Ho-Shik Kim

Prostaglandin A2 and Δ12‐prostaglandin J2 induce apoptosis in L1210 cells

Treatment of L1210 cells with prostaglandin A2 (PGA2) or 9‐deoxy‐Δ9,12‐13,14‐dihydro PGD2 (Δ12‐PGJ2) resulted in significant G2/M arrest and subsequent DNA fragmentation at concentrations that are cytotoxic to the cells. On agarose gel electrophoresis, DNA ladder formation was evident 24 h after the addition of Δ12‐PGJ2 and remained apparent through 72 h, whereas G2/M accumulation was observed 6 h after the treatment. When the morphology of cells was examined by electron microscopy, L1210 cells incubated with a cytotoxic dose of PGA2 or Δ12‐PGJ2 for 24 h showed the characteristic morphological features of apoptosis such as chromatin condensation, nuclear fragmentation and formation of apoptotic body. Cycloheximide blocked the DNA fragmentation and morphological changes induced by Δ12‐PGJ2. Our results suggest that these cyclopentenone PGs caused apoptotic cell death of L1210 cells which is preceded by G2/M accumulation and requires de novo protein synthesis.

Intracellular glutathione level modulates the induction of apoptosis by Δ12-prostaglandin J2

We studied the effect of intracellular glutathione (GSH), which was known to conjugate readily with an alpha, beta-unsaturated carbonyl of 9-deoxy-delta 9,12-13,14-dihydroPGD2 (delta 12-PGJ2), on the cytotoxicity of delta 12-PGJ2. delta 12-PGJ2 caused DNA fragmentation in human hepatocellular carcinoma Hep 3B cells, which was blocked by cycloheximide (CHX). The delta 12-PGJ2-induced apoptosis was augmented by GSH depletion resulted from pretreatment with buthioninine sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase. On the contrary, N-acetyl-cysteine (NAC), a precursor of cysteine, elevated the GSH level and protected cells from initiating apoptosis by delta 12-PGJ2. Sodium arsenite, a thiol-reactive agent, also induced apoptosis, which was potentiated or attenuated by BSO or NAC treatment respectively. These results suggest that the apoptosis-inducing activity of delta 12-PGJ2 is due to thiol-reactivity and intracellular GSH modulates the delta 12-PGJ2-induced apoptosis by regulating the accessibility of delta 12-PGJ2 to target proteins containing thiol groups.

Sox-4 is a positive regulator of Hep3B and HepG2 cells' apoptosis induced by prostaglandin (PG)A2 and Δ12-PGJ2

We reported earlier that expression of Sox-4 was found to be elevated during prostaglandin (PG) A2 and Δ12-PGJ2 induced apoptosis in human hepatocarcinoma Hep3B cells. In this study, the role of Sox-4 was examined using human Hep3B and HepG2 cell lines. Sox-4 induction by several apoptotic inducer such as A23187 (Ca2+ ionophore) and etoposide (topoisomerase II inhibitor) and Sox-4 transfection into the cells were able to induce apoptosis as observed by the cellular DNA fragmentation. Antisense oligonucleotide of Sox-4 inhibited the induction of Sox-4 expression and blocked the formation of DNA fragmentation by PGA2 and Δ12-PGJ2 in Hep3B and HepG2 cells. Sox-4-induced apoptosis was accompanied with caspase-1 activation indicating that caspase cascade was involved in this apoptotic pathway. These results indicate that Sox-4 is involved in Hep3B and HepG2 cells apoptosis as an important apoptotic mediator.

Induction of unfolded protein response during neuronal induction of rat bone marrow stromal cells and mouse embryonic stem cells

When we treated rat bone marrow stromal cells (rBMSCs) with neuronal differentiation induction media, typical unfolded protein response (UPR) was observed. BIP/GRP78 protein expression was time-dependently increased, and three branches of UPR were all activated. ATF6 increased the transcription of XBP1 which was successfully spliced by IRE1. PERK was phosphorylated and it was followed by eIF2α phosphorylation. Transcription of two downstream targets of eIF2α, ATF4 and CHOP/GADD153, were transiently up-regulated with the peak level at 24 h. Immunocytochemical study showed clear coexpression of BIP and ATF4 with NeuN and Map2, respectively. UPR was also observed during the neuronal differentiation of mouse embryonic stem (mES) cells. Finally, chemical endoplasmic reticulum (ER) stress inducers, thapsigargin, tunicamycin, and brefeldin A, dose-dependently increased both mRNA and protein expressions of NF-L, and, its expression was specific to BIP-positive rBMSCs. Our results showing the induction of UPR during neuronal differentiations of rBMSCs and mES cells as well as NF-L expression by ER stress inducers strongly suggest the potential role of UPR in neuronal differentiation.

Induction of p53 and apoptosis by δ 12-PGJ2 in human hepatocarcinoma SK-HEP-1 cells

Human hepatocarcinoma cells (SK‐HEP‐1) were induced to die through apoptosis by treatment with Δ 12‐prostaglandin (PG)J2, as characterized by the appearance of a typical DNA ladder. The induction of apoptosis by Δ 12‐PGJ2 was specifically blocked by cycloheximide (CHX). Western analysis using anti‐p53 or anti‐WAF1 monoclonal antibodies demonstrated that these two protein levels were increased 3 h after Δ 12‐PGJ2 treatment, and accumulated for up to 12 h. The induction of p53 protein seemed to be dependent on the increase of p53 mRNA level, which was inhibited by CHX treatment. However, delayed addition of CHX after Δ 12‐PGJ2 treatment failed to affect both p53 mRNA levels and DNA fragmentation following Δ 12‐PGJ2 treatment, indicating that the inhibition of p53 synthesis may contribute to the protective effect of CHX against Δ 12‐PGJ2‐mediated cytotoxicity. Therefore, our results suggest that the initial events caused by Δ 12‐PGJ2, leading ultimately to SK‐HEP‐1 cell death, involve a certain process required for p53 induction. However, the finding that Δ 12‐PGJ2 is also active against Hep 3B cells which are devoid of a functional p53 indicates that p53 may not be the critical requirement for inducing apoptosis by Δ 12‐PGJ2.

Activation of caspase-8 in 3-deazaadenosine-induced apoptosis of U-937 cells occurs downstream of caspase-3 and caspase-9 without Fas receptor-ligand interaction

3-Deazaadenosine (DZA), a cellular methylation blocker was reported to induce the caspase-3-like activities-dependent apoptosis in U-937 cells. In this study, we analyzed the activation pathway of the caspase cascade involved in the DZA-induced apoptosis using specific inhibitors of caspases. In the U-937 cells treated with DZA, cytochrome c release from mitochondria and subsequent activation of caspase-9, -8 and -3 were observed before the induction of apoptosis. zDEVD-Fmk, a specific inhibitor of caspase-3, and zLEHD-Fmk, a specific inhibitor of caspase-9, prevented the activation of caspase-8 but neither caspase-3 nor caspase-9, indicating that caspase-8 is downstream of both caspase-3 and caspase-9, which are activated by independent pathways. zVAD-Fmk, a universal inhibitor of caspases, kept the caspase-3 from being activated but not caspase-9. Moreover, ZB4, an antagonistic Fas-antibody, exerted no effect on the activation of caspase-8 and induction of apoptosis by DZA. In addition, zVAD-Fmk and mitochondrial permeability transition pore (MPTP) inhibitors such as cyclosporin A (CsA) and bongkrekic acid (BA) did not block the release of cytochrome c from mitochondria. Taken together, these results suggest that in the DZA-induced apoptosis, caspase-8 may serve as an executioner caspase and be activated downstream of both caspase-3 and caspase-9, independently of Fas receptor-ligand interaction. And caspase-3 seems to be activated by other caspses including IETDase-like enzyme and caspse-9 seems to be activated by cytochrome c released from mitochondria without the involvement of caspases and CsA- and BA- inhibitory MPTP.

Silencing of KIF14 interferes with cell cycle progression and cytokinesis by blocking the p27(Kip1) ubiquitination pathway in hepatocellular carcinoma.

Although it has been suggested that kinesin family member 14 (KIF14) has oncogenic potential in various cancers, including hepatocellular carcinoma (HCC), the molecular mechanism of this potential remains unknown. We aimed to elucidate the role of KIF14 in hepatocarcinogenesis by knocking down KIF14 in HCC cells that overexpressed KIF14. After KIF14 knockdown, changes in tumor cell growth, cell cycle and cytokinesis were examined. We also examined cell cycle regulatory molecules and upstream Skp1/Cul1/F-box (SCF) complex molecules. Knockdown of KIF14 resulted in suppression of cell proliferation and failure of cytokinesis, whereas KIF14 overexpression increased cell proliferation. In KIF14-silenced cells, the levels of cyclins E1, D1 and B1 were profoundly decreased compared with control cells. Of the cyclin-dependent kinase inhibitors, the p27Kip1 protein level specifically increased after KIF14 knockdown. The increase in p27Kip1 was not due to elevation of its mRNA level, but was due to inhibition of the proteasome-dependent degradation pathway. To explore the pathway upstream of this event, we measured the levels of SCF complex molecules, including Skp1, Skp2, Cul1, Roc1 and Cks1. The levels of Skp2 and its cofactor Cks1 decreased in the KIF14 knockdown cells where p27Kip1 accumulated. Overexpression of Skp2 in the KIF14 knockdown cells attenuated the failure of cytokinesis. On the basis of these results, we postulate that KIF14 knockdown downregulates the expression of Skp2 and Cks1, which target p27Kip1 for degradation by the 26S proteasome, leading to accumulation of p27Kip1. The downregulation of Skp2 and Cks1 also resulted in cytokinesis failure, which may inhibit tumor growth. To the best of our knowledge, this is the first report that has identified the molecular target and oncogenic effect of KIF14 in HCC.

Apoptosis of L1210 leukemia cells induced by 3-deazaadenosine analogs: Differential expression of c-myc, NF-kappa B and molecular events

A new class of potent apogens (apoptosis-inducing agents) has been identified, consisting of 3-deazaadenosine (DZA), 3-deaza-(+/-)aristeromycin (DZAri) and 1-beta-D-arabinofuranosyl-1H-imidazo[4,5-&cumacr;]pyridine (ara-3-deazaadenine; DZAra-A). They are inhibitors of S-adenosylhomocysteine hydrolase and indirect inhibitors of methylation. Furthermore, they have also been found to form 3-deaza-nucleotide analogs. The DZA analogs, DZA, DZAri, and DZAra-A, induced DNA fragmentation in a dose- and time-dependent manner, reaching a maximum at 250 &mgr;M after 72 h. Cycloheximide at 0.5 &mgr;g/ml completely blocked the DNA fragmentation induced by 250 &mgr;M of each of the analogs. Interestingly, exogenous 100 &mgr;M L-homocysteine thiolactone abrogated the DNA fragmentation caused by DZAri and DZAra-A, but not by DZA. Flow cytometric analysis showed that DZA arrested the cells in the G(2)/M phase, whereas the S phase was arrested by DZAri. Correlated with the effect of DZA was a rapid decrease in the expression of c-myc, whereas nur77 and GAPDH were unaffected. In comparison, there was an elevated expression of IFN-gamma mRNA without apparent change in bax, p53 or GAPDH mRNA after 24 h. After treatment with DZA, there was an elevated expression of NF-kappaB DNA binding activity, which became more pronounced at 24 h. Simultaneously, there was an apparent disappearance of AP-1 activity. Thus, DZA most likely inhibited the RNA synthesis of c-myc, a reduction of which could trigger a cascade of gene transcription leading to apoptosis in L1210 cells. Copyright 1997 S. Karger AG, Basel

Involvement of Sox-4 in the cytochrome c-dependent AIF-independent apoptotic pathway in HeLa cells induced by Delta12-prostaglandin J2.

Δ12-Prostaglandin (PG) J2 is known to elicit an anti-neoplastic effects via apoptosis induction. Previous study showed Δ12-PGJ2-induced apoptosis utilized caspase cascade through cytochrome c-dependent pathways in HeLa cells. In this study, the cellular mechanism of Δ12-PGJ2- induced apoptosis in HeLa cells, specifically, the role of two mitochondrial factors; bcl-2 and apoptosis-inducing factor (AIF) was investigated. Bcl-2 attenuated Δ12-PGJ2-induced caspase activation, loss of mitochondrial transmembrane potential (Δ ψ m), nuclear fragmentation, DNA laddering, and growth curve inhibition for approximately 24 h, but not for longer time. AIF was not released from mitochondria, even if the Δ ψ m was dissipated. One of the earliest events observed in Δ12-PGJ2-induced apoptotic events was dissipation of Δ ψ m, the process known to be inhibited by bcl-2. Pre-treatment of z-VAD- fmk, the pan-caspase inhibitor, resulted in the attenuation of Δ ψ m depolarization in Δ12-PGJ2- induced apoptosis. Up-regulation of Sox-4 protein by Δ12-PGJ2 was observed in HeLa and bcl-2 overexpressing HeLa B4 cell lines. Bcl-2 overexpression did not attenuate the expression of Sox-4 and its expression coincided with other apoptotic events. These results suggest that Δ12-PGJ2 induced Sox-4 expression may activate another upstream caspases excluding the caspase 9-caspase 3 cascade of mitochondrial pathway. These and previous findings together suggest that Δ12-PGJ2-induced apoptosis in HeLa cells is caspase-dependent, AIF-independent events which may be affected by Sox-4 protein expression up-regulated by Δ12-PGJ2.

Induction of apoptosis and c-myc in L1210 lymphocytic leukemia cells by adenosine

High concentrations of adenosine (Ado), when added to L1210 lymphocytic leukemia cells, resulted in apoptosis or programmed cell death. The apoptotic process was accompanied by distinct morphological changes including chromatin condensation and blebbing of plasma membranes. Extensive DNA fragmentation was correlated with Ado concentrations. Furthermore, apoptosis in these cells was preceded by an early but transient expression of c-myc proto-oncogene, and was not influenced by homocysteine thiolactone added to the cells. Since severe combined immunodeficiency (SCID) is associated with a deficiency of adenosine deaminase, leading to defects in both cellular and humoral immunity, Ado-induced apoptosis may thus be a contributing factor in the pathology of SCID. Copyright 1994 S. Karger AG, Basel