José Quintana

Trifolin acetate-induced cell death in human leukemia cells is dependent on caspase-6 and activates the MAPK pathway.

In the present study we demonstrated that the flavonoid derivative trifolin acetate (TA), obtained by acetylation of naturally occurring trifolin, induces apoptosis. Associated downstream signaling events were also investigated. TA-induced cell death was prevented by the non-specific caspase inhibitor z-VAD-fmk and reduced by the presence of the selective caspase inhibitors z-LEHD-fmk (caspase-9), z-DEVD-fmk (caspase-3) and z-VEID-fmk (caspase-6). The apoptotic effect of TA was associated with (i) the release of cytochrome c from mitochondria which was not accompanied by dissipation of the mitochondrial membrane potential (ΔΨm), (ii) the activation of the mitogen-activated protein kinases (MAPKs) pathway and (iii) abrogated by the over-expression of Bcl-2 or Bcl-xL. TA-induced cell death was attenuated by inhibition of extracellular signal-regulated kinases (ERK) 1/2 with U0126 and inhibition of p38MAPK with SB203580. In contrast, inhibition of c-Jun NH2-terminal kinase (JNK) by SP600125 significantly enhanced apoptosis. Although reactive oxygen species (ROS) increased in response to TA, this did not seem to play a pivotal role in the apoptotic process since different anti-oxidants were unable to provide cell protection. The present study demonstrates that TA-induced cell death is mediated by an intrinsic-dependent apoptotic event involving mitochondria and MAPK, and through a mechanism independent of ROS generation.

Induction of G2/M phase arrest and apoptosis by the flavonoid tamarixetin on human leukemia cells

Flavonoids are naturally occurring polyphenolic compounds which display a vast array of biological activities. In this study, we investigated the effects of tamarixetin on viability of human tumor cell lines and found that it was cytotoxic against leukemia cells and in particular P‐glycoprotein‐overexpressing K562/ADR cells. This compound inhibited proliferation in a concentration‐ and time‐dependent manner, induced apoptosis and blocked cell cycle progression at G2‐M phase. This was associated with the accumulation of cyclin B1, Bub1 and p21Cip1/Waf‐1, changes in the phosphorylation status of cyclin B1, Cdk1, Cdc25C and MPM‐2, and inhibition of tubulin polymerization. Moreover, cell death was found to be associated with cytochrome c release and cleavage of caspases and of poly(ADP‐ribose) polymerase, and completely abrogated by the free‐radical scavenger N‐acetyl‐L‐cysteine. The sensitivity of leukemic cells to tamarixetin suggests that it should be considered for further preclinical and in vivo testing.

Naturally occurring asteriscunolide A induces apoptosis and activation of mitogen-activated protein kinase pathway in human tumor cell lines.

Sesquiterpene lactones have attracted much attention because they display a wide range of biological activities, including antitumor properties. Here, we show the effects of the naturally occurring sesquiterpene lactone asteriscunolide A (AS) on viability of human melanoma, leukemia and cells that overexpress antiapoptotic proteins, namely Bcl‐2 and Bcl‐xL. All cell lines were sensitive to this compound, with IC50 values of ∼5 µM. The cytotoxic effects of AS were accompanied by a G2‐M phase arrest of the cell cycle and a concentration‐ and time‐dependent appearance of apoptosis as determined by DNA fragmentation, translocation of phosphatidylserine to the cell surface and sub‐G1 ratio. Apoptosis was associated with caspase‐3 activity and poly(ADP‐ribose) polymerase cleavage and was prevented by the nonspecific caspase inhibitor z‐VAD‐fmk, indicating that caspases are essential components in this pathway. The apoptotic effect of AS was also associated with (i) the release of cytochrome c from mitochondria which was accompanied by dissipation of the mitochondrial membrane potential (ΔΨm) and (ii) the activation of the mitogen‐activated protein kinases (MAPKs) pathway. AS‐induced cell death was potentiated by inhibition of extracellular signal‐regulated kinases (ERK) 1/2 signaling with U0126 and PD98059. Intracellular reactive oxygen species (ROS) seem to play a pivotal role in this process since high levels of ROS were produced early (1 h) and apoptosis was completely blocked by the free radical scavenger N‐acetyl‐L‐cysteine (NAC). The present study demonstrates that AS‐induced cell death is mediated by an intrinsic‐dependent apoptotic event involving mitochondria and MAPKs, and through a mechanism dependent on ROS generation.

5,7,3′‐trihydroxy‐3,4′‐dimethoxyflavone‐induced cell death in human Leukemia cells is dependent on caspases and activates the MAPK pathway

Flavonoids are polyphenolic compounds which display a vast array of biological activities and are promising anticancer agents. In this study we investigated the effect of 5,7,3′‐trihydroxy‐3,4′‐dimethoxyflavone (THDF) on viability of nine human tumor cell lines and found that it was highly cytotoxic against leukemia cells. THDF induced G2–M phase cell‐cycle arrest and apoptosis through a caspase‐dependent mechanism involving cytochrome c release, processing of multiple caspases (caspase‐3, ‐6, ‐7, and ‐9) and cleavage of poly(ADP‐ribose) polymerase. Overexpression of the protective mitochondrial proteins Bcl‐2 and Bcl‐xL conferred partial resistance to THDF‐induced apoptosis. This flavonoid induced the phosphorylation of members of the mitogen‐activated protein kinases (MAPKs) family and cell death was attenuated by inhibition of c‐jun N‐terminal kinases/stress‐activated protein kinases (JNK/SAPK) and of extracellular signal‐regulated kinases (ERK) 1/2. In the present study we report that THDF‐induced cell death is mediated by an intrinsic dependent apoptotic event involving mitochondria and MAPKs, and through a mechanism independent of the generation of reactive oxygen species. The results suggest that THDF could be useful in the development of novel anticancer agents. Mol. Carcinog.

Betuletol 3-methyl ether induces G2-M phase arrest and activates the sphingomyelin and MAPK pathways in human leukemia cells.

Betuletol 3‐methyl ether (BME) is a natural phenylbenzo‐γ‐pyrone that inhibits cell proliferation in human tumor cell lines and induces apoptotic cell death in HL‐60 cells. Here we show that BME displays strong cytotoxic properties in several human leukemia cell lines (U937, K‐562, THP‐1, Jurkat, and Molt‐3) and in cells that over‐express two anti‐apoptotic proteins, namely Bcl‐2 and Bcl‐xL. BME arrested HL‐60 cells at G2‐M phase of the cell cycle, which was associated with the accumulation of cyclin B1 and p21Cip1. Fluorescence microscopy experiments suggest that BME blocked the cell cycle in mitosis. The in vivo tubulin polymerization assay shows that BME inhibits tubulin polymerization and causes similar changes of cellular microtubule network as colchicine. Our results demonstrate that BME‐induced cell death is (i) triggered in human myeloid leukemia cell that over‐express Bcl‐2 and Bcl‐xL, and (ii) associated with loss of inner mitochondrial membrane potential (ΔΨm) and an increase in reactive oxygen species (ROS). Although ROS increased in response to BME, this did not seem to play a pivotal role in the apoptotic process since the anti‐oxidant trolox was unable to provide cell protection. The treatment of HL‐60 cells with BME induces the activation of mitogen‐activated protein kinases (MAPKs) such as c‐Jun N‐terminal kinases, p38 mitogen‐activated protein kinases and extracellular signal‐regulated kinases (ERK)1/2 and stimulates the acid sphingomyelinase with concomitant ceramide generation. The findings of this study suggest that BME could be useful in the development of novel anticancer agents.

Induction of G2-M phase arrest and apoptosis by α-methylene-γ-butyrolactones in human leukemia cells

In this study, we investigated the effect of three synthetic alpha-methylene-gamma-butyrolactones (MBL) on viability of 10 human tumor cell lines and found that these lactones were highly cytotoxic against leukemia cells. Studies performed on HL-60 cells indicate that MBL induce G(2)-M arrest and apoptosis through a caspase-dependent mechanism. Apoptosis was associated to cytochrome c release, cleavage of procaspases-9 and -3, and hydrolysis of PARP. Intracellular reactive oxygen species (ROS) seem to play a key role since high levels of ROS were produced early (<15 min) and apoptosis was completely abrogated by the free radical scavenger N-acetyl-L-cysteine (NAC).

The abietane diterpenoid parvifloron D from Plectranthus ecklonii is a potent apoptotic inducer in human leukemia cells.

BACKGROUND Abietane diterpenes have attracted much attention because they display a wide range of biological activities, including antitumor activities. These compounds are the most diverse of the diterpenoids isolated from species of Plectranthus. Naturally occurring diterpene parvifloron D is the main phytochemical constituent of Plectranthus ecklonii. To examine the therapeutic potential of the plant, we evaluated whether parvifloron D displays cytotoxicity against human tumor cells. METHODS The cytotoxicity was analyzed by colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptosis was evaluated by fluorescent microscopy, transmission electron microscopy, flow cytometric analysis of annexin V-FITC and propidium iodide-stained cells and DNA fragmentation. Protein expression and processing and release of mitochondrial proteins were analyzed by Western blot. Caspase activity was determined using colorimetric substrates. The membrane potential and intracellular reactive oxygen species were detected by flow cytometry. RESULTS Parvifloron D displays strong cytotoxic properties against leukemia cells (HL-60, U-937, MOLT-3 and K-562) and in particular P-glycoprotein-overexpressing K-562/ADR cells, but has only weak cytotoxic effects on peripheral blood mononuclear cells (PBMCs). Overexpression of the protective mitochondrial proteins Bcl-2 and Bcl-xL did not confer resistance to parvifloron D-induced cytotoxicity. Growth inhibition of HL-60 cells that was triggered by parvifloron D was found to be caused by a rapid induction of apoptotic cell death. This apoptosis was prevented by the non-specific caspase inhibitor z-VAD-fmk, and by the selective caspase-9 inhibitor z-LEHD-fmk. Cell death induced by parvifloron D was found to be (i) associated with the dissipation of the mitochondrial membrane potential and the release of cytochrome c, (ii) amplified by inhibition of extracellular signal-regulated kinases (ERKs) 1/2 signaling and (iii) caused by a mechanism dependent on intracellular reactive oxygen species generation. CONCLUSION Parvifloron D is a potent cytotoxic compound against several human tumor cells and also a fast and potent apoptotic inducer in leukemia cells.

Eupatorin-Induced Cell Death in Human Leukemia Cells Is Dependent on Caspases and Activates the Mitogen-Activated Protein Kinase Pathway

Eupatorin is a naturally occurring flavone that inhibits cell proliferation in human tumor cells. Here we demonstrate that eupatorin arrests cells at the G2-M phase of the cell cycle and induces apoptotic cell death involving activation of multiple caspases, mitochondrial release of cytochrome c and poly(ADP-ribose) polymerase cleavage in human leukemia cells. This flavonoid induced the phosphorylation of members of the mitogen-activated protein kinases and cell death was attenuated by inhibition of c-jun N-terminal kinases/stress activated protein kinases. Eupatorin-induced cell death is mediated by both the extrinsic and the intrinsic apoptotic pathways and through a mechanism dependent on reactive oxygen species generation.

The eudesmanolide tanapsin from Tanacetum oshanahanii and its acetate induce cell death in human tumor cells through a mechanism dependent on reactive oxygen species.

In this study the cytotoxicities of two species of Tanacetum were evaluated against human tumor cells. Tanacetum oshanahanii extract was more cytotoxic than Tanacetum ptarmiciflorum. Analyses of both extracts of Tanacetum by ultrahigh performance liquid chromatography-tandem mass spectrometry revealed that T. oshanahanii extract contains the eudesmanolide tanapsin, while T. ptarmiciflorum lacks this sesquiterpene lactone. Tanapsin was cytotoxic against leukemia and melanoma cells, including cells that overexpress Bcl-2 and Bcl-xL, with IC50 values of approximately 10 µM, but not against quiescent or proliferating human peripheral blood mononuclear cells. Treatment of cells with tanapsin induced apoptosis. This was prevented by the non-specific caspase inhibitor z-VAD-fmk, and reduced by the selective caspase-3/7 inhibitor z-DEVD-fmk. Tanapsin acetate was also cytotoxic against leukemia and melanoma cells and a potent apoptotic inducer. Tanapsin-induced cell death was found to be associated with (i) the loss of inner mitochondrial membrane potential (ΔΨm) and release of mitochondrial cytochrome c, (ii) the activation of multiple caspases and the mitogen-activated protein kinase pathway, and (iii) an increase in reactive oxygen species generation. Generation of reactive oxygen species in response to tanapsin seems to play a crucial role in the cell death process since the antioxidant N-acetyl-l-cysteine blocked both ROS generation and cell death.

5,7,3'-Trihydroxy-3,4'-dimethoxyflavone inhibits the tubulin polymerization and activates the sphingomyelin pathway.

Flavonoids are polyphenolic compounds which display a vast array of biological activities and are among the most promising anti‐cancer agents. The derivative of quercetin, 5,7,3′‐trihydroxy‐3,4′‐dimethoxyflavone (THDF), is a natural flavonoid that inhibits cell proliferation and induces apoptosis in human leukemia cells. Here we show that THDF induces cell‐cycle arrest in the M phase and inhibits tubulin polymerization. This was associated with the accumulation of cyclin B1 and p21Cip1, changes in the phosphorylation status of cyclin B1, Cdk1, Cdc25C, and MPM‐2, and activation of the acidic sphingomyelinase (ASMase). Moreover, desipramine attenuated THDF‐mediated cell death, indicating a crucial role of ASMase in the mechanism of cell death. In vivo studies on the athymic nude mouse xenograft model also confirmed that THDF inhibits growth of human leukemia cells and suggest that this compound may have therapeutic value.