Background
Breast and cervical cancers are the most frequent malignancies in women worldwide [
1,
2]. Uncontrolled cell proliferation, which is associated with the loss of the proper cell cycle control, is a prominent feature in these cancers. The cell cycle is controlled by a highly conserved family of cyclin-dependent kinases (Cdks) and their regulatory subunits cyclins. Among the cyclins, cyclin B1 plays a pivotal role as a regulatory subunit for Cdk1, which is indispensable for the transition from G2 phase to mitosis. Overexpression of cyclin B1 has been reported in various human tumors, such as breast cancer, cervical cancer, gastric cancer, colorectal cancer, head and neck squamous cell carcinoma and non-small-cell lung cancer [
3‐
9] and its upregulation is closely associated with poor prognosis in various types of cancers including breast cancer [
6,
10,
11]. Moreover, overexpression of cyclin B1 is involved in the resistance to radiotherapy in head and neck squamous cell carcinoma [
8] and nuclear cyclin B1-positive breast carcinomas are resistant to adjuvant therapy [
11]. More recently, it is reported that both antibodies and T cells are generated in response to aberrant cyclin B1 expression in tumors like breast cancer [
12,
13], indicating that overexpressed cyclin B1 could serve as one of the signals to initiate the communication between cancer cells and their microenvironment.
The mechanisms accounting for overexpressed cyclin B1 are not yet totally understood. It has been reported that the tumor suppressors p53 and BRCA1 negatively regulate the promoter of cyclin B1 [
14‐
17], whereas the oncogene c-Myc positively regulates the expression of cyclin B1 in cooperation with the loss of p53 [
18]. The promoter of cyclin B1 is also upregulated by 17beta-estradiol (E2), insulin-like growth factor I (IGF-I) and prolactin-releasing hormone (PRL), which are considered as the factors contributing to mammary cancer development and progression [
19‐
21]. Moreover, cyclin B1 mRNA is significantly stabilized in cervical cancer cells infected with human papillomavirus type 18 (HPV 18) through upregulating HuR [
22], a ubiquitously expressed member of the Hu family of RNA-binding proteins.
The highly expressed cyclin B1, even in G1 phase, binds to its partner Cdk1, which phosphorylates a series of substrates regardless of the cell cycle phase and contributes to the aggressive proliferation in neoplastic tissues [
23]. In addition, overexpression of cyclin B1 is related to aneuploidy and high proliferation of human mammary carcinomas [
24]. This is consistent with the observation of cyclin B1 overexpression enabling cells to override the G2 DNA damage checkpoint [
16,
25,
26]. Nuclear cyclin B1, together with Cdk1AF, a Cdk1 mutant that cannot be phosphorylated at its inhibitory sites, induced a striking premature mitotic phenotype even after DNA damage [
25,
26], resulting in accumulation of genomic defects, one hallmark of neoplastic development. More strikingly, enforced expression of cyclin B1 induces tetraploidy, either after mitotic spindle inhibition of nocodazole or in the absence of such inhibition if cyclin B1 is coexpressed with c-Myc [
18].
Taken together, deregulation of cyclin B1 is involved in neoplastic transformation and promotes proliferation of tumor cells. Conversely, downregulation of cyclin B1, consequently reducing the activity of Cdk1/cyclin B1, could block the aggressive proliferation of tumor cells. Indeed, our previous data confirm that interfering with cyclin B1 function inhibits proliferation of human tumor cells [
27,
28]. In the present study, we focus on gynecological cancer cell lines and investigate the effect of small interfering RNA (siRNA) induced cyclin B1 knockdown on tumor cell proliferation. Interestingly, the combination of cyclin B1 siRNA with taxol substantially enhanced the inhibitory effect on proliferation of breast cancer cells. Furthermore, while control HeLa cells were progressively growing, the tumor growth of HeLa cells treated with cyclin B1 siRNA prior to inoculation was strongly inhibited in nude mice, indicating cyclin B1 is indispensable for tumor growth
in vivo.
Methods
Cell culture, reagents and cell synchronization
Cervical cancer cell line HeLa and breast cancer cell lines MCF-7, BT-474, SK-BR-3 and MDA-MB-231 were obtained from DSMZ (Braunschweig). Fetal calf serum (FCS) was purchased from PAA laboratories (Cölbe). Opti-MEM I, oligofectamine, glutamine, penicillin, streptomycin and trypsin were obtained from Invitrogen (Karlsruhe). Taxol was from Mayne Pharma (Haar). Cells were synchronized to G1/S boundary by a double-thymidine block. Briefly, cells were treated with 2 mM thymidine (Sigma-Aldrich, Taufkirchen) for 16 h, released into fresh medium for 8 h and subjected again to thymidine for further 16 h. To obtain prometaphase arrest, after initial thymidine incubation and 8 h release cells were exposed to 50 ng/ml nocodazole (Sigma-Aldrich) for 14 h.
Transfection of siRNA and the combined treatment with drugs or irradiation
Four siRNAs targeting cyclin B1 (NCBI accession number of cyclin B1: NM 031966) were synthesized by Dharmacon Research, Inc. (Lafayette), referred to as siRNA1-4. siRNA1 against cyclin B1 corresponds to positions 340–360 of the cyclin B1 open reading frame, siRNA2 to positions 476–496, siRNA3 to positions 776–796 and siRNA4 to positions 1302–1322. Control siRNA targeting green fluorescent protein (siGFP) was also purchased from Dharmacon. All siRNAs were 21 nucleotides in length and contained symmetric 3' overhangs of two deoxythymidines.
Cells were transfected with siRNA using transfection reagent oligofectamine, according to the manufacturer's instructions (Invitrogen). In brief, one day prior to transfection, cells were seeded without antibiotics to a density of 50–60%. In all experiments cells were transfected with siRNA1-4 or siGFP at a concentration of 10 nM. Cells were harvested 48 h after siRNA-treatment for cell cycle evaluation, Western blot analysis and kinase assay. The time kinetics of protein expression were carried out at 24 h, 48 h, 72 h and 96 h and proliferation assays were performed at 24 h, 48 h and 72 h after siRNA transfection in MCF-7 cells.
For chemotherapeutic treatment, MCF-7 cells were at first transfected with siRNA and 4 h later followed by treatment of taxol (3 ng/ml). For irradiation, 6 h post transfection cells were exposed to a single dose of 8 Gy at room temperature by a linear accelerator (SL 75/5, Elekta, Crawley, UK) with 6 MEV photons/100 cm focus-surface distance and a dose rate of 4.0 Gy/min. 48 h after transfection of siRNAs cells were harvested for proliferation assay, cell cycle analysis and apoptosis evaluation.
Western blot analysis and kinase assay in vitro
Cell lysis was performed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% Na-desoxycholate, 0.1% SDS, 1 mM Na
3VO
4, 1 mM phenylmethylsulphonyl-fluoride (PMSF), 1 mM Dithiothreitol (DTT), 1 mM NaF, and protease inhibitor cocktail Complete (Roche, Mannheim)). Total protein was separated by using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to Immobilon-P membranes (Millipore, Bedford, MA). Membranes were exposed to corresponding antibodies for 1 h in PBS containing 5% slim milk, washed with phosphate-buffered saline (PBS) containing 0.2% Tween-20, incubated subsequently with secondary antibodies for 1 h. Finally, the protein bands were visualized with the enhanced chemiluminescence reagent (ECL, Pierce, Rockford). Mouse monoclonal antibodies against cyclin B1 (1:5,000), Cdk1 (1:2,000), anti-mouse secondary antibodies (1:4,000) and anti-rabbit secondary antibodies (1:4,000) were purchased from Santa Cruz (Heidelberg). Rabbit polyclonal antibodies against PARP (poly(ADP-ribose) polymerase, 1: 1000) were from Cell Signaling Technology (Beverly). Mouse monoclonal antibodies against β-actin (1:200,000) were obtained from Sigma-Aldrich. Western blots were quantified by applying a Kodak gel documentation system (model 1D 3.5) and standardized with loading control. For kinase assays
in vitro, antibodies against cyclin B1 (Santa Cruz, Heidelberg) were used for immunoprecipitation from 600 μg of cellular extracts. 0.5 μg histone H1 (Calbiochem, Darmstadt) served as substrate for each reaction. Kinase assays were performed as previously described [
29].
Cell proliferation, cell cycle analysis and apoptosis assay
Cell viability was assessed by trypan blue staining. The proliferation rate of cells was determined at indicated time points by counting cell numbers with a hemacytometer. All experiments were performed in triplicate. For cell cycle analysis, cells were harvested, washed with PBS and fixed in 70% chilled ethanol at 4°C for 30 min, then treated with 1 mg/ml of RNase A (Sigma-Aldrich) and stained with 100 μg/ml of propidium iodide for 30 min. The DNA content of 10,000 cells was determined with a fluorescent-activated cell sorter FACScan (Becton Dickinson Biosciences, Heidelberg). The data were analysed with cell cycle analysis software ModFit LT 2.0 (Verity Software House, Topsham, ME). Most of the experiments were performed in triplicate. Indirect immunofluorescence staining for subcellular cyclin B1 localization and DNA were carried out as previously described [
29]. Apoptosis was assessed using Vybrant™ apoptosis assay kit according to the manufacturer's instructions (Molecular Probes, Leiden).
MCF-7 cells were treated with siRNA1-3 or siGFP for 48 h and harvested for colony formation assays. Briefly, cells were seeded in 24 well-plates at a density of 200 cells/well into culture medium containing 0.3% agar (Roth, Karlsruhe) overlaying 0.5% agar. Cells were cultured at 37°C with 5% CO2, and colonies were counted 4 weeks later using a microscope (Zeiss, Oberkochen). The colony number in control sample was referred as 100% by quantification.
As to experiments in vivo, HeLa cells were treated with siRNA3 or siGFP and harvested after 48 h. HeLa cells (1 × 106) were resuspended in 300 μl of 0.9% NaCl and subcutaneously injected into both flanks of nude mice. Each group contained 4 mice. Three weeks after inoculation the tumor sizes were measured every 3–4 days using callipers and the tumor volumes were calculated according to a standard formula: π/6 × length × width2. The tumor volumes within the group were represented by the mean value. All mice were properly treated in accordance with the guidelines of the local animal committee.
Statistic analysis
For assays in vitro, Student's t-tests were used to evaluate the significance of difference between control cells and siRNAs-treated cells. Differences were considered as statistically significant when p < 0.05. With xenograft mouse model, the significant difference between the siGFP-treated group and siRNA3-treated group was analyzed by Mann-Whitney U test.
Discussion
The prognosis of breast and cervical cancer patients has been improved during recent years, related partly to sophisticated surgery, radiotherapy and adjuvant systemic therapy. Despite these advances, these cancers remain major clinical problems by causing considerable morbidity and mortality in women worldwide. Apart from the standard approaches, novel potent molecular agents for anticancer therapy are in great demand.
In this communication we show that the knockdown of cyclin B1, the regulatory subunit of Cdk1, inhibited cell proliferation and induced apoptosis in various breast and cervical cancer cell lines. Importantly, siRNA mediated cyclin B1 knockdown in combination with chemotherapeutical agent taxol, enhanced the antiproliferative effect on breast cancer cells. Interestingly, the reduction of cyclin B1 in MCF-7 cells impaired colony-forming ability, a hallmark of malignancy in tumor cells. Moreover, while control HeLa cells were progressively growing, the tumor growth of HeLa cells treated with siRNA targeting cyclin B1 prior to inoculation was strongly inhibited in nude mice, indicating cyclin B1 is indispensable for tumor growth in vivo. Taken together, the data strengthen the notion of cyclin B1 being required for the survival and proliferation of breast and cervical cancer cells and depletion/downregulation of cyclin B1 inhibits proliferation of cancer cells in vitro as well as in vivo.
Recent genetic evidence demonstrates that Cdk1 is the only Cdk sufficient to drive the mammalian cell cycle because embryos from Cdk1
-/
- mice fail to develop to the morula and blastocyst stages, whereas mouse embryos lacking all interphase Cdks (Cdk2, Cdk3, Cdk4 and Cdk6) undergo organogenesis and develop to midgestation [
33]. These data underscore that Cdk1 is essential for cell cycle regulation and a major force driving cell proliferation. Cyclin B1, the regulatory subunit of Cdk1, controls the activity of Cdk1 as it associates with and thereby activates Cdk1, regulates its nuclear translocation and passively mediates its inactivation when cyclin B1 is degraded at anaphase transition. Cyclin B1 is fundamental for cell proliferation. Uncontrolled expression of cyclin B1 is associated with neoplastic transformation and gynecological cancer development [
5,
9,
11,
34,
35]. Overexpression of cyclin B1 is believed to confer therapy resistance [
8,
11]. Thus, targeting cyclin B1, leading consequently to the inactivation of Cdk1, could be a promising specific strategy for cell cycle intervention against breast and cervical cancer.
In this work, as a proof-of-concept, the RNA interference was used to downregulate/deplete cyclin B1 and a clear antiproliferative effect was observed in all cancer cell lines studied. Among the breast cancer cell lines investigated, MCF-7 cells exhibited the strongest inhibitory effect on cell proliferation after cyclin B1 siRNA treatment, followed by MDA-MB-231, SK-BR-3 and BT-474 cells (Fig.
2), which possibly correlates with the cyclin B1 level in exponential growing status of each cell line (data not shown). Although the protein level of cyclin B1 in MDA-MB-231 cells was nearly undetectable after siRNA1 or siRNA3 transfection, the inhibitory impact was moderate (Fig.
2D), suggesting the proliferation of MDA-MB-231 cells is not necessarily dependent on the normal level of cyclin B1 and the little amount of remaining cyclin B1 might be sufficient for the survival of MDA-MB-231 cells. Finally, SK-BR-3 and BT-474 cells were also not as sensitive to siRNA treatment (Fig.
2B and
2C) as HeLa or MCF-7 cells. This could be due to the cellular context of SK-BR-3 and BT-474 cells, e.g. Her-2/
neu+, which very often leads to a hormone-independent proliferation of cells. Thus, unlike in MCF-7 cells, targeting Her-2/
neu or other factors promoting G1/S transition could be more effective for inhibiting cell cycle progression in SK-BR-3 and BT-474 cells, which has been shown by our previous study [
36]. On that account, specific targeting of oncogene(s) in individual cancer cell lines, like Her-2/
neu in SK-BR-3 and BT-474 cells, or cyclin B1 in MCF-7, could improve breast cancer therapy. Collectively, downregulation/depletion of cyclin B1 worked effectively in all gynecological cancer cell lines tested. However, only in some cell lines, such as MCF-7 and HeLa, cyclin B1 knockdown resulted in a strong proliferative inhibition, most likely because proliferation in those cell lines is more dependent on high cyclin B1 levels as compared to other cell lines.
Taxane drugs represent the most important class of anticancer agents and are integrated in multidrug-regiments for the therapy of several solid tumors including gynecological cancers. Despite their relevant contribution in ameliorating the quality of life and overall survival of cancer patients, drug resistance and site-effects hamper its wide usage. Therefore, it is desirable to find new ways of lowering drug dosage without losing effectiveness to limit side-effects and possibly also to slow down drug resistance. In this work, cyclin B1 siRNA in combination with taxol, blocking entry into mitosis and targeting the transition of metaphase to anaphase, respectively, demonstrated a high efficacy in inhibiting proliferation of MCF-7 cells. The data suggest that specific targeting of cyclin B1 could sensitize some gynecological cancer cells, like MCF-7 and MDA-MB-231 cells, to conventional chemotherapeutic agents like taxol, thereby reducing their side-effects by lowering their dosage.
Taken together, the data from this work further strengthen the notion that cyclin B1 could be an attractive target for potential anticancer therapy. Inhibiting cyclin B1 function in combination with chemotherapeutic drugs could reinforce the antiproliferative effect in a subset of cancers. As RNA interference still faces the major challenge of systematic delivery [
37], an alternative strategy could be small molecule inhibitors targeting cyclin B, as its crystal structure is recently published [
38]. In parallel to Cdk inhibitors, which have been extensively under clinical investigations, small molecule inhibitors against cyclin B1 could open up a new door for specific molecular cancer therapy by interfering with its protein stability, binding capacity to Cdk1 or its subcellular localization.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
IA and AK conducted cell cycle analyses, proliferation assays, Western blot analyses, apoptosis assays and mouse xenograft experiments in vivo. RY performed the kinetics of MCF-7 cells and soft-agar assays. FR is involved in the combination therapy and assays. MK and RG coordinated this project. KS co-supervised this study and supported the manuscript writing. JY designed and supervised this study, and drafted the manuscript. All the authors read and approved the final manuscript.