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Comparative cytotoxicity of chelidonine and homochelidonine, the dimethoxy analogues isolated from Chelidonium majus L. (Papaveraceae), against human leukemic and lung carcinoma cells.

ABSTRACT

Background: The search for new anticancer compounds is a crucial element of natural products research.

Purpose: In this study the effects of naturally occurring homochelidonine in comparison to chelidonine on cell cycle progression and cell death in leukemic T-cells with different p53 status are described.

Methods: The mechanism of cytotoxic, antiproliferative, apoptosis-inducing effects and the effect on expressions of cell cycle regulatory proteins was investigated using XTT assay, Trypan blue exclusion assay, flow cytometry, Western blot analysis, xCELLigence, epi-fluorescence and 3D super resolution microscopy. A549 cells were used for xCELLigence, donogenic assay and for monitoring microtubule stability.

Results: We found that homochelidonine and chelidonine displayed significant cytotoxicity in examined blood cancer cells with the exception of HEL 92.1.7 and U-937 exposed to homochelidonine. Unexpectedly, homochelidonine and chelidonine-induced cytotoxicity was more pronounced in Jurkat cells contrary to MOLT-4 cells. Homochelidonine showed an antiproliferative effect on A549 cells but it was less effective compared to chelidonine. Biphasic dose-depended G1 and G2/M cell cycle arrest along with the population of sub-G1 was found after treatment with homochelidonine in MOLT-4 cells. In variance thereto, an increase in G2/M cells was detected after treatment with homochelidonine in Jurkat cells. Treatment with chelidonine induced cell cycle arrest in the G2/M cell cycle in both MOLT-4 and Jurkat cells. MOLT-4 and Jurkat cells treated with homochelidonine and chelidonine showed features of apoptosis such as phosphatidylserine exposure, a loss of mitochondrial membrane potential and an increase in the caspases -3/7, -8 and -9. Western blots indicate that homochelidonine and chelidonine exposure activates Chk1 and Chk2. Studies conducted with fluorescence microscopy demonstrated that chelidonine and homochelidonine inhibit tubulin polymerization in A549 cells.

Conclusion: Collectively, the data indicate that chelidonine and homochelidonine are potent inducers of cell death in cancer cell lines, highlighting their potential relevance in leukemic cells.

Keywords:

Homochelidonine

Apoptosis

Biphasic cell cycle arrest

Mitotic block

Introduction

Alkaloids are a rich as well as an important source for searching for pharmacologically active drugs in anticancer treatment. Isoquinoline alkaloids, which currently number more than 2500 members, are isolated mainly from plants of subclasses Magnoliidae and Ranunculidae (Blaschek et al. 2010). Isoquinoline alkaloids are also widely spread in the poppy plant family (Papaveraceae) (Ziegler and Facchini 2008). The core isoquinoline nucleus may be present in plants belonging to the Papaveraceae as such, or as a structural moiety integrated into alkaloids classified as pavines, isopavines, benzophenanthridines, rhoeadines, papaverrubines, protopines, phthalideisoquinolines, protoberberines, aporphines and morphinans (Preininger 1985). Among these isoquinoline alkaloids, the benzophenanthridines have shown a promising cytostatic potential (Mansoor et al. 2013).

Chelidonine and homochelidonine (Fig. 1), B/C-cis-11-hydroxyhexahydrobenzo[c]phenanthridine alkaloids classified as partially hydrogenated-type congeners, were isolated and described as the main natural constituents of Chelidonium majus L. by Schmidt and Selle in the early 20th century (Simanek 1985; Panzer et al. 2001). At first, chelidonine received the most attention. Chelidonine was described as analgesic, antispasmodic, antibacterial, antiviral, antifungal, antioxidant, acetylcholinesterase and butyrylcholinesterase inhibitory (Colombo et al. 1996; Hiller et al. 1998; Gilca et al. 2010; Cahlikova et al. 2010). Later, chelidonine also exhibited cytotoxic, antiproliferative and apoptosis-including activity in diverse cancer cell lines (Kemeny-Beke et al. 2006; Kaminskyy et al. 2008; Paul et al. 2012). It has recently been described that chelidonine causes an increase in DNA damage assayed by [gamma] H2AX in response to 1 and 2h of treatment given a concentration of 3 [micro]g/ml in A-375 and A-375-p53DD malignant melanoma cells (Hammerova et al. 2011). Chelidonine also showed the ability to overcome the multi-drug resistance (MDR) of different cancer cell lines through interaction with ABC-transporters, CYP3A4 and GST, by the induction of apoptosis accompanied by an activation of caspases -3, -8, and -6/9 (El-Readi et al. 2013). Interestingly, much less is known about the biological effects of homochelidonine. Similarly to chelidonine, homochelidonine was found to possess morphine-like properties, acetylcholinesterase and butyrylcholinesterase inhibitory activity (Weber and Hecker 1978, Cahlikova et al. 2010). Contrary to chelidonine, evidence for cytotoxic and apoptosis-inducting activity of homochelidonine is currently missing.

Although evidence of the cytotoxic activities on alkaloids isolated from Chelidonium majus L. of the Papaveraceae family is currently growing, reports indicating biological differences between the main representatives are still very limited. Therefore, the aim of the present study was to characterize the cytotoxic and apoptosis inducing capacity of naturally occurring homochelidonine extracted from Chelidonium majus L. We evaluated the influence of homochelidonine in comparison with chelidonine. The cytotoxicity was evaluated against a mini-panel of human leukemic (MOLT-4, Jurkat, HL-60 and HEL 92.1.7), lymphoma (Raji and U-937), quiescent peripheral blood mononuclear (PBMCs) and healthy primary (MRC-5 and WI-38) cells. Human leukemic T-cells MOLT-4 (p53 wild-type) and Jurkat (p53 deficient) were further used for the evaluation of the viability, proliferation, apoptosis, cell cycle progression, mitotic block and expression of selected cell death- and/or cell cycle arrest-associated proteins. Hypotriploid non-small human lung adenocarcinoma cells A549 (p53 wild-type) were used for real-time continuous analysis of proliferation using the xCELLigence system and for clonogenic survival assay. A549 cells were also employed as a model for indirect immunofluorescence with an anti-([beta]-tubulin antibody due to a lower nuclear-cytoplasmic ratio compared to lymphoblast cells.

Materials and methods

Cell cultures and culture conditions

The experiments were carried out with the Jurkat (p53 mutant E6.1), MOLT-4 (p53 wild-type), Raji (p53 mutant), HL-60 (p53-deficient), U-937 (p53 mutant), HEL 92.1.7 (p53 wild-type), A549 (p53 wild-type), MRC-5 (primary human lung fibroblast) and WI-38 (primary human lung fibroblast) cell lines from the European Collection of Cell Cultures (ECACC, Salisbury, UK). Jurkat and Raji cells were propagated in RPMI 1640 medium supplemented with 10% foetal bovine serum, 2 mM L-glutamine, 1 mM pyruvate, 10 mM HEPES, MEM Non-Essential Amino Acids 10[micro]/ml, 50[micro]g/ml penicillin and 50 [micro]g/ml streptomycin (all reagents from Life Technologies, Grand Island, NY, USA). MOLT-4 cells were cultured in RPMI 1640 medium supplemented with 20% foetal calf serum, 2mM L-glutamine, 1 mM pyruvate, 10 mM HEPES, MEM NonEssential Amino Acids 10 [micro]l/ml, 50 [micro]g/ml penicillin and 50 [micro]g/ml streptomycin (all reagents from Life Technologies, Grand Island, NY, USA). HL-60 cells were cultured in RPMI 1640 medium supplemented with 20% foetal calf serum, 2 mM L-glutamine, 50 [micro]g/ml penicillin and 50 [micro]g/ml streptomycin (all reagents from Life Technologies, Grand Island, NY, USA). HEL 92.1.7 cells were cultured in RPMI 1640 medium supplemented with 10% foetal calf serum, 2 mM L-glutamine, 50 [micro]g/ml penicillin and 50[micro]g/ml streptomycin (all reagents from Life Technologies, Grand Island, NY, USA). U-937 cells were cultured in RPMI 1640 medium supplemented with 10% foetal calf serum, 2mM L-glutamine, 1 mM pyruvate, 10 mM HEPES, 50 [micro]g/ml penicillin and 50 [micro]g/ml streptomycin (all reagents from Life Technologies, Grand Island, NY, USA). A549 cells were cultured in Minimum Essential Medium Eagle with L-glutamine and sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) in the presence of 10% foetal calf serum, 1 mM pyruvate, 10 mM HEPES, 50 [micro]g/ml penicillin and 50 [micro]g/ml streptomycin (all supplements from Life Technologies, Grand Island, NY, USA). MRC-5 and WI-38 cells were cultured in Minimum Essential Medium Eagle with L-glutamine and sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) in the presence of 10% foetal calf serum, 2 mM L-glutamine, MEM Non-Essential Amino Acids 10 [micro]l/ml, 50 [micro]g/ml penicillin and 50 [micro]g/ml streptomycin (all reagents from Life Technologies, Grand Island, NY, USA). The cell cultures were maintained at 37 [degrees]C in a humidified incubator in an atmosphere of 5% C[O.sub.2] - 95% air. The cultures were split every second day by dilution to 2 x [10.sup.5] cells/ml. The CASY Cell Counter and Analyser (Roche, Basel, Switzerland) was used for basic quality control of the cell culture system, for evaluating cell numbers, cell viability and cell debris. Jurkat, MOLT4 and A549 cells in the maximum range of 20 passages and in an exponential growth phase were used for this study.

Isolation of human peripheral blood mononuclear cells

PBMCs were obtained by venous blood draw from healthy male volunteers, from whom informed consents were obtained that their donated blood will be used for isolation of blood cells in order to study the cytotoxic activity of natural compounds chelidonine and homochelidonine. Separation of PBMCs from blood anticoagulated with EDTA was performed using density centrifugation without braking with sterile Histopaque[R]-1077 (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. After isolation, PBMCs were washed three times in Dulbecco's Phosphate-Buffered Saline (DPBS). They were then maintained in RPMI 1640 medium supplemented with 20% foetal calf serum, 2 mM L-glutamine, 1 mM pyruvate, 10 mM HEPES, MEM NonEssential Amino Acids 10 [micro]l/ml, 50 [micro] g/ml penicillin and 50 [micro]g/ml streptomycin (all reagents from Life Technologies, Grand Island, NY, USA) in a humidified atmosphere containing 5% C[O.sub.2] and 95% air.

Cell treatment

Chelidonine and homochelidonine: fresh stock solutions of chelidonine and homochelidonine in concentrations of 50 mM were dissolved in dimethyl sulphoxide--DMSO (Sigma-Aldrich, St. Louis, MO, USA). Stock solutions were freshly prepared before use in the experiments. Chelidonine and homochelidonine were isolated from the dried powdered aerial parts and roots of Chelidonium majus L. The detailed isolation and identification (1D-, 2D-NMR and MS experiments) of both alkaloids is described in Cahlfkova et al. (2010). For the experiments, the stock solution was diluted with the complete culture medium to create final concentrations of 1-20[micro] M, making sure the concentration of DMSO was <0.1% to avoid the toxic effects on the cells. Control cells were sham-treated with a DMSO vehicle only (0.1%; control).

Proliferation and viability

Cell proliferation and viability of Jurkat and MOLT-4 cells were determined 48 h after treatment with 1, 5, 10, 15 and 20[micro] M of chelidonine and homochelidonine. Cell membrane integrity was determined using the Trypan blue exclusion technique--mixing 10 [micro]l of 0.4% Trypan blue and 10 [micro]l of cell suspension. Cell counts were carried out using a Biirker chamber and light microscope Nikon Eclipse E200 (Nikon, Tokyo, Japan).

Cytotoxicity screening using XTT assay

In order to determine cell viability of cells treated with chelidonine and homochelidonine in a broad concentration range we used a standard colorimetric method measuring a tetrazolium salt reduction via mitochondrial dehydrogenase activity. The cells were seeded at previously established optimal density in a 96-well plate. After 48 h incubation cell viability was determined using Cell Proliferation Kit II (XTT, Roche, Germany) according to manufacturer's instructions. XTT-assay was conducted using 200 [micro]l of volume and 100 [micro]l of XTT-labelling mixture. Absorbance was then measured at 480 nm using a 96-multiweil microplate reader Tecan Infinite M200 (Tecan Group Ltd., Mannedorf, Switzerland). Viability was calculated as described in the paper by Havelek and colleagues using the following formula: (%) viability = (A480sample--A480blank)/(A480control--A480blank) x 100, where A480 is the absorbance of utilized XTT formazan measured at 480 nm (Havelek et al. 2012). Data were analysed with GraphPad Prism 5 biostatistics (GraphPad Software, La Jolla, CA, USA) statistical software. Each value is the mean of three independent replicates of each condition.

Analysis of apoptosis

Apoptosis was determined by flow cytometry using an APOPTEST[TM]-FITC kit (Dako, Glostrup, Denmark) according to the manufacturer's instructions. The APOPTEST[TM]-FITC kit employs the property of fluorescein isothiocyanate (FITC) conjugated to Annexin V (Ann-FITC) to bind to phosphatidylserine in the presence of [Ca.sup.2+], and the property of PI to enter cells with damaged cell membranes and to bind to DNA. Measurement was performed immediately using a CyAn (Beckman Coulter, Miami, FL, USA) flow cytometer. Listmode data were analysed using Summit v4.3 software (Beckman Coulter, Miami, FL, USA).

Analysis of the mitochondrial membrane potential

The loss of the mitochondrial membrane potential ([DELTA][[psi].sub.m]) was quantitatively determined by flow cytometry using a JC-1 mitochondria staining kit (Sigma-Aldrich, St. Louis, MO, USA); this assay was performed according to the manufacturer's instructions. Briefly, approximately 1 x [10.sup.6] cells from each condition were collected, washed with PBS, and resuspended in a complete medium at a concentration of 6 x [10.sup.5] cells/ml. The cells were stained with 5 [micro]l JC-1 (1 mg/ml) and incubated in the dark at 37 [degrees]C for 30 min. JC-1 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide is a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green monomers (~525 nm) to orange aggregates (~590nm). Any cell damage event that dissipates the mitochondrial membrane potential prevents the accumulation of the JC-1 dye in the mitochondria and, thus, the dye is dispersed throughout the entire cell leading to a shift from orange to green fluorescence. Fluorescence was measured using a CyAn flow cytometer (Beckman Coulter, Miami, FL, USA). Listmode data were analysed using Summit v4.3 software (Beckman Coulter, Miami, FL, USA).

Cell cycle distribution and internucleosomal DNA fragmentation analysis

Where cell cycle distribution analysis is concerned, the cells were washed with ice cold PBS and fixed with 70% ethanol. In order to detect low molecular-weight fragments of DNA, the cells were incubated for 5 min at room temperature in a buffer (192 ml 0.2 M [Na.sub.2]HP[O.sub.4] + 8 ml of 0.1 M citric acid, pH 7.8) and then labelled with propidium iodide in Vindelov's solution for 1 h at 37[degrees]C. DNA content was determined using the flow cytometer CyAn (Beckman Coulter, Miami, FL, USA) with an excitation wavelength of 488 nm. The data were analysed using Multicycle AV software (Phoenix Flow Systems, San Diego, CA, USA).

BrdU incorporation and DNA content

Cells were pulse-labelled with 10 [micro]M of 5-bromo-2'-deoxyuridine (BrdU) for 30 min prior to assay. After BrdU, pulse cells were washed twice with PBS, resuspended in fresh medium and then sham-(DMSO control), chelidonine or homochelidonine-treated. At various time points post-treatment, the cells were harvested and fixed in cold 70% ethanol. The cells were then stained for both DNA content and BrdU incorporation using the acid denaturation method. Briefly, the cells were washed in a TBS-0.5% BSA solution, denatured by freshly made 2 M HCl and resuspended in 0.1 M sodium metaborate to neutralize HCl. After subsequent washing in TBS-0.5% BSA, we used a fluorescein isothiocyanate (FITC)-conjugated mouse anti-BrdU monoclonal antibody (BD Biosciences, San Jose, CA, USA) for BrdU detection and, finally, cell pellets were resuspended in a TBS-0.5% BSA solution containing propidium iodide (PI) at 10 [micro]g/ml and RNase A at 0.5 mg/ml for DNA content staining. All samples were immediately analysed by a CyAn flow cytometer and the data were plotted using Summit v4.3 software (both from Beckman Coulter, Miami, FL, USA).

Enumeration of mitotic cells, including measurement of histone H3 phosphorylated at Ser10 and DNA content

Histone H3 is phosphorylated at Ser10 when cells enter the mitotic phase and remains unphosphorylated in the other phases of the cell cycle. To determine the number of mitotic cells, Jurkat and MOLT-4 cells treated with 10 [micro]M of chelidonine and homochelidonine and 5 [micro]M of nocodazole (positive control) were fixed and permeabilized by adding ice-cold 100% methanol. Following overnight incubation, the cells were washed with a PBS-0.5% BSA solution and subsequently stained with the Alexa Fluor 488-conjugated anti-pSer10-histone H3 (Cell Signaling Technology, Beverly, MA, USA) antibody diluted with a PBS-0.5% BSA solution in accordance with the manufacturer's instructions. Cells were incubated with agitation for 60 min at room temperature in the dark. Finally, the cells were washed twice with the PBS-0.5% BSA solution and resuspended in a PI staining solution (BD Biosciences, San Jose, CA, USA) at a final volume of 500 pi. All samples were immediately analysed by a CyAn flow cytometer and the data were plotted using Summit v4.3 software (both from Beckman Coulter, Miami, FL, USA).

xCELLigence

The xCELLigence system (Roche, Basel, Switzerland and ACEA Biosciences, San Diego, CA, USA) was used to monitor cell adhesion, proliferation and cytotoxicity. The xCELLigence system was connected and tested by Resistor Plate before the RTCA Single Plate station was placed inside the incubator at 37[degrees]C and 5% C[O.sub.2]. First, the optimal seeding concentration for proliferation experiments of the A549 cells was determined (8000 cells/well). After seeding, the respective number of cells in 190 [micro]l medium per each well of the E-plate 96, the proliferation, attachment and spreading of the cells were monitored every 30 min by the xCELLigence system. Approximately 24 h after seeding, when the cells were in the log growth phase, the cells were exposed in triplicates to 10 [micro]l sterile deionized water containing 5-50 [micro]M of chelidonine and homochelidonine. Controls received sterile deionized water + DMSO with a final concentration of 0.1%. Cells treated with 5% DMSO were used as positive control. Nocodazole, a microtubule depolymerising agent, was used as a reference compound. All experiments were run for 74 h. Growth curves were normalized to the time point of treatment. Evaluations were performed using xCELLigence 1.2.1 software (Roche, Basel, Switzerland and ACEA Biosciences, San Diego, CA, USA).

Clonogenic survival assay

Hypotriploid human lung adenocarcinoma A549 cells were trypsinized to generate a single cell suspension that was seeded in 25 [cm.sup.2] Nunc[TM] cell culture flasks (Thermo Fisher Scientific, Waltham, MA, USA) at a density of 900 cells per flask. On day 1 post-plating, spent medium was replaced with fresh medium. To assess the effect of chelidonine and homochelidonine on cell proliferation and survival, the alkaloids were added into the culture and the cells were incubated for a total of 14 days at various concentrations (1, 5, 10 and 15 [micro]M). Control plates received DMSO <0.1% only. Fourteen days after seeding, colonies were stained with crystal violet (0.5% w/v) in 80% methanol and formaldehyde 37% at a 9:1 ratio. The number of colonies formed in each flask was counted manually on the basis of three separate experiments.

Activity of caspases

The induction of programmed cell death was determined by monitoring the activities of caspase 3/7, caspase 8 and caspase 9 by Caspase-Glo Assays (Promega, Madison, WI, USA) 24 h after treatment with 5 and 10 [micro]M of chelidonine and homochelidonine. The assay provides a proluminogenic substrate in an optimized buffer system. The addition of a Caspase-Glo Reagent results in cell lysis, followed by caspase cleavage of the substrate and the generation of a luminescent signal. A total of 1 x [10.sup.4] cells were seeded per well using a 96-well-plate format (Sigma, St. Louis, MO, USA). After treatment, the Caspase-Glo Assay Reagent was added to each well (50 [micro]l/well) and incubated for 30 min before luminescence was measured using a Tecan Infinite M200 spectrometer (Tecan Group, Mannedorf, Switzerland).

Immunofluorescence staining, epi-fluorescence and 3D super-resolution microscopy

For each condition, 400,000 cells were seeded in 1-well chamber slides under the Nunc[TM] Lab-Tek[TM] II System (Thermo Fisher Scientific, Waltham, MA, USA). After seeding (usually 24 h later), spent medium was replaced with fresh medium and the cells were treated with chelidonine and homochelidonine at 5 and 10 [micro]M. Cells treated with 5 [micro]M of the tubule-disrupting agent nocodazole were used as a reference drug in immunofluorescence analysis. Following 24-h treatment the cells were fixed with 4% freshly prepared paraformaldehyde for 10 min at room temperature, washed with PBS, permeabilized in 0.2% Triton X-100/PBS for 15 min at room temperature and washed with PBS (all reagents from Sigma-Aldrich, St. Louis, MO, USA). Before incubation with the primary antibody (overnight at 4[degrees]C), the cells were incubated with 7% inactivated foetal calf serum + 2% bovine serum albumin in PBS for 30 min at room temperature. Mouse monoclonal anti-[beta]-tubulin (Life Technologies, Grand Island, NY, USA) was used for [beta]-tubulin detection. For the secondary antibody, the affinity pure donkey anti-mouse-Alexa Fluor[R]555-conjugated antibody was purchased from the Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The secondary antibody was applied to each slide (after their pre-incubation with 5.5% donkey serum in PBS for 30 min at room temperature), incubated for 1 h in the dark and washed (3x5 min) with PBS. The nuclei were counterstained with 100 [micro] of 4',6-diamidino-2-phenylindole (DAP1) at 1 [micro] g/ml for 1 h. After the last two washes with PBS, the slides were mounted with an antifading glycerol/n-propyl gallate mounting medium (reagents from Sigma-Aldrich, St. Louis, MO, USA). Images of all of the examined slides were obtained by a Nikon confocal microscope system A1+; the exposure time and dynamic range of the camera in all of the channels were adjusted to the same values for all of the slides to portray quantitatively comparable images. Super-resolution 3D imaging of single isolated nuclei of cells exposed to 10 [micro]M was performed using the Nikon super resolution microscope N-SIM. Images were further processed and merged using NIS-Elements Advanced Research 4.13 (all instruments and software from Nikon, Tokyo, Japan). A minimum of 300 cells were scored to determine percentage of cells arrested in metaphase stage at each alkaloid treatment.

Western blot analysis

Whole-cell lysates (Cell Lysis Buffer, Cell Signaling Technology, Danvers, MA, USA) were prepared 24 h following treatment of Jurkat and MOLT-4 cells with chelidonine and homochelidonine. Cells treated with 0.1% DMSO were used as negative control and cells treated with 5nM of mitoxantrone were used as positive control. Quantification of the protein content was performed using the BCA assay (Sigma-Aldrich, St. Louis, MO, USA). The lysates (20 [micro]g purified protein) were loaded into each lane of a polyacrylamide gel. After electrophoretic separation, the proteins were transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA). Non-specific binding of the membranes was blocked for 1 h in a Tris-buffered saline containing 0.05% Tween 20 (TBS) and 5% non-fat dry milk. The membranes were washed in TBS. Incubation with a primary antibody against specific antigens (p53, p53_serine 15--Exbio, Prague, Czech Republic; [beta]-actin--Sigma-Aldrich, St. Louis, MO, USA; Chk1, Chk1_serine 345, Chk2, Chk2_ threonine 68--Cell Signaling, Danvers, MA, USA) was performed at 4[degrees]C overnight. The following day the membranes were washed 5-times with TBS, each time for 5 minutes, and once with TBS, for 10 min, and then incubated with an appropriate secondary antibody (DakoCytomation, Glostrup, Denmark) for 1 h at room temperature. Band detection was performed using a chemiluminiscence detection kit (Roche, Basel, Switzerland). To ensure equal protein loading, each membrane was reprobed and [beta]-actin was detected.

Statistical analysis

The descriptive statistics of the results were calculated and the charts were made in Microsoft Office Excel 2003 (Microsoft, Redmond, WA, USA) or GraphPad Prism 5 biostatistics (GraphPad Software, La Jolla, CA, USA). In this study all the values were expressed as arithmetic means with S.D. of triplicates, unless otherwise noted. The significant differences between the groups were analysed using the Student's t-test.

Calculation of [IC.sub.50] values

For [IC.sub.50] values calculations data from proliferation determined by use of cell counting and viability determined by use of Trypan blue staining in Jurkat and MOLT-4 cells were further processed by using GraphPad Prism 5 biostatistics (GraphPad Software, La Jolla, CA, USA) software. Chelidonine and homochelidonine concentrations were plotted against number of viable cells and % of viable cells. The [IC.sub.50] values were determined using non-linear regression. For [IC.sub.50] values calculations data from viability determined by use of XTT assay were also processed by using GraphPad Prism 5 biostatistics (GraphPad Software, USA) software. Drug concentrations were plotted against cell viability and the [IC.sub.50] values were determined using non-linear regression.

Results

Cytotoxicity screening of the homochelidonine and chelidonine towards human blood cancer and healthy cells

The homochelidonine and chelidonine were subjected to cytotoxic evaluation against 6 human blood cancer cell lines (Jurkat, MOLT-4, HL-60, Raji, U-937 and HEL 92.1.7), 2 human primary cell lines (MRC-5 and WI-38) and quiescent human PBMCs employing XTT assay. Table 1 demonstrates that blood cancer cells were more sensitive to the cytotoxic activity of homochelidonine (with exception of HEL 92.1.7. and U-937 cells) and chelidonine than the PBMCs. Moreover, PBMCs maintained higher viability at higher evaluated concentrations above 10[micro] M. Chelidonine was more potent in cytotoxicity against leukemic and lymphoma cells than against normal human WI-38 fibroblasts. The MRC-5 fibroblast cells were more affected at lower concentrations of chelidonine treatment; however, at higher evaluated concentrations resist the treatments. Contrary thereto, the both WI-38 and MRC-5 homochelidonine-treated cells had [IC.sub.50] values >10[micro] M (Fig. 2).

Homochelidonine and chelidonine-induced cytotoxicity against Jurkat and MOLT-4 cells was dose-dependent

To further investigate anticancer activity of homochelidonine and chelidonine in leukemic MOLT-4 and Jurkat T-cells, we used the trypan blue exclusion assay, which is based on the principle that live cells will exclude membrane-impermeable Trypan blue, whereas trypan blue will penetrate inside dead cells and stain them. By counting unstained cells using a Burker chamber we showed that homochelidonine decreased the proliferation of Jurkat cells within 48 h of treatment with [IC.sub.50] value 4.8 [+ or -] 0.1 [micro] M. The decrease was statistically significant at 5, 10, 15 and 20p,M. Chelidonine reduced the number (P [less than or equal to] 0.05) of Jurkat cells, after 48 h incubation, at all tested concentrations (1-20 [micro] M) with [IC.sub.50] value 0.7 [+ or -] 0.0[micro] M. By counting the percentage of stained cells using a Burker chamber we showed that compared to the controls, homochelidonine significantly reduced Jurkat cell viability ([IC.sub.50] = 6.4 [+ or -] 0.1 [micro]M) within 48 h of incubation from 10[micro] M and higher. When calculating effect of chelidonine on cell viability, a statistically significant concentration-dependent decrease was observed at 48 h of incubation from 5 [micro] M with [IC.sub.50] value 2.0 [+ or -] 0.2 [micro] M (Fig. 3A).

Parallel experiments were performed with MOLT-4 cells. As shown in Fig. 3B, homochelidonine had a dose-dependent antiproliferative effect ([IC.sub.50] = 9.8 [+ or -] 0.3 [micro] M); the decrease of cell number was statistically significant (P [less than or equal to] 0.05) from 10 [micro]M and higher. Chelidonine exhibited significant antiprolierative effect ([IC.sub.50] = 4.2 [+ or -] 0.2 [micro]M) against these cells from 5 [micro]M and higher. Homochelidonine downregulated the viability of MOLT-4 cells ([IC.sub.50] = 10.1 [+ or -] 0.6 [micro]M) from 5 [micro] M and higher, whereas chelidonine suppressed viability ([IC.sub.50]=4.5 [+ or -] 0.1 [micro]M) from 5 [micro] M and higher. The MOLT-4 cell line was more resistant to the antiproliferative effect of both chelidonine and homochelidonine, when compared to Jurkat cells. Corresponding dose-response curves used for determination of [IC.sub.50] values are shown in the supporting information relating to this paper (Fig. S1A and B, Supporting data).

Homochelidonine and chelidonine induce cell death by apoptosis, which was more pronounced in Jurkat cells than in MOLT-4 cells during 24 h of treatment

In our ongoing experiments to analyse the percentage of viable, early apoptotic and late apoptotic cells induced by chelidonine and homochelidonine treatment we performed flow cytometric quantification of Annexin V and propidium iodide (PI) dual staining. Early apoptosis is characterized by the translocation of phosphatidylserine (PS) from the inner layer of the plasma membrane to the outer surface. Quantification of the Annexin V-FITC bond to externalized PS allows us to clearly distinguish apoptotic cells amongst different cell populations. We used Jurkat and MOLT-4 T-leukemic cells for this assay. Our results demonstrated that chelidonine significantly (P [less than or equal to] 0.001) induced cell death following 24 h exposure. The increase in cell death seems dose-independent for Jurkat cells at concentrations ranging from 5 [micro]M to 20[micro]M (Fig. 4A) and at 10, 15 and 20 [micro]M for MOLT-4 cells (Fig. 4B). In contrast, homochelidonine increased the apoptotic populations of Jurkat (Fig. 4A) and MOLT-4 (Fig. 4B) cells in a dose-dependent manner reaching the plateau at 15 and 20 [micro]M 24 h post treatment. Surprisingly enough, chelidonine and homochelidonine-treated cells had a higher percentage of early and late apoptotic cells in p53-deficinet Jurkat cells than in p53 wild-type MOLT-4 cells (P [less than or equal to] 0.001). For example, MOLT-4 cells treated with chelidonine and homochelidonine at a concentration of 20 [micro]M showed an increase in apoptotic populations by 36% and 38%, respectively. Jurkat cells exposed to chelidonine and homochelidonine at the same concentration as MOLT-4 cells (20 [micro]M) were more sensitive to the effect of both chelidonine and homochelidonine, which induced 56% and 58% of apoptotic cells, respectively. After 48 h of treatment with chelidonine at 10, 15 and 20 [micro] M, the parentage of early (Annexin V single positive) and late (Annexin V and PI double positive) apoptotic cells was higher in MOLT-4 cells when compared to Jurkat cells. On the contrary, the decreased level of apoptosis is also seen in MOLT-4 cells treated with homochelidonine at 5 [micro]M when compared with the Jurkat cells treated with the same concentration. In Jurkat cells exposed to chelidonine and homochelidonine, more cells were early apoptotic in comparison with MOLT-4 cells treated with chelidonine and homochelidonine (Fig. S2A and B, Supporting data).

Homochelidonine and chelidonine treatments decrease the mitochondrial membrane potential ([DELTA][[psi].sub.m]) in Jurkat and MOLT-4 cells

Mitochondria play a crucial role in apoptosis as a rapid and dramatic decrease in the mitochondrial membrane potential ([DELTA][[psi].sub.m]) is observed during apoptosis induction. The drop in [DELTA][[psi].sub.m] is due to permeability transition, it allows molecules to leak out from the mitochondrial matrix and it is believed that the decrease in [DELTA][[psi].sub.m] represents the "point of no return" in a programmed cell death pathway. Thus, we monitored [DELTA][[psi].sub.m] in Jurkat and MOLT-4 cells after chelidonine and homochelidonine treatments using the cationic carbocyanine dye JC-1. Chelidonine induced mitochondrial depolarization (P [less than or equal to] 0.05) in both Jurkat (Fig. 5A) and MOLT-4 (Fig. 5B) cells as measured using the fluorescent monomeric form of JC-1. Analogically as in the Annexin V assay, homochelidonine caused a preferentially concentration-dependent decrease in the percentage of cells displaying intact mitochondria, and then reached plateau at 15 and 20 [mciro]M. Mitochondrial depolarization occurred from 5 [micro] M and higher (P [less than or equal to] 0.05).

Homochelidonine induces biphasic dose-response G1 and G2/M cell cycle arrest in MOLT-4 cells but not in Jurkat cells

To investigate whether chelidonine and homochelidonine could induce cell cycle perturbations in Jurkat and MOLT-4 leukemic T-cells, flow cytometric analyses of PI stained cells were performed. Chelidonine treatment, at all examined concentrations, resulted in a significant increase in the number of cells in the G2/M phase compared to the control in both Jurkat (Fig. 6A) and MOLT-4 (Fig. 6B) cells (P [less than or equal to] 0.001). This effect was concurrently accompanied by a significant (P [less than or equal to] 0.001) decrease in G1 and S phase cells compared to negative control. The sub-G1 fraction of Jurkat cells was increased after chelidonine exposure to (5 [micro] M - 28.3 [+ or -] 1.5%; 10 [micro]M - 23.7 [+ or -] 0.8%; 15 [micro]M - 26.2 [+ or -] 2.9%; 20 [micro]M - 26.2 [+ or -] 1.3%) while in control group it was 0.7% [+ or -] 0.0%. In MOLT-4 cells, application of chelidonine induced increase in percentage of sub-G1 (5 [micro]M - 17.4 [+ or -] 0.4%; 10 [micro] M - 25.3 [+ or -] 1.5%; 15 [micro] M - 26.5 [+ or -] 0.2%; 20 [micro] M - 23.0 [+ or -] 2.6%) compared with the negative control (0.1 [+ or -] 0.0%). Homochelidonine exposure at the lower examined concentrations induced minor changes in cell cycle phase distribution. Application of homochelidonine in Jurkat cells at a concentration of 5 [micro]M decreased the percentage of G1 cells and increased the amount of S-phase cells (P< 0.001) compared to control. A pronounced concentration-dependent increase in the percentage of Jurkat cells in the G2/M phase was detected after exposure to 10[micro]M of homochelidonine. The percentage of Jurkat cells in the G2/M phase had reached the plateau at 15 and 20 [micro]M of homochelidonine and there was a concomitant decrease in the fraction of cells in the G1 and S phases (Fig. 6A). Further experiments revealed that the MOLT-4 cells exhibited biphasic dose-response features in the percentage of cells in the G1 and G2/M phases after homochelidonine treatment. At 5 and 10 [micro] M homochelidonine shows predominant G1 arrest in MOLT-4 cells, while at 15 and 20[micro]M there is a significantly lower amount of G1 cells. The percentage of G2/M-phase MOLT-4 cells decreased at 5 and 10 [micro]M of homochelidonine but a G2/M-phase population predominates at higher concentrations (15 and 20 [micro]M). Besides these, an increase in internudeosomal DNA fragmentation was also observed. Cells with internudeosomal DNA fragmentation (sub-G1 peak) corresponded to 7% at 10 [micro] M, 38% at 15 [micro] M and 33% at 20 [micro]M after homochelidonine treatment (Fig. 6B). In Jurkat cells, compared with control (0.7% [+ or -] 0.0%), the percentage of sub-G1 after homochelidonine treatment was 2.9 [+ or -] 0.4% for 5 [micro] M, 18.8 [+ or -] 1.4% for 10 [micro] M, 28.1 [+ or -] 3.2% for 15 [micro]M and 19.7 [+ or -] 2.0% for 20 [micro]M. The extent of sub-G1 fraction in MOLT-4 cells treated with homochelidonine was diminished to zero values since we detected internudeosomal DNA fragmentation observed as clear sub-G1 peak in MOLT-4 cells.

The effect of chelidonine and homochelidonine-treatment on cell proliferation was cell cycle specific. Cell cycle arrest in the G2/M phase was found in both chelidonine-treated cell lines independently of the concentration used. Inversely, the application of homochelidonine was associated with a significant increase in the fraction of G2/M phase cells only at higher concentrations in p53 deficient Jurkat cells. Exposure of MOLT-4 cells to homochelidonine caused these cells to arrest in G1 at 5 and 10 [micro]M; and in G2 at 15 and 20 [micro]M. Moreover, MOLT-4 cells treated with homochelidonine also displayed DNA fragmentation associated with apoptosis assayed as a sub-G1 population.

Homochelidonine triggers the slowdown of DNA synthesis and G2/M cell cycle arrest, whereas chelidonine preferentially induces a G2/M block alone

To better understand the antiproliferative effects of chelidonine and homochelidonine, particularly those induced by low drug concentrations, Jurkat and MOLT-4 cells were pulse-labelled with BrdU and mock-treated or exposed to chelidonine and homochelidonine at 5 and 10 [micro]M. The progression of S-phase cells was analysed at 8, 16 and 24 h post-treatment by flow cytometry. BrdU pulse labelling demonstrated that homochelidonine decreased the rates of Jurkat (Fig. 7A) and MOLT-4 (Fig. 7B) cells progression through the cell cycle. While mock treated-Jurkat and MOLT-4 cells efficiently progressed through all of the phases of the cell cycle, homochelidonine-treated cells cycled more slowly. In fact, at 24 h, BrdU-labelled cells from control cultures started to appear in the G2/M phase after completion of the S phase. In contrast, only a small percentage of homochelidonine-treated, BrdU-labelled cells recycled to the G2/M phase, the majority of cells still being in the G1 phase at 24 h. Moreover, unlike homochelidonine-treated cultures, the majority of Jurkat and MOLT-4 cells exposed to cheli donine were preferentially blocked at the G2/M phase at 16 h and 24 h post treatment.

Homochelidonine and chelidonine promote caspase activation with a comparable intensity in both Jurkat and MOLT-4 cells

Next, to further confirm whether chelidonine and homochelidonine-treated cells truly underwent apoptosis, caspases -3/7, -8 and -9 activation was quantified by a glow-type luminescent assay. Briefly, caspases -3/7, -8 and -9 activities were significantly (P [less than or equal to] 0.001) increased in both chelidonine and homochelidonine-treated Jurkat (Fig. 8A) and MOLT-4 (Fig. 8B) cells from 5 and 10 [micro]M. The increase seems preferentially dose-dependent in MOLT-4 cells rather than in Jurkat cells. All in all, homochelidonine induced more intense caspase activation in both examined cell lines at 10 [micro]M when compared to chelidonine. Homochelidonine application at 5 [micro]M resulted in less intense caspase activation in MOLT-4 cells and more intense caspase-3/7 and caspase-8 activation in Jurkat cells compared to chelidonine. Caspase-9 was induced with a similar intensity in both homochelidonine and chelidonine-treated Jurkat cells at 5 [micro]M.

Chelidonine and homochelidonine treatments slightly activate checkpoint kinases Chk1, Chk2 and increase phosphorylation of histone H3 at Ser10 in Jurkat and MOLT-4 cells

As we observed cell cycle delay and apoptosis at 5 and 10 [micro]M we decided to evaluate the expression of selected regulatory proteins in Jurkat and MOLT-4 cells 24 h after chelidonine and homochelidonine treatment. We monitored the expression of p53, Chk1, Chk2 and their post-translational modifications using Western blotting. Histone H3 phosphorylation at Ser10, which plays an important role during mitosis, was evaluated using flow cytometry 16 h after treatment. Exposure of p53 wild-type MOLT-4 cells to chelidonine and homochelidonine did not result in an increased accumulation of p53 nor its phosphorylation at Ser15 (Fig. 8D). In follow-up experiments we detected checkpoint kinases 1 and 2 (Chk1 and Chk2) as a key downstream checkpoint regulators activated after DNA damage. Exposure to chelidonine slightly increased the amount of Chkl phosphorylated at Ser345 in both examined cell lines. Another downstream target protein Chk2 was phosphorylated at Thr68 in Jurkat (Fig. 8C) and MOLT-4 (Fig. 8D) cells after exposure to chelidonine or homochelidonine. Our results following from flow cytometry analysis indicated that chelidonine or homochelidonine had a dramatic effect on the cell cycle. We therefore examined whether chelidonine or homochelidonine promoted the phosphorylation of histone H3 (Ser10) in Jurkat and MOLT-4 cells using flow cytometry. Approximately 1.4% and 0.8% of the control population of either Jurkat or MOLT-4 cells, respectively, had a phosphorylated form of histone H3. This population increased to 36.8% and 19.9%, respectively, after exposure to chelidonine at 10[micro]M for 16 h. Also homochelidonine at 10[micro]M after 16 h of treatment significantly increased the formation of phosphorylated histone H3 at Ser10 in Jurkat (32.2%) and MOLT-4 (7.6%) cells. Similarly, a 16 h treatment with nocodazole caused significant G2/M arrest as assessed by PI staining (data not shown), with 43.8% (Jurkat) and 21.6% (MOLT-4) of the cell population staining positive for histone H3 Ser10 phosphorylation, meaning that nocodazole, like chelidonine and homochelidonine, arrests cells in mitosis (Fig. 8E, and F).

Chelidonine and homochelidonine treatment inhibits the proliferation and adhesion degree of A549 cells determined by real-time xCELLigence impedance analysis

The antiproliferative effects of chelidonine and homochelidonine were further monitored by real-time, label-free and continuous measurement of cell proliferation in A549 cells using the xCELLigence system dedicated to adherent cell lines. The xCELLigence system measures cell adhesion, the morphology, viability and number of cells based on impedance, which is displayed as normalized cell index (CI) values. The xCELLigence system uses special 96-well plates integrated with electrodes on the well flat bottom. The more cells that are attached on the electrodes on the well bottom, the larger the increases in electrode impedance and CI value. In addition, the impedance (and also CI) can vary based on the quality of the cell interaction with the electrodes; for example, increased cell adhesion or spreading will lead to an increase in electrode impedance. Thus, electrode impedance, which is displayed as CI values, can be used to monitor cell viability, number, morphology, and also adhesion degree. When cells are not present or are not well-adhered on the electrodes, the CI decreases or are zero. Under other biological conditions such as cell grow, when more cells are attached on the electrodes, the CI values are higher. As shown in Fig. 9A, the application of homochelidonine at 10, 15 and 20 [micro]M decreased the proliferation of A549 cells compared to control. Homochelidonine treatment, at 50 [micro]M, resulted in complete inhibition of cell proliferation. After treatment at 1 and 5 [micro]M of chelidonine, the proliferation of A549 cells was decreased compared to the proliferation of control. The obtained plot shows that exposure to chelidonine at 10, 15, 20 and 50 [micro]M resulted in complete inhibition of cell proliferation compared to vehicle treatment. Cells cultivated in the presence of 5% DMSO were used as positive control. The decrease and subsequent increase of the normalized cell index during the first 24 h of treatment indicates changes in cell morphology and the adhesion degree at early intervals after exposure. A similar plot profile obtained by xCELLigence analysis was discovered when the cells were cultured in the presence of the microtubule depolymerizing agent nocodazole. A549 cells were delayed in proliferation under nocodazole treatment at 1 [micro]M. Independently of the nocodazole applied at concentrations ranging from 5 to 20 [micro]M, the A549 cells were halted in proliferation. The transient decrease and increase in the normalized cell index after 24 h of treatment could be attributed to the microtubule-disrupting activity of nocodazole, chelidonine and higher concentrations of homochelidonine.

Homochelidonine inhibits the clonogenic survival of A549 cells less effectively than chelidonine

To determine whether chelidonine and homochelidonine can also compromise the colony-forming capacity of adherent non-small human lung adenocarcinoma A549 cells, the ability of a single cell to form a colony was evaluated using clonogenic assays. Exposure to chelidonine had a substantial inhibitory effect at a concentration of 5 [micro]M or higher. The capability of A549 cells to generate colonies was reduced (P [less than or equal to] 0.05) at 5 [micro]M of homochelidonine in which case the number of colonies was decreased and the colonies were less macroscopically developed. When a 15 [micro]M concentration of homochelidonine was applied, complete inhibition of the colony formation was observed compared to control (Fig. 9B). A visual representation of this assay documented by photographs is shown in the supporting information relating to this paper (Fig. S3, Supporting data). To the best of our knowledge, this is the first report describing the capability of chelidonine and homochelidonine to suppress the ability of single cells to resist treatments and to grow into a colony.

Homochelidonine and chelidonine applications affect the microtubular structure of A549 cells and induce nuclear fragmentation

After monitoring the transient decrease of the normalized cell index using the xCELLigence impedance system soon after homochelidonine, chelidonine and nocodazole treatment in A549 cells, we investigated the impact of benzophenanthridine alkaloids on microtubules, which play an important role in the proliferation, morphology and quality of cell adhesion. Immunofluorescence staining of microtubules with an anti-[beta]-tubulin monoclonal antibody followed by DAPI counterstaining and epi-fluorescence imaging revealed that mock-treated A549 cells had intact microtubules. Contrary thereto, treatment with chelidonine and homochelidonine resulted in the formation of dense bundles of microtubules around cell nuclei. Dissimilarly, application of nocodazole extensively disassembled microtubule structures with diffuse [beta]-tubulin staining dispersed throughout the cytoplasm (Fig. 10A). When 3D super-resolution microscopy was employed, we observed collapsed aberrant microtubules around cell nuclei in 3-dimensional space after homochelidonine and chelidonine exposure. Cells bearing fragmented nuclei with disrupted microtubules were also frequent between exposed cells (Fig. 10B). Considering blockage of cells mitosis at metaphase a minimum of 300 cells were scored to determine percentage of cells arrested in metaphase stage. We quantified 4.2% accumulation of A549 cells after chelidonine treatment at 5 [micro]M, 10.1% at 10 [micro]M and 0% in 0.1% DMSO vehicle-treated control. Homochelidonine-treated cells were less affected, leading to metaphase arrest in 7.3% of cells at 5 [micro]M and in 7.4% of cells at 10 [micro]M.

Discussion

Our experiments focused on comparing the effects of chelidonine and naturally occurring chelidonine-dimethoxy analogue with open dioxole ring homochelidonine in parallel on leukemic T-cell lymphoblasts with different p53 status. The results presented here showed that chelidonine and homochelidonine affected the proliferation and viability of the human leukemic cells examined. Homochelidonine displayed preferentially concentration-dependent activity, which was not the case of chelidonine in the doses evaluated. Although chelidonine has been found to decrease the proliferation and viability of diverse cancer cells (Panzer et al. 2001; Kemeny-Beke et al. 2006; Kaminskyy et al. 2008), we have demonstrated that chelidonine-induced cytotoxicity was more pronounced in leukemic and lymphoma cells then in human quiescent PBMCs. Induction of apoptosis by chemotherapeutic agents is often mediated through p53-dependent mechanisms; however, p53 is mutated in nearly half of all invasive cancers (Marinelli et al. 2013). While observations proving that the cytotoxicity of chelidonine is associated with PS exposure were also described previously by Kemeny-Beke et al. (2006) and recently also by El-Readi in multidrug resistance cancer cell lines Caco-2 and CEM/ADR5000 (El-Readi et al. 2013), our study reports that homochelidonine shows a significant apoptotic activity against cancer cells from micromolar concentrations. Using leukemic T-cells with different p53 status, we found that chelidonine and homochelidonine possess high antileukemic activity through apoptosis activation in both Jurkat and MOLT-4 cells. Unexpectedly, although MOLT-4 cells were previously described as being hypersensitive to DNA damage-induced apoptosis (Ito et al. 2011), our observations showed that they were less affected by chelidonine and homochelidonine treatment compared to Jurkat cells considered as overall resistant to genotoxic agents (Havelek et al. 2014; Seifrtova et al. 2011). In this study, chelidonine and homochelidonine-induced cell death was found to be accompanied by mitochondrial membrane depolarization. Mitochondria have been described to play a pivotal role in apoptosis induction, as these organelles are the junction of at least two distinct apoptosis pathways (Christensen et al. 2013). Although our paper described the changes in mitochondrial membrane potential in cells after homochelidonine applications, disruption of mitochondria was previously indicated after treatments using the chelidonine-based drug Ukrain. In the study of Habermehl, Ukrain treatment resulted in depolarisation of the mitochondrial membrane in Jurkat cells (Habermehl et al. 2006). In what concerns the effect of chelidonine on the cell cycle, our results are in conformity with the observations published by Panzer and co-workers, who studied the effect of chelidonine on oesophageal cancer cells (Panzer et al. 2001). To the best of our knowledge, this is the first paper to describe the potential of homochelidonine for decreasing the rates of Jurkat and MOLT-4 cell progression through the cell cycle. Our results show that both the extrinsic and the intrinsic signalling pathways would appear to contribute to homochelidonine and chelidonine-induced apoptosis. Our findings correlate well with the ability of the chelidonine-based thiotepa derivate Ukrain to induce caspase activation in Jurkat cells as described previously (Habermehl et al. 2006). In addition to this it was recently reported that chelidonine-induced apoptosis in MDR cells was accompanied by an activation of caspases -3, -8,-6/9 (El-Readi et al. 2013). In this study we evaluated the influence of chelidonine and homochelidonine on the expression and the activation of Chkl and Chk2. Chkl is known to be activated by ATM/ATR kinase-mediated phosphorylation at Ser345, then it phosphorylates Cdc25A/C and finally causes cell arrest in the late S or G2 phase. The activation of Chk2 in checkpoint signalling is initiated by phosphorylation at Thr68 in an ATM/ATR-dependent manner. In addition to the checkpoint regulation of Chkl and Chk2, recent discoveries have highlighted the roles of both kinases in controlling DNA repair and apoptosis (Huang et al. 2008). The p53 is the most important tumour suppressor gene in human cancer. Activation of normal (wild-type) p53 via phosphorylation is observed in stressed cells, such as those exposed to ionizing radiation or chemotherapy (Mirzayans et al. 2012). Our results demonstrated that p53 and its phosphorylated form at Serl5 do not increase after both chelidonine and homochelidonine treatment at 5 and 10[micro]M in MOLT-4 cells. Contrary to our results, the opposite effect of chelidonine at 1, 1.5 and 2mg/ml on p53 expression was observed in malignant melanoma cells by Hammerova and colleagues. Exposure to chelidonine led to a slight increase in the p53 level; however this was to a much lesser extent compared to that of doxorubicin used as positive control in the study (Hammerova et al. 2011). It is reported here that chelidonine and homochelidonine have a growth-inhibitory effect on A549 cells associated with changes in cell morphology and the adhesion degree. The observations that chelidonine had an inhibitory effect on cancer cell proliferation are in conformity with the conclusions published by Hammerova and colleagues. When they analysed the antiproliferative effect of chelidonine at 0.5 [micro]g/ml using the xCELLigence system, they observed immediate decreases in the cell index probably mediated by a detachment of the A375 malignant melanoma cells from the surface of the plate. Detached cells were found to be dead as observed by subsequent PI staining (Hammerova et al. 2011). The antiproliferative effect of chelidonine was also observed in HepG2 cells, in planarian stem cells and MCF-7 cells (Noureini et al. 2009; Isolani et al. 2012; Noureini et al. 2014). Some cytostatic drugs induce their effect by targeting tubulins. Using fluorescence imaging we examined the morphological alteration of the A549 cells after treatment with chelidonine, homochelidonine and nocodazole, focusing on microtubules and nuclei. The results presented in this study suggest that chelidonine and homochelidonine interfere with microtubule depolymerisation. Contrary thereto, treatment with nocodazole disassembled the microtubular structure in cells. In conformity with our data, chelidonine inhibits tubulin polymerisation in HeLa and Vero cells (Panzer et al. 2001). Later, chelidonine was verified as a very potent microtubule-destabilizing agent in another study on micromolar concentrations (Gertsch et al. 2007).

In conclusion, our data indicate that chelidonine and homochelidonine are potent inducers of cell death in examined cancer cell lines. Based on cytotoxicity evaluation against human cancer and noncancer cells, chelidonine and homochelidonine are more active against blood cancer cell lines (with the exception of erythroleukemia HEL 92.1.7 and histiocytic lymphoma U-937 cells in case of homochelidonine treatment) when compared to the human quiescent PBMCs and normal human fibroblasts. The mechanism of anticancer action involves an interaction with tubulin, induction of apoptosis associated with phosphatidylserine exposure, loss of mitochondrial membrane potential and activation of the caspases -3/7, -8 and -9. Exposure to homochelidonine at 5 and 10[micro]M induced G1 cell cycle phase accumulation in MOLT-4 cells and decreased the rates of Jurkat and MOLT-4 cells progression through the cell cycle, whereas the arrest of Jurkat and MOLT-4 cells at G2/M predominated at higher concentrations. Contrary thereto, cell cycle arrest in the G2/M phase was found in both chelidonine-treated leukemic cell lines independently of the concentration used. Treatment with chelidonine and homochelidonine also induced activation of Chkl, Chk2 and phosphorylation of histone H3 at SerlO in Jurkat and MOLT-4 cells. Good activity of naturally occurring homochelidonine which incorporates an open dioxole ring suggests that this structural feature is not essential for cytotoxic activity.

http://dx.doi.org/10.1016/j.phymed.2016.01.001

ARTICLE INFO

Article history:

Received 15 July 2015

Revised 30 December 2015

Accepted 3 January 2016

Conflict of interest

The authors declare that there is no conflict of interest to reveal.

Acknowledgements

The authors would like to thank Ing. Ondrej Sedlak and Mr. Pavel Rozkosny (Nikon spol. s r.o., Czech Republic) for collaboration on the confocal microscopy and super-resolution microscopy imaging experiments. We also wish to thank Ivana Fousova for her skilful technical assistance. This study was financially supported by the ROUTER CZ.1.07/2.3.00/30.0058 Programme of the University of Pardubice and the PRVOUK P37/01 Programme of Charles University in Prague. Radim Havelek is co-financed by the European Social Fund and the state budget of the Czech Republic Project Nos. CZ.1.07/2.3.00/30.0058.

Supplementary Materials

Visual representation of clonogenic assay in non-small human lung adenocarcinoma A549 cells treated with chelidonine and homochelidonine. Dose-response curves for chelidonine and homochelidonine as determined by use of cell counting and Trypan blue exclusion staining.

Apoptosis determined by Annexin V and PI staining 48 hours following treatment with chelidonine and homochelidonine.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.01.001.

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Radim Havelek (a), *, Martina Seifrtova (b), Karel Kralovec (a), Eliska Krocova (a), Veronika Tejkalova (a), Ivan Novotny (c), Lucie Cahlikova (d), Marcela Safratova (d), Lubomir Opletal (d), Zuzana Bilkova (a), Jirina Vavrova (e), Martina Rezacova (b)

(a) Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Studentska 573, Pardubice 532 10, Czech Republic

(b) Department of Medical Biochemistry, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Simkova 870, Hradec Kralove 500 38, Czech Republic

(c) Flow Cytometry and Light Microscopy, Institute of Molecular Genetics of the ASCR, Videnska 1083, Prague 142 20, Czech Republic

(d) ADINACO Research group, Department of Pharmaceutical Botany and Ecology, Faculty of Pharmacy, Charles University in Prague, Heyrovskeho 1203, Hradec Kralove 500 05, Czech Republic

(e) Department of Radiobiology, Faculty of Military Health Sciences, University of Defence in Brno, Trebesska 1575, Hradec Kralove 500 01, Czech Republic

Abbreviations: CH, chelidonine; HCH, homochelidonine; p53, tumour suppressor p53 protein; 3D, three-dimensional; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; DMSO, dimethyl sulphoxide; FITC, fluorescein isothiocyanate; PI, propidium iodide; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide; PBS, phosphate buffered saline; BrdU, 5-bromo-2'-deoxyuridine; TBS, tris-buffered saline buffer; BSA, bovine serum albumin; DAPI, 4',6-diamidino-2-phenylindole; BCA, bicinchoninic acid; Chk1, checkpoint kinase 1; Chk2, checkpoint kinase 2; PS, phosphatidylserine; SD, standard deviation; PBMCs, peripheral blood mononuclear cells; DPBS, Dulbecco's Phosphate-Buffered Saline; CI, cell index.

* Corresponding author. Tel.: +420 466037786.

E-mail address: radim.havelek@upce.cz (R. Havelek).

Table 1
[IC.sub.50] values of the chelidonine and homochelidonine against
mini-panel of human cancer and noncancerous cells with different
tumour suppressor protein p53 gene (TP53) status (a,b,c).

Cell type                      TP53 status

MOLT-4         Cancerous       Wild-type
Jurkat         Cancerous       Mutated
HL-60          Cancerous       Null
Raji           Cancerous       Mutated
U-937          Cancerous       Mutated
HEL 92.1.7.    Cancerous       Wild-type
PBMCs          Noncancerous    Wild-type
MRC-5          Noncancerous    Wild-type
WI-38          Noncancerous    Wild-type

Cell type      Chelidonine         Homochelidonine

MOLT-4         4.6 [+ or -] 0.2    4.8 [+ or -] 0.3
Jurkat         2.2 [+ or -] 0.1    5.6 [+ or -] 0.2
HL-60          4.4 [+ or -] 0.1    8.3 [+ or -] 0.4
Raji           3.2 [+ or -] 0.6    6.8 [+ or -] 0.3
U-937          5.0 [+ or -] 0.2    >10
HEL 92.1.7.    3.4 [+ or -] 0.7    >10
PBMCs          >10                 >10
MRC-5          1.8 [+ or -] 0.2    >10
WI-38          >10                 >10

(a) Results are expressed in [micro]M.

(b) Results are the mean values [+ or -] standard deviations of three
independent replications.

(c) Compounds with [IC.sub.50] value > 10 [micro]M were considered as
not active against evaluated cells.
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Author:Havelek, Radim; Seifrtova, Martina; Kralovec, Karel; Krocova, Eliska; Tejkalova, Veronika; Novotny,
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EXCZ
Date:Mar 15, 2016
Words:10118
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