turmeric for mast cell tumors in dogs

2021/05/21

The majority of these studies have been focused on human and rodent cancer models and the effects of these plant extracts and select compounds vary depending on species and cell origin [23, 24]. Cells were treated the following day with DMSO vehicle control, 6.3g mL1 extract alone, or 3.1g mL1 each extract in combination for 36h. Chemotherapeutic drugs at a 50% inhibitory concentration (IC50) were used as a positive control; 12.5nM toceranib phosphate (Palladia, Zoetis Animal Health, Florham Park, NJ) was used for the C2 cell line, and 0.3 or 0.5M doxorubicin hydrochloride (Sigma Aldrich, St Louis, MO) was used for the CMT-12 and D17 cell lines, respectively. Treatment with 6.3g mL1 TE resulted in an increase from a densitometry value of 1.1 at 12h to 1.5 at 24h in p-SAPK/JNK in the C2 cell line, stable activation from 12h to 24h in the CMT-12 cell line (1.5 and 1.8, respectively). Corri B. Levine, Email: ude.llenroc@64lbc. Careers. Each blot is a representative of three independent experiments. Bethesda, MD 20894, Web Policies Apoptosis and necrosis was measured after 48h treatment using Annexin-V and 7-AAD staining. Datasets used and analyzed during this study are available from the corresponding author upon reasonable request. Consistent with our results, studies have shown that the downstream effects of SAPK/JNK activation are both cell and context dependent: pathway activation can be either pro-apoptotic or pro-proliferative. official website and that any information you provide is encrypted Apoptosis could be, in part, due to overlapping effects on various signaling pathways including SAPK/JNK, ERK 1/2, STAT3, FAK, Src, mTOR, and membrane permeability proteins Bcl-2 and Bax [36, 37]. Within each cell line, means with different letters are significantly different from each other (p<0.05). Digital images were captured using an imaging system (Biospectrum 410; UVP, Upland, CA, USA). The new PMC design is here! Reported data are expressed as meanstandard deviation of 4 independent replicates. All treatments were compared to DMSO vehicle control. CpJun N-terminal kinase (JNK) signaling: recent advances and challenges. After screening several signaling pathways, a consistent increase in the phosphorylated, or active, form of SAPK/JNK was detected with no consistent alterations in any other pathways examined via western blotting. The enhanced susceptibility found in the CMT-12 mammary cancer cell line may be due to the increased accumulation of curcumin when the combination treatment was used. Received 2017 Jun 9; Accepted 2017 Nov 27. The objective of this in vitro study was to determine the effects on canine cancer cell death and possible mechanisms by which TE and RE exert anti-proliferative and cytotoxic effects individually and in combination on canine mastocytoma, mammary carcinoma, and osteosarcoma cell lines. To further assess the oxidative status, western blot analysis for gamma-histone H2A.X phosphorylation status was assessed in the three cell lines with TE, RE or dual treatment showing no phosphorylation in DMSO control or treated cells. TE had a greater effect on inducing cell apoptosis as measured by Caspase 3/7 activation and Annexin-V staining, and the combination treatment using only half the concentration of each extract induced a similar, if not greater, cell response. and transmitted securely. Levine CB, Bayle J, Biourge V, Wakshlag JJ. This obstacle may be overcome through the use of combination treatments with other extracts that improve bioavailability or hinder additional pathways [1416]. Therefore, the possibility that RE could increase the cellular accumulation of the fluorescent compound curcumin was investigated when these compounds were used in combination. Values are expressed as meanstandard deviation of four independent replicates. Tong L, Chuang CC, Wu S, Zuo L. Reactive oxygen species in redox cancer therapy. Generally, these differences may be due to the lower concentrations utilized in our experiments compared to the prior studies. Primary antibodies included mouse anti- phosphorylated-gamma H2A.X and extracellular regulated kinase (ERK) (R&D Biosciences, Boston, MA, USA); mouse anti- Thr202/Tyr204 phosphorylated p44/42 MAPK (ERK1/2) and STAT3 (Cell Signaling Technology, Danvers, MA, USA); rabbit anti- protein kinase B (AKT), Ser473 phosphorylated-AKT, stress-activated protein kinase/jun-N-terminal kinase (SAPK/JNK), Thr183/Tyr185 phosphorylated-SAPK/JNK, focal adhesion kinase (FAK), Tyr397 phosphorylated-FAK, Tyr576/Tyr577 phosphorylated-FAK, Tyr925 phosphorylated-FAK, Src, Tyr416 phosphorylated-Src, Tyr527 phosphorylated-Src, mammalian target of rapamycin (mTOR), Ser2448 phosphorylated-mTOR, Janus kinase 2 (JAK2), Tyr1007/Tyr1008 phosphorylated-JAK2, Ser727 phosphorylated-signal transducer and activator of transcription 3 (STAT3), Tyr705 phosphorylated-STAT3, B-Cell CLL/Lymphoma 2 (BCL2), and BCL2-Associated X Protein (BAX) (Cell Signaling Technology). Caspase 3/7 activation induced by turmeric and rosemary extracts in C2, CMT-12, and D17 cell lines. Davies C, Tournier C. Exploring the function of the JNK (c-Jun N-terminal kinase) signaling pathway in physiological and pathological processes to design novel therapeutic strategies. Julie Bayle, Email: moc.ninaclayor@elyab.eiluj. In this previous literature, there was complete loss or greater than 50% reduction or increase in various portions of the cell cycle warranting further examination of cellular pathways involved. The use of plant extracts has been around for centuries, but investigations into the mechanisms of action across various cancer cell lines are more recent, and appear to be highly variable in cell culture systems [13]. 5D). Kelsey JL, Moore AS, Glickman LT. Epidemiologic studies of risk factors for cancer in pet dogs. Only the CMT-12 cell line showed sustained activation of SAPK/JNK with the combination treatment which may be the underlying reason behind the increased susceptibility of this cell line. After 24h treatment, cells were detached with Accumax dissociation solution (Innovative Cell Technologies), collected and centrifuged for 10m at 500 rcf at 4C. CBL carried out the technical experimentation, performed statistical analysis, and was primary author in the manuscript. Of the three cell lines used in our experiments, only the C2 and D17 cells could be examined as a single cell suspension for cell cycle dynamics, while the CMT-12 cells displayed an artificial accumulation of cells in the G2/M phase. Apoptosis, Canine cancer, Mammary carcinoma, Osteosarcoma, Mastocytoma, Curcumin, Rosemary, {"type":"entrez-nucleotide","attrs":{"text":"DA251471","term_id":"78448130","term_text":"DA251471"}}. Annexin-V 488 conjugate and 7-Aminoactinomycin D (7-AAD) were added to the cell suspensions and incubated for 15m at room temperature.

The addition of RE at the same concentration to TE resulted in a significant increase in GMF of 4.8-fold in the CMT-12 cell line beyond that of TE alone (Fig. For all flow cytometry experiments, 10,000 events were collected per sample and then gated based on a forward-scatter/side-scatter plot. Effects of lycopene on proliferation and death of canine osteosarcoma cells. Federal government websites often end in .gov or .mil. Though there is relatively little primary literature on canine cell lines, one study has shown that a curcumin analog effectively alters STAT phosphorylation and activation in canine osteosarcoma cells [38]. A similar increase in GMF was also seen when half the concentration of each extract was used in combination (data not shown). Tsai CW, Lin CY, Lin HH, Chen JH. The use of natural remedies, or nutraceuticals, in the treatment of cancer and a variety of other diseases appears prevalent in human and veterinary medicine. The use of nutraceuticals is gaining in popularity in human and canine oncology with a relatively limited understanding of the effects in the vastly different tumor types seen in canine oncology. An increase in curcumin fluorescence was seen across the three cell lines with the greatest signal measured in the CMT-12 cell line, especially when the two extracts were used in combination.

After the incubation, ABB was added to the cell suspension and kept on ice until fluorescence analysis. The aim of the current in vitro study was to examine the molecular effects of two natural extracts, turmeric root extract (rich in curcuminoids) and rosemary leaf extract (rich in carnosic acid), previously shown to inhibit proliferation synergistically in three established canine cancer cell lines [11]. Li J, Xiang S, Zhang Q, Wu J, Tang Q, Zhou J, et al. Since the main constituents of TE and RE (curcumin and carnosic acid, respectively) have been implicated as antioxidants, Dihydrorhodamine123 (DHR123; Invitrogen, Carlsbad, CA, USA) assay was used to determine the amount of reactive oxygen species (ROS) present after 12h treatment with each extract according to literature [19]. Cells were treated with indicated concentrations of extracts or DMSO for 48h and the DNA contents were analyzed using propidium iodide staining by flow cytometry. In agreement with our previous proliferation and cytotoxicity results, cell treatment using TE alone was more potent than RE single treatment using the same extract concentrations and experimental conditions. 3E) and from 4% to 13% in the CMT-12 cell line (represented in Fig.

Expression level of Thr183/Tyr185 phosphorylated-SAPK/JNK (p46/p54) and total SAPK/JNK were determined by Western blot analysis. Representative quadrant plots of the CMT-12 cell line treated with (a) DMSO, (b) 6.3g mL1 TE, (c) 6.3g mL1 RE, or (d) 3.1g mL1 TE+3.1g mL1 RE are shown.

Changes in the protein expression levels of SAPK/JNK pathway in turmeric and rosemary-treated cells. Curcumin and cancer cells: how many ways can curry kill tumor cells selectively? Percentages of cells within each cell cycle phase (G1, S, and G2/M) were expressed as meanstandard deviation in (a) C2 and (b) D17 cell lines. Baharuddin P, Satar N, Fakiruddin KS, Zakaria N, Lim MN, Yusoff NM, et al. -Actin was used as a loading control for every blot to ensure even loading of samples. These results demonstrate possible mechanisms behind the observed susceptibility differences across the three cell lines, particularly in light of the heightened response of lesser doses of RE and TE in combination when compared to higher concentrations each extract independently in the CMT12 cell line. SAPK/JNK has been implicated as a therapeutic target in certain contexts and patterns of activation whereby constitutive activation appears to be beneficial towards a pro-apoptotic response in a variety of cell lines and animal models [4749]. Synergy in plant medicines. Khuda-Bukhsh AR, Das S, Saha SK. The following day, samples were centrifuged for 10m at 500 rcf at 4C, resuspended in cold PBS. Lee WH, Loo CY, Young PM, Traini D, Mason RS, Rohanizadeh R. Recent advances in curcumin nanoformulation for cancer therapy. 1Department of Clinical Sciences,Veterinary Medical Center C2-009, Cornell University College of Veterinary Medicine, Ithaca, NY 14853 USA, 2Royal Canin Research Center, Airmargues, France. Nanoparticle encapsulation improves oral bioavailability of curcumin by at least 9-fold when compared to curcumin administered with piperine as absorption enhancer. 2016 Sep 9;8(9). Within each cell line, values with different letters are significantly different from each other (C2 p<0.001; CMT-12 p<0.005; D17 p<0.05), Apoptosis induction by turmeric and rosemary extracts in C2, CMT-12, and D17 cell lines. 1A-B). This value was subtracted from the GMF of stained samples to correct for any shift due to auto-fluorescence of the extract with cells alone. Apoptosis, antioxidant effects, cellular accumulation of curcumin, and perturbation of signaling pathways were assessed. Ravindran J, Prasad S, Aggarwal BB. There were minimal to no cell cycle changes with TE extract alone, therefore further examination of cell cycle pathway analysis was not pursued. Turmeric extract (TE; 88% total curcuminoids, #{"type":"entrez-nucleotide","attrs":{"text":"DA251471","term_id":"78448130","term_text":"DA251471"}}DA251471 Naturex, Avignon, France) and rosemary extract (RE; 67% carnosic acid, #302036 Vitiva, Markovcih, Slovenia) were solubilized in 100% dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) at 20mg mL1. We previously identified two extracts, turmeric extract rich in curcuminoids (TE) and rosemary leaf extract rich in carnosic acid (RE), which were shown to be cytotoxic and reduce proliferation in a synergistic manner in canine mastocytoma, mammary carcinoma, and osteosarcoma cell lines [11]. Values are represented as a ratio of phosphorylated protein to total protein and standardized to DMSO vehicle control at every time point examined. Cells were plated at a density of 4103 cells per well on white walled 96-well tissue culture-treated plates (ThermoFisher Scientific, Waltham, MA, USA) and incubated overnight in complete medium. Three canine neoplastic established cell lines, representing hematopoietic, epithelial, and mesenchymal tumor types were used for all experiments; mastocytoma C2 (Dr. Warren Gold, University of California, San Francisco, USA), mammary gland carcinoma CMT-12 (Dr. R. Curtis Bird, Auburn University, Alabama, USA), and osteosarcoma D17 (#CCL-183; ATCC, Manassas, VA, USA). Gamma-histone H2A.X is a marker of DNA oxidation and initiation of repair and was not detected when compared to UV irradiation as a positive control for DNA damage (data not shown). Membranes were incubated overnight in primary antibody solutions at a dilution of 1:1000 in TBST on a rocking platform at 4C. Plouzek CA, Ciolino HP, Clarke R, Yeh GC. Paclitaxel (Taxol)-induced gene expression and cell death are both mediated by the activation of c-Jun NH2-terminal kinase (JNK/SAPK). In general, early, transient activation of JNK may lead to cell survival, while sustained activation can induce apoptosis and curcumin or rosemary extracts appear to be involved in this constitutive activation of SAPK/JNK [4446]. The dual combination treatment using half the concentration (3.1g mL1 each extract) was as effective as 6.3g mL1 TE alone in all three cancer cell lines (Fig. Observation from previous flow cytometry experiments showed an unexpected increase in the GMF when cells were treated with TE alone when excited at a wavelength of 488nm, whereas no change was observed when RE was used alone (Fig. Raw data from the viability portion of the assay (individual fluorescence values of each well) were normalized to the vehicle alone treatment for each cell line, considered to represent 100% proliferating cells. These experiments were designed to focus on concentrations that may have utility in vivo and concentrations that showed synergistic effects of the compounds in our prior experiments (focusing on synergistic concentrations of 3g/mL of TE and RE versus 6g/mL1 of each extract independently) [11]. Sharma RA, McLelland HR, Hill KA, Ireson CR, Euden SA, Manson MM, et al. probiotics Kumar D, Basu S, Parija L, Rout D, Manna S, Dandapat J, Debata PR. Fluorescence and luminescence was measured using SpectraMax M3 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).

[42, 43] Changes in activation of MAPK/ERK were not observed after treatment in any of the cell lines examined. (Fig.5B;5B; p<0.0001). Bogoyevitch MA, Ngoei KR, Zhao TT, Yeap YY, Ng DC. The cellular accumulation of curcumin was measured by exploiting the auto-fluorescent properties of this compound [20]. Membranes were washed three times with TBST and visualized with a chemi-luminescent reagent (Clarity Western ECL Substrate; Bio-Rad, Hercules, CA, USA). The cell pellet was washed once with PBS before resuspension in DMEM, and filtered before fluorescence analysis when excited at a wavelength of 488nm and then measuring emission using a 530/30 filter. The effects of these purified compounds have been examined in vitro in a variety of human cell lines derived from tumors of the colon, skin, and breast tissue [6], but only a few studies have looked at the effects in canine cancer cells lines [79]. Curcumin improves the efficacy of cisplatin by targeting cancer stem-like cells through p21 and cyclin D1-mediated tumour cell inhibition in non-small cell lung cancer cell lines. Each of the treatment conditions were completed in duplicate and averaged in four independent experiments. The effects of branched-chain amino acids on canine neoplastic cell proliferation and death. Cells were harvested and lysed at 12h and 24h after treatment using Mammalian Lysis Buffer (MLB; 25mM Tris, 100mM NaCL, 1mM EDTA, 1% Triton X-100, 0.004% NaF, 1mM NaVO4, 25mM -glycerophosphoric acid, 100g/ml phenylmethanesulfonyl fluoride, and 1g/ml each aprotinin and leupeptin, pH7.4) and sonication, and then centrifuged for 5m at 14,000 rcf at 4C. The results of this study provides some insights into possible mechanisms by which TE and RE induce apoptosis across three canine neoplastic cell lines. Differences in treatment responses were observed in the three cell lines. The cell pellet was washed once with PBS before resuspension in Annexin Binding Buffer (ABB; 10mM HEPES, 140mM NaCl, 2.5mM CaCl2, pH7.4) at a density of 1106 cell mL1. The supernatant was collected and the protein concentration was determined using the Bradford assay (Coomassie-dye; ThermoFisher Scientific Pierce, Waltham, MA, USA). Verheij M, Ruiter GA, Zerp SF, van Blitterswijk WJ, Fuks Z, Haimovitz-Friedman A, Bartelink H. Role of the stress-activated protein kinase (SAPK/JNK) signaling pathway in radiation-induced apoptosis. Molecular approaches toward targeted cancer prevention with some food plants and their products: inflammatory and other signal pathways.

JW helped conceive the study, supervised the study and helped in manuscript drafting and editing. The global effects of both TE, RE, and the two in combination showed no appreciable alteration in cell cycle kinetics. dog cancer tumor growth earths answers dogs tumor mast canine