turmeric for mast cell tumors in dogs

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.

We have previously shown that turmeric root (TE) and rosemary leaf (RE) extracts can work synergistically to reduce neoplastic cell growth, but the mechanisms are poorly understood and require further elucidation. In our previous study, RE worked in a synergistic manner with TE to decrease cellular proliferation when used in combination. 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. Membranes were incubated overnight in primary antibody solutions at a dilution of 1:1000 in TBST on a rocking platform at 4C. Curcumin and Ellagic acid synergistically induce ROS generation, DNA damage, p53 accumulation and apoptosis in HeLa cervical carcinoma cells. The fold-change data from caspase 3/7 activation, percent of apoptotic cells, intracellular ROS level and curcumin accumulation assays and the ratio data from western blot assay were processed using analysis of variance with Tukeys method for multiple comparisons between all treatment conditions (single, combination and DMSO control). 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]. 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. Although the results of these experiments are promising, the clinical utility is complex due to the absorption, transformation and elimination kinetics of these compounds in general. Chemical genetic analysis of the time course of signal transduction by JNK. Prior studies have shown the autofluorescence of curcumin can be examined with flow cytometry [20]. Y-axis values represent the fold change in geometric mean fluorescence (GMF) of all cells compared to DMSO control.

TE alone was a significantly stronger antioxidant than RE alone using same extract concentration (6.3g mL1) in all the three cell lines (C2, CMT-12 and D17) with TE reducing ROS by about 7590-80%, respectively and RE reducing ROS by about 5040-40%, respectively. Within each cell line, means with different letters are significantly different from each other (p<0.05). Minimally three independent experiments were examined for each treatment through the different assays (percent of gated cells in each phase cycle, percent of apoptotic cells, intracellular ROS and curcumin level) and analyzed with Cell Quest software (BD Biosciences).

(Fig.3).3). Shehzad A, Lee YS. Concentrations that appeared to be most synergistic at inhibiting proliferation from our prior publication [11] were used to observe enhanced or diminished signaling events over extended periods of time from 12 to 24h that might provide insights into the modest apoptotic response. Both extracts had antioxidant effects with RE reducing reactive oxygen species (ROS) by 4050% and TE reducing ROS by 8090%. The use of RE in combination with TE induces a synergistic response to induce apoptosis which is better than either extract alone. Pesakhov S, Khanin M, Studzinski GP, Danilenko M. Distinct combinatorial effects of the plant polyphenols curcumin, carnosic acid, and silibinin on proliferation and apoptosis in acute myeloid leukemia cells. 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. earths answers dogs Minimal change was seen in the D17 cell lines after treatment with 6.3g mL1 RE, 1.1 at both time points. The combination treatment using 3.1g mL1 both extracts induced a small decrease in S phase in only the C2 cell line, and a modest increase in G2/M phase in only the D17 cell line. Treatments which induced a statistically significant change from DMSO within each cell cycle phase are indicated. Cell cycle effects were analyzed after 24h (data not shown) and 48h treatment using propidium iodide staining to label DNA content. 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).

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After examination of several cell signaling pathways, no consistent trend was seen in the phosphorylation status of the variety of signaling proteins, alterations in the mitochondrial proteins involved in apoptosis or markers of DNA damage (data not shown). Pharmacodynamic and pharmacokinetic study of oral curcuma extract in patients with colorectal cancer. Fluorescence and luminescence was measured using SpectraMax M3 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Our results showed an increase in phosphorylated SAPK/JNK after 12h and 24h of treatment with TE alone. Membranes were washed three times with TBST and incubated at room temperature for 1h in the corresponding secondary anti-mouse IgG or anti-rabbit IgG horseradish peroxidase-conjugated antibody at a dilution of 1:2000 (Cell Signaling Technology). Each of the treatment conditions were completed in duplicate and averaged in four independent experiments. tumor mast canine Few studies have been completed in dogs or related cell lines, therefore it is necessary to examine the effects of these compounds in vitro before using them in clinical veterinary trials.

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. Datasets used and analyzed during this study are available from the corresponding author upon reasonable request. Basu S, Kolesnick R. Stress signals for apoptosis: ceramide and c-Jun kinase. For each protein of interest, 30g total proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on gels ranging from 6 to 15% based on the molecular weight of the protein of interest. 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. 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]. Single treatment with 6.3g mL1 TE resulted in a significant decrease in S phase (DNA replication) in the D17 cell line compared to DMSO control. Carnosic acid, a rosemary phenolic compound, induces apoptosis through reactive oxygen species-mediated p38 activation in human neuroblastoma IMR-32 cells. 2016 Sep 9;8(9).

This appears to be related to a variable increased TE uptake in cells and activation of pathways involved in the apoptotic response. official website and that any information you provide is encrypted Changes in the protein expression levels of SAPK/JNK pathway in turmeric and rosemary-treated cells. Three different canine cell lines (C2 mastocytoma, and CMT-12 mammary carcinoma, D17 osteosarcoma) were treated with 6.3g mL1 extract individually, or 3.1g mL1 of each extract in combination based on studies showing synergy of these two extracts. 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].

Wakshlag JJ, Balkman CE. Caspase 3/7 activation was quantified using the ApoLive-Glo Multiplex Assay (Promega, Madison, WI, USA) following manufacturers instructions. Helmerick EC, Loftus JP, Wakshlag JJ. Digital images were captured using an imaging system (Biospectrum 410; UVP, Upland, CA, USA). Apoptosis, antioxidant effects, cellular accumulation of curcumin, and perturbation of signaling pathways were assessed. government site. All statistical analyses were performed using JMP Pro (v. 11.2.1; SAS Institute Inc., Cary, NC, USA). The proteins were then transferred to 0.45m pore size polyvinylidene fluoride membrane (Immobilon-P Membrane; EMD Millipore, Billerica, MA, USA) for 1h at 333mA and then blocked in 5% milk in Tris-buffered saline/0.05% Tween-20 solution (TBST). Synergy in plant medicines. Cell lines were cultured in appropriate complete medium as previously described.11 All culture reagents were purchased from Invitrogen, Carlsbad, CA, USA, unless otherwise indicated. The effects of baicalein on canine osteosarcoma cell proliferation and death. http://creativecommons.org/licenses/by/4.0/, http://creativecommons.org/publicdomain/zero/1.0/, A phase I study investigating the safety and pharmacokinetics of highly bioavailable curcumin (Theracurmin) in cancer patients, Stress activated kinase/jun-N-terminal kinase, Signal transducer and activator of transcription. Royal Canin participated in writing the protocol, analyzing the data, contributing compounds, revising the manuscript for publication. The aforementioned study was funded by Royal Canin (JB and VB) collaborated on the study design and interpretation of data collected. Within each cell line, means with different letters are significantly different from each other (p<0.0001). After images were collected, membranes were washed three times in TBST and incubated with a 1:10,000 dilution in TBST of the house-keeping antibody -Actin (Sigma-Aldrich) for 1h at room temperature. In addition, for measurements of ROS, an unstained control was used to determine the baseline GMF of each extract. All authors have read and accepted the final manuscript.

4). 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. The geometric mean fluorescence (GMF) from each treatment was compared to the DMSO treated samples and represented as fold change for all experiments using GMF due to the differences in fluorescence intensity across cell lines. Concentrations were chosen based on our prior publication surrounding the effective concentrations for synergy between the two extracts of interest [11]. In line with pathway disruption and cell cycle dynamics, TE and RE are often thought to be antioxidants, however curcumin has been shown to cause oxidative damage to DNA and induction of the DNA damage response pathways that are intimately involved in cell cycle alteration or apoptosis [29, 30]. This obstacle may be overcome through the use of combination treatments with other extracts that improve bioavailability or hinder additional pathways [1416]. 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]. C2, CMT-12 and D17 cell lines were harvested and lysed after 12h or 24h treatment with DMSO vehicle control, or 6.3g mL1 Turmeric extract (TE) alone, or 6.3g mL1 Rosemary extract (RE) alone, or combination of 3.1g mL1 each of TE+RE.

JW helped conceive the study, supervised the study and helped in manuscript drafting and editing. The results of this study warrant further investigations into the pharmacodynamics and pharmacokinetics of these extracts in dogs when incorporated into feed to determine if clinical trials are feasible.

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. Baharuddin P, Satar N, Fakiruddin KS, Zakaria N, Lim MN, Yusoff NM, et al. Briefly, cells were detached with Accumax dissociation solutions (Innovative Cell Technologies, San Diego, CA, USA), collected and centrifuged for 10m at 500 rcf at 4C. 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. Chen J, Li L, Su J, Li B, Zhang X, Chen T. Proteomic analysis of G2/M arrest triggered by natural Borneol/Curcumin in HepG2 cells, the importance of the reactive oxygen species-p53 pathway.

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turmeric for mast cell tumors in dogs