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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. 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. JB and VB are employed by Royal Canin. 8600 Rockville Pike 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. sharing sensitive information, make sure youre on a federal 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. 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). 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. 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. Carnosic acid inhibits the growth of ER-negative human breast cancer cells and synergizes with curcumin. All flow cytometric analysis was performed on BD FACSCalibur (BD Biosciences, San Jose, CA, USA). Inhibition of P-glycoprotein activity and reversal of multidrug resistance in vitro by rosemary extract. This obstacle may be overcome through the use of combination treatments with other extracts that improve bioavailability or hinder additional pathways [1416]. Corri B. Levine, Email: ude.llenroc@64lbc. The supernatant was collected and the protein concentration was determined using the Bradford assay (Coomassie-dye; ThermoFisher Scientific Pierce, Waltham, MA, USA). Deng Y, Verron E, Rohanizadeh R. Molecular mechanisms of anti-metastatic activity of Curcumin. 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. Differences in treatment responses were observed in the three cell lines. 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. After the incubation, ABB was added to the cell suspension and kept on ice until fluorescence analysis. 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. Western blots were run in three independent time course experiments and densitometry was completed using ImageJ [21]. 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. government site. This appears to be related to a variable increased TE uptake in cells and activation of pathways involved in the apoptotic response. Prior studies have shown the autofluorescence of curcumin can be examined with flow cytometry [20]. Differences were considered statistically significant at p<0.05. Antioxidant effects of turmeric and rosemary extracts in C2, CMT-12 and D17 cell lines. 3E) and from 4% to 13% in the CMT-12 cell line (represented in Fig. The aforementioned study was funded by Royal Canin (JB and VB) collaborated on the study design and interpretation of data collected. 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. JB and VB conceived the study and participated in its design and participated in manuscript editing. Carnosic acid, the compound of interest in RE, can target a variety of signaling pathways, many of which overlap with those targeted by curcumin. The residuals of all statistical models were found to be normally distributed therefore parametric statistics were utilized. Briefly, cells were detached with Accumax cell dissociation solution (Innovative Cell Technologies, San Diego, CA USA), collected in tubes with 1% fetal bovine serum (FBS) in Phosphate Buffered Saline (PBS) and centrifuged for 5m at 300 rcf at 4C. Minimal change was seen in the D17 cell lines after treatment with 6.3g mL1 RE, 1.1 at both time points. 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. (Fig.1D)1D) cell lines. 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). Carnosic acid, a rosemary phenolic compound, induces apoptosis through reactive oxygen species-mediated p38 activation in human neuroblastoma IMR-32 cells. For all flow cytometry experiments, 10,000 events were collected per sample and then gated based on a forward-scatter/side-scatter plot. TE alone (3.1g mL1) significantly increased the GMF in the C2 and D17 cell lines, 1.7- and 1.8-fold, respectively; while when using RE with TE the increase was 2.2 and 2.3 fold, respectively (Fig. 2), and Annexin-V staining which increased from 6% to 11% apoptotic cells in the C2 (Fig. 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. 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]. Cells were treated the following day with DMSO vehicle control, 6.3g mL1 extract alone, or 3.1g mL1 each extract in combination. 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]. Ravindran J, Prasad S, Aggarwal BB. 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. SAPK mediates doxorubicin-induced differentiation and apoptosis in MCF-7 breast cancer cells. 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. Cell cycle dynamics were analyzed after 24h and 48h of incubation with the different treatments; no significant difference was seen between these two time-points therefore only data from the 48h time point is shown (Fig. Apoptosis, Canine cancer, Mammary carcinoma, Osteosarcoma, Mastocytoma, Curcumin, Rosemary, {"type":"entrez-nucleotide","attrs":{"text":"DA251471","term_id":"78448130","term_text":"DA251471"}}. Cells were treated with the indicated concentrations of extracts for 12h followed by determination of intracellular levels of reactive oxygen species using Dihydrorhodamine123 staining. Next, Caspase-Glo 3/7 Reagent was added to all wells, incubated for 30m at room temperature, and luminescence was measured. Caspase 3/7 activation induced by turmeric and rosemary extracts in C2, CMT-12, and D17 cell lines. 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. 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. 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. Chemical genetic analysis of the time course of signal transduction by JNK. The new PMC design is here! 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. 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]. The cellular accumulation of curcumin was measured by exploiting the auto-fluorescent properties of this compound [20]. The effects of TE and/or RE on cell cycle progression were measured on C2 and D17 tumor cell lines using propidium iodide staining. 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. 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. 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. PMC legacy view FOIA CBL carried out the technical experimentation, performed statistical analysis, and was primary author in the manuscript. and transmitted securely. The cell pellet was washed twice with 1% FBS in PBS, filtered, and resuspended in 70% cold ethanol for overnight fixation. 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]. Changes in the protein expression levels of SAPK/JNK pathway in turmeric and rosemary-treated cells. Cell cycle effects were analyzed after 24h (data not shown) and 48h treatment using propidium iodide staining to label DNA content. The ratio of caspase activation to viable cells is represented as fold increase over DMSO treatment alone. 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]. 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. CpJun N-terminal kinase (JNK) signaling: recent advances and challenges. The global effects of both TE, RE, and the two in combination showed no appreciable alteration in cell cycle kinetics. 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. Concentrations were chosen based on our prior publication surrounding the effective concentrations for synergy between the two extracts of interest [11]. Passos JF, Miwa S, von Zglinicki T. Measuring reactive oxygen species in senescent cells. 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. Treatment with 6.3g mL1 RE induced a significant decrease in G1/G0 phase in the D17 cell line, a reduction in S phase in both cell lines, and an increase in G2/M phase (cell division) in the D17 cell line. Membranes were washed three times with TBST and visualized with a chemi-luminescent reagent (Clarity Western ECL Substrate; Bio-Rad, Hercules, CA, USA). Fluorescence and luminescence was measured using SpectraMax M3 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Effects and synergy of feed ingredients on canine neoplastic cell proliferation. Curcumin and Ellagic acid synergistically induce ROS generation, DNA damage, p53 accumulation and apoptosis in HeLa cervical carcinoma cells. Fresh extract stock solutions were prepared and used for every experiment. Cells were then treated with medium, DMSO vehicle control, extract alone, or extracts in combination. 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]. Lee WH, Loo CY, Young PM, Traini D, Mason RS, Rohanizadeh R. Recent advances in curcumin nanoformulation for cancer therapy. Kumar D, Basu S, Parija L, Rout D, Manna S, Dandapat J, Debata PR. 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. 3A and C lower right quadrant; full analysis Fig. 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. 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. There were some mild alterations in cell cycle in the D17 and C2 cell lines showing decreases in the G1/G0 and increases in G2/M phases with RE and the combination treatment. Quantitative cellular uptake, localization and cytotoxicity of curcumin in normal and tumor cells. 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]. Li J, Xiang S, Zhang Q, Wu J, Tang Q, Zhou J, et al. 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]. Requirement for SAPK-Jnk signaling in the induction of apoptosis by ribosomal stress in REH lymphoid leukemia cells. 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. Baharuddin P, Satar N, Fakiruddin KS, Zakaria N, Lim MN, Yusoff NM, et al. 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). Caspase 3/7 activation was determined as caspase activation per total viable cells for each treatment. Bethesda, MD 20894, Web Policies Molecular approaches toward targeted cancer prevention with some food plants and their products: inflammatory and other signal pathways. Our data at these concentrations only suggest pro-apoptotic responses, suggesting that the mechanisms are unlikely to rely on oxidative damage. 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. 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. Each quadrant represents the number of events considered live (lower left), early apoptotic (lower right), or late apoptotic/necrotic (upper right). Membranes were washed, incubated with mouse secondary antibody at a dilution of 1:2000, and imaged as described. Bioactive molecules derived directly from plants, or modeled after plant compounds, continue to be an active area of cancer research. ImageJ. 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. Sharma RA, McLelland HR, Hill KA, Ireson CR, Euden SA, Manson MM, et al. 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. An official website of the United States government. Curcumin and cancer cells: how many ways can curry kill tumor cells selectively? 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. Effects of lycopene on proliferation and death of canine osteosarcoma cells. 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. Ventura JJ, Hbner A, Zhang C, Flavell RA, Shokat KM, Davis RJ. 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]. 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. Within each cell line, means with different letters are significantly different from each other (p<0.05). 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. This increase in intracellular curcumin levels may play a role in the synergy exhibited when using TE and RE in combination.