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Background α-tocopherol (AT) and γ-tocotrienol (GT3) are vitamin E isoforms considered to have potential chemopreventive properties. AT has been widely studied in vitro and in clinical trials with mixed results. The latest clinical study (SELECT trial) tested AT in prostate cancer patients, determined that AT provided no benefit, and could promote cancer. Conversely, GT3 has shown antineoplastic properties in several in vitro studies, with no clinical studies published to date. GT3 causes apoptosis via upregulation of the JNK pathway; however, inhibition results in a partial block of cell death. We compared side by side the mechanistic differences in these cells in response to AT and GT3. Methods The effects of GT3 and AT were studied on androgen sensitive LNCaP and androgen independent PC-3 prostate cancer cells. Their cytotoxic effects were analyzed via MTT and confirmed by metabolic assays measuring ATP. Cellular pathways were studied by immunoblot. Quantitative analysis and the determination of relationships between cell signaling events were analyzed for both agents tested. Non-cancerous prostate RWPE-1 cells were also included as a control. Results The RAF/RAS/ERK pathway was significantly activated by GT3 in LNCaP and PC-3 cells but not by AT. This activation is essential for the apoptotic affect by GT3 as demonstrated the complete inhibition of apoptosis by MEK1 inhibitor U0126. Phospho-c-JUN was upregulated by GT3 but not AT. No changes were observed on AKT for either agent, and no release of cytochrome c into the cytoplasm was detected. Caspases 9 and 3 were efficiently activated by GT3 on both cell lines irrespective of androgen sensitivity, but not in cells dosed with AT. Cell viability of non-cancerous RWPE-1 cells was affected neither by GT3 nor AT. Conclusions c-JUN is a recognized master regulator of apoptosis as shown previously in prostate cancer. However, the mechanism of action of GT3 in these cells also include a significant activation of ERK which is essential for the apoptotic effect of GT3. The activation of both, ERK and c-JUN, is required for apoptosis and may suggest a relevant step in ensuring circumvention of mechanisms of resistance related to the constitutive activation of MEK1.

Metformin and γ-tocotrienol (GT3) are promising chemopreventative agents with studies showing improved patient outcomes with metformin use in several cancer types, including prostate cancer. However, the cellular mechanisms underlying the improved survival rates in cancer patients treated with these drugs are incompletely understood. Inhibition of mammalian target of rapamycin (mTOR), alteration of p-c-JUN and p-ERK signaling pathways and inhibition of mitochondrial complex 1 have all been implicated as part of metformin's anti-tumorigenesis activities. More recently, metformin was shown to influence the activity of epigenetic modifying enzymes resulting in genome-wide alterations in DNA methylation patterns in breast cancer cell lines. In this study, we investigated whether GT3 and metformin treatment would alter methylation pathways in prostate cancer cells.The prostate cancer cell line PC-3 (androgen independent) and normal prostate control cell line RWPE-1 were treated with varying concentrations of either GT3, metformin, the methylation inhibitor azacitidine, or metformin plus azacitidine for 6 hours. Genomic DNA was isolated, and methylation assessed using the Illumina Infinium methylation EPIC BeadChip array. Analysis revealed treatment of PC-3 cells with metformin promoted hypermethylation of CC2D2B, GHDC, LINC01377, PLPP2, and PTK2 and decreased methylation of TSPY4 as compared to normal prostate controls. qRT-PCR experiments confirmed decreased expression of PTK2 mRNA, consistent with hypermethylation of PTK2. In contrast, GT3 did not affect methylation of any identifiable recognizable genes. This study suggests metformin, but not GT3, may inhibit prostate cancer proliferation partially through epigenetic changes which disrupt downstream focal adhesion kinase (FAK) signaling pathways. Metformin is a promising chemopreventive agent; studies investigating the impact of metformin on cellular adhesion and FAK pathways are ongoing in our laboratory. Citation Format: Christine Ann Moore, Janet Lightner, Michelle Duffourc, Koyamangalath Krishnan. Metformin, not γ-tocotrienol, increases gene methylation and decreases expression of genes responsible for cell signal transduction in prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1274.

Background Ceramide 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. Methods The 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. Results GT3 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. Conclusions Our 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.

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.

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.

Metformin and γ-tocotrienol (GT3) are promising non-cytotoxic drugs for prostate cancer chemoprevention and as adjunctive treatment options for prostate cancer. Multiple mechanistic pathways may be modulated. Studies suggest that GT3, a member of the vitamin E family, and the antidiabetic agent metformin have antineoplastic properties but their effects in prostate cancer are unclear. We compared the abilities of GT3 and metformin in preventing growth of two prostate cancer cells lines (LNCaP and PC-3) and a control prostate cell line (RWPE-1) by quantifying their effects on three signaling pathways known to be pivotal in prostate carcinogenesis (AKT, MAP Kinase [pERK], and p-c-JUN).Two prostate cancer cell lines, LNCaP (androgen dependent) and PC-3 (androgen independent), and prostate control cell line RWPE-1 were treated with increasing concentrations of GT3 and/or metformin in DMEM, and cytotoxicity determined by MTS cell culture experiments. Synergy was determined using the CompuSyn program. IC50 of LNCaP with GT3 and metformin were 76.6 µM and 100.6 mM, respectively. IC50 of PC-3 with GT3 and metformin were 53.8 µM and 73.5 mM, respectively. We found synergy in LNCaP at 5 µM/mM and 10 µM/mM GT3/metformin. We found synergy in PC-3 at 5 µM/mM GT3/metformin. RWPE-1 were unaffected. We determined the effect of GT3 and metformin on signaling pathways by analyzing β-actin, p-AKT, p-c-JUN, and p-ERK in combination via Western immunoblot. Initial experiments with LNCaP and PC-3 cells showed metformin inhibits the expression of p-c-JUN and p-ERK. GT3 inhibits the expression of p-ERK but not p-c-JUN in both prostate cancer cell lines. P-AKT was not activated by GT3 or metformin. GT3 and metformin inhibit cell growth in both prostate cancer cell lines, greater in combination compared to separately (more apparent in LNCaP compared to PC-3). GT3 and/or metformin caused less growth inhibition and no effects on expression of proteins p-AKT, p-c-JUN or p-ERK in the RWPE-1 prostate control cells. There is synergy with GT3 and metformin in inhibiting prostate cancer cell growth. GT3 inhibits expression of p-ERK, and metformin inhibits expression of both p-c-JUN and p-ERK. Although AKT is an important target in prostate cancer therapy, AKT is not modulated by either metformin and GT3 but modulates p-c-JUN and p-ERK. Cytotoxic effects of GT3 and metformin are independent of androgen sensitivity although androgen-independent PC-3 cell lines are more sensitive. GT3 and metformin are promising chemopreventive agents that need to be examined mechanistically. Recent literature suggests metformin influences methylation in breast cancer. We are in the process of analyzing GT3 and metformin and how they alter methylation pathways in prostate cancer. Citation Format: Christine A. Moore, Janet W. Lightner, Michelle Duffourc, Koyamangalath Krishnan. γ-Tocotrienol and metformin are cytotoxic to prostate cancer cell lines and exhibit synergy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3977.

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.

Histone lysine methyl transferase 2 (EZH2) inhibitor GSK126 and a novel deuterated internal standard GSK126-d7 were chemically prepared. We performed in vitro experiments using the prepared GSK126 to: i) confirm in vitro EZH2 inhibitory activity; ii) conduct Sprague-Dawley (SD) rat liver microsomal incubations and identified Phase I metabolites; iii) determine whether or not GSK126 was an Organic Anion Transporter (OAT) substrate; and, iv) determine oral bioavailability by conducting oral and orbital sinus dosing (OSD) experiments and determining blood concentration versus time profiles. GSK126 was shown to decrease the expression of H3K27Me3 protein in medulloblastoma D283 cells and was able to decrease cell viability in KO99L cells, a novel T cell lymphoma cell line. Three in vitro hepatic Phase I mono-oxidative metabolites (L-M1, L-M2 and L-M3) were observed and also detected in rat liver and urine samples from the in vivo studies. GSK126 was found to be an OAT1 and OAT2 substrate, but not an OAT3 or OAT4 substrate. Our Pharmacokinetic (PK) results indicate: 1) GSK126 has very poor oral bioavailability (< 2%); 2) co-administration of probenecid, a prototypical OAT inhibitor, did not significantly alter observed PK; and 3) tissue distribution studies demonstrate that GSK126 predominately distributes to the liver and kidneys after an OSD.

The mevalonate pathway plays an important role in cancer biology and has been targeted with farnesyl transferase inhibitors, although their efficacy is limited due to significant adverse effects. Statins and bisphosphonates inhibit the mevalonate pathway at different steps, thus having negative effects at various levels on cancer cells. A combination of these drugs may result in an amplified cytotoxic effect and allow for use of significantly lower doses of the drugs involved. Statins inhibit the mevalonate pathway at 3-hydroxy-3-methylglutaryl coenzyme A reductase and bisphosphonates at farnesyl pyrophosphate synthase. Our results show that low-dose combinations of simvastatin and alendronate have a synergistic cytotoxic effect on androgen-independent prostate cancer PC-3 cells, but not on androgen-dependent LNCaP or DU 145 prostate cancer cells. These two drugs cause a sequential blockade of the mevalonate pathway and significantly affect survival and apoptotic pathways by down-regulating phospho-AKT and activating c-JUN and ERK.

e13043 Background: A goal of precision chemoprevention is to prevent or delay cancer progression by using minimally toxic agents guided by knowledge of alterations in oncogenic molecular pathways and signaling cascades. Indolent prostate cancer can benefit from chemopreventive agents by delaying growth. Vitamin E is not a single compound and refers to four tocopherols and four tocotrienols. The use of all-racemic-alpha tocopheryl acetate in preventing cancer was studied in the SELECT clinical trial. This trial found it was not chemopreventive and could promote prostate cancer. We compared the abilities of RRR-alpha-tocopherol (AT), RRR-gamma-tocopherol (GT), and gamma-tocotrienol (GT3) in preventing growth of two prostate cancer cell lines (LNCaP and PC-3) and a control prostate cell line (RWPE-1) by quantifying their effects on two signaling pathways known to be pivotal in prostate carcinogenesis, AKT and MAP Kinase (pERK). Methods: LNCaP, PC-3, and RWPE-1 cell lines were treated with increasing concentrations of AT, GT and GT3 in DMEM, and cytotoxicity determined by MTS cell culture experiments. We determined the effect of GT3 on signaling pathways by analyzing B-actin, P-AKT and p-ERK in combination with AKT and/or MEK inhibitors via Western immunoblot and PCR analysis. Results: Initial experiments with LNCaP and PC-3 cells showed GT3 induced the expression of pERK and p-c-JUN, inhibited cell growth and promoted apoptotic cell death. Neither AT nor GT had these effects. AKT was not activated by AT, GT or GT3. Inhibition of AKT activation via MK-2206 did not block GT3 growth inhibition in LNCaP cells. No growth inhibition was found with RWPE-1 control cells in presence of AT, GT or GT3 yet GT3 induced pERK expression. AT did not interfere with cancer growth inhibition and did not block the anticancer effects of GT3. Conclusions: GT3 induces the activation of pERK but this effect is not sufficient to account for the in vitro chemopreventive effects of this form of vitamin E. The AKT pathway is not modulated by GT3 and does not appear to be relevant to GT3 killing of PC-3 or LNCaP. Of five signaling pathways implicated in prostate carcinogenesis, we explored AKT and pERK. Future research will explore B-catenin, mTOR and PI3K.

Peroxiredoxins (PRDXs) are highly abundant antioxidant enzymes overexpressed in many cancers, e.g., breast cancer, prostate cancer, lung cancer. About 90% of cellular peroxides are reduced by PRDXs. Melanoma cells, and cancer cells in general, often have a high level of oxidative stress which is thought to drive the expression of many cancer phenotypes. AKT activation (i.e., phosphorylation) is a key mechanism giving rise to increased oxidative stress and production of reactive oxygen species (ROS) in cancer cells. The degree of AKT activation in cancer cells often correlates with cancer aggressiveness. We sought to determine if increasing AKT1 activation in human melanoma cell line SK-MEL-28 by transfection with pBABE-puro-MF-AKT1 (constitutively active AKT1) would cause a compensatory increase in PRDXs. Isobaric tags for relative and absolute quantitation (iTRAQ) with tandem mass spectrometry was utilized to measure the relative abundance of proteins in transfected (SK-MEL-28-AKT1) and non-transfected control cells (SK-MEL-28). Our results show that (P This research was supported in part by NIH Grant C06RR0306551 and the East Tennessee State University Robert W. Summers Pediatric Research Endowment.

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.

Targeting the mutant BRAF protein is an accepted approach to the treatment of metastatic melanoma. Potent and specific BRAF inhibitors like vemurafenib and dabrafenib are superior to chemotherapy in treatment of BRAF mutant melanomas which represent nearly 50% of all melanomas. Previous studies have shown that certain isoforms of vitamin E and statins can have synergistic anti-cancer activity. We determined whether a combination of delta-tocotrienol (DT3), an unsaturated vitamin E isoform, and simvastatin, an HMG-CoA reductase inhibitor, can exert an anti-neoplastic activity on BRAF-mutated SK-MEL-28 and BRAF-wild type SK-MEL-2 melanoma cell lines and whether a differential effect would be evident. MTS assays were used to analyze cytotoxicity. SK-MEL-28 and SK-MEL-2 cells were cultured in MEM media containing 10% serum and plated in 96-well culture plates for 24 hours then treated with DT3 (0-40 μM), simvastatin (0-5 μM), or a combination and dosed again at 48 hours. SK-MEL-28 and SK-MEL-2 cells grown in 60 mm plates and were treated with DT3 at concentrations of 40, 30, 20 μM, simvastatin at a concentrations of 20, 10, 5 μM or dissolution vehicle as a control for 6 h. Protein concentration of cell lysates was measured spectrophotometrically (GLO Max Multi+, Promega), using a BCA protein assay kit. The samples were run in SDS PAGE and blotted onto nitrocellulose membranes. Membranes were incubated with antibodies against Hsp 70 (Enzo Life Sciences, Farmingdale, NY), Hsp 90 (Santa Cruz, Dallas, TX), pS6 and pBAD (Cell Signaling, Danvers, MA). Using MTS assay, we found that DT3 (IC50 38.8 μM) and simvastatin (IC50 22.7μM) have cytotoxic effects on melanoma cell line SK-MEL-28, but on the SK-MEL-2 cells DT3 does not have an effect at the concentrations studied (10-40 μM DT3) yet simvastatin (IC50 16.9 μM) does have cytotoxicity. Further studies determined that combinations of these drugs display a synergistic effect on SK-MEL-28 by inhibition of pS6 and pBAD and subsequent apoptosis. However, these effects are not observed in SK-MEL-2 cells; treated SK-MEL-2 cells show over-expression of Hsp70 and Hsp90 suggestive of a rescue effect leading to lesser cytotoxic activity. The selective cytotoxicity observed in BRAF-mutated cells and not in wild type BRAF melanoma cell lines by both DT3 and simvastatin warrants further research into the potential therapeutic use of these combinations. This observation has added importance in the light of recent findings that show the acquisition of BRAF mutation is an early event in melanogenesis and hence these compounds may have a key role in chemoprevention approaches to melanoma. Citation Format: Kelley Cross, Victoria Palau, Marianne Brannon, Janet Lightner, Megan Dycus, William Stone, Koyamangalath Krishnan. Delta-tocotrienol and simvastatin induce cytotoxicity and synergy in BRAF mutant SK-MEL-28 but not in wild type BRAF SK-MEL-2 melanoma cancer cells. [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 3568.

Michael F Wempe,1 Janet W Lightner,2 Bettina Miller,1 Timothy J Iwen,1 Peter J Rice,1 Shin Wakui,3 Naohiko Anzai,4 Promsuk Jutabha,4 Hitoshi Endou51Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA; 2Department of Pharmacology, East Tennessee State University, Johnson City, TN, USA; 3Department of Toxicology, Azabu University School of Veterinary Medicine, Chuo Sagamihara, Kanagawa, Japan; 4Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, Mibu, Shimotsuga, Tochigi, Japan; 5Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Mitaka, Tokyo, JapanAbstract: 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.Keywords: benzbromarone analogs, bioavailability, glucuronidation, oxidation, structure–activity relationship

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.

Many primary human tumors and tumor cell lines highly express human L-type amino acid transporter 1 (hLAT1); cancerous cells in vivo are strongly linked to LAT1 expression. Synthetic chemistry and in vitro screening efforts have afforded a variety of novel and highly hLAT1 selective compounds, such as JPH203 1. In a recent report, we demonstrated that 1 has potent in vitro and in vivo activity. JPH203 was intravenously administered to produce significant growth inhibition against HT-29 tumors transplanted in nude mice. The current work develops a robust LC/MS-MS method to monitor 1 and its major Phase II metabolite N-acetyl-JPH203 2 from biological samples. We have conducted in vitro and in vivo experiments and the major scientific findings are: i) the major route of biotransformation of 1 is Phase II metabolism to produce 2; ii) metabolite 2 is formed in various organs/tissues (i.e. blood, liver, kidney); and iii) as dogs, which are deficient in NAT genes, do not produce 2, the dog will not be an appropriate toxicological model to evaluate 1.

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.

Summary 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. Methods Idebenone and idebenone linoleate were investigated in pig ear skin and melanoma (B16: F10 mouse) cells. Diffusion experiments were conducted at 37 °C (bath temperature) using Franz diffusion cells. Authentic metabolite samples were synthetically prepared. Samples were analyzed using liquid chromatography-mass spectrometry/mass spectrometry. Cell viability was determined via the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Results Idebenone was shown to permeate across viable porcine ear tissue; there was no evidence that idebenone linoleate permeated across porcine ear tissue after 4 h. Idebenone was metabolized to idebenone acid in both pig ear and mouse melanocytes; only minor idebenone linoleate metabolism was observed. Idebenone displayed delayed in vitro toxicity (via MTT assay) in melanocytes, while idebenone linoleate displayed no such in vitro toxicity. Conclusions The in vitro metabolism and cytotoxicity results suggest that metabolic activation of idebenone is the likely culprit that activates the skin irritation mechanism via idebenone in vivo usage. An idebenone ester (e.g. idebenone linoleate) appears to provide a superior in vitro safety profile over idebenone.

Metformin, an anti-diabetic biguanide, has been shown to have cytotoxic and chemopreventive properties in in vitro and epidemiological studies, respectively. Metformin's predominant cytotoxic functions may be through activation of 5′ adenosine monophosphate-activated protein kinase (AMPK) with subsequent cell apoptosis. Ceramides, a group of waxy lipids found in the cellular membrane, are key players in many elements of cell signaling, including the regulation of cell differentiation, proliferation, and apoptosis. While traditionally thought of as being pro-apoptotic, this notion has been questioned with the discovery of pro-survival ceramides, as well as the demonstration that certain species of ceramides are increased in breast cancer cells. Metformin reduces ceramides in insulin-resistant mouse myoblasts and may reduce the vascular complications of diabetes mellitus. MCF-7 and MDA-MB-231 breast cancer cell lines were treated with increasing concentrations of metformin and cytotoxicity was determined by MTT cell culture experiments after 72 hours of drug exposure. Metformin induces cytotoxicity in these breast cancer cells at a lowest concentration of 1.25 mM, and percentage cytotoxicity increases in a dose-dependent manner. We then determined the effect of metformin on cell ceramide synthesis by analyzing key ceramide synthetic enzymes. Using western immunoblot and polymerase chain reaction (PCR) analysis, we determined that metformin induces a reduction in serine palmitoyl transferase, ceramide synthase, dihydroceramide desaturase, and neutral sphingomyelinase in MCF-7 and MDA-MB-231 breast cancer cells. We also analyzed the distribution of ceramides in treated breast cancer cells using polyclonal antibodies against ceramides. By confocal microscopy, ceramides are globally decreased in MCF-7 and MDA-MB-231 cells treated with metformin. This data suggests that the pro-apoptotic effect of metformin may be partly mediated through its disruption of the balance of ceramides and/or through the reduction of pro-survival ceramides within breast cancer cells. It is also possible that the predominant ceramides in MCF-7 and MDA-MB-231 breast cancer cells are pro-survival and metformin suppresses these ceramides. Further work is necessary to characterize the ceramide content of MCF-7 and MDA-MB-231 cancer cells before and after metformin treatment. Citation Format: Daniel Wann, Victoria Palau, Janet Lightner, Marianne Brannon, William Stone, Koyamangalath Krishnan. Metformin decreases cellular ceramides in MCF-7 and MDA-MB 231 breast cancer cell lines by inhibition of ceramide synthetic enzymes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4639. doi:10.1158/1538-7445.AM2015-4639

Introduction Statins and tocotrienols modulate the cholesterol biosynthesis pathway by inhibiting the 3-hydroxy-3- methylglutaryl coenzyme A (HMG-CoA) reductase. Tocotrienols modulate HMG-CoA reductase by post-transcriptional downregulation. In addition, tocotrienols contain a farnesol moiety in its side-chain that triggers degradation of HMG-CoA reductase. These effects lead to suppression of cell proliferation, cell cycle arrest, and apoptosis. Several studies have shown that statins have suppressive effects in in vitro experiments on acute myelocytic leukemia cell lines. Since both statins and gamma-tocotrienol are associated with decreased cholesterol biosynthesis, we hypothesized that if the cytotoxicity of these drugs on cancer cells is related to impaired biosynthesis of cholesterol, combination of them could synergize in cytotoxicity on leukemic cells. Materials and Methods K-562 and HL-60 leukemia cells were grown in Iscove's Modified Dulbecco's medium with penicillin/streptomycin, 10% fetal bovine serum and 20% fetal bovine serum added respectively. K-562 and HL-60 leukemia cells were seeded in 96 well plates, grown overnight, and treated for 24, 48 and 72 hours with simvastatin in concentrations of 1,2,4, and5 µM; gamma-tocotrienol in concentrations of 20, 40, and 80 µM; and a combination of the two drugs in the same concentrations. For 24 hour dose, cells were seeded at a density of 5000/well and for 48 and 72 hour doses, at 3500/well. Following the treatment, MTS/PMS reagent (Promega, Madison, WI), [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] mixed with and an electron coupling reagent (phenazine methosulfate), was added at 40 µg/well and incubated at 37°C for 2 hours. We measured the solubilized formazan crystals at 450 nm as an indicator of cytotoxicity. The presence of ATP as an indicator of cell viability was measured with the CellTiter Glo® assay (Promega). Cells were again seeded, grown overnight, and dosed as indicated above. Following treatment, the assay was conducted as specified by the manufacturer. Results Both simvastatin and gamma-tocotrienol induce cytotoxicity in K-562 and HL-60 cell lines by the MTS and Cell Titer Glo Assays. The IC50 at 72 hour incubation are as follows 1) HL-60: simvastatin - 16.543 uM, gamma tocotrienol - 36.297 uM; 2) K-562: simvastatin - 5.235 uM, gamma tocotrienol - 34.947 uM. We used CompuSyn to calculate the IC50. When combined, simvastatin and gamma-tocotrienol exhibit synergy at lower concentrations when examined by isobologram analysis. Conclusion Gamma-tocotrienol, an isoform of vitamin E, and simvastatin, a cholesterol lowering drug exhibit synergy in induction of cytotoxicity in K-562 and HL-60 leukemia cell lines. Rescue experiments and mechanistic pathway analysis are being explored to confirm these observations. Disclosures: No relevant conflicts of interest to declare.

Pancreatic cancer is the fourth leading cause of cancer-related death in the United States. Treatment options are limited and novel therapeutic agents that can inhibit signaling pathways implicated in the proliferation and survival of pancreatic cancer cells are of interest. Tocotrienols are members of the vitamin E family but, unlike tocopherols, possess an unsaturated isoprenoid side-chain that confers superior and mechanistically different anti-cancer properties on pancreatic cancer cells. The ability of tocotrienols to selectively inhibit the HMG CoA reductase pathway through post-translational degradation of HMG CoA reductase, inhibit the PI3/Akt, ERK pathways through downregulation of Her2/ErbB2 receptors as well as suppression of the activity of transcription factor NF kappa B, could be the basis for some of these properties. Recent studies have shown increase in cellular ceramide levels in cancer cells treated with tocopherols, leading to arrest in tumor proliferation and apoptosis. The actual mechanism of increase in ceramide levels is not clearly understood and is thought to be due to increased synthesis. We explored the hypothesis that the increase in ceramide on the cellular membrane could be due to increased transport from the ER to the membrane. The ARV1 (ACAT-related enzyme-2 required for viability) gene encodes an ER- localized protein involved in lipid homeostasis and ceramide transport in yeast and mammals. Deletion of the ARV-1 gene in yeast causes accumulation of ceramide in cytoplasm. Our previous work indicated that gamma-tocotrienol (GT3) has potent anti-proliferative activity in k-ras mutated pancreatic cancer cell (MIA-Paca-2) through the inhibitory effect on Akt, Ras/Raf/Erk and ErBb2 receptor as well as induction of apoptotic pathways through Fox-03 and GSK-3b. Our current data shows GT3 may decrease expression of caveolin, a cell survival marker, and cause up-regulation of ARV-1 that encodes a ceramide transport protein. We further show via HPLC (Babraham Bioscience Technologies Ltd, Cambridge, U.K) that total ceramide levels remain unchanged in the cell. There may be a change in ceramide (monoclonal anti-ceramide IgM, Sigma Chemicals) cellular distribution as indicated by immunofluorescence data. Upregulation of ARV-1 may be involved in increase in cellular ceramide transport or redistribution of ceramide to the cell membrane. Transport of ceramide to membrane lipid rafts or mitochondrial membrane channels may cause loss of survival signaling and induce apoptosis. Thus it is likely that GT3 apoptotic effect on k-ras mutated MIA-Paca2 cells is related not to changes in the total ceramide levels, but rather to its increased transport and redistribution. Further work is necessary in order to better understand the role of GT3 in ceramide redistribution in pancreatic cancer cells. Citation Format: Kanishka Chakraborty, Victoria P. Ramsauer, Janet W. Lightner, William L. Stone, Koyamangalath Krishnan. Gamma-tocotrienol upregulates the ceramide transporter, Arv-1, in pancreatic cancer cells. [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 2106. doi:10.1158/1538-7445.AM2013-2106

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

Abstract: Pancreatic cancer is the fifth leading cause of cancer death in the United States. Alternate strategies to treat and prevent this cancer are urgently required. In this regard, novel therapeutic agents that can inhibit signaling pathways implicated in the proliferation and survival of pancreatic cancer cells are of immense interest. 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 post-translational degradation of HMG CoA reductase and to suppress the activity of transcription factor NFκβ could be the basis for some of these properties. Data: Our studies indicate that γ- and Δ-tocotrienol have potent anti-proliferative activity in pancreatic cancer cells (Panc-28, MIA-PACA-2 and BXPC-3). Indeed both tocotrienols induced cell death (> 50%) by the MTT cell viability assay in all three 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 S6k) in addition to suppressing the activation of protein kinase, Akt. Suppression of activation of Akt by gamma-tocotrienol led to downstream upregulation of Fox-03 and GSK-3b. 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. Conclusion: 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/Akt and ERK/MAP kinases via downregulation of Her2/ErbB2 expression. The molecular components for this mechanism are not completely elucidated and needs further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4490. doi:10.1158/1538-7445.AM2011-4490

The mevalonate pathway plays an important role in cancer biology and has been targeted previously with farnesyl transferase inhibitors, although their efficacy is limited due to significant adverse effects. We hypothesize that combination of Simvastatin with Alendronate may have significant synergistic anti-carcinogenic potential in prostate cancer due to sequential blockade in the mevalonate pathway and will serve as a safer and better tolerated alternative than previously tried drugs. Simvastatin and alendronate sequentially inhibit HMG Co-A reductase and farnesyl pyrophosphate synthase enzymes respectively in the mevalonate pathway. This inhibition subsequently reduces isoprenoid synthesis including farnesyl pyrophosphate and geranylgeranylpyrophosphate. Prenylation (isoprenoid attachment) of G protein subunits and nuclear laminins help facilitate protein interactions in addition to termination of small GTPases’ activity. These proteins have important biological roles, including transmembrane signal transduction (RAS), cytoskeletal reorganization (RHO), gene expression (RAS), microtubule organization and nucleocytoplasmic transport. Inhibiting this pathway may have important anti-oncogenic effects through inhibition of processes such as tumor cell adhesion, migration, invasion, proliferation and induction of apoptosis. To test the hypothesis, we plated a predetermined number of 5000 PC3 cells into a 96 well plate and allowed them to adhere to the wells for an initial 24 hour incubation period using standard color free medium. Simvastatin and alendronate were prepared in various dose concentrations (simvastatin; 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0µM and alendronate: 1, 2.0, 5.0. 10.0. 20.0, and 40.0µM). The cells were treated with simvastatin or alendronate alone, and then in various dose combinations. The cells with drug free medium were used as control. After 96 hour incubation, the quantity of living cells were determined via standard MTT assay and spectrophotometric analysis at 570nm and compared to the control group. Significant inhibition of PC3 cell growth was observed using simvastatin and alendronate alone as well as in combinations at low doses. Isobologram analysis was utilized as visual assessment of the drugs’ interaction with independent statistical analysis from calculations using total dose in a fixed-ratio combination and with calculated additive total dose for the same effect. Our results demonstrate synergistic inhibition of PC3 cell growth in vitro at various low dose combinations of simvastatin with alendronate. The fact that both of these drugs are commonly used in the population with no reported severe adverse effects from their combination offers a safe, effective and synergistic treatment option in the future treatment of prostate cancer. In vivo studies must be undertaken to explore these effects further. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4407. doi:10.1158/1538-7445.AM2011-4407