Abstract
Background: Aloe-emodin is an anthraquinone with potential pharmacological properties, including numerous antitumor properties. The purpose of the study was to determine whether aloe-emodin induces mitotic death in cervical cancer cells. Materials and Methods: Analysis of morphological changes as surrogate mitotic death indicators in HeLa cells was carried out using optical, fluorescence and electron microscopy. Viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide reduction assay. Cell-cycle analysis was performed using flow cytometry. Results: Aloe-emodin increased the number of multinucleate cells, giant and micronuclear cells. There was a concentration-dependent decrease in the mitotic index with a predominance of cells in the metaphase of the mitotic process and inhibition of division in the G2/M phase of the cell cycle. The presence of cells with abnormal mitosis and cells with injury to the division spindle was also demonstrated. Conclusion: Aloe-emodin induces mitotic catastrophe in cervical cancer cells.
For many years, apoptosis has been recognized as the main type of cell death. Recent studies have shown that treatment of cancer apoptosis induced by chemotherapy drugs (including plant origin) in addition to programmed cell death, may induce alternative types of death in cancer cells, including mitotic catastrophe (1, 2). Compounds inducing mitotic catastrophe in HeLa cells is the subject of our research.
Aloe-emodin (1,8-dihydroxy-3-hydroxymethyl-9,10-anthrachinone) is on anthraquinone found in the leaves and roots of Rheum palmatum L. (3), Rhamnus frangula L., Rhamnus cathartica L. (4), Aloe barbadensis Mill., and Aloe arborescens Mill. (5). Numerous studies indicate that aloe-emodin is a compound with multiple biological activities, and its antitumor mechanism of action is based on proapoptotic and antiproliferative properties, and has still not been fully elucidated (6, 7).
The aim of the study was to evaluate the effect of aloe-emodin on morphological and biochemical changes in cervical cancer cells with particular attention to changes that could indicate cell death alternative to apoptosis, i.e. mitotic catastrophe.
Materials and Methods
In vitro culture conditions. HeLa cells were cultured in dishes (Nunc) at 37°C and with 5% carbon dioxide in a DirectHeat CO2 incubator (Thermo Fisher Scientific, Waltham, MA, USA). The culture was carried out on Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), with the addition of 1% of a mixture of antibiotics containing penicillin, streptomycin and amphotericin B. Reagents were from Thermo Fisher Scientific. Aloe-emodin (C15H10O5) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The cells were exposed to aloe-emodin at concentrations of 1, 15, 30, 60 and 100 μM for 24 and 48 h.
Preparation of cells for morphological analysis. The cells (control and test) were grown on sterile cover slides in culture dishes. After 24 and 48 hours of exposure to aloe-emodin, the cells were fixed in methanol, stained with Harris hematoxylin (Sigma-Aldrich) followed by eosin solution (Sigma-Aldrich). The preparations were dehydrated with increasing series of alcohol, cleared in xylene and immersed in Histokitt (Glaswarenfabrik Karl Hecht GmbH & Co KG, Germany). The experiment was performed in three independent experiments (three repetitions for individual concentrations and incubation times, including the control group).
Evaluation of cell mitotic index and morphological changes. Analysis of control cells and cells treated with aloe-emodin was performed using the Nikon Eclipse 80i light microscope with Nikon Instruments NIS Elements D 3.10 (Nikon Instruments, Amsterdam, the Netherlands). In each of the preparations 5,000 cells were counted in three independent experiments (15,000 cells/concentration/time).
Based on the obtained results, the mitotic index and the number of cells with morphological changes defining mitotic death were determined, including the presence of giant cells, multinucleated cells, cells containing micronuclei in cytoplasm, and the presence of cells inhibited in metaphase (8, 9). Cell analysis for micronuclei was performed based on the morphological characteristics reported by Fenech et al. (10). In addition, the number of apoptotic cells was determined.
Analysis of the cell cycle. The effect of aloe-emodin on cell distribution in the cell cycle was assessed by flow cytometry. The cells were incubated with aloe-emodin for 48 h at a concentration of 1 μM and 100 μM. After detaching the cells with trypsin, they were fixed with ice-cold 70% ethanol. The cells were rinsed in phosphate-buffered saline and then labeled with a suspension containing 0.1% NP-40, 10 μg/ml of DNAase-free RNAse and 5 μg of propidium iodide. After 15 min of incubation, the cells were analyzed using FACSCanto II and the FACSDiva program (BD Biosciences, San Jose, CA, USA). A total of 10,000 events were analyzed in each sample. The percentage of cells in individual phases of the cycle was estimated using the ModFit LT 4.1.7 program (Verity Software House, Topsham ME, USA).
Evaluation of ultrastructural changes. After 48 hours' incubation, cells for transmission electron microscopy were prepared according to the modified method of Marzella and Glauman (11). Cells were fixed in 3% glutaraldehyde and 2% osmium tetroxide (SPI Supplies, West Chester, PA, USA), and embedded in Epon 812 resin (Serva Electrophoresis, Heidelberg, Germany). Specimen for the TECNAI G2 Spirit microscope (FEI Company, Hillsboro, OR, USA) were prepared using EM UC7 ultramicrotome (Leica Microsystems, Wetzlar, Germany).
Labeling of cell nuclei with fluorochrome 4’,6-diamidine-2-phenylindole (DAPI). After 48 hours of incubation of the cells in the basal medium (control cells) and medium with aloe-emodin at 100 μM (test cells), staining was performed with 10 μg/ml DAPI solution (Sigma Aldrich). The prepared preparations were analyzed using a Nikon TiE fluorescence microscope (Nikon Instruments) using a DAPI dichroic filter block (358 nm excitation, emission above 461 nm).
Cell viability assay 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazole bromide (MTT) reduction assay. Cells plated on 96-well plates (Nunc) were incubated at 37°C for 48 hours in a medium containing aloe-emodin at concentrations of 1-100 μM. The medium was then removed and the cells were incubated for 2 h with a solution of MTT (Sigma-Aldrich) according to the modified Mossmann method (12). After removing the medium from MTT, precipitate (containing formazan crystals) was dissolved in DMSO and shaken for a period of 10 min. The absorbance was read on a Synergy 2 multimode microplate reader (BioTek, Winooski, VT, USA) at a wavelength of 570 nm and 690 nm. The experiment was performed in three independent experiments.
Statistical analysis. The significance of the obtained differences was determined using statistical analysis, carried out using Statistica 10.0 software (StatSoft, Krakow, Poland). The results obtained were evaluated using a non-parametric Chi-squared test. The cell viability analysis was supported by the Tukey's test and the cell-cycle distribution by Newman-Keul's test. The differences were statistically confirmed at p<0.05.
Results
Aloe-emodin inhibits cell division. Exposure of HeLa cells to increasing aloe-emodin concentration for 24 and 48 hours caused a highly statistically significant reduction in the mitotic index. The highest inhibition of division, to 3.50% (Figure 1), was demonstrated after 48-hour activity of aloe-emodin at 100 μM. It should be emphasized that cells in the metaphase stage (Figure 2) comprised 85.25% (24-hour incubation) and 88.68% (48-hour incubation) of the dividing cells.
From measurements made using flow cytometry, it appears that aloe-emodin induces cell-cycle arrest in HeLa cells (Figure 3). The most significant (F(2,6)=239,09, p<0.001, η2=0.988) increase in the percentage of cells was 60.78% in the G2/M phase as a result of treating cells with 100 μM aloe-emodin compared with 14.18% cells in the G2/M phase after aloe-emodin at a concentration of 1 μM and control (6.47%). At the same time, with 100 μM aloe-emodin, a reduction in cells in the G0/G1 phase to 26.33% (F(2,6)=279,084, p<0.001, η2=0.989) was demonstrated. Control cells in this phase accounted for 75.22%. The results indicate that the encumbrance of HeLa cells with aloe-emodin results in a concentration-dependent significant increase in the cell population in the G2/M phase.
Aloe-emodin induces abnormalities of mitosis and mitotic death. With respect to control values, treatment with aloe-emodin led to a highly statistically significant increase in the number of multinucleated cells, cells with micronuclei located near the nucleus and giant cells was demonstrated (Figure 4). Giant cells in the interphase stage contained several nuclei varying in size and shape, often with one or more micronuclei (Figure 5B and 6E). Cells with abnormal chromosomal segregation at various stages of mitotic division, with the chromosomes remaining in the cytoplasm, not pulled to the poles of the cell were also found (Figure 5D and 6C). The presence of cells with abnormal metaphase (bipolar, tripolar, and multipolar) was also characteristic of treatment with aloe-emodin (Figure 6B). The dispersal of the Golgi apparatus in the form of numerous vesicles in the cytoplasm was also observed in the cells (Figure 5C).
Aloe-emodin reduces viability and induces apoptosis of HeLa cells. After culture of cells for 48 hours in a medium supplemented with different concentrations of aloe-emodin, a highly statistically significant reduction in cell viability was observed (Figure 7B). The half-maximal inhibitory concentration (IC50 value) for aloe-emodin was determined by the MTT assay as 66.40 μM, while the IC90 (concentration inhibiting cell growth by 90%) as 75.21 μM. The highest reduction in cell viability (to 7.30%) was found at 100 μM aloe-emodin. At the same time, a highly significant increase in the number of apoptotic cells was observed after 24 (74.7%) and 48 (78.3%) hours of exposure to the test compound at 100 μM (Figure 7A). Apoptotic cells (labeled with DAPI fluorochrome) were characterized by clear chromatin condensation and nuclear fragmentation (Figure 7C).
Discussion
Among the existing models of cell death, such as apoptosis, necrosis, autophagy, oncosis, mitotic catastrophe is least described. It is the death of the cell in which anomalies occur during the process of mitosis. Mitotic catastrophe is a consequence of the dysfunction of cell-cycle checkpoints, and consequently improper chromosomal segregation and cell division. Morphologically, it is reflected by fragmentation of the nucleus, and the formation of large cells with one nucleus (13). Characteristic features may also be the lack of a nucleus or the presence of two or more nuclei in the cell, the formation of cells with a micronucleus, multinucleated cells, and giant cells, which are recognized as mitotic death markers (13). Cell death due to mitotic catastrophe occurs during mitosis, or as a result of its arrest in metaphase regardless of p53 or during abnormal mitotic division depending on p53 (1, 2, 14).
As a consequence of the 24- and 48-h action of the anthraquinone studied here on HeLa cells, an increase in the number of cells with micronuclei that arise during mitotic cell division was observed. The main mechanism contributing to their formation are chromosomal breakdown and dysfunction of the mitotic apparatus, the effect of which is their not being moved to the poles of the dividing cells and staying in the cytoplasm (15).
A statistically significant increase in the population of cells in the G2/M phase of the cell cycle, with simultaneous reduction of cells in the G0/G1 phase (Figure 3D), the retention of cell division mainly at the metaphase stage (Figure 2) and the presence of mitotic death markers, i.e. cells with abnormal mitosis (Figure 6), as well as an increase in the number of multinucleated and giant cells (Figure 4), testify to the sensitivity of tumor cells to the effects of aloe-emodin. Numerous literature data indicate that cells undergoing abnormal mitosis may undergo subsequent apoptotic death (8, 9). It is also believed that apoptosis may occur during mitotic catastrophe but through caspase-independent pathways (16). The highly statistically significant increase in the number of apoptotic cells revealed in the studies here is shown in Figure 7A and C. Our team's research revealed that aloe-emodin can stimulate the death of HeLa cells during mitosis through the process of mitotic catastrophe (Figure 4B and D, Figure 6).
As can be seen from the available literature, chemotherapeutics that affect the mitotic spindle may be the most effective in killing cancer cells, especially those resistant to apoptosis (16). In these cells, the signaling pathways leading to apoptosis are blocked (17).
It should be noted that the morphological indicators of death mitotic analyzed here were also observed in cells treated with other anticancer compounds, such as vinca alkaloids (vinflunine, vinorelbine, vincristine, vinblastine), taxanes (paclitaxel, docetaxel) (17-23), oxaliplatin (24), cisplatin (25), and doxorubicin (26).
A characteristic morphological change after aloe-emodin is also visible in Figure 4C, the dispersal of the Golgi apparatus. Numerous data indicate that the cytoskeleton plays a key role in the organization of the Golgi apparatus (27, 28). The changes revealed (Figure 5C) suggest that aloe-emodin induces disorganization of the cytoskeleton of the cell and at the same time indicates its antiproliferative properties.
Of note is the lack of cells in the anaphase, telophase and cytokinesis stage in cells treated with aloe-emodin at a concentration of 100 μM, which may additionally indicate its effect on the dividing spindle (Figure 2). Similar morphological changes have been demonstrated in cells treated with antimitotic drugs such as vincristine (29), colchicine (30, 31) and taxol (32, 33).
As shown in our studies, depending on concentration and exposure time (Figures 5, 6 and 7), the death of HeLa cells on the path of mitotic catastrophe induced by aloe-emodin can be an alternative to apoptosis. In our research, we also showed that another anthraquinone, emodin, is a compound that promotes the death of cervical cancer cells through mechanism that occurs with involvement of the lysosomal compartment (34).
Aloe-emodin showed a concentration-dependent and time-dependent induction of mitotic death in HeLa cell lines. This compound has similar effect to those currently used in the oncological treatment of plant-derived cytostatic. The changes in HeLa cells induced by aloe-emodin indicate its great potential as an anticancer compound.
Acknowledgements
This work was supported by Jan Kochanowski University, grant: 066/R/11.
Footnotes
Conflicts of Interest
All Authors have read the Journal's policy on disclosure of potential conflicts of interest and have none to declare.
- Received January 24, 2018.
- Revision received February 16, 2018.
- Accepted February 23, 2018.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved