Sodium selenite alters microtubule assembly and induces apoptosis in vitro and in vivo
© Shi et al.; licensee BioMed Central Ltd. 2013
Received: 19 November 2012
Accepted: 9 January 2013
Published: 17 January 2013
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© Shi et al.; licensee BioMed Central Ltd. 2013
Received: 19 November 2012
Accepted: 9 January 2013
Published: 17 January 2013
Previous studies demonstrated that selenite induced cancer-cell apoptosis through multiple mechanisms; however, effects of selenite on microtubules in leukemic cells have not been demonstrated.
The toxic effect of selenite on leukemic HL60 cells was performed with cell counting kit 8. Selenite effects on cell cycle distribution and apoptosis induction were determined by flow cytometry. The contents of cyclin B1, Mcl-1, AIF, cytochrome C, insoluble and soluble tubulins were detected with western blotting. Microtubules were visualized with indirect immunofluorescence microscopy. The interaction between CDK1 and Mcl-1 was assessed with immunoprecipitation. Decreasing Mcl-1 and cyclin B1 expression were carried out through siRNA interference. The alterations of Mcl-1 and cyclin B1 in animal model were detected with either immunohistochemical staining or western blotting. In situ detection of apoptotic ratio was performed with TUNEL assay.
Our current results showed that selenite inhibited the growth of HL60 cells and induced mitochondrial-related apoptosis. Furthermore, we found that microtubule assembly in HL60 cells was altered, those cells were arrested at G2/M phase, and Cyclin B1 was up-regulated and interacted with CDK1, which led to down-regulation of the anti-apoptotic protein Mcl-1. Finally, in vivo experiments confirmed the in vitro microtubule disruption effect and alterations in Cyclin B1 and Mcl-1 levels by selenite.
Taken together, the results from our study indicate that microtubules are novel targets of selenite in leukemic HL60 cells.
Microtubules have important roles in many cell behaviors such as cell division, organelle positioning, vesicular transport and cell-shape determination [1–3]. Previous studies have showed that microtubule dynamics are necessary for these functions in vivo[2, 4–6]. Therefore, chemicals affecting microtubule dynamics often impact these functions in vivo. On that basis, many anti-tumor agents have been developed for their effects on microtubule dynamics and cell-cycle distribution [7–12].
Selenium (Se) is an essential trace element , and appropriate selenium intake is necessary for the body to synthesize selenoproteins. Some researchers have shown that selenite concentrations that are within the nutritional range inhibit tumor formation by acting on antioxidants and in the inhibition of DNA adduct formation, the promotion of cell cycle progression and DNA repair [14–16]. However, super-nutritional levels of selenite induce endoplasmic reticulum stress, mitochondrial-related apoptosis, DNA strand breaks and cell-cycle arrest [15–19]. Therefore, many molecules, such as Akt, GADD153, P53, ERK, P38, Bad, Bim and Bax [20–24], have been reported to be involved in high-dose selenite-induced apoptosis. Additionally, super-nutritional selenite intake has been shown to be toxic to drug-resistant cancer cells and effective on tumor xenografts, which suggests that selenite has potential therapeutic effects [23–25].
In an in-depth study of selenium, selenite was reported to have strong inhibitory effects on sulfhydryl-containing proteins  such as tubulins, which composed microtubules [26, 27], but the effects of selenite on microtubules in cancer cells had not been proven. Based on our proteomics study, proteins linked to microtubule dynamics were thought to have roles in selenite-triggered apoptosis . Therefore, our study aimed to investigate the role of selenite in microtubule assembly and induction of apoptosis.
Selenium is an essential trace element for animals, and it has been shown that super-nutritional selenite intake has anti-tumor activity [14, 18, 19, 27, 29]. Several reports have also proved the anti-tumor effects of selenite in vivo. However, the effects of selenite on microtubule dynamics in cancer cells have not been demonstrated. Our study indicated that microtubules were a novel target of selenite.
Leynadier D et al. first discovered that selenite could directly interact with the sulfhydryl groups of β-tubulin and could inhibit microtubule polymerization in vitro. To our knowledge, we are the first to discover that selenite also induces microtubule depolymerization in HL60 cells and in vivo. However, because microtubules reorganized in Jurkat but not in HL60 cells, the apoptotic mechanisms of the two cell lines differed. We mainly investigated the mechanisms by which selenite induced apoptosis. Because tumor cells have a strong ability to replicate themselves and tubulins, which compose spindles, are essential for this process, we speculate that selenite-induced apoptosis is at least partly dependent on the effects of selenite on microtubules. Therefore, the growth inhibitory effect of selenite on cultured HL60 cells was assessed, and we discovered that 20 μM of sodium selenite significantly inhibited cell growth. Furthermore, Annexin V-FITC/PI double staining assay proved that selenite-induced apoptosis occurred and nuclear fragmentation was witnessed in selenite-treated cells. Last, we discovered that cytochrome C and AIF were released from the mitochondria to the cytoplasm, which suggested that selenite-induced apoptosis in HL60 cells might be associated with the mitochondrial apoptotic pathway. Cell cycle-related proteins that were consistently altered with microtubule dynamics could regulate Bcl-2 family members, which were located in the mitochondria. Therefore, we speculated that selenite inhibited HL60 cell growth through its effects on microtubules.
Several reports suggested that microtubule-interfering drugs affected cell cycle distribution by regulating the activity of CDKs and, therefore, altering protein phosphorylation at different cell cycle phases . Mcl-1, a Bcl-2 family member, is regulated by the Cyclin B1/CDK1 complex and is linked to the mitochondrial apoptotic pathway by binding and inhibiting pro-apoptotic proteins [12, 30, 31]. Our current study proved that selenite could induce cell cycle arrest and remarkable alterations of Cyclin B1 and Mcl-1 levels in HL60 cells through its effect on microtubule depolymerization. Interestingly, a combination treatment of Colchicine and selenite in Jurkat cells up-regulated Cyclin B1 and down-regulated Mcl-1. The observations in Jurkat cells also supported the relationship between microtubule destruction and alterations in Cyclin B1 and Mcl-1 after selenite exposure. Cyclin B1 is necessary for the activity of CDK1, which phosphorylates and destabilizes Mcl-1 [32–36]. We observed that Cyclin B1 interacted with CDK1. Furthermore, either siRNA interference of Cyclin B1 or inhibition of the CDK1/Cyclin B1 complex with Roscovitine rescued the decrease of Mcl-1. Further investigation confirmed the protective role of Mcl-1 on HL60 cells and suggested that the growth inhibitory effects of selenite might be associated with the down-regulation of Mcl-1. Finally, a combination of siRNA targeting Mcl-1 and selenite treatment caused a higher apoptotic ratio than selenite treatment alone. These results supported our conclusion that selenite altered microtubule assembly and inhibited HL60 cell growth through cell cycle arrest and decrease in Mcl-1 levels.
The above-described experiments indicated that selenite altered microtubule assembly and induced cell cycle arrest in HL60 cells. To identify the therapeutic activity of selenite in vivo, we established a HL60-cell-bearing nude mice model. Experiments in vivo showed that selenite inhibited tumor growth and induced nucleus pyknosis. Furthermore, we also found that selenite depolymerized microtubules in vivo. Additional experiments demonstrated that alterations of Cyclin B1 and Mcl-1 levels in the nude mice model were similar to those findings in vitro, which suggested that the mechanisms demonstrated in vitro were also active at the tissue level.
In conclusion, the microtubule destruction that was induced by selenite stimulated the apoptotic pathway by up-regulating Cyclin B1, which interacted with CDK1 and destabilized the anti-apoptotic protein Mcl-1. We also found that sodium selenite had therapeutic functions in a HL60-cell-bearing nude mice model through its microtubule destruction effects. Importantly, this investigation explored the effects of selenite on apoptosis in a distinct way.
Roscovitin, anti-β-Tubulin (2-28-33) and anti-β-Actin (AC-15) antibodies were obtained from Sigma-Aldrich. Anti-Cyclin B1 and anti-Mcl-1 antibodies, which were used for western blotting, were obtained from Cell Signaling Technology. For immunohistochemical staining, an anti-Cyclin B1 antibody was purchased from Excell, an-anti CD33 antibody was purchased from BIOSS and an anti-Mcl-1 antibody was purchased from Santa Cruz. The Cdc2 (1/Cdk1/Cdc2) antibody was purchased from BD Biosciences Pharmingen. HRP-conjugated anti-mouse and anti-rabbit antibodies were purchased from ZSGB-BIO. A FITC-conjugated anti-mouse antibody was purchased from Jackson.
HL60 and Jurkat cells were grown in RPMI 1640 medium containing 10% advanced fetal bovine serum, 100 units/mL penicillin and 100 units/mL streptomycin and incubated in a humidified, 5% CO2 incubator that was set at 37°C.
HL60 cells (8 × 105 total) were harvested. The cells were transferred to slides, fixed in 4% paraformaldehyde and permeabilized using 0.1% Triton X-100. After the slides were blocked with 2% BSA, the cells were incubated with β-tubulin antibody overnight at 4°C. After washing with PBS three times, the cells were incubated with FITC-conjugated secondary antibody for 60 min at room temperature. After a second round of washing, the cells were stained with DAPI for approximately 5 min, and the slides were washed three times and mounted in anti-fading medium. Images were visualized using a Zeiss microscope (Carl Zeiss, Jena, Germany).
Approximately 1 × 106 cells were collected for each treatment. After washing with ice-cold PBS, the cells were resuspended in RIPA lysis buffer (20 nM Tris, pH 7.5; 1 mM EDTA; 1 mM EGTA; 150 mM NaCl; 1% Triton X-100; 2.5 mM sodium pyrophosphate; 1 mM β-glycerolphosphate; 1 mM Na3VO4; 1 mM PMSF; and 1 μg/mL leupeptin) and were submitted to ultrasonication on ice. The lysates were centrifuged at 12,000 × g for 20 min at 4°C, and equal amounts of proteins were separated by SDS-PAGE. The proteins on the PAGE gel were then transferred to a nitrocellulose membrane. After being blocked with 5% non-fat milk, the membranes were washed with TBST and incubated overnight with primary antibody at 4°C. After being washed three times with TBST, the membranes were incubated with a HRP-conjugated secondary antibody for approximately 1 h at room temperature. Subsequently, the membranes were washed another three times and probed with supersignal chemiluminescent substrate.
Cells (1 × 107) were harvested and washed twice with ice-cold PBS. The pellets were resuspended in RIPA buffer and lysed on ice for 30 min. Subsequently, the lysates were centrifuged at 12,000 × g for 20 min at 4°C. A suitable amount of cdc2 antibody was added to the protein lysate (200 μg) and rotated overnight at 4°C, while the remaining protein was used as input. Protein A + G was added, and the mixture was rotated for another 3 h at 4°C; then, the samples were washed with RIPA buffer three times. Finally, the beads were resuspended in 3 × SDS loading buffer and boiled for 10 min. After a short centrifugation step, the supernatant was collected.
siRNAs targeting Cyclin B1 (5′-CCAAACCTTTGTAGTGAAT-3′), Mcl-1 (5′-GGACTGGCTAGTTAAACAA-3′) and negative controls for each sequence were synthesized by GenePharma. Approximately 1 × 107 cells were collected and washed with Opti-MEM medium (Gibco). Then, the cells were transfected with 200 nM siRNA and RNAiMAX in Opti-MEM. After transfection for approximately 12 h, the cells were treated with sodium selenite for 24 h.
Approximately 1 × 106 cells were collected and fixed in 70% ethanol overnight at 4°C. Each sample was centrifuged at 1,000 × g for 10 min at room temperature and washed with ice-cold PBS. Subsequently, the cells were incubated with 50 μg/ml RNase in PBS for 30 min at 37°C. After adding PI to the cells at a final concentration of 50 μg/mL, we detected the absorption at 620 nm by flow cytometry.
The cells (1 × 106) were harvested and washed twice with ice-cold PBS. Subsequently, the cells were stained with AnnexinV-FITC in binding buffer in the dark for 15 min. After being centrifuged at 1,000 × g for 10 min, the cells were resuspended in binding buffer containing PI. Finally, the apoptotic ratio was determined by an Accuri C6 flow cytometry. We calculated the apoptotic ratio by calculating the sum of Annexin V+/PI- cells’ ratio and Annexin V+/PI+ cells’ ratio.
HL60 cells were seeded into a 96-well plate at a concentration of 40,000 cells per well. After treatment with varying concentrations of sodium selenite for 24 h, cell viability was assessed using CCK-8 kits (Dojindo Molecular Technologies, Tokyo, Japan).
An established method was modified and used to separate insoluble tubulin from soluble tubulin . Approximately 2 × 106 cells were collected and washed twice. The cells were then resuspended in hypotonic buffer at 37°C for 5 min. After centrifugation at 14,000 × g for 10 min at 25°C, the supernatants, which contained soluble tubulin, were collected, and the pellets containing insoluble tubulin were resuspended in RIPA buffer and subjected to ultrasonication on ice. The lysates were centrifuged at 12,000 × g for 10 min at 4°C, and the supernatants were collected.
At the beginning of the experiment, 4-week-old female mice were chosen and divided into control or selenite-treated group randomly. Each group was marked and put into its own box. The two groups lived in the same context and were fed with the same food and water. Leukemia HL60 cells were inoculated subcutaneously into female nude mice. After tumors were palpable, an intraperitoneal injection of sodium selenite dissolved in PBS was given to each mouse every 2 days (1.5 mg/kg/day) for 3 weeks, and the control group was treated with PBS for the same period time. At the end of the experiment, the mice were sacrificed, and the tumors and spleens were rapidly removed and weighed. The Declaration of Helsinki and the guide for Laboratory Animal Care and Use were maintained.
The slides were deparaffinized in xylene and dehydrated with decreasing concentrations of ethanol. After the slides were washed with running water for 2 min, endogenous peroxidase was blocked with 3% peroxide that was dissolved in methanol. Subsequently, the slides were immersed in boiled sodium citrate for antigen retrieval, and after being washed three times with 0.01 M PBS for 5 min, the slides were incubated with either anti-Mcl-1 or anti-Cyclin B1 antibody overnight at 4°C. The slides were incubated with secondary antibody at room temperature for 3 h, treated with DAB, stained with Mayer’s hematoxylin for 2 min and washed with running water. Slides were then dehydrated with increasing concentrations of ethanol and clarified with xylene. Finally, the slides were mounted with medium.
After dehydration with decreasing concentrations of ethanol as described above, the slides were stained with Harriss hematoxylin for 15 min and washed for 3 min. Then, the slides were immersed in 1% hydrochloric acid dissolved in 75% ethanol for 30 s. Before dehydration, the slides were stained with eosin for 10 min. Finally, the slides were clarified with xylene and mounted with medium.
A FragEL™ DNA Fragmentation Detection Kit was purchased from MERCK. The slides were deparaffinized in xylene and dehydrated with decreasing concentrations of ethanol. After washing with 1 × TBS for 2 min, the slides were incubated with 20 μg/ml proteinase K for 20 min at room temperature. The slides were then washed with 1 × TBS and incubated with 1 × TdT buffer for approximately 30 min at room temperature. Subsequently, the slides were incubated with 57 μl of mix buffer and 3 μl of TdT enzyme for 60 min at 37°C. After being washed three times with 1 × TBS, the slides were mounted with medium.
The values were represented as mean ± SEM. Two-tailed students’ t-tests were used for two groups comparison analysis, and P < 0.05 was considered to be significant. The bar charts were used to reflect the alterations of experimental data [18, 24, 37, 38].
First author: Kejian Shi.
First of all, we would like to specially thank Dr. Yun Shen, Dr. Qun Xu and Master Yang Yang for their selfless aids. This work was supported by the National Natural Science Foundation of China (No. 31170788 and No. 30970655), the National Natural Science Foundation for Young Scholars of China (No. 31101018), the Natural Science Foundation of Beijing (No. 5082015), the State Key Laboratory Special Fund (No. 2060204) and the Ministry of Education of China Doctor Training Unit (No. 20091106110025).
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