Marianne Brannon

Protective effect of liposome-encapsulated glutathione in a human epidermal model exposed to a mustard gas analog.

Sulfur mustard or mustard gas (HD) and its monofunctional analog, 2-chloroethyl ethyl sulfide (CEES), or “half-mustard gas,” are alkylating agents that induce DNA damage, oxidative stress, and inflammation. HD/CEES are rapidly absorbed in the skin causing extensive injury. We hypothesize that antioxidant liposomes that deliver both water-soluble and lipid-soluble antioxidants protect skin cells from immediate CEES-induced damage via attenuating oxidative stress. Liposomes containing water-soluble antioxidants and/or lipid-soluble antioxidants were evaluated using in vitro model systems. Initially, we found that liposomes containing encapsulated glutathione (GSH-liposomes) increased cell viability and attenuated production of reactive oxygen species (ROS) in HaCaT cells exposed to CEES. Next, GSH-liposomes were tested in a human epidermal model, EpiDerm. In the EpiDerm, GSH-liposomes administered simultaneously or 1 hour after CEES exposure (2.5 mM) increased cell viability, inhibited CEES-induced loss of ATP and attenuated changes in cellular morphology, but did not reduce caspase-3 activity. These findings paralleled the previously described in vivo protective effect of antioxidant liposomes in the rat lung and established the effectiveness of GSH-liposomes in a human epidermal model. This study provides a rationale for use of antioxidant liposomes against HD toxicity in the skin considering further verification in animal models exposed to HD.

Identification of oxidized protein hydrolase as a potential prodrug target in prostate cancer

BackgroundEsterases are often overexpressed in cancer cells and can have chiral specificities different from that of the corresponding normal tissues. For this reason, ester prodrugs could be a promising approach in chemotherapy. In this study, we focused on the identification and characterization of differentially expressed esterases between non-tumorigenic and tumorigenic prostate epithelial cells.MethodsCellular lysates from LNCaP, DU 145, and PC3 prostate cancer cell lines, tumorigenic RWPE-2 prostate epithelial cells, and non-tumorigenic RWPE-1 prostate epithelial cells were separated by native polyacrylamide gel electrophoresis (n-PAGE) and the esterase activity bands visualized using α-naphthyl acetate or α-naphthyl-N-acetylalaninate (ANAA) chiral esters and Fast Blue RR salt. The esterases were identified using nanospray LC/MS-MS tandem mass spectrometry and confirmed by Western blotting, native electroblotting, inhibition assays, and activity towards a known specific substrate. The serine protease/esterase oxidized protein hydrolase (OPH) was overexpressed in COS-7 cells to verify our results.ResultsThe major esterase observed with the ANAA substrates within the n-PAGE activity bands was identified as OPH. OPH (EC 3.4.19.1) is a serine protease/esterase and a member of the prolyl oligopeptidase family. We found that LNCaP lysates contained approximately 40% more OPH compared to RWPE-1 lysates. RWPE-2, DU145 and PC3 cell lysates had similar levels of OPH activity. OPH within all of the cell lysates tested had a chiral preference for the S-isomer of ANAA. LNCaP cells were stained more intensely with ANAA substrates than RWPE-1 cells and COS-7 cells overexpressing OPH were found to have a higher activity towards the ANAA and AcApNA than parent COS-7 cells.ConclusionsThese data suggest that prodrug derivatives of ANAA and AcApNA could have potential as chemotherapeutic agents for the treatment of prostate cancer tumors that overexpress OPH.

Myosin content of individual human muscle fibers isolated by laser capture microdissection.

Muscle fiber composition correlates with insulin resistance, and exercise training can increase slow-twitch (type I) fibers and, thereby, mitigate diabetes risk. Human skeletal muscle is made up of three distinct fiber types, but muscle contains many more isoforms of myosin heavy and light chains, which are coded by 15 and 11 different genes, respectively. Laser capture microdissection techniques allow assessment of mRNA and protein content in individual fibers. We found that specific human fiber types contain different mixtures of myosin heavy and light chains. Fast-twitch (type IIx) fibers consistently contained myosin heavy chains 1, 2, and 4 and myosin light chain 1. Type I fibers always contained myosin heavy chains 6 and 7 (MYH6 and MYH7) and myosin light chain 3 (MYL3), whereas MYH6, MYH7, and MYL3 were nearly absent from type IIx fibers. In contrast to cardiomyocytes, where MYH6 (also known as α-myosin heavy chain) is seen solely in fast-twitch cells, only slow-twitch fibers of skeletal muscle contained MYH6. Classical fast myosin heavy chains (MHC1, MHC2, and MHC4) were present in variable proportions in all fiber types, but significant MYH6 and MYH7 expression indicated slow-twitch phenotype, and the absence of these two isoforms determined a fast-twitch phenotype. The mixed myosin heavy and light chain content of type IIa fibers was consistent with its role as a transition between fast and slow phenotypes. These new observations suggest that the presence or absence of MYH6 and MYH7 proteins dictates the slow- or fast-twitch phenotype in skeletal muscle.

Sodium Pyruvate Modulates Cell Death Pathways in HaCaT Keratinocytes Exposed to Half-Mustard Gas

2-Chloroethyl ethyl sulfide (CEES) or half-mustard gas, a sulfur mustard (HD) analog, is a genotoxic agent that causes oxidative stress and induces both apoptotic and necrotic cell death. Sodium pyruvate induced a necrosis-to-apoptosis shift in HaCaT cells exposed to CEES levels ≤ 1.5 mmol/L and lowered markers of DNA damage, oxidative stress, and inflammation. This study provides a rationale for the future development of multicomponent therapies for HD toxicity in the skin. We hypothesize that a combination of pyruvates with scavengers/antioxidants encapsulated in liposomes for optimal local delivery should be therapeutically beneficial against HD-induced skin injury. However, the latter suggestion should be verified in animal models exposed to HD.

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.

In vitro evaluation of novel N-acetylalaninate prodrugs that selectively induce apoptosis in prostate cancer cells

BackgroundCancer cell esterases are often overexpressed and can have chiral specificities different from that of the corresponding normal cells and can, therefore, be useful targets for activating chemotherapeutic prodrug esters. Prodrug esters are inactive compounds that can be preferentially activated by esterase enzymes. Moreover, cancer cells often exhibit a high level of intrinsic oxidative stress due to an increased formation of reactive oxygen species (ROS) and a decreased expression of some enzymatic antioxidants. Prodrugs designed to induce additional oxidative stress can selectively induce apoptosis in cancer cells already exhibiting a high level of intrinsic oxidative stress. This study focused on the in vitro evaluation of four novel prodrug esters: the R- and S- chiral esters of 4-[(nitrooxy)methyl]phenyl N-acetylalaninate (R- and S-NPAA) and the R- and S- chiral esters of 4-[(nitrooxy)methyl]naphth-1-yl N-acetylalaninate (R- and S-NQM), which are activated, to varying extents, by oxidized protein hydrolase (OPH, EC 3.4.19.1) yielding a quinone methide (QM) intermediate capable of depleting glutathione (GSH), a key intracellular antioxidant. OPH is a serine esterase/protease that is overexpressed in some human tumors and cancer cell lines.MethodsTo evaluate the chiral ester prodrugs, we monitored cellular GSH depletion, cellular protein carbonyl levels (an oxidative stress biomarker) and cell viability in tumorigenic and nontumorigenic prostate cancer cell lines.ResultsWe found that the prodrugs were activated by OPH and subsequently depleted GSH. The S-chiral ester of NPAA (S-NPAA) was two-fold more effective than the R-chiral ester (R-NPAA) in depleting GSH, increasing oxidative stress, inducing apoptosis, and decreasing cell viability in tumorigenic prostate LNCaP cells but had little effect on non-tumorigenic RWPE-1 cells. In addition, we found that that S-NPAA induced apoptosis and decreased cell viability in tumorigenic DU145 and PC3 prostate cell lines. Similar results were found in a COS-7 model that overexpressed active human OPH (COS-7-OPH).ConclusionsOur results suggest that prostate tumors overexpressing OPH and/or exhibiting a high level of intrinsic oxidative stress may be susceptible to QM generating prodrug esters that are targeted to OPH with little effect on non-tumorigenic prostate cells.

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.

Abstract 3568: 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

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.

Reply to “Letter to the editor: Comments on Stuart et al. (2016): ‘Myosin content of individual human muscle fibers isolated by laser capture microdissection’”

reply: Dr. Schiaffino is a highly respected and experienced investigator who has been very involved in muscle fiber type characterization for nearly three decades. In his comments ([2][1]) about our previous publication ([4][2]), he made several points. He is concerned about our data showing that