oligonucleosomal cleavage observed in apoptotic HL-60 cells. In K-562 populations, chromosomal territories were located separately either in one apoptotic body or underwent disassembly into chromosomal segments dispersed into single and/or several apoptotic bodies. The apoptotic disorganization of chromosomal territories was irregular, leading mainly to chromosomal segments of different sizes and, consequently, chromosomal disassembly was not observed at specific sites. In comparison with the control, an increased number of centromeric FISH signals were observed in prolonged confluence-treated K-562 cells induced to apoptosis. This finding can be explained either as a consequence of apoptosis or by polyploidization. Sequential staining of the same apoptotic nuclei by the FISH and TUNEL techniques revealed that chromosomal territory segmentation precedes the formation of nuclear apoptotic bodies.
Cell Mol Life Sci 2003 May
PMID:Chromosomal territory segmentation in apoptotic cells. 1282 85

Protein kinase C delta (PKC delta) plays an important role in the regulation of apoptosis in response to diverse anticancer agents. PKC delta is cleaved irreversibly to a catalytically active fragment in response to apoptotic stimuli; however, little information is available about the regulation of PKC delta gene expression. In this study, we found that the amount of steady-state PKC delta mRNA and protein was increased by etoposide in mouse L1210 leukemia cells. The transcriptional rate of the PKC delta gene and the stability of PKC delta mRNA were increased by treatment with etoposide, resulting in the accumulation of PKC delta protein. Rottlerin inhibited etoposide-induced PKC delta gene expression significantly, while Go6976, LY294002 and PD98059 had no effect. Further, both stable and adenovirus-mediated expression of a dominant negative PKC delta(KR) abrogated etoposide-induced PKC delta expression. Etoposide-stimulated PKC delta transcription but not PKC delta mRNA stability was blocked completely by pretreatment with rottlerin. Our data reveal a novel mechanism whereby PKC delta gene is regulated at the transcriptional and post-transcriptional level in the L1210 leukemia cell line.
J Mol Biol 2004 Jul 16
PMID:Transcriptional and post-transcriptional regulation of the PKC delta gene by etoposide in L1210 murine leukemia cells: implication of PKC delta autoregulation. 1522 13

Etoposide is a podophyllotoxin semiderivative that is used in a variety of chemotherapy treatments, including therapy for children tumors. This drug promotes the formation of a ternary DNA-topoisomerase II-etoposide complex that triggers apoptosis. The purpose of this work was to analyze the occurrence of apoptosis in the seminiferous epithelium of prepubertal, pubertal, and adult rats treated with 10, 20, and 40 mg/Kg of etoposide during the prepubertal phase, as well as the role of apoptosis in etoposide-induced testicular damage. The rat testes were fixed in Bouin's liquid, and the apoptotic cells were quantified by means of the hematoxylin and eosin (H&E) technique (all groups) and the terminal dUTP nick end labeling (TUNEL) method (prepubertal groups only). The results obtained from both the H&E and TUNEL methods showed an increased frequency of apoptosis in the seminiferous epithelium of treated animals, except for the subgroup that received the 10-mg/Kg dose and was sacrificed 12 hr after the treatment and for the etoposide-treated pubertal group, that did not show cells suggesting apoptosis during H&E analysis. The labeled cells were mainly primary spermatocytes and differentiated spermatogonia. The prepubertal rats showed an etoposide-dose-dependent diminution of differentiated spermatogonia. Etoposide treatment during the prepubertal phase increases the frequency of apoptosis in the seminiferous epithelium, and causes serious harm to male fertility. 2004.
Anat Rec A Discov Mol Cell Evol Biol 2004 Jul
PMID:Apoptosis and testicular alterations in albino rats treated with etoposide during the prepubertal phase. 1522 3

The resistance of hypoxic cells to conventional chemotherapy is well documented. Using both adenovirus-mediated gene delivery and small molecules targeting hypoxia-inducible factor-1 (HIF-1), we evaluated the impact of HIF-1 inhibition on the sensitivity of hypoxic tumor cells to etoposide. The genetic therapy exploited a truncated HIF-1alpha protein that acts as a dominant-negative HIF-1alpha (HIF-1alpha-no-TAD). Its functionality was validated in six human tumor cell lines using HIF-1 reporter assays. An EGFP-fused protein demonstrated that the dominant-negative HIF-1alpha was nucleus-localized and constitutively expressed irrespective of oxygen tension. The small molecules studied were quinocarmycin monocitrate (KW2152), its analog 7-cyanoquinocarcinol (DX-52-1), and topotecan. DX-52-1 and topotecan have been previously established as HIF-1 inhibitors. HT1080 and HCT116 cells were treated with either AdHIF-1alpha-no-TAD or nontoxic concentrations (0.1 microM; <IC(10)) of KW2152 and DX-52-1 and exposed to etoposide in air or anoxia (<0.01% oxygen). Topotecan inhibited HIF-1 activity only at cytotoxic concentrations and was not used in the combination study. Etoposide IC(50) values in anoxia were 3-fold higher than those in air for HT1080 (2.2 +/- 0.3 versus 0.7 +/- 0.2 microM) and HCT116 (9 +/- 4 versus 3 +/- 2 microM) cells. KW2152 and DX-52-1 significantly reduced the anoxic etoposide IC(50) in HT1080 cells, whereas only KW2152 yielded sensitization in HCT116 cells. In contrast, AdHIF-1alpha-no-TAD (multiplicity of infection 50) ablated the anoxic resistance in both cell lines (IC(50) values: HT1080, 0.7 +/- 0.04 microM; HCT116, 3 +/- 1 microM). HIF-1alpha-no-TAD expression inhibited HIF-1-mediated down-regulation of the proapoptotic protein Bid under anoxia. These data support the potential development of HIF-1 targeted approaches in combination with chemotherapy, where hypoxic cell resistance contributes to treatment failure.
Mol Pharmacol 2006 Feb
PMID:Reversing hypoxic cell chemoresistance in vitro using genetic and small molecule approaches targeting hypoxia inducible factor-1. 1625 58

PDK-1 activates PI3-kinase/Akt signaling and regulates fundamental cellular functions, such as growth and survival. NF-kB is involved in the induction of a variety of cellular genes affecting immunity, inflammation and the resistance to apoptosis induced by some anti-cancer drugs. Even though the crucial involvement of the PI3-kinase/Akt pathway in the anti-apoptotic activation of NF-kB is well known, the exact role of PDK-1 as well as PI3-kinase/Akt in NF-kB activation is not understood. Here we demonstrate that PDK-1 plays a pivotal role in transcriptional activation of NF-kB by dissociating the transcriptional co-repressor HDAC1 from the p65 subunit of NF-kB. The association of CBP with p65 was not directly modulated by PDK-1 or by PI3-kinase. Etoposide activated NF-kB through PI3-kinase/Akt, and the transcription activation domain (TAD) of p65 was further activated by wild-type PDK-1. Overexpression of a dominant negative PDK-1 mutant decreased etoposide-induced NF-kB transcription and further down-regulated the ectopic HDAC1-mediated decrease in NF-kB transcriptional activity. Thus activation of PDK-1 relieves the HDAC1-mediated repression of NF-kB that may be related to basal as well as activated transcription by NF-kB. This effect may also explain the role of the PI3-kinase/PDK-1 pathway in the anti-apoptotic function of NF-kB associated with the chemoresistance of cancer cells.
Mol Cells 2005 Oct 31
PMID:PI3-kinase and PDK-1 regulate HDAC1-mediated transcriptional repression of transcription factor NF-kappaB. 1626 99

We have previously shown that the protein subunit of telomerase, hTERT, has a bonafide N-terminal mitochondrial targeting sequence, and that ectopic hTERT expression in human cells correlated with increase in mtDNA damage after hydrogen peroxide treatment. In this study, we show, using a loxP hTERT construct, that this increase in mtDNA damage following hydrogen peroxide exposure is dependent on the presence of hTERT itself. Further experiments using a dominant negative hTERT mutant shows that telomerase must be catalytically active to mediate the increase in mtDNA damage. Etoposide, but not methylmethanesulfate, also promotes mtDNA lesions in cells expressing active hTERT, indicating genotoxic specificity in this response. Fibroblasts expressing hTERT not only show a approximately 2-fold increase in mtDNA damage after oxidative stress but also suffer a 10-30-fold increase in apoptotic cell death as assayed by Annexin-V staining, caspase-3 activation and PARP cleavage. Mutations to the N-terminal mitochondrial leader sequence causes a complete loss of mitochondrial targeting without affecting catalytic activity. Cells carrying this mutated hTERT not only have significantly reduced levels of mtDNA damage following hydrogen peroxide treatment, but strikingly also do not shown any loss of viability or cell growth. Thus, localization of hTERT to the mitochondria renders cells more susceptible to oxidative stress-induced mtDNA damage and subsequent cell death, whereas nuclear-targeted hTERT, in the absence of mitochondrial localization, is associated with diminished mtDNA damage, increased cell survival and protection against cellular senescence.
Hum Mol Genet 2006 Jun 01
PMID:Mitochondrial localization of telomerase as a determinant for hydrogen peroxide-induced mitochondrial DNA damage and apoptosis. 1661 1


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