Kiyoshi Tanabe

Apoptosis Induced by Cadmium in Human Lymphoma U937 Cells through Ca2+-calpain and Caspase-Mitochondria- dependent Pathways

Apoptosis induced by cadmium has been shown in many tissues in vivo and 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-xL and 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 μm and 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 Δψm were 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.

Enhancement of hyperthermia-induced apoptosis by a free radical initiator, 2,2′-azobis (2-amidinopropane) dihydrochloride, in human histiocytic lymphoma U937 cells

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.

The role of intracellular Ca2+ in apoptosis induced by hyperthermia and its enhancement by verapamil in U937 cells

PURPOSE The relationship between apoptosis induced by 42 degrees C and 44 degrees C hyperthermia alone or in combination with verapamil and changes in intracellular Ca(2+) concentration ([Ca(2+)]i) was investigated in U937 cells. METHODS Apoptosis induced by hyperthermia was assessed according to DNA fragmentation, nuclear morphologic changes, and expression of phosphatidylserine on the outside plasma cell membrane. These changes were measured by flow cytometry. The [Ca(2+)]i of individual cells after hyperthermia was monitored by a digital image-analyzing technique using Fura-2. RESULTS Hyperthermia-induced apoptosis reached a plateau after 6 h and was found to be both time and temperature-dependent. DNA fragmentation was maximum at 44 degrees C after 30 min. Verapamil enhanced the apoptosis induced by 42 degrees C and 44 degrees C hyperthermia in normal cells and by 44 degrees C hyperthermia in thermotolerant cells. The number of cells containing higher [Ca(2+)]i (more than 200 nM) was significantly increased by hyperthermia and further elevated by the addition of verapamil in both normal and thermotolerant cells. Apoptosis induced by hyperthermia was markedly decreased by an intracellular Ca(2+) chelator, BAPTA-AM, in a dose-dependent manner. CONCLUSION These results indicate that [Ca(2+)]i increase plays a crucial role in apoptosis induced by hyperthermia and the combined treatment with verapamil in normal and thermotolerant U937 cells. Furthermore, hyperthermia-combined drug therapy has potential significance in cancer therapy.

A Free Radical Initiator, 2,2′-azobis (2-aminopropane) Dihydrochloride Enhances Hyperthermia-induced Apoptosis in Human Uterine Cervical Cancer Cell Lines

Hyperthermia-induced apoptosis and its enhancement in the presence of a temperature-dependent free radical initiator, 2,2′-azobis (2-aminopropane) dihydrochloride (AAPH) were examined in human uterine cervical cancer cell lines, CaSki and HeLa. When both cell lines were treated with hyperthermia at 44°C for 60 min, minimal apoptosis was observed. When combined with nontoxic AAPH (50 mM), significant enhancement of apoptosis was observed, where the initial rate of free radical formation was about twice as high than that at 37°C. Augmentation of the growth delay, lipid peroxidation (LPO), activation of caspase-3 and increase in [Ca2+]i were also observed after the combined treatment. A water-soluble vitamin E, Trolox, blocked the increase in [Ca2+]i and an intracellular Ca2+ chelator, BAPTA-AM, prevented the DNA fragmentation induced by the combination. Cytochrome c release was also revealed by fluorescence microscopy. However, no significant change in mitochondrial membrane potential and expression of Bax and Bcl-2 was observed. A slight increase in Fas expression was observed only in CaSki cells after the combined treatment. These results indicate that hyperthermia and AAPH induce enhanced apoptosis and subsequent cell killing via two pathways; a pathway dependent on increase in LPO and [Ca2+]i, and a pathway associated with cytochrome c release and subsequent caspase activation without changes of mitochondrial membrane potential and Bax/Bcl-2 expression in these cell lines. Since it is known that cancer cells are generally resistant to physical and chemical stress-induced apoptosis, free radical generators like AAPH appear to be a useful thermosensitizer for hyperthermic cancer therapy.

A Specific Inhibitor of Factor Xa, DX-9065a, Exerts Effective Protection against Experimental Tumor-Induced Disseminated Intravascular Coagulation in Rats

(+)-2S-2-[4-[[(35S)-1-acetimidoyl-3-pyrrolidinyl]oxy]phenyl]-3-[7- amidino-2-napthyl]propanoic acid hydrochloride pentahydrate (DX-9065a) is an antithrombin III-independent, selective inhibitor of activated blood coagulation factor X (FXa). We investigated the protective effects of DX-9065a against tumor-bearing experimental disseminated intravascular coagulation (DIC) induced by the inoculation of AH-109A cells into rats. DX-9065a was subcutaneously administered at doses of 0.03 and 0.1 mg/kg/hour through an osmotic pump transplanted immediately after the inoculation of the tumor cells during the observation period. Platelet count decreased 12 days after the inoculation, concomitant with an increase in the thrombin-antithrombin III complex and fibrin and fibrinogen degradation products. Doses of 0.03 and 0.1 mg/kg/hour of DX-9065a significantly inhibited the decrease in plasma fibrinogen concentration and platelet count 13 days after the inoculation, respectively. These findings suggest that direct, selective inhibition of FXa by DX-9065a improves the hypercoagulable state induced by the progress of solid tumor.