Contrasting effects of α-tocopheryl succinate on cisplatin- and etoposide-induced apoptosis

Targeting mitochondria is a promising strategy in tumor cell elimination. d-α-tocopheryl succinate (α-TOS), a redox-silent analog of vitamin E, is a potentially powerful tool for fighting tumors by directly affecting mitochondria. However, when used at low concentrations it can suppress apoptosis induced by the conventionally used anticancer drug cisplatin. In cells treated with cisplatin, 30 μM α-TOS prominently attenuated the manifestation of characteristic features of apoptosis — release of cytochrome c from mitochondria, caspase-3-like activity, and cleavage of poly(ADP-ribose) polymerase. In contrast, cell death induced by etoposide was not inhibited but rather stimulated by α-TOS. Thus, co-treatment with α-TOS and conventional antitumor drugs should be carried out with caution.     

Superoxide is responsible for apoptosis in rat vascular smooth muscle cells induced by α-tocopheryl hemisuccinate

We investigated the mechanism of cell toxicity of α-tocopheryl hemisuccinate (TS). TS concentration- and time-dependently induced the lactate dehydrogenase release and DNA fragmentation of rat vascular smooth muscle cells (VSMC). Exogenous addition of superoxide dismutase, but not catalase, significantly inhibited the cell toxicity of TS. The NADPH-dependent oxidase activity of VSMC was stimulated by TS treatment. The cell toxicity of TS was inhibited by NADPH oxidase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride. Consequently, TS-induced apoptosis of VSMC was suggested to be caused by exogenous O₂⁻ generated via the oxidase system activated with TS.     

Syntheses of α-tocopheryl glycosides by glucosidases

Enzymatic syntheses of water-soluble alpha-tocopheryl glycosides were carried out in di-isopropyl ether using amyloglucosidase from Rhizopus mold or beta-glucosidase isolated from sweet almond. Optimum conditions for the amyloglucosidase were: alpha-tocopherol 0.5 mmol, D-glucose 0.5 mmol, 400 activity unit (AU) amyloglucosidase, 0.2 mM pH 7 phosphate buffer and 72 h; and for the beta-glucosidase: alpha-tocopherol 0.5 mmol, D: -glucose 0.5 mmol, 110 AU beta-glucosidase, 0.1 mM pH 6 phosphate buffer and 72 h. Out of 11 carbohydrates employed, amyloglucosidase reacted only with D-glucose to give 50% of 6-O-(alpha-D-glucopyranosyl)alpha-tocopherol. However, the beta-glucosidase gave 6-O-(beta-D-glucopyranosyl)alpha-tocopherol, 6-O-(alpha-D-galactopyranosyl)alpha-tocopherol, 6-O-(beta-D-galactopyranosyl)alpha-tocopherol, 6-O-(alpha-D-mannopyranosyl)alpha-tocopherol and 6-O-(beta-D-mannopyranosyl)alpha-tocopherol in yields ranging from 10-25%. Water solubility of 6-O-(alpha-D-glucopyranosyl)alpha-tocopherol was 26 g/l at 25 degrees C. alpha-Tocopheryl glycosides showed antioxidant activities with IC(50) values from 0.5 to 1 mM and angiotensin-converting enzyme (ACE) inhibitory activity with IC(50) values from 1.3 to 2.6 mM.     

Mechanism of action of α-galactosidase

The effect ofpH on K_m and V_max values of coconut α-galactosidase indicates the involvement of two ionizing groups with pK_a values of 3.5 and 6.5 in catalysis. Chemical modification has indicated the presence of two carboxyl groups, a tryptophan and a tyrosine, at or near the active site of α-galactosidase. Based on these facts a new mechanism of action for α-galactosidase is proposed in which the ionizing group with a pK_a of 3.5 is a carboxyl group involved in stabilizing a carbonium ion intermediate and the ionizing group with a pK_a of 6.5 is a carboxyl group perturbed due to the presence of a hydrophobic residues in its vicinity which donates a H⁺ ion in catalysis.     

5,5′-dimethyloxazolidine-2,4-dione is a strong inducer of differentiation of human promyelocytic leukemia (HL-60) cells

5,5′-Dimethyloxazolidine-2,4-dione (DMO), a weak non-metabolizable acid, is commonly utilized for determining intracellular pH. In these studies, DMO was tested as an inducer of differentiation on the basis that its uptake and subsequent dissociation might transiently raise intracellular pH and activate ion-fluxes critical for triggering maturation. After 5 days of exposure to 40 mM DMO, >60% of HL-60 cells displayed phenotypic and functional changes characteristic of mature granulocytes. As with other inducers of HL-60 cell differentiation, commitment to differentiation required culture in the presence of DMO for more than 24 h, indicating that if transient effects on pH or ion-fluxes occurred, they were not sufficient to trigger this process.DMO was either weak or inactive as an inducer of murine erythroleukemia cell (FLC) differentiation. Although other weak acids and bases triggered differentiation of both HL-60 cells and FLC, the spectrum of response differed markedly between the two lines. These results suggest that: (1) a number of common buffering agents have the potential to alter cell phenotype, and (2) their effects must be evaluated for each individual cell type.     

The effect of α-phenyl-N-t-butylnitrone on ionizing radiation-induced apoptosis in U937 cells

Ionizing radiation induces the production of reactive oxygen species (ROS), which play an important causative role in apoptotic cell death. α-Phenyl-N-t-butylnitrone (PBN) is one of the most widely used spin-trapping compounds for investigating the existence of free radicals in biological systems. We investigated the effects of PBN on ionizing radiation-induced apoptosis in U937 cells. Upon exposure to 2 Gy of γ-irradiation, there was a distinct difference between the control cells and the cells pre-treated with 2 mM PBN for 2 h in regard to apoptotic parameters, cellular redox status, mitochondria function and oxidative damage to cells. PBN effectively suppressed morphological evidence of apoptosis and DNA fragmentation in U937 cells exposed to ionizing radiation. The [GSSG]/[GSH+GSSG] ratio and the generation of intracellular ROS were higher and the [NADPH]/[NADP⁺+NADPH] ratio was lower in control cells compared to PBN-treated cells. The ionizing radiation-induced mitochondrial damage reflected by the altered mitochondrial permeability transition, the increase in the accumulation of ROS, and the reduction of ATP production were significantly higher in control cells compared to PBN-treated cells. PBN pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax and p53, and down-regulation of Bcl-2 compared to control cells upon exposure to ionizing radiation. This study indicates that PBN may play an important role in regulating the apoptosis induced by ionizing radiation presumably through scavenging of ROS.     

β-Elemene piperazine derivatives induce apoptosis in human leukemia cells through downregulation of c-FLIP and generation of ROS

β-Elemene is an active component of the herb medicine Curcuma Wenyujin with reported antitumor activity. To improve its antitumor ability, five novel piperazine derivatives of β-elemene, 13-(3-methyl-1-piperazinyl)-β-elemene (DX1), 13-(cis-3,5-dimethyl-1-piperazinyl)-β-elemene (DX2), 13-(4-ethyl-1-piperazinyl)-β-elemene (DX3), 13-(4-isopropyl-1-piperazinyl)-β-elemene (DX4) and 13-piperazinyl-β-elemene (DX5), were synthesized. The antiproliferative and apoptotic effects of these derivatives were determined in human leukemia HL-60, NB4, K562 and HP100-1 cells. DX1, DX2 and DX5, which contain a secondary amino moiety, were more active in inhibiting cell growth and in inducing apoptosis than DX3 and DX4. The apoptosis induction ability of DX1 was associated with the generation of hydrogen peroxide (H(2)O(2)), a decrease of mitochondrial membrane potential (MMP), and the activation of caspase-8. Pretreatment with the antioxidants N-acetylcysteine and catalase completely blocked DX1-induced H(2)O(2) production, but only partially its activation of caspase-8 and induction of apoptosis. HL-60 cells were more sensitive than its H(2)O(2)-resistant subclone HP100-1 cells to DX1-induced apoptosis. The activation of caspase-8 by these compounds was correlated with the decrease in the levels of cellular FLICE-inhibitory protein (c-FLIP). The proteasome inhibitor MG-132 augmented the decrease in c-FLIP levels and apoptosis induced by these derivatives. FADD- and caspase-8-deficient Jurkat subclones have a decreased response to DX1-induced apoptosis. Our data indicate that these novel β-elemene piperazine derivatives induce apoptosis through the decrease in c-FLIP levels and the production of H(2)O(2) which leads to activation of both death receptor- and mitochondrial-mediated apoptotic pathways.     

Mechanism of the cyanide-catalyzed oxidation of α-ketoaldehydes and α-ketoalcohols

Cyanide catalyzes the reduction of dioxygen or of ferricytochrome c by dihydroxyacetone phosphate. The rapid initial phase of these reactions, but not the subsequent slow phase, was augmented by incubating the triose phosphate aerobically or anaerobically at pH 9.0 prior to adding the cyanide. The aerobic incubation, which was most effective, was associated with a decline in enediol, whereas the less effective anaerobic incubation was accompanied by an increase in enediol content. This suggested that the α-ketoaldehyde product of autoxidation of the enediol, rather than the enediol itself, was responsible for the rapid phase reaction which followed addition of cyanide. This was confirmed by exploring the cyanide-catalyzed oxidation of the α-ketoaldehyde, phenylglyoxal. The inhibitory effect of the manganese-containing superoxide dismutase indicated that O₂⁻ was a kinetically important intermediate of the rapid phase reaction. A reaction mechanism is proposed which is consistent with the results presented.     

Mitochondrial targeting of α-tocopheryl succinate enhances its anti-mesothelioma efficacy

Abstract

Malignant mesothelioma (MM) is a fatal neoplastic disease with no therapeutic option. Therefore, the search for novel therapies is of paramount importance.

Methods

Since mitochondrial targeting of α-tocopheryl succinate (α-TOS) by its tagging with triphenylphosphonium enhances its cytotoxic effects to cancer cells, we tested its effect on MM cells and experimental mesotheliomas.

Results

Mitochondrially targeted vitamin E succinate (MitoVES) was more efficient in killing MM cells than α-TOS with IC₅₀ lower by up to two orders of magnitude. Mitochondrial association of MitoVES in MM cells was documented using its fluorescently tagged analogue. MitoVES caused apoptosis in MM cells by mitochondrial destabilization, resulting in the loss of mitochondrial membrane potential, generation of reactive oxygen species, and destabilization of respiratory supercomplexes. The role of the mitochondrial complex II in the activity of MitoVES was confirmed by the finding that MM cells with suppressed succinate quinone reductase were resistant to MitoVES. MitoVES suppressed mesothelioma growth in nude mice with high efficacy.

Discussion

MitoVES is more efficient in killing MM cells and suppressing experimental mesotheliomas compared with the non-targeted α-TOS, giving it a potential clinical benefit.     

Inhibition of ornithine decarboxylase by α-difluoromethylornithine induces apoptosis of HC11 mouse mammary epithelial cells

Summary. The effect of α-difluoromethylornithine (DFMO) on the apoptosis of HC11 mouse mammary epithelial cells was investigated. The involvement of reactive oxygen species (ROS) and Bcl-2 protein in the mechanism of apoptosis induced by ornithine decarboxylase (ODC) inhibition was also assessed. DFMO (0.1, 1 and 5 mM) induced apoptosis of HC11 cells in dose- and time-dependent manner. Apoptosis manifests itself with morphological features like: cell shrinkage, condensation of chromatin, pyknosis and fragmentation of nucleus, followed by secondary necrosis (putrosis). The decrease in the nuclear DNA contents appearing as the hypodiploidal peak sub-G₁ in the DNA histogram was not dependent on the presence of prolactin (5 μg/ml) in DFMO-treated cultures. Apoptosis induced by ODC inhibition was associated with a rapid increase in ROS concentration in HC11 cells observed within 1 h after DFMO treatment. The down-regulation of Bcl-2 as a decrease in cell number expressing bcl-2 and a lowered Bcl-2 protein content in cells expressing this protooncogene was also noted. The administration of putrescine (50 μM) lowered the number of early-apoptotic, late-apoptotic and necrotic cells. Moreover, it increased the number of cells expressing bcl-2. In conclusion, the disturbance of cellular polyamine homeostasis by inhibition of their synthesis enhances mammary epithelial cell susceptibility to apoptosis. It may occur in the mammary gland at the end of lactation, when the depletion of circulating lactogenic hormones and activation of intra-mammary apoptogenic factors expression take place. Received May 6, 1999; Accepted December 15, 1999     

Cryptotanshinone enhances TNF-??-induced apoptosis in chronic myeloid leukemia KBM-5 cells

Cryptotanshinone is a biologically active compound from the root of Salvia miltiorrhiza. In the present study, we investigated the molecular mechanisms by which cryptotanshinone is in synergy with tumor necrosis factor-alpha (TNF-α) for the induction of apoptosis in human chronic myeloid leukemia (CML) KBM-5 cells. The co-treatment of cryptotanshinone with TNF-α reduced the viability of the cells [combination index (CI) < 1]. Concomitantly, the co-treatment of cryptotanshinone and TNF-α elicited apoptosis, manifested by enhanced the number of terminal deoxynucleotide transferase-mediated dUTP-nick-end labeling (TUNEL)-positive cells, the sub-G1 cell populations, and the activation of caspase-8 and -3, in comparison with the treatment with either drug alone. The treatment with cryptotanshinone further suppressed TNF-α-mediated expression of c-FLIP(L), Bcl-x(L), but the increased level of tBid (a caspase-8 substrate). Furthermore, cryptotanshinone activated p38 but not NF-κB in TNF-α-treated KBM-5 cells. The addition of a specific p38 MAPK inhibitor SB203580 significantly attenuated cryptotanshinone/TNF-α-induced apoptosis. The combination treatment of cryptotanshinone and TNF-α also stimulated the reactive oxygen species (ROS) generation. N-acetyl-L-cysteine (NAC, a ROS scavenger) was not only able to block cryptotanshinone/TNF-α-induced ROS production but also the activation of caspase-8 and p38 MAPK. Overall, our findings suggest that cryptotanshinone can sensitize TNF-α-induced apoptosis in human myeloid leukemia KBM-5 cells, which appears through ROS-dependent activation of caspase-8 and p38.     

Involvement of integrin α5β1 in apoptosis and drug resistance of human breast carcinoma cells

The doxorubicin-resistant MCF-7Dox cell line derived from the drug-sensitive MCF-7 human breast carcinoma cell line, differs from the latter by a strongly reduced expression of the α2β1 integrin and a highly increased ex-pression of the α5β1 integrin. Silencing of this integrin in the MCF-7Dox cells by transfection with α5-specific siRNA markedly stimulated anoikis and increased sensitivity of these cells to doxorubicin. α5β1 silencing was also accompanied by a significant inhibition of the activity of signal protein kinases Akt and Erk2. Our results suggest involvement of common integrin-mediated signal mechanisms controlling apoptotic cell death under various stress conditions.     

The effect of α2,6-linked sialic acid on anti-IgM antibody-induced apoptosis in Ramos cells

Apoptosis in B cells is induced through the B cell antigen receptor (BCR) and affects the sialic acid recognition molecules on B cells. We investigated the effects of ₁-acid glycoprotein (AGP), which mainly contains 2,6-linked sialic acid, on anti-IgM antibody (Ab)-induced apoptosis in Ramos cells, which are derived from Burkitt's lymphoma. When Ramos cells were incubated with anti-IgM-Ab in plates coated with AGP, neuraminidase-digested AGP (asAGP) or 2,3-sialylated AGP (2,3AGP), apoptosis was suppressed only in those coated with AGP. We also studied the effects of CD22, which is expressed on the surface of mature B cells and binds to sugar chains containing 2,6-linked sialic acid, with anti-CD22 monoclonal antibody (mAb). Anti-CD22mAb enhanced anti-IgM Ab-induced apoptosis in Ramos cells. These contradictory results suggested that the recognition molecules for 2,6-linked sialic acid on AGP, which inhibits B-cell apoptosis, is distinct from CD22, or that different binding domains of CD22 between 2,6-linked sialic acid and anti-CD22 mAb exert opposite functions of suppression or enhancement to anti-IgM Ab-induced B cells.