Treatments which induced a statistically significant change from DMSO within each cell cycle phase are indicated. 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.
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. Shaikh J, Ankola DD, Beniwal V, Singh D, Kumar MN.
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 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].
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. 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).
government site. Membranes were incubated overnight in primary antibody solutions at a dilution of 1:1000 in TBST on a rocking platform at 4C.
Next, Caspase-Glo 3/7 Reagent was added to all wells, incubated for 30m at room temperature, and luminescence was measured.
Our data at these concentrations only suggest pro-apoptotic responses, suggesting that the mechanisms are unlikely to rely on oxidative damage. Markers of apoptosis, antioxidant capabilities, and changes in the activation of common cell signaling pathways were analyzed after treatment.
The effects of these two compounds in combination have been examined in acute myeloid leukemia cells and breast cancer cells [17, 18], showing synergy in anti-proliferative effects and increased pro-apoptotic signaling. The C2 (a), CMT-12 (b), and D17 (c) cell lines were treated with the indicated concentration of extracts for 24h and then cellular accumulation of curcumin was quantified by flow cytometry.
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.
All treatments were compared to DMSO vehicle control.
After 24h treatment, cells were detached with Accumax dissociation solution (Innovative Cell Technologies), collected and centrifuged for 10m at 500 rcf at 4C. The influence of natural products upon drug discovery. 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.
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.
Effects and synergy of feed ingredients on canine neoplastic cell proliferation.
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. 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.
Generally, these differences may be due to the lower concentrations utilized in our experiments compared to the prior studies. Requirement for SAPK-Jnk signaling in the induction of apoptosis by ribosomal stress in REH lymphoid leukemia cells.
Quantitative cellular uptake, localization and cytotoxicity of curcumin in normal and tumor cells.
Douglass BJ, Clouatre DL.
Unfortunately, the use of TE in vivo has been limited by its poor bioavailability and efforts are still underway to increase the absorption and bioavailability of the curcuminoids found in this extract [13].
6) were detected in the three cancer cell lines.
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. Evaluation of the protective effects of all-trans-astaxanthin on canine osteosarcoma cell lines. In our previous study, RE worked in a synergistic manner with TE to decrease cellular proliferation when used in combination.
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. 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. Corri B. Levine, Email: ude.llenroc@64lbc. Carnosic acid inhibits the growth of ER-negative human breast cancer cells and synergizes with curcumin. 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.
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. 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). The cell pellet was washed twice with 1% FBS in PBS, filtered, and resuspended in 70% cold ethanol for overnight fixation.
All authors have read and accepted the final manuscript.
Li J, Xiang S, Zhang Q, Wu J, Tang Q, Zhou J, et al. Synergy in plant medicines. The residuals of all statistical models were found to be normally distributed therefore parametric statistics were utilized. Tong L, Chuang CC, Wu S, Zuo L. Reactive oxygen species in redox cancer therapy. The research leading to these results was supported by Royal Canin SAS. Concentrations were chosen based on our prior publication surrounding the effective concentrations for synergy between the two extracts of interest [11].
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].
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.
Digital images were captured using an imaging system (Biospectrum 410; UVP, Upland, CA, USA). Cells were treated the following day with DMSO vehicle control, 6.3g mL1 extract alone, or 3.1g mL1 each extract in combination. Apoptosis, Canine cancer, Mammary carcinoma, Osteosarcoma, Mastocytoma, Curcumin, Rosemary, {"type":"entrez-nucleotide","attrs":{"text":"DA251471","term_id":"78448130","term_text":"DA251471"}}. 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.
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].
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. 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]. 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]. 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. 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.
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. Shehzad A, Lee YS.
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 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.
National Library of Medicine
CBL carried out the technical experimentation, performed statistical analysis, and was primary author in the manuscript.
Chemical genetic analysis of the time course of signal transduction by JNK.
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Ravindran J, Prasad S, Aggarwal BB.
[42, 43] Changes in activation of MAPK/ERK were not observed after treatment in any of the cell lines examined.
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].
Careers. Lee LF, Li G, Templeton DJ, Ting JP. Moore J, Yousef M, Tsiani E. Anticancer Effects of Rosemary (. Cells were plated on 60mm tissue culture-treated plates (LPS, Rochester, NY) and incubated in complete medium until 60% confluent. 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. Effects of lycopene on proliferation and death of canine osteosarcoma cells.
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 effective compounds of interest have been purified from a variety of plants and are used in treating various diseases, including cancer [4].
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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 site is secure. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Berginc K, Trontelj J, Basnet NS, Kristl A. Physiological barriers to the oral delivery of curcumin. Upon further examination, it was found that RE treatment caused a significant increase in the cellular accumulation of curcumin by approximately 30% in the C2 and D17 cell lines, and by 4.8-fold in the CMT-12 cell line.
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. 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).
In addition, sustained activation and signaling through the SAPK/JNK pathway may play a role in this cell lines increased sensitivity to apoptosis.
Molecular mechanisms of curcumin action: signal transduction. Ramos S. Effects of dietary flavonoids on apoptotic pathways related to cancer chemoprevention.
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).
The
Within each cell line, means with different letters are significantly different from each other (p<0.05).
Bioactive molecules derived directly from plants, or modeled after plant compounds, continue to be an active area of cancer research. 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).
Although there were similar increases in SAPK/JNK activation with TE and dual treatment of TE/RE (half doses) in C2 and D17 cell lines, these were not significantly different from DMSO control at 12h or 24h incubation time points. Bogoyevitch MA, Ngoei KR, Zhao TT, Yeap YY, Ng DC.
Cells were treated for 12h (reactive oxygen species generation), 24h (curcumin accumulation), or 48h (Apoptosis/Necrosis, Cell Cycle). 2), and Annexin-V staining which increased from 6% to 11% apoptotic cells in the C2 (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. Changes in densitometry compared to DMSO control with significance of p<0.05 represented by *.
The new PMC design is here! 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.
