Combined use of hyperthermia and irradiation cause antiproliferative activity and cell death to human esophageal cell carcinoma cells--mainly cell cycle examination

In clinically hyperthermia and irradiation therapy for malignant neoplasms are known that they have antiproliferative activity and cell death (including apoptosis) inducing activity. However not only mechanisms of cell death induction but treatment effects of them still have been unclear. In this time we showed that cell cycles from G0/G1 phase to S-G2/M phase were delayed by hyperthermia and G2/M phase accumulation were caused immediately by irradiation. And we also demonstrated that the combination treatments of hyperthermia and irradiation induced synergistic antiproliferative effects and strong effects of cell death to human esophageal carcinoma cell lines. Although treatments of hyperthermia and irradiation were mild individually, combination treatment of hyperthermia and irradiation were useful for esophageal carcinoma treatment.     

Delta 12-prostaglandin J2 mimics heat shock in inducing cell cycle arrest at G1 phase

Using a human neuroblastoma cell line GOTO, the effects of delta 12-prostaglandin (PG) J2 on the modulation of cell cycle progression and protein synthesis were examined in comparison with those caused by heat shock (HS). delta 12-PGJ2 induced G1 arrest, the peak of which was obtained at 24 h and continued for 72 h. HS was found to induce G1 arrest earlier than delta 12-PGJ2. Furthermore, sequential HS could maintain G1 arrest. delta 12-PGJ2 induced the synthesis of several heat shock proteins (HSPs) in a manner similar to HS. Using immunoblot analysis, HSP72 was detected prior to inducing G1 arrest and accumulated during the subsequent 72h. The content of HSP72 induced by HS also correlated well with the induction, release, and maintenance of G1 arrest. In addition, both delta 12-PGJ2 and HS induced HSP72 mRNA and simultaneously suppressed N-myc mRNA expression. These results suggest that delta 12-PGJ2 and HS regulate cell cycle progression of GOTO cells via similar mechanisms.     

Induction of heme oxygenase by delta 12-prostaglandin J2 in porcine aortic endothelial cells.

delta 12-Prostaglandin (PG)J2 stimulated the synthesis of a 31,000-dalton protein (termed p31) and the induction of cellular heme oxygenase activity in porcine aortic endothelial cells. A good correlation was observed between the time courses and dose dependencies of the induction of p31 synthesis and that of heme oxygenase activity by delta 12-PGJ2. Hemin, a known inducer of heme oxygenase, also induced p31 synthesis as well as heme oxygenase activity in the cells. On two-dimensional gel electrophoresis, p31 induced by delta 12-PGJ2 exhibited an isoelectric point of 5.4, which coincided exactly with that induced by hemin. These results indicate that the p31 induced by delta 12-PGJ2 in porcine aortic endothelial cells is heme oxygenase.     

Cycloheximide reduces PGD2 or delta 12-PGJ2 cytotoxicity on NCG cells

To study the precise mechanism of cytotoxic activity of PGD2 or delta 12-PGJ2 (a biologically active metabolite of PGD2), we examined the effect of various compounds on PGD2 or delta 12-PGJ2 cytotoxicity, using a human neuroblastoma cell line (NCG). Cycloheximide (CHM) specifically protected PGD2 cytotoxicity on NCG cells. When delta 12-PGJ2 was tested, CHM exhibited a similar rescue effect. Puromycin, mitomycin C, and alpha-amanitin did not affect PGD2 or delta 12-PGJ2 cytotoxicity. Emetine showed a variable and no consistent rescue effect CHM may have been active at the primary site where PGD2 or delta 12-PGJ2 exerts its cytotoxicity. This is the first report indicating that CHM reduces the cytotoxicity induced by PGD2 or delta 12-PGJ2.     

Induction of 68,000-dalton heat shock proteins by cyclopentenone prostaglandins. Its association with prostaglandin-induced G1 block in cell cycle progression

Cyclopentenone prostaglandins (PGs) such as PGA2 and delta 12-PGJ2 act specifically on cells in the G1 phase and induce block of cell cycle progression (Ohno, K., Sakai, T., Fukushima, M., Narumiya, S., and Fujiwara, M. (1988) J. Pharmacol. Exp. Ther. 245, 294-298). In this study, we characterized proteins induced by these PGs in HeLa S3 cells of synchronized growth and examined its association with the cell cycle block. HeLa S3 cells transiently expressed two 68-kDa proteins of isoelectric points of 5.5 and 5.6 in the G1 phase of cell cycle. When G1-enriched cells were incubated with either PGA2 or delta 12-PGJ2, synthesis of these proteins was markedly enhanced. Enhancement by delta 12-PGJ2 was persistent and irreversible, whereas that by PGA2 was reversible. delta 12-PGJ2 also enhanced the synthesis of two additional 68-kDa proteins with isoelectric points of 5.8 and 5.9. On two-dimensional gel electrophoresis, these proteins overlapped exactly with the 68-kDa heat shock proteins induced in cells treated at 43 degrees C for 90 min. They were also indistinguishable from the heat shock proteins in limited proteolysis. When delta 12-PGJ2 was incubated with G2/M phase cells, it induced only a small and transient increase in the 68-kDa proteins. These results suggest that cyclopentenone PGs extensively induce 68-kDa heat shock proteins in the G1 phase HeLa S3 cells and this induction is closely associated with the G1 block of cell cycle progression caused by these PGs.     

delta 12-Prostaglandin J2, an ultimate metabolite of prostaglandin D2 exerting cell growth inhibition

L-1210 murine leukemia cells were exposed to prostaglandin D2 (PGD2), 10 micrograms/ml, in culture medium for various time, and subsequent cell growth was observed. More than 24 h exposure to PGD2 was required to inhibit cell growth almost completely. During this period, PGD2 degraded time-dependently into several products. The major product was identified as delta 12-PGJ2 by TLC, UV and mass spectra. When delta 12-PGJ2 was added to cells instead of PGD2, it evoked growth inhibition with much shorter contact time than PGD2. In addition, when the medium containing PGD2 was preincubated at 37 degrees C for 24 h, it elicited growth inhibition with only 6 h contact with cells. Furthermore, when the medium containing PGD2 was changed every 6 h during 24 h exposure time to cells, no significant growth inhibition was observed. These results suggested that PGD2 per se has little, if any, growth inhibitory activity, and delta 12-PGJ2 is an ultimate metabolite exerting growth inhibition. This action appears to be independent of cAMP, since delta 12-PGJ2 was virtually inactive in raising intracellular cAMP levels.     

Selective synthesis and retention of 66k protein in a human neuroblastoma cell line (NCG) treated with a cytotoxic dosage of delta 12-prostaglandin J2

delta 12-prostaglandin(PG)J2 (7.5 micrograms/ml) significantly inhibited protein synthesis and cell growth in a human neuroblastoma cell line (NCG), decreasing these factors by 31.5% and 78.2% of the control values, respectively. Two protein synthesis inhibitors, cycloheximide (CHM) and emetine, exhibited a dose-dependent protective effect for neuroblastoma cells against delta 12-PGJ2 cytotoxicity. At a concentration of 15 micrograms/ml CHM, the number of viable cells increased from 21.8% to 36.7% of the control value (p less than 0.01). The sodium dodecyl sulfate-polyacrylamide gel analysis of [35S]methionine-incorporated proteins revealed an increased synthesis of 86k, 70k and 66k proteins in the delta 12-PGJ2-treated NCG cells under the condition that delta 12-PGJ2 exerts cytotoxicity. Of these proteins, the amount of 66k protein was particularly increased in cell cytosol; however, its synthesis did not occur when CHM prohibited the delta 12-PGJ2 cytotoxic effect. When emetine was used instead of CHM, similar results were obtained. These results strongly suggest that the 66k protein plays a critical role in the delta 12-PGJ2 cytotoxicity.     

Occurrence of 9-deoxy-delta 9,delta 12-13,14-dihydroprostaglandin D2 in human urine

We have developed a highly sensitive and specific solid-phase enzyme immunoassay for 9-deoxy-delta 9,delta 12-dihydroprostaglandin D2 (delta 12-PGJ2) and studied the occurrence of this novel PGD2 metabolite in human urine. The assay detected delta 12-PGJ2 over the range of 2-200 pg, and the antiserum showed 2% cross-reaction with PGJ2 and less than 0.2% with other PGs. We used this assay and purified the delta 12-PGJ2-like immunoreactive substance from human urine. Purification consisted of chromatographies on a Sep-Pak C18 cartridge, a silicic acid column, reversed-phase high-performance liquid chromatography, and finally an affinity column of anti-delta 12-PGJ2 antibody. As a result, about 850 ng of delta 12-PGJ2-like immunoreactive substance were recovered from 60 liters of human urine. The purified material was identified as delta 12-PGJ2 by gas chromatography/high resolution-selected ion monitoring using the molecular ion m/z 448[M]+. and ions [M - 15]+, [M - 43]+, [M - 100]+., and [M - 143]+. The amounts of delta 12-PGJ2 in the urine from normal, volunteer men and women were 151.5 +/- 20.0 and 65.6 +/- 5.4 ng/24 h (mean +/- S.E., n = 5), respectively. The delta 12-PGJ2 amount in urine did not alter significantly during storage for at least 24 h or by the addition of authentic PGD2 to urine samples, suggesting that the delta 12-PGJ2 we determined was not derived from the decomposition of PGD2 in the urine during storage or purification. Moreover, when a single dose of PGD2 (1 mg/kg) was injected intravenously into cynomolgus monkeys, the urinary level of delta 12-PGJ2 increased 20- to 180-fold over the normal levels, whereas the delta 12-PGJ2 level decreased by 40-50% of the normal levels, following the administration of indomethacin at a dose of 1 mg/kg. These results indicate that delta 12-PGJ2 is formed naturally in the body and excreted as a urinary PGD2 metabolite.     

15-deoxy-delta12,14-prostaglandin J2-induced apoptosis in amnion-like WISH cells.

Apoptosis at the site of rupture has been proposed to play a role in premature rupture of the fetal membranes, a condition associated with increased risk of neonatal sepsis and preterm birth. We investigated the ability of peroxisome proliferator-activated receptor (PPAR)-gamma ligands 15-deoxy-delta12,14PGJ2 (15d-PGJ2), delta12PGJ2, ciglitizone and rosiglitazone to induce apoptosis in the amnion-like WISH cell line. 15d-PGJ2 (10 microM) induced morphological characteristics of apoptosis within 2 h, with biochemical indices (caspase activation and substrate cleavage) following shortly after; maximum cell death (approximately 60%) was observed by 16 h, with an EC50) of approximately 7 microM 15d-PGJ2. Delta12-PGJ2 also induced apoptosis but was less potent and acted at a much slower rate. While ciglitizone also induced apoptosis, rosiglitazone had no effect on cell viability. The mechanism of induction of apoptosis by 15d-PGJ2 and delta12PGJ2, which may be independent of PPAR-gamma activation, requires further elucidation.     

Differential effects of serum constituents on apoptosis induced by the cyclopentenone prostaglandin 15-deoxy-delta12,14-prostaglandin J2 in WISH epithelial cells

Cyclopentenone prostaglandins, delta12-PGJ2 and 15d-PGJ2, have potent anti-tumour and anti-inflammatory activities, and have been shown to induce apoptosis in amnion-derived WISH cells. In this study, we have investigated the protective effects of serum and its constituents (growth factors and albumin) on delta12-PGJ2 and 15d-PGJ2-induced apoptosis in WISH cells. Serum (0.5% w/v) was protective against both delta12-PGJ2 and 15d-PGJ2-induced apoptosis. This was not due to the presence of serum-derived growth factors (EGF, IGF-1 and IGF-2), since they had no significant effect on 15d-PGJ2-induced cell death. In contrast, IGF-1 partially inhibited etoposide-induced apoptosis, confirming the presence of a functional IGF-1 receptor signalling system. Albumin was identified as the key survival factor in serum, since albumin and delipidated albumin exhibited the same level of protection from 15d-PGJ2-induced apoptosis as serum itself. The potential for serum albumin to regulate the bioactivity of cyclopentenone PGs may be of considerable importance in pathological conditions where roles for cyclopentenone PGs have been identified.     

PGJ2 and delta 12PGJ2 inhibit cell growth accompanied with inhibition of phosphoinositide turnover in human astrocytoma cells

PGJ2 and delta 12PGJ2 (1 microM to 30 microM) inhibited the growth of human astrocytoma cells (1321N1) in a time-dependent manner within 48 hrs, determined by [3H]thymidine incorporation into acid-insoluble fraction or amounts of protein. The EC50 values for PGJ2 and delta 12PGJ2 were approximately 8 microM and 6 microM, respectively. [3H]Thymidine incorporation to acid insoluble fraction was inhibited by these PGs within 1 hr, indicating that these PGs rapidly affect cell functions. Although it has been reported that an increase in cyclic AMP inhibits cell growth, PGJ2 and delta 12PGJ2, but not PGE1, reduced isoproterenol (10 microM)-induced accumulation of cyclic AMP, suggesting that PGJ2 and delta 12PGJ2 may disturb adenylate cyclase system, which might be independent on cell growth. On the other hand, these PGs inhibited the incorporation of [3H]inositol into phospholipid fraction within 6 hrs. Furthermore, PGJ2 and delta 12PGJ2 inhibited carbachol- and/or histamine-induced accumulation of inositol phosphates with a similar dose-dependency to their inhibitions of cell growth. In membrane preparations, however, PGJ2 and delta 12PGJ2 failed to inhibit GTP gamma S (10 microM)- nor Ca2+ (1 mM)-induced accumulation of inositol phosphates. The site of PGJ2 or delta 12PGJ2 in inhibition of inositol phosphate accumulation would not be phospholipase C nor a putative GTP binding protein involved in activation of phospholipase C. The present results indicate that PGJ2 and delta 12PGJ2 inhibit cell growth in human astrocytoma cells and the inhibition of phosphoinositide turnover by these PGs might be involved in the inhibition of cell growth.     

Delta 12-prostaglandin J2, a plasma metabolite of prostaglandin D2, causes eosinophil mobilization from the bone marrow and primes eosinophils for chemotaxis

PGD(2), a major mast cell mediator, is a potent eosinophil chemoattractant and is thought to be involved in eosinophil recruitment to sites of allergic inflammation. In plasma, PGD(2) is rapidly transformed into its major metabolite delta(12)-PGJ(2), the effect of which on eosinophil migration has not yet been characterized. In this study we found that delta(12)-PGJ(2) was a highly effective chemoattractant and inducer of respiratory burst in human eosinophils, with the same efficacy as PGD(2), PGJ(2), or 15-deoxy-delta(12,14)-PGJ(2). Moreover, pretreatment of eosinophils with delta(12)-PGJ(2) markedly enhanced the chemotactic response to eotaxin, and in this respect delta(12)-PGJ(2) was more effective than PGD(2). delta(12)-PGJ(2)-induced facilitation of eosinophil migration toward eotaxin was not altered by specific inhibitors of intracellular signaling pathways relevant to the chemotactic response, phosphatidylinositol 3-kinase (LY-294002), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (U-0126), or p38 mitogen-activated protein kinase (SB-202190). Desensitization studies using calcium flux suggested that delta(12)-PGJ(2) signaled through the same receptor, CRTH2, as PGD(2). Finally, delta(12)-PGJ(2) was able to mobilize mature eosinophils from the bone marrow of the guinea pig isolated perfused hind limb. Given that delta(12)-PGJ(2) is present in the systemic circulation at relevant levels, a role for this PGD(2) metabolite in eosinophil release from the bone marrow and in driving eosinophil recruitment to sites of inflammation appears conceivable.     

热疗联合一氧化氮供体硝酸异山梨酯诱导Hep-A细胞凋亡的研究

To investigate the apoptosis of Hep-A cells induced by hyperthermia combined with Nitric Oxide donor (Isosorbide dinitrate, ISDN) and its mechanism. The inhibitory effect on the growth of Hep-A cells was measured by MTT assay. Apoptosis of Hep-A cells was observed by electron microscopy and flow cytometry. The levels of Bcl-2 were detected with Western blot assay. It showed stronger antiproliferative ability in three experimental groups than that in control, and hyperthermia combined with ISDN group had better inhibitory effect than other groups (p < 0.05). With electron microscopy, marked changes of cell apoptosis were observed, including microvilli disappearance or reduction, cell shrinkage, chromatin condensation or margination and the presence of "apoptosis bodies". The apoptotic ratio induced by hyperthermia and ISDN group was higher than other groups, furthermore, the levels of Bcl-2 were decreased in three experimental groups. The present study indicated that hyperthermia combined with ISDN could induce apoptosis of Hep-A cells and be more effective than either hyperthermia or ISDN, which may be related to expression decreased Bcl-2.     

Phospholipase C delta 1 regulates cell proliferation and cell-cycle progression from G1- to S-phase by control of cyclin E-CDK2 activity

In the present study, we examined the role of PLC delta 1 (phospholipase C delta 1) in the regulation of cellular proliferation. We demonstrate that RNAi (RNA interference)-mediated knockdown of endogenous PLC delta 1, but not PLC beta 3 or PLC epsilon, induces a proliferation defect in Rat-1 and NIH 3T3 fibroblasts. The decreased proliferation was not due to an induction of apoptosis or senescence, but was associated with an approx. 60% inhibition of [(3)H]thymidine incorporation. Analysis of the cell cycle with BrdU (bromodeoxyuridine)/propidium iodide-labelled FACS (fluorescence-activated cell sorting) demonstrated an accumulation of cells in G(0)/G(1)-phase and a corresponding decrease in cells in S-phase. Further examination of the cell cycle after synchronization by serum-starvation demonstrated normal movement through G(1)-phase but delayed entry into S-phase. Consistent with these findings, G(1) cyclin (D2 and D3) and CDK4 (cyclin-dependent kinase 4) levels and associated kinase activity were not affected. However, cyclin E-associated CDK2 activity, responsible for G(1)-to-S-phase progression, was inhibited. This decreased activity was accompanied by unchanged CDK2 protein levels and paradoxically elevated cyclin E and cyclin E-associated CDK2 levels, suggesting inhibition of the cyclin E-CDK2 complex. This inhibition was not due to altered stimulatory or inhibitory phosphorylation of CDK2. However, p27, a Cip/Kip family CKI (CDK inhibitor)-binding partner, was elevated and showed increased association with CDK2 in PLC delta 1-knockdown cells. The result of the present study demonstrate a novel and critical role for PLC delta 1 in cell-cycle progression from G(1)-to-S-phase through regulation of cyclin E-CDK2 activity and p27 levels.     

Prostaglandin J2 and its metabolites promote neurite outgrowth induced by nerve growth factor in PC12 cells

Although A- and J-type prostaglandins (PG's) arrest the cell cycle at the G1 phase in vitro and suppress tumor growth in vivo, their effects on neuronal cells have not so far been clarified. Here, we found promotion of neurite outgrowth as a novel biological function of PGJ's. In PC12h cells, PGJ's (PGJ2, Delta12-PGJ2 and 15-deoxy-Delta12,14-PGJ2) promoted neurite outgrowth in the presence of nerve growth factor (NGF), whereas they themselves did not show such a promotion. The potency of promoting neurite outgrowth was PGJ2 < Delta12-PGJ2 < 15-deoxy-Delta12,14-PGJ2. However, troglitazone, an activator of peroxisome proliferator-activated receptorgamma (PPARgamma), and other PG's including PGA1, PGA2 and PGD2 did not promote neurite outgrowth. These results suggest that PGJ's promote neurite outgrowth independently of PPARgamma activation.     

Delta12-Prostaglandin J2 inhibits the ubiquitin hydrolase UCH-L1 and elicits ubiquitin-protein aggregation without proteasome inhibition

To investigate molecular mechanisms linking inflammation with neurodegeneration, we treated neuronal cultures with prostaglandins (PGs), which are mediators of inflammation. PGA1, D2, J2, and Delta12-PGJ2, but not PGE2, reduced the viability and raised the levels of ubiquitinated proteins in the neuronal cells. PGJ2 and its metabolite, Delta12-PGJ2, were the most potent of the four neurotoxic PGs tested in inducing both effects. To address the mechanism by which these agents lead to the accumulation of ubiquitinated proteins, we tested their effects on neuronal ubiquitin hydrolases UCH-L1 and UCH-L3 as well as on proteasome activity. Notably, Delta12-PGJ2 inhibited the activities of UCH-L1 (K(i) approximately 3.5 microM) and UCH-L3 (K(i) approximately 8.1 microM) without affecting proteasome activity. Intracellular aggregates containing ubiquitinated proteins were detected in Delta12-PGJ2-treated cells, indicating that these aggregates can form independently of proteasome inhibition. In conclusion, impairment of ubiquitin hydrolase activity, such as triggered by Delta12-PGJ2, may be an important contributor to neurodegeneration associated with accumulation of ubiquitinated proteins and inflammation.     

IFN-beta pretreatment sensitizes human melanoma cells to TRAIL/Apo2 ligand-induced apoptosis

All human melanoma cell lines (assessed by annexin V and TUNEL assays) were resistant to apoptosis induction by TRAIL/Apo2L protein. TRAIL/Apo2L activated caspase-8 and caspase-3, but subsequent apoptotic events such as poly(ADP-ribose) polymerase cleavage and DNA fragmentation were not observed. To probe the molecular mechanisms of cellular resistance to apoptosis, melanoma cell lines were analyzed for expression of apoptosis regulators (apoptotic protease-associated factor-1, FLIP, caspase-8, caspase-9, caspase-3, cellular inhibitor of apoptosis, Bcl-2, or Bax); no correlation was observed. TRAIL/Apo2L was induced in melanoma cell lines by IFN-beta and had been correlated with apoptosis induction. Because IFN-beta induced other gene products that have been associated with apoptosis, it was postulated that one or more IFN-stimulated genes might sensitize cells to TRAIL/Apo2L. Melanoma cell lines were treated with IFN-beta for 16-24 h before treatment with TRAIL/Apo2L. Regardless of their sensitivity to either cytokine alone, >30% of cells underwent apoptosis in response to the combined treatment. Induction of apoptosis by IFN-beta and TRAIL/Apo2L in combination correlated with synergistic activation of caspase-9, a decrease in mitochondrial potential, and cleavage of poly(ADP-ribose) polymerase. Cleavage of X-linked inhibitor of apoptosis following IFN-beta and TRAIL/Apo2L treatment was observed in sensitive WM9, A375, or WM3211 cells but not in resistant WM35 or WM164 cells. Thus, in vitro IFN-beta and TRAIL/Apo2L combination treatment had more potent apoptotic and anti-growth effects when compared with either cytokine alone in melanoma cells lines.     

Resveratrol Induces Cell Cycle Arrest and Apoptosis in Malignant NK Cells via JAK2/STAT3 Pathway Inhibition

Natural killer (NK) cell malignancies, particularly aggressive NK cell leukaemias and lymphomas, have poor prognoses. Although recent regimens with L-asparaginase substantially improved outcomes, novel therapeutic approaches are still needed to enhance clinical response. Resveratrol, a naturally occurring polyphenol, has been extensively studied for its anti-inflammatory, cardioprotective and anti-cancer activities. In this study, we investigated the potential anti-tumour activities of resveratrol against the NK cell lines KHYG-1, NKL, NK-92 and NK-YS. Resveratrol induced robust G0/G1 cell cycle arrest, significantly suppressed cell proliferation and induced apoptosis in a dose- and time-dependent manner for all four cell lines. In addition, resveratrol suppressed constitutively active STAT3 in all the cell lines and inhibited JAK2 phosphorylation but had no effect on other upstream mediators of STAT3 activation, such as PTEN, TYK2, and JAK1. Resveratrol also induced downregulation of the anti-apoptotic proteins MCL1 and survivin, two downstream effectors of the STAT3 pathway. Finally, resveratrol induced synergistic effect on the apoptotic and antiproliferative activities of L-asparaginase against KHYG-1, NKL and NK-92 cells. These results suggest that resveratrol may have therapeutic potential against NK cell malignancies. Furthermore, our finding that resveratrol is a bonafide JAK2 inhibitor extends its potential benefits to other diseases with dysregulated JAK2 signaling.