However, changes in Thr183/Tyr185 phosphorylated-SAPK/JNK (p-SAPK/JNK; Fig. 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. 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. 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). 6) were detected in the three cancer cell lines. RE induced a significant increase in phosphorylation after 12h and 24h of treatment in the CMT-12 cell line, while in the C2 cell line this increase was only seen at 12h and returned to baseline by 24h. The dual combination treatment had the greatest effect in the CMT-12 cell line, resulting in phosphorylated SAPK/JNK at levels greater than either extract alone (even using twice the concentration). 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. Li J, Xiang S, Zhang Q, Wu J, Tang Q, Zhou J, et al. 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). sharing sensitive information, make sure youre on a federal The site is secure. This anti-oxidant property has been thought to be involved in cell survival and possible resistance to chemotherapeutic intervention [31]. When the dual combination treatment (3.1g mL1 TE+3.1g mL1 RE) was used, a significant increase in Annexin-V positive cells compared to vehicle control was seen in the 3 cell lines, but this was not significant compared to 6.3g mL1 TE alone in C2 cell line. Densitometry values represent a ratio of phosphorylated protein to total protein and normalized to DMSO vehicle control of the same time point (mean of three separate experiments). 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. A treatment with 6.3g mL1 RE alone resulted in a statistically significant increase of 1.4-fold in Caspase 3/7 activation in all three cell lines when compared to vehicle control. There were minimal to no cell cycle changes with TE extract alone, therefore further examination of cell cycle pathway analysis was not pursued. Caspase 3/7 activation was quantified using the ApoLive-Glo Multiplex Assay (Promega, Madison, WI, USA) following manufacturers instructions. Fossey SL, Bear MD, Lin J, Li C, Schwartz EB, Li PK, et al. 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). Bioactive molecules derived directly from plants, or modeled after plant compounds, continue to be an active area of cancer research. The greatest increase in p-SAPK/JNK was seen with the combination of 3.1g mL1 each of TE and RE in the CMT-12 cell line where densitometry values increased from 1.0 with DMSO treatment to 4.3 after 12h incubation and 4.8 after 24h incubation (p<0.05 from DMSO treatment). Samples were equilibrated to a common volume (g L1) in MLB and 5 laemmili loading buffer (300mM Tris-HCl pH6.8, 10% Sodium dodecyl sulfate, 50% glycerol, 12.5% -Mercaptoethanol, 0.025% Bromophenol blue). 3A and C lower right quadrant; full analysis Fig. The cell pellet was washed twice with 1% FBS in PBS, filtered, and resuspended in 70% cold ethanol for overnight fixation. Markers of apoptosis, antioxidant capabilities, and changes in the activation of common cell signaling pathways were analyzed after treatment. Curcumin suppresses proliferation of colon cancer cells by targeting CDK2. Fluorescence and luminescence was measured using SpectraMax M3 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Corri B. Levine, Email: ude.llenroc@64lbc. Further examination of the cellular effects into cell signaling pathways previously implicated in TE and RE treatment were performed [2535]. The use of TE and its major compound of interest, curcumin, has been extensively studied to treat a variety of diseases and ailments, perhaps due to its ability to bind and interact with a variety of cellular proteins [12]. Samples were then incubated for 30m at room temperature and analyzed with an excitation of wavelength of 488nm and emission of 617nm. 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. 8600 Rockville Pike JB and VB conceived the study and participated in its design and participated in manuscript editing. Membranes were incubated overnight in primary antibody solutions at a dilution of 1:1000 in TBST on a rocking platform at 4C. Wakshlag JJ, Kallfelz FA, Wakshlag RR, Davenport GM. Inhibition of P-glycoprotein activity and reversal of multidrug resistance in vitro by rosemary extract. 
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. 4). Changes in densitometry compared to DMSO control with significance of p<0.05 represented by *. (Fig.1D)1D) cell lines. All statistical analyses were performed using JMP Pro (v. 11.2.1; SAS Institute Inc., Cary, NC, USA). Paclitaxel (Taxol)-induced gene expression and cell death are both mediated by the activation of c-Jun NH2-terminal kinase (JNK/SAPK). 2), and Annexin-V staining which increased from 6% to 11% apoptotic cells in the C2 (Fig. The influence of natural products upon drug discovery. Einbond LS, HA W, Kashiwazaki R, He K, Roller M, Su T, et al. Prior studies have shown the autofluorescence of curcumin can be examined with flow cytometry [20]. Under our cell culture conditions and extract concentrations used, we could not elicit a pro-oxidative response from TE or RE in any cell line used. Berginc K, Trontelj J, Basnet NS, Kristl A. Physiological barriers to the oral delivery of curcumin. 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). Digital images were captured using an imaging system (Biospectrum 410; UVP, Upland, CA, USA). 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. Values are expressed as meanstandard deviation of four independent replicates. 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]. Shehzad A, Lee YS. 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. Cul J, Zhang M, Zhang YQ, JNK XZH. Induction of apoptosis through the activation of SAPK/JNK followed by the expression of death receptor Fas in X-irradiated cells. Our results indicate that different tumor types are likely to have a differential response to such interventions. Shaikh J, Ankola DD, Beniwal V, Singh D, Kumar MN. Antioxidant effects of turmeric and rosemary extracts in C2, CMT-12 and D17 cell lines. Within each cell line, means with different letters are significantly different from each other (C2 p<0.05; CMT-12 and D17 p<0.0001). In addition, for measurements of ROS, an unstained control was used to determine the baseline GMF of each extract. 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. 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). This was attributed to cell clumping in this cell line due to the fixation method used and use of propidium iodide causing cell-doublets to be inappropriately represented as G2M phase. In the case of cell cycle dynamics, Dunnetts method was used to control for multiple comparisons when studying the percent of gated events difference between single treatment or dual combination and DMSO control only at each time point. [42, 43] Changes in activation of MAPK/ERK were not observed after treatment in any of the cell lines examined. However, in the CMT-12 and D17 cell lines, the combination treatment induced a significantly greater percentage of apoptotic cells, 40% and 13%, respectively, compared to 6.3g mL1 TE alone (13% and 7%) and 6.3g mL1 RE alone (5%) (Fig. Cells were incubated with the indicated treatments for 48h and the induction of apoptosis was detected by Annexin V-FITC and 7-AAD staining followed by flow cytometric analysis. This obstacle may be overcome through the use of combination treatments with other extracts that improve bioavailability or hinder additional pathways [1416]. Briefly, after 36h of treatment, viability reagent was added to the wells and incubated at 37C for 30m and fluorescence was measured at 400Ex/505Em. Caspase 3/7 activation was determined as caspase activation per total viable cells for each treatment. Quantitative cellular uptake, localization and cytotoxicity of curcumin in normal and tumor cells. 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. Expression level of Thr183/Tyr185 phosphorylated-SAPK/JNK (p46/p54) and total SAPK/JNK were determined by Western blot analysis. Carnosic acid, the compound of interest in RE, can target a variety of signaling pathways, many of which overlap with those targeted by curcumin. Kelsey JL, Moore AS, Glickman LT. Epidemiologic studies of risk factors for cancer in pet dogs. The role of Nutraceuticals in pancreatic cancer prevention and therapy: targeting cellular signaling, MicroRNAs, and Epigenome. In our previous study, RE worked in a synergistic manner with TE to decrease cellular proliferation when used in combination. Previous literature has shown a synergistic effect between these two extracts, specifically the cleavage of poly ADP-ribose polymerase and Caspase-8, 9, and 3 on human cell lines [17]. 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. This appears to be related to a variable increased TE uptake in cells and activation of pathways involved in the apoptotic response. JJW has received compensation from Nestle Purina, Mars, Annamaet Pet Food Company, and Veterinary Recommend Solutions for consultation. Within each cell line, means with different letters are significantly different from each other (p<0.05). will also be available for a limited time. Tong L, Chuang CC, Wu S, Zuo L. Reactive oxygen species in redox cancer therapy. 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. Chemical genetic analysis of the time course of signal transduction by JNK. 
Cells were plated on 60mm tissue culture-treated plates (LPS, Rochester, NY) and incubated in complete medium until 60% confluent. The following day, samples were centrifuged for 10m at 500 rcf at 4C, resuspended in cold PBS. 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. Gilbert B, Alves LF. 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. Douglass BJ, Clouatre DL. The Cells were then treated with medium, DMSO vehicle control, extract alone, or extracts in combination. Kim J, Freeman MRJNK. These cell lines were chosen for initial screening as representative cell lines of the three major cell lineages of cancer in dogs in hopes of finding a similar global effect across different cell lineages. Joseph J. Wakshlag, Phone: 607 253-4389, Email: ude.llenroc@73wj. Mosieniak G, Sliwinska MA, Przybylska D, Grabowska W, Sunderland P, Bielak-Zmijewska A, Sikora E. Curcumin-treated cancer cells show mitotic disturbances leading to growth arrest and induction of senescence phenotype. Levine CB, Bayle J, Biourge V, Wakshlag JJ. Carnosic acid bioavailability from rosemary extract, though good, has not been studied extensively; however there is rapid glucuronidation, methylation, and oxidation of carnosic acid and related molecules from rosemary extract, and the bioactivity of these modified derivatives are unknown at this time[51]. 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. 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. The ratio of caspase activation to viable cells is represented as fold increase over DMSO treatment alone. Fig.3E).3E). Cell cycle effects were analyzed after 24h (data not shown) and 48h treatment using propidium iodide staining to label DNA content. Niedzwiecki A, Roomi MW, Kalinovsky T, Rath M. Anticancer Efficacy of Polyphenols and Their Combinations. Received 2017 Jun 9; Accepted 2017 Nov 27. The benefit of using these plant extracts to treat cancer is the potential synergy of multiple compounds found within a single extract whereby the major compound may have one or more targets, while other molecules in the extract may be affecting other targets or influencing absorption kinetics [5]. Varying degrees of susceptibility were detected across the three cancer cell lines used, with the CMT-12 cell line being the most susceptible to these treatments, perhaps due to a greater increase in intracellular curcumin accumulation as shown by flow cytometry. The transient nature of activated SAPK/JNK in the C2 and D17 cell lines lead us to believe this may be involved in the diminished proliferation, however other pathways may be involved in the induction of apoptosis in these cancer cell lines. CpJun N-terminal kinase (JNK) signaling: recent advances and challenges. Molecular approaches toward targeted cancer prevention with some food plants and their products: inflammatory and other signal pathways. Events labeled only Annexin-V positive were considered to represent apoptotic cells; events labeled Annexin-V positive and 7-AAD positive were considered to represent necrotic cells. An official website of the United States government. 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]. The .gov means its official. The cellular accumulation of curcumin was measured by exploiting the auto-fluorescent properties of this compound [20]. Carnosic acid, a rosemary phenolic compound, induces apoptosis through reactive oxygen species-mediated p38 activation in human neuroblastoma IMR-32 cells. Lee LF, Li G, Templeton DJ, Ting JP. Therefore, the possibility that RE could increase the cellular accumulation of the fluorescent compound curcumin was investigated when these compounds were used in combination. The new PMC design is here! 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. Annexin-V 488 conjugate and 7-Aminoactinomycin D (7-AAD) were added to the cell suspensions and incubated for 15m at room temperature. 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. Synergy in plant medicines. Evaluation of the protective effects of all-trans-astaxanthin on canine osteosarcoma cell lines. After treating cells for 36h, TE alone (6.3g mL1) resulted in a significant increase in apoptotic cells in the C2 and CMT-12 cell lines as determined by Caspase 3/7 activation, 2- and 2.5-fold, respectively (Fig. The TE+RE combination treatment resulted in Caspase 3/7 activation and apoptosis in all cell lines, beyond the effects of TE alone with the CMT-12 cell line being most susceptible. e Percent early apoptotic cells (lower right quadrant of Annexin V positive and 7-AAD negative cells) are represented as meanstandard deviation (three independent replicates). 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. Bethesda, MD 20894, Web Policies In the D17 cell line only a minor non-significant increase was observed (1.2 at 12h, 1.1 at 24h). Lim TG, Lee SY, Huang Z, Lim DY, Chen H, Jung SK, et al. Both extracts had antioxidant effects with RE reducing reactive oxygen species (ROS) by 4050% and TE reducing ROS by 8090%. 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. 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. 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. Julie Bayle, Email: moc.ninaclayor@elyab.eiluj. We are grateful to the University of California San Francisco, especially Dr. Warren Gold and Dr. George Caughey, for supplying the C2 canine mastocytoma cell lines and to Auburn University, especially Dr. R. Curtis Bird, for supplying CMT-12 canine mammary gland carcinoma cell line. Apoptosis, Canine cancer, Mammary carcinoma, Osteosarcoma, Mastocytoma, Curcumin, Rosemary, {"type":"entrez-nucleotide","attrs":{"text":"DA251471","term_id":"78448130","term_text":"DA251471"}}. Gonzlez-Vallinas M, Gonzlez-Castejn M, Rodrguez-Casado A, Ramrez de Molina A. Dietary phytochemicals in cancer prevention and therapy: a complementary approach with promising perspectives. DMSO was compared against cells in media alone showing no significant differences, therefore DMSO treated cells were used as the control sample for all comparisons. Helmerick EC, Loftus JP, Wakshlag JJ. Cells were treated the following day with DMSO vehicle control, 6.3g mL1 extract alone, or 3.1g mL1 each extract in combination. Membranes were washed, incubated with mouse secondary antibody at a dilution of 1:2000, and imaged as described. Curcumin and cancer cells: how many ways can curry kill tumor cells selectively? 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. Cell lines were grown on tissue culture-treated plates (Laboratory Product Sales, Rochester, NY, USA) at 37C and 5% CO2 for all experiments and passage of cells, unless otherwise noted. Only C2 and D17 cell lines were analyzed due to the presence of frequent doublets with CMT-12 cells resulting in an artificial accumulation in the G2/M phase. Samples were centrifuged again for 5m at 300 rcf at 4C and resuspended in DNA staining solution [2% propidium iodide (Sigma Aldrich), 0.1% Triton X-100 (Sigma Aldrich), in PBS]. Prior literature has shown that curcumin can have significant effects on cell cycle dynamics through the upregulation of cyclins or cyclin dependent kinase activity [2628]. Passos JF, Miwa S, von Zglinicki T. Measuring reactive oxygen species in senescent cells. Combination of curcumin and bicalutamide enhanced the growth inhibition of androgen-independent prostate cancer cells through SAPK/JNK and MEK/ERK1/2-mediated targeting of NF-B/p65 and MUC1-C. Kuwabara M, Takahashi K, Inanami O. Molecular mechanisms of curcumin action: signal transduction. Cells were treated with the indicated concentrations of extracts for 12h followed by determination of intracellular levels of reactive oxygen species using Dihydrorhodamine123 staining. Within each cell line, means with different letters are significantly different from each other (p<0.0001). The aforementioned study was funded by Royal Canin (JB and VB) collaborated on the study design and interpretation of data collected. Baharuddin P, Satar N, Fakiruddin KS, Zakaria N, Lim MN, Yusoff NM, et al. 5A, C; p<0.0001). Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. pii: E552. CBL carried out the technical experimentation, performed statistical analysis, and was primary author in the manuscript. Tai J, Cheung S, Wu M, Hasman D. Antiproliferation effect of rosemary (Rosmarinus Officinalis) on human ovarian cancer cells in vitro. 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. FOIA Moore J, Yousef M, Tsiani E. Anticancer Effects of Rosemary (. 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 safety of these commonly used feed ingredients and continual synergy between the extracts make them candidates for inclusion in the diet as a potential adjuvant treatment for dogs diagnosed with neoplasia, if appropriate serum concentrations can be achieved. 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. 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. The cell suspension was then incubated at 37C for 30m, pelleted, and resuspended in 1mL DMEM and filtered before fluorescence analysis of cells. These differences across cell lines suggest the complexity in cellular response and potential susceptibility of various cell lines to treatment, further clarifying the need to understand what types of cancer may be more responsive to these interventions. official website and that any information you provide is encrypted The effects of baicalein on canine osteosarcoma cell proliferation and death. Our assessment of antioxidant status after treatment unanimously indicated that the cells are under less oxidative stress after treatment with either TE or RE alone or in dual combination within 12h of incubation. Ramos S. Effects of dietary flavonoids on apoptotic pathways related to cancer chemoprevention. SAPK mediates doxorubicin-induced differentiation and apoptosis in MCF-7 breast cancer cells. 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. 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). In addition, sustained activation and signaling through the SAPK/JNK pathway may play a role in this cell lines increased sensitivity to apoptosis. The use of RE in combination with TE induces a synergistic response to induce apoptosis which is better than either extract alone. Treatments which induced a statistically significant change from DMSO within each cell cycle phase are indicated. Plouzek CA, Ciolino HP, Clarke R, Yeh GC. Johnson CR, Jiffar T, Fischer UM, Ruvolo PP, Jarvis WD. and transmitted securely. 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). The research leading to these results was supported by Royal Canin SAS. In addition RE treatment enhanced the c-jun N-terminal kinase (JNK) activity in the C2 cell line and TE+RE exposure increased activated JNK by 45 times in the CMT-12 cell line. Our data at these concentrations only suggest pro-apoptotic responses, suggesting that the mechanisms are unlikely to rely on oxidative damage. Each of the treatment conditions were completed in duplicate and averaged in four independent experiments. 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. Effect of rosemary extract on intracellular accumulation of curcumin in canine tumor cell lines. Generally, these differences may be due to the lower concentrations utilized in our experiments compared to the prior studies. 1A-B). 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. Background fluorescence and luminescence was measured in wells containing treatments but no cells. 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. Before Ventura JJ, Hbner A, Zhang C, Flavell RA, Shokat KM, Davis RJ. Nanoparticle encapsulation improves oral bioavailability of curcumin by at least 9-fold when compared to curcumin administered with piperine as absorption enhancer. Cells were treated with indicated concentrations of extracts or DMSO for 36h. Activated caspase 3/7 per viable cells was expressed as mean fold change from DMSO control values standard deviation from three independent replicates. Reported data are expressed as meanstandard deviation of 4 independent replicates. Differences in treatment responses were observed in the three cell lines. 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. 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. National Library of Medicine 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. Bar graphs represent the average of three individual experiments performed in duplicate and representative cell cycle histograms of DMSO control treated (c) C2 and (d) D17 cell lines are shown at 48h of treatment. A previous study in the human breast cancer cell line, MCF-7, showed an increase in intracellular accumulation of various chemotherapeutic drugs which was attributed to competitive inhibition of transmembrane transport pump P-glycoprotein by rosemary extract [25]. 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]. 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. 
- Worldwide Stamps Usps
- Group Spa Day Fort Lauderdale
- Woodstock Way Hotel Restaurant
- Hampton Inn Bellevue / Nashville-i-40 West
- Nike Mini Swoosh Oversized Joggers Asos
- 360 Degree Camera For Car Installation
- Data Management And Storage In Research
- Bluetooth Stereo System
- Pouring Pitcher For Candle Making
- Layered Pearl Necklace Choker
- Ferry From Barcelona To Mallorca How Long
- Intuitive Eating A Revolutionary Anti-diet Approach
- Uniweld Nitrogen Regulator
- Santa Maria Melograno Perfume
