Janet Lightner

Tocotrienols inhibit AKT and ERK activation and suppress pancreatic cancer cell proliferation by suppressing the ErbB2 pathway.

Tocotrienols are members of the vitamin E family but, unlike tocopherols, possess an unsaturated isoprenoid side chain that confers superior anti-cancer properties. The ability of tocotrienols to selectively inhibit the HMG-CoA reductase pathway through posttranslational degradation and to suppress the activity of transcription factor NF-κB could be the basis for some of these properties. Our studies indicate that γ- and δ-tocotrienols have potent antiproliferative activity in pancreatic cancer cells (Panc-28, MIA PaCa-2, Panc-1, and BxPC-3). Indeed both tocotrienols induced cell death (>50%) by the MTT cell viability assay in all four pancreatic cancer cell lines. We also examined the effects of the tocotrienols on the AKT and the Ras/Raf/MEK/ERK signaling pathways by Western blotting analysis. γ- and δ-tocotrienol treatment of cells reduced the activation of ERK MAP kinase and that of its downstream mediator RSK (ribosomal protein S6 kinase) in addition to suppressing the activation of protein kinase AKT. Suppression of activation of AKT by γ-tocotrienol led to downregulation of p-GSK-3β and upregulation accompanied by nuclear translocation of Foxo3. These effects were mediated by the downregulation of Her2/ErbB2 at the messenger level. Tocotrienols but not tocopherols were able to induce the observed effects. Our results suggest that the tocotrienol isoforms of vitamin E can induce apoptosis in pancreatic cancer cells through the suppression of vital cell survival and proliferative signaling pathways such as those mediated by the PI3-kinase/AKT and ERK/MAP kinases via downregulation of Her2/ErbB2 expression. The molecular components for this mechanism are not completely elucidated and need further investigation.

Inhibiting efflux with novel non-ionic surfactants: Rational design based on vitamin E TPGS.

Tocopheryl Polyethylene Glycol Succinate 1000 (TPGS 1000) can inhibit P-glycoprotein (P-gp); TPGS 1000 was not originally designed to inhibit an efflux pump. Recent work from our laboratories demonstrated that TPGS activity has a rational PEG chain length dependency. In other recent work, inhibition mechanism was investigated and appears to be specific to the ATPase providing P-gp energy. Based on these observations, we commenced rational surface-active design. The current work summarizes new materials tested in a validated Caco-2 cell monolayer model; rhodamine 123 (10microM) was used as the P-gp substrate. These results demonstrate that one may logically construct non-ionic surfactants with enhanced propensity to inhibit in vitro efflux. One new surfactant based inhibitor, Tocopheryl Polypropylene Glycol Succinate 1000 (TPPG 1000), approached cyclosporine (CsA) in its in vitro efflux inhibitory potency. Subsequently, TPPG 1000 was tested for its ability to enhance the bioavailability of raloxifene - an established P-gp substrate -in fasted male rats. Animals dosed with raloxifene and TPPG 1000 experienced an increase in raloxifene oral bioavailability versus a control group which received no inhibitor. These preliminary results demonstrate that one may prepare TPGS analogs that possess enhanced inhibitory potency in vitro and in vivo.

γ-Tocotrienol induces growth arrest through a novel pathway with TGFβ2 in prostate cancer

Regions along the Mediterranean and in southern Asia have lower prostate cancer incidence compared to the rest of the world. It has been hypothesized that one of the potential contributing factors for this low incidence includes a higher intake of tocotrienols. Here we examine the potential of γ-tocotrienol (GT3) to reduce prostate cancer proliferation and focus on elucidating pathways by which GT3 could exert a growth-inhibitory effect on prostate cancer cells. We find that the γ and δ isoforms of tocotrienol are more effective at inhibiting the growth of prostate cancer cell lines (PC-3 and LNCaP) compared with the γ and δ forms of tocopherol. Knockout of PPAR-γ and GT3 treatment show inhibition of prostate cancer cell growth, through a partially PPAR-γ-dependent mechanism. GT3 treatment increases the levels of the 15-lipoxygenase-2 enzyme, which is responsible for the conversion of arachidonic acid to the PPAR-γ-activating ligand 15-S-hydroxyeicosatrienoic acid. In addition, the latent precursor and the mature forms of TGFβ2 are down-regulated after treatment with GT3, with concomitant disruptions in TGFβ receptor I, SMAD-2, p38, and NF-κB signaling.

Anti-Neoplastic Activity of Two Flavone Isomers Derived from Gnaphalium elegans and Achyrocline bogotensis

Over 4000 flavonoids have been identified so far and among these, many are known to have antitumor activities. The basis of the relationships between chemical structures, type and position of substituent groups and the effects these compounds exert specifically on cancer cells are not completely elucidated. Here we report the differential cytotoxic effects of two flavone isomers on human cancer cells from breast (MCF7, SK-BR-3), colon (Caco-2, HCT116), pancreas (MIA PaCa, Panc 28), and prostate (PC3, LNCaP) that vary in differentiation status and tumorigenic potential. These flavones are derived from plants of the family Asteraceae, genera Gnaphalium and Achyrocline reputed to have anti-cancer properties. Our studies indicate that 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxy flavone) displays potent activity against more differentiated carcinomas of the colon (Caco-2), and pancreas (Panc28), whereas 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone) cytototoxic action is observed on poorly differentiated carcinomas of the colon (HCT116), pancreas (Mia PaCa), and breast (SK-BR3). Both flavones induced cell death (>50%) as proven by MTT cell viability assay in these cancer cell lines, all of which are regarded as highly tumorigenic. At the concentrations studied (5–80 µM), neither flavone demonstrated activity against the less tumorigenic cell lines, breast cancer MCF-7 cells, androgen-responsive LNCaP human prostate cancer line, and androgen-unresponsive PC3 prostate cancer cells. 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxy flavone) displays activity against more differentiated carcinomas of the colon and pancreas, but minimal cytotoxicity on poorly differentiated carcinomas of these organs. On the contrary, 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone) is highly cytotoxic to poorly differentiated carcinomas of the colon, pancreas, and breast with minimal activity against more differentiated carcinomas of the same organs. These differential effects suggest activation of distinct apoptotic pathways. In conclusion, the specific chemical properties of these two flavone isomers dictate mechanistic properties which may be relevant when evaluating biological responses to flavones.

Potent human uric acid transporter 1 inhibitors: in vitro and in vivo metabolism and pharmacokinetic studies

Human uric acid transporter 1 (hURAT1; SLC22A12) is a very important urate anion exchanger. Elevated urate levels are known to play a pivotal role in cardiovascular diseases, chronic renal disease, diabetes, and hypertension. Therefore, the development of potent uric acid transport inhibitors may lead to novel therapeutic agents to combat these human diseases. The current study investigates small molecular weight compounds and their ability to inhibit 14C-urate uptake in oocytes expressing hURAT1. Using the most promising drug candidates generated from our structure–activity relationship findings, we subsequently conducted in vitro hepatic metabolism and pharmacokinetic (PK) studies in male Sprague-Dawley rats. Compounds were incubated with rat liver microsomes containing cofactors nicotinamide adenine dinucleotide phosphate and uridine 5′-diphosphoglucuronic acid. In vitro metabolism and PK samples were analyzed using liquid chromatography/mass spectrometry-mass spectrometry methods. Independently, six different inhibitors were orally (capsule dosing) or intravenously (orbital sinus) administered to fasting male Sprague-Dawley rats. Blood samples were collected and analyzed; these data were used to compare in vitro and in vivo metabolism and to compute noncompartmental model PK values. Mono-oxidation (Phase I) and glucuronidation (Phase II) pathways were observed in vitro and in vivo. The in vitro data were used to compute hepatic intrinsic clearance, and the in vivo data were used to compute peak blood concentration, time after administration to achieve peak blood concentration, area under the curve, and orally absorbed fraction. The experimental data provide additional insight into the hURAT1 inhibitor structure–activity relationship and in vitro–in vivo correlation. Furthermore, the results illustrate that one may successfully prepare potent inhibitors that exhibit moderate to good oral bioavailability.

A specific molecular beacon probe for the detection of human prostate cancer cells.

The small-molecule, water-soluble molecular beacon probe 1 is hydrolyzed by the lysate and living cells of human prostate cancer cell lines (LNCaP), resulting in strong green fluorescence. In contrast, probe 1 does not undergo significant hydrolysis in either the lysate or living cells of human nontumorigenic prostate cells (RWPE-1). These results, corroborated by UV-Vis spectroscopy and fluorescent microscopy, reveal that probe 1 is a sensitive and specific fluorogenic and chromogenic sensor for the detection of human prostate cancer cells among nontumorigenic prostate cells and that carboxylesterase activity is a specific biomarker for human prostate cancer cells.

Investigating idebenone and idebenone linoleate metabolism: in vitro pig ear and mouse melanocyte studies

Objective  The aim of this study was to investigate inherent in vitro permeability, metabolism, and cytotoxicity of idebenone – an active used to protect skin as an anti‐aging agent – and compare it to idebenone linoleate.

Abstract 4351: Gamma-tocotrienol not alpha-tocopherol is cytotoxic to prostate cancer cells through modulation of phospho-c-Jun and phospho-Erk.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Chemical differences in gamma-tocotrienol including the presence of an isoprenoid side chain are responsible for differences in cytotoxic potency of gamma-tocotrienol compared to alpha-tocopherol. Alpha-tocopherol has been shown to be ineffective in prostate cancer chemoprevention in the SELECT trial; in fact, there was an increased incidence of prostate cancer in the alpha-tocopherol arm indicative of deleterious effects. Therefore, we explored the cytotoxic effects of alpha-tocopherol (AT), gamma-tocopherol (GT) and gamma-tocotrienol (GT3) in prostate cancer cell lines, PC-3 (androgen independent) and LNCaP (androgen dependent). Prostate cancer cell lines were grown in culture and treated with various concentrations and duration with the vitamin E isoforms. Cytotoxicity was determined by MTT and cell viability assays. We also explored possible molecular mechanisms to further understand differences in effects between the tocopherols and tocotrienols. GT3 showed cytotoxic effects in a dose and time-dependent manner, in both cell lines. PC-3 cells were noted to be more sensitive to growth suppression effects of GT3 than LNCaP cells. GT did not show significant cytotoxicity on cell growth at any given dose or time end point, whereas AT may promote prostate cancer cell growth. We also found that the combination of AT (40um) with increasing dose concentration of GT3 showed less pronounced cytotoxicity at 24 hours, when compared with various concentrations of GT3 alone, again suggesting a possible antagonistic role of AT. Exposure of PC-3 and LNCaP cells to increasing dose of GT3 was also examined for p-AKT, p-Erk, p-ERBB2 and phospho-c-Jun activity via Western blot at various time points. Our results show a dose dependent up regulation of pc-Jun and p-Erk in GT3 treated cells. No effects were noted on p-Akt or p-ERBB2 at the time points and concentrations studied. Some earlier studies found JNK and MAPK/Erk activation as playing a central role in the tocopherol induced apoptosis. C-Jun is downstream effector in JNK signaling pathway, activates transcription factors, which in turn modulate gene expression, to generate appropriate biological responses leading to apoptosis. In contrast to Erk recognized role in cancer cells proliferation we found Erk 1/2 up-regulation in GT3 induced apoptosis. This finding has also been reported earlier in some studies on prostate and other cancer cell lines. Our work shows that GT3 has cytotoxic effects on prostate cancer cells and induces cellular apoptosis through the modulation of phospho-c-Jun and MAPK pathways, while AT has either none or promotes prostate cancer cell growth in culture. Further studies are under way to dissect the molecular mechanisms of c-Jun and Erk activation by gamma-tocotrienol in prostate cancer cells. Citation Format: Rashid Mahboob, Victoria P. Ramsauer, Janet W. Lightner, William L. Stone, Koyamangalath Krishnan. Gamma-tocotrienol not alpha-tocopherol is cytotoxic to prostate cancer cells through modulation of phospho-c-Jun and phospho-Erk. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4351. doi:10.1158/1538-7445.AM2013-4351

γ-Tocotrienol induces apoptosis in pancreatic cancer cells by upregulation of ceramide synthesis and modulation of sphingolipid transport

BackgroundCeramide synthesis and metabolism is a promising target in cancer drug development. γ-tocotrienol (GT3), a member of the vitamin E family, orchestrates multiple effects that ensure the induction of apoptosis in both, wild-type and RAS-mutated pancreatic cancer cells. Here, we investigated whether these effects involve changes in ceramide synthesis and transport.MethodsThe effects of GT3 on the synthesis of ceramide via the de novo pathway, and the hydrolysis of sphingomyelin were analyzed by the expression levels of the enzymes serine palmitoyl transferase, ceramide synthase-6, and dihydroceramide desaturase, and acid sphingomyelinase in wild-type RAS BxPC3, and RAS-mutated MIA PaCa-2 and Panc 1 pancreatic cancer cells. Quantitative changes in ceramides, dihydroceramides, and sphingomyelin at the cell membrane were detected by LCMS. Modulation of ceramide transport by GT3 was studied by immunochemistry of CERT and ARV-1, and the subsequent effects at the cell membrane was analyzed via immunofluorescence of ceramide, caveolin, and DR5.ResultsGT3 favors the upregulation of ceramide by stimulating synthesis at the ER and the plasma membrane. Additionally, the conversion of newly synthesized ceramide to sphingomyelin and glucosylceramide at the Golgi is prevented by the inhibition of CERT. Modulation ARV1 and previously observed inhibition of the HMG-CoA pathway, contribute to changes in membrane structure and signaling functions, allows the clustering of DR5, effectively initiating apoptosis.ConclusionsOur results suggest that GT3 targets ceramide synthesis and transport, and that the upregulation of ceramide and modulation of transporters CERT and ARV1 are important contributors to the apoptotic properties demonstrated by GT3 in pancreatic cancer cells.

Abstract 3567: Metformin suppresses synthesis of pro-survival sphingolipid, sphingosine-1-phosphate, by inhibition of sphingosine kinase-1, in MCF-7 and SK-BR-3 breast cancer cell lines

The antidiabetic drug, Metformin, may possess anti-cancer properties. Metformin has been shown to suppress proliferation of breast cancer cells primarily through activation of AMP-activated protein kinase (AMPK) and its suppression of downstream signaling pathways, such as mTOR, involved in cell replication. Other mechanisms may also play a role. Sphingolipids have a role in apoptosis and survival. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator, promotes cell survival, proliferation, migration, angiogenesis, lymph angiogenesis, and immune response. S1P is involved in both intracellular and extracellular functions and regulates proliferation and survival. Blocking S1P synthesis inhibits cellular proliferation. Sphingosine kinase (SphK) is a lipid kinase that catalyzes formation of S1P from the precursor sphingosine. SphK is known to be upregulated in cancer cells, promoting tumor progression. S1P has a critical role in cancer progression and is considered a viable target for cancer therapeutics. Our previous studies show that metformin has an effect on the synthesis of pro-apoptotic ceramides. We hypothesized that metformin induces cytotoxicity by reducing levels of the pro-survival sphingolipid, S1P. Firstly, MCF-7 and SK-BR-3 breast cancer cell lines were treated with increasing concentrations of metformin, and cytotoxicity was determined by MTT cell culture experiments after 24 hours of drug exposure. Metformin induces cytotoxicity in these breast cancer cells at a lowest concentration of 2.5mM, and percentage cytotoxicity increases in a dose-dependent manner. We utilized liquid chromatography and mass spectrometry and determined that cellular S1P levels are decreased in MCF-7 cells treated with 2.5mM metformin when compared with the control group. Finally, we treated MCF-7 and SK-BR-3 breast cancer cells with metformin, SK I/II (a known SphK inhibitor), and an untreated control group for 2, 4 and 6 hours. The dose of metformin was 10mM, which was chosen from a dose-response curve using MTT assay. The dose of SK I/II was 20uM, chosen based on the IC50 given. All treatments were done using low glucose media. Using the lysates from the harvested cells, gel electrophoresis and western blots using antibodies to SphK and S1P were run. Our results showed that metformin decreased the cellular levels of SphK and S1P. Thus, metformin exhibits anticancer properties via inhibiting the production of pro-survival lipid S1P. This data suggests that the pro-apoptotic effect of metformin may be partly mediated through its disruption of synthesis of S1P in breast cancer cells. Further work is necessary to characterize the sphingolipid content of MCF-7 and SK-BR-3 cancer cells before and after metformin treatment. Citation Format: Ashley Rose, Daniel Wann, Janet Lightner, Marianne Brannon, William Stone, Victoria Palau, Koyamangalath Krishnan. Metformin suppresses synthesis of pro-survival sphingolipid, sphingosine-1-phosphate, by inhibition of sphingosine kinase-1, in MCF-7 and SK-BR-3 breast cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3567.