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Radiation exposure such as A-bomb or radiation therapy is considered a major health-risk factor for cardiovascular disease. In order to understand the molecular mechanisms underlying the inflammatory reaction frequently encountered in the vascular system after exposure to ionizing radiation, we carried out a global scale microarray and computational gene expression analyses on human umbilical endothelial cells (HUVECs) exposed to X-ray (2.5Gy). The gene ontology analysis revealed that the down-regulated genes were associated with cell cycle regulation, whereas the up-regulated genes were associated with inflammatory responses, in particular, the type 1 interferon response. The computational analysis using ingenuity pathway analysis also identified a gene network containing the interferon response factor 7 (IRF7) and its transcriptional targets such as interferon-induced transcripts (IFITs) and Mx1, which have been known to be associated with inflammation in endothelial cells. The up-regulated genes and the gene network identified here may explain the inflammatory response induced by X-irradiation. These findings uncover part of the molecular basis of the mechanism(s) of the inflammatory disorder in response to X-irradiation in HUVECs. The dataset is publicly available at the Gene Expression Omnibus (GEO) repository (http://www.ncbi.nlm.nih.gov/geo/) with accession number GSE76484.

Reactive oxygen species (ROS) act as a mediator of ionizing radiation-induced cellular damage. Previous studies have indicated that MnSOD (SOD2) plays a critical role in protection against ionizing radiation in mammalian cells. In this study, we constructed two types of stable HeLa cell lines overexpressing SOD2, HeLa S3/SOD2 and T-REx HeLa/SOD2, to elucidate the mechanisms underlying the protection against radiation by SOD2. SOD2 overexpression in mitochondria enhanced the survival of HeLa S3 and T-REx HeLa cells following γ-irradiation. The levels of γH2AX significantly decreased in HeLa S3/SOD2 and T-REx HeLa/SOD2 cells compared with those in the control cells. MitoSox(TM) Red assays showed that both lines of SOD2-expressing cells showed suppression of the superoxide generation in mitochondria. Furthermore, flow cytometry with a fluorescent probe (2',7'-dichlorofluorescein) revealed that the cellular levels of ROS increased in HeLa S3 cells during post-irradiation incubation, but the increase was markedly attenuated in HeLa S3/SOD2 cells. DNA microarray analysis revealed that, of 47,000 probe sets analyzed, 117 and 166 probes showed more than 2-fold changes after 5.5 Gy of γ-irradiation in control and HeLa S3/SOD2 cells, respectively. Pathway analysis revealed different expression profiles in irradiated control cells and irradiated SOD2-overexpressing cells. These results indicate that SOD2 protects HeLa cells against cellular effects of γ-rays through suppressing oxidative stress in irradiated cells caused by ROS generated in the mitochondria and through regulating the expression of genes which play a critical role in protection against ionizing radiation.

A drug of two halves: New artificial compounds composed of a macrosphelide core skeleton and an epothilone side chain were designed and synthesized. These compounds were more potent inducers of apoptosis than the parent natural‐type macrosphelides.

Hybrid compounds of macrosphelides and epothilones, both of which are natural macrolides having a 16-membered skeleton, were designed and synthesized using a ring-closing metathesis (RCM) strategy. Some of these hybrids were found to exhibit notable apoptosis-inducing activity against human lymphoma cells with higher potency than parent natural macrosphelides, and to be a promising lead compound for development of a new antitumor agent.

To determine the effect of 2,2′‐azobis(2‐amidinopropane) dihydrochloride (AAPH) on ultrasound (US)‐induced cell killing, human monocytic leukemia cells (U937) were incubated at various temperatures (25.0, 37.0 and 40.0°C) for 1 min in air‐saturated phosphate‐buffered solution (PBS) containing 50 mMAAPH before exposure to nonthermal 1 MHz US for 1 min at an intensity of 2.0 W/cm2. Cell viability was determined by means of the Trypan blue dye exclusion test immediately after sonication. Apoptosis was measured after 6‐h incubation post‐sonication by flow cytometry. Free radicals generated by AAPH, a temperature‐dependent free radical generator, or US or both were also investigated using electron paramagnetic resonance (EPR) spin trapping. The results showed that US‐induced cell lysis and apoptosis were enhanced in the presence of AAPH regardless of the temperature at the time of sonication. At 40.0°C, US alone induced increased cell killing, while AAPH alone is capable of inducing significant but minimal apoptosis at this temperature. Although free radicals were increased in the combined treatment, this increase did not correlate well with cell killing. The mechanism of enhancement points to the increased uptake of the agent during sonication rather than potentiation by AAPH. These findings suggest the clinical potential of temperature‐dependent free radical generators in cancer therapy with therapeutic US.

The combined effects of hyperthermia at 44 °C and local anesthetics on apoptosis in human histiocytic lymphoma U937 cells were investigated. When the cells were exposed to hyperthermia for l0 min marginal DNA fragmentation and nuclear fragmentation were observed. In the presence of amide-type local anesthetics further enhancement was found depending on concentration. The order of the concentration required for maximum induction was the reverse order of the lipophilicity (prilocaine > lidocaine > bupivacaine). Western blotting revealed that in hyperthermia there was initial release of Ca2+from the intracellular store site as indicated by increased expression of the type 1 inositol-1,4,5-trisphosphate receptor. However, the combination with lidocaine did not induce any further enhancement. Lidocaine enhanced the decrease in ATP content and the increase in intracellular Ca2+concentration in individual cells induced by hyperthermia. In addition, superoxide formation, decrease in the mitochondrial membrane potential, and activation of intracellular caspase-3 were found in the cells treated with hyperthermia and lidocaine. All of these were suppressed in part in the presence of the intracellular Ca2+ion chelator BAPTA-AM (bis-(O-aminophenoxy)-ethane-N, N, N′,N′-tetraacetic acid-acetoxymethyl). The present results indicate that local anesthetics at optimal concentrations enhance hyperthermia-induced apoptosis via Ca2+- and mitochondria-dependent pathways. Initial release of Ca2+from intracellular store sites caused by hyperthermia and followed by the subsequent increase in the intracellular Ca2+concentration and the additional activation of the mitochondrial caspase-dependent pathway (partly regulated by intracellular Ca2+concentration) plays a crucial role in the enhancement of apoptosis induced by the combination of hyperthermia and lidocaine.

To elucidate the mechanism how a free radical initiator, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), induces cell death at hyperthermic temperatures, apoptosis in a human histiocytic lymphoma cell line, U937, was investigated. Free radical formation deriving from the thermal decomposition of AAPH was examined by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). An assay for DNA fragmentation, observation of nuclear morphological changes, and flow cytometry for phosphatidylserine (PS) externalization were used to detect apoptosis and revealed enhancement of 44.0°C hyperthermia-induced apoptosis by free radicals due to AAPH. However, free radicals alone derived from AAPH did not induce apoptosis. Hyperthermia induced the production of lipid peroxidation (LPO), an increase in intracellular Ca2+ concentration ([Ca2+]i) and enhanced expression of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). The effects of hyperthermia on LPO and [Ca2+]i were enhanced markedly by the combination with AAPH. A significant decrease in Bcl-2 expression, increase in Bax expression, a loss of mitochondrial membrane potential (ΔΨm) and a marked increase in cytochrome c expression were found only in cells treated with hyperthermia and AAPH. Although an intracellular Ca2+ ion chelator, BAPTA-AM, completely inhibited DNA fragmentation, water-soluble vitamine E, Trolox, only partially suppressed DNA fragmentation and the increase in [Ca2+]i. In contrast, LPO was inhibited completely by Trolox, but no inhibition by BAPTA-AM was found. These results suggest that apoptosis induced by hyperthermia alone is due to the increase in [Ca2+]i arising from increased expression of IP3R1 and LPO. Additional increase in [Ca2+]i due to increased LPO and the activation of mitochondria-caspase dependent pathway play a major role in the enhancement of apoptosis by the combination with hyperthermia and AAPH.

Apoptosis induced by cadmium has been shown in many tissues in vivoand in cultured cells in vitro. However, its molecular mechanism is not fully understood. When the human histiocytic lymphoma cell line U937 was treated with cadmium for 12 h, evidence of apoptotic features, including change in nuclear morphology, DNA fragmentation, formation of DNA ladder in agarose gel electrophoresis, and phosphatidylserine externalization, were obtained. Moreover, loss of the mitochondrial membrane potential (Δψm) was observed in the cadmium-treated cells and was inhibited by a broad caspase inhibitor (Z-VAD-FMK). Caspase inhibitors suppressed the DNA fragmentation in the order of Z-VAD-FMK > caspase-8 inhibitor > caspase-3 inhibitor. Expression of Bcl-xLand Bid decreased significantly in the cadmium-treated cells, although no apparent change in Bcl-2 and Bax expression was found. Tetrakis-(2-pyridylmethyl) ethylendiamine, a cell-permeable heavy metal chelator, partially reversed the increase of fluorescence of Fura-2 in the cadmium-treated cells. In addition, verapamil (70 μm), a voltage-dependent Ca2+channel blocker, inhibited the DNA fragmentation induced by cadmium less than 100 μmand decreased the fluorescence of Fura-2. Cadmium up-regulated the expression of type 1 inositol 1,4,5-trisphosphate receptor (IP3R) but not type 2 or type 3 IP3R. Calpain inhibitors I and II partially prevented DNA fragmentation. No effects of Z-VAD-FMK on the expression of type 1 IP3R or of calpain inhibitors on the loss of Δψmwere observed. These results suggest that cadmium possibly induced apoptosis in U937 cells through two independent pathways, the Ca2+-calpain-dependent pathway and the caspase-mitochondria-dependent pathway.

Ultraviolet light (UV) induced rapid apoptosis of U937 leukemia cells, concurrent with DNA fragmentation and cleavage of poly(ADP-ribose)polymerase (PARP) by activated caspase-3. Thein vitroreconstitution of intact HeLa S3 nuclei and apoptotic U937 cytosolic extract (CE) revealed that (i) Ca2+/Mg2+-dependent, Zn2+-sensitive endonuclease activated in the apoptotic CE induced DNA ladder in HeLa nuclei at pH 6.8–7.4, (ii) activated caspase-3 cleaved PARP in HeLa nuclei, and (iii) when the apoptotic CE was treated with the caspase-3 inhibitor (1 μM Ac-DEVD-CHO) or the caspase-1 inhibitor (10 μM Ac-YVAD-CHO), the former, but not the latter, caused a 50% inhibition of DNA fragmentation and the complete inhibition of PARP cleavage in HeLa nuclei. Similarly, Ac-DEVD-CHO (100 μM) inhibited apoptosis and DNA ladder by 50% and PARP cleavage completely in UV-irradiated U937 cells, but Ac-YVAD-CHO (100 μM) did not. Thus, UV-induced apoptosis of U937 cells involves the Ca2+/Mg2+-dependent endonuclease pathway and the caspase-3–PARP cleavage–Ca2+/Mg2+-dependent endonuclease pathway. The former pathway produced directly 50% of apoptotic DNA ladder, and the latter involved activated caspase-3 and PARP cleavage, followed by formation of the remaining 50% DNA ladder by the activated endonuclease. In UV-irradiated B-cell lines, further, p53-dependent increase of Bax resulted in a greater caspase-3 activation compared to its absence. However, UV-induced activation of JNK1 and p38 was not affected by the caspase-1 and -3 inhibitors in U937 cells, so that caspases-1 and -3 do not function upstream of JNK1 and p38.