Paul W. Sylvester

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.

Optimization of the Tetrazolium Dye (MTT) Colorimetric Assay for Cellular Growth and Viability

The MTT colorimetric assay is an established method of determining viable cell number in proliferation and cytotoxicity studies. This assay is based on the cleavage of the yellow tetrazolium salt, MTT, to form a soluble blue formazan product by mitochondrial enzymes, and the amount of formazan produced is directly proportional to the number of living, not dead cells, present during MTT exposure. Since the MTT assay is rapid, convenient, and economical, it has become a very popular technique for quantification of viable cells in culture. However, various parameters have been identified that can affect cellular metabolism and other factors, which significantly modify MTT-specific activity and can result in calculated false high or false low cell counts. Therefore, it is essential to establish assay parameters with the proper controls for each cell line and/or drug treatment in order to optimize assay conditions and minimize confounding effects. These parameters should include determining appropriate cell densities, culture medium, optimal concentrations and exposure times for MTT, fresh culture medium at the time of assay to avoid nutrient depletion, and controlling for drug treatment effects that may influence cellular metabolism. By controlling these important parameters, the MTT colorimetric assay provides accurate and reliable quantification of viable cell number.

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

Studies were conducted to determine the comparative effects of tocopherols and tocotrienols on normal mammary epithelial cell growth and viability. Cells isolated from midpregnant BALB/c mice were grown within collagen gels and maintained on serum-free media. Treatment with 0–120 μM α-and γ-tocopherol had no effect, whereas 12.5–100 m μM tocotrienol-rich fraction of palm oil (TRF), 100–120 μM δ-tocopherol, 50–60 μM α-tocotrienol, and 8–14 μM γ- or δ-tocotrienol significantly inhibited cell growth in a dose-responsive manner. In acute studies, 24-h exposure to 0–250 μM α-, γ-, and δ-tocopherol had no effect, whereas similar treatment with 100–250 μM TRF, 140–250 μM α-, 25–100 μM γ- or δ-tocotrienol significantly reduced cell viability. Growth-inhibitory doses of TRF, δ-tocopherol, and a-, γ-, and δ-tocotrienol were shown to induce apoptosis in these cells, as indicated by DNA fragmentation. Results also showed that mammary epithelial cells more easily or preferentially took up tocotrienols as compared to tocopherols, suggesting that at least part of the reason tocotrienols display greater biopotency than tocopherols is because of greater cellular accumulation. In summary, these findings suggest that the highly biopotent γ- and δ-tocotrienol isoforms may play a physiological role in modulating normal mammary gland growth, function, and remodeling.

Role of caspase-8 activation in mediating vitamin E-induced apoptosis in murine mammary cancer cells.

The vitamin E family of compounds is divided into two subgroups, tocopherols and tocotrienols. However, tocotrienols display more potent apoptotic activity in mammary cancer cells. Although the mechanism(s) mediating tocotrienol-induced apoptosis is presently unknown, apoptosis is carried out by activation of initiator caspases (caspase-8 or -9) that subsequently activate effector caspases (caspase-3, -6, or -7). Studies were conducted to determine whether tocotrienol-induced apoptosis is mediated by activation of the caspase-8 and/or caspase-9 pathway. Highly malignant +SA mouse mammary epithelial cells were grown in culture and maintained on serum-free media. Treatment with tocotrienol-rich-fraction of palm oil (TRF) and γ-tocotrienol, but not α-tocopherol, induced a dose-dependent decrease in +SA cell viability. TRF- and γ-tocotrienol-induced cell death resulted from apoptosis, as determined by DNA fragmentation and positive TUNEL assay staining. Additional studies showed that treatment with 50 μM TRF or 20 μM γ-tocotrienol increased intracellular activity and levels of processed caspase-8 and -3 but not caspase-9. Furthermore, treatment with specific caspase-8 or -3 inhibitors, but not caspase-9 inhibitor, completely blocked the tocotrienol-induced apoptosis in +SA cells. These findings demonstrate that tocotrienol-induced apoptosis in +SA mammary cancer cells is mediated through activation of the caspase-8 signaling pathway and is independent of caspase-9 activation.

Gamma-tocotrienol inhibits neoplastic mammary epithelial cell proliferation by decreasing Akt and nuclear factor kappaB activity.

Tocotrienols, a subgroup within the vitamin E family of compounds, have been shown to display potent anticancer activity and inhibit preneoplastic and neoplastic mammary epithelial cell proliferation at treatment doses that have little or no effect on normal cell growth and function. However, the specific intracellular mechanisms mediating the antiproliferative effects of tocotrienols are presently unknown. Because Akt and nuclear factor κB (NFκB) are intimately involved in mammary tumor cell proliferation and survival, studies were conducted to determine the effects of γ-tocotrienol on Akt and NFκB activity in neoplastic +SA mammary epithelial cells in vitro. Treatment with 0–8 μM γ-tocotrienol for 0–3 days caused a dose-responsive inhibition in +SA cell growth and mitotic activity, as determined by MTT colorimetric assay and proliferating cell nuclear antigen immunocytochemical staining, respectively. Studies also showed that treatment with 4 μM γ-tocotrienol, a dose that inhibited +SA cell growth by more than 50% compared with that of untreated control cells, decreased intracellular levels of activated phosphotidylinositol 3-kinase-dependent kinase (PI3K)-dependent kinase 1 (phospho-PDK-1) and Akt, and reduced phospho-Akt kinase activity. Furthermore, these effects were not found to be associated with an increase in either phosphatase and tensin homologue deleted from chromosome 10 (PTEN) or protein phosphatase type 2A phosphatase activity. In addition, γ-tocotrienol treatment was shown to decrease NFκB transcriptional activity, apparently by suppressing the activation of IκB-kinase-α/β, an enzyme associated with inducing NFκB activation, in summary, these findings demonstrate that the antiproliferative effects of γ-tocotrienol result, at least in part, from a reduction in Akt and NFκB activity in neoplastic +SA mammary epithelial cells.

Interaction between opiates and hypothalamic dopamine on prolactin release.

Abstract Opiate stimulation of prolactin (PRL) release appears to involve a hypothalamic mechanism(s). The present study utilized both central acting drugs and direct measurement of hypothalamic dopamine (DA) to investigate this problem. Administration of L-dopa, the precursor of DA; piribedil, a DA agonist; or amineptine, a DA reuptake inhibitor, each decreased serum PRL concentrations. Morphine sulfate (MS) and haloperidol (HAL) significantly increased serum PRL levels. L-dopa and piribedil reversed the stimulatory effect of MS on serum PRL concentrations by increasing dopamine activity. MS blocked the inhibitory effects of amineptine on serum PRL release, possibly by decreasing the concentration of DA available for reuptake. Injection of subeffective doses of HAL concurrently with a subeffective dose of MS increased serum PRL concentrations, by an additive inhibitory action on dopaminergic activity. β-endorphin, an endogenous opioid peptide, decreased the rate of DA turnover in the median eminence, and increased serum PRL levels approximately 10 - fold. These observations indicate that opiates stimulate PRL release by decreasing DA activity in the median eminence.

γ‐Tocotrienol inhibits ErbB3‐dependent PI3K/Akt mitogenic signalling in neoplastic mammary epithelial cells

Abstract.  The antiproliferative effects of γ‐tocotrienol are associated with suppression in epidermal growth factor (EGF)‐dependent phosphatidylinositol‐3‐kinase (PI3K)/PI3K‐dependent kinase‐1 (PDK‐1)/Akt mitogenic signalling in neoplastic mammary epithelial cells. Studies were conducted to investigate the direct effects of γ‐tocotrienol treatment on specific components within the PI3K/PDK‐1/Akt mitogenic pathway. +SA cells were grown in culture and maintained in serum‐free media containing 10 ng/ml EGF as a mitogen. Treatment with 0–8 µmγ‐tocotrienol resulted in a dose‐responsive decrease in the +SA cell growth and a corresponding decrease in phospho‐Akt (active) levels. However, γ‐tocotrienol treatment had no direct inhibitory effect on Akt or PI3K enzymatic activity, suggesting that the inhibitory effects of γ‐tocotrienol occur upstream of PI3K, possibly at the level of the EGF‐receptor (ErbB1). Additional studies were conducted to determine the effects of γ‐tocotrienol on ErbB receptor activation. Results showed that γ‐tocotrienol treatment had little or no effect on ErbB1 or ErbB2 receptor tyrosine phosphorylation, a prerequisite for substrate interaction and signal transduction, but did cause a significant and progressive decrease in the ErbB3 tyrosine phosphorylation. Because ErbB1 or ErbB2 receptors form heterodimers with the ErbB3 receptor, and ErbB3 heterodimers have been shown to be the most potent activators of PI3K, these findings strongly suggest that the antiproliferative effects of γ‐tocotrienol in neoplastic +SA mouse mammary epithelial cells are mediated by a suppression in ErbB3‐receptor tyrosine phosphorylation and subsequent reduction in PI3K/PDK‐1/Akt mitogenic signalling.

Counteraction of gonadal steroid inhibition of luteinizing hormone release by naloxone.

The effect of naloxone on the negative feedback action of gonadal steroids on luteinizing hormone (LH) release was studied in castrated male and female rats. The reduction by estradiol benzoate or testosterone propionate of elevated serum LH levels in rats ovariectomized for 4 weeks was reversed by a single injection of naloxone. Injection of estradiol benzoate, together with progesterone, similarly reduced serum LH levels in ovariectomized rats, and this inhibition was partially reversed by naloxone. A single injection of testosterone propionate decreased serum LH levels in male rats castrated 48 h earlier, and naloxone completely blocked the androgen-induced inhibition of LH secretion. Chronic administration of either testosterone propionate or morphine sulfate to castrated male rats prevented the postcastration rise of serum LH. The decrease of hypothalamic LHRH in castrated rats was completely blocked by testosterone propionate or morphine sulfate administration. These results are believed to indicate that hypothalamic opiates are involved in gonadal steroid feedback inhibition of LH release.

Tocotrienol-Induced Caspase-8 Activation Is Unrelated to Death Receptor Apoptotic Signaling in Neoplastic Mammary Epithelial Cells

Tocotrienols, a subclass in the vitamin E family of compounds, have been shown to induce apoptosis by activating caspase-8 and caspase-3 in neoplastic mammary epithelial cells. Since caspase-8 activation is associated with death receptor apoptotic signaling, studies were conducted to determine the exact death receptor/ligand involved in tocotrienol-induced apoptosis. Highly malignant +SA mouse mammary epithelial cells were grown in culture and maintained in serum-free media. Treatment with 20 μM γ-tocotrienol decreased +SA cell viability by inducing apoptosis, as determined by positive terminal dUTP nick end labeling (TUNEL) immunocytochemical staining. Western blot analysis showed that γ-tocotrienol treatment increased the levels of cleaved (active) caspase-8 and caspase-3. Combined treatment with caspase inhibitors completely blocked tocotrienol-induced apoptosis. Additional studies showed that treatment with 100 ng/ml tumor necrosis factor-α (TNF-α), 100 ng/ml FasL, 100 ng/ml TNF-related apoptosis-inducing ligand (TRAIL), or 1 μg/ml apoptosis-inducing Fas antibody failed to induce death in +SA cells, indicating that this mammary tumor cell line is resistant to death receptor-induced apoptosis. Furthermore, treatment with 20 μM γ-tocotrienol had no effect on total, membrane, or cytosolic levels of Fas, Fas ligand (FasL), or Fas-associated via death domain (FADD) and did not induce translocation of Fas, FasL, or FADD from the cytosolic to the membrane fraction, providing additional evidence that tocotrienol-induced caspase-8 activation is not associated with death receptor apoptotic signaling. Other studies showed that treatment with 20 μM γ-tocotrienol induced a large decrease in the relative intracellular levels of phospho–phosphatidylinositol 3-kinase (PI3K)-dependent kinase 1 (phospho-PDK-1 active), phospho-Akt (active), and phospho-glycogen synthase kinase-3, as well as decreasing intracellular levels of FLICE-inhibitory protein (FLIP), an antiapoptotic protein that inhibits caspase-8 activation, in these cells. Since stimulation of the PI3K/PDK/Akt mitogenic pathway is associated with increased FLIP expression, enhanced cellular proliferation, and survival, these results indicate that tocotrienol-induced caspase-8 activation and apoptosis in malignant +SA mammary epithelial cells is associated with a suppression in PI3K/PDK-1/Akt mitogenic signaling and subsequent reduction in intracellular FLIP levels.

Effects of Morphine and Naloxone on Inhibition by Ovarian Hormones of Pulsatile Release of LH in Ovariectomized Rats

The purpose of this study was to determine the effects of morphine and naloxone on pulsatile release of LH in ovariectomized rats treated (or untreated) with ovarian steroids. Ovariectomized rats were given a subcutaneous injection of estradiol benzoate (20 micrograms) or estradiol benzoate (20 micrograms) and progesterone (10 mg) 3 days prior to experimentation. The rats were then given intravenous injections of naloxone (2 mg/kg), morphine (5 mg/kg), or 0.87% NaCl every hour for 3 h. For LH assays, 0.3 ml blood was collected via an atrial cannula 15 min after drug treatment and every 15 min thereafter for 3 h. Pulsatile LH release was suppressed by estradiol benzoate or the combination of estradiol benzoate and progesterone. Naloxone was able to counteract inhibition of pulsatile LH release by these steroids. These results suggest that the endogenous opioid peptides are involved in the negative feedback exerted by estrogen and progesterone on pulsatile LH release. Morphine had no effect on steroid inhibition of pulsatile LH release.