Tocotrienol-induced cytotoxicity is unrelated to mitochondrial stress apoptotic signaling in neoplastic mammary epithelial cells.

Tocotrienols and tocopherols represent the 2 subgroups within the vitamin E family of compounds, but tocotrienols display significantly greater apoptotic activity against a variety of cancer cell types. However, the exact mechanism mediating tocotrienol-induced apoptosis is not understood. Studies were conducted to determine the effects of tocotrienols on mitochondrial-stress-mediated apoptotic signaling in neoplastic +SA mammary epithelial cells grown in vitro. Exposure for 24 h to 0-20 micromol/L gamma-tocotrienol resulted in a dose-responsive increase in +SA cells undergoing apoptosis, as determined by flow cytometric analysis of Annexin V staining. However, tocotrienol-induced apoptosis was not associated with a disruption or loss of mitochondrial membrane potential, or the release of mitochondrial cytochrome c into the cytoplasm, as determined by JC-1 flow cytometric staining and ELISA assay, respectively. Interestingly, apoptotic +SA cells showed a paradoxical decrease in mitochondrial levels of pro-apoptotic proteins Bid, Bax, and Bad, and a corresponding increase in mitochondrial levels of anti-apoptotic proteins, Bcl-2 and Bcl-xL, suggesting that mitochondrial membrane stability and integrity might actually be enhanced for a limited period of time following acute tocotrienol exposure. In summary, these findings clearly demonstrate that tocotrienol-induced apoptosis occurs independently of mitochondrial stress apoptotic signaling in neoplastic +SA mammary epithelial cells.     

The Fas-associated death domain protein/caspase-8/c-FLIP signaling pathway is involved in TNF-induced activation of ERK

The cytokine TNF activates multiple signaling pathways leading to cellular responses ranging from proliferation and survival to apoptosis. While most of these pathways have been elucidated in detail over the past few years, the molecular mechanism leading to the activation of the MAP kinases ERK remains ill defined and is controversially discussed. Therefore, we have analyzed TNF-induced ERK activation in various human and murine cell lines and show that it occurs in a cell-type-specific manner. In addition, we provide evidence for the involvement of the signaling components Fas-associated death domain protein (FADD), caspase-8, and c-FLIP in the pathway activating ERK in response to TNF. This conclusion is based on the following observations: (I) Overexpression of FADD, caspase-8, or a c-FLIP protein containing the death effector domains only leads to enhanced and prolonged ERK activation after TNF treatment. (II) TNF-induced ERK activation is strongly diminished in the absence of FADD. Interestingly, the enzymatic function of caspase-8 is not required for TNF-induced ERK activation. Additional evidence suggests a role for this pathway in the proliferative response of murine fibroblasts to TNF.     

Antiproliferative and apoptotic effects of tocopherols and tocotrienols on preneoplastic and neoplastic mouse mammary epithelial cells

Studies were conducted to determine the comparative effects of tocopherols and tocotrienols on preneoplastic (CL-S1), neoplastic (-SA), and highly malignant (+SA) mouse mammary epithelial cell growth and viability in vitro. Over a 5-day culture period, treatment with 0-120 microM alpha- and gamma-tocopherol had no effect on cell proliferation, whereas growth was inhibited 50% (IC50) as compared with controls by treatment with the following: 13, 7, and 6 microM tocotrienol-rich-fraction of palm oil (TRF); 55, 47, and 23 microM delta-tocopherol; 12, 7, and 5 microM alpha-tocotrienol; 8, 5, and 4 microM gamma-tocotrienol; or 7, 4, and 3 microM delta-tocotrienol in CL-S1, -SA and +SA cells, respectively. Acute 24-hr exposure to 0-250 microM alpha- or gamma-tocopherol (CL-S1, -SA, and +SA) or 0-250 microM delta-tocopherol (CL-S1) had no effect on cell viability, whereas cell viability was reduced 50% (LD50) as compared with controls by treatment with 166 or 125 microM delta-tocopherol in -SA and +SA cells, respectively. Additional LD50 doses were determined as the following: 50, 43, and 38 microM TRF; 27, 28, and 23 microM alpha-tocotrienol; 19, 17, and 14 microM gamma-tocotrienol; or 16, 15, or 12 microM delta-tocotrienol in CL-S1, -SA, and +SA cells, respectively. Treatment-induced cell death resulted from activation of apoptosis, as indicated by DNA fragmentation. Results also showed that CL-S1, -SA, and +SA cells preferentially accumulate tocotrienols as compared with tocopherols, and this may partially explain why tocotrienols display greater biopotency than tocopherols. These data also showed that highly malignant +SA cells were the most sensitive, whereas the preneoplastic CL-S1 cells were the least sensitive to the antiproliferative and apoptotic effects of tocotrienols, and suggest that tocotrienols may have potential health benefits in preventing and/or reducing the risk of breast cancer in women.     

Futile caspase-8 activation during the apoptotic cell death induced by DNA damaging agents in human B-lymphoblasts

Caspase-8 plays an essential role in apoptosis induced by Fas activation. Moreover, caspase-8 can be processed also in response to exposure to genotoxic agents. To decipher the role of caspase-8 in DNA damaging agent (DDA)-induced apoptosis as well as the pathway(s) leading to its activation in response to genotoxic stress, we investigated caspase-8 processing induced by ionizing radiation (IR) or mitomycin C (MMC) treatment in human B-lymphoblasts. Altogether, our observations establish that caspase-8 is actively processed in both receptor-mediated and DDA-induced cell death. However, while Fas-dependent apoptosis absolutely required caspase-8 activity, it is not necessary for completion of the apoptotic program induced by IR and MMC. Experiments performed to understand the molecular pathway(s) of the caspase-8 activation after DDA demonstrated that for both IR and MMC, the Fas/Fas-L interaction is dispensable. Data obtained from caspase inhibitors and from lymphoblasts carrying mutations in ATM and FANCC proteins, involved in DDA response, clearly showed that distinct mechanisms are responsible for caspase-8 activation by IR and MMC in B-lymphoblasts. IR-dependent processing of caspase-8 involves ATM, mitochondrial collapse, FANCC, and caspase-3 activation. Caspase-8 activation by MMC evokes the mitochondrial pathways involving FANCC but not ATM. Collectively, our data indicate that caspase-8 activation is essentially a bystander effect and not a major determinant of the behavior of DDA-exposed cells.     

Retinoic acid induces caspase-8 transcription via phospho-CREB and increases apoptotic responses to death stimuli in neuroblastoma cells

Caspase-8 is frequently deleted or silenced in neuroblastoma and other solid tumor such as medulloblastoma and small cell lung carcinoma. Caspase-8 expression can be re-established in neuroblastoma cell lines by treatment with demethylating agents or with IFN-gamma. Here we show that four different retinoic acid (RA) derivatives also increase caspase-8 protein expression in neuroblastoma, medulloblastoma and small cell lung carcinoma cell lines. This increase in protein expression is mirrored by an increase in RNA expression in NB cells. However, the promoter region of the caspase-8 gene was not responsible for the induction of caspase-8 expression. Rather, we identified another intronic region containing a CREB binding site that was required for maximal induction of caspase-8 via RA. DNA-protein interaction assays revealed increased phospho-CREB binding to this response element in RA-treated NB cells. Furthermore, mutations of the CREB binding site completely blocked caspase-8 induction in the luciferase reporter system assay and transfection of dominant-negative form of CREB repressed the up-regulation of caspase-8 by RA. Importantly, RA-released cells maintained caspase-8 expression for at least 2-5 days and were more sensitive to doxorubicin and TNFalpha. Thus, RA treatment in conjunction with TNFalpha and/or subsets of cytotoxic agents may have therapeutic benefits.     

HER/ErbB receptor interactions and signaling patterns in human mammary epithelial cells

Background  

Knowledge about signaling pathways is typically compiled based on data gathered using different cell lines. This approach implicitly assumes that the cell line dependence is not important. However, different cell lines do not always respond to a particular stimulus in the same way, and lack of coherent data collected from closely related cellular systems can be detrimental to the efforts to understand the regulation of biological processes. To address this issue, we created a clone library of human mammary epithelial (HME) cells that expresses different levels of HER2 and HER3 receptors in combination with endogenous EGFR/HER1. Using our clone library, we have quantified the receptor activation patterns and systematically tested the validity of the existing hypotheses about the interaction patterns between HER1-3 receptors.     

B cell receptor cross-linking triggers a caspase-8-dependent apoptotic pathway that is independent of the death effector domain of Fas-associated death domain protein.

We have previously reported that B cell receptors, depending on the degree to which they are cross-linked, can promote apoptosis in various human B cell types. In this study, we show that B cell receptors can trigger two apoptotic pathways according to cross-linking and that these pathways control mitochondrial activation in human Burkitt's lymphoma cells. Whereas soluble anti-mu Ab triggers caspase-independent mitochondrial activation, cross-linked anti-mu Ab induces an apoptotic response associated with a caspase-dependent loss of mitochondrial transmembrane potential. This B cell receptor-mediated caspase-dependent mitochondrial activation is associated with caspase-8 activation. We show here that caspase-8 inhibitors strongly decrease cross-linking-dependent B cell receptor-mediated apoptosis in Burkitt's lymphoma BL41 cells. These inhibitors act upstream from the mitochondria as they prevented the loss of mitochondrial membrane potential observed in B cell receptor-treated BL41 cells. Caspase-8 activation in these cells was also evident from the detection of cleaved fragments of caspase-8 and the cleavage of specific substrates, including Bid. Our data show that cross-linked B cell receptors induced an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and the activation of caspase-9 and caspase-3. Cells expressing a dominant negative mutant of Fas-associated death domain protein were sensitive to cross-linked B cell receptor-induced caspase-8 activation and apoptosis; therefore, this caspase-8 activation was independent of the death effector domain of Fas-associated death domain protein.     

TNF-alpha-induced cell death in ethanol-exposed cells depends on p38 MAPK signaling but is independent of Bid and caspase-8

Alcoholic liver disease is associated with an increase in the number of necrotic and apoptotic liver parenchymal cells. Part of this injury is mediated by TNF-alpha. Ethanol exposure sensitizes cells to the cytotoxic effects of TNF-alpha. This may be due, in part, to the increased propensity of the mitochondria in ethanol-exposed cells to induction of mitochondrial permeability transition (MPT) by various agents, including the proapoptotic protein Bax. This idea is supported by the observation that increased cell death induced by TNF-alpha in ethanol-exposed cells was dependent on development of the MPT. In the present study, we elucidate the pathways through which ethanol exposure enhances TNF-alpha induction of the MPT and the resulting cytotoxicity. Specifically, ethanol-exposed cells display caspase-8- and Bid-independent cell killing during TNF-alpha treatment. Moreover, the ethanol-enhanced pathway is dependent on p38 MAPK signaling, which brings about caspase-3 activation, mitochondrial depolarization, accumulation of cytochrome c in the cytosol, and the translocation of Bax to the mitochondria. Additionally, ethanol-exposed cells display a blunting of TNF-alpha-induced Akt activation and Bcl-2 antagonist of cell death phosphorylation that may account, in part, for the increased sensitivity of the mitochondria to Bax-mediated damage.     

Se-methylselenocysteine activates caspase-3 in mouse mammary epithelial tumor cells in vitro

Se-methylselenocysteine (MSC) inhibits mouse mammary epithelial tumor cell (TM6) growth. When synchronized TM6 cells were exposed to 50 microM MSC, either for 30 minutes or continuous, the 116 kDa poly(ADP-ribose)polymerase (PARP) was cleaved to an 85 kDa fragment indicative of cells undergoing apoptosis. The earliest cleaved PARP appears at 24 hr time point followed by elevated levels of 85 kDa fragment at 34 hr and 48 hr time points when the cells were exposed to continuous treatment with MSC. Results also showed that MSC increased caspase-3 activity at 24 hr time point. In addition, continuous treatment with MSC induced DNA fragmentation at 34 hr and 48 hr time points with caspase-3 gene expression moderately increased at 16 hr and 24 hr time points. Caspase-6 and -8 were also involved in the MSC-induced apoptosis but to a lesser extent. These results suggest that MSC mediates cleavage of PARP and apoptosis by activating one or more caspases in synchronized TM6 cells and the events are dependent on the duration of treatment.     

Transferrin receptor activity in rat mammary epithelial cells

The binding of 125I-transferrin to rat mammary cells isolated by collagenase and hyaluronidase digestion has been investigated. Surface binding was determined at 4 degrees C and total binding also at 4 degrees C but in the presence of 0.1% w/v saponin. KD values between 20 and 25 nM were obtained. Binding assays at 37 degrees C showed the internalisation of the receptor and the bound transferrin was occurring but also provided evidence for an impaired recycling of the receptors to the cell surface in the freshly isolated cells. No differences in total binding were observed in cells prepared at different stages of lactation with a mean value of 29 fmol transferrin bound/micrograms cellular DNA, equivalent to 180,000 receptors per cell.     

Caspase-3 is a component of Fas death-inducing signaling complex in lipid rafts and its activity is required for complete caspase-8 activation during Fas-mediated cell death

Since its discovery, caspase-8 has been placed at the apex of the proteolytic cascade triggered by death receptor (DR) cross-linking. Because of its capacity to interact with the cytoplasmic portion of DR, it has been suggested that caspase-8 acts independently of other caspases in the initiation of Fas and other DR signaling. In this study, we demonstrate that in Jurkat cells, caspase-3 cleavage is an early step during Fas-induced apoptosis. We show that caspase-3 processing into its p20 occurs rapidly after Fas cross-linking, in the absence of mitochondrial depolarization and caspase-9 activation. Moreover, caspase-3 is present in lipid rafts of untreated Jurkat cells and peripheral T lymphocytes. Caspase-3, caspase-8, and Fas-associated death domain are further recruited to lipid rafts of Jurkat cells following anti-Fas treatment. Fas immunoprecipitation reveals that caspase-3 is a component of the death-inducing signaling complex, suggesting that this cysteine protease is in close proximity to caspase-8. Furthermore, transduction of Jurkat cells with a caspase-3 dominant-negative form inhibits caspase-8 processing and results in inhibition of apoptosis, suggesting that caspase-3 activity is required for caspase-8 activation. Overall, these findings support a model whereby caspase-3 is a component of the death-inducing signaling complex located in lipid rafts, and as such, is involved in the amplification of caspase-8 activity by the mitochondrion.     

ATP-induced apoptotic cell death in porcine ovarian theca cells through P2X7 receptor activation

Folliculogenesis modulation via distinct neurotransmitters is a well-documented phenomenon. Intraovarian purinergic signaling mechanisms have been identified previously in different species. However, the molecular elements involved and the physiological role of this purinergic signaling remain to be elucidated. Here, studies using RT-PCR amplification, immunoblotting, and immunofluorescence microscopy showed that murine and porcine ovaries express the P2X7 subtype receptor, a cationic receptor-channel operated by ATP. Using immunofluorescence it was demonstrated that P2X7 protein expression, in both mouse and pig, occurs specifically in the theca cells from antral follicles. Isolated porcine theca cells maintained in primary cultures and tested with 1 mM ATP or 250 microM Bz-ATP, a specific agonist of P2X7, responded with an increase in intracellular calcium concentration, as demonstrated in cells loaded with fluo-4 as calcium indicator. This strongly suggested that P2X7 receptors in theca cells are functional. Moreover, application for 24 hr of 1 mM ATP or 250 microM Bz-ATP induced apoptotic cell death as indicated by the DNA fragmentation pattern, positive TUNEL test, and annexin V binding. This ATP effect was antagonized by 300 microM PPADS and 200 microM oxidized ATP. Also, addition of 5 mM EGTA in the external medium to chelate free Ca++ decreased death cell to 24% of that produced by 200 microM Bz-ATP, suggesting that Ca++ influx participates in the phenomenon. The highly specific and functional expression of P2X7 receptors in theca cells suggest a role for ATP in modulating follicular physiology.     

Bid is cleaved upstream of caspase-8 activation during TRAIL-mediated apoptosis in human osteosarcoma cells

TRAIL induces apoptosis in many malignant cell types. In this study, we used the human papilloma virus (HPV) 16 E6 protein as a molecular tool to probe the TRAIL pathway in HCT116 colon carcinoma cells and U2OS osteosarcoma cells. Intriguingly, we found that while E6 protected HCT116 cells from TRAIL, U2OS cells expressing E6 remained sensitive to TRAIL. Furthermore, silencing FADD and procaspase-8 expression with siRNA did not prevent TRAIL-induced apoptosis in U2OS cells. However, siBid provided significant protection from TRAIL, and the cleavage kinetics of Bid and caspase-8 revealed that Bid was cleaved prior to the activation of caspase-8. Cathepsin B activity in U2OS cells was significantly activated shortly after exposure to TRAIL, and the cathepsin B inhibitor, CA074Me, inhibited both TRAIL- and anti-DR5-mediated apoptosis and delayed the cleavage of Bid. These findings suggest that TRAIL activates a pathway dependent on Bid, but largely independent of FADD and caspase-8, in U2OS cells.     

Endogenous prostaglandin production by established cultures of neoplastic rat mammary epithelial cells

Summary The production and release of prostaglandins (PGs) into the growth medium by established cultures of neoplastic, mammary epithelial cells derived from (a)N-nitrosomethyl-urea (NMU)-induced and (b) 7,12-dimethylbenz(a) anthracene (DMBA)-induced mammary tumors, was assessed using radioimmunoassay techniques. Prostaglandin production was determined, to a considerable extent, by in vitro conditions and the tumor line analyzed. In medium supplemented with bovine calf serum (10%), NMU cells synthesized and released nanogram quantities of PGF₂, PGE₁, and PGF_2α (6.7, 4.7, and 1.7 ng/10⁶ cells per 48 h, respectively). Concentrations of the two stable protanoid metabolites, 6-keto-PGF_1α and TXB₂, were indistinguishable from controls. In cells derived from the DMBA-induced tumor (RBA cells), no net production of immunoreactive PGs was detected. In contrast, in media supplemented with fetal bovine serum (10%), both RBA and NMU cells synthesized and released nanogram quantities of PGE₂ (1 and 4 ng/10⁶ cells per 48 h, respectively). PGE₂ production by both NMU and RBA cells was inhibited by ibuprofen, an inhibitor of cyclooxygenase (EC 1. 14. 99.1). The pattern of PG inhibition by ibuprofen differed in the two cell lines. In NMU cells, a linear dose-response inhibitory pattern was discernable, whereas in RBA cells a biphasic pattern was observed; PGE₂ levels incresed at low concentration of ibuprofen and then decreased at higher concentration. At 100 μg/ml ibuprofen, PG synthesis and release was inhibited by 90 and 100% and cell growth by 64 and 66% in NMU and RBA cells, respecively. There was no obvinous dse-response relationship between ibuprofen concentration and cell growth inhibition in either cell line. These results underline the importance of the serum component of growth medium when analyzing PG production in vitro and suggest that the epithelial cell components of experimental mammary tumors are capable of producing physiogically relevant amounts of PGs. This work was supported by Grant CA 29602 from the National Cancer Institute, Bethesda, MD, and Grant PDT-208 from the American Cancer Society.