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ORIGINAL ARTICLE
Year : 2017  |  Volume : 9  |  Issue : 3  |  Page : 221-229  

Uncaria tomentosa (Willd. ex Schult.) DC (Rubiaceae) sensitizes THP-1 cells to radiation-induced cell death


1 Program in Biomolecular Science, Laurentian University, Sudbury, ON P3E 2C6; Health Sciences North, Sudbury, ON P3E 5J1, Canada
2 Health Sciences North, Sudbury, ON P3E 5J1, Canada
3 Program in Biomolecular Science, Laurentian University, Sudbury, ON P3E 2C6; Health Sciences North, Sudbury, ON P3E 5J1; Division of Medical Science, Northern Ontario School of Medicine, Sudbury, ON P3E 2C6; Health Sciences North Research Institute, Sudbury, ON, P3E 5J1, Canada

Date of Web Publication12-Jul-2017

Correspondence Address:
Robert M Lafrenie
Health Sciences North Research Institute, 41 Ramsey Lake Road, Sudbury, ON P3E 5J1
Canada
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pr.pr_83_16

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   Abstract 

Background: Uncaria tomentosa (Willd. ex Schult.) DC (Rubiaceae), known as Cat's Claw or Uña de gato, is a traditionally used medicinal plant native to Peru. Some studies have shown that U. tomentosa can act as an antiapoptotic agent and enhance DNA repair in chemotherapy-treated cells although others have shown that U. tomentosa enhanced apoptosis. Objective: To determine if treatment with U. tomentosa can significantly enhance cell death in THP-1 cells exposed to ionizing radiation. Materials and Methods: THP-1 monocyte-like cells were treated with ethanolic extracts of U. tomentosa in the presence or absence of bacterial lipopolysaccharide and then exposed to ionizing radiation. Cell proliferation was assessed by MTT and clonogenic assays and the effects on cell cycle measured by flow cytometry and immunoblotting. Changes in cell signaling were determined by immunoblotting and cytokine ELISA and activation of apoptosis measured by caspase activation and DNA fragmentation analysis. Results: Treatment of THP-1 cells with U. tomentosa had a small effect on cell proliferation. However, when the U. tomentosa-pretreated cells were also subjected to 5–9 Gy ionizing radiation, they showed a significant decrease in cell proliferation and increased cellular apoptosis as measured by DNA fragmentation and caspase activation. Treatment with U. tomentosa also decreased the expression of Cyclin E and Cyclin B, key regulators of normal cell cycle progression, and decreased the phosphorylation of various stress-activated, cell survival proteins including p38, ERK, and SAP/JNK kinase. Conclusions: These results suggest that U. tomentosa could be useful in enhancing cell death following anticancer therapies including ionizing radiation.

Keywords: Cell signaling, cell survival, cytokine, monocyte, Uncaria tomentosa, X-rays


How to cite this article:
Allen L, Buckner A, Buckner CA, Cano P, Lafrenie RM. Uncaria tomentosa (Willd. ex Schult.) DC (Rubiaceae) sensitizes THP-1 cells to radiation-induced cell death. Phcog Res 2017;9:221-9

How to cite this URL:
Allen L, Buckner A, Buckner CA, Cano P, Lafrenie RM. Uncaria tomentosa (Willd. ex Schult.) DC (Rubiaceae) sensitizes THP-1 cells to radiation-induced cell death. Phcog Res [serial online] 2017 [cited 2017 Nov 21];9:221-9. Available from: http://www.phcogres.com/text.asp?2017/9/3/221/210327

Summary

  • Treatment of THP-1 cells with Uncaria tomentosa increases their susceptibility to X-rays. The combination of Uncaria tomentosa and X-ray exposure strongly inhibits cell signaling and promotes apoptosis.




Abbreviations Used: LPS: Lipopolysaccharide, TNF: Tumor necrosis factor: IL-1, Interleukin-1: SDS: Sodium dodecylsulphate, TBS: Tris-buffered saline.


   Introduction Top


Uncaria tomentosa (Willd. ex Schult.) DC (Rubiaceae) or Uña de Gato is a Peruvian plant that the Ashaninka Indians of South America have used for generations to treat various medical ailments including arthritis, cancer, and premenstrual syndrome.[1],[2] The woody vine is prepared and served in a hot water tea-like concoction. The discovery that U. tomentosa treatment of monocytes can inhibit the lipopolysaccharide (LPS)-dependent expression of tumor necrosis factor-alpha (TNF-α) highlights its potential as a natural anti-inflammatory agent.[3],[4],[5],[6],[7],[8],[9]

We previously showed that treatment of THP-1 monocyte-like cells with U. tomentosa decreases LPS-dependent production of TNF-α by more than 50% while augmenting the production of interleukin 1 beta (IL-1β) by more than 25%.[9] Treatment with U. tomentosa was shown to inhibit the LPS-dependent activation of all AP-1 subunits and to inhibit p65 and the classical nuclear factor-kappa B (NF-κB) pathway while promoting activation of the p52 nonclassical NF-κB pathway.[10] Inhibition of the p50 subunit of NF-κB, with SN50, partially restored TNF-α secretion in U. tomentosa-treated cells but did not affect IL-1β secretion suggesting U. tomentosa is more specific for the classical NF-κB pathway.[10] Inhibition of the classical NF-κB pathway may be important for the prevention and treatment of cancer [11],[12] while elevated p52 can enhance cell survival without promoting tumourigenesis.[13],[14],[15] Treatment with U. tomentosa has been shown to improve outcomes for animals or patients treated with chemotherapeutics or radiation. In some studies, this improvement was associated with a decrease in immune responsiveness to therapy [16],[17],[18],[19],[20] while other studies showed the benefit did not involve immune function.[21],[22],[23] Some studies have even shown that U. tomentosa can enhance cellular recovery following DNA damage by promoting the repair of both single-strand and double-strand DNA breaks.[24],[25],[26]

In the current studies, we report that the treatment of THP-1 cells with U. tomentosa sensitized them to ionizing radiation-induced cell death. Treatment of THP-1 cells with U. tomentosa alone or in combination with LPS had only modest effects on cell viability. We had previously shown that treatment with LPS-promoted activation of cell signaling pathways associated with cell survival but that inclusion of U. tomentosa could inhibit some of these pathways.[9] However, treatment with ionizing radiation following U. tomentosa pretreatment inhibited cell signaling, inhibited the expression of cyclin E and cyclin B, prevented accumulation of the cells at any of the cell cycle checkpoints, and increased the frequency of apoptotic cell death.


   Materials and Methods Top


Cell culture and treatment

THP-1 cells,[27] obtained from the American Type Culture Collection (ATCC Manassas, VA, USA), were maintained in RPMI 1640 media supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Login, Utah) and 1% antibiotic/antimycotic solution (Invitrogen, Burlington, ON, Canada) in 5% CO2 at 37°C. For all experiments, the cells were treated with suspending media or 20–160 μg/ml U. tomentosa extract for 24 h. In some experiments, the cells were also co-treated with 2.5 μg/ml bacterial LPS (Escherichia coli Serotype 0127, Sigma-Aldrich Chemical, St. Louis, MO, USA) for 24 h. The cells were then treated with 0–15 Gy ionizing radiation using a Gulmay Medical X-ray machine (Scarborough, ON, Canada) and collected for analysis after various incubation times.

Preparation and characterization of Uncaria tomentosa extracts

U. tomentosa (Willd.) DC (Rubiaceae) was obtained as a powdered preparation of the plant's root as identified and provided by Dr. Rosaria Rojas, Lima. Peru. Extracts were prepared through exhaustive percolation with 95% ethanol (100 mg/ml to create the stock concentration) as described.[9] Different preparations of U. tomentosa were used and compared by high-performance liquid chromatography (HPLC) to normalize for the quantity of marker components. This resulted in the use of two different final concentrations based on the amount of ground root material used to create the extract. The U. tomentosa extract was analyzed using HPLC on a Breeze 2 chromatography system (Waters Inc., Toronto, ON, Canada) fitted with a 4.6 mm × 100 mm Sunfire C18 column 3.5 μm resin [Figure 1]. The solvents used were: (A) 60 volumes 10 mM phosphate buffer, pH 6.6; 20 volumes acetonitrile; and 20 volumes methanol; and (B) 30 volumes 10 mM phosphate buffer, pH 6.6; 25 volumes acetonitrile; and 25 volumes methanol. The solvent gradient was applied at 1 ml/min starting with 100% A and finishing with 100% B over 40 min followed by 10 min with 100% B. The marker components, corresponding to uncarine C, uncarine D, uncarine E, mitraphyline, isomitraphyline, and rhynchophylline, were detected at 245 nm and compared to a group of standards (Planta Analytical) and were very similar to our previous preparations.
Figure 1: High-performance liquid chromatography analysis of Uncaria tomentosa extracts. The root powder was extracted with 95% ethanol and subjected to high-performance liquid chromatography as described. The identification of the indicated peaks is based on the retention times of the indicated standard compounds analyzed under the same conditions

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Microscopic visualization of THP-1 cells

THP-1 cells were treated with media U. tomentosa and/or LPS for 24 h and then exposed to ionizing radiation, as indicated. The cell morphology was examined each day using a Zeiss-Axiophot 100 phase-contrast microscope (Zeiss Canada, Toronto, ON) and images obtained using Northern Eclipse software.

Quantitation of cytokines

THP-1 cells (106/ml) were treated with media U. tomentosa and/or LPS for 24 h and then exposed to ionizing radiation, as indicated. Following 24 h incubation, the conditioned media were collected and analyzed for the amount of secreted IL-1β and TNF-α using ELISA kits from R and D Systems (Minneapolis, MN, USA).[10] The experiments were performed in triplicate and analyzed using a Students' t-test. Similar results were obtained for three independent experiments.

Analysis of cell proliferation using the MTT assay

THP-1 cells (103 cells/well) were plated on 96 well plates in quintuplicate and treated with media, U. tomentosa and/or LPS for 24 h and then exposed to ionizing radiation, as indicated. On each day of culture, 5 pg of thiazolyl blue tetrazolium bromide (Sigma M2128) was added to each well and incubated at 37°C for 4 h. The media were removed, the MTT crystals dissolved in 200 ml of dimethyl sulfoxide, and the absorbance read at 540 nm each day. Comparisons in the values between treatment conditions were evaluated using a one-way ANOVA. The percent inhibition was reported as the mean ± standard deviation (SD) for three independent experiments.

Clonogenic assays

THP-1 cells (106/ml) were treated with media U. tomentosa and/or LPS for 24 h and then exposed to ionizing radiation, as indicated. The treated cells (105 per sample) were suspended in methylcellulose culture media (20% FBS and 2.8% methylcellulose in Iscove's Modified Dulbecco's Media [Invitrogen]) and 3 ml plated on six well plates. Following 14 days of incubation at 37°C and 5% CO2, to allow proliferation of the cells into colonies, a minimum of five microscopic fields/sample were counted. Only those colonies that contained more than fifty cells were counted. The experiment was performed in triplicate, and the results show the mean ± SD for the combination of three independent experiments analyzed using a Students' t-test.

Cell cycle analysis by flow cytometry

THP-1 cells (106/ml) were treated with media U. tomentosa and/or LPS for 24 h, exposed to ionizing radiation, and then incubated for 4–24 h, as indicated. The cells were fixed by suspension in 70% isopropanol at −20°C, and nuclear DNA was stained by incubation in 0.1% propidium iodide in methanol as described.[28] The fluorescent cell population profiles were created on a Beckman-Coulter Epics Elite flow cytometer (Beckman-Coulter, Mississauga, ON, Canada). The percent cells reported in each phase are the mean of three independent experiments.

Protein isolation and immunoblot analysis

THP-1 cells (106/ml) were treated with media U. tomentosa and/or LPS for 24 h, exposed to ionizing radiation, and incubated for 24 h, as indicated. Cells were collected by centrifugation and lysed by incubation in RIPA buffer (1% Triton X-100, 0.5% sodium dodecyl sulfate [SDS], 0.5% sodium deoxycholate, 150 mM NaCl, 50 mM Tris–HCl, and pH 7.6), supplemented with 5 mM NaF, 2 mM sodium orthovanadate (Sigma–Aldrich), and protease inhibitors (Roche, Laval, QB).[29] The proteins (30 mg/lane) were subjected to electrophoresis on 10% polyacrylamide gels containing SDS and transferred to nitrocellulose membranes: four identical blots were created from each group of samples and probed independently. The membranes were blocked by incubation in 5% bovine serum albumin (BSA) in Tris-buffered saline, pH 7.5, and 0.1% Tween-20 (TBST) and then incubated with antibodies against phospho-ERK1/2 (titer 1:1000, Cell Signaling Lake Placid, NY, USA), phospho-AKT (titer1:1000, Cell Signaling), phospho-SAP/JNK (titer 1:1000, Cell Signaling), P38 (titer1:1000, Cell Signaling), Cyclin B (titer 1:250, Santa Cruz, Biotechnology, Santa Cruz, CA), Cyclin E (titer 1:500, Santa Cruz Biotechnology), poly ADP-ribose polymerase (PARP) (titer 1:250, Santa Cruz Biotechnology), or caspase-3 (titer 1:1000, Cell Signaling) in 1% BSA and TBST. The blots were washed and then incubated in appropriate secondary antibody - horseradish peroxidase (HRP) conjugates in 1% BSA and TBST. HRP was detected by incubation in ECL reagent (Pierce Chemical Co. Rockford, IL, USA) and exposed to ECL X-ray film (Amersham, Baie-d'Urfe, QC). Densitometry of the signal intensity was determined for each blot, normalized to the intensity of β-tubulin staining in a duplicate blot, and the mean relative intensity (to the control set at 1.0) reported for three independent blots.

DNA laddering assay

THP-1 cells (3 × 105/ml) were treated with media, U. tomentosa and/or LPS for 24 h, exposed to ionizing radiation, and incubated for 6 h. The cells were collected by centrifugation at 400 ×g for 10 min and the pellet lysed by incubation in TE/Triton buffer (0.2% Triton X-100, 10 mM Tris, pH 8.0, and 1 mM ethylenediaminetetraacetic acid) on ice for 10 min.[30] The lysate was subjected to centrifugation at 13,000 ×g for 15 min at 4°C. The low molecular weight DNA-containing supernatant was transferred to a fresh microcentrifuge tube treated sequentially with 15 μg/ml RNase A (Sigma-Aldrich) for 1 h at 37°C and 0.025% SDS and 0.10 μg/ml proteinase K for 1 h at 50°C. The DNA was precipitated using cold isopropanol and the pellet resuspended in gel loading buffer (0.01% bromophenol blue and 20% sucrose in TBE) and subjected to electrophoresis on a 2% agarose-TBE gel containing 0.001% ethidium bromide.

Nuclear morphology

THP-1 cells (106/ml) were treated with media U. tomentosa and/or LPS for 24 h and then exposed to ionizing radiation, as indicated. The cells were collected at 24, 48, and 72 h postirradiation, stained for 10 min in 2.5 μg/ml Hoechst stain, fixed in 3% paraformaldehyde in PBS, pH 7.4, and examined using a Zeiss–Axiophot 100 fluorescence microscope and Northern Eclipse software.


   Results Top


Effect of Uncaria tomentosa and ionizing radiation on cell growth and morphology

The morphology of THP-1 cell cultures treated with U. tomentosa and LPS for 24 h and then exposed to ionizing radiation and cultured for 48 h is shown in [Figure 2]a. Exposure to 9 Gy ionizing radiation had very little effect on the morphology of THP-1 cells. The cells were normal in appearance and were able to divide and replicate even after being exposed to doses of ionizing radiation up to 15 Gy (not shown). THP-1 cells treated with 160 mg/ml U. tomentosa were smaller and fewer in number but were able to survive and replicate. Treatment of THP-1 cells with both LPS and U. tomentosa resulted in fewer cells but those that survived appeared normal and were able to replicate. We have previously shown that LPS treatment alters THP-1 cell signaling,[10] and it has been shown that LPS can change susceptibility to cell death in response to treatment with other agents. Cells treated with U. tomentosa, in the presence or absence of LPS, and then exposed to 5 or 9 Gy ionizing radiation, demonstrated significant cell fragmentation, and loss of viability.
Figure 2: Treatment with Uncaria tomentosa and ionizing radiation inhibits cell proliferation. (a) Microscopy of THP-1 cell suspensions treated with Uncaria tomentosa, lipopolysaccharide, and/or ionizing radiation for 48 h. (b) The relative number of THP-1 cells (mean ± standard deviation, n = 3) was determined using an MTT assay. Each panel shows cells treated with Uncaria tomentosa (the gray line shows the number of untreated THP-1 cells). The different panels show cells that were also untreated or treated with lipopolysaccharide, 9 Gy radiation, or 9 Gy radiation and lipopolysaccharide

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The survival of THP-1 cells after exposure to ionizing radiation and treatment with U. tomentosa and/or LPS was determined using MTT assays. Relative cell number was determined each day and growth curves plotted [Figure 2]b. Untreated THP-1 cells showed an increase in cell number for the first 5 days when it reached a plateau. Treatment of cells with 20 μg/ml U. tomentosa had very little effect on cell growth, while treatment with 160 μg/ml U. tomentosa delayed cell growth by 58% ± 11% on day 6 (based on three independent experiments), although by day 8 treated cell number reached >80% of control. Treatment with both LPS and 160 μg/ml U. tomentosa significantly reduced THP-1 growth although the extent was variable between experimental replicates (not shown). Treatment of THP-1 cells with LPS and 9 Gy ionizing radiation also reduced THP-1 cell growth by 16% ± 9% by day 6. The addition of 20 μg/ml U. tomentosa to the radiation-treated cells strongly inhibited cell growth by 36% ± 14% while inclusion of 160 μg/ml U. tomentosa almost completely inhibited growth.

THP-1 cell growth as determined by clonogenic assay

The ability of single cells to proliferate into colonies was measured using clonogenic, colony-forming assays after treatment with U. tomentosa, LPS, and/or ionizing radiation. Treatment of THP-1 cells with U. tomentosa decreased colony formation: treatment with 80 μg/ml U. tomentosa decreased colony formation by 40% ± 22% (based on three independent experiments), and treatment with 80 μg/ml U. tomentosa and LPS decreased colony formation by 46% ± 12% [Figure 3]. Treatment with 5–15 Gy ionizing radiation also decreased colony formation by >35% which was further decreased with the addition of LPS. Treatment with both U. tomentosa and ionizing radiation resulted in very large decreases in colony formation, and at higher doses of U. tomentosa (80 and 160 μg/ml), colony formation was completely inhibited [Figure 3].
Figure 3: Effect of Uncaria tomentosa and ionizing radiation on clonogenic growth. THP-1 cells were treated with Uncaria tomentosa extract in the presence of absence or lipopolysaccharide for 24 h and then with ionizing radiation. The treated cells were suspended in methylcellulose growth media and allowed to grow into colonies. The number of colonies (mean ± standard deviation for the combination of three independent experiments) was counted per high power field

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Cell cycle analysis of THP-1 cells treated with Uncaria tomentosa and ionizing radiation

To determine if the effects on cell growth were mediated through alterations in cell cycle progression, flow cytometry was performed on unsynchronized THP-1 cells stained with propidium iodide. As shown in [Figure 4]a, there were no significant changes in cell cycle parameters following treatment with U. tomentosa for 48 h. Similar results were seen at 6, 12, 24, and 36 h posttreatment in the presence and absence of LPS treatment (not shown). Exposure of THP-1 cells to only 9 Gy ionizing radiation showed a small increase in the number of cells in G2 (but not the expected, strong G2 arrest usually induced by ionizing radiation). The combination of 20 or 100 μg/ml U. tomentosa and ionizing radiation increased the sub-G1 population of cells by 2.0 or 3.5 fold (P < 0.05), compared to untreated controls (based on average values for three independent experiments). The addition of LPS slightly increased the number of cells in the sub-G1 DNA peak (not shown). In addition, the treated THP-1 cells showed a small decrease in the proportion of cells in the G2/M phase and S phases.
Figure 4: Effect of Uncaria tomentosa and ionizing radiation on cell cycle and cyclin expression. (a) THP-1 cells were treated with Uncaria tomentosa for 24 h and/or ionizing radiation for a further 24 h and then stained with propidium iodide. The average percent of cells (n = 3 experiments) in each of the cell cycle peaks is shown. (b) Immunoblot analysis of cyclin B and cyclin E expression levels for THP-1 cells treated with Uncaria tomentosa and/or lipopolysaccharide for 24 h and/or 9 Gy ionizing radiation for a further 24 h. The relative difference in band intensity, normalized for β-tubulin expression, was averaged for three experiments and is shown below each band

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To identify potential cell cycle targets of U. tomentosa, the effects on cyclin expression were examined by immunoblot analysis. Treatment of THP-1 cells with LPS increased cyclin E expression by 1.2 fold (based on densitometry of three independent blots) but had little effect on cyclin B [Figure 4]b. Treatment of THP-1 cells with only U. tomentosa caused a small decrease in the amount of cyclin E (0.8 fold) and cyclin B (0.7 fold) expression, and treatment with only 9 Gy ionizing radiation caused an increase in cyclin B (1.8 fold) and a small decrease in cyclin E (0.8 fold). However, in cells exposed to both ionizing radiation and U. tomentosa treatment, cyclin B and cyclin E levels were strongly downregulated by 20 μg/ml of U. tomentosa and completely blocked by 100 μg/ml of U. tomentosa.

Effects of treatment and activation on cell signaling

To identify potential mechanism by which treatment with U. tomentosa and ionizing radiation affected cell proliferation, we examined the activation of survival pathway proteins. Treatment of THP-1 cells with LPS increased the activation of the MAP kinase pathways as shown by enhanced phosphorylation of the p38, pERK, and SAP/JNK kinase proteins and the phosphorylation of AKT [Figure 5]. Treatment with U. tomentosa decreased p-Akt, pERK, and SAP/JNK phosphorylation in a dose-dependent manner both in the presence and absence of LPS. Treatment of THP-1 cells with 20 μg/ml and 100 μg/ml of U. tomentosa inhibited p38 phosphorylation although 100 μg/ml of U. tomentosa was required to inhibit LPS-dependent p38 phosphorylation. Exposure to 9 Gy ionizing radiation resulted in a general inhibition of MAP kinase and AKT phosphorylation. In cells treated with ionizing radiation, U. tomentosa was able to further inhibit ERK, p38, and AKT with the same pattern as nonirradiated cells. However, SAP/JNK phosphorylation was activated in cells exposed to ionizing radiation and LPS, both in the presence and absence of U. tomentosa.
Figure 5: Effect of Uncaria tomentosa and ionizing radiation on the activation of stress signaling molecules. Immunoblot analysis of pERK, pAKT, pJNK, and pP38, expression levels for THP-1 cells treated with Uncaria tomentosa and/or lipopolysaccharide for 24 h and/or 9 Gy ionizing radiation for a further 24 h. The relative difference in band intensity, normalized for β-tubulin expression, was averaged for three experiments and is shown below each band

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Cytokine expression as determined by ELISA assay

The activation of cell signaling pathways in response to ionizing radiation was also examined by measuring the effects on cytokine secretion. TNF-α and IL-1β secretion was measured in the conditioned media of THP-1 cells treated with U. tomentosa, LPS, and/or ionizing radiation. Consistent with previous results'[10] treatment with LPS increased the secretion of both TNF-α and IL-1β (P < 0.01) [Figure 6]. Exposure to 5 or 9 Gy ionizing radiation blocked LPS-dependent TNF-α secretion by 50% (based on three independent experiments). Treatment with 40, 80, or 160 μg/ml U. tomentosa almost completely inhibited the LPS-dependent secretion of TNF-α (P < 0.01) in the presence or absence of ionizing radiation. In contrast, the secretion of IL-1β was augmented by treatment with U. tomentosa. Treatment of cells with both LPS and 80 μg/ml U. tomentosa increased IL-1β secretion by 2.1 fold (from three independent experiments). However, exposure to ionizing radiation caused a dose-dependent decrease in IL-1β secretion by THP-1 cells. In cells treated with all doses of U. tomentosa and LPS, ionizing radiation significantly blocked IL-1β secretion by 25%–70% (P < 0.05).
Figure 6: Effect of Uncaria tomentosa and ionization radiation on tumor necrosis factor-alpha and interleukin 1 beta secretion. THP-1 cells were treated with Uncaria tomentosa and/or lipopolysaccharide for 24 h and/or ionizing radiation and then incubated in serum-free media for a further 24 h. The amount of cytokine (mean + standard deviation, n = 3) was measured in the conditioned media using ELISA analysis for tumor necrosis factor-alpha and interleukin 1 beta

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Apoptosis

To determine if the decrease in cell number following exposure to U. tomentosa, LPS, and ionizing radiation was due to an increase in cell death, we examined classical markers of apoptosis including caspase cleavage, PARP cleavage, and DNA fragmentation. Caspase-3 cleavage, which correlates with increased apoptosis, was most significant in cells treated with 100 μg/ml U. tomentosa, LPS, and ionizing radiation for 24 h [Figure 7]a. PARP cleavage was present in cells treated with 100 μg/ml U. tomentosa or combinations of U. tomentosa, LPS, and/or ionizing radiation, U. tomentosa and LPS, and U. tomentosa, LPS, and ionizing radiation for 24 h.
Figure 7: Treatment with Uncaria tomentosa and ionizing radiation promotes cellular apoptosis. THP-1 cells were treated with Uncaria tomentosa and/or lipopolysaccharide for 24 h and/or ionizing radiation and then incubated for a further 24 h. (a) Cell lysates were obtained and duplicate blots probed for caspase-3 or PARP with antibodies which detect both the cleaved and uncleaved forms. (b) DNA fragments were isolated from the cells, precipitated, and then subjected to electrophoresis on agarose gels stained with ethidium bromide. M shows the DNA marker. (c) Treated cells stained with Hoechst 33342 and fixed on glass coverslips were visualized on a fluorescence microscope

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THP-1 cells treated for 24 h with a combination of 100 μg/ml U. tomentosa, LPS, and ionizing radiation showed a much greater amount of DNA laddering than treatment with any of the individual agents [Figure 7]b. This confirmed the flow cytometry results that showed treatment of THP-1 cells with 100 μg/ml U. tomentosa, LPS, and ionizing radiation resulted in 3-fold increase in cells in the sub-G1 peak [Figure 4]a. In addition, the nuclear morphology of Hoechst-stained THP-1 cells showed that the nuclei of THP-1 cells treated with only LPS or 9 Gy ionizing radiation appeared intact and that treatment with all of U. tomentosa, LPS, and ionizing radiation was required to promote significant nuclear fragmentation typical of apoptosis [Figure 7]c.


   Discussion Top


Patients frequently include natural products, such as U. tomentosa, in treatment regimens for a variety of medical conditions, including inflammatory diseases and cancer.[1],[2] We have previously shown that U. tomentosa inhibits the expression of the potent pro-inflammatory cytokine TNF-α (and augments IL-1β) and differentially regulates activation of NF-κB transcription factor subunits suggesting that U. tomentosa elicits its cellular response through the NF-κB-mediated pathway.[10] The inhibition of NF-κB has been shown to enhance apoptosis in malignant cells treated with chemotherapy and/or radiation treatment.[11],[20],[31],[32],[33],[34] Further, treatment with U. tomentosa was shown to induce apoptosis in sarcoma, medullary thyroid, and breast cancer cells.[35],[36],[37],[38],[39] However, others have reported that U. tomentosa can enhance cell survival after radiation-induced DNA damage.[25],[26]

The results of the current experiments showed that treatment of THP-1 cells with U. tomentosa (and LPS) followed by exposure to ionizing radiation significantly enhanced cellular apoptosis. We discovered that THP-1 cells were extremely resistant to exposure to ionizing radiation or to treatment with U. tomentosa. The cells remained viable after exposure to 15 Gy radiation or following treatment with 20 μg/ml U. tomentosa (treatment with 160 μg/ml delayed growth for several days). However, when the THP-1 cells were pretreated with U. tomentosa and then treated with ionizing radiation, they showed a profound decrease in cell growth and an increase in apoptosis which was further enhanced by the addition of LPS. The increase in apoptosis was measured as cleavage and activation of caspase-3, cleavage of PARP, and DNA laddering (nucleosome laddering was best seen at early time points and DNA appeared as “smears” at later time points, likely due to the ability of THP-1 monocyte-like cells to phagocytose and further degrade any apoptotic body generated during the experiment).

Combining U. tomentosa treatment and ionizing radiation had a potent effect on cell signaling pathways which contribute to apoptosis. Treatment of THP-1 cells with both U. tomentosa and ionizing radiation had a much stronger effect on blocking activation of ERK, p38, and AKT phosphorylation and on inhibiting cytokine expression. In addition, LPS treatment appeared to increase the sensitivity of THP-1 cells to cell death in response to U. tomentosa and/or ionizing radiation. Treatment of THP-1 cells with U. tomentosa inhibited the phosphorylation of ERK, p38, and Akt (and decreased cyclin B and E expression), and this was exaggerated by LPS treatment. Although LPS treatment was shown to promote IL-1β and TNF-α secretion, likely through activation of NF-kB,[10] secretion of TNF-α was potently inhibited by U. tomentosa consistent with our previous results.[9],[10] Previous studies showed ionizing radiation alone can activate TNF-α and IL-1β through activation of NF-κB.[40] However, our studies showed that ionizing radiation only marginally enhanced cytokine expression. The most significant effect of ionizing radiation in this study was its ability to inhibit phosphorylation of ERK, p38, and Akt and decrease secretion of both IL-1β and TNF-α induced by LPS. Therefore, THP-1 cells treated with all of U. tomentosa, ionizing radiation, and LPS showed strong inhibition of ERK, p38, and Akt and the highest levels of apoptosis. Previous results have shown that inhibition of NF-kB can inhibit prosurvival and antiapoptotic pathways [15],[41],[42],[43] and that inhibition of the MAP kinase and AKT survival pathways can result in the enhancement of cell death following exposure to ionizing radiation.[44] Exposure of THP-1 cells to only ionizing radiation inhibited SAP/JNK phosphorylation but treatment with ionizing radiation and U. tomentosa preserved LPS-dependent SAP/JNK phosphorylation. This is consistent with previous studies that showed a combination of pERK inhibition and SAP/JNK activation could activate caspase-3 and promote apoptosis.[45],[46],[47]

THP-1 cells treated with ionizing radiation do not show the typical arrest of the cell cycle at the G2/M phase [40],[48] and show only a small accumulation of cells in G2 6–8 h posttreatment. Pretreatment with U. tomentosa completely blocked even this small accumulation in G2 and cells treated with U. tomentosa and ionizing radiation showed an increase in the number of cells in the sub-G1 peak which correlates to cellular apoptosis. An examination of cyclin E (which is enhanced in cells in G1[49]) and cyclin B (which is enhanced in G2/M [50]) expression supports the idea that treatment of THP-1 cells with both U. tomentosa and ionizing radiation is required to have significant effects on cell cycle regulation and apoptosis. Treatment of THP-1 cells with U. tomentosa had a minor effect on cyclin E and cyclin B suggesting normal progression through the cell cycle and cells treated with only ionizing radiation showed a small accumulation of cyclin B which indicates an accumulation at G2. However, exposure to ionizing radiation and U. tomentosa completely inhibited the expression of both cyclin E and cyclin B. The lack of cyclin E and B protein expression combined with the increase in the sub-G1 peak suggested that the THP-1 cells did not complete the mitotic cycle and underwent apoptosis.


   Conclusion Top


We previously showed that U. tomentosa has the potential to be an effective anti-inflammatory agent and can inhibit TNF-α secretion and AP-1 and NF-κB activation in THP-1 cells.[10] We now show that treatment with U. tomentosa can promote radiation-induced apoptosis in THP-1 cells. While treatment of THP-1 cells with 20 μg/ml U. tomentosa or 9 Gy ionizing radiation did not significantly change cell growth, exposure to both U. tomentosa and ionizing radiation significantly decreased cell growth and promoted apoptosis. The observation that even high doses of U. tomentosa have relatively low toxicity until combined with ionizing radiation suggest that it may have some promise in future studies as a therapeutic.

Financial support and sponsorship

This study was financially supported by Northern Cancer Foundation (Sudbury); Cancer Therapeutics Research Institute (Sudbury).

Conflicts of interest

There are no conflicts of interest.

 
   References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]



 

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