The sorcin-annexin VII calcium-dependent interaction requires the sorcin N-terminal domain

Dolichyl monophosphate (Dol-P) has been found to induce apoptosis in human leukemia U937 cells. During this apoptotic execution, the increase of plasma membrane fluidity (5–20 min), caspase-3-like protease activation (2–4 h), chromatin condensation and DNA ladder formation (3–4 h) were observed successively. Here, we report that reduction in mitochondrial transmembrane potential and translocation of apoptosis-inducing factor (AIF) are early events (1–3 h) in the apoptotic process induced by Dol-P in U937 cells. The AIF was concentrated around nuclei and partly translocated to the nuclei, which was confirmed by immunocytochemistry using specific anti-AIF antibody. Both caspase-8 and caspase-3 inhibitors blocked only DNA fragmentation but not mitochondrial processes, AIF migration and chromatin condensation. These results indicate that mitochondrial changes are an early step in the apoptosis induced by Dol-P and AIF is one of the important factors which induce chromatin condensation in nuclei.     

Biosynthesis of lipid-linked oligosaccharides by microsomes from virus induced Hepatoma MC-29. Dependence of some glycosyl transferases on dolichyl phosphates of different chain length

Dolichyl phosphates of various chain length ranging from 7 to 22 isoprene units were tested as lipid acceptors in transglycosylation reactions in chicken liver and Hepatoma MC-29. In the presence of exogenous dolichyl phosphate mixture (18 and 19 isoprene units) the synthesis of dolichyl pyrophosphate N-acetylglucosamine and dolichyl phosphate mannose increased 3 times both in the liver and Hepatoma MC-29, while the formation of dolichyl phosphate glucose was 4 fold higher in the liver and 6-fold higher in Hepatoma MC-29. In liver microsomes the maximum rate of the stimulation of glycosylation was achieved by exogenous dolichyl phosphates, containing 18 and 19 isoprene units, while glycosyl transferases in microsomes from Hepatoma MC-29 did not show any structural requirements to the chain length of dolichyl phosphates.     

Protein kinase C activation modulates pro- and anti-apoptotic signaling pathways

Activation of protein kinase C (PKC) by TPA in human U937 myeloid leukemia cells is associated with induction of adherence, differentiation, and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these differentiating cells about 25% of U937 cells accumulated in the subG1 phase after TPA treatment. This effect proved to be phorbol ester-specific, since other compounds such as retinoic acid or vitamin D3 failed to induce apoptosis in conjunction with differentiation. Only a specific inhibitor of PKC, GF109203X, but not the broad-spectrum kinase inhibitor staurosporine or a tyrosine kinase inhibitor genistein could reverse the induction of apoptosis. Bryostatin-1, another specific PKC activator with distinct biochemical activity failed to induce apoptosis. Moreover, bryostatin-1 completely abolished the induction of apoptosis in U937 cells even if added 8 hours after TPA treatment. Apart from apoptosis induced by various chemotherapeutic drugs, TPA-related cell death is not mediated by an autocrine Fas-FasL loop and could not be prevented by a blocking antibody to the Fas receptor. However, a 75% reduction in the number of apoptotic cells after TPA stimulation was achieved by preincubation with a blocking antibody to the TNFalpha receptor. Tetrapeptide cleavage assays revealed a four-fold increase in the DEVD-cleavage activity in U937 cells compared to a three-fold increase in TUR cells. Immunoblotting demonstrated that TUR cells did not activate significant levels of caspase-3 or -7, whereas in U937 cells a 20-kDa cleavage product corresponding to activated caspase-3 was detectable after 3 d TPA exposure. Moreover, immunoblots revealed a strongly reduced expression of the adaptor molecule APAF-1, which is required for cytochrome c-dependent activation of caspase-9 and subsequently caspase-3. APAF-1 proved to be inducible after PKC activation with phorbol ester in U937, but not in TUR cells. Thus, APAF-1 expression may, at least in part, be regulated by PKC activity and reduced APAF-1 levels are associated with resistance to various inducers of apoptosis. Furthermore, TPA exposure of U937 cells is associated with increased levels of the pro-apoptotic proteins Bak and Bcl-xs, whereas simultaneously a decline in the Bcl-2 expression was noticable.     

Jolkinolide B from <Emphasis Type="Italic">Euphorbia fischeriana Steud</Emphasis> induces apoptosis in human leukemic U937 cells through PI3K/Akt and XIAP pathways

Jolkinolide B, a bioactive diterpene isolated from the roots of Euphorbia fischeriana Steud, is known to induce apoptosis in cancer cells. However, the molecular mechanism of its anti-cancer activity has not been fully elucidated. In the present study, we found that Jolkinolide B reduced cell viability and induced apoptosis in a dose- and timedependent manner in human leukemic U937. The induction of apoptosis was also accompanied by the downregulation of PI3K/Akt and the inhibitor of apoptosis protein (IAP) family proteins. Moreover, we observed that Jolkinolide B treatment resulted in activation of caspase-3 and -9, which may partly explain the anti-cancer activity of Jolkinolide B. Taken together, our study for the first time suggest that Jolkinolide B is able to enhance apoptosis of U937 cells, at least in part, through downregulation of PI3K/Akt and IAP family proteins. Moreover, triggering of caspase-3 and -9 activation mediated apoptotic induction.     

Generation of reactive oxygen species is an early event in dolichyl phosphate-induced apoptosis

The mechanism of induction of apoptosis by dolichyl phosphate (Dol-P) was investigated in U937 cells. Studies using isolated mitochondria revealed that the respiratory complex II activity was almost completely inhibited by 20 microg/ml of Dol-P but not by the same concentration of dolichol. Activities of complex I and III were also inhibited by Dol-P, but nearly 50% of activity still remained at 20 microg/ml. Dol-P induced release of cytochrome-c from the isolated mitochondria. Fluorometric microtiter plate assay revealed that generation of reactive oxygen species (ROS) increased in a time-dependent manner. Flow cytometric analysis also indicated that Dol-P caused loss of mitochondrial membrane potential (Deltapsi(m)) and increased ROS generation. The addition of the antioxidant pyrrolidine dithiocarbamate (PDTC) significantly inhibited Dol-P-induced ROS generation and activation of caspase-3. A specific inhibitor of respiratory complex II, thenoyltrifluoroacetone (TTFA), increased ROS generation, potentially mimicking the consequence of inhibition of electron flow at complex II by Dol-P in U937 cells. Electron microscopy revealed that mitochondria became swollen and spherical in shape by the treatment with Dol-P. Neither the tyrosine kinase inhibitor k252a nor mitogen activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitors PD98059 and U0126 inhibited the Dol-P-induced apoptosis. Together, these results suggest that the direct disruption of mitochondrial respiratory complexes and the consequent ROS generation play a critical role in the initiation of Dol-P-induced apoptosis.     

Phloretin-induced apoptosis in B16 melanoma 4A5 cells and HL60 human leukemia cells.

The dihydrochalcone phloretin induced apoptosis in B16 mouse melanoma 4A5 cells and HL60 human leukemia cells. Phloretin was suggested to induce apoptosis in B16 cells mainly through the inhibition of glucose transmembrane transport. The phloretin-induced apoptosis in B16 cells was inhibited by actinomycin D, Ac-YVAD-CHO caspase-1-like inhibitor, and Ac-DEVD-CHO caspase-3-like inhibitor. During the induction of apoptosis by phloretin, the expression of Bax protein in B16 cells increased and the levels of p53, Bcl-2, and Bcl-XL proteins did not change. Our results suggested that phloretin induced apoptosis through the promotion of Bax protein expression and caspases activation. On the other hand, phloretin may induce apoptosis in HL60 cells through the inhibition of protein kinase C activity because phloretin inhibited protein kinase C activity in HL60 cells more than that in B16 cells. The phloretin induced-apoptosis in HL60 cells was not inhibited by actinomycin D and the caspase-1-like inhibitor, but slightly inhibited by the caspase-3-like inhibitor. Phloretin reduced the level of caspase 3 protein in HL60 cells, but not the level of the Bcl-2 protein. Phloretin did not increase the level of Bax protein. Phloretin was suggested to induce apoptosis in HL60 cells through the inhibition of protein kinase C activity, followed by the pathway, which is different from that in B16 cells.     

Involvement of protein kinase C-regulated ceramide generation in inostamycin-induced apoptosis

Activation of caspases is commonly involved in the apoptosis induced by various anticancer drugs. However, the upstream events leading to the activation of caspases seem to be specific to each anticancer drug. In the present study, we examined the possible involvement of protein kinase C (PKC) and ceramide generation in caspase-3(-like) protease activation induced by inostamycin, a phosphatidylinositol synthesis inhibitor. Treatment of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of PKC, suppressed the release of cytochrome c from mitochondria and the activation of caspase-3(-like) proteases in inostamycin-treated cells, but not in other anticancer drug-treated cells. Inostamycin induced the elevation of intracellular ceramide levels, and fumonisin B1, an inhibitor of ceramide synthase, inhibited inostamycin-induced cytochrome c release, caspase-3(-like) protease activation, and apoptosis. Moreover, TPA also inhibited inostamycin-induced ceramide synthesis. Taken together, our results suggest that inostamycin-induced apoptosis is mediated by PKC-regulated ceramide generation, leading to the activation of a caspase cascade.     

Limited proteolysis of filamin is catalyzed by caspase-3 in U937 and Jurkat cells

Members of the caspase family have been implicated as key mediators of apoptosis in mammalian cells. However, few of their substrates are known to have physiological significance in the apoptotic process. We focused our screening for caspase substrates on cytoskeletal proteins. We found that an actin binding protein, filamin, was cleaved from 280 kDa to 170, 150, and 120 kDa major N-terminal fragments, and 135, 120, and 110 kDa major C-terminal fragments when apoptosis was induced by etoposide in both the human monoblastic leukemia cell line U937, and the human T lymphoblastic cell line Jurkat. The cleavage of filamin was blocked by a cell permeable inhibitor of caspase-3-like protease, Ac-DEVD-cho, but not by an inhibitor of caspase-1-like protease, Ac-YVAD-cho. These results suggest that filamin is cleaved by a caspase-3-like protease. To examine whether caspase-3 cleaves filamin in vitro, we prepared a recombinant active form of caspase-3 directly using a Pichia pastoris overexpression system. When we applied recombinant active caspase-3 to the cell lysate of U937 and Jurkat cells, filamin was cleaved into the same fragments seen in apoptosis-induced cells in vivo. Platelet filamin was also cleaved directly from 280 kDa to 170, 150, and 120 kDa N-terminal fragments, and the cleavage pattern was the same as observed in apoptotic human cells in vivo. These results suggest that filamin is an in vivo substrate of caspase-3.     

The antidepressants imipramine, clomipramine, and citalopram induce apoptosis in human acute myeloid leukemia HL-60 cells via caspase-3 activation.

Some widely used antidepressants such as imipramine, clomipramine, and citalopram have been found to possess antineoplastic effects. In the present study, these compounds were found to induce apoptotic cell death in human acute myeloid leukemia HL-60 cells. Apoptosis induced by the antidepressants was identified by electron microscopy and conventional agarose gel electrophoresis and was quantitated by propodium iodide staining and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) via flow cytometry. Treatment with apoptosis-inducing concentrations of the antidepressants (80 microM imipramine, 35 microM clomipramine, or 220 microM citalopram) caused induction of caspase-3/caspase-3-like activity, which was monitored by the cleavage of poly(ADP-ribose) polymerase (PARP), the loss of the 32 kD caspase-3 (CPP32) precursor, and the cleavage of the fluorescent CPP32-like substrate PhiPhiLux. Pretreatment with a potent caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone (zVAD-fmk) inhibited antidepressant-induced CPP32/CPP32-like activity and apoptosis. Furthermore, activation of caspase induced by the antidepressants was preceded by the hypergeneration of intracellular reactive oxygen species (ROS). These results suggested that the antidepressants may induce apoptosis via a caspase-3-dependent pathway, and induction of apoptosis by the antidepressants may provide a clue for the mechanism of their antineoplastic effects.     

Mechanism of UV-Induced Apoptosis in Human Leukemia Cells: Roles of Ca/Mg-Dependent Endonuclease, Caspase-3, and Stress-Activated Protein Kinases

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.     

Interferon-gamma sensitizes human myeloid leukemia cells to death receptor-mediated apoptosis by a pleiotropic mechanism

The role of interferon (IFN)-gamma as a sensitizing agent in apoptosis induced by ligation of death receptors has been evaluated in human myeloid leukemia cells. Incubation of U937 cells with IFN-gamma sensitized these cells to apoptosis induced by tumor necrosis factor-alpha, agonistic CD95 antibody, and tumor necrosis factor-related apoptosis-inducing ligand. Other human myeloid leukemic cells were also sensitized by IFN-gamma to death receptor-mediated apoptosis. Treatment of U937 cells with IFN-gamma up-regulated the expression of caspase-8 and potently synergized with death receptor ligation in the processing of caspase-8 and BID cleavage. Concomitantly, a marked down-regulation of BCL-2 protein was also observed in cells incubated with IFN-gamma. Furthermore, the caspase-dependent generation of a 23-kDa fragment of BCL-2 protein, the release of cytochrome c from mitochondria and the activation of caspase-9 were also enhanced upon death receptor ligation in IFN-gamma-treated cells. Ectopically expressed Bcl-2 protein inhibited IFN-gamma-induced sensitization to apoptosis. In summary, these results indicate that IFN-gamma sensitizes human myeloid leukemic cells to a death receptor-induced, mitochondria-mediated pathway of apoptosis.     

Role of fatty acid chain length on the induction of apoptosis by newly synthesized catechin derivatives

The catechins, a family of polyphenols found in tea, can evoke various responses, including apoptosis. In this study we investigated whether the chemical modification of (?)-epigallocatechin gallate (EGCG) could enhance its apoptosis activity.We found that one of the catechin conjugated with capric acid [(2R,3S)-3′,4′,5,7-tetrahydroxyflavan-3-yl decanoate; catechin-C10] was most potent to induce apoptosis in U937 cells. C10 treatment resulted in a significant increase in reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP) loss, cytochrome c release caspase-9 and caspase-3 activation. In addition to this C10 also activated extrinsic pathway significantly as evident by time-dependent increase in Fas expression and caspase-8 activity. C10 mediated cleavage of Bid may be an important event for cross talk between intrinsic and extrinsic signaling. Moreover, pre-treatment of cells with anti-oxidant N-acetyl-l-cysteine (NAC) significantly prevented C10-induced apoptosis but did not protect MMP loss. Treatment of cells with pan-caspase inhibitor significantly inhibited apoptosis indicating that caspases are playing key role. In addition to this C10 was found to induce apoptosis in human colon cancer (HCT116) cells while it showed resistance to human keratinocytes (HaCat).In short our results showed that the optimal fatty acid side chain length is required for the apoptosis inducing activity of catechin derivatives in U937 cells.     

Characterization of the Saccharomyces cerevisiae cis-prenyltransferase required for dolichyl phosphate biosynthesis

The prenyltransferase involved in the biosynthesis of dolichyl phosphate has been characterized in Saccharomyces cerevisiae. Although the enzyme is predominantly membrane-bound, a significant percentage was found in the soluble fraction. The prenyltransferase preferentially utilizes farnesyl pyrophosphate as the allylic substrate and isopentenyl pyrophosphate as cosubstrate with half-maximal velocities obtained at 25 and 6.7 microM, respectively. The enzymatic activity is sensitive to sulfhydryl reagents and is inhibited by all detergents tested, except 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate at concentrations less than 5 mM. The product of the reaction has been characterized as an alpha-unsaturated polyprenyl pyrophosphate, containing 12-15 isoprene units, approximately two isoprene units shorter than the endogenous yeast dolichyl phosphate. The stereochemistry of addition of isoprene units by the prenyltransferase was shown to be cis by a comparison of the HPLC retention time for a pentadecaprenyl phosphate derived from the in vitro reaction product with that for an authentic mixture of alpha-cis- and alpha-trans-pentadecaprenyl phosphates.     

Manganese(II) induces apoptotic cell death in NIH3T3 cells via a caspase-12-dependent pathway

Under physiological conditions, manganese(II) exhibits catalase-like activity. However, at elevated concentrations, it induces apoptosis via a non-mitochondria-mediated mechanism (Oubrahim, H., Stadtman, E. R., and Chock, P. B. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9505-9510). In this study, we show that the Mn(II)-induced apoptosis, as monitored by caspase-3-like activity, in NIH3T3 cells was inhibited by calpain inhibitors I and II or the p38 MAP kinase inhibitor, SB202190. The control experiments showed that each of these inhibitors in the concentration ranges used exerted no effect on activated caspase-3-like activity. Furthermore, caspase-12 was cleaved in Mn(II)-treated cells, suggesting that the Mn(II)-induced apoptosis is mediated by caspase-12. This notion is confirmed by the observations that pretreatment of NIH3T3 cells with either caspase-12 antisense RNA or dsRNA corresponding to the full-length caspase-12 led to a dramatic decrease in caspase-3-like activity induced by Mn(II). The precise mechanism by which Mn(II) induced the apoptosis is not clear. Nevertheless, Mn(II), in part, exerts its effect via its ability to replace Ca(II) in the activation of m-calpain, which in turn activates caspase-12 and degrades Bcl-xL. In addition, the dsRNA(i) method serves as an effective technique for knocking out caspase-12 in NIH3T3 cells without causing apoptosis.     

Roles of Bcl-2 family members,PI3K and NF-κB pathways in <Emphasis Type="Italic">Escherichia coli</Emphasis>-induced apoptosis in human monocytic U937 cells

We previously showed that infection of human monocytic U937 cells with nonpathogenic Escherichia coli (E. coli) induced rapid apoptosis in a dose- and time-dependent manner. We also found that E. coli increase p38 mitogen-activated protein Kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK), and decrease extracellular-Regulated Kinase1/2 (ERK1/2) phosphorylation and increase caspase-3 and -9 activity in U937 cells. The current study determines if Bcl-2, Bax, the phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor kappa B (NF-κB) regulates E. coli–induced U937 cell apoptosis. Studying the underlying mechanisms we found that the E. coli-induced apoptosis in U937 cells was associated with a more prominent reduction in expression of Bcl-2, levels of P-Akt and NF-κB. Because levels of inhibition of apoptosis protein (cIAP), and X-chromosomelinked inhibitor of apoptosis protein (XIAP) are regulated by NF-κB, E. coli decreased the levels of these proteins in U937 cells through inhibition of NF-κB. Moreover, E. coli markedly elevated Bax expression and cytochrome c redistribution. LY294002, PDTC and Embelin, specific inhibitors of PI3K, NF-κB and XIAP, induced U937 cell apoptosis and the apoptosis is dependent on activity of caspase-3 and -9 in E. coli-treated U937 cells. Through using LY294002 and western blotting, we identified NF-κB was the downstream Akt target regulated by E. coli. Taken together, these results clearly indicate reduced activation of NF-κB via impaired PI3K/Akt activation could result in increased apoptosis of U937 cells infected by E. coli. Moreover, E. coli can induce apoptosis with an increased expression of Bax and a reduced expression of Bcl-2, which resulted in increased levels of cytochrome c release and increase caspase-3 and -9 in U937 cells.     

Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation

Heat shock protein 70 (HSP70) has been shown to act as an inhibitor of apoptosis. We have also observed an inhibitory effect of HSP70 on apoptotic cell death both in preheated U937 and stably transfected HSP70-overexpressing U937 (U937/HSP70) cells. However, the molecular mechanism whereby HSP70 prevents apoptosis still remains to be solved. To address this issue, we investigated the effect of HSP70 on apoptotic processes in an in vitro system. Caspase-3 cleavage and DNA fragmentation were detected in cytosolic fractions from normal cells upon addition of dATP, but not from preheated U937 or U937/hsp70 cells. Moreover, the addition of purified recombinant HSP70 to normal cytosolic fractions prevented caspase-3 cleavage and DNA fragmentation, suggesting that HSP70 prevents apoptosis upstream of caspase-3 processing. Because cytochrome c was still released from mitochondria into the cytosol by lethal heat shock despite prevention of caspase-3 activation and cell death in both preheated U937 and U937/hsp70 cells, it was evident that HSP70 acts downstream of cytochrome c release. Results obtained in vitro with purified deletion mutants of HSP70 showed that the carboxyl one-third region (from amino acids 438 to 641) including the peptide-binding domain and the carboxyl-terminal EEVD sequence was essential to prevent caspase-3 processing. From these results, we conclude that HSP70 acts as a strong suppressor of apoptosis acting downstream of cytochrome c release and upstream of caspase-3 activation.     

Dolichyl phosphate-dependent glycosyltransferases utilize truncated cofactors.

Synthetic truncated dolichyl phosphates of chain lengths from four to thirteen isoprene units (Jaenicke L. and Siegmund H.-U., Chem. Phys. Lipids 51 (1989) 159-170) were assayed for their cofactor activity in the enzymatic transfer of hexoses and hexosamines. The enzymes were microsomal preparations from the green alga Volvox carteri, baker's yeast, and mammalian liver cells. Under saturating conditions, the acceptor activities of the truncated dolichyl phosphates increased from zero to full strength as compared to the mixture of long-chain dolichyl phosphates from natural sources with growing chain length from five to nine isoprene units. Km determinations confirmed the results. Of the geometric isomers of dolichyl 7-phosphate (35 carbon atoms), the 14-trans compound has unchanged acceptor activity; all-trans dolichyl 7-phosphate, however, was almost inactive. The data suggest that hydrophobicity may be an important, but not the only criterion for the binding of the isoprene moiety to the active sites of the transferase enzyme(s) and that the geometry of more than only one double bond in the dolichols is recognized.     

Establishment of persistent infection with HIV-1 abrogates the caspase-3-dependent apoptotic signaling pathway in U937 cells

Treatment of 26L cells, a subclone obtained from U937 cells, with TNF-alpha or DNA-damaging agents such as teniposide (VM26) and camptothecin (CPT) induced morphologically and biochemically typical apoptotic changes, including the activation of procaspase-3. The cells persistently infected with HIV-1 (26L/HIV), however, showed a marked resistance to VM26 and CPT, whereas they hardly lost the sensitivity to TNF-alpha. TNF-alpha-induced apoptosis of 26L/HIV cells proceeded without the increase in caspase-3 activity, indicating that signaling for apoptosis in the infected cells proceeded through an alternative caspase-3-independent pathway which could respond to TNF-alpha but not to VM26 and CPT. The evidence that p-toluenesulfonyl-l-lysine chloromethyl ketone (a trypsin-like serine protease inhibitor) blocked VM26- and CPT-induced apoptotic changes but not TNF-alpha-induced apoptosis also supported the existence of the alternative TNF-alpha-inducible pathway. The results also suggest that a TLCK-sensitive protease is involved upstream of the procaspase-3 activation process and that the protease is essential for the progress of VM26- and CPT-induced apoptosis. The similar effect of HIV-1-productive infection on the apoptosis induced by the DNA-damaging agents was also confirmed by utilizing U1 cells, which are latently HIV-1-infected U937 cells. The cells became resistant to these agents after induction of the viral production by pretreatment with PMA. These results suggest that persistent HIV-1 infection blocks an apoptotic pathway triggered by DNA damaging agents through the inhibition of the procaspase-3 activation process.     

Isolation of tryptophol as an apoptosis-inducing component of vinegar produced from boiled extract of black soybean in human monoblastic leukemia U937 cells

We isolated a novel apoptosis-inducing component, tryptophol, from vinegar produced from boiled extract of black soybean (black soybean vinegar). Compound-6 purified from an ethyl acetate extract of black soybean vinegar using high performance liquid chromatography (HPLC) induced fragmentation of DNA and the development of apoptotic bodies (characteristic physiological features of apoptosis) in U937 cells. By analysis of chemical structure, this active compound was identified as tryptophol. Tryptophol induced apoptosis involving caspase-8 and -3 activation, followed by cleavage of poly (ADP-ribose) polymerase (PARP), as shown by measurement of enzyme activity and immunoblot analysis. The cell viability of normal lymphocytes separated from human blood was less affected by tryptophol, and fragmentation of DNA was not induced in normal lymphocytes. These results indicate that tryptophol isolated from black soybean vinegar inhibited the proliferation of U937 cells by inducing apoptosis via a pathway involving caspase-8 followed by caspase-3, without affecting normal lymphocytes.