Tehranolide inhibits cell proliferation via calmodulin inhibition, PDE, and PKA activation
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Tehranolide, natural sesquiterpene lactone with an endoperoxide group, has been shown to inhibit cell growth in cancer cells. Tehranolide was purified from Artemisia diffusa. To detect cell viability and proliferation, MTT assay was performed. In order to determine the role of tehranolide on calmodulin (CaM) structure and activity, its effects were evaluated with fluorescence emission spectra and CaM-mediated activation of phosphodiesterase (PDE1), in comparison with artemisinin. In fact, PDE1 inhibition, cAMP accumulation, and cAMP-dependent protein kinase A (PKA) activation were examined. The inhibitory effect of tehranolide on CaM structure is more than artemisinin. The kinetic analysis of tehranolide–CaM interaction has shown that this agent competitively inhibited the activation of PDE1 without affecting Vmax. Tehranolide increased Km value in higher amounts compared with artemisinin. Moreover, tehranolide had a cytotoxic effect on K562 cell line but not on normal human lymphocytes. Additionally, PDE inhibition and consequent cAMP accumulation and PKA activity were required for inhibiting cancer cell growth by tehranolide. Our results show that tehranolide significantly reduces cell proliferation in a time and dose-dependent manner in K562 cells via CaM inhibition, following PDE inhibition, cAMP accumulation, and consequent PKA activity.
KeywordsTehranolide Calmodulin Phosphodiesterase cAMP PKA
cAMP-dependent protein kinase A
Cell proliferation is regulated by a complex array of signaling pathways. cAMP acts as an important second messenger in many pathways regulating cell growth, differentiation, and apoptosis . The elevation of cAMP subsequently activates cAMP-dependent PKA that can phosphorylate various proteins, thus regulating a cascade of transcriptional events implicated in the inhibition of cancer cell growth [2, 3, 4]. PDEs degrade cAMP and promote the return of PKA to its inactive tetrameric state . PDE is an isozymic family composed of many different types, grouped into seven broad gene families including Ca2+CaM-dependent (PDE1) .
PDE inhibitor-induced apoptosis is selective towards malignant cells without affecting normal cells . CaM has been shown to play roles in regulating apoptosis . CaM antagonists induce apoptosis in various tumor models. Thus, CaM is involved in the regulation of biological processes such as cell proliferation  and has been extensively used as anticancer agents, but the molecular mechanism of CaM antagonist-mediated apoptosis is poorly understood [3, 10].
Materials and methods
Isolation and purification of tehranolide
One thousand grams of A. diffusa were collected from East of Iran. Dry plant was extracted by a mixture of n-hexane/ethyl acetate/methanol (1:1:1) and kept in room temperature for 24 h. The extract was passed through Wattman filter paper. The filtrate was evaporated by vacuum rotary evaporator at 45 °C for 2 h and kept in freezer. The concentrated filtrate was run through a silica gel column chromatography at different solvent polarities starting from a nonpolar solvent (n-hexane) to a medium polarity (n-hexane/ethyl acetate) and ended with ethyl acetate alone. Then, mixtures of different concentrations of n-hexane/ethyl acetate/methanol with increasing concentration of methanol towards the higher polarity were used. Fractions were collected from these processes, and the purity of the components in each fraction was evaluated by thin layer chromatography (TLC). Fractions 35, 36, 37, 38, 39, 40, 41, and 42 exhibited a single band on TLC. Afterwards, tehranolide was identified by the 125 MHz 13C NMR spectra, using CDCl3 as a solvent.
Fluorometric measurements were carried out using a Shimadzu Model RF-5000 spectrofluorometer. The instrument was operated in the energy mode, and the emission spectrum of CaM was studied at λex = 277 nm and λem = 310 nm. Tehranolide and artemisinin had no fluorescence activity at these wavelengths.
Assay for PDE-1 activity
A modification of a three-step PDE1 assay was used to determine CaM-dependent activation of PDE1 and its inhibition by tehranolide. 3,5–cyclic–nucleotide PDE, 2-mM cyclic AMP, and 100 μM CaM in 0.5 ml of Tris buffer solution (40 mM Tris-chloride, 0.1 mM MnCl2, and 0.01 mM CaCl2 in a glass of distilled water at pH 7.5) were incubated alone, with increasing concentrations of native tehranolide (1 × 10−6 to 9 × 10−6 M) for 10 min at 30 °C. The reaction was stopped by placing the test tubes in a boiling water bath for 2 min and then cooling. The 5-AMP in the reaction product was cleaved into adenosine and inorganic phosphate by incubation with 5'-nucleotidase (100 μl) for 10 min. The reaction was stopped by adding 0.05 ml of trichloride acid (55 %, weight to volume) and 0.15 ml molybdic acid solution and centrifuged until clear. The clear supernatants were decanted into the test tubes with Fiske-Subbarow reagent. Blue color reaction was allowed to develop in the presence of inorganic phosphorus for 10 min. Absorbance at 660 nm was measured with spectrophotometry.
Determination of the kinetic parameters
Where R is the gas constant, T is the absolute temperature, Yobs is the observed fluorescence emission intensity of CaM at each concentration of artemisinin, and tehranolide Yn and Yd are the values for the native and denatured states, respectively.
Where ∆G (H2O) is the value of ∆G at zero concentration of ligand, m is a measure for the dependence of ∆G on ligand concentration, and [D] is the concentration of ligand.
A kinetic analysis of the inhibition of CaM-dependent PDE by tehranolide and artemisinin resulted in Lineweaver–Burk plots. Km values were calculated in the absence and presence of inhibitors from Lineweaver–Burk plots of data. The point at which the lines crossed the y-axis represents the theoretical maximum velocity (Vmax) of the activated PDE1 in the presence and absence of inhibitors.
The human erythroleukemic cell line (K562) was purchased from Pasteur Institute, Tehran, Iran. In this study, the cells were cultured in RPMI 1640 medium (Gibco/BRL) supplemented with 10 % FBS (GIBCO/BRL), 100 unit/ml penicillin/100 mg/ml streptomycin, and 1 % L-glutamine (Gibco/BRL). The cells were grown in a humidified atmosphere containing 5 % CO2 at 37 °C. Normal lymphocytes were collected from healthy persons. Human peripheral blood was obtained from healthy volunteers (aged 25–50 years). Blood samples were obtained by venipuncture of the vein, and blood was collected in sterile EDTA K3-containing tubes.
PBMCs were obtained by centrifugation over 1.077 g/ml Ficoll–Hypaque gradient (Sigma, UK).
Cells were washed in saline and finally resuspended in RPMI 1640 medium and cultured (5 × 104 cells/ml) in 96-well flat-bottom culture plates in medium supplemented with different doses of tehranolide, 10 % fetal calf serum, 2 mm L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich), and the cell viability analysis was measured.
Cell viability analysis
Analysis of cell viability was performed with MTT (3-[4, 5-methylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide) assay. Briefly, growing cells were cultured (5,000 cells/well) overnight in 96-well tissue culture plates. Different doses of tehranolide were added to the respective wells, after 24, 48, and 72 h, 20 μL MTT (Sigma-Aldrich, USA) was directly added to the media with a final concentration of 2 mg/ml. After 4-h incubation, the medium containing MTT was discarded, and 100 μL DMSO (Sigma-Aldrich, USA) was added. MTT crystals were completely solubilized with libration for 10 min. Absorbance was measured in an ELISA reader at 570 nm, and viability was expressed as the percentage of untreated controls.
Quantitative determination of cAMP concentration
The intracellular cAMP content was determined using cAMP enzyme immunoassay kit (Stratagene, La Jolla, CA). Briefly, a total of 2 × 105 cells were washed with phosphate-buffered saline (PBS) and harvested with centrifugation. Cell pellets were resuspended in lysis reagent provided by the kit. The lysed cells were immediately assayed for cAMP according to the manufacturer's protocol. The assay was based on competition between unlabeled cAMP and a fixed quantity of peroxidase-labeled cAMP for a limited number of binding sites on a cAMP-specific antibody.
Measurement of PKA activity
PKA activity was measured using the colorimetric PKA assay kit. The treated cells were washed with ice-cold PBS twice, resuspended in lysis buffer (50 mM Tris–HCl, 2.5 mM EDTA, 1 mM MgCl2, 10 mM NaF, 10 % glycerol, pH 7.2), sonicated, and centrifuged. Supernatants were taken as lysates. Thirty micrograms of cell lysate was used to measure PKA activity. PKA activity assays were performed following the manufacturer's instructions included with the colorimetric PKA assay kit.
Blocking PKA activation on cell growth inhibition
Rp-cAMP (Adenosine 3,5-cyclic monophosphorothioate triethyammonium salt hydrate), a specific inhibitor of cAMP-dependent protein kinase A (Biolog Life Science Institute, Bremen, Germany), was purchased from Sigma and diluted in supplemented RPMI to the required concentrations. Cells were preincubated with Rp-cAMP for 20 min before treatment with tehranolide.
At first, normality of the data and homogeneity of variances were tested with K–S and Levene's statistical tests, respectively. Then, one-way analysis of variance or Mann–Whitney nonparametric test was used to determine the statistical significance (p < 0.05) between values of test and control groups. Data were analyzed using SPSS software version 16, and results were expressed as the mean ± standard error (mean ± SE).
The purified material was characterized by 13C NMR spectroscopy. The structure of tehranolide has been shown in Fig. 1.
Interaction of tehranolide with calmodulin
Inhibition of CaM-dependent cAMP PDE-1 by tehranolide
An increased concentration of tehranolide was tested based on the measurement of enzyme activity. The method involved the hydrolysis of cAMP to AMP that was quantified with the spectrophotometer method. PDE1 activity was plotted against the concentrations of tehranolide (Fig. 2b).
Kinetic analysis of PDE1 activity and inhibition of PDE1 by tehranolide
The effect of tehranolide on calmodulin stability
Calculated parameters of calmodulin stability against artemisinin and tehranolide
∆G (H2O) (kCal/mol)
Measurement of cell growth
To measure the effect of tehranolide on growth of tumor cells, different doses of tehranolide were tested; the results indicated that tehranolide reduced the growth of K562 cell line in a concentration-dependent manner.
The effect of tehranolide on intracellular levels of cAMP and PKA activity in K562 cells
To determine the level of cAMP accumulation and PKA activity in the K562 cells in response to tehranolide, cAMP content and PKA activity were measured using enzyme immunoassay. In response to tehranolide and artemisinin (PDE1 inhibitors), intracellular cAMP accumulation were observed in K562 cells (Fig. 5b, c). Additionally, tehranolide-treated group showed a significant increase in PKA activity comparing with control group (Fig. 5d).
Activation of PKA by cAMP is required for tehranolide-induced cell growth inhibition
To confirm that the effect of tehranolide on cell growth inhibition was mediated by PDE inhibition and the consequent increase of cAMP and PKA activation, cells were preincubated for 20 min with specific inhibitor of PKA (Rp-cAMP) (10 μM) and then treated with tehranolide (35–75 μM); afterwards, MTT assay was performed.
CaM, a Ca2+-dependent regulatory protein, has been shown to overexpress in malignant and transformed cells [24, 25], resulting in alterations in the interaction of CaM with its target proteins . Furthermore, CaM antagonists have been shown to inhibit tumor cell invasion in vitro . Moreover, PDE inhibitors induce apoptosis in malignant cells, without affecting normal cells . PDE inhibitors are also currently available or in development for treatment of a variety of other pathological conditions. In the recent years, it has been shown that artemisinin and its derivatives have anticancer effects [28, 29, 30].
According to the previous results, our team follows up the research in two steps: In the first step, tehranolide was purified from A. diffusa, its effect on CaM structure was investigated by spectrofluorometry. Our findings indicated that tehranolide induced some conformational changes on the CaM and increased fluorescence emission of CaM; the inhibitory effect of tehranolide was higher than artemisinin, as shown in Fig. 2a.
Subsequently, the effects of CaM activation on PDE1 in the absence (control) and presence of artemisinin or tehranolide were studied, and the kinetic analysis of PDE1 activity was performed. It was demonstrated that tehranolide is a more potent inhibitor of PDE1 activity than artemisinin (Fig. 2b). The plot of CaM-dependent PDE activity versus tehranolide and artemisinin concentrations (Fig. 2b) demonstrated that tehranolide and artemisinin could both inhibit the CaM-dependent activity of PDE1. However, tehranolide acted more potent than artemisinin. It was found that tehranolide has a higher Ki constant (6.1 μM) compared with artemisinin (10 μM), as shown in Fig. 3a.
On the other hand, tehranolide and artemisinin could act synergistically and increase the Km value of PDE1, while Vmax was constant in the presence or absence of both compounds. The data confirmed that these agents competitively inhibited the activation of PDE1 without affecting Vmax. This parameter was used to estimate CaM stability after the complex formation with artemisinin or tehranolide.
As shown in Fig. 4, the calculated ∆G (H2O) values are in agreement with our abovementioned findings. The decrease in ∆G (H2O) value is the main reason for the decrease in protein stability because of its interaction with the mentioned ligands. However, the level of decrease in ∆G (H2O) value appeared to be in the order of tehranolide > artemisinin, and this means that tehranolide induces more instability in CaM structure, indicated by lower ∆G (H2O).
In addition, the concentration required for tehranolide to interact with CaM is lower than artemisinin, suggesting that tehranolide has higher affinity for interaction with CaM, which is related to its structure. This interpretation has been confirmed with the higher m-value which is a measure of protein sensitivity to ligand , and the lower D1/2, which is the ligand concentration that induces instability in half of the protein molecules (Table. 1).
In the second step, the cytotoxic activity of tehranolide was investigated. It was found that tehranolide inhibited growth of K562 cells, while it had no cytotoxic effect on the growth of peripheral blood lymphocytes, as shown in Fig. 5a. In an attempt to elucidate the role of raised levels of cAMP and PKA activity in cell growth inhibition by tehranolide and artemisinin, the intracellular cAMP content by enzyme immunoassay were measured, as shown in Fig. 5b, c.
It is hypothesized that cell growth inhibition by tehranolide is via a PKA-mediated mechanism.
To test this hypothesis, the effect of tehranolide on PKA activity was evaluated. Our results have shown that tehranolide leads to increased activity of PKA, as shown in Fig. 5d.
Finally, to address whether PKA plays a role in the cytotoxic activity of tehranolide, cells have been pretreated with a specific PKA inhibitor (Rp-cAMP) in the presence of different concentration of tehranolide and the cytotoxicity indicated by MTT assay, as shown in Fig. 5e. As a conclusion, treatment of cells with tehranolide in the presence of pRp-cAMP strongly decreases the inhibitory effects of tehranolide in K562 cells.
Through this study and based upon our pervious results, it was confirmed that tehranolide, as a potent CaM antagonist, causes an increase in the cAMP concentration following PDE1 inhibition. Tehranolide significantly reduces cell proliferation in a time and dose-dependent manner in K562 cells which probably involves modulating cAMP/PKA signaling pathways via CaM and PDE inhibition. Thus, in short, tehranolide has a great potency as an antitumor agent.
Conflicts of interest
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