SAPK mediates doxorubicin-induced differentiation and apoptosis in MCF-7 breast cancer cells. 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. Paclitaxel (Taxol)-induced gene expression and cell death are both mediated by the activation of c-Jun NH2-terminal kinase (JNK/SAPK). Tsai CW, Lin CY, Lin HH, Chen JH. Values are expressed as meanstandard deviation of four independent replicates. CBL carried out the technical experimentation, performed statistical analysis, and was primary author in the manuscript. and transmitted securely. 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. Each blot is a representative of three independent experiments. d Representative histogram of emission intensity in CMT-12 cell line after 24h treatment with DMSO, 3.1g mL1 TE alone, 3.1g mL1 RE alone, or 3.1g mL1 TE+RE combination is shown. [42, 43] Changes in activation of MAPK/ERK were not observed after treatment in any of the cell lines examined. Cells were plated on 60mm tissue culture-treated plates (LPS, Rochester, NY) and incubated in complete medium until 60% confluent. 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 and necrosis was measured after 48h treatment using Annexin-V and 7-AAD staining. official website and that any information you provide is encrypted Background fluorescence and luminescence was measured in wells containing treatments but no cells. Fossey SL, Bear MD, Lin J, Li C, Schwartz EB, Li PK, et al. Within each cell line, means with different letters are significantly different from each other (p<0.05). Learn more 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. 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. 5D). Our results indicate that different tumor types are likely to have a differential response to such interventions. 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). Our data at these concentrations only suggest pro-apoptotic responses, suggesting that the mechanisms are unlikely to rely on oxidative damage. 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. Cells were treated with the indicated concentrations of extracts for 12h followed by determination of intracellular levels of reactive oxygen species using Dihydrorhodamine123 staining. This obstacle may be overcome through the use of combination treatments with other extracts that improve bioavailability or hinder additional pathways [1416]. Gilbert B, Alves LF. Concentrations were chosen based on our prior publication surrounding the effective concentrations for synergy between the two extracts of interest [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. The new PMC design is here! Helmerick EC, Loftus JP, Wakshlag JJ. The https:// ensures that you are connecting to the Our results showed an increase in phosphorylated SAPK/JNK after 12h and 24h of treatment with TE 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]. This anti-oxidant property has been thought to be involved in cell survival and possible resistance to chemotherapeutic intervention [31]. Activated SAPK/JNK increased from 12h to 24h in the C2 cell line (1.8 and 2.1, respectively) and the CMT-12 cell line (1.2 at 12h to 1.5 at 24h) which was not significant over time. 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]. Apoptosis, antioxidant effects, cellular accumulation of curcumin, and perturbation of signaling pathways were assessed. Niedzwiecki A, Roomi MW, Kalinovsky T, Rath M. Anticancer Efficacy of Polyphenols and Their Combinations. 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). 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. Membranes were incubated overnight in primary antibody solutions at a dilution of 1:1000 in TBST on a rocking platform at 4C. Julie Bayle, Email: moc.ninaclayor@elyab.eiluj. 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. Antioxidant effects of turmeric and rosemary extracts in C2, CMT-12 and D17 cell lines. Caspase 3/7 activation was determined as caspase activation per total viable cells for each treatment. This pathway has been implicated in driving cells to apoptosis when faced with environmental stressors such as oxidative stress, inhibition of protein synthesis, changes in the cell-matrix interaction, or signaling from inflammatory cytokines [3941]. (Fig.5B;5B; p<0.0001). Khuda-Bukhsh AR, Das S, Saha SK. Careers. Therefore, the possibility that RE could increase the cellular accumulation of the fluorescent compound curcumin was investigated when these compounds were used in combination. 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. Corri B. Levine, Email: ude.llenroc@64lbc. 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]. Annexin-V 488 conjugate and 7-Aminoactinomycin D (7-AAD) were added to the cell suspensions and incubated for 15m at room temperature. Reported data are expressed as meanstandard deviation of 4 independent replicates. 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. 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. ImageJ. 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]. 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. HHS Vulnerability Disclosure, Help National Library of Medicine 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. 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). 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. Vincent Biourge, Email: moc.ninaclayor@egruoib.tnecniv. Minimal change was seen in the D17 cell lines after treatment with 6.3g mL1 RE, 1.1 at both time points. 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. 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. The effective compounds of interest have been purified from a variety of plants and are used in treating various diseases, including cancer [4]. Li Y, Go VL, Sarkar FH. 2), and Annexin-V staining which increased from 6% to 11% apoptotic cells in the C2 (Fig. 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. Cells were treated for 12h (reactive oxygen species generation), 24h (curcumin accumulation), or 48h (Apoptosis/Necrosis, Cell Cycle). (Fig.1D)1D) cell lines. The residuals of all statistical models were found to be normally distributed therefore parametric statistics were utilized. 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. Next, Caspase-Glo 3/7 Reagent was added to all wells, incubated for 30m at room temperature, and luminescence was measured. 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 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. Lim TG, Lee SY, Huang Z, Lim DY, Chen H, Jung SK, et al. -Actin was used as a loading control for every blot to ensure even loading of samples. Requirement for SAPK-Jnk signaling in the induction of apoptosis by ribosomal stress in REH lymphoid leukemia cells. While these differences were significant, the mild alterations in cell cycle in the D17 and C2 of 510% decrease in the G1/G0 and increase in G2/M phases with RE and less consistent yet similar changes with both TE and RE combined were not considered large enough to continue examining pathways related to cell cycle arrest as observed in prior studies using these cell lines [22]. A similar increase in GMF was also seen when half the concentration of each extract was used in combination (data not shown). All authors have read and accepted the final manuscript. Cells were then treated with medium, DMSO vehicle control, extract alone, or extracts in combination. 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. 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. Caspase 3/7 activation induced by turmeric and rosemary extracts in C2, CMT-12, and D17 cell lines. Shaikh J, Ankola DD, Beniwal V, Singh D, Kumar MN. 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. 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. 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. Wakshlag JJ, Balkman CA, Morgan SK, McEntee MC. 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). 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. Joseph J. Wakshlag, Phone: 607 253-4389, Email: ude.llenroc@73wj. For all flow cytometry experiments, 10,000 events were collected per sample and then gated based on a forward-scatter/side-scatter plot. Wakshlag JJ, Balkman CE. Moore J, Yousef M, Tsiani E. Anticancer Effects of Rosemary (. 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. Baharuddin P, Satar N, Fakiruddin KS, Zakaria N, Lim MN, Yusoff NM, et al. 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. Fluorescence and luminescence was measured using SpectraMax M3 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). U.S. National Institutes of Health, Bethesda, Maryland, USA. Bioactive molecules derived directly from plants, or modeled after plant compounds, continue to be an active area of cancer research. 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). All statistical analyses were performed using JMP Pro (v. 11.2.1; SAS Institute Inc., Cary, NC, USA). 8600 Rockville Pike