Induction of apoptosis in human leukaemic cells by IPENSpm, a novel polyamine analogue and anti-metabolite.
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ABSTRACT:
SUBMITTER: Fraser AV
PROVIDER: S-EPMC1222862 | biostudies-other | 2002 Oct
REPOSITORIES: biostudies-other
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Project description:IntroductionID1, founding member of the inhibitor of differentiation (ID) family, is involved in cell population growth, apoptosis and tumourigenesis.Methods and resultsWe investigated mRNA levels of ID1 in human myeloid leukaemic cell lines and in specimens of patients with acute myeloid leukaemia (AML), using semiquantitative reverse transcription-polymerase chain reaction, and protein levels of ID1 in human myeloid leukaemic cell lines using Western blot analysis. Six of seven AML cell lines and 12 of 15 AML patient samples were found to have barely detectable ID1 mRNA. All of these cell lines showed the same levels of protein in proportion to levels of mRNA. Two of the AML cell lines with low ID1 expression, KG1 and KG-1a, were chosen for treatment with either the DNA demethylation reagent, 5-aza-2'-deoxycytidine (DAC), or the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). These treatments were alone or in combination, and ID1 expression was induced by both DAC and TSA. No hypermethylated ID1 gene promoter was detected in the majority of the cell lines and patient specimens, by methylation-specific polymerase chain reaction, suggesting that induction of ID1 in KG1 and KG-1a was not due to direct demethylation of the ID1 gene promoter. Chromatin immunoprecipitation showed that accumulation of acetyl-histone H3 and release of HDAC1 were correlated with ID1 induction by these drugs. Flow cytometric assay demonstrated more apoptosis induced by TSA or TSA in combination with DAC, in both KG-1 and KG-1a cell lines. Increase of intracellular reactive oxygen species was observed when treated with TSA.ConclusionMost AML cell lines and human AML samples have very low levels of expression of ID1. TSA or TSA in combination with DAC is able to restore ID1 expression in low ID1-expressing AML cell lines by re-activating the aberrantly deacetylated promoter, and this also results in more apoptotic cell death, in which ID1 and the redox pathway may be involved.
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Project description:The topoisomerase II inhibitor etoposide induced apoptosis in HL-60 cells within 4 h of exposure to the drug, as measured by changes in morphology, DNA fragmentation and cytotoxicity assays. Etoposide-induced apoptosis was accompanied by an increase in polyamine efflux from the cells and a decrease in total polyamine content during the first 24 h of exposure to the drug. Although both enzyme activities increased slightly, there were no significant changes in spermidine/spermine N1-acetyltransferase activity or polyamine oxidase activity. After longer exposures (48-72 h), significant induction of spermidine/spermine N1-acetyltransferase activity and loss of polyamine content occurred. These results suggest that polyamine oxidation and the resultant hydrogen peroxide produced may be associated with the initiation of apoptosis, while induction of the acetyltransferase and overall loss of intracellular polyamines may be involved in the final, possibly necrotic, stages of cell death.
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Project description:The HMGA2 (high-mobility group AT-hook) protein has previously been shown as an oncoprotein, whereas ectopic expression of HMGA2 is found to induce growth arrest in primary cells. The precise mechanisms underlying this phenomenon remain to be unravelled. In the present study, we determined that HMGA2 was able to induce apoptosis in WI38 primary human cells. We show that WI38 cells expressing high level of HMGA2 were arrested at G2/M phase and exhibited apoptotic nuclear phenotypes. Meanwhile, the cleaved caspase 3 (cysteine aspartic acid-specific protease 3) was detected 8 days after HMGA2 overexpression. Flow cytometric analysis confirmed that the ratio of cells undergoing apoptosis increased dramatically. Concurrently, other major apoptotic markers were also detected, including the up-regulation of p53, Bax and cleaved caspase 9, down-regulation of Bcl-2; as well as release of cytochrome c from the mitochondria. We further demonstrate that the shRNA (small-hairpin RNA)-mediated Apaf1 (apoptotic protease activating factor 1) silencing partially rescued the HMGA2-induced apoptosis, which was accompanied by the decrease of cleaved caspase-3 level and a decline of cell death ratio. Our results also reveal that γH2A was accumulated in nuclei during the HMGA2-induced apoptosis along with the up-regulation of cleaved caspase 2, suggesting that the HMGA2-induced apoptosis was dependent on the pathway of DNA damage. Overall, the present study unravelled a novel function of HMGA2 in induction of apoptosis in human primary cell lines, and provided clues for clarification of the mechanistic action of HMGA2 in addition to its function as an oncoprotein.
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Project description:BackgroundCandidaspongiolide (CAN), a novel polyketide from a marine sponge, is the active component of a mixture that was found to be potently cytotoxic in the National Cancer Institute's 60-cell-line screen.MethodsEffects of CAN on U251 glioma and HCT116 colorectal cancer cells and on normal fibroblasts were assessed using radiolabeling studies to measure protein synthesis, clonogenic assays to measure cell survival, flow cytometry of annexin V- and propidium iodide-stained cells to measure apoptosis, and western blots in the presence or absence of specific inhibitors to assess accumulation and phosphorylation of potential downstream target proteins.ResultsCAN inhibited protein synthesis and potently induced apoptosis in both U251 and HCT116 cells, the latter in part by a caspase 12-dependent pathway. For example, 25%-30% of U251 or HCT116 cells became apoptotic after 24 hours of treatment with 100 nM CAN. CAN also rapidly induced sustained phosphorylation of eukaryotic translation initiation factor-2 (eIF2)-alpha at Ser51 and of the translation elongation factor eEF2 at Thr56, which could contribute to its dose-dependent inhibition of protein synthesis. Stable expression of dominant-negative eIF2alpha was sufficient to prevent CAN-induced eIF2alpha phosphorylation and induction of apoptosis but insufficient to prevent inhibition of protein synthesis. CAN induction of eIF2alpha phosphorylation did not occur by a classic endoplasmic reticulum stress pathway. However, an inhibitor of and small-interfering RNAs to the double-stranded RNA-dependent protein kinase PKR prevented CAN-mediated eIF2alpha phosphorylation and apoptosis, respectively. Although CAN inhibited protein synthesis in both cancer cells and normal human fibroblasts, it induced eIF2alpha phosphorylation and apoptosis only in cancer cells.ConclusionsCAN triggers PKR/eIF2alpha/caspase 12-dependent apoptosis and inhibits protein synthesis in cancer cells but only inhibits protein synthesis in normal cells.
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Project description:BACKGROUND:Glioblastoma is the most aggressive form of brain tumors showing resistance to treatment with various chemotherapeutic agents. The most effective way to eradicate glioblastoma requires the concurrent inhibition of multiple signaling pathways and target molecules involved in the progression of glioblastoma. Recently, we obtained a series of 1,2,3,4-tetrahydroisoquinoline alkaloids with potent anti-cancer activities, including ecteinascidin-770 (ET-770; the compound 1a) and renieramycin M (RM; the compound 2a) from Thai marine invertebrates, together with a 2'-N-4"-pyridinecarbonyl derivative of ET-770 (the compound 3). We attempted to characterize the molecular pathways responsible for cytotoxic effects of these compounds on a human glioblastoma cell line U373MG. METHODS:We studied the genome-wide gene expression profile on microarrays and molecular networks by using pathway analysis tools of bioinformatics. RESULTS:All of these compounds induced apoptosis of U373MG cells at nanomolar concentrations. The compound 3 reduced the expression of 417 genes and elevated the levels of 84 genes, while ET-770 downregulated 426 genes and upregulated 45 genes. RM decreased the expression of 274 genes and increased the expression of 9 genes. The set of 196 downregulated genes and 6 upregulated genes showed an overlap among all the compounds, suggesting an existence of the common pathways involved in induction of apoptosis. We identified the ErbB (EGFR) signaling pathway as one of the common pathways enriched in the set of downregulated genes, composed of PTK2, AKT3, and GSK3B serving as key molecules that regulate cell movement and the nervous system development. Furthermore, a GSK3B-specific inhibitor induced apoptosis of U373MG cells, supporting an anti-apoptotic role of GSK3B. CONCLUSION:Molecular network analysis is a useful approach not only to characterize the glioma-relevant pathways but also to identify the network-based effective drug targets.
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