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Aspirin has been confirmed as an effective antitumor drug in various cancers. However, the relationship between aspirin and uterine leiomyoma is still underexplored. Here, we explored the effects of aspirin on human uterine leiomyoma cells and provide insights into the underlying mechanisms. Cell Counting Kit-8 (CCK-8) and flow cytometry analysis showed that aspirin treatment inhibited cell proliferation and promoted cell cycle arrest at G0/G1 phase in a dose- and time-dependent manner of human uterine leiomyoma cells. Further studies revealed that aspirin blocked the interaction between K-Ras and p110α by co-immunoprecipitation and immunofluorescence. Western blotting demonstrated K-Ras-p110α interaction was required for the effects of aspirin-induced inhibition on cell growth and cell cycle transition via cell cycle regulators, including cyclin D1 and cyclin-dependent kinase 2 (CDK2). PI3K/Akt/caspase signaling pathway was involved in human uterine leiomyoma cell growth under aspirin treatment. Taken together, these results suggest that aspirin inhibited human uterine leiomyoma cell growth by regulating K-Ras-p110α interaction. Aspirin which targeting on interaction between K-Ras and p110α may serve as a new therapeutic drug for uterine leiomyoma treatment.
Uterine leiomyoma (fibroid) is one of the most common benign smooth muscle tumors with an estimated incidence of 75% in reproductive aged women and ~25% of fertile women bear clinical symptoms, such as heavy or abnormal uterine bleeding, pelvic pain and infertility (
Aspirin, the most commonly used non-steroidal anti-inflammatory agent for the treatment of fever, pain or other inflammatory conditions, has been identified as a potential chemopreventive drug supported by epidemiological data or clinical trials (
Kirsten rat sarcoma-2 viral (v-Ki-ras2) oncogene homologue (K-Ras) mutations represent genetic defect found in various human cancers. Activated K-Ras signaling contributed to promoting tumor initiation and cell proliferation (
In this study, we used human uterine leiomyoma cells to investigate the anti-proliferation function of aspirin and investigated the underlying mechanisms.
Human uterine leiomyoma (UtLM) cells (GM10964) were obtained from Coriell Institute for Medical Research (Camdern, NJ, USA). Cells were incubated in culture medium: Medium 199 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco), endothelial cell growth supplement (PromoCell, Heidelberg, Germany), heparin (Sigma-Aldrich, St. Louis, MO, USA), 105 U/l penicillin and 100 mg/l streptomycin (Gibco) at 37°C, in 5% CO2. Upon reaching 70–80% confluence, cells were subcultured with 0.1% trypsin with 0.02% EDTA (Gibco). Human uterine leiomyoma cells were used for the following experiments by aspirin (Sigma-Aldrich) treatment.
Aspirin with a purity exceeding 98% was purchased from Sigma-Aldrich. It was dissolved in distilled water at the desired concentrations.
Cell viability was assessed by Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Kumamoto, Japan). Cells (5×103 cells/well) were seeded in a 96-well plate. Plates were pre-incubated for 24 h in a humidified incubator. Then the cells were serum starved for another 24 h. Forty-eight hours after different treatments, 10% CCK-8 solution was added to each well of the plate and the plates were incubated for 2 h in the incubator. The absorbance was measured at 450 nm by BioTek microplate reader (Winooski, VT, USA).
Cell cycle was evaluated by flow cytometry. After appropriate treatment, cells were harvested, rinsed twice with phosphate-buffered-saline (PBS) and fixed in 70% ethanol for 24 h. Cells were incubated with 100 µl RNase A solution (KeyGen Biotech, Nanjing, China) for 30 min at 37°C and were added with 400 µl PI solution (KeyGen Biotech) for staining for 30 min in the dark. DNA content of cell distribution was analyzed at 488 nm by flow cytometry (EPICS XL-MCL; Beckman Coulter, Miami, FL, USA).
Total proteins were prepared from cultured human uterine leiomyoma cells. Protein was extracted with RIPA lysis buffer (Beyotime Biotechnology, Nantong, China) and protease inhibitor cocktail (Sigma-Aldrich). The protein content was evaluated using BCA Protein assay kit (Beyotime Biotechnology) and bovine serum albumin as the standard. Equal amounts of total protein were boiled and separated on SDS-PAGE gels (Beyotime Biotechnology) and transferred to PVDF membrane (Millipore, Bedford, MA, USA). The membranes were incubated with the blocking solution at room temperature for 1 h. Then the membranes were incubated with primary antibodies against K-Ras (mouse monoclonal, 1:1,000; Sigma-Aldrich), p110α (rabbit monoclonal, 1:1,000; Cell Signaling Technology, Beverly, MA, USA), p85 (rabbit monoclonal, 1:1,000; Cell Signaling Technology), p-p85 (Tyr-458, rabbit polyclonal, 1:1,000; Cell Signaling Technology), Akt (rabbit polyclonal, 1:1,000; Cell Signaling Technology), p-Akt (Ser-473, rabbit polyclonal, 1:1,000; Cell Signaling Technology), cyclin D1 (rabbit monoclonal, 1:1,000; Cell Signaling Technology), CDK2 (rabbit monoclonal, 1:1,000; Cell Signaling Technology), caspase-3 (rabbit monoclonal, 1:1,000; Cell Signaling Technology), caspase-9 (rabbit monoclonal, 1:1,000; Cell Signaling Technology), caspase-8 (rabbit monoclonal, 1:1,000; Cell Signaling Technology), caspase-12 (rabbit monoclonal, 1:1,000; Cell Signaling Technology), PARP (rabbit monoclonal, 1:1,000; Cell Signaling Technology), α-tubulin (mouse monoclonal, 1:1,000; Sigma-Aldrich), his (mouse monoclonal, 1:1,000; Sigma-Aldrich), flag (rabbit monoclonal, 1:1,000; Sigma-Aldrich) for 24 h at 4°C. Then the membranes were subsequently probed with respective secondary antibodies (1:2,000, Cell Signaling Technology) for 1 h at room temperature. The protein band signals of target bands were detected by Bio-Rad Molecular Imager ChemiDoc XRS plus System (Bio-Rad, Richmond, CA, USA). Quantification of band intensities was measured via ImageJ software (National Institute of Health, Bethesda, MD, USA).
The cell lysates were extracted using non-denaturing lysis buffer. Co-immunoprecipitation (co-IP) was done using the Thermo Scientific Pierce co-IP kit. The antibodies were first immobilized for 2.5 h using AminoLink Plus coupling resin. The resin was then washed and incubated with cell lysate overnight. Then the resin was washed and protein eluted using elution buffer. Samples were resolved on SDS-PAGE gels (Beyotime Biotechnology) and transferred onto PVDF membranes (Millipore). The bound proteins were determined by immunoblotting with the indicated antibodies.
Human uterine leiomyoma cells were seeded at a density of ~1×105/ml. After 24 h, pEnter-his vector, pEnter-his-K-Ras and pEnter-flag-p110α (Vigene Biosciences, Jinan, China) were transfected into the cells with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Plasmids and Lipofectamine 2000 reagent were diluted in serum and antibiotics free Opti-MEM® I (Invitrogen) to form the transfection complexes, respectively, for 20 min at room temperature and then added to the cells. After incubation for 6 h at 37°C, transfection complexes were replaced with DMEM containing 20% fetal calf serum. Cells were prepared for following experiments after 48 h.
The siRNA duplexes against K-Ras gene synthesized by Ribobio (Guangzhou, China) were transiently transfected with Lipofectamine 2000 reagent (Invitrogen). Scramble RNA was used as negative control. Scramble RNA and K-Ras siRNA strands and Lipofectamine 2000 reagent were diluted in serum and antibiotic-free DMEM to form the transfection complexes for 10 min at room temperature and then were added to cells. After incubation for 6 h at 37°C, transfection complexes were replaced with DMEM containing 20% fetal calf serum (Gibco). Cells were prepared for following experiments after 48 h. The siRNA sequences for K-Ras are as follows: negative control: sense-5′-UUCUCCGAACGUGUCACGU-3′; antisense-5′-ACGUGACACGUUCGGAGAA-3′. KRAS siRNA: sense-5′-GUCUCUUGGAUAUUCUCGA-3′; antisense-5′-UCGAGAAUAUCCAAGAGAC-3′.
Immunofluorescence was performed to detect the effects of aspirin on the p110α/K-Ras interaction. Human uterine leiomyoma cells cotransfected with his-K-Ras and flag-p110α in confocal well were incubated with primary his and flag antibodies (1:100, Sigma-Aldrich) at 4°C overnight, followed by the incubation with secondary Cy3 anti-mouse or FITC anti-rabbit antibodies (1:200, Beyotime Biotechnology) at room temperature for 1 h. Nuclei were stained with Hoechst (1:200, Beyotime Biotechnology) for 5 min. Cells were photographed by confocal system (Olympus, Tokyo, Japan).
Raw data were applied directly in statistical analysis. Statistical analysis was calculated using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Collected data were presented as the mean ± SD. Datasets with three or more groups were analyzed by one-way ANOVA. Comparisons of two groups were analyzed by Student's two-tailed t-test. The level for statistical significance was 0.05.
Human uterine leiomyoma (UtLM) cells (GM10964) obtained from Coriell Institute for Medical Research have been used widely for investigating cell proliferation of uterine leiomyoma in research as
To investigate the effects of aspirin on the growth of human uterine leiomyoma cells, human uterine leiomyoma cells were exposed to six different concentrations of aspirin (0.2, 1, 2, 4, 8 and 10 mmol/l) for 24 and 48 h. CCK-8 analysis was used to assess the effects of aspirin on cell viability. Aspirin induced a dose- and time-dependent inhibition in cell viability (
To investigate the underlying mechanisms of cell growth inhibition induced by aspirin in human uterine leiomyoma cells, we examined the effect of aspirin on cell cycle distribution. Different concentrations of aspirin (0.2, 1, 2, 4, 8 and 10 mmol/l) were used for 48 h in human uterine leiomyoma cells (
As reported, K-Ras was involved in various carcinomas and the interaction between PI3K p110α and Ras was required in tumorigenesis (
Western blotting showed that pEnter-K-Ras plasmid transfection increased K-Ras expression and anti-K-Ras siRNA (40 mmol/l) transfection significantly decreased endogenous K-Ras protein expression in human uterine leiomyoma cells (
The association between K-Ras and p110α was remarkably increased by K-Ras overexpression which could be inhibited by aspirin treatment (4.23 mmol/l) for 48 h (
In human uterine leiomyoma cells with increased K-Ras-p110α interaction induced by K-Ras overexpression, the cell viability was enhanced to 132.19±4.34% which was significantly reduced to 52.91±7.87% under the treatment of aspirin (4.23 mmol/l) for 48 h (
Cell cycle distribution was quantified by flow cytometry. The S-phase cell fraction in K-Ras overexpressed cells, with increased K-Ras-p110α interaction, was increased from 28.45±4.01 to 36.77±6.09% and the G0/G1-phase cell fraction was reduced from 65.59±7.52 to 58.73±4.67%, while the S-phase cell fraction was reduced to 12.67±1.09% and the G0/G1-phase cell fraction was increased to 82.34±9.01% under aspirin treatment (
Cell cycle transition is positively regulated by cyclins and cyclin-dependent kinases. To explore the molecular mechanism by which K-Ras-p110α interaction affects G1/S transition, we analyzed the proteins regulating cell cycle progression including cyclin D1 and CDK2. We found that the expression of cyclin D1 and CDK2 was enhanced in K-Ras overexpression group in which the interaction of K-Ras and p110α was increased while the protein expression was remarkably reduced under aspirin treatment (
In K-Ras overexpressed cells with the increase in K-Ras-p110α interaction, the levels of phosphorylated PI3K p85 and phosphorylated Akt were enhanced while the phosphorylated protein expression was decreased in aspirin treatment (
Caspase family members have been proved to be associated with PI3K/AKT signaling pathway in cell growth (
In the present study, we found that aspirin inhibits the proliferation and promote cell cycle arrest via cell cycle regulators of human uterine leiomyoma cells. These effects are at least in part by downregulation of K-Ras-p110α interaction and the subsequent modulation of PI3K/Akt/caspase signaling pathway.
Recent studies have demonstrated a wide variety of positive effects of aspirin on cancer therapy, however, little is known about the effects of aspirin on uterine leiomyoma. Thus, we showed that aspirin inhibited human uterine leiomyoma cell proliferation
PI3K along with Ras family small GTPases, mediating cell growth, differentiation, proliferation and multiple other cellular processes, are one of the most important early signaling components (
The K-Ras protein has been considered as a major target in the discovery of antitumor drugs because K-Ras mutation is commonly observed in various cancers and it lies at the apex of numerous growth regulatory cascades (
In conclusion, aspirin inhibits the proliferation of human uterine leiomyoma cells
This study was supported by National Natural Science Foundation of China (no. 81302771), Natural Science Foundation of Guangdong Province (no. 2014A030313087), Science and Technology program of Guangzhou City (no. 201607010255), and the Fundamental Research Funds for the Central Universities (no. 17ykzd02).
Aspirin inhibits human uterine leiomyoma cell growth. CCK8 analysis was used to measure the cell viability of human uterine leiomyoma cells after treatment with aspirin (0.2, 1, 2, 4, 8 or 10 mmol/l) for 24 h (A) and 48 h (B) (n=5–6, *P<0.05 vs con). (C) Inhibitory dose-response curves to determine the IC50 for aspirin on cell viability after 48-h treatment was calculated by non-linear regression using four-parameter logistic curves on GraphPad Prism 5 software (n=5–6, *P<0.05 vs con). All values represent mean ± SD.
Aspirin inhibits G1/S transition in human uterine leiomyoma cells. (A) Flow cytometry analysis of cell cycle distribution induced by various concentrations of aspirin for 48 h. (B) Percentage of cell number in different phases in cell cycle was determined by quantitative analysis (n=6–7, *P<0.05 vs con). All values represent mean ± SD.
Aspirin blocks K-Ras-p110α interaction in human uterine leiomyoma cells. (A) Western blot analysis of protein expression of human uterine leiomyoma cells under aspirin treatment (4.23 mmol/l) for 48 h. Human uterine leiomyoma cell lysates were harvested for co-immunoprecipitated assays to detect the endogenous (B) and exogenous (C) interaction (n=5, *P<0.05 vs con). (D) Confocal images showed K-Ras-p110α interaction induced by aspirin. Green is for flag-p110α and red is for his-K-Ras and yellow is for merge of green with red which accounted for the K-Ras-p110α interaction (n=3). All values represent mean ± SD.
K-Ras-p110α interaction is regulated by K-Ras expression under aspirin treatment. (A) Western blot analysis showed that pEnter-his-K-Ras plasmid transfection increased K-Ras expression in human uterine leiomyoma cells (n=6, *P<0.05 vs con). (B) Anti-K-Ras siRNA (40 mmol/l) transfection for 48 h significantly decreased endogenous K-Ras protein expression in human uterine leiomyoma cells (n=6, *P<0.05 vs con). (C) The interaction between K-Ras and p110α was increased in K-Ras overexpressed cells which was inhibited by aspirin treatment (n=6, *P<0.05 vs con). (D) The K-Ras-p110α interaction was blocked by K-Ras knockdown and aspirin had no further inhibitory effects (n=6, *P<0.05 vs con). All values represent mean ± SD.
Effects of K-Ras-p110α interaction on cellular proliferation under aspirin treatment. (A) CCK8 results showed that K-Ras overexpression promoted cell growth which was inhibited by aspirin treatment (n=5, *P<0.05 vs con). (B) K-Ras deficiency inhibited cellular proliferation and aspirin had no more inhibitory effects (n=6, *P<0.05 vs con). (C) Flow cytometry analysis showed that G1/S transition was promoted by K-Ras overexpression which was inhibited by aspirin treatment (n=6, *P<0.05 vs con). (D) K-Ras knockdown inhibited the cell cycle transition with no more inhibitory effects under aspirin treatment (n=6–7, *P<0.05 vs con). All values represent mean ± SD.
Effects of K-Ras-p110α interaction on cell cycle regulatory proteins. (A) Representative western blot images and densitometric analysis showed that K-Ras cDNA transfection significantly increased cyclin D1 and CDK2 expression which was reduced by aspirin treatment in human uterine leiomyoma cells (n=5, *P<0.05 vs con). (B) Knockdown of K-Ras reduced protein expression of cyclin D1 and CDK2 which aspirin had no more effects on (n=6, *P<0.05 vs con). All values represent mean ± SD.
Aspirin inhibits human uterine leiomyoma cell proliferation through K-Ras/PI3K/Akt signaling pathway. (A) Western blot analysis showed K-Ras cDNA transfection significantly increased the phosphorylation of PI3K p85 and Akt which was inhibited by treatment of aspirin (n=5, *P<0.05 vs con). (B) K-Ras deletion reduced the protein expression of phosphorylated PI3K p85 and Akt with no more inhibitory effects under aspirin treatment in human uterine leiomyoma cells (n=7–8, *P<0.05 vs con). All values represent mean ± SD.
Caspase activation and PARP cleavage induced by aspirin are dependent on K-Ras/PI3K/Akt signaling pathway. (A) Western blot results showed in K-Ras overexpression cells, the expression of cleaved caspase-3, −9, −8, −12, and PRAP were increased under aspirin treatment (n=4, *P<0.05 vs con). (B) Western blot results showed that cleaved caspase-3, −9, −8, −12, and PRAP were increased in K-Ras deletion human uterine leiomyoma cells and aspirin had no more enhancement effects on caspase activation and PARP cleavage (n=4–5, *P<0.05 vs con). All values represent mean ± SD.